Abstract:The photoreceptor cells and the angle of minimum resolution(AMR)for four species of coral-reef teleosts,Sebastes hubbsi,Sebastes nigricans,Hexagrammos otakii and Hexagrammos agrammus have been studied by means of common histological methods.The main results obtained are as follows:(1)Cone photoreceptors in the retinas of H.otakii and H.agrammus are arranged in a mosaic:four double cones are arranged in a square around a single central cone.The double cones of S.hubbsi and S.nigricans form highly organized rows and the single cones are distributed around four double cones occasionally.Compared with S.hubbsi and S.nigricans,the arrangement of cones for H.otakii and H.agrammus is more complex,and the ratio of single cones to double cones is greater in the retinas of H.otakii and H.agrammus,which may indicate that H.otakii and H.agrammus have a better color vision.(2)The ratio of rod cells to cones of S.hubbsi and S.nigricans is greater than 7：1,but for H.otakii and H.agrammus the ratio is less than 3：1.It is speculated that S.hubbsi and S.nigricans are nocturnal,while H.otakii and H.agrammus are diurnal.(3)The areas of greatest cone density for S.hubbsi,S.nigricans,H.otakii and H.agrammus are found in the ventral-temporal region,ventral region,ventral-temporal region and dorsal-temporal region,respectively,which is adapted to their ecology and feeding behavior.(4)For the four experimental species of fish,the AMR with S.hubbsi,S.nigricans,H.otakii and H.agrammus decrease in sequence.The preliminary analysis suggests that even in the same habitat,the visual characteristics for different species of fish vary a lot,which may provide some beneficial reference for the development of high selective fishing gear.

Abstract:The complete cDNA sequence of luteinizing hormone receptor(LHR)gene in turbot(Scophthalmus maximus L)was cloned by degenerate primer PCR amplification and RACE technology,and provided a bioinformatics foundation and tissue expression for the study of LHR genetic characters.The full length of LHR gene is 3 814 bp,encodes 685 amino acids and shares a higher degree of homology with the Hippoglossus hippoglossus.The amino acid sequence analysis revealed that the LHR gene encoded hydrophobic protein and its relative molecular weight was 76.54 ku,isoelectric point was 7.22,and contained signal peptides and transmembrane domains.Subcellular localization of LHR was in endoplasmic reticulum and cell membrane.Meanwhile,the amino acids sequence contains thirty-three phosphorylation sites,five glycosylation sites,6 leucine-rich repeats,SCOP,PDB and seven conserved transmembrane helix domains.Furthermore,the secondary structure of LHR was mainly composed of random coil.The tertiary structure of domain area of LHR protein showed α-helix structure.In addition,we found LHR protein probably plays a key role in transport,translation and metabolism process.Tissue expression analysis showed LHR mRNA was preferentially expressed in ovary,whereas strong amplification signal was also detected in liver.Therefore,the above results laid a genetic information foundation for further study of the LHR gene function in the turbot gonads development,providing the theoretical basis for fish breeding in marine aquaculture.

Abstract:In order to select suitable sea gulfs from the coastal waters of Qingdao for the growing of released Fenneropenaeus chinensis,we studied the species composition of fishery resources,stock biomass and growth characteristics of released F.chinensis in the Aoshan Bay from June to September 2010.The results showed that:(a)five bottom trawl surveys showed that the Oratosquilla oratoria had the highest catch,being 46.66% of all species' catches,while the catches of Cryptocentrus hexanema and Platycephalus indicus that are the predators of F.chinensis were 1.28%.The evaluation grade to biomass of phytoplankton was Ⅴ;(2)The trawl surveys showed that the recapture rate of F.chinensis was 0.10%,while the recapture rate of F.chinensis from production investigation of fisherman was 1.20%,a little lower than the results of most surveys in Bohai Sea.(3)The F.chinensis had fast growth rate,the male and female F.chinensis had different growth pattern during their growth period,the maximal dW_t/dt of female F.chinensis was 0.88,and male was 0.47 g.The growth rate of male was faster than that of the female at early growth period.In late growth period,the growth rate of male and female F.chinensis ran counter to that of male and female F.chinensis in the early growth period.The body length of female F.chinensis was larger than that of male F.chinensis in catchable time.The body length asymptotic values of female and male F.chinensis were L_∞=200.83 mm and L_∞=172.64 mm respectively,while the weight asymptotic values of female and male F.chinensis were W_∞=104.05 g and W_∞=38.81 g respectively.The average weights of male and female F.chinensis in catchable time were 34.20 and 43.25 g respectively,reached the optimum catchable size.According to the species composition of fishery resources and the evaluation grade to biomass of phytoplankton in the Aoshan Bay,the growth characteristics and stock biomass of the released F.chinensis,we think that the Aoshan Bay is the suitable growth area for releasing F.chinensis.

Abstract:Annealing control primer(ACP)system was applied to find candidate genes related to muscle growth of Nile tilapia by screening differentially expressed genes in the muscle tissues of male and female fish.This study randomly selected five male and five female fish from Nile tilapia populations cultured under the same conditions to build a RNA pool,and differentially expressed genes of two groups were analyzed by annealing control primer system.Eight differentially expressed genes were identified and sequenced by amplification with 20 arbitrary primers.In these genes,five were already known as variant 3(LOC100691543),60S ribosomal protein L3(RL3),Parvalbumin beta-like,muscle-type creatine kinase M2-CK and transcription factor Sox4 gene,and others,while three were unknown.Base on quantitative real-time PCR,the relative expression levels of transcription variant 3(LOC100691543) and ACP6-Y in the muscle of male Nile tilapia were significantly higher than those in female fish(P<0.01),while the relative expression levels of ACP3-X,60S ribosomal protein L3(RL3),Parvalbumin beta-like,ACP15-X,muscle-type creatine kinase M2-CK and transcription factor Sox4 in the muscle of female Nile tilapia were significantly higher than those in male fish(P<0.01).All the 8 ESTs were screened by annealing control primer system,which may have participated in regulating muscle growth of male and female Nile tilapia.This study laid a foundation for further screening of muscle growth related genes.

Abstract:Based on the data of lake survey conducted during 2007—2010,an mass-balanced ecosystem model of Lake Chaohu was constructed using Ecopath with Ecosim 6.1,with the aim of characterizing its ecosystem structure and functioning,which would help making ecosystem-based management.The model comprised 16 functional groups including primary producers,the main species of fishes,non-fish vertebrates,and detritus.Results showed that the functional groups are organized into four trophic levels.The system throughout,production and consumption are relatively large,with the values of 41 003.08,17 937.42 and 4 486.67 t/(km²·a),respectively.Flows from trophic Ⅱ and Ⅲ dominated the ecosystem.Considering Odum's theory of ecosystem development,Lake Chaohu was placed on a low developmental stage with higher net primary production(NPP),total primary production/total respiration(TPP/R)and lower connectance index(CI),system omnivory index(SOI),Finn's cycling index(FCI)and Finn's mean path length(FMPL).The cause of the degeneration of Lake Chaohu ecosystem was considered to be the high biomass and production of primary producer,phytoplankton and its low rate of utilization.

Abstract:The study aimed to better understand the constitution and expression pattern of common carp(Cyprinus carpio)globin family,and their contributions to hypoxia adaptation of the species.Complete gene sequence of common carp neuroglobin(Ngb)was obtained by BLAST searching in draft genome sequence and screening in full-length cDNA library,proving that Ngb gene co-existed in common carp genome with myoglobin-2(Mb-2),a unique paralog of globin previously reported to be specifically expressed in brain.Quantitative real-time PCR results indicated that common carp Ngb gene expression was brain-specific and hypoxia responsive.Ngb expression was up-regulated during the aquatic surface respiration,and followed by a decline when the fish fell into coma,then no significant change in Ngb expression was observed during the reoxygenation recovery.Analysis on gene structure and phylogeny indicated that teleost Ngb genes were highly conserved,suggesting that common carp Ngb might have similar protein structure and biological functions to its zebrafish ortholog,while distinct from the paralogous Mb-2.Expression quantity was significantly different between two common carp strains with distinct hypoxia tolerance.Nearly 10 times higher Ngb expression was detected in the hypoxia tolerant mirror carp.These results suggested that Ngb and Mb-2 might serve the brain in different ways,to accomplish better oxygen delivery and neuron protection against hypoxia,playing an important role in hypoxia adaptation of common carp.

Abstract:In order to study eukaryotic plankton community succession,and the relationship between eukaryotic plankton community and physicochemical factors in the ponds with Carassius auratus gibelio in coastal mud flat,water samples were collected monthly in 2011.We measured the relevant physicochemical factors,analyzed the diversity of eukaryotic plankton community by 18s rDNA PCR-DGGE technique,and analyzed the relationship between the plankton community and physicochemical factors by canonical correspondence analysis(CCA)method.The results showed that the concentration of total phosphorus(TP)ranged from 0.17 to 1.12 mg/L all the year round,and presented its peak value in July.The range of phosphate(PO₄-P)concentration was from 0.04 to 0.30 mg/L,and the peak values occurred in July and August.The range of nitrite(NO₂-N),ammonia-nitrogen(NH₄⁺-N)and nitrate(NO₃-N)concentration was 0.02-0.57 mg/L,0.20-2.37 mg/L,0.04-10.47 mg/L,and their peak values occurred in December,May and November,respectively.DGGE profiles of 18S rRNA gene fragments from the pond revealed that the diversity of eukaryotic plankton assemblages was highly variable.A total of 73 observable bands were amplified from January to December,and the average was 24.67.Shannon-Wiener index of pond was higher in May,June,August and September.The cluster analysis(UPGMA)dendrograms of eukaryotic plankton revealed remarkable seasonality except for March,July and November.August,September and October clustered into one group.April,May and June clustered into one group,and January,February,December clustered into one group.CCA revealed that temperature,total phosphorus and ammonia-nitrogen played a key role in the structure of the eukaryotic plankton community in pond,which can provide a theoretical basis for ecological regulation of aquaculture and optimization of aquaculture model.DGGE method was a cost-effective way to gain insight into seasonal dynamics of eukaryotic plankton communities in culture ponds.

Abstract:In this paper,the MAPKK(mitogen-activated protein kinase kinase)was isolated from the mud crab,Scylla paramamosain using RT-PCR and RACE methods.The obtained full-length cDNA of MAPKK was 1 558 bp with an open reading frame of 1 224 bp encoding a putative peptide of 407 amino acids.By alignment,the amino acid sequence of S.paramamosain MAPKK showed high homology with those of some other animals.It suggested MAPKK was highly conservative.Real-time PCR showed that the MAPKK gene was expressed in various tissues,and highly expressed in brain ganglion and ovary.The MAPKK mRNA profiles during ovarian development indicated that the expression of MAPKK was significantly high at developing stage.We inferred that MAPKK might play a stimulative role in the ovarian development of the mud crab.

Abstract:: The 3×2 two factors random animal experiment was conducted for 56 days to evaluate the effects of dietary fat and protein on digestive enzymes activities in alimentary canal and liver,nonspecific immunity of Atlantic salmon and the water environment reared at recirculation aquaculture systems(RAS).The trial fish(initial weight 650±45.5 g)were randomly allocated into 6 triplicated treatments and fed six kinds of diets in a factorial design containing three fat levels(18%,21%,24%,labeled as F18,F21,F24)and two protein levels(38%,48%,labeled as P38,P48).Results show that:(1)the stomachal,intestinal and hepatic lipase activities in medium fat level were significantly higher than that in low fat level by 11.52%(P>0.05),14.63%(P<0.05),4.31%(P>0.05)respectively;trypsin showed much higher activity than pepsin in gut and liver,and activities of trypsin in gut and liver were enhanced at high protein level by 8.23%(P>0.05)and 8.39%(P<0.05);(2)diet low fat and high protein levels could be good to promote the intestinal canal AKP activity of Atlantic salmon,intestinal AKP activity was significantly improved at low fat level by 18.61%,31.70%(P<0.05)than that at high and medium fat levels respectively,and was also enhanced by high protein level by 13.69%(P<0.01).(3)Atlantic salmon had a highest serum SOD activity at medium fat level and a lowest MDA activity at high fat level;high protein level was beneficial to increase the serum LZM activity and C3 concentration by 9.49%(P>0.05)and 5.93%(P<0.05).(4)diet low protein level can significantly reduce the content of ammonia nitrogen and nitrate in water by 61.70%(P<0.05)and 28.36%(P<0.01),diet high fat level could reduce water ammonia nitrogen content by 10.00%(P>0.05)and 8.20%(P>0.05)than medium and high level.This research revealed that appropriately increasing diet fat level could benefit the lipase activity,increasing diet protein level could benefit immunity,and the combination of low protein and high fat could effectively reduce the ammonia nitrogen excretion of Atlantic salmon.

Abstract:Heat shock protein(Hsp90),representing an important molecular chaperone in eukaryotic cells,plays particularly important roles in a variety of stress responses,development and signal transduction of plants.In this study,based on unigene sequences which were obtained from whole transcriptome sequencing of P.haitanensis,two full-length PhHsp90 genes were obtained by rapid amplification of cDNA ends(RACE),and named PhHsp90-1 and PhHsp90-2.The full-length cDNA of the PhHsp90-1 gene comprised 2 572 nucleotides and contained an open reading frame of 2 427 bp(GenBank accession:KF732652),encoding a protein of 809 amino acid residues with the predicted molecular weight of 90.2 kDa and theoretical isoelectric point of 4.79;and the full-length cDNA of the PhHsp90-2 gene comprised 2 510 nucleotides and contained an open reading frame of 2280bp(GenBank accession:KF732651),encoding a protein of 760 amino acid residues with the predicted molecular weight of 86.2 kDa and theoretical isoelectric point of 4.81.On the basis of conserved motifs and phylogenetic tree analysis,PhHsp90-1 belongs to the endoplasmic reticulum subfamily of Hsp90 and PhHsp90-2 belongs to the cytoplasmic subfamily of Hsp90.The expressions of the two PhHsp90 genes,as measured by real-time quantitative PCR,were significantly induced by high-temperature stress and desiccation stress,but had different expression patterns.Under high-temperature stress,the expression levels of the two PhHsp90 genes all significantly increased first and then decreased.However,during desiccation,the expression levels of PhHsp90-1 and PhHsp90-2 were significantly increased only when the water loss was >60%.These results suggested that the two PhHsp90s play important roles in the response to high-temperature stress and extreme desiccation stress.

Abstract:In summer of 2011,harmful algae bloom(HAB)caused by picoplanktonic alga Aureococcus anophagefferens broke out again along the coast of Qinhuangdao Island,China.In order to understand the toxicity and the impact on human health,acute toxicity of the bloom water against Artemia sinica and the feeding rate of A.sinica on A.anophagefferens were studied under laboratory conditions.Stimulation effects of A.anophagefferens monoclonal culture on skins and eyes of rabbits were also studied.In addition,acute oral toxicity tests were carried out on mice.The results showed that the bloom water has no impact on the survival of A.sinica within 48 h.A.sinica could feed on A.anophagefferens,and the feeding rate increased with cell density of A.anophagefferens.The highest feeding rate of A.sinica within 8 h was up to 3 287 cells/ind/h.A.anophagefferens had no discernible effect on rabbits' skins and eyes,and also had no effect on mice.It is relatively safe for people working or swimming in the sea during the A.anophagefferens bloom.

Abstract:To explore the probable function of segment 10(S10)encoded protein of grass carp reovirus(GCRV)HZ08 strain,the ORF of S10 gene was amplified and cloned into the prokaryotic expression vector pET-32a(+),and the obtained recombinant expression vector was named pET32a-S10.The recombinant vectors were transformed into BL21 competent cells.The Escherichia coli containing recombinant vector expressed fusion protein of approximately 53 ku after induction by IPTG.The recombinant protein was purified through Ni-chelating affinity chromatography,and the purity was above 97.4% explained by SDS-PAGE gel scan analysis.Polyclonal antibody against S10 encoded protein was generated by immunization of female Kunming mice with purified recombined protein.The antibody titers in sera of the immunized mice were detected by ELISA and the specificity of the antibody was identified by Western blot and indirect immunefluorescence assay(IFA).The results showed that the antibody titer cloning is 1：10⁶ and can specially identify the GCRV-HZ08.This means the S10 encoded protein is one of the structural protein of GCRV-HZ08.

Abstract:The gamete quality of F₁-generation Chinese sturgeon(Acipenser sinensis)was evaluated by a series of biological indexes of eggs(diameter,weight,fertilization rate and hatching rate)and sperm(the highest activation rate,life time and fast movement time).And the growth characteristics of F₂-generation(2012-11-2013-07)were analyzed by following up the development situation.Results showed that the eggs of F₁-generation were full shape and the average diameter was 3.71 mm,which was less than that of wild population.The fertilization rate(60.1%)and hatching rate(36.8%)were both at lower-middle level when compared with data over the years.Sperm motility in 2012 and 2013 showed that the effective moving time(circinate movement time+fast movement time)were longer than those of wild population.It indicated that eggs quality of F₁-generation was lower than that of wild population,and sperm quality was higher yet.In comparison with F₁-generation cultured by intensive farming system in the same aquaculture base over the years,F₂-generation showed obvious growth phase differences.That is,the growth rate was slower in larvae phase but faster obviously in juvenile and young fish phase.Consequently,it was effective to get high quality gamete and F₂-generation by improving the gonad development perfection of cultured F₁-generation in order to prevent the degradation of germplasm.And it was vitally important for Chinese sturgeon species conservation.

Abstract:Red crucian carp(abbreviated as RCC)with 100 chromosomes belonged to the Cyprininae subfamily,and the blunt snout bream(Megalobrama amblycephala)(abbreviated as BSB)with 48 chromosomes belonged to the Cultrinae subfamily.In the first generation of RCC(♀)×BSB(♂),we obtained triploid hybrids(abbreviated as 3nRB)and tetraploid hybrids(abbreviated as 4nRB).In this study,we made the analysis of karyotype of the chromosomes,FISH of the chromosomes and the gonadal development in 3nRB.The results are as follows:(1)3nRB hybrids had 124 chromosomes with a karyotype of 31m+45sm+26st+22t,which possessed two sets of RCC-derived chromosomes,and one set of BSB-derived chromosomes.(2)The species-specific repetitive sequences probe hybridized to 100 chromosomes in RCC but none in BSB.In 3nRB,the probe hybridized to 100 chromosomes,suggesting that they possessed two sets of RCC-derived chromosomes.(3)The gonad of 3nRB displayed developmental anomaly,which was considered as a retrogression trait.This paper discussed the formation mechanism of 3nRB,and provided experimental data for study of distant hybridization and polyploidy in fish,which is of importance in genetic breeding.

Abstract:Ichthyophthirius multifiliis(Ich)is one of the most pathogenic ciliated protozoan parasitizing the gills and skin of fishes,and can cause mortality of a large number of cultured fishes,subsequently resulting in a serious economic loss in aquaculture.So it is necessary to control the parasitic ciliate in aquaculture.In the past several decades,malachite green was frequently used to treat ichthyophthiriasis in fish farms.However,malachite green has been banned nowadays for use in food fish due to its carcinogenic and genotoxic effects on humans.So far,there is none of both effective and safe therapeutants to treat Ich,therefore,there is an urgent need to find both efficacious and safe parasiticides to control Ich.Previous studies showed some Chinese medicinal plants contained anti-Ich compounds,which were easily degradable,and safe for human and water environment,and could further be developed into promising therapeutants against Ich,but the anti-Ich activity of Cinnamonum cassia Presl,a Chinese medicinal plant,was unknown.The present study was designed to investigate the anti-Ich activity and compound of C.cassia Presl with grass carps under in vitro conditions.The powdered C.cassia were extracted primarily with 95% ethanol.Then the crude extract was sequentially extracted with petroleum ether,ethyl acetate and methanol,respectively.The anti-Ich efficacy of petroleum ether fraction was the best among the 3 extracts.Thereby,bioactivity-guided fractionation and isolation of the compound responsible for anti-Ich activity were carried out with the petroleum ether extract,and one purified compound with anti-Ich activity was obtained,and subsequently identified as cinnamaldehyde by means of MS,¹H NMR,¹³C NMR spectroscopic analyses.The anti-Ich compound,cinnamaldehyde,could cause 100% mortality of trophonts at 50 mg/L,and theronts at 8 mg/L for 4 h exposure.The 4-h EC₅₀ values of cinnamaldehyde against trophonts and theronts were 13.9 mg/L and 1.8 mg/L,respectively.The compound also stopped reproduction of all encysted tomonts at 50 mg/L.These data confirmed that cinnamaldehyde is effective against I.multifiliis,and promising for development of a new,safe and efficacious antiparasitic agent.

Abstract:Ridgetail white prawn(Exopalaemon carinicauda)is an important commercially exploited species in China,however,its speedy cultural development suffered the seedling shortage because of the absence of standard protocol in artificial seedling.To provide the basic knowledge needed in artificial reproduction,a histological study on the gonadal development of E.carinicauda was performed by the paraffin section technique and HE staining method.The results show that the female reproductive system consists of ovary,oviduct and oviporus.Oogenesis could be classified into five different phases and the ovary development could be divided into five stages accordingly.The average gonad index varied from 0 to 10%.The female could spawn several times in the reproductive season.The male reproductive system is composed of testis,vas deferens and male gonopore.The testis consists of many seminiferous tubules,in which there are numerous sperms at different development stages.The spermatogenesis could be classified into five phases,i.e.,spermatogonium,primary spermatocyte,secondary spermatocyte,spermatid and sperm.The vas deferens where the spermatophore forms could be separated into anterior,medial,posterior and terminal segments.

Abstract:The Japanese scallop(Mizuhopecten yessoensis),naturally occurring in Russia,Japan and North Korea,was introduced to China in 1980s.It is economically important and has become a major aquaculture species in the northern Yellow Sea.Recently,"Summer mortality syndrome" becomes a serious problem for scallop growers.During warm summer months,episodes of high mortality coincide with periods of elevated water temperature,low dissolved oxygen and disease outbreak.Mortalities appear to result from a poorly-understood interaction between high temperature,high density aquaculture,genetic depression and opportunistic infection by microorganisms.Resistance to summer mortality may be heritable in scallops,and phenotypic selection has been used to produce resistant strains in other bivalves.However,the broodstock of M.yessoensis used remains unselected in China.We initiated a family selection program for summer mortality resistance in 2011.Selective breeding programs for improving the scallop stocks are expensive,labor-intensive,and typically rely on lengthy field trials in which selection for survival is compromised by inherent stochasticity of outbreaks of "summer mortality syndrome".Reliable laboratory assays that identify and eliminate poor-performing families prior to planting could improve selection efficiency.We tested the hypotheses that juvenile survival after heat shock predicts adult survival at harvest for full-sib families of M.yessoensis.We heat-shocked(18 and 23 ℃)juveniles from each of 10 families,and monitored their survival for 5-20 days,then classified A08 and A09 families as high-surviving and the others as low-surviving.We also deployed replicated groups of siblings from all 10 families in coastal waters of Dachangshan Island,Dalian for one year.At harvest,we estimated family-specific average survival(%)and average individual shell height.We found that the high survival rates in both A08(34.3%±3.5%)and A09(46.5%±5.3%)families were identical with the predicted values after heat shock,and there was no family-level correlation between juvenile survival and shell height.We conclude that assays measuring heat tolerance in early juveniles hold promise of predicting performance of M.yessoensis families planted in coastal waters.

Abstract:In order to study the dual endocrine effect on the gonadotropin release of pituitary in the ancient Chondrostei fish,luteinizing hormone releasing hormone analog(LHRH-A)and dopamine(DA)were used in vitro to research their effect on gonadotropin release of pituitary fragment in Amur sturgeon(A.schrenckii).Two experiments were designed to study how the LHRH-A and DA affect the GtH release of pituitary fragment:Three times repetitive pulse stimulation of 10,100 and 1 000 nmol/L different dosages of LHRH-A were introduced every one hour for five minutes,perfusion;And then after two hours,perfusion of 200 nmol/L DA,1 000 nmol/L LHRH-stimulation was introduced for five minutes.The perfusate was collected every five minutes to test the GtH content using Radio Immunoassay(RIA)method.The results showed that repetitive pulse administration of LHRH-A was able to be stimulating GtH release from the pituitary fragments in vitro in Amur sturgeon,without dose-dependent manner at the dosage from 10 nmol/L to 1 000 nmol/L.It seemed LHRH-A at high concentration restrained the release of GtH by itself.Dopamine(DA)had no obvious effect on basal GtH secretion of pituitary while it could restrain the GtH release of pituitary fragment induced by LHRH-A.It proved that dual endocrine regulation of GtH release also existed in sturgeon.

Abstract:To provide a better understanding of the role of hemocytes in internal defence and to expand the knowledge of environmental immunotoxicology in P.viridis,it is essential to explore the fundamental knowledge of hemocytes.Three major types of hemocytes were identified in the hemolymph,including granulocyte,semi-granulocyte(small and large sizes)and hyalinocyte.Granulocytes were intermediate-sized cells with diameter of (7.49±1.32) μm and accounted for 40.4%±8.4%,containing numerous granules in the cytoplasm.Semi-granulocytes were the largest cells with diameter of (12.45±2.21) μm and percentage of 34.6%±9.4%,containing some granules.Small semi-granulocytes were only different from semi-granulocytes in the cell size with diameter of (6.64±1.05) μm.Hyalinocytes were the smallest[diameter,(4.69±1.01) μm]and least abundant(15.6%±4.8%)with few granules.Flow cytometry has revealed that the dense-granulocytes are the most active in cell phagocytosis,whereas the dense-granulocytes and semi-granulocytes have similar activities in terms of esterase activity and reactive oxygen species(ROS)generation.In contrast,the hyalinocytes showed the lowest levels in phagocytosis,enzymatic activities and ROS production.Hyalinocyte mortality was higher compared with the other two hemocytes.The immune functions assessed by the flow cytometry indicated that the granulocytes are the main hemocytes involved in the cellular defence in the green-lipped mussel.