Abstract:The Cynoglossidae fish is an important resource of germplasm in marine fish.In this study,21 measurable characters of four groups of Cynoglossus fish including one cultured population of Half-smooth tongue sole Cynoglossus semilaevis(Günther,1873)(Group Cs),one wild population of Roule's tongue-sole Cynoglossus roulei(Wu,1932)(Group Cr)and two wild populations of Three-lined tongue sole Cynoglossus abbreviatus(Gray,1832)(Group Cas,which was divided into Group Ca1 and Group Ca2)were compared using One-way ANOVA and Student-Newman-Keuls multiple comparison method.The results showed that there is no significant difference between Group Ca1 and Group Ca2,while significant difference existed between any other two groups.Furthermore,the gene fragments of cytochrome oxidation enzyme I subunits(COⅠ)and the cytochrome b(Cyt b)in mitochondria(mtDNA)were cloned and analyzed from the four tongue sole groups respectively.The intraspecific and interspecific genetic distances of the four tongue sole groups were further analyzed based on the COⅠ and Cyt b genes.Both morphological and phylogenetic analysis proved that Half-smooth tongue sole and Roule's tongue sole had a closer relationship,while it was far from Three-lined tongue sole.N-J phylogenetic evolutionary analysis indicated that COⅠ and Cyt b genes can be used as DNA barcoding in germplasm identification of these three tongue sole species.This study provided a clear genetic background for cross breeding,selective breeding and system development research for the three species of tongue sole fish.

Abstract:The ridgetail white prawn,Exopalaemon carinicauda supports a large yield of polyculture ponds in eastern China.However,the artificial breeding technique for E.carinicauda is not quite advanced,which limits the further development of the E.carinicauda culture,and there have been few studies on the mechanism of gonadal development and embryonic development.Recent studies have shown that ecdysteroid receptor(EcR)is not only involved in molting and growth of Arthropod but also plays important roles in ovarian development and embryogenesis.To study the regulatory role of EcR in ovarian development and embryogenesis of E.carinicauda,the full-length EcR cDNA of E.carinicauda(EcEcR)was cloned by the methods of degenerated oligonucleotide primers and rapid amplification of the cDNA ends(RACE),and quantitative real-time PCR was used to quantify the relative expression level of EcEcR in different tissues,ovarian development and embryogenesis of E.carinicauda.The results showed that the full-length EcEcR(GenBank accession number:KC506600)was 2 638 bp,containing an open reading frame(ORF)of 1 713 bp encoding a 570 amino acid polypeptide.Multi-sequence alignment analysis showed that EcEcR shared the high identities with Crangon crangon,Macrobrachium nipponens,Homarus americanus,however,it shared lower identity with EcRs from Marsupenaeus japonicas and Litopenaeus vannamei.EcEcR was expressed in various tissues and the highest in the heptopancreas.The EcEcR mRNA levels in ovary and heptopancreas of E.carinicauda reached a maximum at the stage Ⅲ and stage Ⅳ,respectively,and the EcEcR expression levels were higher in heptopancreas than in ovary during the ovarian development.However,the EcEcR mRNA levels in ovary at stages IV and V were significantly higher than those at stages Ⅰ and Ⅱ.Another very important finding was that significantly positive correlations existed between the EcEcR expression level and total vitellogenin content.During embryogenesis,the levels of EcEcR expression were higher at stages of gastula,protozoea and metazoea than those at other stages.The results indicated that the relationships displayed in the phylogenic tree of EcRs may correspond to their breeding practice,and EcEcR mRNA was expressed highly in hepatopancreas.EcEcR was involved in vitellogenin synthesis during ovarian development of E.carinicauda, and the EcEcR expression level in hepatopancreas was consistent with the gonadosomatic index.EcEcR played an important role in the ovarian maturation,and may play key roles in cell differentiation and organogenesis during embryo development.

Abstract:As the “energy factory”,the mitochondria play an important role in the metabolism of cells.However,it is little known whether or not the mitochondria in shrimps mediate the energy metabolism-related gene expressions by methylating the motochondrial genome DNA sequences.In this study,in oder to analyze the methylation profile of the mitochondria in response to starvation in the ridgetail white prawn Exopalaemon carinicauda,we designed 51 BSP(bisulfite sequencing PCR)primer pairs where PCR amplification zones covered the whole mitonchondial genome.Of the 51 primer pairs,10 primer pairs could get the predicted PCR products,but only 5 primer pairs were selected for further studies because of their repeatable PCR amplification merits.The methylation profile of different individuals at 10 d,20 d and 30 d after starvation stress were analyzed,using the above 5 primer pairs.Our results showed that the average death time of tolerating starvation for the E.carinicauda was 19.8 days,and the longest tolerating time was 48 days.The DNA methylation in mitochondria genome was found at each starvation stress stage.But,to our surprize,the methylation ratio decreased following the elongation of starvation time.The individuals with DNA methylation were the highest on 10^th d,second on 20^th d,and the least on 30^th d.The DNA methylation was mainly found in the ND genes(NADH dehydrogenase gene)whose expression production was responsible for maintaining the respiratory electron-transport chain in mitochondria.So,we speculated that the funciton of methylation is to control the energy transfer in mitochondia.

Abstract:In order to find more functional genes with EF-hand domain in Hyriopsis cumingii,we cloned the full-length cDNA of EF-hand calcium-binding domain-containing protein 1(EFCB1)by using RACE-PCR technique,and performed bioinformatics analysis;EFCB1 gene expression levels were analyzed in different tissues from Hyriopsis cumingii(mantle pallial,gill,visceral mass,adductor muscle,axe foot,gonad,liver,gut,haemolymph and kidney)and in different periods of the mantle and visceral mass after inserting nucleus.The results showed that the EFCB1 cDNA sequence length was 981 bp,containing a 531 bp open reading frame(ORF)encoding 176 amino acid residues,239 bp 5'-untranslated region(UTR)and 211 bp 3'-UTR.The molecular weight of the peptide was predicted 19.9 ku,with an isoelectric point of 4.70.Amino acid sequence analysis showed that there was a transmembrane region.Results of the ProtScale online analysis showed that the protein was hydrophilic protein.There were two typical EF-hand calcium binding domains,respectively DLNDDKLISPEE(98-109)and DTNGDDKLDGEE(129-140).Phylogenetic analysis indicated that the EFCB1 of H.cumingii was closely related to seawater bivalve Crassostrea gigas,followed by that of fish and then insect.Real-time Q-PCR results showed that the EFCB1 mRNA is ubiquitously expressed in various tissues(mantle paillial,gill,visceral mass,adductor muscle,axe foot,gonad,liver and gut),with the highest level in the gut and gill(P<0.05),and it is also highly expressed in the pearl culture tissues(mantle and visceral mass).These data suggested that EFCB1 plays an important role in the absorption process of Ca²⁺,and there might be an intrinsical relationship between EFCB1 and pearl formation of H.cumingii.Furthemore,expression differences were found among different periods of the mantle and visceral mass after inserting nucleus in the H.cumingii,and the expression level of EFCB1 in the mantle was significantly higher than that in visceral mass(P<0.05),after nucleus inserted 20 d it was significantly higher than other periods(P<0.05),suggesting EFCB1 has an important function in the formation process of pearl sac and nacre secretion,indicating EFCB1 might be an important regulator of pearl formation.The results of present study may provide useful information for further studies on regulation mechanism of calcium metabolism and pearl formation.

Abstract:The 70 ku HSPs are responsible for intracellular chaperone and extracellular immunoregulation,which play crucial roles in protecting cells against environmental stresses,such as heat shock,heavy metals,drought,disease and parasitization.In this researchpartial cDNA sequence of HSP70 was cloned from Mytilus coruscus.BLAST analysis and phylogenetic relationship strongly suggested that this sequence was a member of HSP70 family and highly homologous with other known HSP70 of bivalve,especialy with Mytilus galloprovincialis(96%),and they gathered into the same branch in the evolutionary tree.In heat shock of 37 ℃,the expression of HSP70 in gill and hepatopancreas was up-regulated rapidly and was 30 times higher than that of control mussels after 2 h.Then the expression increased as time went on,and reached the maximum peak approximately 100-fold in hepatopancreas,which showed that HSP70 of M.coruscus was thermal induced protein.The mRNA of HSP70 in hepatopancreas and gills after infection by Escherichia coli and Bacillus subtilis,which were common microorganisms in water,was detected based on RT-PCR analysis.The temporal expression of HSP70 mRNA was upregulated,especially in the group of E.coli challenge.The maximum level in hepatopancreas and gills appeared at 8 h post-injection with 4.0-fold in gills and 4.5-fold in hepatopancreas,and then dropped back to the original level at 48 h post-injection.In Bacillus subtilis infection,the transcripts of HSP70 in hepatopancreas was induced to the highest at 6 h after post-injection with 2.5-fold,which was similar to the effect of PBS injection.In gills the mRNA of HSP70 increased slowly to the highest at 8 h with 4-fold,then decreased quickly,and returned gradually to physiological level at the end of this experiment.All these results indicated that HSP70 in M.coruscus played a significant role in regulating the environmental stress and immune response.

Abstract:Matrix metalloproteinases(MMPs)could degrade the extracellular matrix and participate in various physiological processes,such as homeostasis and immune response.MMP-17 is one membrane type MMP that attaches to the cell surface by Glycosyl-phosphatidyl inositol(GPI),and participates in various physiological processes,such as organic homeostasis and immune defense.The pearl oyster Pinctada martensii is the main species cultured for marine pearl production in China.To deliberate the function of MMP-17 in the immune response of P.martensii,in this study,using rapid amplification of cDNA ends(RACE)technology,we have obtained the full length sequence of Pinctada martensii MMP(Pm-MMP-17)cDNA and analyzed its sequence characteristics and function.Our results showed that the cDNA full length of Pm-MMP-17 is 2 794 bp,including 156 bp of 5'UTR,715 bp of 3'UTR,and 1 923 bp of open reading frame(ORF)encoding 640 amino acid residues with an estimated molecular mass of 73.11 ku and theoretical isoelectric point of 8.98.Multi-sequence alignment and phylogenetic analysis results showed that Pm-MMP-17 was highly homologous with that from other species and had 82% sequence identity with Crassostrea gigas MMP-17.Amino acid sequence analysis showed Pm-MMP-17 had five conservative domains:signal peptide,propeptide,catalytic domain,hinge region and hemopexin-like domain.Meanwhile,real-time PCR analysis showed that Pm-MMP-17 was ubiquitously expressed in all tissues detected,including adductor muscle,pearl sac,foot,mantle,hemolymph,hepatopancreas,gonads and gills,with the highest expression level in hemolymph,followed by adductor muscle and gill.Previous research has showed that Pm-MMP-17 was highly expressed in the eosinophils and involved in the regulation of cell migration,growth and differentiation.We supposed that the highly expression of Pm-MMP-17 gene in the hemolymph was crucial for the migration of hemolymph in the body of pearl oyster.After LPS stimulation,the expression level of Pm-MMP-17 began to increase and reached the highest level at 12 h,and then gradually declined to the normal level(P<0.05),indicating again the important function of Pm-MMP-17 gene in the immune response of pearl oyser P.martensii.Therefore,our data showed Pm-MMP-17 participated in the immune response in P.martensii and provided the basis for further research in the mechanism underlying the immune defense in pearl oyster.

Abstract:The specific dynamic action in intestine is an important factor affecting transit time of food and feeding frequency,and can even influence the growth and development of decapod crustaceans.Moreover,that the midgut and hindgut of crustaceans perform different functions has also been proved.Nowadays,it is widely accepted that acetylcholinesterase(AchE)and nitric oxide synthase(NOS)positive neurons play an important role in the enteric nervous system of mammals.However,such studies in crustaceans are still rare.The aim of the present study was to clarify the distribution and morphological characteristics of AchE and NOS positive neurons throughout the E.sinensis midgut and hindgut using histochemistry,and to analyse the content of Ach and NO and Na⁺,K⁺-ATP enzyme activity of midgut and hindgut in E.sinensis by biochemical method.As far as we know,this is the first study in which whole-mount preparations of intestinal walls of midgut and hindgut in E.sinensis were respectively made and stained with H&E.The results showed that muscle layers,submucosa and basement-membrane layers were successfully obtained and observed in this study.Compared to the hindgut,the midgut muscle layer consisted of thin muscle fiber with obvious muscle fiber spacing.Submucosas of midgut and hindgut were made up of various cells.These cells were more abundant in the submucosa of hindgut than in midgut.Without obvious cell structure,basement-membrane was thin and transparent.AchE-positive neurons and NOS-positive neurons were distributed densely only in the submucosa,especially in hindgut.The AchE-positive neurons were colored brown,which ranged from 3 to 10 μm in size.The shapes of AchE-positive neurons in midgut were various,most of them were round,oval or spindle shape.The NOS-positive neurons with different sizes and shapes were colored blue.A few of these cells with cytoplasmic projections could be observed.The contents of Ach and NO and Na⁺,K⁺-ATP enzyme activity in midgut were obviously different from those in hindgut.The contents of Ach(1.28±0.41 μg/mg prot)and NO(1.84±0.25 μg/mg prot)in midgut were significantly lower than those in hindgut(Ach,1.62±0.27 μg/mg prot;NO,2.10±0.25 μg/mg prot)(P<0.05).However,Na⁺,K⁺-ATP enzyme activity of midgut(1.12±0.17 μmolPi/mg prot/h)was significantly higher than that of hindgut(0.62±0.18 μmolPi/mg prot/h)(P<0.05).In conclusion,AchE-positive neurons and NOS-positive neurons were detected mainly in the submucosa of E·sinensis intestine.There are obvious differences in the morphology of neurons and the content of neurotransmitter in midgut and hindgut of E.sinensis.

Abstract:In this study,marine bivalve molluscs species,Sinonovacula constricta,were exposed to benzo[a]pyrene(BaP)and phenanthrene(PHE)due to their ubiquitousness and bioavailability in the marine aquatic environment.Kinetic parameters(uptake rate constant(k₁),elimination rate constant(k₂),accumulation factor(BCF),and biological half-life(B_1/2))of the BaP and PHE bioaccumulation and depuration were calculated by two-compartment kinetic model for 15-day uptake and subsequent 9-day depuration periods.The results showed that the bioaccumulation ability of BaP was higher than PHE in S.constricta during 15-day uptake period.The increase of BaP was 0.45 ng/(kg·d)(40 μg/L groups),0.33 ng/(kg·d)(20 μg/L groups),0.23 ng/(kg·d)(10 μg/L groups),respectively.The increase of PHE was 0.33 ng/(kg·d)(30 μg/L groups),0.25 ng/(kg·d)(20 μg/L groups),0.18 ng(kg·d)(10 μg/L groups),respectively.The uptake and depuration periods of BaP and PHE were mainly in the initial stage of experiment.Those values of k₁ of BaP and PHE were calculated as varying from 0.39 to 0.52 and from 0.30 to 0.43,meanwhile,the average values of k₁ were 0.44 and 0.36,respectively.Those values of k₁ decreased with the increase of BaP and PHE exposure concentration in ambient seawater.Those values of k₂ of BaP and PHE were calculated as varying from 0.001 6 to 0.001 7 and from 0.002 8 to 0.003 3,meanwhile,the average values of k₂ were 0.001 6 and 0.003 0,respectively.There was no obvious variation trend of those values of k₂.Those values of C_Amax of BaP and PHE were calculated as varying from 3 062.76 to 9 758.25 μg/kg and from 1 308.54 to 3 171.84 μg/kg,meanwhile,the average values of C_Amax were 5 969.08 and 2 287.71 μg/kg,respectively.Those values of B_1/2 of BaP and PHE were calculated as varying from 407.73 to 433.22 d and from 210.68 to 248.06 d,meanwhile,the average values of B_1/2 were 421.20 d and 232.04 d,respectively.Those values of C_Amax and B_1/2 increased with the increase of BaP and PHE exposure concentration in ambient seawater.

Abstract:To investigate the seasonal variance of carbonic exchange in different grass carp ponds and explore the relationship between carbonic fluxes and environment factors of overlying water,three poly-culture systems,i.e.(1)GSB group:grass carp Ctenopharyngodon idellus,silver carp Hypophthalmictuthys molitrix and big head carp Aristichthys nobilis,(2)GSBL group:grass carp,silver carp,big head carp and Litopenaeus vannamei,(3)GSBC group:grass carp,silver carp,big head carp and carp Cyprinus carpio,were studied by simulation in the laboratory from April to September,2013.The changes of benthic fluxes of carbon across the sediment-water interface with time were studied,as well as the diurnal variation of carbon in the overlying water in August.The results showed that dissolved inorganic carbons(DIC)fluxes ranged from 0.65 mg/(m²·d)to 16.90 mg/(m²·d),while dissolved organic carbons(DOC)fluxes and particulate organic carbon(POC)fluxes ranged from 0.16 mg/(m²·d)to 3.49 mg/(m²·d),-2.29 mg/(m²·d)to 3.32 mg/(m²·d),respectively.Different carbon fluxes were all maximum in August for GSB and GSBC groups,but in July for GSBL group.During our experiments,the ex-fluxes of DIC and DOC were observed in every month.POC was up-taken by the sediments from April to June,but released from the sediments in other months.In the mid-to-late cultivation time,the benthic fluxes of DIC,DOC and POC in GSBL and GSBC groups were significantly higher than those in GSB group(P<0.05).The diurnal variation of carbons had obvious characters in August,with the content of DIC and DOC being maximum at 4:00 am.The content of POC in the GSBL got the maximum value at 4:00 am and the minimum value at 12:00 am,which was opposite to that of GSBC group.The results demonstrated a significantly negative correlation of carbon fluxes with DO and pH in overlying water,but a significantly positive correlation with overlying water temperature.

Abstract:A feeding trial was conducted to evaluate the optimum ratio at which dietary sodium dihydrogen phosphate(NaH₂PO₄)was replaced with citric acid in the diets of juvenile yellow catfish Pelteobagrus fulvidraco.Seven diets were formulated:positive control group(DP)that contained only 2% NaH₂PO₄ and negative control group(DN)that consisted of neither NaH₂PO₄ nor phytase;and five treatment groups supplemented with 2 g/kg citric acid and 20,15,10,5,0 g/kg concentrations of NaH₂PO₄.Thus,dietary NaH₂PO₄ was replaced with citric acid at ratios of 0%,25%,50%,75%,and 100%,respectively.The groups were desjgnated as D0,D25,D50,D75,and D100.Triplicate tanks were each filled with 50 juvenile ind.(3.5±0.1)g,which were fed with one of the seven experimental diets.After eight weeks,growth performance,apparent digestibility,body proximate composition,vertebra minerals and antioxidative capacity were investigated.The results showed that:(1)Citric acid supplementation enhanced weight gain and specific growth rate,reduced the feed conversion ratio compared with the negative group,but there were no significant differences with the positive group.;(2)Citric acid supplementation significantly enhanced the apparent digestibility of dry matter,protein and phosphorus and decreased fecal phosphorus and nitrogen content compared with negative group,while no significant difference with the positive group.The content of fecal nutrients decreased with the increasing of replacing level;(3)there were no differences among control groups and treatment groups in body moisture,crude protein,crude lipid and ash(P>0.05).Compared with the negative control,citric acid supplementation significantly improved the retention of Ca,Mg,P and Cu in the whole body.The highest retention of these nutrients was observed between 25% and 75% replacement levels.(4)Antioxidant enzymes activity(CAT,AKP and SOD)reached the highest between 25% and 75% replacement levels compared with the negative group while no differences were observed in serum MDA and IgM content.In conclusion,in order to reduce nitrogen and phosphorus output in the aquaculture water bodies,the replacing level of sodium dihydrogen phosphate with citric acid in yellow catfish diet between 25% and 50% will not affect fish growth performance and will help to improve nutrients utilization under the current conditions.

Abstract:To evaluate the effects of dietary available phosphorus(AP)levels on growth performance,body composition and biochemical indices of GIFT tilapia(Oreochromis niloticus),seven practical diets(isonitrogenous and isocaloric)were formulated to contain 0.52%(basal diet,control),0.61%,0.70%,0.78%,0.87%,0.96% and 1.05% of AP supplied with monocalcium phosphate(MCP).Each diet was fed to triplicate groups of 25 fish with an initial average body weight of(29.40±0.15)g for 56 d.The results showed that dietary AP levels significantly affected weight gain rate(WGR),specific growth rate(SGR),feed conversion ratio(FCR)and viscerosomatic index(VSI)(P<0.05).Quadratic regression analysis projected the optimal dietary AP level of tilapia to 0.80% based on WGR.With the increase of dietary AP level,the total phosphorus and crude ash contents of whole body markedly increased and then leveled off(P<0.05),while moisture contents of whole body were not significantly affected(P>0.05),whereas crude lipid contents of the whole body,mesentery and liver significantly decreased(P<0.05).Broken-line analysis showed that dietary AP requirement of tilapia were 0.81% and 0.80% respectively,based on the total phosphorus and crude ash contents of whole body.With increasing dietary AP level,hepatic activities of malate dehydrogenase(MDH),pyruvate kinase(PK),lipoprotein lipase(LPL)and hepatic lipase(HL)markedly increased(P<0.05).With an increment of dietary AP level,activities of serum glutamic-pyruvic transaminase(GPT)significantly decreased(P<0.05),but alkaline phosphatase(AKP)markedly increased(P<0.05).Serum superoxide dismutase(SOD)activity initially increased and then decreased,but liver MDA concentration showed contrary trends to that of SOD as dietary AP level increased.Highest hepatic SOD activity and the lowest liver MDA concentration were both recorded at 0.78% AP.In conclusion,the overall results in this study suggest that the optimal dietary AP level for GIFT tilapia(30-150 g)was 0.80%.

Abstract:In order to provide a basis for the research and development of formulated cuttlefish feeds for juvenile cuttlefish,the nutrient composition was investigated in 800 yolks of fertilized eggs of S.pharaonis.The contents of moisture,ash,crude protein,crude fat,amino acids and fatty acids of yolk of S.pharaonis fertilized eggs were determined by adopting national standard method.The results showed as follows:The content of crude protein in yolk of fertilized eggs of S.pharaonis was 76.33%(dry weight basis),and the contents of total amino acid(TAA)and essential amino acids(EAA)were 71.22% and 32.38%(dry weight basis).The ratio of EAA to TAA was 45.46%,and Glu was 9.97%.The content of crude fat was 12.71%(dry weight basis).A total of 17 fatty acids were found,including 8 saturated fatty acids SFA,5 monounsaturated fatty acids(MUFA),and 4 polyunsaturated fatty acids(PUFA).And they accounted for 43.47%,7.54% and 49.25% of total fatty acids,respectively.Moreover,the content of EPA and DHA in yolk was 32.80%,7.70%,respectively.The ratio of EPA to DHA was 4.26.The yolk was rich in Ca,Mg,Zn and Al.The content of Zn.Al and Fe in yolk was 0.77,0.71 and 0.43 mg/kg(fresh weight basis),respectively.In conclusion,the yolk of fertilized eggs of S.pharaonis has high contents of protein,low content of fat,various kinds and high contents of amino acids,and lots of PUFA,especially DHA.The formulated cuttlefish feeds should be high in protein,low in fat and rich in EAA and n-3 polyunsaturated fatty acids.For example,protein requirement is 76.33%,Lys requirement is 5.49%,fat requirement is 12.71%,DHA requirement is 4.17%,EPA requirement is 0.98%,and Cu requirement is 0.19 mg/kg,and Zn requirement is 2.77 mg/kg(dry weight basis).

Abstract:To provide information for controlling bacterial disease of aquatic animals,this study reveals the diversity and resistance of the drug-resistant bacteria around Anguilla rostrata and the farming ponds.Drug-resistant bacteria in the samples from intestine,epidermis,gill of A.rostrata and the pond water were screened by five antimicrobials.Their antibiotic resistance was determined by the Kirby-Bauer disc diffusion method and taxonomic status was identified by phylogenetic analysis of 16S rDNA sequences.The results showed that 108 resistant strains were isolated and classified into 20 genera,such as Aeromonas,Citrobacter,and Acinetobacter.The percentage of multiple resistance to three or more varieties of antibiotics was 93.5%,while the rate judged by three or more classes of antibiotics was 86.1%.The frequency of resistance to amoxicillin(90.7%)was high,and so with tetracycline,rifampicin,sulfonamides,and amphenicols(60%-80%).On the contrary,resistance to cefotaxime,neomycin and quinolones(less than 20%)was unfrequent.The multiple antibiotic resistance(MAR)indexes of isolates from intestine,epidermis,gill,and water were 0.40,0.41,0.42 and 0.47 respectively,which showed the high risk of MAR in every ecological sample,especially in the water sample.Among genera,MAR indexes of Citrobacter(0.58)and Klebsiella(0.61)were high,while that of Acinetobacter(0.21)was low relatively.Multi-drug resistant bacteria turned out to be ubiquitous in A.rostrata and the farming water,to which enough attention should be paid.Resistant bacteria from A.rostrata and water environments were mostly sensitive to some fishery drugs such as norfloxacin and neomycin.So they could be preferential drugs to treat the bacterial disease in aquaculture.

Abstract:: To explore the pathogenesis and pathogenesis regularity of edema disease and determine the clinical diagnosis of the disease,7 soft-shelled turtles with typical clinical symptoms were chosen as subjects,which were selected from some suspected cases with “Pelodiscus sinensis spleen-and-kidney-induced edema” in Hangzhou and Haiyan County.The study went through by clinical anatomy and making paraffin section,using H.E.staining.At the part of vivisection,the cases were intact at surface but with anorexia,less activity and edema in neck and limbs with varying degrees.Genitals of 2 cases prolapsed.Oropharyngeal cavity was found with congestion,inflammation and less mucus.Symptoms of nasal and oral hemorrhage,liver congestion,gastrointestinal wall edema and hyperemia were found in some cases.The number of erythrocyte was increased,lymphocytic aggregated,eosinophil infiltration in spleen.Necrosis appeared in some area of spleen.Erythrocyte was congested with eosinophil infiltration in renal interstitial and glomerular.Granular degeneration and vacuolar degeneration happened in distal convoluted tubule and the proximal tubule,and seriously even necrosis.In a summary,a diagnosis of spleen-and-kidney-induced edema could be made in pathology.Artery in villus of oropharyngeal cavity was expanded and congested.Erythrocyte exuded into oropharyngeal cavity and even serious necrosis.Goblet cells closely packed in the epithelial of oropharyngeal cavity.The number of lymphocyte increased and eosinophil infiltrated in submucosa of oropharyngeal cavity.It can be seen that oropharyngeal cavity pathological changes were closely associated with the disease.Pathological changes of other tissues are not serious.The results also show that there is no gland like salivary gland or parotid in soft-shelled turtle.The disease name “swollen neck disease”and“mumps”should be abolished.

Abstract:Sea cucumbers are easy to undergo autolysis after harvest or under physical stimulations.Proteinases in sea cucumber viscera are supposed to play important roles in the degradation of major structural protein collagen.In the present study,crude proteinases were extracted from sea cucumber viscera and their hydrolysis ability on gelatin was investigated by zymography.The degradation of gelatin by crude proteinases was strongly inhibited by serine proteinase inhibitor PMSF and metalloproteinase inhibitors,including EDTA and EGTA.It was of interest to notice that the gelatinolytic activity of an enzyme with molecular weight of 33.2 ku was activated by Ca²⁺ and inhibited by EDTA,indicating that it may belong to metalloproteinase.This proteinase was further purified to homogeneity from sea cucumber viscera by 80% ammonium sulfate fractionation and a combination of chromatographic steps including DEAE-Sepharose ion exchange,Sephacryl S-200 gel filtration,Phenyl Sepharose Fast Flow hydrophobic interaction and Mini Q Sepharose ion exchange.The optimum temperature and pH of the finally purified proteinase were 30 ℃ and 7.5,respectively.Its enzymatic activity was almost completely suppressed by metalloproteinase proteinase inhibitors(EDTA,EGTA)and serine proteinase inhibitors(PMSF,Pefabloc SC).Furthermore,the enzyme effectively hydrolyzed native sea cucumber collagen both at 4 and 37 ℃, suggesting its important role in the autolysis of sea cucumber.

Abstract:The purpose of this study was to investigate the effects of prepared hydrolysis peptides-Fe²⁺ complexes(TPH-Fe²⁺)on the depuration of cadmium(Cd²⁺)from red swamp crayfish(P.clarkii)tissues.The results indicated that the concentration of Cd²⁺ in crayfish tissues from high to low was visceral mass>intestine>gill>abdomen muscle>cheliped muscle respectively,all of which were not significantly decreased during the 12 days of depuration.However,significant differences were observed in groups feeding by 400,800,and 1 200 mg/kg TPH-Fe²⁺added into basal feed after 12 day's feeding.The Cd²⁺ concentrations in intestine,visceral mass,gill,and abdomen muscle were significantly decreased by 40.60%,36.13%,33.36%,and 10.15%,respectively.Nevertheless,the variables of Cd²⁺ concentrations in heliped muscle treated with 400-1 200 mg/L TPH-Fe²⁺ showed no significant differences from controls at any point during the experimental period.For these reasons,TPH-Fe²⁺ can be recommended as depuration agent in crayfish feed to decrease the Cd²⁺ concentrations in tissues.

Abstract:With the fast development of aqua-feed industry,fish oil has seen a great shortage,which limits the sustainable development of aquaculture industry.Alternative oils are thought to be the key to solve this problem.However,decreased immune response and disease resistance have been found as the substitution level increased.The nature of fish oil replacement is actually the fatty acid replacement.Thus,a review was made on the effects of fatty acids on immune response and mechanism involved in this process.Dietary saturated fatty acids could decrease fish immunity,while optimal inclusion of n-3 long chain polyunsaturated fatty acids(LC-PUFA)and conjugated fatty acid(CLA)or enhancing the ratio of n-3 to n-6 polyunsaturated fatty acids(PUFA)were beneficial to nonspecific immune responses of fish species.There have been a lot of studies which aimed to elucidate mechanism about how dietary fatty acid regulates fish immunity.Overall,dietary fatty acid could regulate fish immunity through several pathways such as membrane structure,signal transmission,eicosanoid,cytokine and steroid etc.These indicated that fatty acid is a critical factor to regulate fish immunity.Greater efforts should be made to study mechanism involved in this process.Future studies should be emphasized from the following perspectives:how dietary fatty acid regulate transcription factor about immunity;Relationship between fatty acid composition of intestine,bacterial community structure,and immune responses;Effects of environmental factors on fatty acid metabolism and immunity of fish species;how non-fatty acid nutrients(minerals and vitamins)regulate fatty acid metabolism and fish immunity.