Abstract:Cdc2(Cyclin Dependent Kinase, namely CDK1) encoded by cdc2 gene and CyclinB combination regulates G2/M transition. To find out molecular mechanism that diploid hybrid fish could produce diploid gamete, the full length cDNAs of cdc2 in the third gynogenetic generation (G3), red crucian carp( Carassius auratus red var.), triploid crucian carp and allotetraploid were obtained by PCR and rapid amplification of cDNA ends. Our data showed that all the cDNAs of cdc2 gene in the four different ploidy fishes encode a protein of 302 amino acids containing a domain (PSTAVRE) which combinate Cyclins. A high homology of 97.6% of the Cdc2 protein can be drawn by comparing the amino acid sequences in this four fishes, which indicating the higher conservative function and evolution of Cdc2 protein in this four fishes. A comparative expression pattern of cdc2 in early-stage gonads of G3 and different ploidy fishes was carried out by Real-time PCR using specific primers against the same sequences of coding regions in the four fishes. The results showed that the expression of cdc2 in the ovary of G3 was higher than those of red crucian carp and triploid crucian carp, while lower than that of allotetraploid, which, at the molecular level, indicates existence of polyploid oogonia in early-stage gonads of G3. The higher expression of cdc2 in G3 suggests that consecutive S-phase replication may occur without intervening mitosis, which might be related to the formation mechanisms for the diploid eggs generated by diploid hybrids.

Abstract:To investigate the impact effects of 17a-methyl testosterone on the body length (BL), body weight (BW), body health index (CF), and the anal fin 3rd fin section length (FL), the section number (FJ), and the most wide department width (FW), as well as the level of ARα mRNA and VTGα mRNA expression in female mosquitofish (Gambusia affinis). A total of 250 juvenile female mosquitofish (after birth about 18 d) were exposed to different concentrations of MT (0.5, 5, 50 and 500 nM) in the experimental group, and set the control group and the parallel groups. Exposure experiment lasted for 21 days. The results showed that, the BL and CF in female juvenile exposed to different concentrations of MT for 21 d were not significantly different, only the BW in fish exposed to 50 nM and 500 nM of high-concentration groups was significantly decrease (p<0.05) when compared with the control group; the FJ in 3rd fin of anal fin showed a significant increase, FL was along with extending and FW also showed a significant increase in width (p<0.05); Female juveniles were exposed to different concentrations of MT after 21 d, ARα mRNA expression level was dose-related increase significantly (p<0.05 ), however, VTGα mRNA expression level showed dose-related declinesignificantly (p<0.05 ). The results indicated that the androgenic effects of MT was very obvious, resulting in the morphological masculinization in female mosquitofish during the growth and development process; VTGα and ARα mRNA expression levels could be used as the ideal biomarkers for monitoring the aquaculture water androgen pollutants.

Abstract:The suborder Stromateoidei (Teleoste: Perciformes) currently comprises 6 families, 16 genera, and about 70 species. They occur worldwide in coastal and oceanic waters of tropical to temperate regions. Fishes of Stromateoidei are one of the most economically important in the world. However, due to the highly diversity of morphological characters, the taxonomic status and phylogenetic relationships of Stromateoidei has been long-lasting confused. In this study, the mitochondrial 16S rRNA gene fragment of 8 species of Stromateoidei collected from Chinese coastal waters, were amplified using PCR techniques. The homologous sequences of other Stromateoidei fishes from GenBank were also included in this study. The genetic information indexes, including base composition, sequence variation and Kimura-2 parameter genetic distance were examined. Phylogenetic trees were constructed based on neighbor-joining (NJ) and maximum-parsimony (MP) methods. Of the 458 aligned base pairs, with 119 variable sites, 114 parsimony informative sites, 5 singleton sites, were detected in the analyzed Stromateoidei fishes (5 families, 13 genera, and 32 species). The average contents of T, C, A and G were 22.2%, 24.5%, 30.0% and 23.3% respectively for COI analysis. The genetic distances among families, genera and species were respectively range from 0.060 to 0.120, 0.009 to 0.125, and 0.000 to 0.163. The phylogenic analysis placed Centrolophidae in the most basal position of the Stromateoidei, Pampus (Stromateidae) at the top of the two trees, Ariommatidae, Tetragonuridae, Nomeidae in a sister position to the clade of Peprilus and Stromateus (Stromateidae). Based on the results of molecular analysis and morphology data, the several conclusions were drawn as follow: (1) family Centrolophidae, which was early diverged, is the most primitive monophyly group in the suborder Stromateoidei; (2) Ariommatidae, Tetragonuridae are the two monophyletic groups, which showed close relationship with the non-monophyletic group of Nomeidae; (3) the paraphyletic group of Pampus is the most recently diverged species, which is in accordance with the fact that it is also the most flourishing genus in Stromateoidei. There are two parallel evolutionary clades in genus Pampus. The species within the two clades seem to be relevant to their biogeographic distributions. In addition, the application of 16S rRNA gene for phylogenetic study in the suborder Stromateoidei is discussed in this study.

Abstract:The cavefish Triptophysa xiangxiensis is a species of loach belonging to family Cobitidae, order Cypriniformes. It is endemic to longshan county of western Hunan Province of China. In this study,A total of 103 individuals from three T. xiangxiensis populations which obtained from three caves in wulongshan mountain were studied using 16 pairs of microsatellite markers. Using polymorphism information content (PIC), mean heterozygosity (H), number of effective alleles and F-statistics, the genetic diversity and genetic differentiation were evaluated. A total of 83 different alleles were detected in all examined loci．The number of alleles ranged from 3 to 8,with an average number of about 5 per locus．The observed ( HO) and expected heterozygosity( HE) ranged from 0.3625 to 0.9465 and from 0.5386 to 0.9065,respectively．The polymorphism information content for these three populations were 0.2632、0.2313、0.3035. All of which revealed that the genetic diversity in these three T. xiangxiensis populations were low．The analysis of molecular variance( AMOVA) indicated that almost majority of the variance in the T. xiangxiensis was within stocks(92.84%),and 7.16% was among stocks．The result of AMOVA 、F-statistics, Nei’s genetic distance and genetic identity indicated that genetic difference was relatively small and genetic differentiation was low,with high genetic identity between all two populations．The information generated in this study will contribute to the conservation of this endangered T. xiangxiensis species.

Abstract:In 2011，seven batches of Litopenaeus vannamei, introduced from American and Singapore were collected to evaluate the growth performance.. The nest design was adopted and 130 full-sib families (17 hybridized combinations and 7 inbreed combinations) were obtained by artificial insemination. Offspring of the 130 families were cultured separately through the larval and juvenile stages, and were fluorescence-tagged when they reached a body length of 3 cm. The shrimps were tagged with a unique family code by injecting different colours of “Visible Implant Fluorescent Elastomers” (VIE) before being stocked in one grow-out pond. All families were randomly divided into two groups and then cultured in both Huanghua, Hebei and Aoshan, Qingdao, simultaneously. Body weight (g) of these shrimps was measured after 153 d. growth performance of every population and combination was evaluated by analyzing the least squares means (LSM) and heterosis for body weight in Litopenaeus vannamei. The results showed that, at 153 d, the variation coefficient of body weight of the Litopenaeus vannamei stocks was 13%~26%. The LSM for body weight of the UA5 batch, UA4 batch and SIN batch were higher than average by 5.34%, 2.51% and 1.67%, respectively. So these 3 batches could be used as parents for high quality watermelon cultivars. A growth performance analysis showed the average LSM of hybridized combination (18.14 g) was higher than the average LSM of inbreed combinations (17.17 g). The highest LSM occurred in the cross between the UA4 batch and the UA5 batch, with 7.61 percent higher than average. Heterosis analysis showed, they ranged from a low of -1.77% to a high of 11.72%, with a mean of 5.45%, positive heterotic vigor was detected in most hybridized combination. The highest heterosis occurred in the cross between the UA1 batch and the UA2 batch. Our study provides insight into methods for improving the accuracy of genetic selection for growth traits in Litopenaeus vannamei，and for genetics and breeding research into quantitative traits in other aquatic animals.

Abstract:The Australian redclaw crayfish, Cherax quadricarinatus, is a crustacean belonging to the order Decapoda, family Parastacidae. It is currently one of the most important commercially farmed fresh crayfish in the world due to its many advantages, including polyphagia, fast growth and easy culture, as well as being rich in protein and low in cholesterol. During the last two decades, the environmental and health problems in redclaw crayfish culture have resulted in the outbreak of infectious diseases with the constantly expanding scale of aquaculture and the rapid development of intensive culture techniques of C. quadricarinatus in China.White spot syndrome virus (WSSV) as the pathogenic agent caused acute and lethal infect ion of shrimp, resulting in mass economic losses to shrimp aquaculture industry all around the world especially in China. In recent years, the cultivation of redclaw crayfish (Cherax quadricarinatus) is developing, and the disease of C. quadricarinatus was one of the major factors in its culture and even caused redclaw crayfish to die. The viral disease was found in polyculture of redclaw crayfish with Penaeus vannamei. To explore the pathogenic mechanism of C. quadricarinatus infected by WSSV (white spot syndrome virus) , a prophenoloxidase gene（CqproPO） was cloned from haemocytes of C. quadricarinatus by Rapid Amplification Complementary DNA Ends(RACE) method, the proPO gene expression patterns in different tissues and the mRNA expression of proPO gene in hemocyte, hepatopancreas and gill tissues of C. quadricarinatus infected by WSSV artificially were studied. The results indicated that the full length cDNA of CqproPO consisted of 2962bp with a 1998 bp Open Reading Frame(ORF)，which encoded 665 amino acids，and a predicted signal peptide of 38 amino acids, and the predicted molecular mass was 75.86KDa. Sequence analysis showed CqproPO contained two conserved copperbinding sites; The secondary and tertiary structure assay also showed that the CqproPO has α-helices and β-strands, which is delimiting a cavity where the hydrophobic ligands are bound just as other HCs. ORF contains two tyrosine kinase phosphorylation sites, 13 casein kinase II phosphorylation sites, 7 protein kinase C phosphorylation sites, one dependent on cAMP-and cGMP protein kinase phosphorylation sites and three N-glycosylation sites, which these sites were structural basis of the physiological functions completed. The deduced amino acids sequence of CqproPO sharing 79% homology with Procambarus clarkii and 74%, 69%, 67%, 67% with Pacifastacus leniusculus, Nephrops norvegicus, Homarus americanus, Homarus gammarus respectively; Phylogenetic analysis revealed that CqproPO and prophenoloxidase from P. clarkii, P. leniusculus, N.norvegicus, H.americanus, H. gammarus and Panulirus longipes were in the same phylogenetic branches; The Realtime-PCR results showed that CqproPO was widely distributed, with the highest expression level in haemocytes, small amount of expression in intestine, antennal gland, gills, ovary and hepatopancreas, detectable expression level in stomach and muscle, while expression was almost undetectable in testis; The expression level of prophenoloxidase (proPO) in haemocytes, hepatopancreas and gills from C. quadricarinatus were studied and compared by means of artificial WSSV infection. The results indicated that the expression level of CqproPO in the non-immunized infected group (Group II) and immunized infected group (Group III) reached the maximum at 12 h and 24h, which was 1.3-2.55 times higher than that in the control group, and was noticeable higher than the controls (p <0.05). But the expression level of prophenoloxidase gene had sharply declined with the time extending of the infection. The expression level of proPO gene from crayfishes injected by immune polysaccharides before an infected virus (GroupⅢ) were higher than the directly affected groups (GroupⅡ) in haemocytes, hepatopancreas and gills. These observations indicated that the immunopotentiator could improve the innate anti-viral ability of the crustaceans against WSSV.

Abstract:Glyceraldehyde-3-phosphatede hydrogenase(GAPDH)is one of the basic enzymes that maintain the life activities.In this paper,gapdh gene was isolated from the mud crab,Scylla paramamosain using RT-PCR and RACE methods.The obtained full-length cDNA of GAPDH was 1 440 bp with an open reading frame of 1 008 bp encoding a putative peptide of 335 amino acids.By alignment,the amino acid sequence of S.paramamosain GAPDH showed high homology with those of some other animals.It suggested gapdh was highly conservative.Real-time PCR showed that the gapdh gene was expressed in various tissues,and highly expressed in thoracic ganglia,eyestalk ganglia,ovary and epidermis.During the ovarian development,the expression of gapdh mRNA was significantly higher at early-developing stage(Ⅱstage)and lowest at ripe stage(Ⅴstage).It indicated that GAPDH might play an important role in cell mitosis in ovary of the mud crab.

Abstract:Dmrt is a family of genes related to the sexual regulators Doublesex and Mab-3.Our previous studies showed that EsDmrt-like transcripts were detectable only in testis of Chinese mitten crab,Eriocheir sinensis.In order to verify whether EsDMRT-like protein is also specifically expressed in testis,the prokaryotic expression plasmid pET-32a-EsDmrt-like was constructed according to the sequence of EsDmrt-like.The recombinant plasmid was transferred into host bactria Escherichia coli BL21(DE3),and the recombinant protein Trx-EsDMRT was expressed under the induction of IPTG.SDS-PAGE analysis showed that the fusion protein was highly expressed as inclusion body with the molecular weight of 46 ku.The recombinant protein was purified by affinity for preparing EsDMRT-like antibody.The resultant antibody can specifically recognize the recombinant protein and endogenous EsDMRT-like protein in the testis as revealed by Western-blotting analysis.EsDMRT-like was specifically expressed in the testis,with molecular weight of 52 ku,which is twice as those of the monomer.After denatured treatment by urea and DTT,the protein displayed two forms of polypeptides:52 and 34 ku.Both of them can be recognized by antiserum raised against EsDMRT-like protein,indicating that the form of 34 ku was generated from 52 ku polypeptide and the EsDMRT-like protein exists as a dimer in testis.This result suggested that EsDmrt-like may regulate the male sex development/differentiation by dimerization in the crab.

Abstract:EST-SSR is one of important methods to isolate microsatellite loci,which is low cost,high transferability among phylogenetically related species and a probability of being associated with functional regions of the genome.34 polymorphic EST-SSR markers were isolated and characterized in the blood clam,Tegillarca granosa,from the ESTs generated by 454 sequencing.The number of alleles for the 34 SSR markers in 30 individuals of Zhejiang Fenghua population varied from 2 to 7,with an average of 3.59 alleles per locus.The observed heterozygosity ranged from 0.000 to 0.600,while the expected heterozygosity varied from 0.078 to 0.771.Polymorphic information content ranged from 0.106 to 0.718,and 13 SSR loci deviated from Hardy-Weinberg equilibrium.25 EST-SSR identified from annotated genes were expected to be useful for mapping these genes in linkage maps.Interspecific transferability of the 34 markers revealed that 8 were polymorphic in Anadara craticulata,resulting in a transferability rate of 23.53%.To our knowledge,this is the first report on the survey of SSR transferability in blood clams.These novel polymorphic EST-SSR markers should be particularly useful for comparative mapping,gene tagging and QTL mapping among blood clams.

Abstract:Effects of different inoculation densities on growth and toxin production of Alexandrium minutum were studied.Experiments were conducted under conditions of varying inoculation densities(0.05×10⁴,0.10×10⁴,0.15×10⁴,0.30×10⁴cells/mL).Higher inoculation densities always resulted in shorter lag phase of A.minutum,and an earlier setting in of the steady growth phase.However,the specific growth rate and maximum cell concentration of A.minutum decreased with increasing inoculation densities.The multiplication models developed correlated well with the observations,as the inoculation density increased,the initial-density dependent parameter a also deceased and the reduction of environmental capacity,instantaneous multiplication included.According to MBA analysis,toxicity of A.minutum is typically highest when cells are growing fast in early exponential phase and lowest as growth slows and eventually stops in plateau phase.The PSP-toxin congeners,GTX1(2.14 fmol/cell),GTX2(2.08 fmol/cell),GTX3(4.97 fmol/cell),GTX4(5.04 fmol/cell)were analysed by high-performance liquid chromatography with fluorescence diction(HPLC-FLD).Result of this study implied that toxin production of the dinoflagellate A.minutum is better in 0.10×10⁴-0.15×10⁴ cells/mL than other condition of inoculations densities.

Abstract:To illustrate the application prospect of nonviable probiotics in marine fish aquaculture,the present study evaluated the effect of dietary administration of viable and heat-inactivated autochthonous probiotic Psychrobacter sp.SE6 on the growth performance and serum immune parameters of grouper(Epinephelus coioides).225 juvenile fish with an initial weight of(14.6±0.2) g were randomly divided into 3 groups(groups T0,T1 and T2)with 3 replicates per group and 25 fish per replicate.The fish were fed for 60 d with basal diet containing 0 cfu/g(control group T0),1×10⁸ cfu/g viable Psychrobacter sp.SE6(group T1)and 1×10⁸ cfu/g heat-inactivated Psychrobacter sp.SE6(group T2),respectively.The results showed that,in the early period(0-30 d),the weight gain rate(WGR)and specific growth ratio(SGR)of the two probiotic groups were significantly higher than the control group(P＜0.05),and the feed conversion ratios(FCR)of two probiotic groups were lower than those of the control group.During the whole experimental period(0-60 d),the WGR and SGR of the two experimental groups were significantly higher than those of the control group(P＜0.05),the FCR of the two experimental groups was lower than that of the control group,and the FCR of group T1 was significantly lower than that of the control(P＜0.05).At day 30,the serum immunoglobulin M(IgM)and complement C3 contents in the experimental groups were higher than the control group,but not significantly(P>0.05).At day 60,the serum T-SOD activity,complement C3 and IgM contents in the group T1 were significantly higher than those of the control(P＜0.05),while the activities of T-SOD and lysozyme,complement C3 and IgM contents in the group T2 were higher than those of the control,but not significantly(P>0.05).In conclusion,both the viable and heat-inactivated Psychrobacter sp.SE6 played positive roles in promoting the growth performance and feed utilization,improving specific and nonspecific immune functions in E.coioides.Although better effect was observed in the viable probiotic group,the heat-inactivited probiotics have several advantages,such as high safety,stable quality and convenient processing and storage.Therefore,further study is needed to evaluate the application of heat-inactivated probiotcs in marine fish aquaculture.

Abstract:The aim of the present experiment was to study the effects of quorum quenching enzyme AI-96 against Aeromonas hydrophila NJ-1 attack with the bath challenge in zebrafish. The experiments set two dietary groups for zebrafish,1) basal diet as control, 2) enzyme-containing diet with 3 U/g food of AI-96. Zebrafish tissues (gill, liver, kidney and intestine) were sampled at day 0.5, 1, 3, 7 and 14 after co-bath challenge with live Aeromonas hydrophila NJ-1 at two doses (high 2.5×10 8 cfu/mL, low 0.7×10 8 cfu/mL). The populations of NJ-1 colonizing in the tissues were quantified by real time PCR, and accumulated mortality of zebrafish were recorded everyday. Results showed that after challenge, the NJ-1 were detectable in the tissues of the attacked zebrafish in the total population order of intestine> gill > liver > kidney. More NJ-1 colonized in the tissues in the high dosage challenge treatment than the low group. Interestingly, the diets containing AI-96 enzmye significantly decreased the colonizing populations of NJ-1 in the zebrafish (P < 0.05), compared with the counterparts fed with basal diets when the fish was chanllenged with 2.5×108 cfu/mL NJ-1. In 0.7×10 8 cfu/mL challenge treatment, the NJ-1 number colonizing in gill (3d), intestine (0.5, 1, 3, 7, 14 d), liver (3 d) and kidney (7 d) were also lower in the fish treated with AI-96 containing diets than the ones with basal diets (P < 0.05). Moreover, these effect was more obviously in 0.7×10 8 cfu/mL challenge treatment after 7 d (P < 0.05). Therefore, the dietary AI-96 enzyme could improved the survival rate against NJ-1 attack in both challenge levels, For this reason, quorum quenching enzyme could effectively protect zebrafish against A. hydrophila at 0.7×10 8 cfu/mL level in zebrafish.

Abstract:Exopalaemon carinicauda is one of the marine aquaculture species with high economic value in China.This paper studied the effects of cryopreservation(-196 ℃)on the enzyme activity(SDH,LDH,Na+K+-ATP,SOD,CAT,GR and Acrosin)in E.carinicauda spermatozoa,in order to provide a theoretical basis for cryopreservation effect on E.carinicauda spermatozoa,and lay a foundation for artificial propagation of E.carinicauda.We set up a control group(without cryoprotectant),and three experimental groups(DMSO(V/V)10%,12.5%,15%),detected the enzyme activities on day 0,1,3,5,7,15.The result showed that,after cryopreservation,in addition to GR,the other enzyme activities decreased significantly(P<0.05),with the biggest drop in the control group.GR activity significantly increased in 7 days,and the control group was significantly higher than the experimental group(P<0.05),declining again in 15 days.The group of 15% DMSO measured enzyme activities were higher than other groups,indicating that 15% DMSO had protective effect on enzyme of spermatozoa.15 days later,under the condition of 15% DMSO,the activities of SDH,LDH and Na⁺/K⁺-ATPase decreased from(28.500±1.453)U/mL,(1290.836±27.603)U/L and(2.605±0.232)μmol/(mg?h)to(15.300±0.950)U/mL,(363.713±13.943)U/L and(0.542±0.186)μmol/(mg?h)respectively;the activities of SOD and CAT decreased from(106.497±7.217)U/mL,(383.632±4.731)U/g to(17.036±0.321)U/mL and(166.940±1.910)U/g respectively;the activities of acrosin decreased from(3.521±0.010)μIU/106 to(1.212±0.043)μIU/10⁶;but the activities of GR increased from(217.042±6.962)U/L to(302.787±24.558)U/L.From the decline range,cryopreservation had the biggest influence on SOD,followed by Na⁺/K⁺-ATPase.

Abstract:A strain of grass carp reovirus,named GCRV-GD108 was isolated from sickened grass carp with symptoms of haemorrhage in Guangdong Province.Its whole genomic sequence was obtained.This reovirus consisted of 11 dsRNA segments which encoded 11 proteins instead of 12 proteins that belonged to Aquareovirus(AQRV).Sequence comparison showed that it possessed only 7 homologous proteins to grass carp reovirus(GCRV)(with 17.6%-45.8% identities),but 9 homologs to MRV(with 15%-46% identities).The virus structure protein VP5,which was encoded by M5 gene of GCRV-GD108,shared a high homology and a conserved motif for NTP binding with the μ2 protein of mammalian reovirus(MRV),suggesting that the VP5 protein is functional homologue of μ2,and possesses NTPase activity.A prokaryotic expression vector of M5 gene had been constructed previously,and the engineering bacteria pET30c-M5/BL21(DE3)was selected.According to the requirements of experience,we had chosen appropriate purification method of VP5 protein.The NTPase activity of the purified recombinant VP5 protein was analyzed,using the method of malachite green/ammonium molybdate reagent for quantification of the released Pi of NTP.The result showed that the recombinant protein possessed NTPase activity.The activity of VP5 is dependent on the cations Mg²⁺ or Na⁺/K⁺,but its activity was inhibited with the presence of Ca²⁺.DNAstar software was used to predict the antigenicity of M5 gene encoded protein.Online prediction based on the analysis results of the three indexes,hydrophilicity,surface probability and antigenic index,showed that 86 regions in the M5 encoding protein potentially form epitopes,suggesting that VP5 possesses strong immunogenicity.The purified recombinant VP5 protein was used to immunize the healthy grass carp,and its protection role was tested by artificial infection of the immunized fish.The serum of the immunized grass carp was analyzed by ELISA and the mRNA level of IgM from the head kidney of grass carp was analyzed by qRT-PCR.These results indicated that recombinant protein VP5 could induce the immunized grass carp to produce high titer antibodies and the expression levels of IgM were significantly improved.However,this protein could not provide protection for GCRV infection.The NTPase activity of GCRV-GD108 VP5 protein was confirmed for the first time,but this protein could not provide immune protection for grass carp.

Abstract:Channel catfish reovirus(CCRV)is the pathogen of the hemorrhage of channel catfish(Ictalurus punctatus Rafinesque),which propagates in the kidney of channel catfish(CCK)cell line.In this study,the evaluation of whether the CCRV could induce the apoptosis of CCK cells was examined by Hoechst33258 staining,DNA fragmentation assay,TUNEL reaction,and flow cytometry analysis using JC-1,etc.The infection test indicated that CCRV caused typical cytopathic effect(CPE)in CCK cells.Chromatin condensation,nuclei marginalization and the apoptotic bodies were observed in Hoechst 33258 staining,and the apoptotic rate increased with the time going of CCRV infection.The DNA fragmentation assay demonstrated that fragmentation was first noted at 12 h post-infection and reached the peak at 72 h post-infection.In addition,genomic DNA was broken and lots of 3′-termianl free-hydroxyl group(-OH)were generated in infected CCK in TUNEL assay.The hypo-diploid fraction was shown in the sub-G1 cells analysis and the apoptotic rate was 53.44% at 48 h post-infection.Furthermore,the change of the mitochondrial membrane potential(MMP)was investigated by flow cytometric analysis with JC-1 fluorescent labeling and the result showed that the membrane permeability and MMP of CCK cells changed significantly at 24 h post-infection.These results demonstrated that CCRV induced apoptosis in CCK cells.Besides,CCRV-induced apoptosis did require the viral replication as the apoptosis was blocked in CCK cells by both heat-inactivated and UV-inactivated virus.

Abstract:In order to study the immunogenic and protective effects of DNA vaccine,plasmid DNA encoding flagellin flaC gene(designated as pcDNA-flaC)was used as a DNA vaccine to immunize red snapper(Lutjanus sanguineus).The distribution,expression and immunoprotection of the DNA vaccine were analyzed in tissues of the red snapper by PCR,RT-PCR and challenge test.PCR results indicated that pcDNA-flaC was distributed in liver,spleen,kidney,gill and injection site muscle at 7-28 days after vaccination.RT-PCR results indicated that the flaC gene was expressed in all above tissues of vaccinated fish at 7-28 days after vaccination.These results demonstrated that the DNA vaccine was distributed and flaC gene was expressed in various tissues of vaccinated fish.Red snapper immunized with DNA vaccine showed higher serum antibody levels at 7-28 days after vaccination,compared to fish vaccinated with the control eukaryotic expression vector pcDNA3.1 and PBS.In addition,fish immunized with DNA vaccine developed a protective response to live V.alginolyticus challenge 28 days post-inoculation,as demonstrated by increased survival of vaccinated fish over the control fish.This study indicates that pcDNA-flaC is an effective vaccine candidate against V.alginolyticus infection.

Abstract:In this study,the effect of crosslinking method on biological properties of grass carp skin collagen sponge was discussed.Collagen was extracted from skin of grass carp(Ctenopharyngodon idellus)and collagen sponge was prepared from this collagen.Then,this collagen sponge was crosslinked with different methods,such as UV,dehydrothermal,EDC/NHS and glutaraldehyde crosslinking processes.At the same time,the biological and mechanical properties of those collagen sponges,including degree of crosslinking,denaturation temperature,tensile strength and enzymatic sensitivity in vitro,were evaluated and compared.Experiment results indicated that the grass carp skin collagen was type Ⅰ collagen.The degree of crosslinking of different crosslinking methods decreased in the order of glutaraldehyde(72.0%)＞EDC/NHS(32.5%)＞dehydrothermal(29.9%)＞UV(15.6%).Compared with control collagen sponges,the denaturation temperature(67.4 ℃),tensile strength(125.6 kPa)and enzymatic sensitivity in vitro of collagen sponges crosslinked by glutaraldehyde were significantly improved(P＜0.05);EDC/NHS crosslinking could lead to obvious increasing in denaturation enthalpy(6.86 J/g)and moderate improving on tensile strength(98.6 kPa)and enzymatic sensitivity for collagen sponge(P＜0.05).The changes of biological and mechanical properties of collagen sponges after being crosslinked by dehydrothermal and UV crosslinking processes were not notable.The results of FTIR showed that glutaraldehyde crosslinking could improve properties of collagen sponges by forming new covalent bond in triple helix structure of collagen,while EDC/NHS crosslinking do that by forming new hydrogen bonds among collagen molecules.This research shows that glutaraldehyde and EDC/NHS crosslinking could lead to obvious improvement in properties of collagen sponges but the influences on properties of collagen sponges after being crosslinked by dehydrothermal and UV crosslinking processes were limited.

Abstract:Successive green tides that happened in South Yellow Sea in recent years have caused widespread concern.In order to provide the basis for making integrated use of green tide algae,and according to national standards,this study analyzed basic nutrients,chlorophyll,amino acids,mineral elements and heavy metals of green algae collected from Jiangsu and Shandong in 2010.The results indicated that:carbohydrates were the main nutritional components of these green tide algae,accounting for 35.82%-52.43%;the crude protein contents varied greatly,from 31.04% to 12.11%;the fat contents were low and all less than 1%;there was a significant difference in the contents of chlorophyll,the contents decreased significantly when the collection dates delayed;the amino acid contents were high and the percent age of essential amino acids was up to 37.45%,the flavor amino acids aspartic acid,glutamic acid,glycine and alanine were considerably high;these algae contained abundant mineral elements,the contents of Ca,Fe,Zn were distinctly high,but the heavy metals Pb,Hg and Cd were all lower than the limited requirements in national standards.The composition of attached samples did not change significantly.

Abstract:Historical investigation and the achievements of resources biology of main marine demersal food fishes along the coastal waters of China,with special regard to the ribbon fish(Trichiurus japonicas),small yellow croaker (Larimichthys polyactis),large yellow croaker (Larimichthys crocea) and black scraper (Thamnaconus modestus), are reviewed in this paper.This review covers the identification and classification of fish geographical populations and their spawning stocks,historical pattern changes,the structure of populations and stocks,age composition and growth characteristics,feeding habits,sex mature cycles,gonadosomatic index(GSI),fecundity of the spawning stocks,spawning grounds,feeding grounds,wintering grounds,migration routes,assessment of amounts of nature resources and catches,adjustment of fishing boats,and restricted measures for fishing effort.The prospects and problems of resource biology of main marine demersal food fishes along the coastal waters of China are also discussed.