Abstract:Acute viral necrosis virus(AVNV)was reported as one causative agent responsible for mass mortality of adult Chlamys farreri,which is widely cultured along northern China coast.To explore its pathogenesis at the molecular level,a gene was cloned which was predicted to encode AVNV primase based on the genomic sequence of AVNV completed by our laboratory.The gene encodes a protein of 350 aa with a predicted molecular mass of 60 ku.To obtain AVNV ORF 024,which probably encodes AVNV primase,a pair of specific primers was designed based on the genomic sequence of AVNV.Then this paper amplified the expected DNA by PCR,and used the total genomic DNA extracted from infected C.farreri tissues as template.Amplified PCR fragments were cloned into the prokaryotic expression vector pET-32a(+).After that,the plasmid pET32a-prim was transformed into E.coli BL21(DE3)stain,and AVNV primase was expressed under IPTG induction.SDS-PAGE analysis showed that the two induced recombinant proteins’ molecular mass was about 60 and 55 ku,the Western-blotting and mass spectrometry analysis proved that the expressed protein(60 ku)was the primase,while another expressed protein(55 ku)had some of the primase peptide fragments.Meanwhile,the experiments showed that Pico-Green(it was able to specifically bind to a random short segments of RNA)as the fluorescent dye which had a stable fluorescence intensity in 30 min.So the 30 min was the termination time for the enzymatic activity analysis of the recombined primase.In the presence of template of the poly(d C),the analysis of the enzymatic activity indicated that the recombinant primase could specifically catalyze the hydrolysis of GTP.The enzymatic activity of primase was enhanced by 0.1 mmol/L Zn ²⁺ but inhibited by 1 mmol/L Mn²⁺ and EDTA.

Abstract:A swimming crab is one of the most economically important edible marine crabs in the East Asia area,and it is widely distributed in the coastal waters of China,Japan and Korea.To assess the level of genetic diversity and genetic variation of swimming crab populations along China coasts,the species from the six geographical populations from China Sea(Dalian,Dongying,Lianyungang,Zhangzhou,Zhoushan and Zhanjiang)were studied based on mitochondrial D-loop whole gene sequences.There were 1 141 bp,185 variation sites and 129 parsimony informative sites in D-loop whole gene sequences.And there were 48 haplotypes among 60 swimming crab individuals.Nucleotide diversity index and Haplotype diverstity index together revealed a high genetic diversity and low selective effect for swimming crab populations in China Sea.The genetic differential index F_ST of total six populations was 0.189 72,which meant that the genetic differentiation among populations was moderate and significant(P<0.05)and the inferred differentiation time was about(196.8-260.5)×10³ years.Low gene flow indicated that there were genetic differentiations among the fifteen groups,except between LYG and DY,ZJ,ZZ,respectively,in addition between ZJ and ZZ(N_em>5).Especially,there were pronounced levels of genetic differentiation between ZS and the others and a moderate genetic differentiation between DL and LYG,DY,ZJ and ZZ,respectively.The genetic distance had no significant correlation with geographical distance,so there may be more intricate reasons in population occurrence and spread.

Abstract:Matrix protein is one of the structure proteins of infectious haematopoietic necrosis virus(IHNV),and its synthesis leads to apoptosis or programmed cell death in the transfected cells.To study its structure and function,the open reading frame of matrix protein gene was amplified by RT-PCR from IHNV-Sn isolate and cloned into plasmid pET27b(+)vector.The structure and characteristics of the gene were studied by means of bioinformatics software.The results are described as follows:the length of the M gene,encoding 195 aa,was 588 bp.The molecular weight was 21.88 ku and the isoelectric point was 9.35.The matrix protein was rich in serine,threonine and alkaline amino acid,and was composed of plenty of α-helix,extended β and coil.The protein is hydrophobic with no signal peptide and transmembrane regions.The antigenicity of matrix protein was good.According to the protein structure prediction,there might be no N-glycosylation sites,and there were 7 O-glycosylation sites and 15 phosphorylation sites in matrix protein.Phylogenetic analysis showed that the IHNV-Sn isolate was in the same branch with the isolates from America.The study established the foundation for genetic background information,pathogenesis,molecular epidemiology research of IHNV.

Abstract:EST-SSR was used to detect the hereditary difference in wild and cultured populations of the Betaphycus gelatinae,the result showed that nine individuals from each population were used of the 5 EST-SSR primers amplified,a total of 195 reproducible loci ranging from 250 to 2 500 bp were amplified from all the 18 individuals.The average percentages of polymorphic loci in wild and cultured populations were 74.29% and 70%.The intra-population genetic distance was 0.46 in wild population and 0.22 in cultured population.The cluster analysis showed that 18 individuals were clustered into two groups base on 0.49 of GS; wild populations were divided into 3 branches and cultured populations were divided into 2 branches base on 0.66; wild populations were divided into 5 branches and cultured populations were divided into 3 branches base on 0.73.The higher the value,the more branches the wild populations have.The research shows that genetic diversity of wild populations ampler than cultured.

Abstract:The diseased hybrid snakehead(Channa maculate ♀×Channa argus♂)were collected for analysis of the pathogen both in bacteriology and virology to make the pathologic agent clear.None of pathogenic bacteria was obtained using routine bacteria separation method.One rhabdovirus was isolated using cell culture technique,regression tests,electron microscopy,molecular biology techniques and others,which assigned hybrid snakehead rhabdovirus -C1207(HSHRV-C1207).HSHRV-C1207 could replicate and proliferate in EPC,FHM,GSB,SFC,CIK cells,and cause the significant cytopathic effect(CPE).The regression tests in which separated viruses were injected in the abdominal cavity were all presented with symptoms similar to the natural incidence,and the final mortality was 90%.Electron microscopy observation of ultra-thin section samples of specimen-infected EPC cells revealed that the bullet-shaped virus replicated in cytoplasm with regular arrangement like other fish rhabdovirus,and had a non-enveloped structure with a diameter and length of approximately 60 nm and 145 nm respectively.The OIE recommended primers for Infectious haematopoietic necrosis virus(IHNV),spring viraemia of carp virus(SVCV),viral haemorrhagic septicaemia virus(VHSV)and three pairs of specific primers were designed by G gene conserved sequences of snakehead rhabdovirus(SHRV),siniperca chuatsi rhabdovirus(SCRV)and hirame rhabdovirus(HIRRV)respectively,were applied to the RT-PCR amplification for detecting the possible rhabdovirus in diseased fish and virus-infected cells.A specific ladder product about 350 bp only was amplified with primers of SCRV.Sequencing analysis of the G gene of HSHRV-C1207 showed that this gene shares the sequence identities 93.8% and 20.4% with the respective genes of SCRV and SHRV respectively,and the corresponding deduced amino acid sequence identity 93.7% and 18.3% respectively.Phylogenic relationship with other rhabdoviruses using the corresponding amino acid sequences of G protein indicates that HSHRV C1207 is most closely related to the vesiculovirus genus and shares the highest identity with SCRV,but was far from the snakehead rhabdovirus(SHRV)and the other rhabdovirus.The above results indicated that the causes of hybrid snakehead diseases ceaseless breaking out was the rhabdovirus,and this new isolates HSHRV-C1207 belongs to vesiculovirus genus.It may be a novel variant SCRV isolate or a new member.

Abstract:Porphyra dentata is one of the main species of natural resources of Porphyra,which has high economic and medicinal value.It is meaningful to develop and increase new Porphyra cultivation by studying the ecological characteristics of P.dentate.In order to understand physiological responses of P.dentate cultured in different conditions,we studied the responses of P.dentate to different temperature,salinity and light intensity by determining the change about growth,photosynthetic pigments contents,soluble protein contents,malondialdehyde(MDA)contents and the peroxidase activity.The experimental results showed that,thallus was able to maintain a high relatively growth rate under 17-20 ℃,when temperature was higher than 23 ℃,the growth of thallus was inhibited,it turned to redness and appeared to fester.At salinity of 25-35,the thallus grew quickly under culture conditions,when culture salinity was lower than 25,the content of photosynthetic pigment,soluble protein,malondialdehyde(MDA)and the activity of peroxidase(POD)and catalase(CAT)of thallus was significantly changed,and thallus became thinner and whitish,its edge displayed decay phenomenon.The thallus had a high growth rate under culture light intensity of 3 000- 6 000 lx ,when the culture light intensity reached 9 000 lx,content of chlorophyll a of thallus was low,but the content of soluble protein and MDA and the activity of POD were significantly increased,the thallus began to fester after several days’ culture.According to the result,we know that suitable culture conditions for P.dentata thallus are temperature of 17-20 ℃,salinity of 25-35,and light intensity of 6 000 lx.

Abstract:Endogenous spoilage bacteria of Litopenaeus vannamei are the bacterial floras living in Litopenaeus vannamei body which cause the Litopenaeus vannamei spoilage.It is one of the most important factors which cause spoilage while the Litopenaeus vannamei are stored up and in transportation process.In order to find out the endogenous dominant spoilage bacteria of Litopenaeus vannamei under refrigeration,the bacteria separation is applied in experiment and therefore the endogenous spoilage bacteria are gained,and then the bacterial strains of high capability of decomposing protein were obtained after protein comparison experiment.Once more,auto-biochemical identification system and 16S rRNA sequencing are applied in experiment process.The result shows that the endogenous dominant spoilage bacteria of Litopenaeus vannamei are of Brevibacterium, and the semblance of auto-biochemical identification and 16S rRNA sequencing is 98.833% and 99.065% respectively.Researches show that the endogenous dominant spoilage bacteria of Litopenaeus vannamei under refrigeration are of Brevibacterium,and it is possible to research on endogenous freshness preservation technology of Litopenaeus vannamei by controlling Brevibacterium bacteria.

Abstract:Dinoflagellate genus Cochlodinium Schütt has been documented to form blooms along the coastlines of many countries and caused mass mortalities of fish or shellfish.From October to November,2009,harmful algal blooms(HABs)caused by dinoflagellate Cochlodinium geminatum broke out in the coastal waters in Zhuhai,Guangdong.The bloom covers more than 300 km².In order to understand the toxicity of the bloom water,and provide some basis to the algae bloom management and further study of this species,acute toxicity of the bloom water against larvae of zooplankton Artemia salina,fish Scatophagus argus and shrimp Penaeus vannamei was investigated under room conditions.Moreover,feeding rate of A.salina on C.geminatum was also investigated in this paper.The results showed that LC₅₀(Median Lethal Concentration)of C.geminatum against A.salina larvae was 9.55×10⁴/mL within 24 h.LT₅₀ of C.geminatum against A.salina larvae decreased with the increasing cell density.LT₅₀ of C.geminatum with cell density of 2.5×10³/mL against A.salina larvae was 48.5 h.No adverse effects of C.geminatum on the survival of fish and shrimp larvae was observed within 60 h.On the other hand,it was found that both A.salina and S.argus larvae could feed on C.geminatum.But the feeding rate of A.salina larvae was low.Overall,the bloom water of C.geminatum caused an impact on the survival of A.salina.C.geminatum with high cell density could cause mass mortality of A.salina larvae.However,A.salina could live on low cell densities of C.geminatum.Causes of the death of A.salina larvae may not be due to the ingestion of C.geminatum.C.geminatum caused acute toxicity neither against the S.argus larvae nor against the P.vannamei larvae.

Abstract:In order to understand the population dynamics of Antarctic krill(Euphausia superba Dana)in the South Shetland Islands,the main Chinese fishing ground of Antarctic krill fishery,using the biological data collected by the fishery observers,the present study analyzes the spatial-temporal characteristics of population composition of Antarctic krill based on dividing the study area into small scale units with the latitude-longitude spatial resolution of 10′ and 10′.The results show that,in the 23 units analyzed,the proportion of immature females is highest(42.1%),followed by mature females(35.6%),the proportion of males is below 20% and all individuals are immature,the proportion of juvenile individuals is only 2.7%.Compared with other periods,the individuals in juvenile stage is higher(9.6%)in the middle of December 2010 and the proportion of immature males(M₂)is higher than that of mature females(F₃).The similarity of maturity stage composition can be found in the last ten-day period of December 2010 and the first ten-day of January 2011,the juveniles can not be found in the samples and the proportion of M₂ is decreasing and the proportion of F₃ is significantly increasing.The distribution patterns of M₂,F₂ and F₃ are similar in the different water layers(PSI=90.9-97.7).The juveniles are mainly distributed in the 60 to 80 m layer(66.9%)and the individuals of F₂,M₂ and F₃ are located in the 40 to 60 m layer.

Abstract:The present work was carried out to study the effect of temperature on some metabolic enzyme activities in the female swimming crab(Portunus trituberculatus)during its calcification process in laboratory.Activities of PK,HK and SDH were measured in hepatic,muscular and branchial tissue of the swimming crab at 1,2,8,16 h after molting and the completely hardening stage at different temperatures(18 ℃,22 ℃,26 ℃),respectively.The primary results are recorded as belows:①Compared to moderate temperature(22 ℃),PK activity in muscle of P.trituberculatus was found to be significantly increased during 1-8 h after molting at higher temperature(26 ℃)and lower temperature(18 ℃),indicating that the glycolysis rate increased in muscular tissue of the swimming crab at higher and lower temperatures; PK dynamics was found to be peaked in hepatic and branchial tissues of P.trituberculatus at 1-2 h after molting which showed a greater rate of glycolysis in these two tissues.②HK activity in the hepatic tissue of P.trituberculatus decreased during the calcification process in all treatments.HK activity in the liver at 1-16 h was significantly higher than the activity of calcified status,indicating a gradually decreased utilizing capacity of glucose in liver of P.trituberculatus during the calcification process.However,the branchial HK activities showed an opposite dynamic trend compared with that in hepatic tissue,which projected an increased utilizing capacity of glucose levels in gills of P.trituberculatus during the calcification process.The difference may be related to different capabilities to use glucose in different tissues of the swimming crab during calcification process.③SDH activity in hepatic tissue of P.trituberculatus in the all treatments was found to be gradually increased within 16h after molting,indicating that a gradual strengthening of the citric acid cycle in the liver during calcification process.In addition,SDH dynamics was found to be peaked in muscular and branchial tissues of crab occurring within 8h after molting in the all treatments.The activity of SDH at low temperature(18 ℃)was significantly lower than that at high temperature(26 ℃).The statistics described above showed an aerobic strengthening in musclar and branchial tissues of P.trituberculatus at high temperature compared to the low temperature.In summary,glycolysis process under low temperature conditions did not change significantly,while the citric acid cycle had been significantly weakened during the calcification process.The activity of SDH in muscle and branchial tissues of P.trituberculatus at lower temperature was significantly lower than that at higher temperature,indicating that lower temperature can indeed extend the time of its soft-shell stage after molting.

Abstract:The experiment studied the change of enzymes in mild and acute drop of temperature.The activities of CAT,SOD,POD and the content of MDA in “Shuiyuan No.1” sea cucumber and cultured sea cucumber were analyzed.The results show that:(1)In terms of mild drop in temperature,the activity of CAT were trend to decreased and then increased in “Shuiyuan No.1” sea cucumber and Dalian cultured sea cucumber,the activities of CAT were 402.78-424.32 U/mL and 409.81-430.08 U/mL in “Shuiyuan No.1” sea cucumber and Dalian cultured sea cucumber; the activity of SOD and POD first increased and then decreased and then increased in “Shuiyuan No.1” sea cucumber and Dalian cultured sea cucumber; the activities of SOD were 0.019-0.086 U/mL and 0.014-0.069 U/mL in “Shuiyuan No.1” sea cucumber and Dalian cultured sea cucumber,the activities of POD were 0.000 45-0.000 89 U/mL and 0.000 24- 0.000 66 U/mL in “Shuiyuan No.1” sea cucumber and Dalian cultured sea cucumber; the activity of MDA first increased and then reduced in “Shuiyuan No.1” sea cucumber and Dalian cultured sea cucumber,the content of MDA were 5.83-9.79 nmol/mL and 7.57-9.90 nmol/mL in “Shuiyuan No.1” sea cucumber and Dalian cultured sea cucumber.(2)In terms of acute drop in temperature,the activity of CAT,SOD and MDA first increased and then reduced in “Shuiyuan No.1” sea cucumber and Dalian cultured sea cucumber,the activities of CAT were 382.51-409.71 U/mL and 383.86-399.42 U/mL in “Shuiyuan No.1” sea cucumber and Dalian cultured sea cucumber; the activities of SOD were 0.023-0.038 U/mL and 0.025-0.035 U/mL in “Shuiyuan No.1” sea cucumber and Dalian cultured sea cucumber; the content of MDA were 5.83-9.79 nmol/ml and 5.57-9.90 nmol/mL in “Shuiyuan No.1” sea cucumber and Dalian cultured sea cucumber; the activity of POD were first increased and then decreased and then increased again to reduce,the activities of POD were 0.000 20-0.000 70 U/mL and 0.000 18-0.000 40 U/mL in “Shuiyuan No.1” sea cucumber and Dalian cultured sea cucumber.(3)Both the activities of CAT,SOD,POD and the content of MDA were changed in the different ways of dropping temperature and the changing in “Shuiyuan No.1” sea cucumber was more sensitive than in Dalian cultured sea cucumber in the terms of mild and acute drop in temperature.The activities of CAT and POD was higher in “Shuiyuan No.1” sea cucumber than in Dalian cultured sea cucumber; the content of MDA was larger in the terms of acute drop than in the terms of mild drop in temperature.These results revealed that there are different immune responses to sea cucumber in different drops of temperature patterns.“Shuiyuan No.1” sea cucumber was more sensitive and has higher capacity of antioxidant than Dalian cultured sea cucumber in the low temperature environment.

Abstract:This experiment was conducted to study the effect of dietary lipid level on the body index,lipid deposition and lipid metabolic enzyme activities of juvenile Onychostoma sima.We randomly assigned fish(n=900)with average initial body weight of(0.78±0.05)g into 6 groups consisting of 3 replicates that each replicate was 50 fish.Six isonitrogenous and isoenergetic diets(40% CP and 16.30 MJ/kg GE)were formulated containing 0(control group),2%,4%,6%,8%or 10% soybean oil respectively,Those actual lipid level was 2.83%,4.52%,6.68%,9.14%,11.35% and 14.07%,respectively.The experiment lasted for 60 days.The results showed that the condition factor(CF)of Onychostoma sima initially increased with increasing lipid levels,but then decreased,the CF was highest(2.64%)when the dietary lipid level was 9.14%,but there was no significant differences when lipid level were 6.68%-14.07%,(P＞0.05 ).The hepatosomatic index(HSI)increased with increasing dietary lipid levels(P<0.05),while viscerosomatic index(VSI)had no significant differences(P＞0.05).The crude lipid content of muscle and liver were easily affected by dietary lipid levels,and the crude lipid content of muscle and liver increased with increasing dietary lipid levels.There was no significant effect of dietary lipid level on nitrogen gain(P＞0.05),energy gain was highest when the dietary lipid level was 14.07%,at the same time,the nitrogen and energy retention initially increased with increasing lipid levels,but then decreased,those were highest when the dietary lipid level was 11.35% and 9.14% respectively.Lipid gain of Onychostoma sima increased with increasing dietary lipid levels,but the lipid retention decreased(P<0.05).The activities of intestinal lipase initially increased with increasing lipid levels,but then decreased,and the activities of lipoprotein lipase,hepatic lipase and total lipase first increased,then decreased as the level of dietary lipid increased,those were all highest(296.03 U/g,30.59 U/g,34.28 U/g and 64.87 U/g respectively)when the dietary lipid level was 9.14%.At the same time,fatty acid synthetetase(FAS)decreased with the increase of contents.Results of above show that suitable dietary lipid level could improve the utilization rate of feed nutrients and reduce body lipid deposition,but when dietary lipid level reaches or exceeds 11.35%,the lipid deposition in liver would increase,which is bad for health and growth of Onychostoma sima.

Abstract:A feeding trial was conducted to determine the effect of dietary dextrin level on growth,activities of digestive enzyme and blood biochemical indices of juvenile obscure puffer,Takifugu obscurus.Five iso-nitrogenous(42%)and iso-lipidic(9%)diets containing graded levels of dextrin(10%,15%,20%,25% and 30%)were prepared.The fish［average weight(10.25±0.51 g)］were stocked in triplicate tanks during a feeding period of 60 days.The results showed that best specific growth rate and feed efficiency occurred for fish fed the diet with dietary dextrin level at 25%.Viscerosomatic index and hepatosomatic index of 30% group were significantly higher than those of 10% group(P<0.05),meanwhile liver lipid content of 30% group was also significantly higher than other groups(P<0.05).Liver glycogen and total amino acids content in plasma of 10% group were significantly lower than those of other groups(P<0.05).Compared with the 10% and 15% groups,amylase activity in hepatopancreas and intestine of other groups was significantly increased(P<0.05),while trypsin activity in hepatopancreas of the 30% group decreased significantly(P<0.05); total cholesterol and triglycerides content in plasma of the 25% and 30% groups increased significantly(P<0.05),while blood urea nitrogen in plasma of other groups decreased significantly(P<0.05).Red blood cell counts of 15% and 20% groups were significantly higher than other those of groups(P<0.05),while apoptosis ratio,reactive oxygen species and calcium ions content of 30% group were significantly higher than those of other groups(P<0.05).However,plasma glucose concentration,aspartate aminotransferase and alanine aminotransferase activity as well as lipase activity in hepatopancreas and intestine of obscure puffer were not affected by dietary dextrin levels.The present results showed that the optimum dextrin level in diets is at 20% -25% for juvenile obscure puffer under the present research conditions.

Abstract:The purpose of this study was to investigate the effects of vitamin E(V_E)on serum biochemical indices and antioxidant capacity of juvenile Megalobrama amblycephala under acute high temperature stress and recovery.180 healthy fish(45.00±0.50)g were selected and randomly divided into three groups:a control group was fed with basal diet,and two treatment groups fed with basal diet supplemented with 50 and 400 mg/kg V_E,respectively.Each group has four replications.After 90 days’ feeding trial,all fish were exposed to high temperature stress(32 ℃)for 1 day and recovered at 25 ℃ for 7days in a controlled temperature aquarium.The results showed that the diet supplemented with 50 mg/kg V_E significantly increased the activity of intestine glutathione compared with the control group(P<0.05)prior to the stress.After high temperature stress,the diet supplemented with 50 mg/kg V_E significantly decreased the levels of serum aspertate aminotransferase(ALT)activity and cortisol(COR)content at 1d after stress,reduced the levels of serum alanine aminotransferase(AST)activity,triiodothyronine(T₃)and COR content at 7 d recovery after stress(P<0.05),and improved the serum alkaline phosphatase(ALP)activity,intestine glutathione(GSH)activity and superoxide dismutase(SOD)activity at 7d recovery after stress(P<0.05)compared with the control group(P<0.05).The diet supplemented with 400 mg/kg V_E significantly reduced the levels of serum ALT and intestine malonaldehyde(MDA)content at 1 day after stress and 7 d recovery after stress,COR content at 7 d recovery after stress(P<0.05)and increased the levels of serum ALP and T₃at 1 day after stress concentration compared with the control group(P<0.05).So it is suggested that ingestion of a basal diet supplemented with V_E(50-400 mg/kg diet)could help regulate the change of blood lipids,improve antioxidant capacity,alleviate the change of blood biochemical parameters in high temperature stress condition,reduce lipid peroxidation,and enhance resistance against high temperature stress in Megalobrama amblycephala.

Abstract:In order to investigate the effects of injecting phosphatidyl serine(PS)on hemocyanin synthesis and hemocyanin-derived phenol oxidase(Hd-PO)and probe into the pathway and activating mechanism of PS as a possible native elicitor for Hd-PO in white shrimp(Litopenaeus vannamei),we injected adult white shrimps different PS with 5,10,20 μg/mL,and assayed hemocyanin concentration in plasma,expression of hemocyanin subunits(p75 and p77)and mRNA in hepatopancreas,along with Hd-PO activity in plasma from white shrimp.They were investigated by spectrophotometry,native-PAGE,SDS-PAGE and RT-PCR in vivo with different sampling time at 0,6,12,24,36,48 and 60 h respectively,and we set the control group and parallel group.The results showed that hemocyanin concentration declined slightly during 6 h after injecting PS and afterwards changed as a peak by 36^th h after injection with a maximum at 24^th h(P<0.05);expression of hemocyanin subunits and mRNA all had peak changes during 36 h after injecting PS,and likewise,all reaching their ceiling values at 12^th h(P<0.05);Hd-PO activity had a peak change within 36 h after injecting PS,with its climax at 12^th h(P<0.05).The research revealed that PS could activate hemocyanin to show phenol oxidase activity with significant time-dose effects,and meanwhile brought down hemocyanin concentration in a short time at first and subsequently started the hemocyanin synthesis,indicating that PS is the elicitor of Hd-PO in white shrimp,which lays the theoretical foundation for the study on immunological function of hemocyanin in shrimp.

Abstract:Interleukin 8(IL-8),belonging to CXC family,is the first discovered chemokine,and is closely associated with non-specific immunity of fish.In this study,a full-length cDNA sequence of the IL-8 gene was cloned from crucian carp(Carassius auratus)in Qihe River using RT-PCR and RACE techniques.The IL-8 cDNA sequence consisted of 641 bp nucleotides,including 102 bp at 5＇UTR,242 bp at 3′UTR and an open reading frame with 297 bp nucleotides.This cDNA sequence encoded a precursor protein of 98 amino acids and the former 22 amino acid residues formed a signal peptide.The putative peptide contained four cysteine residues,and the former two cysteine residues were separated by an arginine residue and consequently formed a CXC structure.Furthermore,similar to the most fishes,IL-8 of crucian carp lacked the completed ELR(Glu-Leu-Arg)motif in front of the CXC structure,which was commonly present in mammalian and chicken IL-8 molecules.Therefore,DPR motif was substituted for ELR motif in IL-8 of crucian carp.Comparison of the amino acid sequences showed that the crucian carp IL-8 shared the highest similarity with Cyprinus carpio(94%).Phylogenetic analysis also demonstrated that the crucian carp IL-8 had the close relationship with Cyprinidae species such as Cyprinus carpio(94%),Hypophthalmichthys nobilis(81%),Ctenopharyngodon idella (81%)and Hypophthalmichthys molitrix(80%).IL-8 mRNA expression in eight different tissues of crucian carp was determined by Real-time Quantitative PCR technique.The results displayed that the level of IL-8 mRNA was highest in muscle,followed by spleen,head kidney and brain.The research would be beneficial to the regulation of non-specific immunity in crucian carp,and also provides a certain reference value for improving the health breeding level of crucian carp.

Abstract:Eight isonitrogenous,isoenergetic and isolipidic diets were formulated to evaluate the feasibility of partial replacement of white fish meal(WFM)by dehulled soybean meal(DSM)or enzyme-treated dehulled soybean meal(EDSM),and to determine suitable inclusion levels of white fish meal and two kinds of soybean meal in diets for juvenile largemouth bass(Micropterus salmoides),based on growth performance,feed efficiency,body composition,non-specific immunity and hematological parameters.In four diets(D1-D4),inclusion level ratios of WFM to DSM were 45∶5,40∶12,35∶18 and 30∶25,respectively,and in the other four diets(E1-E4),the same inclusion level ratios were kept,only substituting EDSM for DSM.Each diet was randomly assigned to triplicate tanks of 35 fish［average initial weight(5.32±0.05)g］with an indoor temperature-controlled recirculating freshwater system.Fish were fed to apparent satiation twice daily(8:00 and 16:00)over 63 days.During the experimental period,water temperature was maintained at(28±1)℃ and dissolved oxygen content kept nearly saturated.The natural light cycle was adopted.The results suggested that no differences in survival rate and specific growth rate were found among treatments.Feed efficiency and protein efficiency ratio,however,occurred to a significant declining tendency when inclusion levels of DSM were higher than 5%,or EDSM higher than12% in diets.With decreasing in WFM inclusion level and increasing in DSM or EDSM inclusion level in diets,apparent digestibility coefficients of both protein and lipid were found to be higher.Meanwhile,protein and moisture concentrations in whole body,muscle and viscera of tested fish decreased significantly,but lipid concentrations became higher.With above changes in dietary WFM and DSM or EDSM levels,hepatosomatic index and liver glycogen content occurred to be lower.Serum lysozyme activity and serum protein content became lower althought serum complement activity(CH50)showed no significant difference among the treatments.While no difference in red cell count was found,the blood parameters of haemoglobin and haematocrit were affected significantly and negatively.In conclusion,it is feasible to use 45% WFM and 5% DSM as main protein sources in diet for juvenile largemouth bass,based on a comprehensive judgment of growth rate,survival rate,body composition,feed efficiency and health of tested fish.Inclusion levels of 40% WFM and 12% EDSM can achieve a similar performance,substituting EDSM for DSM in the diet,by which 11% of WFM can be replaced with EDSM.The effectiveness of enzyme treatment in removing the deleterious effects of antinutrients from soybean meal is confirmed in this study.

Abstract:In order to study the underlying molecular mechanisms for growth of Epinephelus septemfasciatus,the insulin-like growth factorⅠ(IGF-Ⅰ)mature peptide sequence was amplified from E.septemfasciatus liver with RT-PCR method.It was predicted that the mature peptide was composed of 210 base pairs,which encodes 70 amino acid residues and consists of B-C-A-D four functional domains.The matured peptide fragment was subcloned into the expression vector pET-28a and was successfully expressed in E.coli BL21(DE3)cell.The result of SDS-PAGE analysis indicated that the fusion protein expressed in the form of inclusion bodies with molecular weight of 11 ku and maximally amounted to 51.8% of the whole protein in the E.coli cell 3 hours after being induced with IPTG.The purified recombinant protein was obtained through denaturation,purification and refolding.The Western-blotting indicated that the recombinant protein had specifically been recognized by 6×His antibody.The proliferation experiment showed that the purified IGF-Ⅰ fusion protein could significantly promote the proliferation of breast cancer cells MDA231,and this indicated that it has biological activity.

Abstract:In order to establish a procedure to induce artificial gynogenesis for genetic improvement and sex control studies in Nibea albiflora,ultraviolet irradiation was used to genetically inactivate the sperm and cold shock was used to induce the chromosome duplication of the eggs.Relative DNA content in newly-hatched larvae of candidate haploid gynogens,candidate diploid gynogens and the control groups from normal fetilization were mensurated,and paternity test was made using SSR markers for confirmation of gynogenesis.The gynogenetic diploid was successfully induced by activating egg development with UV-irradiated sperm combined with cold shock to prevent extrusion of the second polar body.UV irradiation time range was preliminarily determined by an insemination trial with the sperms irradiated by different doses of UV.The fertilized egg hatchability exhibited typical Hertwig effect when sperm irradiation time was 0-100 seconds at a UV intensity of 3 800 μW/(cm²·s).All newly-hatched larvae exhibited typical haploid syndrome when the UV irradiation time was over 60 seconds.In addition,we designed an orthogonal experiment to further optimize UV irradiation time,cold shock initiation time and cold shock duration time.The orthogonal experiment results showed that the hatching rate was highest(16%)in the following conditions:sperm irradiation time was 60 seconds,cold shock temperature was 3-4 ℃,cold shock initiation time was 2 minutes after fertilization and cold shock duration time was 10 minutes.The larvae of the optimal group have the same morphological characteristics and cellular DNA content with normal diploids.A further paternity test with five compatible SSR primers of Larimichthys crocea proved that the larvae in the optimal group were all gynogenetic diploids without paternal specific alleles.