Abstract:The cell lines from different organs of half-smooth tongue sole were the ideal material for researches on fish virology,genetic screening and functional analysis,etc.In this study,the researches of the spleen and ovary cells from female individual in vitro culture were developed by trypsin digestion method.The results showed that the spleen and ovary cells were cultured in minimum essential medium(MEM)supplemented with 20% fetal bovine serum(FBS),50 mmol/L 2-Mercaptoethanol(2-Me),100 U/mL penicillin,100 μg/mL streptomycin,1 mmol/L sodium pyruvate and 10 ng/mL basic fibroblast growth factor(bFGF),and microscopically,the cells were composed of fibroblast-like cells.The spleen cells were subcultured for 9 passages.Up to now,the ovary cells had been subcultured for 11 passages.Chromosome analysis revealed that the spleen and ovary cells maintained normal diploid number(2n=42),containing the heterogametic W chromosome.In subculture,the suitable concentration of FBS for the ovary cells was 20%.The spleen cells had been successfully transformed with red fluorescent protein(RFP)reporter plasmid.

Abstract:The changes in mortalities ofPenaeus monodon(with 1×10⁵ copies/g WSSV),WSSV loads in muscle,THC and the percentage of different types of haemocyte were studied by injection with WSSV solution containing 3×10³,6×10²,2×10²copies/mL virus and PBS.Real-time PCR,and microscopical examination were used in this experiment.The results showed that the cumulative mortalities were 93.33%±2.89%,56.67%±5.77%,45.00%±5.00% respectively,when the concentration of WSSV solution were 3×10³,6×10²,2×10² copies/mL.No death was observed in the PBS group.When the concentration of WSSV solution was 3×10³copies/mL,the virus load in the muscle reduced to minimum(3.54×10⁴copies/g)at 30 min postinjection,and reached its maximum(3.12×10⁸copies/g)at 48 h postinjection,then decreased again.For the other two experiment groups,the minimal virus load(1.11×10⁵,9.54×10⁴copies/g)in the muscle was observed at 1 h post-injection and the highest virus load(1.48×10⁷,5.46×10⁶copies/g)occurred at 48 h post-injection,but a second highest virus load(1.58×10⁵,1.11×10⁵copies/g)appeared at 6 h post-injection.No significant changes were found in the WSSV load of the control group throughout the experiment.The fluctuation range and the peak time of total haemocyte count(THC)were different between different dosage of WSSV solution.Especially THC reduced to minimum(3.48×10⁶,4.05×10⁶/mL)at 12 h and 48 h post-injection with 3×10³ copies/mL solution.The variation of THC and WSSV load were negatively correlated except individual time points.Under all groups,the percentage of semi-granular cells showed upward trend obviously early post infection,but later remained at a high level although fluctuated; the percentage of granular and hyalinocyte cells showed downward trend early postinjection significantly,while increased slightly in the middle period,but the percentage of granular cells at the later stage of post-infection was significantly lower than that of early period,while the levels of hyaline cells between later and early stages of post-injection have no significant difference.

Abstract:White spot syndrome virus(WSSV)can infect many kinds of crustaceans and thus has caused huge economic losses to the crustacean farming.In order to detect WSSV precisely and sensitively,quantify WSSV conveniently,and further investigate the WSSV multiplication patterns in crustaceans,a practical real-time PCR for the quantification and diagnosis of WSSV is required to be developed.However,currently,the harvested quantitative real-time PCR for WSSV was either costly for those which were developed based on the Taqman probe technology,or insensitive for a latest method which was set up with the SYBR green dye.In this study,a practical,sensitive,and specific quantitative real-time PCR for WSSV based on SYBR Green was developed,and the tissue distributions and the profiles of WSSV proliferation in ridgetail white prawns(Exopalaemon carinicauda)were examined with this method.The details were as follows.First,a pair of primers(F1,R1)was designed to amplify the target fragment,which would be used to construct WSSV standard plasmid,and then another pair of primers(RF2,RR2)located inside the above target fragment was synthesized to produce amplicons in real-time PCR reaction,thereby developing a new diagnostic and quantitative real-time PCR for WSSV with SYBR Green dye.The results demonstrated that this quantitative method for WSSV was specific,sensitive,and precise.Subsequently,we further investigated WSSV distribution and proliferation pattern in ridgetail white prawns.The results of WSSV distribution showed that WSSV infected all detected tissues,among which,the connective tissues under the carapace possessed the highest virus load(1.1×10⁷ copies/μg tissue),and the second highest tissue were gills with the virus load of 4.1×10⁶ copies/μg tissue; The data of WSSV proliferation pattern illustrated that the copies of WSSV began to increase dramatically at 48 h after challenge,but before that there was no obvious change for virus copies.With the increase of infection time,the copies of WSSV increased more and reached the highest numbers at 120h post infection.In addition,we found that during this process the mortality of ridgetail white prawns caused by WSSV was approximately 20%.All these results suggest that WSSV is a deadly pathogen to ridgetail white prawns,and the new quantitative real-time PCR for WSSV is practica.

Abstract:Bio-Floc consists of phytoplankton,bacteria,aggregates of living and dead particulate organic matter,and grazers of the bacteria.It not only can regulate water quality,but also can supply the food protein for breeding animals.For the study of bacterial community structure of BioFloc in freshwater culture systems,the key technologies of Israeli Avnimelech team about Bio-Floc in the field of aquaculture were used in this paper.Glucose was added as the carbon source which makes a C∶N ratio of 20∶1 be maintained during the experiment.The bacterial communities of BioFloc were analyzed by using the PCR-DGGE(PCR-denaturing gradient gel electrophoresis)on the 5th,10th,and 15th day,respectively.The results indicated that the similarity of bacterial communities was highest on the 5th day and 10th day which was 67.4%,and was lowest on the 5th day and 15th day which was 40.5%.Diversity of bacterial species in the Bio-Floc was maximal on the 10th day and minimal on the 15 day.The acquired sequences of 24 bands in DGGE gel were performed by BLAST searches against NCBI database,and the sequences including the closely related sequences were aligned with Clustal W program in BioEdit software for phylogenetic analysis.The main microbes represented by 24 main bands in DGGE gel were Alphaproteobacteria,Gammaproteobacteria,Betaproteobacteria,Actinobacteria,Bacilli and Bacteroidetes.Among these bacteria,the α-,β-,and γ-proteobacteria are the major bacterial groups,but Actinobacteria bacteria exist on the 10th day and the 15th day.α-proteobacteria was the dominant bacteria during the entire process.The specific bacteria at different stages were usually the flocculant-producing bacteria:Acidovorax,Aeromonas,Agrobacterium only for 5th day;Bacillus,Rhodococcus for 10th day and 15th day,respectively.This study first found that the composition and diversity of bacteria of the Bio-Floc used in freshwater aquaculture systems are extremely rich.The study of functional microbial composition of the Bio-Floc may help to lay the foundation for the further application of the bio-floc technology in actual aquaculture production.

Abstract:The aim of the present study was to detect the levels of NH⁺₄-N,NO^-₂-N and NO^-₃-N,and to determine the numbers of ammonifying,nitrifying,and denitrifying bacteria,which are responsible for the nitrogen cycling process in four representative grass carp(Ctenopharyngodon idellus)ponds.After sampling water from 4 grass carp ponds,one hundred strains selected randomly from each sample were tested by qualitative color reaction,and on the basis of the test,11 strains of heterotrophic bacteria were selected for 16S rRNA sequence analysis.Results showed that the mean levels of NH⁺₄-N and NO^-₂-N was 5.597 mg/L and 0.135 mg/L,respectively.The cultivable heterotrophic bacteria density in fish pond was 3.26×10⁵ cfu/mL and 89.75% of them were involved in nitrogen cycle,65.75% and 31% of which were involved in NH⁺₄-N production and degrading,respectively.This result indicated that the overwhelming majority of nitrite-producing bacteria resulted in high level of nitrite in grass fish pond.The identification results of 11 strains of heterotrophic bacteria were as follows:six of them were related to Stenotrophomonas,three of them were related to Pseudomonas,and the other two strains were Klebsiella and Enterobacter,respectively.Moreover,there might be strain differences on utilization of nitrogen resourses.

Abstract:To investigate the applicability of the transposon piggyBac in Cyprinidae and Cobitidae fish, the expression element of DsRed under the control of PAβ promoter was inserted into the plasmid pigA3GFP to generate transgenic vector pigA3GFP-AβDsRed. The results of the CIK cell transfected with pigA3GFP-AβDsRed showed that the transposon piggyBac could transfer exogenous genes into CIK genome, and both of the promoter A3 from silkworm and the promoter CMV from CMV have the bioactivity. Moreover, the pigA3GFP-AβDsRed and pigA3GFP were mixed with helper plasmid helper-pigA3 respectively, and then were introduced into the eggs of goldfish and loach by sperm-mediated transfer. The results of fluorescence observation, PCR and Dot blotting verified that both transgenic goldfish and loach were obtained. Therefore, PB transposon was considered to be active in some fish of Cyprinidae and Cobitidae. Therefore, PB transposon was considered to be active in some fish of Cyprinidae and Cobitidae.

Abstract:The seasonal changes of bacteria community composition in digestive diverticula of 2 age scallopsChlamys farreri were studied.The scallop samples were collected every month since June 2009 to June 2010(except Feb.)from Shazikou Liuqinghe sea area of Qingdao,which was the scallop culture area.The genomic DNA was extracted from digestive diverticula of scallop using SDS schizolysis method.Bacterial V3-V5 region of 16S rDNA was amplified with the 358f and 907r primers.Then,denaturing gradient gel electrophoresis(DGGE)was used to analyze and discriminate the amplified fragments.The DGGE fingerprint was analyzed by software Quantity One.The results showed that DGGE profiles exhibited 11 distinguishable bands,and 9 bands existed in all 12 months,the proportion of co-existed bands was 81.82%,the results indicated that the bacterial community composition was relatively stable in the digestive diverticula.Only 9 DGGE bands were successfully re-amplified,cloned,sequenced and aligned with BLAST.The result showed that the bacterial sequences shared high similarities(97%-99%)with sequences logged on NCBI.And the 9 bands belong to 4 major groups:Proteobacteria,Firmicute,Cyanobacteria and Actinobacteria.The predominant bacteria species in digestive diverticula of scallops were Massilia,Bacillus,Synechococcus,Exiguobacterium,Rhodococcus and uncultured bacteria.

Abstract:In the present study,full-length cDNA sequences of histocompatibility complexⅠα antigen(MHCⅠα)was cloned by rapid amplification of cDNA ends technique (RACE)from humphead snapper(Lutjanus sanguineus).Full-length cDNA sequence of MHCⅠα is 1 369 bp,encoding 354 amino acids.BLAST analysis revealed that the amino ids sequence of MHCⅠα shared high identity(84%)with other MHCⅠ.Phylogenetic tree was constructed by the NeighborJoining method,and the results suggested MHCⅠα of humphead snapper shared the closest genetic relationship with the MHCⅠof Epinephelus coioides.The results of fluorescent real time quantitative RTPCR showed that the expression of MHCⅠα mRNA could be detected in head kidney,and the maximum expression appeared in 6-15 h post infection.MHCⅠα was subcloned into pET32a(+)to construct expression plasmids pET32-MHCⅠα.SDS-PAGE and Western blot analysis indicated that the recombinant proteins were successfully expressed in Escherichia coli BL21(DE3).Then the recombinant proteins were purified and the antiserum was obtained by immunizing rabbits with the purified recombinant proteins emulsified with adjuvant.ELISA analysis showed that the titer of the antiserum prepared in this study was 1∶40 000.The results of the Western blot revealed that specific antigenantibody reaction occurred between the antiserum and the recombinant proteins.

Abstract:Microbiological investigation was performed to determine the mariculture impact on the ecological distribution of bacteria in the sediment of the laver culture area and cage culture area of Xiangshangang Bay in January and October,2006,and the results were compared with those in adjacent non-cultured area of cage and laver culture.Bacterial quantity presents a series of characters in sediment:Higher bacterial number was observed in Oct.than that in Jan.,and the horizontal distribution of bacterial quantity displayed that Cage culture area［average of(9.6×10⁴±2.0×10⁵) cfu/g］>Reference site near cage［average of(1.5×10⁴±2.4×10⁴) cfu/g］>Laver culture area［average of(4.3×10³±1.6×10³) cfu/g］and Reference site near laver culture［average of(4.7×10³±3.0×10³) cfu/g］.109 strains of heterotrophic bacteria isolated from laver culture area mainly belonged to 17 genera,and Bacillus and Coryneforms were the dominant genera.95 strains isolated from reference site near laver culture mainly belonged to 18 genera,andBacillus and Coryneforms were the dominant genera.136 strains isolated from cage culture area mainly belonged to 11 genera,andBacillus,Coryneforms,Pseudomonas,Acinetobacter and Vibrio were the dominant genera.Bacteria in sediment under laver culture and reference site near laver culture were mostly common ones that appeared in marine sediment,and the microbial densities in these two areas were higher than that in cage culture area and reference site near cage.Because of longterm drainage of organic matters,cage culture sediment’s microbial density had reduced,its bacterial composition was similar to that in muddy sea areas,and the effect on bacterial community induced by culture has spread to its adjacent area.It is clear that there exist differences in the characters of bacterial ecological distribution in laver and cage culture areas,and bacterial quantitative distribution and community structures were closely related to self-pollution status of mariculture.

Abstract:Several recent studies suggesting that respiration and calcification activities by shellfish represent a net source of CO2. In order to the assess the effect of shellfish aquaculture on the sea-air CO2 flux, a Pacific oyster Crassostrea gigas aquaculture area which is located in East Gongjia Island, Rushan City, were selected as the investigation area. Based on the investigation data of pH, total alkalinity (TA), chla, etc. in October 2011, the concentration and horizontal distribution of dissolved inorganic carbon (DIC) system parameters were calculated. Moreover, sea-air CO2 fluxes and the contribution of phytoplankton to the sea-air CO2 flux were estimated. Results showed that the DIC concentration of surface sea water ranged between 1953.20 μmol/L to 2130.74 μmol/L, with an average of 2048.73 ± 57.19 μmol/L; the main component of DIC was HCO3-, which occupied 88.25%; the pCO2 ranged from 220.08 μatm to 262.29 μatm, with an average of 246.46 ± 23.00 μatm; the sea-air flux of CO2 ranged from -53.78 mmol/(m2.d) to -21.93 mmol/(m2.d), with an average of -42.09 mmol/(m2.d), which represented that this area acted as a strong sink of atmosphere CO2 in autumn; Carbon fixed strength of phytoplankton ranged between 460.27 to 725.64 mg/(m2.d), with an average of 593.27 ± 91.98 mg/(m2.d); the higher sea-air flux of CO2 was mostly contributed by the photosynthesis of phytoplankton; there was no significant difference between culture area and non-cultured area of sea-air flux of CO2, which indicated that the respiration and calcification activities of oyster had little effect on the CO2 flux.

Abstract:Chinese shrimp(Fenneropenaeus chinensis)is distributed mainly along Chinese inshore areas,and is one of the most important farmed shrimp in China.The studies on innate immune responses of shrimps,especially on immune defense against the main crustacean pathogens,will provide more knowledge of shrimp immunity to prevent infectious Invertebrates do not possess an adaptive immune system based on highly specific antibodies and antigen receptors.They must rely on efficient immune defenses capable of protecting them against invading microorganisms.The chief issue of crustacean immunity should concern non-self-recognition mechanisms.Proteins that specifically bind to certain carbohydrate components on the surface of microorganisms play an important role in non-self-recognition and cleaning up of the invading microorganisms.Such proteins are known as pattern recognition receptors(PRRs).Lectins exist in almost all living organisms.Due to their ability of binding to terminal sugars on glycoproteins and glycolipids,lectins are primary candidates for pattern recognition receptors in innate immunity.C type Lectin is regarded as a potential molecule involved in immune recognition and phagocytosis through opsonization in crustacean.In the preliminary study,a novel C-type lectin was cloned from hemocytes of Chinese shrimp,(Fenneropenaeus chinensis).It contains two tandem carbohydrate recognition domains(CRDs)/C-type lectinlike domains.Both of the CRDs contain a QPD(Gln-Pro-Asp)motif that has a predicted binding specificity for galactosetype sugar.In this research,two recombinant target proteins(rFclectinCRD1 and rFclectinCRD2)were expressed by prokaryotic expression system.The result showed that fusion protein was expressed in the form of inclusion bodies.The LC-ESI-MS analysis showed that two peptide fragments of rFclectin-CRD1 and rFclectin-CRD2 were identical with the corresponding sequence of F.chinensis C-type lectin.Recombinant protein was purified by immobilizedmetal affinity chromatography and Ni-NTA technology.The concentrations of purified target proteins were 0.4 g/L.rFclectin-CRD1 and rFclectinCRD2 had agglutinating and antimicrobial activity against main pathogens in aquaculture in a calciumdependent manner.The agglutinating activity can be inhibited by multiple carbohydrates,such as galactose,peptidoglycan and lipopolysaccharide.These results suggest thatFclectin,as a dependent carbohydrate-recognition protein,is one of the important PRRs.It might play a crucial role in the innate immunity of the shrimp and is expected to be applied in disease control.

Abstract:he experiment was conducted to evaluate the effects of partial replacement of fish meal(FM)with silkworm pupae(SWP)on juvenile Cyprinus carpio var.specularis［(12.10±0.89) g］.FM diet(fish meal 10%,silkworm pupae 0%,CP 36.18%,EE 5.18%)and SWP diet(fish meal 5%,silkworm pupae5.5%,CP 35.38%,EE 5.06%)were formulated to feed the fish three times daily to visual satiety in indoor circulating water system for 61 days.The results showed that final body weight(FBW),weight gain rate(WGR),special growth rate(SGR),protein efficiency ratio(PER),feed conversion ratio(FCR),survival rate(SR)of the SWP group and FM group were not significantly different(P>0.05),while no difference was found in condition factor(CF),viscerossomatic index(VI),hepatosomatic index(HI),spleen index(SI),intestine index(II),as well as the content of moisture,crude lipid,crude protein,crude ash of the fish tissue(P>0.05).The contents of Lys in whole body and Tyr in muscle of SWP group were significantly lower than those in FM group(P<0.05).The contents of C14∶0,C16∶1n-7 and C18∶1n-7 in muscle of SWP group were significantly lower than those in FM group,and the contents of C18∶3n-3 and ∑n-3PUFA in muscle of SWP group were significantly higher than those in FM group(P<0.05).On the other hand,the contents of C18∶3n-3 and C22∶5n-3 in hepatopancreas of SWP group were significantly higher than those in FM group(P<0.05).No significant difference was found in serum biochemical indices,index of atherogenicity(IA)and index of thrombogenicity(IT)between the two groups(P>0.05).There are no significant differences in mucosa amount,mucosa height,mucosa width and submucosa thickness of foregut and midgut(P>0.05),however,the muscular coat thickness of foregut was significantly lower in SWP group than FM group(P<0.05).It is concluded that silkworm pupae is an acceptable alternative animal protein source,which can replace up to 50% of fish meal protein inC.carpiovar.specularisdiet without significant adverse impact on the health status,althought it affects the tissue amino acid and fatty acids profiles,and has a tendency to promote the growth performance and feed utilization.