Abstract:The razor clam, Sinonovacula constricta, which belongs to Mollusca,Bivalvia,Veneroida,Solecurtidae,is a common shellfish as human seafood,living in the lower-to-mid intertidal zones along the coast of the West Pacific Ocean.The genetic structure of the razor clam has not been reported along the coast of Zhejiang and Fujian Provinces,the major aquaculture regions in China following the widespread culture.In this study,genetic structure of eight populations of S.constricta from Zhejiang and Fujian Provinces(NH,SM,YH,YQ,CL,HJ,HA and SS)was estimated by using microsatellite loci and mitochondria COⅠ sequences.Based on microsatellite loci,the effective allele gene(N_e)number ranged from 6.2 to 9.0,and the expected heterozygosity(H_e)was high,ranging from 0.806 to 0.875.Based on mitochondria COⅠ,haplotype diversity(Hd)ranged from 0.942 to 1.000 and nucleotide diversity(P)varied from 0.005 6 to 0.011 5. The analysis of genetic differentiation revealed that the F_ST values ranged from 0.001 4 to 0.063 8 based on microsatellite loci and from 0.000 9 to 0.368 0 based on mitochondria COⅠ.The phylogenetic tree analyses showed that the genetic relationships of eight populations were not consistent with a model of isolation by distance(IBD),but the populations within one bay could be clustered first.It is suggested that the razor clam contains high genetic diversity,but gene flow exists between Zhejiang populations and Fujian populations based on the genetic relationship.

Abstract:Porphyra haitanensis is an economically important marine crop in southern China.To study the molecular mechanism of high temperature tolerance in P. haitanensis,the technology of annealling control primer(ACP) was used to screen the differentially expressed genes in F₄ generation gametophytic blades of a high temperature tolerance line Z-61.The primer combination of dT-RSL and RSL7 amplified one differentially expressed gene fragment.After sequence analysis,the gene fragment was proved to be the full length sequence of ribosomal protein S7 gene,and then it was named Phrps7 (acceession number JF719273).The Phrps7 gene of 702 bp contains an open reading frame of 585 bp encoding a PhRPS7 protein of 195 amino acid.The PhRPS7 protein assembled by 5 helix,6 sheet and 6 cycle shared high sequence identity with RPS7 proteins from other organisms(>55%),and its molecular formula was C₉₈₄H₁₆₀₄N₂₉₄O₂₈₃S₂.Phylogenetic analysis showed that the PhRPS7 protein has closer relation with RPS7 proteins from epiphyte than those from other organisms.The results of real-time quantitative PCR indicated that the expressed level of Phrps7 has high relationship with high temperature stress.At the beginning of high temperature stress(0-6 d),the expression of Phrps7 gene was up-regulated significantly,but at the later stage of high temperature stress(>6 d),the expression of Phrps7 gene was down-regulated,however,it was still higher than that at the beginning.

Abstract:Haematococcus pluvialis, a unicellular green alga,is one of the potent biological sources for valuable secondary metabolite astaxanthin production.Methyl jasmonate(MeJA),a naturally-occurring plant cellular signal molecular,was found to induce secondary metabolites production.In this paper,the effects of MeJA on the cell growth,astaxanthin production and dxs gene expression of Haematococcus pluvialis were investigated in detail,which provides a theoretical foundation for cosmically manufacturing astaxanthin.The results showed that,the addition of different concentrations of MeJA restrained the cell growth of H.pluvialis and decreased total astaxanthin production also.Addition of MeJA could promote unit cell astaxanthin accumulation,and with the increased MeJA concentrations,the unit cell astaxanthin content showed the trendency that was first increased and then decreased.The optimum addition concentration of MeJA for astaxanthin production was 800 μmol/L.The highest yield of the unit cell astaxanthin content in H.pluvialis was 1.75?10^-9 mg,which increased by 23.24% compared with 1.42?10^-9 mg without MeJA.This study provides a simple means to increase astaxanthin production.RT-PCR analysis showed that dxs gene expression was induced by MeJA.With the treatment of 800 μmol/L MeJA, dxs gene expression levels were the highest.The correlation analysis results showed that,the astaxanthin content of H. pluvialis and the amount of dxs gene expression were in positive correlation,thus it can be seen that the dxs gene expression level can reflect the astaxanthin production of H. pluvialis, and dxs gene was a key enzyme gene of astaxanthin synthesis of H.pluvialis.

Abstract:Mycosporine-like amino acids(MAAs)found in marine organisms,especially in the algae were a class of ultraviolet absorption substances.To study the constituents and proportion of MAAs from Gracilaria changii of Zhanjiang,MAAs were separated and purified by C-18 Sep-Pak SPE and semi-preparative HPLC.Four mycosporine-like amino acids were found and were identified as palythine,shinorine,palythinol and porphyra334,with an approximate ratio of 4.9∶1∶1.2∶9.9,based on the characteristics UV absorption and spectral data.This paper compared the accuracy of UV and HPLC determination for the content of porphyra-334 which was the main component of G.changii, and established fitting curves of two methods.Verified by experiment,the HPLC detection for the main components of MAAs was ultimately determined.This research laid the foundation for the development and use of the UV radiation and anti-oxidative substances in the economic algae produced in our country.

Abstract:Response surface methodology(RSM)and Central Composite Rotatable Design(CCD)were used to study the combined effects of ammonia(0.02-2.00 mg/L)and density(1-5 ind/10 L)on the specific growth rate(SGR)and antioxidant activities in the liver of juvenile tilapia(Oreochromis niloticus).The objectives were to find out the linear,quadratic or reciprocal effects of ammonia and density on the growth and antioxidant activities in the liver of juvenile tilapia by the regression equation.The results showed that the linear and quadratic effects of ammonia and density on the SGR were significant(P<0.05),SGR increased at the beginning and then decreased with the increased concentrations of the ammonia,and the density effects showed the same trend.There was a significant interactive effect between ammonia and density on growth(P<0.05).Higher SGR was observed when the density was 1-2 ind/10 L and ammonia was 0.02-0.20 mg/L.When concentrations of external ammonia were higher than 0.2 mg/L and density were around 3 indiv/10 L,SGR was also higher.With the increased concentrations of ammonia and density,the content of MDA in the liver increased gradually,while the activities of SOD and CAT increased at the beginning and then decreased.The linear effect of ammonia and density had significant effects on MDA,SOD and CAT(P<0.05),the quadratic effects of ammonia and density had very significant effects on the SOD and CAT(P<0.01).There was a significant interactive effect between ammonia and density on CAT activity.High concentrations of external ammonia and high density inhibited SOD and CAT activities.Model equations of the effects of ammonia and density on SGR,MDA,SOD and CAT were established,with the R² being 0.972 4, 0.913 2,0.938 9 and 0.969 2 respectively(P<0.01),which could be used for prediction.Growth and activities of antioxidant enzymes were more influenced by ammonia than by density.In the cultural practice,it is suggested that the density should be controlled reasonably and the dissolved oxygen be maintained adequate to reduce ammonia in order to improve growth and disease resistance of tilapia.

Abstract:In the present study,the effect of ascorbic acid on primary cultured hepatocytes viability and antioxidant capability under trichlorfon stress were investigated in Carassius auratus gibelio. The hepatocytes were cultured with media containing 0,50,100,200,400 and 800 μmol/L concentration ascorbic acid.Cell viability,the changes in hepatocytes lactate dehydrogenase(LDH)activity and ascorbic acid concentration were assayed.In addition,cellular intracellular total antioxidant capacity(T-AOC),glutathione-S-ePoxide transferase(GST)and butyrylcholinesterase(B-ChE)activities and cytochrome P450(CYP450)concentration under trichlorfon stress were determined.The results showed that hepatocyte viability was significantly increased(P<0.05)when cell was cultured with 100 μmol/L concentrations ascorbic acid in vitro compared with control.Cellular ascorbic acid concentration was significantly increased with media contained 50,100,200,400 and 800 μmol/L concentration ascorbic acid.There were no significant changes in cellular LDH activity with media containing 50,100,200,400 μmol/L concentration ascorbic acid.But LDH activity was markedly(P<0.05)increased when ascorbic acid concentration was 800 μmol/L.Under trichlorfon tress,the cellular antioxidant capability was increased significantly(P<0.05)at 50,100 and 200 μmol/L concentration ascorbic acid treatments.In conclusion,ascorbic acid facilitated hepatocytes viability and function of anti-oxidative stress when cells were cultured with 50-200 μmol/L concentrations ascorbic acid.

Abstract:Nutritional components in the meat of Pangasius bocourti from Yingshan of Hubei Province were analyzed and nutritive quality was evaluated.The contents of general nutrients,amino acids,fatty acids and mineral elements were determined with the current national standard method.The results showed that the contents of moisture,crude protein,crude fat and ash in the meat of P. bocourti were 75.36%?1.07%, 17.90%?0.43%,5.13%?0.18% and 0.98%?0.09% respectively.The total content of eighteen common amino acids was(69.01?0.07)g/100 g(dry weight),among which 5 kinds of delicious amino acids and 8 kinds of essential amino acids for human body accounted for 41.70%, and 48.68% respectively.The ratio of essential amino acids to non-essential amino acids was 95.06%.The constitutional rate of the essential amino acids accorded with the Food and Agriculture Organization of the United Nations/World Health Organization(FAO/WHO)Standards.According to nutrition evaluation in amino acid score(AAS) and chemical score(CS),the first limited amino acid was Tryptophan(Trp)and the second limited amino acid was Valine(Val).There were 24 kinds of fatty acids in crude fat,among which the content of oleic acid was the highest 38.41%?0.29% and the ratio of total mono-unsaturated fatty acids content to total fatty acids content in this fish was 42.68%?0.10%.The ratio of n-6/n-3 polyunsaturated fatty acids(PUFA)was 1.48.Potassium(K)was the most predominant mineral element found in meat and its content was(1 118.51?30.67) mg/100 g(dry weight).In trace elements,the contents of iron(Fe)and zinc(Zn)were(1.19?0.10) mg/100 g(dry weight),(1.18?0.05) mg/100 g(dry weight).Compared with other fish species,the meat of P.bocourti had a high quality in protein with well-balanced essential amino acid composition and was rich in n-3 polyunsaturated fatty acids and mineral elements. It indicated that P. bocourti was one of freshwater fishes with high nutritional value.

Abstract:The retinoid X receptor(RXR)is a member of nuclear hormone receptor family proteins and plays an important role in the regulation of intracellular receptor signaling pathways.Three retinoid X receptor subtypes(RXRα,RXRβ and RXRγ)cDNAs were obtained from Lateolabrax japonicus by RT-PCR and RACE.The obtained cDNA sequence of RXRα was 1 686 bp in length,containing a 5′-untranslated region(UTR)of 364 bp and an uncompleted CDS of 1 322 bp,and encoded a putative protein of 440 amino acids.The full length cDNA sequence of RXRβ was 1 741 bp in length,containing a 5′ UTR of 150 bp,3′UTR of 256 bp and an ORF of 1 335 bp which encoded a putative protein of 444 amino acids with an estimated molecular weight of 49.5 ku.The RXRγ cDNA was 1 847 bp in length with 88 bp of 5′-UTR,397 bp of 3′-UTR and an ORF of 1 362 bp which encoded a putative protein of 453 amino acids with an estimated molecular weight of 49.9 ku.Like other RXRs,the DNA-binding domain(DBD)of L.japonicus RXRα, RXRβ and RXRγ contain P box and D box and the ligand binding domain(LBD)contains 12 α-helices and 2 β-sheets with a ligand-dependent activation function AF-2 in the COOH terminus of the H12. In the phylogenetic tree, L. japonicus RXRα, RXRβ and RXRγ were clustered with corresponding subtypes respectively.The tissue-specific expression of each RXR gene was explored using RT-PCR.The mRNA of RXRα were expressed in all tissues examined with low level.The mRNA of RXRβ were expressed homogeneously in all tested tissues excepting a low expression in heart. RXRγ mRNA were also expressed in all tissues tested with high levels in muscle, intestine,kidney,fat and spleen.The results lay the foundation for further study of the functions of RXRα, RXRβ and RXRγ.

Abstract:In order to better understand the important role of sediment microorganisms in material cycle and energy flow of aquaculture pond system,three shrimp(Litopenaeus vannamei)ponds covered with plastic sheet in the bottom in Hengshan,Ningbo were chosen and phospholipid fatty acid(PLFA)analysis methods were used to investigate the sediment microbial community structure and its relationship with physical-chemical factors during a three-month period(from June to September,2010).The results showed that the sediment PLFAs were mainly contributed by the saturated fatty acid,branched fatty acid and monounsaturated fatty acid; the amount of polyunsaturated fatty acid and cyclopropane fatty acid was relatively smaller.The total microbial biomass,bacterial biomass and fungi biomass increased significantly with culture time.The principal component analysis(PCA)of the characteristic PLFAs in different periods showed the total of PC1 and PC2 explained the composition of sediment microbial community structure of 81.4%.Although the majority of microorganisms in the sediment were bacteria,bacteria/fungi ratio decreased significantly with culture time due to the better adaptation of fungi to the sediment habitat.The ratios of G /G^- bacteria in the later culture period were smaller compared to the mid-period,but did not change obviously from later period to final-period.Redundancy analysis(RDA)showed that the total microbial biomass,bacterial biomass and fungi biomass were significantly correlated with the total nitrogen and G bacterium biomass, G^- bacterium biomass were significantly correlated with the NO₂-N,NO₃-N. All of them were negatively correlated with the secchi depth(SD).The experimental results indicated that throughout the whole culture process,environmental factors of this new model ponds showed good consistency and stability.Simultaneously,the diversity of the sediment microbial community and the coordination between the structure variation and the environment are also relatively reasonable.The results also demonstrated that the PLFA technique well explained the microbial community according to the variation of PLFAs’ composition in different periods,and overcame the shortcomings of traditional culturing analyzing methods to a certain extent.

Abstract:The complete cDNA sequence of luteinizing hormone receptor(LHR)was cloned through degenerate primer PCR amplification and Smart^TM Race technology.The length of the cDNA of yellow catfish(Pelteobagrus fulvidraco) LHR gene is 2 524 bp and encodes the protein of 701 amino acids.This gene belongs to glycoprotein hormone receptor(GpHR)family,which contains the conserved seven transmembrane helix domains(TM helix).Meanwhile the amino acids sequence contains nine Leucine-rich repeats(LRRs).Potential N-linked glycosylation sites are ²⁹NFTC, ⁸²NVSR, ²⁰³NGSR, ⁵⁴⁸NLTV in yellow catfish LHR gene and phosphorylation site predictions identified 24 Ser,6 Thr and 5 Tyr potential phosphorylation sites.By Clustal X and MEGE 4.0 alignment revealed the highly conserved in GpHRs’ specific signature sequences: ²⁸⁷CCAF, ⁴⁷¹ERW, ⁵⁸⁴FTD and ⁶⁵⁷NPFLY.The only one protein kinase C phosphorylation site is ⁴⁰⁰S.Tissue distribution pattern showed preferential expression of LHR in ovary,and trace amounts were detected in brain,kidney,heart,liver and intestine(brain>kidney>heart>liver>intestine).Plasma 17β-Estradiol(E₂)levels in reproductive cycle were detected by radio immunoassay(RIA).The result showed that the highest peak in reproductive cycle is stage Ⅳ.The high transcript level of LHR in ovary was revealed in the reproductive phase of ovarian cycle,which was the relatively high expression from Ⅲ to Ⅴ phase and the highest level in stage Ⅴ.And we also analyzed the brain expression level in reproductive cycle,found the highest level in stage Ⅳ consistent with the level of E₂.Results demonstrated the gene of LHR in brain and ovary might also be involved in the processes which preferentially promote vitellogenesis and regulate ovulation maturation and ovulate either indirectly or directly.

Abstract:Cyanobacterial bloom has been a hot issue and the microbial community is one of the important functional groups in the decomposition process of cyanobacteria accumulation.In this study,restriction fragment length polymorphism(RFLP)was applied to analyze the bacterial diversity of different depths in the cyanobacterial accumulation of lakefront at northern shore of west region of Chaohu Lake.The total DNA of the microorganisms from three different layers of cyanobacterial accumulation(Layer A:0-2 cm,Layer B:2-10 cm,Layer C:10-20 cm)were extracted by the direct method.The DNA extracted was amplified using bacterial universal primers 27F and 1492R.PCR products were ligated into the pMD-18T Vector and ligations were transformed into Escherichia coli DH5α to construct 16S rDNA clone library of microbes in the cyanobacterial accumulation.120 positive clones each layer from the library were screened and their 16S rDNA fragments were reamplified.The fragments were digested with HhaⅠ and RsaⅠ respectively and their fingerprints were analyzed.The results showed that the layers from top to bottom include 51,32,27 restriction endonnuclease types and the coverage(C- value)of the clone library were 80.8%, 87.5%, 89.2%, respectively.Margalef indices(d_Ma), Shannon-Wiener indices(H)and Simpson indices(D)all decreased with the depth increasing.Phylogenetic analysis suggested that the dominant bacteria in different layers of cyanobacterial accumulation belonged to Firmicutes and Proteobacteria.With the depth increasing,the species of Proteobacteria decreased,but those of Firmicutes increased.Some bacterial species in Layer A also belonged to Cyanobacteria,Actinobacteria,Planctomycete,and Spirochaetes.And some in Layer C belonged to Bacteroidetes.The results revealed the changes of bacterial diversity and species composition during the decomposition of cyanobacteria accumulation.This study on the bacterial diversity not only provided microbiology information for understanding the decomposition process of cyanobacteria accumulation,but also supplied the theoretical basis for using the resources of unknown bacteria in the cyanobacteria accumulation.

Abstract:To study its structure and function,the ORF81 gene was amplified by PCR from koi herpesvirus China’s Jilin strains(KHV-CJ)infected.Mirror carp primary fin cells were then cloned into the vector pMD18-T to construct the recombinant plasmid.The structure and function of the gene were studied by means of bioinformatics software,including phylogenetic tree analysis compared with other three KHV strains published in GenBank.The results showed that the length of the ORF81 gene,encoding 256 aa,was 771 bp.The molecular weight was 28 246.50 u and the isoelectric point was 8.404.The protein is hydrophobic.The most likely signal peptide cut was in 29 bit(Ser).ORF81 has four transmembrane regions.The antigenicity of ORF81 was good.According to the protein structure analysis,there may be no Nglycosylation sites,6 O-glycosylation sites and 11 phosphorylation sites.Phylogenetic analysis showed that he ORF81 gene of KHV-CJ was in the same branch with the Israel strain.Above all, ORF81 gene may have good immunogenicity.The study established the foundation for genetic background information,pathogenesis,molecular epidemiology and genetic engineering vaccine research of koi herpesvirus.

Abstract:To understand the responses of hairtail(Trichiurus japonicus)catches to fishing and climate variability in the East China Sea(ECS),the time series of hairtail catches between 1956 and 2006 in the ECS was analyzed,which could be partitioned into inter-annual variation and internal trend.The internal trend was attributed to the monotonic growth of fishing efforts,the result of which was fitted by the Fox model showed that the relation between hairtail catch and fishing efforts was significant(P<0.01).After detrending the catch,variation in the catch was greatly affected by the climate fluctuation,which mainly included land precipitation,monsoon wind speeds,sea surface temperature and a proposed index of tropical cyclones(P<0.05).Variation of those main climatic factors and fishing efforts were significantly correlated with the annual catch(P<0.01).In conclusion,the land runoff brought nutrients to the coastal water,and the monsoons controlled the distribution and availability of the nutrients.In addition,the increase of water temperature was not only positive for the gonad development and maturity of hairtail,but also helpful to the availability of baits for hairtail.Thirdly,the tropical cyclones mobilized nutrient elements and enhanced biological production in many ways such as water mass moving,wind mixing,and upwelling.The paper concluded that both fishing efforts and climate change had influence on the variation of hairtail catch,and the climate changes in the future could be helpful to the increase of hairtail catch in the ECS,but also may result in a much bigger amplitude of catch variation.

Abstract:A microsatellite-enriched library of the ridgetail white prawn(Exopalaemon carinicauda)was constructed using the method of magnetic bead hybridization enrichment.Genomic DNA of ridgetail white prawn was digested with the restriction endonuclease Hae Ⅲ.DNA fragments from 400 to 1 200 bp were recycled and ligated to Hae Ⅲ adaptors.The ligated DNA fragments were hybridized with biotinylated probe(AG)₁₅.Then,enriched DNA fragments were ligated to pMD18-T vector.947 clones were randomly picked and screened by colony PCR.667 clones were positive.184 clones were randomly selected and sequenced from positive clones.150 clones were found to contain 199 microsatellite sequences.Among these microsatellites,79.4% belonged to perfect type,13.6% belonged to imperfect type and 7.0% belonged to compound type.From the primers designed for the 119 microsatellite loci,64 could amplify expected PCR products and 26 were found to be polymorphic.The number of alleles at each locus ranged from 3 to 16.The observed and expected heterozygosities varied from 0.111 to 0.929 and from 0.246 to 0.932,respectively.The PIC value ranged from 0.231 to 0.909.These new loci will be useful in the study of population genetic structure and conservation in this species.

Abstract:Edible gelatin films based on shark skins were successfully prepared.The effect of protein concentration,glycerol content and environmental humidity on the properties of edible gelatin films was investigated.As a result,white and transparent gelatin films could be produced using shark skins.With increasing protein content of film-forming solution,elongation at break(EAB),water vapor permeability(WVP)and transparency value of films increased slightly,but no differences in tensile strength(TS)were observed.Nevertheless,TS generally decreased with increasing glycerol concentration from 10 to 70%.The thermal stability of gelatin films was analyzed using DSC.It is revealed that the mechanical properties of films were not changed and polymeric materials exist in a glassy state when water activity(Aw)<0.33.Conversely,as Aw increased above 0.44,glass transition temperature(T_g)starting point of films below 25 ℃ was observed and EAB was increased,while TS of films decreased dramatically.In the case of Aw 0.92,gelatin films even dissolved into sol.These results clearly indicate that gelatin films prepared from shark skins could only be applied in dry environment(Aw<0.44).

Abstract:In April 2010,thirty-six families were established by selecting mature breeders in the third generation selected line.In November 2011,four families were randomly sampled from the thirty-six families and subjected to the experiment where genetic diversity and kinship of the sampled families were analyzed using thirteen microsatellite markers.Thirty-nine alleles were detected at thirteen microsatellite loci in the four families and the number of the alleles at each locus ranged from two to five.The average observed and expected heterozygosity of the four families varied from 0.531 to 0.597 and from 0.474 to 0.507,respectively.The genetic differentiation index(F_ST)among the four families was 0.15.Based on the genotypes of offspring,all parental genotypes of the four families were successfully deduced. A dendrogram was constructed using UPGMA based on genetic similarity matrix.The UPGAM dendrogram of the 120 individuals indicated that 98.3% of the individuals from each family could be fully clustered and the classification results were consistent with the source of genealogy.Results show that there exists high genetic diversity among the four families and microsatellite marker is a useful tool for genealogical identification of pearl oyster.

Abstract:The effects of high pressure processing(HPP)(0,50,150,300,450 and 600 MPa for 15 min)on the sensory quality,color,firmness,pH,amino nitrogen,Ca² -ATPase activity and ultrastructure of Aristichthys nobilis muscle were investigated.The odor,taste and chewiness were modified by HPP,and the sensory quality improved significantly after treatment at 300,450 or 600 MPa(P＜0.05). L* and b* value increased while a* reduced after HPP.The firmness of the muscle increased pronouncedly(P<0.05)with pressure beyond 150 MPa.HPP treated samples had higher content of amino nitrogen(P<0.05)compared to the control with increases of 15.19% and 17.89% for 50 and 300 MPa pressurized samples,respectively.pH showed a positive correlation with the pressure.The activity of Ca² -ATPase was inhibited by HPP,and pressures beyond 150 MPa inactivated Ca² -ATPase more rapidly.Moreover,HPP had a great impact on ultrastructure of A. nobilis muscle.After treatment at 150 MPa,sarcomeres contracted and myofibrils looked thicker than the control.In addition,disappearance of Z-line, M-line and H-zone and breakage of A-band and I-band were observed.The structure of sarcomeres was completely destroyed,intermyofibrillar space disappeared and gelation of myofibrils was observed when pressurized by 450 MPa.Our results indicated that the HPP could serve as a promising technique for commercial quality enhancement of A. nobilis muscle.