Abstract:Chub mackerel (Scomber japonicus) is an important commercial fish species in the East China Sea and Yellow Sea. Since 1997, the annual catch of Chub mackerel has been decreasing. It is necessary to assess its stock size and manage it properly. The stock assessment and risk analysis of the management strategy for Chub mackerel was carried out using a Bayesian surplus production model, and the stock status was determined. The stock biomass, annual total allowable catch and probability that the stock collapsed in the next 5 years were also estimated. The results indicated that the stock of Chub mackerel in the East China Sea and the Yellow sea was in the state of “overfishing”, but not overfished in 2006. The decision analysis showed that harvest rate of 0.3 was the optimum management regulation with precautionary approach, under this harvest rate the average stock biomass would increase from 451 thousand tons in 2006 to 871 thousand tons in 2011, the probability that stock biomass in 2011 reaching BMSY would be 0.47, and the probability of having an overfished stock would be 0.

Abstract:The common carp (Cyprinus carpio L.) is the most extensively cultured fish in the world, as well as in China. Construction of genetic linkage map of common carp is necessary to increase the efficiency of selective breeding, especially complicated traits. So, it is the basis of keeping on the genetic study of a species with construction of full genetic map and location of quantitative trait. In this study，a genetic linkage map has been constructed for the common carp (C.carpio L.) using the mode of outbreeding fullsib family in OneMap software firstly. The segregation of 923 polymorphic markers, including 911 fAFLP (fluorescent amplification fragment length polymorphism, fAFLP) markers and 12 gene markers were studied in a panel with 110 F₂ individuals derived from a single hybrid F₁, which was produced by crossing of redpurse carp（C.carpio var.wuyuanensis）and bighead carp (C.pellegrini Tchang). The results showed that the framework of genetic linkage map consisted of 238 fAFLP markers and 8 EST markers in 50 linkage groups that ranged in size from 2.01 cM to 437.46 cM and consisted of between 2 and 48 markers. The total linkage distance spanned by these markers was 2 876.64 cM with an average of 14.68 cM for the whole framework map, and the map coverage is 66.56%.This genetic linkage map is an improvement of further quantitative traits loci (QTL), fine mapping, marker assisted selection(MAS) linked to important economic traits and comparative genome mapping in common carp.

Abstract:Fishes and macroinvertebrates(FM) were sampled respectively in ShangSanheng artificial reef area(Marked by B) in May，Sept. of 2007 and Mar.，May，Jul. of 2009 by trawlnet, and XiaSanheng artificial reef area(Marked by A) from Jan. to Aug. of 2009 by multimesh gillnets around Shanheng artificial reef waters, in order to assess resource enhancement effects of the deployed artificial reefs(ARs). Several control sites were chosen for better explanation on habitat restoration function by ARs. CPUE, Margalef species richness index, commercial species variation and Whilm species diversity index were used to discuss the change of FM resource. Result shows that:(1) A area: average CPUE’s significant differences were found neither between ARs and soft bottom habitat nor between ARs and rocky reef habitat(P＞0.05). However, the same index value from Feb. to Aug. between ARs and soft bottom habitat, was quite different(P＜0.05). species richness level of ARs was obviously higher than the soft bottom habitat by 60.2%(P＜0.05), no differences existed among ARs habitat and Rocky reef habitat(P＞0.05). Both ARs habitat and Rocky reef habitat share more commercial species than soft bottom habitat(P＜0.05), and they seemed to be equal by chance to attract commercial species(P＞0.05). As to species diversity, it was exactly the same as commercial species differences explained above, namely, which is equal between ARs habitat and Rocky reef habitat, and better than soft bottom habitat.(2) B area: CPUE in AR area was obviously improved by 2.05 times after deployment(P＞0.05) and 2 times higher than control site(P＞0.05).Species richness level has been improved by 25.3% after reef deployment, though no significant difference were found between them. However, it did have a better level than control site by 31.3% higher(P＜0.05).5 more commercial species were sampled in AR area after reef deployment compared to its originally number(P＜0.05), and about 4 more species were sampled in AR area than control site after reefs were set. The diversity index in AR area have improved to a certain level better than before and control site, though they appeared to be not so quite different(0.05＜P＜0.1). It is concluded that resource level and community structure have been improved a lot in both AR areas by comparisons to its originally level and control site level. The Sanheng artificial reefs have become a new habitat for more species and more individuals.

Abstract:Anti-mullerian hormone (AMH), (also called mullerianinhibiting substance, MIS), a member of the transforming growth factorβ family (TGF-β), is a peptide growth factor，which is the regression of the Müllerian duct in the male foetus during early testis differentiation. To study the function of the AMH protein and the distribution of AMH in differentiating tilapia testis and ovary, the partial AMH cDNA was cloned using reverse transcription polymerase chain reaction (RT-PCR) and expressed in E.coli and polyclonal antibody was prepared for the further study. The antigenicity of AMH was first predicted by using DNAstar software in which the 22-243 amino acids have strong antigenicity and immunogenicity. at the same time, the special primers were designed by PRIMER 5.0 and the cDNA encoding AMH was amplified from total RNA of O.aurea gonad by RT-PCR，and blasted against other AMH cDNA sequences in the GenBank．The analysis of the sequence data indicated that the coding region of the cDNA fragment，which encoded 221 amino acid residues, was about 663 bp in size．The amplified cDNA fragment was cloned into the prokaryotie expression vector，pGEX5x1，to produce the expression vector pGEXAMH．The recombinant plasmid was transformed into E.coli BL21. AMHGST fusion protein was obtained after the addition of IPTG into the growth media. SDSPAGE analysis revealed that the AMHGST was expressed after induction with IPTG for 4h．A protein band of 49 ku appeared on SDSPAGE gel and was proved by Western blot．The mass production of the recombinant protein was about 38.7％ of total bacteria protein．After purification and cleavage of the fusion protein, purified AMH protein could be obtained．Then the fusion protein was used to immunise some ICR mice to produce antiAMH antibody． This fusion protein could significantly elicit specific antibody response in immunized mice compared with the blank groups, and the peak of serum reached 0.672±0.411 at the 5th week after immunization．These results demonstrated that recombinant protein could induce high AMH antibody responses in laboratory animals．In this study, we report the expression of partial AMH gene and preparation of polyclonal antibody of recombinant AMH protein. For nearly two decades, research on AMH had been focused almost exclusively on mammals and birds. There were no reports of an AMH orthologue in teleost fish, and there might even have been some doubts about its existence given the name of this hormone and the fact that modern teleost do not have Müllerian ducts. Our study first reports that an AMH exists in tilapia and the polyclonal antibody was raised. In present study, the GSTrap FF column was used to purify the recombinant protein and the high purity AMH protein was obtained. Prokaryotic recombinant protein expression systems have several advantages. These include ease of culture, and very rapid cell growth. Expression can be induced easily in bacterial protein expression systems using IPTG. Also, purification is quite simple in prokaryotic expression systems and there are a lot of commercial kits available for recombinant protein expression. In conclusion, our results showed that partial AMH gene was cloned and polyclonal antibody was prepared for further research.

Abstract:Sea cucumber Apostichopus japonicus is one species of deposit feeders with abundant food resources. The analysis of fatty acid biomarkers provides an alternative method of investigating diets, which is not subject to the biases of stomach content analysis. In the present study, 16:1(n-7)/16:0 and EPA ［20:5(n-3)］ were selected as diatom fatty acid biomarkers according to the fatty acid composition in body wall tissues of A. japonicus. DHA ［22:6(n-3)］, ∑［18:2(n-6)+18:3(n-3)］ and 20:4(n-6) were used as fatty acid biomarkers of flagellate or protozoan alga, Chlorophyta and brown alga respectively; Odd & branched FAs and 18:1(n-7) were the fatty acid biomarkers of heterotrophic bacteria. Seasonal variations of food sources in sea cucumber A. japonicus, sampled from a critical cofferdam aquaculture pond, were determined by fatty acid biomarkers analysis. The results showed the food sources consisted of diatoms, flagellate or protozoan, bacteria, brown alga and Chlorophyta originated organic matters. The sea cucumber A. japonicus was characterized by significant diatom markers such as high ratio of 16:1(n-7)/16:0 (0.731.82) and EPA contents (7.24%-14.45%), so diatoms were speculated as one of the main diets. And the two diatom markers showed significant variations during the period the experiments (F=8.871, P<0.001; F=22.882, P<0.001; respectively). The low contents of DHA (2.41%-4.45%) and DHA/EPA ratio (0.29-0.40) showed the flagellate or protozoan provided low proportions of diets for A. japonicus in the year. The food contribution of Chlorophyta was also low with the biomarker ［∑18:2(n-6)+18:3(n-3)］ changing from 1.51% to 2.28% in the duration of the study. However, the relative contents of brown seaweed biomarker 20:4(n-6) were also found high in the year (4.88%-8.16%) with the peak values appearing in autumn and winter. Fatty acid biomarkers specific to bacteria CytophagaFlavobacteria and Proteobacteria were also found in considerable amounts in the sea cucumber tissue, which suggested that there were substantial bacterial input into the food of the species. The principal component analysis showed the main food sources of A. japonicus changed with seasons. Diatoms, flagellate or protozoan, brown alga and bacteria were the main diets of the sea cucumber in January. The contribution of diatoms, flagellate or protozoan and Chlorophyta were comparatively high in March. Chlorophyta was the most important diets for A. japonicus in June. The diets of sea cucumber were mainly bacteria and Chlorophyta in July. Bacteria were the most important food source of A. japonicus in August and September, and the brown alga and bacteria supplied considerable amounts of food for the sea cucumber from October to November. The present study provides alternative materials for feed research of sea cucumber A. japonicus.

Abstract:In order to further research the role of Sox9 in the development of the Siberian sturgeon (Acipenser baerii Brandt)’s lateral line neuromast, the full-length Sox9 cDNA of Siberian sturgeon had been successfully cloned by RT-PCR and rapid amplification of cDNA ends (RACE) methods. The full-length Sox9 cDNA of Siberian sturgeon had 1 409 bp, which consisted of a 1 083 bp open reading frame (ORF) encoding 360 amino acids, a 19 bp 5′untranslated region (5′UTR) and a 307 bp 3′untranslated region (3′UTR). The molecular weight of this mature peptide was estimated to be 41 221.7U. Neighbor-Joining (NJ) method had been used for multiple alignment analyses, and Siberian sturgeon was closest to Amur sturgeon (Acipenser schrenckii). The Siberian sturgeon’s Sox9 gene was high conservative to other species. These results were much similar to other studys that had already been done in Blue tilapia (Oreochromis aurea), Yellow catfish (Pelteobagrus fulvidraco) and Swamp eel (Moncpterus albus)〖WTB1〗. Meanwhile, in order to study the connection between the feature of the expression of the Sox9 to the development of the Siberian sturgeon’s lateral line neuromast, The fluorescent quantitative RT-PCR analysis had been used for detecting Sox9 relative expression levels in the different tiusses and different embryonic development stages. Sox9 had relatively low expression levels in liver, pancreas, kidney and muscle. However, in the brain, Sox9 had very high expression levels, and the relative expression levels of the brain reached to 8.9 times the quantity of the relative expression in the pancreas and to 21 times the quantity of the relative expression in the kidney. In the different stages of the Siberian sturgeon embryonic development, Sox9 expression had different relative expression levels. Its relative expression had no major differences and reached to relatively high in gastrula stage, neurula stage and formation of visual bubble. Especially in the gastrula stage and neurula stage, Sox9 relative expression reached to 1.2 times and 1.4 times the quantity of formation of heart. All of these results were nearly same as the resaeches of zebrafish (Danio rerio), chicken (Gallus gallus) and mouse (Mus musculus). At the same time，the expression features showed Sox9 might play a very important roles in the development of the neuromast of the Siberian sturgeon which already had found in the development processes of the zebrafish neuromast. This research will lay a foundation for the future study on the role of Siberian sturgeon’s Sox9 gene in the differentiation of lateral line ganglion development, cells migration processes and so on. At the same time, the study on the role of the Sox9 in the cell signaling patheway of the development of the Siberian sturgeon’s lateral line neuromast is required too.

Abstract:The yellow catfish Pelteobagrus fulvidraco has become a valuable aquaculture species in China and the possible use of sterile triploids is an interesting option for its culture. The optimal conditions for the induction of triploidy for P. fulvidraco by means of cold and heat shocks were investigated and compared in the present study. Three treatment variables were considered: the time after fertilization when the shock was applied, the temperature and the duration of the shock. Ploidy was determined by chromosome counting and flow cytometric analysis. The diploid, triploid, haploid and aneuploid (or heteroploid) were all identified in the P. fulvidraco eggs (or juvenile individuals) after cold and heat shocks. The survival rate in stages of gastrulae and post hatch, the rates of triploidy induction and deformity under different treatment methods were evaluated. The results indicated that optimal treatment conditions for cold shocks were 5 ℃ for 20 min at 2 min after fertilization, which resulted in about 70% triploidy embryos, 50% relative survival at hatching stage, and 25% triploidy individuals in juvenile period. The optimal treatment conditions for hot shocks were 40 ℃ for 2 min at 2 min after fertilization, which results in 58% triploidy embryos, 39% relative survival at hatching stage, and 40% triploidy individuals in juvenile period. The orthogonal design analysis showed that the time after fertilization when the shock applied was the most important factor to the rates of hatching and triploid induction at embryonic stage in coldshock groups, while in heatshock groups temperature was the most important factor to the inducing rates of triploids, deformities and relative survival at hatching stage. The results demonstrated that cold shock were less effective in inducing triploidy in P. fulvidraco for its seriously injury to embryos and lower survival rate after hatching compared with heat shock. In conclusion, this paper presents the optimal conditions for triploidy induction in P. fulvidraco using cold and heat shocks and indicates that it is possible to massproduce triploids of this commercially important species.

Abstract:The genetic diversity of silver carp (Hypophthalmichthys molitrix) populations around the world was thoroughly evaluated by AFLP at three different levels: populations within Yangtze River(Hanjiangsection, YZHJ and Laohesection, YZLH), among Chinese native populations(Yangtze River, YZ, Pearl River, PR overseas Amur River, AMU) and between Chinese native populations and overseas transplanted populations (Danube River, DAU and Mississippi River, MIS). The results showed Nei’s genetic diversity (H) of native populations was 0.048 1±0.115 1, 0.065 9±0.133 3, 0.051 0±0.115 5, 0.066 1±0.136 4 for YZHJ, YZLH, PR, AMU, respectively; while it was 0.057 6±0.125 0, 0.054 0±0.122 1 for the transplanted populations of DAU and MIS, respectively. The whole genetic diversity of the Chinese native populations(0.072 9±0.129 5) was higher than that of the transplanted populations. AMOVA showed the variance within population(91.86%) was larger than that of interpopulation(8.14%). The genteic divergence index within Yangtze River group, Chinese native groups, Chinese native groups and overseas groups was 0.070 1(P<0.01), 0.070 4(P<0.01) and 0.042 4(P<0.01), respective, which showed there are significant differences in three different group levels. These results would lay a base for long observation on trend of genetic variation of silver carp native populations and transplanted populations.

Abstract:As one of the oldest subcold water fish, Acipenser barei is valuable in term of fish research and economy. In order to provide information that may help with the improvement of artificial propagation and evolutional development study, the morphological observation during embryonic development of A.barei were studied under rearing conditions. Under 15.5-18.0 ℃, it took 133 h from fertilization to hatching with the average cumulation temperature of 2 173-2 369 ℃·h. The embryonic development process could be divided into 7 stage, including fertilized egg stage, cell division stage, blastula stage, gastrula stage, neurula stage, organ formation stage and hatching stage, and contained thirtyfour developing phases, each of which the characteristics were described. The cleavage pattern is radial holoblastic cleavage, and is similar to that of amphibian, but not obviously different with many other fishes; however, the disc cleavage is something like that of bony fish.

Abstract:Decapterus maruadsi in Fujian coastal waters has been facing considerable fishing pressure because the resources of main economic demersal fishes have been exhausted. The information of stock structure of different populations will give us theoretical guidance in planning management and conservation of natural resources and in genetic improvement programs. In this study, amplified fragment length polymorphism (AFLP) was used to analyze the genetic diversity and variation of two populations of D. maruadsi from Eastern and southern Fujian coastal water. AFLP bands were scored for presence (1) or absence (0) and transformed into 0/1 binary character matrix. AMOVA and mismatch distribution analysis were performed in Arlequin, Nei genetic diversity and Shannon genetic diversity index were conducted in POPGEN, genetic distance between individuals were computed in Phyltools and the UPGMA tree was generated in MEGA. A total of 563 loci ranging in size from 100 bp to 450 bp were detected from 62 individuals using eight primer combinations, of which 364 were polymorphic. The number of bands per primer combination varied from 54 to 84 and the polymorphic bands per primer combination ranged from 45.83% to 84.44%. The proportion of polymorphic loci, the Nei genetic diversity and Shannon genetic diversity index of these two populations were 62.70 % and 58.97 %, 0.187 5 and 0.180 9, 0.287 8 and 0.2763, respectively, revealing no significant difference (P>0.05) between the two populations. The results showed that the genetic diversity of these two seemed at the same level. Compared with other fishes, the genetic diversity of D. maruadsi in the coastal waters of Fujian province is above the middle level, with a higher genetic diversity, indicated that the population genetic structure of D. maruadsi had not been destroyed. Wide range of habitats, unbiased sex ratio and the characteristics of its distribution could be the reasons for the higher genetic diversity. G_st value, Shannon genetic diversity index and AMOVA analysis indicated that the genetic variation mainly came from individuals within populations. The UPGMA tree based on genetic distance between individuals exhibited no correlation between clades and geographic distribution. N_mshowed that there was frequent gene flow between the two populations. Mismatch distribution analysis and dominant gene frequency revealed that these two populations have the same genetic population structure. The results of AFLP analysis indicated that there is no significant genetic differentiation between two populations. Therefore, it draws a conclusion that D. maruadsi in the costal waters of Fujian province can be assigned to the same management unit. Annual migrations and larval drift in the ocean currents could be the reasons for little genetic structure in the studied area. However, AFLP markers are inherited as dominant markers, further studies utilizing codominant markers are needed for a better understanding of the genetic diversity of this fish.

Abstract:Rubisco (Ribulose 1,5bisphosphate Carboxylase / Oxygenase) is the first key enzyme in the photosynthesis pathway. In this study, the full length cDNA, encoding Rubisco large subunit (RbsL) was cloned from Ulva linza for the first time. Partial rbcL cDNA sequence of 1 101 bp was first cloned by RTPCR from total RNA, then, both sequences at 3′ and 5′ ends were amplified using 3′ RACE and 5′ RACE technologies that yielded 579 bp and 371 bp DNA fragments respectively. A full length cDNA sequence of 1472 bp was obtained from these cloned sequences and was assigned to a new NCBI accession number DQ813496. It contained the 1 425 bp full coding sequence for Rubisco large subunit (RbsL) and a leader sequence of 47 bp, and it encoded a protein of 474 amino acids. Through bioinformatic analysis and BLAST search, the full cDNA sequence in Ulva linza showed high homologies to known full length cDNAs of the same gene from other green algae where rbcL cDNAs were previously cloned. The identity ranged from 82.07% to 85.78%, and 88.00% to 94.11%, respectively, at DNA and amino acid levels. The highest homology of the protein was obtained between Ulva linza and Chlorella pyrenoidosa. Based on the newly identified cDNA sequence, multiple alignment was performed by using bioinformatic software among amino acid sequences of Ulva linza and other two higher plants and one green alga. Then, the secondary and threedimensional structures for Rubisco large subunit in Ulva linza were also predicted and analyzed with online programs.

Abstract:Microsatelliteenhanced genomic library of oriental river prawn Macrobrachium nipponense of Tai Lake was constructed using repeatenrichment method with biotinlabeled oligos ［(CA)₁₅ and (CT)₁₅］ and streptavidin magnetic beads. From a total of randomly selected 120 clones, 83 clones with more than ten motif repeats were positive. And the sequence size ranges from 191 to 580 bp by with an average of 311 bp. The (CA/GT)_n and (AG/TC)_n were the abundant microsatellite motifs with a percentage of 59.09% and 20.45% in the isolated microsatellites. Several other 17 types of repeat sequences were detected, including repeats consisting of (GC)_n, (AAG)_n, (ACAA)_n and (GGCAGA)_n. Characterization of these microsatellites showed that 25.75% sequences were classified as perfect type, 20.45%as imperfect type and 53.80% as compound type. Genetic diversity of 4 wild stocks of M. nipponense in 4 sites of Tai Lake, i.e. Wujiang (WJ), Binhu (BH), Yixing (YX) and Wuxing (WX), were investigated using 12 microsatellite DNA loci from the isolated primers . The allele numbers, effective allele numbers, observed heterozygosity and expected heterozygosity under HardyWeinberg equilibrium were estimated to characterize genetic diversity. The result showed that all the 12 loci in the 4 stocks were highly polymorphic. Tests of departures from Hardy Weinberg equilibrium indicated the loci except Mni86,Mni103 and Mni114 in each stock with significant heterozygosity deficiency. F_ST and AMOVA analysis across all stocks and loci indicated that there was low level of divergence among 4 stocks. The UPGMA clustering tree based on D_A genetic distance demonstrated that Wujiang and Wuxing stock have near relationship, and they have a distant relationship with Wuxi and Yixing stock.

Abstract:Ribosomal DNA is usually selected as molecular marker for phylogenetic study and the sequence variances especially in IGS region can affect the growth rate of organisms to certain extent. A complete rDNA of Chinese mitten crab (Eriocheir sinensis) was isolated in this study. The rDNA is 11 660 bp in size and consists of 18S(1 873 bp), ITS1(317 bp), 5.8S(163 bp), ITS2(614 bp), 28S(4 461 bp) and IGS(4 240bp). The (AT) contents of different regions range from 40.1% to 48.6%, which are lower than those in Drosophila melanogaster (55.8%-80.0%) and higher than Cyprinus carpio (22.0%-43.7%).The SSR loci per 100 nucleotides in the sequence of descending are ITS-2(0.98%), IGS(0.49%), 28S(0.38%), ITS-1(0.32%), 18S(0.21%), 5.8S(0%).The results of ITS2 intra-variances analysis showed that two types of ITS-2 existed in Chinese mitten crab, with a 12 bp(CGCAGGACCACC) insertion at 405 nt. The IGS region is 4 240 bp in size and there are 136-139 bp speciesspecific repeats that contain an Xho I restriction site. And a stemloop structure (182 pairs stem and 21 nucleotides loop) is located following the specific repeats. The sequence of rDNA in the Chinese mitten crab will provide the foundation of studying the variances among different geographical populations and exploring the molecular marker associated with the fast growth rate of the Chinese mitten crab.

Abstract:A kind of aqueous extracts of Porphyra yezoensis was extracted with distilled water, and its inhibitory activity against four species of red tide microalgae, Amphidinium hoefleri, Karenia mikimitoi, Skeletonema costatum and Alexandrium tamarense was tested. The extracts were extracted with five solvents (methanol, acetone, ethyl acetate, chloroform and petroleum ether), respectively, to probe their inhibitory effects on the growth of those microalgae. Based on the observation of microalgal morphology and density, cell size and the contents of some key physiological indicators (chlorophyll, proteins and polysaccharides), the inhibitory effect of the solvent extract that had strongest inhibition on all tested microalgae was investigated. Thereafter, the extracts mentioned above were separated again with petroleum ether phase, ethyl acetate phase, nbutanol phase and distilled water phase by the method of liquidliquid fractionaction. The results showed that the growth of the four microalgae was significantly inhibited by the aqueous extracts of P. yezoensis at the concentration of 16 g/L. The inhibitory effect of the aqueous extracts of P.yezoensis for A. hoefleri and K.mikimitoi was stronger than that for A.tamarense and S.costatum. The methanol extracts of P.yezoensis dry powder had the strongest effect, and at the concentration of 16 g/L its growth inhibitory effect on A. hoefleri, K.mikimitoi, A.tamarense and S.costatum was 70.5%, 79.9%, 67.1% and 65.1% on day 12, respectively. At the same time, the cell size and athletic ability of the cells decreased, and cavities, pieces and pigment faded in cells were caused. The further investigation found that under the effect of the methanol extracts the contents of chlorophyll, proteins and polysaccharides in the cells of those microalgae significantly decreased. The methanol extracts were partitioned to petroleum ether phase, ethyl acetate phase, nbutanol phase and distilled water phase by liquidliquid fractionaction, among which those extracted with petroleum ether and ethyl acetate had the strongest effect.