Abstract:To study the isolation rate and toxin type of Clostridium perfringens in fresh Meretrix meretrix Linnaeus, one hundred gut samples of Meretrix meretrix Linnaeus bought from market were examined bacteriologically for the occurrence of C. perfringens. Isolates were examined by polymerase chain reaction (PCR) for genes encoding the four lethal toxins (α, β, ε and ι) for classification into toxin type and for genes encoding enterotoxin and the novel β2 toxin for further subclassification. The amplified complete α toxin gene was cloned and sequenced and compared with strains isolated from other living environment. Then, alpha toxin gene detected from Meretrix meretrix Linnaeus muscle (C. perfringens positive in gut)was performed for food safety assessing. The results indicated that C. perfringens could be isolated in 71 gut samples (71%) from Meretrix meretrix Linnaeus, 49 strains (69%) were C. perfringens toxin type C (α and β toxin positive), 22 strains (31%) were toxin type A (α toxin positive). In addition, genes encoding for β2 toxin and enterotoxin were found in the both toxin types. These amplified toxin gene fragments were cloned and sequenced and compared with the corresponding genes in GenBank, and the identity exceeded 98%. The homology of complete α toxin gene was 98.64%-99.58％ when compared with strains isolated from other living environment. This is the first report of C. perfringens toxin genes in Meretrix meretrix Linnaeus in general, the origin of this bacterium and its importance to human food poisoning are discussed.

Abstract:Chinese shrimp (Fenneropenaeus chinensis) is one of the leading cultured species in China. But shrimp farming is currently threatened by pathogen, such as virus, bacteria, etc, among which the white spot syndrome virus (WSSV) is the most serious. The efficient method to avoid or diminish the influence of the virus may lie in the diet, as evidences had revealed the main way that the WSSV was spread in Chinese shrimp was caused by food. This study was initiated to assess the effect of earthworm and housefly larva on growth and survival rate of F. chinensis after WSSV infection. Commercial feed and clam were control groups, and earthworm and housefly larva were assigned into four treatment groups. Three repetitions were made for each diet treatment with 130 individuals in each repetition. The body length and the body weight were measured before and after the experiment, respectively. The survival rate was also calculated when the shrimp were challenged with different gradient WSSV by oral infection, and the dead shrimp were removed every two hours. The descending order of the gain of body weight and body length in the four diet treatment was clam, earthworm, housefly larva and commercial feed. There was significant difference between the body length (P<0.05) and very significant difference between the body weight of each group (P<0.01). Fixed linear model was applied to test the survival rate of F. chinensis by diet variation and different WSSV dosage. The results showed that the survival rate of F. chinensis fed on earthworm and housefly larva was significantly higher than that of the clam and commercial diet fed groups, and the survival rate of group fed on housefly larvae was significantly higher than that of group fed on artificial feed. The results indicated that earthworm and housefly larvae can obviously enhance the growth and disease resistance of Chinese shrimp, and are good food for Chinese shrimp. Results detected by NestPCR showed none of shrimp was infected by WSSV prior to the experiment, and the positive rate of 96.94% of shrimp mortality was possibly caused by infection.

Abstract:Extracellular products (ECP) play an important role in the pathogenicity of Vibrio alginolyticus. In some studies, it has been observed that ECP of V. alginolyticus could cause haemolysis. However, there are few reports regarding the types of haemolysins from V. alginolyticus and the contribution of the haemolysins to the pathogenicity of V. alginolyticus. In the present study, based on reported haemolysin gene sequences from Vibrio, universal primers were designed and used for detecting the distribution of haemolysin gene among V. alginolyticus strains. 74 out of 96 V. alginolyticus strains produced amplification segments of haemolysin gene (vah). vah segment from pathogenic strain ZJ0451 was sequenced and the primers for reverse PCR were designed based on obtained sequence. Through reverse PCR, and the following gene cloning and sequencing, complete vah gene and flanking sequences were obtained. By blast, it was confirmed that the vah from V. alginolyticus has high similarity with TLH haemolysin gene from various vibrios. The predicted peptide strand has a signal peptide at its Nend. With pET32a vector, vah expression vectors were constructed, containing fusion tags with different lengths. They were successfully expressed in Escherichia coli Bl21 (DE3). At 26 ℃, induced proteins reached the relatively highest amount. vah expressed protein has a signal peptide and it can be cut out in the prokaryotic expression system.

Abstract:The activity of agarase depended on the bacterial growth conditions such as growth medium formula. In this study, we used marine bacterial strain Brevundimonas sp. DT-7 as an extracellular agarase producer and optimized the growth medium formula to produce maximum agarase that can be used for producing oligosaccharides from agar on a commercial scale in the future. Eight components in the bacterial culture medium (X₁ agar powder，X₂ NaCl, X₃ FePO₄，X₄ CaCl₂，X₅ K₂HPO₄，X₆ FeSO₄·7H₂O，X₇ yeast extract， X₈ peptone) were selected for the optimization. PlackettBurman design was used in the experiments and the significance of each component (X₁-X₈) contributing to activity of agarase (Y₁) was determined. The significant levels were defined at P<0.05. The data show that among the eight components, three medium components (X₁ agar powder, X₄ CaCl₂ and X₈ peptone) are significant factors for production of agarase from DT7. The three significant factors (X₁ agar powder, X₄ CaCl₂ and X₈ peptone) were selected for further analysis using BoxBehnken design. A regression model was obtained by response surface regression method (SAS 8.2) as follows: Y₁=499.6+19.45X₁+13.99X₄－1.213X₈－33.16X₁²－5.3X₁X₄－3.45X₁X₈－39.64X₄²－10.23X₄X₈－15.79X₈². The results demonstrate the optimum culture medium components (w/v): 0.536 % agar powder, 2.5% NaCl, 0.1% FePO₄, 0.052% CaCl₂, 0.1% K₂HPO₄，0.03% FeSO₄·7H₂O, 1% yeast extract, and 0.484% peptone, respectively. Using those optimized culture components and combined with other culture conditions ［50 mL culture medium in 250 mL flask, initial pH 7.5, shaken at 120 r/min, incubated at （23±1）℃ for 28 h］, the agarase activity of 503.3 U/mL was obtained, which was significantly higher than that of 372.0 U/mL when the culture medium was not optimized. We conclude that (1) using SAS software is a very effective and quick way to optimize a bacterial growth culture medium; (2) the culture medium optimized in this study is simply formulated and can be used for production of high activity of agarase at low cost; (3) with further modification and improvement, Brevundimonas sp. DT7 can be a source bacterium for production of agarase on a commercial scale. This is a preliminary study. Further research will be required to evaluate Brevundimonas sp. DT-7 in commercial application.

Abstract:Cathepsin C is a member of the papain superfamily of lysosmal cysteine protease. Our previous studies showed that the expression of cathepsin C was involved in the final stages of oocyte maturation of the kuruma prawn (Marsupenaeuzs japonicus). In this study, the developmental process of the prawn oocytes was examined with light microscope and the cathepsin C activity was measured during oogenesis and final oocyte maturation. The prawn oocyte development was classified into 9 phases, which include (1) oogonium phase, (2) plasmosome phase, (3) peripheral nucleolus phase, (4) primary vitellogenesis phase, (5) secondary vitellogenesis phase, (6) early cortical rod phase, (7) middle cortical rod phase, (8) late cortical rod phase, and 9) ovulation phase. The germinal vesicle breakdown occurred before ovulation. Immunolgical blot analysis showed that cathepsin C protein was expressed exclusively at the cortical rod stages in final oocyte maturation. However, the cathepsin C activity was low and there was no significant difference at cortical rod stages as compared with noncortical rod stages, suggesting that cathepsin C might have a crucial role in regulating CR release and/or formation of a jelly layer during fertilization.

Abstract:Tissue structure and rheological properties of both raw and heated turban shell foot with different time (5, 20, 60 min) were investigated, and texture profile analysis and sensory evaluation were experimented, which would provide theoretic basis and scientific gist for opening up the production of turban shell foot and optimizing process. The structural changes were observed using a light microscope, the rheological parameters and texture profile analysis were determined by texture analyzer. The relationship between texture profile analysis and sensory evaluation was evaluated. The results show that heating and its time had made great difference on the tissue structure and rheological properties of turban shell foot, and there is a correlation between rheological parameters and texture structure. The hardness became lower when turban shell was heated, while both elasticity and adhesiveness increased. On the view of sense, heating for 20 min was more enjoyable. The hardness, springiness and chewiness of TPA significantly correlated with those of sensory evaluation, and there was a good correlation between sensory evaluation and TPA parameter on adhesiveness.

Abstract:Sensitivity to 0-1 mmol/L ATP of K_ATP channel in the neurosecretory cells of MTXO in the eyestalks of Eriocheir sinensis was observed with insideout patchclamp method and properties of K_ATP channels were studied with selective blocker and steroids. With symmetrical 200 mmol/L K⁺ on both sides of the excised membrane, the singlechannel conductance was approximately 68 pS and the reversal potential was observed. These channels had a weak inward rectifying property. ATP applied inhibited channel activity in a concentrationdependent manner. The K_ATP currents can’t be reduced by depolarized stimulation when concentration of ATP is greater than 0.1 mmol/L. Moreover, opening activity of K_ATP was blocked when 1 mmol/L ATP was applied. The K_ATP channels were not obviously affected by cholesterol, but they were completely blocked by 0.1 mmol/L glybenclamide. The adenosine triphosphatesensitive potassium channel, termed K_ATP channel, is characterized by a strong inhibition of channel activity by intracellular ATP thereby coupling the metabolic state of cells to their excitability and regulating a variety of functions. These results indicate that metabolic state of MTXO has feedback regulation on the secretion of neuropeptides hormone. Cholesterol can’t control secretion of eyestalks hormone by affecting K_ATP channels.

Abstract:The provision of fishing right in Article 123 of Property Right Law of the People′s Republic of China is to entail reforming China′s marine fisheries management with a view to establishing marine fishing right regimes. Such regimes bear the objective of sustainable utilization of fisheries resources. Read together with Article 8 of General Principles of the Civil Law of the People′s Republic of China and Article 46 of Property Right Law of the People′s Republic of China, the spatial scope of marine fishing right regime, under Article 123 of Property Right Law of the People′s Republic of China, should be construed as within sea areas over which China has sovereignty, such as the territorial sea and the internal sea, not including Exclusive Economic Zone (EEZ), while other marine fishing right regimes with different legal nature could be established within EEZ. In the light of existing fisheries management measures in China, there are three alternatives based on which marine fishing right regimes could be established, namely fishing license, horsepower index of vessel engine and fishing quota. The paper proposes it would be the better choice to establish marine fishing right regimes based on horsepower index of vessel engine, considering the development tendency of international fisheries management and governmental reform and community construction in China. As for allocation of fishing rights, fishing rights within landward limit of the territorial sea and fishing rights within EEZ should be dealt with separately due to their different legal characters. With respect to the former rights, they should be allocated by the government to local fishing communities equally with a view to achieving social equity, where local fishermen and fishing vessel owners should have priority over others, such owners of fishing companies or investors from other parts of China. In contrasting to the former rights, the latter rights could be auctioned to all potential buyers with a view to achieving economic efficiency, taking into account of fishing vessels having already operated there. In so doing, it will help realize the objectives of fisheries management, introduce output control management system into China′s fisheries management, and improve the performance of fisheries management.

Abstract:Based on the cited hydrographical data from the East China and Yellow Sea, the blooming process and required conditions of Enteromorpha prolifera were studied. The results indicated that the frequency of red tides was decreased significantly in the Changjiang Estuary (CE) during recent years. In May, 2008, E. prolifera gradually developed in the CE, since there were abundant nutrients and favorable ecological environment such as optimum temperature (20-25 ℃) in the CE. Subsequently, the population was carried from the CE northeastward to the southern Yellow Sea by the Changjiang Diluted Water (CDW). The flow of CDW slowed down in the southern Yellow Sea, and the population of E. prolifera aggregated there. Then small scale E. prolifera populations were developed in the southern Yellow Sea. Thereafter, the populations were dispersed and carried to central Yellow Sea due to the effect of sea surface current. In the Yellow Sea, the surface current runs northward, but in the process of movement, it was hindered by the Shandong Peninsula, forming E. prolifera aggregation further. Then current rip formed between the surface current and the Yellow Sea Coastal Water. Consequently, E. prolifera was bloomed in waters off Shandong Peninsula. Moreover, the outline of the Jiaozhou Bay is concave. All these were beneficial to the aggregation of E. prolifera in the bay. Therefore, E. prolifera was massively bloomed in Jiaozhou Bay. That’s why the E. prolifera was aggregated and bloomed in the waters off Qingdao in the year 2008.

Abstract:The mass mortality of cultured scallops Chlamys farreri often occurs in summer and brings huge loss to farmers. However, the loss could be reduced greatly by transferring and renewing scallop cages or harvesting scallops before the occurrence of the massive death of scallops. A model for predicting the death of scallops by using the principle of Artificial Neural Network (ANN), the method of Back Propagation (BP) network and MATLAB software has been developed. The data to build the prediction model were acquired from a two year (2002 & 2003) investigation on temperature, salinity, pH, NH₃-N, NO₂-N of seawater and protein concentration, acid phosphatase activity, alkaline phosphatase activity, superioxide dismutase activity, catalase activity of scallops serum in Sanggou Bay, Rongcheng, Shandong Province. We debugged the model repeatedly by changing the key parameters, input layer node number, hidden layer node number, sample number and epoch number. After 300 times of studies and training, the sumsquared error of the prediction model decreased from 67.46 to 0.009 1. The model was tested, and the prediction accuracy was 87.5%. It is the first time that ANN was used in the aquaculture disease prediction. This model has many strong points, such as data adapting well, study momentarily and predicting accurately. The present study presents a new way for disease prediction and control of aquaculture animals.

Abstract:One hundred and fifty seven individuals of Japanese Spanish mackerel Scomberomorus niphonius were collected from five geographic populations in the northern Yellow Sea (Dongying and Weihai) and East China Sea (Zhoushan, Ningbo and Wenzhou), and 20 individuals of IndoPacific king mackerel (S. guttatus) were also collected from Guangzhou, Guangdong Province, as outgroup. One way ANOVA analysis, principal component analysis and cluster analysis were applied in this study. The result of one way ANOVA revealed significant difference (P<0.05) in means of SP/FL, VA/FL, pectoral fin rays and gill rakes between the Yellow Sea and East China Sea populations, and there are significant differences between two species in most of morphological characteristics. According to Mayr 75% rule, the differences among the five populations were still below the population level. By principal components analysis, three principal components were established, with their contributory ratios being 32.32%, 11.92% and 7.67%，respectively, and the cumulative contributory ratio was 51.91%. The plot of first and second component showed that there were significant differences between the Yellow Sea and East China Sea. The results of cluster analysis revealed that the two populations from the Yellow Sea were pooled into one group, while the other three populations from the East China Sea were pooled into another group, and the significant geographical variances were detected in this species.

Abstract:Samples of Leptomedusae were collected from the Taiwan Strait at 20.849°-27.056°N，114.920°-121.241°E, during January and April, 2007. Two new species of Leptomedusae, Aequorea atrikeelis sp. nov. and Lovenella sinousa sp. nov. are described in the present paper based on the type specimens from the Taiwan Strait. The specific diagnosis of the two new species are as follows:Aequorea atrikeelis sp. nov. (Fig.1) , Umbrella flat, slightly convex; stomach usually half as wide as umbrella; mouth with 23 lips, simple, lamellar, about less 2 times than radial canals; with 47 radial canals; gonads linear along whole length of radial canals; with 14 marginal tentacles, base bulbs broad and conical, with an abaxial keel, its abaxial swelling covering black pigmented patch; usually with 3 (2-4) rudimentary marginal warts between successive tentacles; all base bulbs and marginal warts with an excretory papillae on the abaxial bulbs; with 1 statocyst between successive radial canals. Lovenella sinousa sp. nov. (Fig.2), Umbrella flatter than hemispherical; with 4 gonads linear, longitudinally divided, with irregular sinuate folds on either side of each gonad, running from the nearly base manubrium to the vicinity of the ring canal; 4 perradial marginal tentacles with a large basal bulbs, bulge and prominence in abaxial part, with black pigmented patch along abaxial bulbs, each flanked by 8-10 pairs of lateral cirri; usually with 6-7 rudimentary marginal warts and 7-8 statocysts between successive tentacles; all marginal warts without lateral cirri, but with a distinct black pigmented spot on the extreme tip; each statocyst with 2-3 concretions.

Abstract:1-［hydroxyl-diethyl-malonate］-isopropyl dodecenoic acid (C₂₂H₃₈O₇), a type of growthinhibitor, was successfully isolated and purified from cultural liquid of Isochrysis galbana. A study was conducted to investigate the inhibitory effects of HDMA on the growth of Isochrysis galbana. Results showed that exogenously added HDMA inhibited the growth of this algae. HDMA significantly decreased the chlorophyll contents of algal cells. And it had a very significant effect on the ratio of extracellular protein to polysaccharide and activities of NR (nitrate reductase), SOD (superoxide dismutase) and POD (peroxidase) of algal cells. But it was obvious to increase MDA (malonyldialdehyde) contents of algal cells. The results gave hints to elucidate the speciesspecific antialgal mechanisms of HDMA. Inhibition mechanism of HDMA on Isochrysis galbana is to decrease the chlorophyll contents, and decrease or increase activities of NR, SOD and POD of algal cells, and change the ratio of extracellular protein to polysaccharide, thus increasing ratio of extracellular protein to polysaccharide leads to the increase in hydrophobicity of algal cells, finally algal cells flocculate and subside. Affecting uptake of inorganic nitrogen by cells and photosynthesis might play a major role in the inhibitory mechanism. And the excess active oxygen was produced under HDMA stress and active oxygen participated in the damage of HDMA to this algae. It indicated that the physiological metabolism of Isochrysis galbana was inhibited by HDMA.

Abstract:Microsatellite DNA fingerprinting technology was used to investigating the genetic variation and germplasm identification within three tilapias of Oreochromis aureus，O. niloticus and red O. mossambicus. 82 pairs of primers which could give stable and polymorphic amplification profiles were screened out from 103 microsatellite loci and used to analyze the genetic structure of three varieties. Totally, 605 alleles of microsatellite loci were obtained and the average number of alleles was 7.37. Additionally, microsatellite DNA fingerprinting database of three tilapias was established based on the results of alleles of microsatellite loci by Popgen32 software package and illustrative patterns were constructed by EXCEL. The results showed that, the mean value of observed heterozygosity of O. aureus, O. niloticus and O. mossambicus was 0.179 6, 0.530 1, 0.312 2, the mean value of expected heterozygosity was 0.203 2, 0.678 6, 0.291 3, and the polymorphism information content (PIC) was 0.075 4, 0.608 3, 0.152 8, respectively. It indicated that, the genetic diversity of O. niloticus was the highest and O. aureus was the lowest. Then 16 pairs of primers that can distinguish one variety tilapia from the others were screened out as the core primers for tilapia germ plasm identification. The amplified data from 16 pairs of special primers were transformed to the application platform of digital microsatellite DNA fingerprinting, which could be used to identify tilapia species. This result could provide a technique for solving the problem of germplasm farrago for tilapia.

Abstract:Bioremediation of polluted aquaculture sediment by immobilized and free microorganisms (FR-M) was studied in pond enclosures by measuring multiple water qualities of overlying water, total organic carbon (TOC) of sediment, value of bioremediation of sediment (G-value), heterotrophic bacteria and antisulfuration bacteria. Immobilized microorganisms included microorganisms immobilized by fibrous net active carbon (FC-M), biological active carbon (BC-M), zeolite (ZE-M) or diatomite (DI-M). The results were as follows: immobilized microorganisms indirectly impacted water qualities of overlying water in experimental enclosures. Concentrations of nitrate of overlying water in ZE-M, DI-M and FC-M were significantly higher than those in control enclosures during final experimental period(P＜0.05). Chemical oxygen demands (COD) of overlying water in BC-M and ZE-M were significantly lower than those in FR-M and Control(P＜0.05), indicating that immobilized microorganisms effectively remediated polluted materials in bottom water in experimental enclosures; FC-M, DI-M and ZE-M degraded sediment organic material more effectively compared to FR-M and Control. Effects of immobilized microorganisms on bioremediation value of sediment (G-value) were apparent. G-values in BE-M, DI-M and ZE-M were significantly higher than those in FR-M and Control(P＜0.05), which suggested that immobilized microorganisms accelerated bioremediation of polluted sediment in experimental enclosures. Immobilized microorganisms did not significantly affect the amount of heterotrophic bacteria(P＞0.05), which probably could be explained by the reasons that many other factors influenced the community of microorganisms. Immobilized microorganisms changed the amount of antisulfuration bacteria significantly(P＜0.05). Immobilized microorganisms (in particular in ZE-M and BC-M) more effectively reduced the amount of anti-sulfuration bacteria compared to Control and FR-M. All these results obtained in the present study showed that immobilized microorganisms could improve bioremediation of polluted aquaculture sediment significantly with best results in ZE-M and BC-M.

Abstract:In this paper, genetic diversity of five wild silver carp（Hypophthalmichthys molitrix）populations, collected from the Yangtze River, Pearl River and Heilongjiang River was analyzed using 34 microsatellite markers. The observed(H_o) and expected heterozygosity (H_e)，polymorphism information content(PIC) and number of effective alleles(A_e) were all determined．The genetic similarity coefficient and Nei'S standard genetic distance were computed based on the allele frequencies．The HardyWeinberg equilibrium was checked by X₂ test．Genetic differentiation and hierarchical partition of genetic diversity were evaluated by F_STand N_m．A dendrogram was constructed based on NJ method using MEGA3 software package .For each locus, 1-9 alleles were amplified with an average of 4.666 7,adding up to 154 alleles in all five populations, of which the mean valid alleles were 2.948 9.We found the genetic variability level was moderate in the five populations, as shown by H_o(0.329 5-0.461 9),H_e (0.500 2-0.552 9) and PIC(0.443 5-0.493 0),respectively. Based on the genetic similarity coefficients and genetic distance, the genetic diversity between the two groups of Yangtze and Pearl Rivers(D_s=0.1852) was higher than that of the two populations of Heilongjiang and Pearl Rivers(D_s=0.1558). There were no obvious relations between genetic distance and geographical distribution of the five populations.

Abstract:Growth and nitrogen budgets of young cobia (average initial body weight 10.0 g), yellow grouper (average initial body weight 5.5 g) and ovate pampano (average initial body weight 7.7 g) at different rations (from starvation to satiation) were studied, and the relationships between growth as well as nitrogenous excretion and ration were established in this paper. The results showed that as ration increased, specific growth rate (SGR) of young cobia increased with a decelerating pattern and there was no significant difference of SGR between satiation and subsatiation (9% per day) ration, but SGR of young yellow grouper and ovate pampano increased with a linear pattern. Among three fish species SGR of young yellow grouper and ovate pampano were similar but much slower than that of young cobia. As ration increased, feed conversion efficiency (FCE) of young cobia first increased, peaked at 9% per day ration and then decreased, but FCE of young yellow grouper and ovate pampano increased along and peaked at satiation ration. Among three fish species, FCE of young cobia and yellow grouper at satiation ration were similar, but much higher than that of young ovate pampano. Food nitrogen (C_N), faeces nitrogen (F_N), growth nitrogen (G_N) and excretion nitrogen (U_N) all increased with increased ration for three fish species. C_N and U_N were highest for young cobia and lowest for young yellow grouper. The nitrogen budgets of young cobia, yellow grouper and ovate pampano at satiation and subsatiation ration were 100 C_N = 7.7(6.0)F_N + 22.6(31.3)G_N + 69.7(62.68)U_N、100C_N = 2.7(2.8) F_N + 20.9(8.4)G_N + 76.4(88.8) U_N和100C_N = 1.8(1.4)F_N + 12.8(9.3) G_N + 85.4(89.3) U_N (the data in bracket indicated those at subsatiation ration), respectively. Both the proportions of food nitrogen lost in faeces and the variations of the proportions for three fish species were small. For young cobia at subsatiation ration (9% per day) the proportion of food nitrogen stored as growth was more than, and the proportion of food nitrogen lost in nitrogen was less than those at satiation ration, but contrary for young yellow grouper and ovate pampano. So among three fish species, nitrogenous excretion and nitrogen budget had an interspecific difference and increased food consumption was one of the major reasons for increased excretion nitrogen when diets contained approximate nitrogen content. Based on the specific growth rate, feed conversion efficiency, nitrogenous excretion and nitrogen budget, it could be concluded that the suitable feeding level was subsatiation ration (9% per day) or about 70% of satiation ration for young cobia and satiation ration for young yellow grouper and ovate pampano at this growth stage.

Abstract:Fish in which gynogenesis has been induced have all their chromosomes inherited from the mother and, if females are the homogametic sex, they usually are all females. Because barfin flounder Verasper moseri females grow faster than males, the production of allfemale populations is highly desirable. Due to the low volumes of semen produced by male flounder, and to eliminate any potential genetic contribution by homologous sperm, activation of flounder eggs with heterologous sperm was also necessary. To test methods for inducing diploid gynogenesis in barfin flounder using homologous sperm, the cryopreservative sperm of Lateolabrax japonicas was used to activate eggs and cool shock was used to prevent extrusion of the second polar body. Four treatments, named for their expected outcome, were employed: hybrid, haploid, triploid, and gynogenetic diploid to prove the ability of Lateolabrax japonicas sperm activating flounder eggs. Diploid gynogenesis was induced by activating egg development with UV irradiated sperm (80 MJ/cm²) for 7-9 min in seawater, and then subjecting the eggs to cold shock in -1-1.5 ℃ seawater for 60-90 min. The hybrid of could not survive to hatching, thus the offspring was allogynogenesis only and the result of microsatellite markers analysis proved this result. This work provides procedures for induction of diploid gynogenesis in barfin flounder using heterologous sperm.

Abstract:Anti-Mullerian hormone (AMH), also known as Mullerian inhibiting substance (MIS), is a peptide growth factor and member of the large TGF-β family of growth and differentiation factors. Using RT-PCR and RACE, this paper isolated two AMH cDNA from Oreochromis aureus which was different in 3′UTR. One was 621 bp, the other was 168 bp. They differed by only 2 bp excluding the different sequence length. The same 5′ UTR was 27 bp and the same ORF was 1 545 bp, which encoded 514 amino acids containing the two domains characteristic of the AMH protein family: the TGF-β domain 93 amino acids and the AMH-N domain 237 amino acids. Homology analysis showed that AMH shared little conservation. O. aureus was more similar to the orthologous regions in other fishes (32%-60%) than to the corresponding regions of mammalian AMH genes (19%-21%). However, the TGF-β domain shared relatively high conservation. The similarity was 54%-72% between O. aureus and other fishes. Comparing the sequence of cDNA to its genome revealed AMH gene consisted of six introns, which was similar in zebrafish and different in mouse and human due to the absence of two additional introns (intron Ⅰand intron Ⅵ). Analysis of O. aureus AMH promoter showed the presence of several putative transcription factor binding sites: SRY, SF-1, GATA-4, Sox5, Sox9. Real time RT-PCR (Taqman probe) was used to detect the AMH expression in adult fish different tissues including testis, ovary, brain, liver, muscle and heart, and the AMH expression level in different development stages including larva, juvenile, adult fish. The results showed that AMH was only expressed in ovary and testis. Comparing the AMH expression level in the three development stages, we concluded that AMH was not expressed in larva, and the amount of gene transcripts was significantly higher in male than in female in juvenile and adult fish, and it was 10 times in male juvenile as in male adult fish.

Abstract:An eightweek net pen experiment was conducted to evaluate the potential to replace fish meal with three oil seed meals, soybean meal, rapeseed meal and cottonseed meal, in diets for 11.3 g sutchi catfish (Pangasius sutchi). Seven isonitrogenous and isocalific diets were established. The control diet was formulated to contain 45% fish meal, whereas in the other six diets, soybean meal was added at 31% and 46% to replace 50% and 75% of the fish meal, or rapeseed meal added at 20% and 40% to replace 25% and 50% of the fish meal, or cottonseed meal added at 19% and 39% to replace 25% and 50% of the fish meal. Results of the experiment indicated dietary fish meal level could be reduced from 45% to 23% by adding soybean meal at 31%, or from 45% to 34% by adding rapeseed meal at 20% or cottonseed meal at 19%, without significantly negative effects on survival, feed intake, weight gain, specific growth rate (SGR), feed conversion ratio (FCR), protein retention efficiency, viscerosomatic index and haematocrit value (Hct). Feed intake, weight gain and SGR significantly decreased when dietary fish meal level was reduced to 11% by adding soybean meal at 46%. Reducing fish meal level to 23% by adding rapeseed meal at 40% resulted in the increase of FCR and decrease of energy retention efficiency, while reducing fish meal level down to 23% by adding cottonseed meal at 39% resulted in the decrease of weight gain, SGR and Hct. These results suggest fish meal level in sutchi catfish diets could be reduced to 23% by using soybean meal (added at 31%), and to 34% by using rapeseed meal (added at 20%) or cottonseed meal (added at 19%), as fish meal substitutes.

Abstract:A study was conducted to investigate the effects of dietary calcium (Ca) and phosphorus (P) on juvenile jade perch, Scortum bacoo, and to characterize P deficiency signs. Six isoenergetic and isonitrogenous purified diets （D₁: 0.0% Ca, 0.0% P; D₂: 0.5% Ca, 0.0% P; D₃: 0.0% Ca, 0.6% P; D₄: 0.5% Ca, 0.6% P; D₅: 1.0% Ca, 0.61% P; D₆: 1.5% Ca, 0.6% P） were prepared using casein and gelatin as the main protein sources, Calactate and NaH₂PO₄·2H₂O as the Ca, P source , respectively. A design of six treatments with three replicates per treatment was used. Each group contained 20 juveniles placed randomly in net cages. Jade perch with an initial weight of 13.42±0.68 g were fed to satiation with one of the six diets for 60 days. At the end of the experiment, growth permance, whole body and muscle composition, and mineral (Ca, and P) deposition in vertebrae were measured. Fish fed diets without P supplement（D₁ and D₂） showed reduced final weight, special growth rate and mineral (Ca and P) deposition in vertebrae, and an increase in feed conversion rate , whole body and muscle lipid content than fish fed diets with P supplement (P<0.05). When P was not supplemented, 0.5% Ca supplement had no significant effect on final weight, special growth rate , feed conversion rate , whole body and muscle composition, ash content and mineral (Ca, and P) deposition in vertebrae (P>0.05). When diets were supplemented with 0.6% P, Ca supplement from 0.0% to 1.5%had no significant effect on final weight, special growth rate , whole body composition, muscle composition (moisture, crude protein and ash), vertebrae composition (ash, P and CaP ratio ) (P>0.05) . Excess Ca supplement (1.5%) had a negative effect on vertebrae Ca deposition (P<0.05). Fish fed diets with P supplement（D₃-D₆） showed an decrease in whole body and muscle lipid content, an increase in ash content and mineral (Ca, and P) deposition in vertebrae (P<0.05) . Ca and P supplement had no significant effect on Ca-P ratio ranged from1.78 to 1.82 in vertebrae (P>0.05). If growth performance and vertebrae mineralization are taken into account, 0.5% Ca supplement might be optimum when diet was supplemented with 0.6% P.

Abstract:In order to investigate heterosis of two lines of Manila clam (Ruditapes philippinarum) with different shell shapes ,the diallel cross of wideshell (W) and plateshell(P) was conducted in June,2007. The experiments consisted of two hybrid groups ［PW（P♀×W♂）,WP(W♀×P♂)］and two selffertilized groups ［WW(W♀×W♂), PP(P♀×P♂)］. The results show that the shell width/shell length(W/L) and numbers of radiocostae of hybrid offspring are between those of offspring of two selffertilized groups. At planktonic stage, instead of the larval growth heterosis, the larval survival heterosis and maternal effect of larval size are observed, the survival heterosis of offspring of PW and WP are (3.43±0.54) and (4.21±0.55) ,respectively. At nursing stage indoor, both growth and survival heterosis are found, and the maternal effects decrease ,the growth heterosis of offspring of PW and WP are (10.76±2.25) and (8.28±1.88), the survival heterosis are (5.52±0.62) and (11.10±2.41). At grown out stage outdoor, the growth and survival heterosis of offspring is significant, the maternal effects disappeared. The growth heterosis of offspring of PW and WP are (16.22±0.23) and (14.80±1.50), and the weight heterosis are (50.29±1.13) and (35.27±2.43), respectively; the survival heterosis are (12.30±1.37) and (17.45±0.75). The above results indicate crossing of Manila clam with different shell shapes has heterosis which increase with development.The study will lay theoretic basis for utilization of heterosis of Manila clam with different shell shapes.

Abstract:Genomic library with highmolecular weight inserts is an essential and powerful tool for genomic studies. An unbiased fosmid library of Porphyra yezoensis was constructed and characterized. The library comprised 23 040 clones with 100％ recombination frequency. The range of insert size of most bulk of clones is from 28 kb to 40 kb and the average size is about 35 kb. Therefore, the library coverage is about 2.78 genomeequivalent. The theoretical coverage was verified by successfully screening out 4 randomly selected genes, 18S rRNA, atpA, h2A and rbcL, once at least from the super pools which covered the whole library clones. No variations were observed by comparing the original clones with those of the hundredth generation cultivated successively, indicating the good fidelity and stability of the fosmid library. This study laid the foundations for gene cloning and genomic research in P. yezoensis.

Abstract:We analyzed the nutritive compositions of 3 preys (Acanthogobius ommaturus, Exopalaemon annandalei, Corbicula fluminea), artificial feed, wild and farmed juvenile Acipenser sinensis. By calculating the ratio of essential amino acids in the 3 preys and artificial feed to that of wild and farmed juvenile A. sinensis (a/A) and essential amino acid index (EAAI), we evaluated the protein nutritive value of the 3 preys and artificial feed. By analyzing the fatty acid contents of 3 preys, artificial feed, wild and farmed juvenile A. sinensis, we evaluated the fatty acid nutritive value of the 3 preys and artificial feed. The results showed that the content of crude protein was 80.57%, 67.84%, 51.88%, 54.20%, 91.83%, and 83.40% respectively. Using the muscle of wild juvenile A. sinensis as the reference protein, EAAI of 3 preys and artificial feed was 0.938, 0.913, 0.918, and 0.956 respectively. And using the muscle of farmed juvenile A. sinensis as the reference protein, EAAI were 0.940, 0.913, 0.915, and 0.953 respectively. Protein analysis of 3 preys and artificial feed showed that they are all highquality protein material for the wild and farmed juvenile A. sinensis. By analyzing the fatty acid contents of 3 preys and artificial feed, we found that the contents of n-3 PUFA were rich in A. ommaturus, E. annandalei and C. fluminea, but were poor in artificial feed. n-3 PUFA in artificial feed could not meet the needs for the migration of juvenile A. sinensis. As the nutritive compositions of 3 preys and artificial feed are different, the selection and development of artificial feed for juvenile A. sinensis according to the natural nutritive compositions characteristics of their preys are quite necessary and we should supplement the needed nutritive components to the artificial feed in order to meet the different needs of their survival, growth and migration.