Abstract:Pond ecosystem is a complicated system with the characteristic of dynamic state, multiple factor and unrepresentative. It was difficult to give out results for cultivation problem via single factor statistic traditionally. The paper studied the model optimization of relationship between Siniperca chuatsi health index and seventeen aquatic ecological factors of Siniperca chuatsi culturing pond. The ecological factors involved pH value, ammonia nitrogen content, nitrate content, CO₂ content, Mg^2＋ content, total alkalinity and general bacterial population, et al. Sampled water with plexiglass water sampler every two weeks between 8:00 a.m. to 9:00 a.m. Siniperca chuatsi health examination was done at the same time as water sampling. Employing the statistics as weight factor、F inspection value、Partial regression square-sum and correlation coefficient et al., the superiority model factors were obtained by mathematical statistics to 87 group of experiment data. A non-linear model between the superiority seven factors(pH、NH^4＋-N、NO^3--N 、CO₂ 、total alkalinity、Mg₂₊、bacteria counts) and the siniperca chuatsi health comprehensive indicators were regressed and expressed as y＝a0＋ ＋ ＋ . Its precision was 92.1%. By means of the model, optimization cultivation conditions were analyzed, in order to do favor to ecological culture. The most effect factors on y which coming from the model were bacteria counts、CO2、pH and NH^4＋-N, and their best range took turns 13~27×104 cfu*L^-1、less than 3 mL*L^-1、7.5~8.0 and 0.5 mL*L^-1。

Abstract:A cDNA library from the brain of Andrias davidianus was constructed using the CloneMinerTM cDNA Library Construction Kit. The titer of the primary library was 1.5×105 cfu·mL^-1, and the recombined efficiency of cDNA library was 91.70%. The average insert size was 0.791 kb, while the titer of amplified library was 4.0×109 cfu·mL^-1. The thyroid-stimulating hormone β-subunit (TSHβ) cDNA was obtained through sequencing clones picked randomly, suggesting some full length cDNAs were contained in the library. TSHβ was 705 bp in length, including an open reading frame of 417 bp. The deduced TSHβ subunit contains 138 amino acids consisting of a putative signal peptide of 19 amino acides and a putative mature protein of 119 amino acides. The deduced amino acides of the Chinese giant salamander TSHβ mature protein shares identities of 53.6%, 52.2% and 60.3% separately with bullfrog, Japanese toad and xenopus. The vertebrate TSHβ protein sequences had been used to construct Neighbor-joining (NJ) and Maximum parsimony (MP) phylogenetic tree. As analyzed, Andrias davidianus derived from amphibia firstly, which could reflect its original evolutionary status. RT-PCR analysis indicated that the TSHβ mRNA was expressed abundantly not only in pituitary but also in gonads.

Abstract:900 carps with an average initial weight of 9.18±0.21 g, were divided into 6 groups, feeding diets which were formulated with vegetable protein enzymatic hydrolysates (VPEH) 0%、20%、40%、60%、80%、100% of intact protein respectively for 60d. The results indicated that when the VPEH in diet protein is 40%, the weight gain, feed intake, protein and ash productive values were higher than other groups, the feed conversion ration was lower. Intestinal weight and folds height, lipase, alkaline phosphatase and Na⁺,K⁺-ATPase activities enhanced with VPEH levels increasing, until to 60%(P<0.05). When VPEH was over 60%, the weight gain、protein and ash productive values, enzymes activities and folds height of intestine decreased(P<0.05), while the glutamine-oxaloacetate transaminase in hepatopancrease、ammonia level in plasma and lipid productive values increased (P<0.05). It suggested that VPEH could promote Jian carp cyprinus carpio Jian growth, hepatopancreas and intestine development, increase digestive enzymes activities.

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Abstract:Oriental river prawn Macrobrachium nipponense is the most important commercial species in China. Mitochondrial cytochrome oxidase subunit I (COI) gene was used to analyze the genetic diversity and genetic structure. A fragment of the COI gene was amplified and sequenced from 100 wild M.nipponense individuals obtained from 5 lakes of China: Poyang Lake (PY), Dongting Lake (DT), Tai Lake (TH), Chao Lake(CH) and Hongze Lake(HZ). Forty nine nucleotide variable positions were detected in analyzed 607 bp COI sequences, and 35 haplotypes were defined among five populations, but only seven shared haplotypes was observed. Each population had good haplotypic diversity and nucleotide diversity, and among them PY shows the best genetic diversity. The fixation indices (Fst) analyzed by AMOVA was total to 0.31873 (P<0.05), which showed significant genetic fixation among the five populations. The Kimura 2-paramter genetic distance calculated by the MEGA version 3.1 between the population of PY and CH was highest up to 0.0191, while that between the population of HZ and CH was the lowest to 0.0051. The neighbor-joining trees and UPGMA trees from haplotypes and populations of M.nipponense indicated that, the population of PY and DT might have originated from one clade, while population of TH, HZ and CH might have originated from another clade.

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Abstract:In this study, α2M gene from Hyriopsis cumingii was cloned. The accession number in the Genbank is DQ993157.1.The full-length cDNA spans 5124bp, which contains an open reading frame of 4836bp, 5’ untranslated region of 35bp and 3’untranslated region of 253bp. The open reading frame encodes a protein of 1611 amino acids. The first 23 amino acids in the N-terminus is recognized as a signal peptide. The relative molecular weight of the deduced protein is 177571.8D, isoelectric point is 5.49, and instability index is 39.53, which is classified as stable. Furthermore, Expression of α2M in different tissues of Hyriopsis cumingii was determined by RT-PCR and the result showed no expression was detected in mantel, conductor muscle, intestine, sexual gland except hemocyte. After injecting by Escherichia coli and Aeromonas hydrophila, α2M expression level had a certain amount of rise 12h later, which fully proved the existence of α2M immune system in Hyriopsis cumingii.

Abstract:To investigate the in vitro immunostimulating effects of Lentinan and Astragalus polysaccharides in cellular and molecular levels in the Cyprinus carpio, the macrophages and the peripheral blood leukocytes were isolated from the head kidney and blood of Cyprinus carpio respectively through Percoll gradient density centrifugation, cultured and exposed to different concentrations of Astragalus polysaccharides and Lentinan. The proliferation of the peripheral blood granulocytes, respiratory burst of macrophage and IL-1βmRNA expression of macrophages were analyzed by MTT ,NBT reduction, Griess reaction and Real time PCR respectively. Results showed that proliferation of peripheral blood leukocytes after 24 h incubation with Astragalus polysaccharides at 10,100µg·mL^-1 and Lentinan at 100,1000µg·mL^-1 was markedly stimulated. Lentinan at 1, 100,1000µg·mL^-1 could induce superoxide anion in macrophages markedly, while Astragalus polysaccharides had no effect. Reduction of nitric oxide of head kidney macrophages (HK macrophages) after 96 h incubated by polysaccharides showed Astragalus polysaccharides at 10µg·mL^-1 had conspicuous effect on nitrogen burst activity of macrophages but Astragalus polysaccharides had no effect, moreover both Astragalus polysaccharides and Lentinan at 1000µg·mL^-1 inhibited the nitrogen burst activity at higher doses. The expression of interleukin-1βin the HK macrophages after 24 h stimulation by the two polysaccharides showed Astragalus polysaccharides and Lentinan stimulated IL-1βexpression in carp head kidney . It is suggested that Astragalus polysaccharides and Lentinan could modulate the immune response of Cyprinus carpio.

Abstract:As a result of a successful work on intergeneric crossing in fish during the years of 2006 and 2007 in Zhejiang Institute of Freshwater Fisheries, a hybrid F₁ of Erythroculter ilishaeformis♀×Megalobrama amblycephal ♂ were obtained. Further comparative analysis of morphological characteristics, karyotypes and gene groups were carried out to clarify the differences of morphological and genetic traits between the hybrid and its parents. The results are as follows, (1) both fertilizing rates and hatching rates by crossing of E.ilishaeformis♀× M.amblycephal ♂ were above 90%, which indicates a good kin ability between female E.ilishaeformis and male M.amblycephal; (2) most numeric traits of the hybrid F₁ appeared to be medium. Among ten countable traits, the numbers of gill rake and lateral scale are within the range of its parents’, the average hybrid index is calculated to be 54.56, which interprets that the countable traits approach to the middle values and slightly incline to its male brood’s. Of seventeen measurable traits, nine traits are significantly different to its parents’, including four traits incline to paternity, three to maternity and two deviate from its parents’. The average hybrid index is calculated to be 49.59, which indicates the measurable traits are also close to the middle values. Cluster analysis and discriminant analysis of body frame indexes denote that the body shape of hybrid F1 has obvious differences from its parents, appears to be of mid-type and be affected more by its maternity; (3) Chromosome numbers (2n) of the hybrid F1 are counted to be 48 and karyotype formula are described as 18m+26sm+4st (NF=92), which is similar to those of its parents. The observations specify that the hybrid F1 is a diploid with fertility; (4) Most RAPD enlargement stripes of the hybrid F1 could be found in both parents and some are solely from either paternity or maternity, which shows a hybrid in nature. The hybrid F1 has a relative heredity distance of 0.4327 from the maternity and 0.2312 from its paternity. The former is farther than the later, which elucidate that the hybrid F1 has an unequal heredity difference to its parents and a genetic inclination to its paternity. This is also supported by UPGMA.

Abstract:To find out the pattern of toxicity dynamics of snail Nassarius spp., snail samples were collected from Hanjiang, Tong’an and Xiapu of Fujian province once a week from March to September in 2006, and the toxicity of the samples was screened by the mouse bioassay method. The snail sample with the highest toxicty was then picked out and analyzed for paralytic shellfish poisoning toxins (PSPs) and tetrodotoxins (TTXs) with high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), respectively. It was found that most of the snail samples collected were Nassarius semiplicatus, and toxic samples were detected in all the three sampling sites during the sampling season. The ratios of toxic samples to the whole samples collected were 14%, 43% and 20%, respectively, in Hanjiang, Tong’an and Xiapu. The highest toxicity was 1620MU/100g in a snail sample collected from Xiapu on Jul. 12th. Most of the toxic samples in Hanjiang were found in March, but in the other two sampling sites, toxic samples were found both in March and in a period from June to July. It was suggested that the dynamics of snail toxicity was affected by both seasonal factors and geographical factors. There were no PSP toxins detected in the snails sample with the highest toxicity. However, TTX and its derivative, trideoxyTTX, were detected in this sample, suggesting that TTX and its derivatives should be responsible for the toxicity in snail Nassarius.

Abstract:China is a big country with seaweed production, and seaweed is the peoples favorite health food. However, since the new sanitation standard and testing standards were issued, in accordance with the new testing standards, we found that the over-standard of inorganic arsenic was very serious in the seaweed. In order to explore its cause, this paper surveyed the inorganic arsenic content of some seaweed products such as laver and kelp which come from home and abroad, and we also carried out tracking detection the determination of inorganic arsenic of the different harvest period fresh laver and the laver products, and aquaculture regional water. We found that in accordance with the new sanitation standard and testing standards, the over-standard of inorganic arsenic was a common problem; it was related to the inorganic arsenic content of aquaculture regional water, but not a linear relationship. In the experimentation, we used two methods of the GB/T5009.11 to determinate the inorganic arsenic in seaweed, the distinction of data was very greatly. And these two method were all not suitable to determinate the inorganic arsenic in seaweed.

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Abstract:Viral nervrou necrosis virus (VNNV) causes the pathogenic effects in the nerve tissues of many marine fish, which caused great economic loss in mariculture of many countries. In this study, the capsid protein (CP) gene of VNNV 0603 Strain was amplified successfully by reverse-transcription polymerase chain reaction (RT-PCR), and inserted into plasmid pFastBacⅠof the Baculovirus expression system Bac-To-Bac to develop plasmid pFastBac-cp. Through homologous recombination between pFastBac-cp and Bacmid which was the modified genome of Autogragha california nuclear polyhedrosis virus (AcNPV), the recombinant shuttle vector named Bacmid-cp was obtained in E. coli DH10Bac. After tranfection with Cellfectin Reagent, recombinant Baculovirus (AcNPV-cp) was formed in Sf9 cells. The specific 37kD protein band and the hybridizing reaction between expressed protein and positive serum for VNNV were showed by analysis of SDS-PAGE and Western blotting, which indicated that VNNV CP protein had been expressed in insect cells Sf9 with immunological activity. The self-assembled virus-like particles (VLPs) were observed in the negative staining CP crude extract and in the ultra thin section of infected Sf9 cells with electron microscopy, which were similar to VNNV whole virion, and became a crystal array in cytoplasm. On the base of present study, the particle vaccine of VNNV would be developed and put into practice in the near future.

Abstract:Studies on hybridization between Mytilus galloprovincialis and Mytilus coruscus and pilot-scale breeding were carried out from 2005 to 2007. F₁ were obtained with technologies of intensive culture of parental mussel, identification of male and female, amelioration of waterbodies exchange of breeding, feeding compound tiny algae, et at. The results show that the growth characters are positive cross F₁> _{M. galloprovincialis}> _M.coruscus> negative cross F₁. 377 million juvenile mussels of positive cross F₁ with shell length of 1.13 mm, shell height of 0.78 mm were obtained. The survival ratio reaches 37%. Advantages of positive cross F₁ exist in shell length, shell height, survival ratio, when compared with M.galloprovincialis and M. coruscus. But the hatching rate is lower. No obvious advantages were found in negative cross F₁.

Abstract:The variation of morphology and ultrastructure between fresh Siberian sturgeon sperm and frozen-thawed sperm were observed by transmission and scanning electron microscopy in this paper. The results showed that the sperm morphology and ultrastructure changed greatly after frozen-thawed. The length of acrosome, the ratio of middle head width to anterior head width and the width of midpiece increased significantly (P<0.05). The length of midpiece and posterolateral projections became short. there was significant difference between fresh and thawed sperm (P<0.05). About 30.5% of the sperms were damaged after frozen-thawed. The ultrastructure exhibition of freezing injure focused on: the posterolateral projections mixed with nuclear and the content of acrosome lost; the arrangement between acrosomal vesicle, subacrosome and nucleus became slack; the plasma membrane of sperm swelled, folded and disrupted; the membrance of endonuclear canals unclear; the membrane of mitochondria was cracked, dispersed and even mitochondria lost; the membrane of flagellum changed slack and released from flagellum. Some of the damage sperms lost the acrosome, midpiece and flagellum. The flagellum regularly disrupted from the posterior part of midpiece. Most damaged parts focused on the membrane-system of sperm, microtubular-system was still well. Freezing and thawing procedure were the key of cryopreservation. Study of Siberian sturgeon sperm ultrastructure would direct us choose the proper cryopreservant, extender and right method, including freezing and thawing procedure, to conserve the sperm.

Abstract:Extracted and purified by using fractional precipitation, as contrast with same stage diploid, the vitellogenin cytochemistry allocation of triploid rainbow trout was made by detecting gonad, liver, blood and intestines at different developmental stage with immunohistochemical method. The testing result showed that: The purified protein was a kind of Vitellogenin, showing peculiar female. The vitellogenin were not measured in the examined organizations of tiploid rainbow trout atⅠ-Ⅴstage. The reaction of the vitellogenin were positive in blood and liver of female diploid rainbow trout at Ⅲ stage, and in ovaries at Ⅳ-Ⅴstage Vg, but it was negative reaction in bowels atⅠ-Ⅴstage. The ectogenic vitellogenin of diploid rainbow trout was produced in the liver of mature female, under estrogen′s control, before being secreted into the bloodstream and later incorporated into growing oocytes. The liver of triploid rainbow trout can not synthesize adequacy vitellogenin which was necessary to oocytes development; this was one reason that the triploid female rainbow trout was sterile.

Abstract:Tryptase has been considered as a cellular marker of mast cells in the tissuse of human and some mammalian species To detect frog mast cells whether or not contain tryptase in thire cytoplasm, a murine monoclonal antibody (AA1), raised against human mast cell tryptase , was used in an indirect immunoperoxidase technique to stain the paraffin sections of intestine, spleen and tongue collected from bullfrogs（Rana catesbeiana） and sqamous cell carcinoma tissue of esophagus (as a positive control) from an adult man. It was firstly demonstrated that the frog mast cells contain tryptase in their cytoplasm and an excellent cross-reaction of the antibody AA1 with frog mast cell tryptase was obtained from the Boun’s fluid fixed frog tissues. In frog tissues the tryptase-positive mast cells mainly lie in lamina propria of intestine, a little were distributed in the base of intestinal villus and around submucosal glands of tongue and none were found in the spleen. There were lots of tryptase-positive mast cells in the mesenchyme of the human carcinoma tissue of esophagus. In conclusion, like human mast cells, bullfrog mast cells also contain the special enzyme, tryptase and it was likely that bullfrog can be used as an ideal experimental animal for the study of mast cell biology.

Abstract:It is very important to establish a kind of simple and useful marking method to evaluate multiplication and releasing effect and the current situation of fisheries resource. The golden cuttlefish were chosen to be the study matieral in this article. The larval golden cuttlefish hached after 15d were immersed in 6.0 ~ 8.0 ×10^-3 % alizarin complexone (ALC) for 24 h. The tagged group and control group were reared in1.5m×1.5 m×1.5 m indoor pond for 30 d, and then they were removed to 2667 m² outdoor pond. After 60d, their mean doral mantle length grew to 91.4 mm (control group) and 87.6 mm (tagged group) respectively, they were removed to indoor pond for over winter. During 210d experiment, the golden cuttlefish were sampled randomly in order to examine the growth and the dye-retention of the tagged cuttlebone after tagged 15、30、45、60、90、210 d. The results of the experiment showed that this marking method is very effective, and the survival rate of the tagged golden cuttlefish is 100%. There are no significant differences between the growth of the tagged group and the control group (P>0.05). The half tagged lavender ring is very clear in the cuttlebone even after tagged 210d. Additionally, according to this method, we could tag lots of samples easily in one time, and the retention of marker can last long period. Due to the maneuverability and easy to distinguish tagged ones, it is suggested that this method could be a good marking method for golden cuttlefish.

Abstract:Fish-scale-deposition amount (SDA) of 47 surface sediment samples and 6 sediment cores from central Yellow Sea, changjiang estuary, fujian and zhejiang coastal area have been investigated in this study, to reveal fish-scale-deposition information and spatial distribution in typical fishery sea area. This study proved the feasibility of applying fish-scale-deposition rates (SDR) to trace fish population dynamic changes in China typical fishery sea area. Results showed that the species composition of fish-scale-deposition in central yellow sea was simple. Anchovy (Engraulis japonicus) and small yellow croaker (Pseudosciaena polyactis) were the major fish in Yellow Sea. In surface sediment anchovy made up 75.0% and small yellow croaker made up 25.0%. The positions where found scales, distributed on northwest and southeast edge of central Yellow Sea approximately, and SDA was high around 10594 position (34°59.9′N, 122°29.9′E) and 12694 position (33°59.9′N, 123°59.0′E). The vertical distributions of SDA of 3 positions in central Yellow Sea were inhomogeneous with depth increasing. The same change trend and an obvious identical abundance peak of SDA appeared in the vertical distributions of 3 positions in central Yellow Sea. In changjiang estuary SDA per unit volume was lower than that in central yellow sea, and the species composition of fish-scale-deposition was complex. 68.1% scales were identified in changjiang estuary surface sediment; small yellow croaker, acropoma japonicum and anchovy were the major fish, made up 29.2%, 12.5% and 8.3%respectively. The positions where found scales, distributed along central Zhoushan Fishing Ground and paralleled to the coastal line approximately. SDA was high around H-28 position (28°09.1′N, 122°55.3′E). The vertical distributions of SDA in changjiang estuary were irregular, but the same change trend and the major fish that had continuous change trend, did not appear in the vertical distributions of 3 positions. Compared with changjiang estuary, central Yellow Sea is the more appropriate study area for applying SDR to trace fish population dynamic changes.

Abstract:Heavy metal pollution in benthic filter-feeding bivalves has been received more and more attention. With regard to the studies in this field, the two-compartment kinetic model is a new developed mathematic model. In the present study, accumulation and elimination of three heavy metals (Cu, Pb, and Cd) in Tegillarca granosa Linnaeus were investigated in a semi-static system. Kinetic parameters of bioconcentration were obtained form the two-compartment model by nonlinear curve fitting, including uptake rate constant (K¹), elimination rate constant (K²), bioconcentration factor (BCF), and biological half-life (B1/2). It was found that the metal concentration data were fitted to the two-compartment model, and good agreement was found between the predicted and observed metal concentration using the goodness-of-fit test. The modeling results showed that K1 ranged from 7.318 to 29.928, K2 ranged from 0.0064 to 0.0176, BCF ranged from 457.3 to 3303.6, and B1/2 ranged from 39.4 to 108.3. The results also indicated that the uptake rate constant (K¹) and bioconcentration factor (BCF ) generally decreased with the increase of heavy metal exposure concentration in ambient seawater, the bioaccumulation ability order of T. granosa to the three heavy metals was Pb,Cd,Cu, the biological half-life of Pb in T. granosa was longer than those of Cd and Cu, maximal content in the organism (CAmax) at the theoretic equilibrium increased and was basically directly proportiona1 with increasing metal concentration in the outside water. So it could be regarded that the heavy metal concentration in T. granosa faithfully reflected the contaminations status and process of the ambient surroundings, accordingly T. granosa could be regarded as the indicator of the three heavy metals in coastal waters of Zhejiang province and other waters with the distribution of T. granosa.

Abstract:Myostatin (MSTN) gene was isolated from Japanese flounder(Paralichthys olivaceus) by homology cloning strategy and genome walking .By sequence analysis and verified in cDNA level, Japanese flounder MSTN gene included three exons and two introns, encoding a 374-amino-acid protein. Eight TATA box, one CAAT box and six E box located in 5` flanking region. A polyA signal existed in 3` flanking region. Nine conserved cysteine residues and an RXXR proteolytic cleavage domain were detected in the C terminal of protein. MSTN in Japanese flounder was high homologous with MSTN genes in fish via phylogeny analysis. The RT-PCR analysis demonstrated that MSTN was undetectable during embryo development and its transcription was influenced by environment factors in adult.