Abstract:Insulinlike growth factor I （IGF-I） is a conserved peptide expressed ubiquitously, which shows highly homologous diverse effects on development, growth, and metabolism. With RT-PCR, the fragment encoding the turbot （Scophthalmus maximus） mature IGF-I peptide was amplified. It was predicted that the mature peptide was composed of 70 amino acids including 6 cysteines which may form 3 disulfide bonds. The target fragment was then successfully subcloned into the express vector pGEX-4T-1 and was highly expressed in E.coli BL21（DE3）plysS. The result of SDS-PAGE showed that the fusion protein expressed in the form of inclusion bodies with molecular weight of 34 ku and maximally amounted to 59 % of the whole protein in the E.coli cell 4 hours after being induced with IPTG. The western blotting indicated that recombinant protein had the antigenicity to antiGST antibody. The inclusion bodies were dissolved in 6 mol/Lguanidine chloride followed by pulse renaturation in refolding buffer containing 0.5 mol/L LArginine, 1.0 mol/L GSH and 0.2 mol/L GSSG. Then the renatured recombinant protein was purified by GSTrap FF affinity chromatography. The effect of purified GST-IGF-I on turbot kidney cells was analysed, and it indicated that the recombinant GST-IGF-I can stimulate the proliferation of the cells.

Abstract:Two subtype estrogen receptor β (ERβ1/β2) cDNAs were obtained from blue tilapia, Oreochromis aureus by Reverse Transcription Polymerase Chain Reaction (RTPCR) and RACE. The full length cDNA sequence of ERβ1 was 4 262 bp in length, containing a 5′ Untranslated Regions (UTR) of 239 bp, 3′ UTR of 2 349 bp and an open reading fame (ORF) of 1 674 bp which encoded a putative protein of 557 amino acids with an estimated molecular weight of 62 ku. The ERβ2 cDNA was 2 506 bp in length with 393 bp of 5′ UTR, 109 bp of 3′ UTR and an ORF of 2 004 bp which encoded a putative protein of 668 amino acids with an estimated molecular weight of 74 ku. The deduced amino acid sequence of ERβ1 of blue tilpia possessed 99.1% similarity with the counterpart of nile tilapia, O. niloticus, and similarities with other Perciformes fishes were from 82.6% to 94.2%. The ERβ2 amino acid sequences of blue tilpia shared 98.7%, 81.8%, 76.3%, 64.7% and 55.0% identities with nile tilapia, Micropterus salmoides, Oncorhynchus mykiss, Fundulus heteroclitus and Danio rerio respectively. Blue tilpia ERβ1/β2 were clustered with nile tilapia ERβ1/β2 respectively in the phylogenetic tree. ERβ1/β2 mRNA expressions between male and female were compared by RealTime PCR (RTPCR). ERβs were expressed ubiquitously and differently in ten tested tissues. ERβ1 was significantly expressed in ovary, testis, liver, kidney and intestinal tissues compared with other tissues (P<0.05). ERβ2 was highly expressed in testis, liver and kidney (P<0.05). In ovary, testis, intestine and hypothalamus, expression of ERβ1 was significantly higher than that of ERβ2, and it was 164 times of ERβ2 in ovaries. After 17 betaestradiol (E₂) was injected for 24 h, ERα/β1 mRNA in hypothalamus of male blue tilpia was increased, of which ERα increased significantly. Expression of ERα in hypothalamus increased significantly in 7.5 mg/kg E₂ treated group, while no significant change was observed for ERβ2. In pituitary, expression of ERα/β1/β2 in the low and middle E₂ concentrations (2.5, 5.0 mg/kg) was downregulated, but upregulated in high E₂ concentration group (7.5 mg/kg), and significant difference was found for ERα(P<0.05) . ERβ2 expression was increased in testis significantly (P<0.05) in middle and high E₂ concentration groups (5.0, 7.5 mg/kg). No significant change was observed for ERα/β1 expression. The different distribution of the ERβ1/β2 mRNAs and different regulation of ERα/β1/β2 mRNA in hypothalamus, pituitary and testis by E₂ indicated that various biological functions existed among these three ER subtypes in blue tilpia.

Abstract:The genetic diversity of cultured and wild silver pomfret (Pampus argenteus) populations was analyzed based on mtD-loop and COI gene. The results showed that the average A, T, C and G contents in D-loop were 40.00%, 30.55%, 16.75% and 12.70%, respectively, and the contents of A + T were 70.55%, higher than those of G + C. In the COI gene, the average A, T, C and G contents in Dloop gene were 25.85%, 33.90%, 21.30% and 18.85%, respectively, and the contents of A + T were 59.75%, also higher than those of G + C. The total variable sites, number of haplotypes (h), haplotype diversity (Hd), nucleotide diversity (π) and mean pairwise nucleotide differences (k) of two populations based on D-loop were 19, 15, 0.895, 0.007 and 2.505, respectively. The same parameters based on COI gene were 33, 17, 0.713, 0.004 and 2.239, respectively. Based on mtD-loop and COI gene, the genetic diversity of cultured population was lower than that of wild population. The Hd of cultured population based on mtD-loop and COI gene were 0.562 and 0.571, respectively. And the Hd of wild population were 0.891 and 0.801, respectively. The AMOVA analysis based on D-loop showed that the significant genetic divergence existed in cultured and wild populations, while there was no significant genetic divergence when it was analyzed based on COI gene. In conclusion, both D-loop and COI gene were effective molecular markers for analyzing the genetic diversity of silver pomfret population, while the sensitivity of D-loop in detecting the genetic diversity among populations was higher than that of COI gene.

Abstract:In order to build base populations for selective breeding, ninety families in nine groups were constructed using a full diallel cross design with parents of Pinctada fucata from three different places ［Beihai (B), Xuwen (X) and Sanya (S)］. Comparisons of the breeding value and general evaluation among families were studied after 60 days culture in outdoor pond. The results showed that the Kung breeding values of shell length were over 20 in eight families (BX2, BX9, BB4, BB5, BB8, BS9, XS3 and SB8). The family XS3 had the largest breeding values in shell length and shell width and comprehensive value, whereas XX3 had the smallest ones. The overall values of BB4 and XS3 were over one. General combining abilities (GCA) in shell length for Sanya, Xuwen and Beihai parents were 17.40, 17.45 and 18.27, respectively while GCAs in shell height were 14.74, 14.79 and 15.54, respectively. Specific combining abilities (SCA) in shell length were 17.75, 18.27 and 18.12 for Sanya(♂)Xuwen(♀), SanyaBeihai and XuwenBeihai matings, respectively while SCAs in shell height were 14.98, 15.51 and 15.46, respectively. Average heteroses in shell length were 1.41, 0.96 and 0.67 for SanyaXuwen, SanyaBeihai and XuwenBeihai matings, respectively while average heteroses in shell height were 1.13, 1.60 and 1.19, respectively. This study suggested that the parents from Beihai are preferable.

Abstract:A total of 11 halfsib families and 33 fullsib families were generated by using the method of unbanlanced nest design,in Sept. 2007. Phenotypic traits of different families, such as egg sizes, fertilization rates, embryonic hatching rates,larval and juvenile growth, survival and metamorphosis were analyzed. The results showed that the differences in egg sizes and fertilization rates among the fullsib families were not significant, but those in embryonic hatching rates were significant（P＞0.05，n=90）. The growth of each family was not the same at different stages.The fastest growth family named C₂ was significantly larger than the slowest growth family named F₂ by 28.24% in average shell length （P＜0.05，n=90）at 9 days of larval stage. D₃ whose absolute growth was the largest of all families was bigger than the average absolute growth by 37.24%.The fastest growth family I₁ at 40 days of nursing stage was significantly larger than the slowest growth family B3 by 78.29% in average shell length（P＜0.05，n=90）. Moreover, I₁ was the largest of all families in absolute growth,and it was bigger than the average absolute growth by 87.61%, the distributions of shell length between 400-500 μm and above 500 μm accounted for 30% and 53.33% of total shell length among I₁ , respectively. However, the growth trait depression families B₁ and B₃ involved in miniaturization, the distributions of shell length below 300 μm accounted for 83.33% and 90% of total shell length of B₁ and B₃ , respectively. The survival of each family was different at the same stage. The highest suevival family I₂ was significantly larger than the average survival by 94.14%, and the lowest growth suevival family F₂ was obviously smaller than the average survival by 72.65% at 9 days of larval stage. The survivals of all families at 40 days of nursing stage were higher than 85%. The study of fullsib families at metamorphosis stage presented some phenomena,such as metamorphism time delay and metamorphism individual miniaturization. The metamorphosis time was delayed from 15 to 22 days. The average metamorphic size was （193.18±12.15） μm . The smallest metamorphic size family was K₁ with（183.00±14.42） μm of shell length, and the metamorphic sizes of three families which were generated through each female mated with the same male named K were generally smaller than other families, and this was mainly due to paternal effect.The metamorphosis rates were significantly different among the fullsib families.The highest metamorphic rate family was E₃ with the rate of (94.33%±0.58%), and the lowest metamorphic rate family was B₂ with the rate of (7.33%±2.08%) . These results suggest that I₁ is a family with the best early growth and development traits, and it could be used to produce highquality seed which presents short culture cycle,and higher production in artificial selection breeding.

Abstract:Parvalbumin is the major allergen of fish species. Investigation into this protein is beneficial not only to detecting allergen, but also to producing aquatic products with low allergenicity. In the present study, parvalbumin from the skeletal muscle of silver carp (Hypophthalmichthy molitrix) was first purified to homogeneity by homogenization, centrifugation, heat treatment extraction and gel filtration chromatography on Superdex 75. Purified parvalbumin revealed three protein bands with molecular mass of 12, 14 and 24 ku, respectively, as detected by Tricine-SDS-PAGE under nonreducing conditions while under reducing conditions, only a band corresponding to 12 ku was detected. Westernblotting using antifrog parvalbumin monoclonal antibody (PARV-19) positively reacted with the three protein bands of 12, 14 and 24 ku, suggesting they are different forms of parvalbumin. A polyclonal rabbit antisilver carp parvalbumin antibody was prepared and immunoglobulin G (IgG) was purified by Protein A Sepharose affinity column. Dot-blot revealed that the antibody reacted with parvalbumin even after dilution to 1/51200, suggesting its higher titer. Westernblotting analysis indicated that parvalbumins from four fishes (common carp, silver carp, crucian carp, sea bream) can all be detected by the polyclonal antibody specifically.

Abstract:In order to clone and analyze the differentially expressed genes of gonads of domesticated broodstock Penaeus monodon Fabricius，total RNA was extracted from the ovary and testis，and differential display technique was performed to screen the differentially expressed genes by using a set of 3 anchored primers for cDNA synthesis and 8 arbitrary primers in the PCR. The differential display DNA bands were separated by denaturing ureapolyacrylamide gel electrophoresis (PAGE) and displayed by silver staining. Finally 173 cDNA fragments were isolated，and 44 cDNA fragments were excised. After cloning and sequencing，11 differences in expression of the screened cDNA fragments were confirmed by real-time RT-PCR，and 10 cDNA fragments proved to be real difference. Results show that these 10 fragments were expressed differently between ovary and testis，7 fragments were expressed significantly higher level in ovary，two fragments were expressed significantly higher level in testis，one fragment was particularly expressed in testis. The nucleotide sequence analysis revealed that two fragments were not homologous to of the known genes or ESTs，may be novel genes. TC1 was only expressed in testis，and there was no any difference between female and male DNA level. TG6-3 revealed a preferential expression level in testes than ovaries，and it was only expressed in male brain but no expression in female brain. This research provided a foundation for further studies of the molecular mechanism of sexual differentiation and gonadal development of Penaeus monodon.

Abstract:Chinese shrimp (Fenneropenaeus chinensis) is one of the most economically valuable and widely cultured species in China. During the last decade its development has been seriously affected on a regular basis by the outbreak of viral and bacterial diseases. To analyze the immune defense mechanisms of shrimp F. chinensis, a prophenoloxidase gene (FCproPO) was cloned from haemocytes of Chinese shrimp by Rapid Amplification Complementary DNA Ends (RACE) method. The full length cDNA of FCproPO consisted of 2 311 bp with a 2 061 bp Open Reading Frame(ORF)，which encoded 687 amino acids，and the predicted molecular mass was 78.71 ku. It showed 84% and 71% homology with those of Litopenaeus vannamei and Marsupenaeus japonicas. The Realtime-PCR results showed that the expression level of FCproPO in haemoeytes was highest among seven studied tissues．Sequence analysis showed FCproPO contained two conserved copperbinding sites. Phylogenetic analysis revealed that FCproPO and prophenoloxidase from L. vannamei, M. japonicus and P. monodon were in the same phylogenetic branches. Realtime-PCR analysis showed that the expression of FCproPO was upregulated distinctly in the hepatopanereas of shrimp when they were challenged by Vibrio anguillarum and white spot syndrome virus (WSSV). FCproPO showed different expression profiles in the hepatopancreas during V. anguillarum or WSSV virus infection．The result implied that FCproPO might play an important role in the shrimp immune system．

Abstract:Samples of Hydractinia collected from the Taiwan Strait at 20.85°N -27.06°N, 114.92°E -121.24°E during January and July 2007, and Beibu Bay at 17.06°N -21.57°N, 107.40°E -110.10°E during July 2006 to April 2007. Two new species of Hydractinia spiralis sp. nov. and Hydractinia recurvatus sp. nov. are described in the present paper based on the type specimens from the Taiwan Strait and Beibu Bay. The specific diagnosis of the two species is as follows: Hydractinia spiralis sp. nov.  Umbrella without apical projection; manubrium cylindrical, about 2/3 of length of manubrium; oral arms simple, each with one terminal cnidocyst cluster; gonads very large, ellipticlike, covering whole interradial on manubrium, no medusa bud; with 6 tentacles, 4 perradial, 2 opposite interradial, all basal bulbs elongate conical, equal size without abaxial pigmented patch, tentacles very long, in the upper 2/3 of the proximal part of tentacle, thick and stout, evenly covered cnidocysts, and suddenly in the 1/3 of distal part of tentacles, tapered thin, with encircling spiral nematocysts. Hydractinia recurvatus sp. nov.  Umbrella without apical projection; manubrium short and small, quadraticshaped with a conspicuous gastric peduncle, about 1/2 of length of manubrium; mouth with 4 perradial lips elongated to form very short, recurvate oral arms, each with terminal cnidocyst cluster; with 4 oval gonads interradial on manubrium wall, and about 1/2 of gonads ovalshaped attached on interradial position of manubrium at the place between recurvate oral arms, no medusa; up to 16 solid marginal tentacles with swollen, conical basal bulbs, and approximately from the same size, no ocelli; tentacle very long, evenly covered ring endocysts and a distinct terminal knob.

Abstract:The sex and gonad development in cultured Acipenser baerii was scanned by ultrasonic scanning. The results of sex and development stages of scanned A. baerii were identified by Minimally Invasive Surgical technique and histological examination. By comparative study, the results showed that there was higher accuracy of sex identification in A. baerii by ultrasonic method, the accuracy of identification in female was 95% higher than that in male, which was 87%. Success of gonad development identification by ultrasonic method was decided by gonad development, the accuracy of gonad development in early stages (stages Ⅰ-Ⅱ) was 65.25% lower than that in later stages (stages Ⅲ-Ⅴ), which was 88.65%.The speed of sex identification using ultrasonic technology was faster, according to the developmental status of A. baerii， the duration time was only 30 s.

Abstract:With designed genespecific primers on basis of Clone 18 sequence screened out of a suppressive subtracted cDNA library from the male gametophyte of Laminaria japonica, a full length cDNA (GenBank accession No: EF490312) was cloned by use of rapid amplification of cDNA ends (RACE). It was composed of 2087 bp in length including 118 bp 5′-untranslated region (UTR), 694 bp 3′-UTR with poly A at this end and 1 275 bp open reading frame (ORF). Its homology with Guillardia theta nucleomorphencoding CbbX protein (GenBank accession number: CAB65663) reached 66% in peptide sequence. The deduced protein of L. japonica cbbX gene contained 424 amino acids with the first 19 ones from N-terminal on constituting a signal peptide. The matured protein was composed of 405 amino acids after endonuclease restriction digestion with a putative molecular weight of 45.26 ku and pI at 5.28. The coding region of cbbX gene was interrupted by eight introns with all splicing sites well matching a GT-AG rule. There was no difference between female and male gametophyte cbbX genes in their cDNA or DNA sequences. From 184 Gly of the encoded protein, there was a Walker ATPbinding motif. The deduced CbbX from L. japonica gametophytes was clustered with cryptophyte nucleomorphand rhodophyte nucleusencoding CbbX which was significantly different from the chloroplastencoding one according to the constructed neighborjoining phylogenetic tree. It was supposed, therefore, that the cloned cbbX gene from L. japonica gametophytes was possibly encoded by nucleus genome. The transcription level of cbbX gene in male gametophytes was proven significantly higher than that in females by quantitative real-time PCR, and the diurnal transcription patterns were a little different between males and females, both of which confirmed that cbbX gene was a differentially expressed one in L. japonica gametophytes. This research lays a foundation for the function identification and subcellular location of cbbX gene.

Abstract:In order to clarify the ionic regulation methodology for shrimp farming with inland saline waters, a feeding trial was conducted to investigate the effects and mechanism of ambient Mg²⁺ and Ca²⁺ concentrations on the growth and energy budget of juvenile Litopenaeus vannamei using artificial seawater at salinity 30. Eight treatments were set: R1, R2, R3, R4, R5, R6, R7 and R8, and the Mg²⁺/Ca²⁺ ratios and Mg²⁺,Ca²⁺ concentrations (mg/L) were 0.11(35, 330), 0.53(175, 330), 1.59(525, 330), 2.42(800, 330), 3.36(1110, 330), 4.78(1110, 232), 7.87(1110, 141) and 11.81(1110, 94), respectively. The experiment lasted 40 days, but Treatment R8 just continued for 30 days because of shrimp mortality. During the experiment, the growth, molting, food conversion efficiency and energy budget of L. vannamei were all significantly affected by both Mg²⁺and Ca²⁺ concentrations in the water (P<0.05). However, shrimp food consumption was just significantly influenced by Ca²⁺ concentration (P<0.05), not by Mg²⁺. It indicated that L. vannamei had powerful ability to endure very low ambient Mg²⁺ concentration, but it was vulnerable to low Ca²⁺ concentration. In conclusion, if using Mg²⁺ or Ca²⁺ deficient saline groundwater for shrimp farming, we only need to complement the Mg²⁺ concentration to at least 15% of its normal level or Ca²⁺ concentration to 60% of its normal level in seawater by adding magnesium or calcium salt, and the shrimp growth could be normal.

Abstract:In order to investigate the actual oxygen transfer effect of micropore aerator, a comparative experimental study on micropore aerator and aerator was carried out for their aeration ability utilizing the laboratory 10 m diameter standard tank containing clean water as a testing platform, at the water temperature 20 ℃, atmospheric pressure 101.325 kPa and the initial DO level 0 mg/L, and following the standard testing procedure“SC/T 6009-1999, the test method of oxyenenriched capacity for aerator”. The result shows micropore aerator can efficiently increase the DO level at the bottom area of aquaculture waters. Compared with the 1.5Kw aerator with the average aerating capacity of 0.66 kg/h, the average aeration capacity of micropore aerator with 20 m laying pipe is 0.25 kg/h, 0.40 kg/h for 42 m, 1.12 kg/h for 98 m, and 1.55 kg/h for 200 m. Its oxygenenriching capacity can exceed jet aerator at the pipe laying density of our current experiment. In the case of the same air inlet pressure, the oxygenenriching velocity of micropore aerator increases with the increasing of the pipe length.

Abstract:Apparent digestibility coefficients (ADC) of seven selected feed ingredients, petfood poultry byproduct meal (P-BMB), feedgrade poultry byproduct meal (F-PBM), blood meal (BM), feather meal (FEM), soybean meal (SBM), rapeseed meal (RSM), silkworm pupa (SWP) and a blend (MM) of P-PBM, F-PBM, BM and FEM, for Japanese sea bass, Lateolabrax japonicus, were measured. A reference diet was formulated to contain 42.0 % crude protein and 18.9 MJ/kg gross energy, and eight test diets were formulated by combining the reference diet and each of the tested ingredients at a ratio of 70∶30. Chromic oxide was added at 1% in the reference diet and test diets as inert marker. During the experiment, the fish were fed the reference diet and test diets twice daily, and fecal sample was collected by siphon two hours after each feeding. Results of the experiment showed dry matter ADC of the tested ingredients ranged within 61%-87%, and protein ADC within 80%-96%, and energy ADC within 75%-93%. ADC of protein of SBM was the highest, and ADC of dry matter and energy of BM was the highest, among the tested ingredients. ADC of protein of MM was the lowest, while ADC of dry matter and energy of RSM was the lowest among the tested ingredients.

Abstract:In order to classify the haemocytes of adult Neomysis japonica, light and electron microscopical studies were carried out. According to the presence or the absence of cytoplasmic granules, nucleus/cytoplasm(N/C)ratio, the number of granules with different sizes and electrondensity of their cytoplasmic granules, three types of haemocytes were recognized in adult Neomysis japonica: type 1 hyaline cells(no cytoplasmic granule), type 2 semigranular cells(a few cytoplasm granule), type 3 granular cells(abundant granules in cytoplasm). The proportion of the three types were analyzed depending on Wright’s smears, and the result indicated that the proportion of haemocytes(47.53%±0.02) was the largest, while that of hyalinocytes(21.24%±0.02) the smallest. The size was increased from type 1 to type 3, however, the N/C rate was decreased. The activites of acid Phosphatase(ACP), alkaline phosphatase(AKP), lysozyme(LYZ), and phenoloxidase(PO) were analyzed by biological and chemistry methods in total body of Neomysis japonica on 0 d，10 d， 20 d， 30 d after being hatched，respectively. The activities of ACP， AKP, LYZ, PO were decreasing with the growth of individuals．The activity of LYZ was 305.20 U/mg Prot on average, the highest in the three enzymes； the second is ACP, at 0.236 5 U/mg Prot； The activities of PO and ACP were almost the same at 0.129 5 U/mg Prot in adult N. japonica．In conclusion, hyaline cells were mainly haemocytes type in adult N. japonica．In addition, lysozyme played a siginificant role in immune process of N. japonica．The activities of ACP, AKP, LYZ, PO were decreasing gradually with the individual development．

Abstract:Lipovitellin(Lv) is one of the major proteolytic products of vitellogenin(Vtg) after it is taken up by growing ovary in the manner of receptormediated endocytosis，which provides nutrient storage for the developing embryos and larvae．In this paper，we purified the Lv from ovarian extract of the Pelteobagrus vachelli in stage Ⅳ by two steps of chromatography elution method．The first step of purification was to use Sephacryl-S-300 column，and 3 elution peaks were showed．We are suspicious that the 3th elution peak contained Lv through analysing the Native-PAGE chart and elution curve of stage Ⅳovarian extract．The second step of purification was to use DEAEcellulose column in ionexchange chromatography elution，and there were 2 elution peaks．We found that the 2th elution peaks(in 0.2 mol/L NaCl gradient) contained single high concentration of protein by Native-PAGE analysing of the sample．The Lv characterized as a phosphorlipoglycoprotein by NativePAGE and stained of gels for carbohydrates with periodic acidSchiff’s reagent，for phosphorus with Rhodamine B，and for lipids with Sudan black B．The molecular weight of Lv is about 230 ku detected by Native-PAGE．The Lv broke into 2 same subunits in Sodium dodecyl sulphatePAGE，each with a molecular weight of 106 ku．In 190-1 000 nm continuous spectrum scanning，the purified Lv of P. vachelli obviously showed absorb peak in 273 nm，and it demonstrated that the Lv contained carotenoid．NativePAGE analysis of the purified Lv of P. vachelli show that there is no disulfide bond．It is relatively stabilized by heated treatment．We prepared the rabbit polyclonal antiserum against P. vachelli Lv by using purified Lv．The titre for Lv polyclonal antisera was 1∶32 in double immunodiffusion assay detection．It was showed that the purified Vtg which was induced by E₂ in male P. vachelli serum could react with the rabbit antisera against P. vachelli Lv and the purified Lv could react with the rabbit antisera against P. vachelli Vtg and a single immunoprecipitin line was formed．The plasma from the control male P. vachelli have no reaction．The normal existence of the femalespecific reactivity for P. vachelli Lv can be confirmed．The test also demonstrated that both purified Vtg and stage Ⅳovarian extract from the P. vachelli can crossreact with the rabbit Lv polyclonal antiserum by the analyses of Native-PAGE and Westernblotting．We are sure that the rabbit Lv polyclonal antiserum has preferable specificity with Vtg and Lv．

Abstract:Tryptase has been considered as a cellular marker of mast cells in the tissues of human and some mammals. In order to detect whether or not fish mast cells contain tryptase in their cytoplasm, a murine monoclonal antibody (AA1), raised against human mast cell tryptase , was used to stain the paraffin sections of the digestive tract tissue collected from Tilapia nilotica，Anguilla japonica and Anguilla anguilla and gastric cancer tissue(as a positive control) from an adult man by Elivision^TMplus immunohistochemical techniques, Alcian blue Safranin O staining and the modified toluidine blue staining were used as histochemical staining at the same time. The results showed: Nile tilapia mast cells were displayed better by Alcian blue Safranin O staining and the modified toluidine blue staining, while the effects of A. japonica and A. anguilla were not very good by Alcian blue Safranin O staining, and the mast cells could not be examined in A. japonica and A. anguilla by the modified toluidine blue staining. It was firstly demonstrated that Nile tilapia mast cells contain tryptase in their cytoplasm by Elivision^TMplus immunohistochemical techniques with a murine monoclonal antibody AA1 raised against human mast cell tryptase. Small amount of the positive tryptase cells were found in Nile tilapia tissues and mostly lie in the base of mucosal epithelial cells and lamina propria in the sections of digestive tract. While the tryptasepositive mast cells had not been examined in A. japonica and A. anguilla. It showed that there was difference of biochemical element in cytoplasm granules among different fish mast cells. There were lots of tryptasepositive mast cells in the gastric carcinoma interstitium of human.

Abstract:The inorganic arsenic(iAs) in seaweed food is an important safety index in seafood quality detection. High performance liquid chromatographyhydride generationatomic fluorescence spectrometry(HPLC-HG-AFS) was used to determine the iAs in seaweed food. The concentration of extraction reagents, the extraction time and instrument conditions were optimized. A new method to determine the content of inorganic arsenic in seaweed food using HPLC-HG-AFS was obtained: firstly the seaweeds were extracted with 1.2 mol/L HCl at 70 ℃ for 1 h , then the extracts were oxidized with hydrogen peroxide, and finally were analyzed by HPLCHGAFS under the condition of 15 mmol/L(NH₄)₂HPO₄ flow phase(pH=6.0). The average recovery rates of 0.10 mg/kg and 1.00 mg/kg AsⅢ in samples were all above 92% and the relative standard deviations were all below 4% with a high accuracy. The present study proved that HPLC-HG-AFS was a reliable method to determine the iAs in seaweed food and provide enough evidences for the establishment of the standard for iAs in seaweed food.

Abstract:To investigate the possibility of flaB as a candidate antigen for vaccine production, primers were designed based on flaB gene sequences published in GenBank. The flaB gene of Vibrio alginolyticus strain HY9901, the causative agent of vibriosis in Lutjanus sanguineus, was amplified by PCR and cloned into pMD19-T vector. Sequence analysis revealed that flaB gene is 1 134 bp and encodes a putative protein of 377 amino acids. The amino acid sequence of FlaB of V. algonilyticus showed highest identity to V. parahaemolytus (92%). The flaB gene was linked into prokaryotic vector pET-32a(+), and the HisFlaB fusion protein with 60.5 ku molecular mass was successfully expressed in E. coli BL21. The soluble recombinant protein was highly expressed under induction conditions of exposure to IPTG (0.4 mmol/L) at 28 ℃ for 10 h and successfully purified on Ni²⁺ IDA column. The purified fusion protein was injected into SPF mice to produce antiFlaB serum. Western blot analysis revealed that the prepared antiserum not only specifically reacts to the FlaB fusion protein, but also specifically reacts to natural total protein extracted from V. alginolyticus. This result indicates that the FlaB may be one of the important protective antigens of V. alginolyticus, which could provide a basis for further study on the immunogenecity of FlaB and vaccine preparation.

Abstract:Using immobilized mucosal model in vitro, combined with the isotope tracing method, the adhesion characteristics of Enterococci,Citrobacter isolated from carp intestines and Bacillus isolated from water to the carp foregut mucus were investigated. Results showed that after the modification with proteolytic enzymes or sodium metaperiodate, the relative adhesion percentages of all five bacteria were declined extremely significantly after they were treated with sodium metaperiodate(P<0.01), but it was affected less by the proteolytic enzymes treatment（P>0.05）, indicating that main lectins existing on the cell surface of the five bacteria are glycoprotein compounds. After the modification with proteolytic enzymes or sodium metaperiodate of mucus, the relative adhesion percentages of part of five bacteria were declined significantly after the mucus was treated with proteolytic enzymes(P<0.05), but it was affected significantly by the sodium metaperiodate treatment(P<0.05), indicating that the special receptors of the microorganisms are protein compounds. According to the interactions such as exclusion, competition, and displacement, the five probiotic bacteria can reduce the relative adhesion percentages of part pathogens significantly. The inhibition of displacement to the pathogens was best, but the disparity was led by the different strain.

Abstract:The researches on antibacterial peptides from Mytilus coruscus, an important Mytilus in aquaculture, have significant value helping people to understand the mechanism of innate immune system of this mussel. Here, three peptides with antibacterial activity were purified from Mytilus coruscus serum by multidimensional high performance liquid chromatography(HPLC). The three peptides exhibited complementary antimicrobial properties against both grampositive and gramnegative bacteria. The mass and the Nterminal sequences of these peptides were analyzed by a combination of Edman degradation and Mass Spectrometry. Based on the results of sequential BLAST, these antibacterial peptides from Mytilus coruscus serum belong to Mytilin family and are named Mytilin1, Mytilin2 and Mytilin3, respectively; The molecular mass of these antibacterial peptides are 3885.17 u，3993.26 u and 3991.39 u, respectively. Among them, Mytilin1 was characterized as a 34residues peptide including eight cyctines formed four disulfides. The cDNA sequence coding for the Mytilin-1 precursor was obtained by screening PCR from the cDNA library of Mytilus coruscus blood cell. The precursor of Mytilin-1 contains a putative signal peptide of 22 residues, a processing peptide sequence of 34 amino acids, and a Cterminal extension of 46 residues rich in acidic residues. This study lays the foundation for further research about the molecular diversity and the mechanism of these antibacterial peptides from Mytilus coruscus serum.

Abstract:In comparative fishing method, one of important experimental methods in research on size selectivity of trawls, the control codend mesh size is crucial and a possible upper limit is generally suggested to be half the mesh size of the test codend. However, the suggestion is unavailable when the body size of target specie of trawl is small enough, especially in shrimp or prawn trawl fisheries. In order to discuss how to choose a rational mesh size of control codend, experiments, in which test codends with different mesh sizes (20, 30, 35 and 40 mm) were rigged in a same multicodends beam trawl and fished simultaneously, were carried out in Lvsi Fishing Ground. The catch frequency of Parapenaeposis hardwickii, Pseudosciaena polyactis and Collichthys lucida retained by codends were fitted to a selectivity analysis model in which the selectivity curves of different codends were regarded as geometrical similar under two contrary hypotheses (hypothesis 1: the 20 mm mesh size codend is regarded as test codend and selective for catch species and, hypotheses 2: as control codend and nonselective for species). The result demonstrated that the goodness of model fit and the selective parameters of different test codends were not affected significantly by the two contrary hypotheses. Taking the difference in relative fishing intensity and statistical method of analyzing size selection between multiconends trawl and conventional trawls into account, authors suggest that the assumption of nonselectivity of 20 mm mesh size control codend is acceptable in analysis of size selectivity in the experiemts, which means that the general upper limit of control codend mesh size could be broken.