Abstract:Japanese flounder (Paralichthys olivaceus) is one of the most important fishes for mariculture in China as it is fast growing and has high market value. However, in recent years, the Japanese flounder aquaculture encountered many problems such as disease, decreased growth rate and germplasm degradation etc. So, it is very important to breed new strains with high growth rate and disease resistance in flounder aquaculture. In the present study, the growth characteristics of 63 flounder families developed previously in our lab were compared. The body length and body weight of 80-230 d individuals from these families were measured and the results showed that the significant growth differences among families have been found. The average body length and average body weight of individuals from family with the fastest growth rate were 1.47 and 3.08 times than those of family with the slowest growth rate, respectively, and the absolute growth rate among these families ranges from 0.138 to 0.478. The multivariate analysis of variance (MANOVA) to family factors showed that the growth days of flounder family did not have significant influence on the body length and weight (P>0.05). The one way AVOVE and SNK analysis for 63 family growth characterization revealed that all families could be divided into 5 groups including the fastest growth, fast growth, normal growth, slow growth and the slowest growth group with significant difference (P<0.01). From these groups, eight fast growth families numbers of 19, 36, 51, 21, 69, 61, 64 and 81 were selected. The offspring growth characterization of seven cross combinations including RS♂×JS♀, RS♂×RS♀, RS♂×YS♀, JS♂×RS♀, JS♂×JS♀, YS♂×RS♀ and YS♂×JS♀ was analyzed with SNK method, and the results revealed that the JS♂×RS♀ hybridization family grew faster (P<0.05) and the obvious significant difference in the growth speed of family from different stock cross and same stock cross (P<0.05). The progeny testing of halfsib family suggested the JS29, JS12, RS41 paternity and RS75, RS28, RS86, JS21, JS22 maternity possessed the excellent growth genes owing to their offspring fast growth speed (P<0.05). Finally, based on these body length and weight data of the fastest and slowest growth family, the body length and weight relationship was constructed with correlation coefficient R²>0.5, the similar a value and b<3. And this showed that the family individual body length and weight had close relation and two kinds of families in stable growth environment were in allometric growth period.

Abstract:To better understand the relation of UCP1,UCP2 to the physiology mechanism of Chinese perch (Siniperca chuatsi),UCP1,UCP2 gene sequence and exon/intron structure were obtained from Chinese perch by PCR and RACE method. Using βactin as external control, six tissues UCP1,UCP2 gene expression specificity of Chinese perch were also studied by RTPCR. The UCP1,UCP2 gene sequences were 3 146 bp, 2 890 bp in length. ORF were 942 bp, 939 bp and the deduced protein sequences are 313, 312 amino acids respectively.UCP1 included 5 introns and 6 exons and UCP2 has 7 introns and 8 exons. In the deduced amino acid sequence of UCP1,UCP2, three mitochondrial carrier protein motifs, the UCP signature motifs, six transmembrane αhelix domains and the putative purinenucleotide binding domain (PNBD) both were found. The UCP1 mRNA is primarily expressed in liver, and low level of expression can also be detected in intestine. The UCP1 mRNA was not detectable in spleen, muscle, brain and adipose tissue. In contrast, the UCP2 mRNA is ubiquitous in four selected tissues. The high abundance was observed in adipose tissue, muscle and intestine, and low abundance was observed in liver. Brain has no expression. The data indicated that tissue specificity of Chinese perch UCP1,UCP2 mRNA probably relates to their feed habits, physiology role, and mRNA expression level may be greatly affected by ROS production in different tissues.

Abstract:This study investigated population genetics and phylogenetics of Liniparhomaloptera disparis (Cypriniformes: Homalopteridae) among river drainages on Hainan Island and in coastal western South China, using nucleotide sequences in the mtDNA control region. Samples were taken from 3 major river systems on Hainan Island (Province), 2 major branches of the Pearl River system in Guangdong Province, and 7 independent coastal river systems in Guangdong and Guangxi Provinces. Hainan Island and coastal western South China were connected at the last glacial maximum about 10 thousand years ago. This study thus contributes to the understanding of phylogeography of different fishes from Hainan Island and the South China mainland. A total of 87 individuals from these 12 drainages were used in sequencing; we found 95 variable nucleotide sites among 477 base pairs, with 39 singleton sites and 56 parsimoniously informative sites.Neighborjoining (NJ) analysis with 20 haplotype nucleotides in the control region revealed 3 monophyletic clades. The Pearl River system and the independent coastal drainages in western Guangdong Province are closely related (clade I); the independent coastal drainages in eastern Guangxi Province group tightly with the Changhuajiang River on Hainan Island (clade II); the Wanquanhe and Nandujiang River drainages on Hainan Island form a lineage distinctly separate from the others (clade III). This indicates that populations of L. disparis on Hainan Island are genetically differentiated into 2 lineages, one similar to those in Guangxi Province, and the other unique. The variation within a clade (0.583%-1.775%) was much lower than that between clades (4.205%-5.715%) or that between clades and outgroups (11.825%-13.73%), demonstrating that L. disparis in these 12 drainage systems in coastal western South China and Hainan Island has divided into 3 genetically distinguishable groups. Analysis of molecular variance (AMOVA) revealed that most of the genetic variation resided among populations within a geographic region (65.04%) with little variation between geographic regions (2.24%), rejecting our null hypothesis based on current disjunct geography that populations from the independent drainages in coastal western South China were closely related to those from the Pearl River but distantly related to those from Hainan Island. Variation in the mtDNA control region may have occurred due to drainage separation by montane uplift in the Tertiary, and cannot postglacial disjunction of Hainan Island from the mainland in the Quaternary. This geographic barrier has not existed long enough to effect reproductive isolation, leaving close relationships of populations from the Changhuajiang River on Hainan Island with those from the independent coastal drainages in eastern Guangxi Province. Glacial maxima sea level recessions facilitated connections of different river systems, allowing those from the independent coastal drainages in western Guangdong Province to remain closely related to those from the Pearl River system. Our AMOVA is consistent with the small variation within a clade revealed by the NJ analysis. Speciation within L. disparis and phylogenetic positions among clades require further study.

Abstract:Fullsib family is an important material for genetic analysis. Sixtytwo fullsib families of the first generation of Pacific white leg shrimp, Litopenaeus vannamei, are constructed by natural mating and seventeen of them are studied in growth traits. Results indicate no significant correlation of hatchability and survival rate of postlarvae to the weight of female parent. Seven families are significantly faster in growth than others in the 40 weeks of growth traits, and the order of their weight is A02＞A11＞A01＞A17＞A06＞A08＞A16. These seven faster families are 23.2% higher than the mean value of all 17 families in growth rate (g/week) during the 40 weeks traits (P<0.05). By comparison of each family, families A01, A02 and A11 are 25.5%, 31.6% and 31.0% larger than the mean value of the seventeen families respectively. Moreover, these three families display the smaller coefficient of variation (CV), and the CV values in the middle of growth period are 11.58%, 9.95% and 8.48% respectively. The CV values of the three families are <5% in the later growth period. Therefore the results indicate that they are three families with excellent economic properties. These results are helpful for further selective breeding of fastgrowth line of Litopenaeus vannamei.

Abstract:Porphyra haitanensis is an important economic marine crop, and has been widely cultivated along the coasts of South China. The correct identification of species or forma of Porphyra is necessary for ensuring the wellbred cultivation and the quality of production. However, as the gametophytic blade of Porphyra is morphologically simple and marked variations occur as environmental conditions change, it is very difficult to identify the species or forma of Porphyra based only on their morphological characteristics. In order to find a new way to discriminate the forma of P. haitanensis, the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (including 5.8S rDNA) of 10 germplasm materials of wildtype P. haitanensis were amplified and sequence analyzed by NCBI blast. The sequence length of the amplified fragments that ranged from 1 208 bp to 1 219 bp, can be divided into ITS1, 5.8S and ITS2 three regions. The 5.8S rDNA region of the 10 materials was of an identical size, 160 bp. The ITS1 and ITS2 regions varied slightly by two or three bps. By multiple sequences alignment, the result showed that the sequences of ITS region (including ITS1 and ITS2) of the 10 materials are different from each other and identical sequence pair was not found among these strains, sequence homology was 95.82％-99.73％, so it is easy to discriminate 10 germplasm materials of P. haitanensis by comparing the sequences of their ITS regions. And the sequences of 5.8S region of the 10 materials are identical, but difference with other Porphyra species, sequences homology was 79.7％-95.0％, so the sequences of 5.8S rDNA can be used to discriminate the species of Porphyra. In other words, the sequence of 5.8S rDNA-ITS region of P. haitanensis was alternate array by conservative region and high variational region, and the mode of sequence array of 5.8S rDNAITS regions can be a powerful tool in variety identification of P. haitanensis. The phylogenetic analyses based on these data also support the results.

Abstract:The Tarim River system lies in the border of QinghaiTibetan plateau, it is typical arid desert climate, and the species and distribution of aboriginal fish are very special. To master the compositon and distribution of aboriginal fish in this water system, the authors carried out a comprehensive survey on the seven water systems of Tarim River. The researchers set up 200 collection points, collected 3000 fish samples, and identified 1 order, 2 families 5 genera, 12 species, which all belong to Schizothorax and Triplophysa of central Asian mountain complex. The differences in horizontal and vertical distribution of aboriginal fish are obvious. The horizontal distribution shows an obvious diversity and regional variety, and the vertical distribution shows an obvious zonal variety. The first grade national protected animal Aspiorhynchus laticeps only distributs below 1 500 m altitude, Schizothorax maily inhabits at 1 300 - 2 700 m altitude, Gymnodiptychus mainly inhabits at 1 500- 2 900 m altitude, and Diptychus mainly inhabits at 2 700-3 700 m altitude. Triplophysa at the similar altitude of Schizothorax, but up to 3 700 m. The largescale and large spatial distribution pattern of aborginal fish in Tarim River shows a trend toward narrow, the habitats of Aspiorhynchus laticeps become islandized highly. This article reports the situation of aboriginal fish in Tarim River system comprehensively and systematically for the first time, it provides full and accurate information for comprehensive management of Tarim River system and protection of aboriginal fish.

Abstract:From August 25 to October 10 in 2008, four groups of inbreed offspring of Cyprinus carpio haematopteru (HL),inbreed offspring of Cyprinus carpio var. jian (JL),offspring of Cyprinus carpio haematopteru ♀×Cyprinus carpio var. jian ♂(HJ), offspring of Cyprinus carpio haematopteru ♂ × Cyprinus carpio var. jian ♀(JH) were cultured in cages for 45 days. Then, eight serum biochemical indices and seven hematological general parameters of two hybrid and two inbreed populations were determined and compared in order to analyze the changes of hematological indices due to crossbreed and discuss the biological significance. The results show that there are significant differences on a serum biochemical index (glucose) (P<0.01) and a hematological general parameter (hemoglobin) (P<0.05) between offspring of Cyprinus carpio haematopteru ♀×Cyprinus carpio var. jian♂ and inbreed offspring of Cyprinus carpio haematopteru; there are significant differences on a serum biochemical index (total protein) (P<0.01) and a hematological general parameter (haematocrit) (P<0.05) between offspring of Cyprinus carpio haematopteru ♀×Cyprinus carpio var. jian ♂ and inbreed offspring of Cyprinus carpio var. jian. Furthermore, there are significant differences on there serum biochemical indices (total protein; glutamicoxalacetic transaminase; cholesterol) (P<0.01) and a hematological general parameter (hemoglobin) (P<0.05) between offspring of Cyprinus carpio haematopteru ♀ ×Cyprinus carpio var. jian ♂ and inbreed offspring of Cyprinus carpio haematopteru; there are significant differences on four serum biochemical indices (glutamic oxalacetic transaminase; glucose; triglyceride) (P<0.01) between offspring of Cyprinus carpio haematopteru ♀×Cyprinus carpio var. jian ♂ and inbreed offspring of Cyprinus carpio var. jian, but there is no significant difference on hematological general parameter between the two populations. The differences on hematological indices between two hybrid and two inbreed populations reflect that the level of metabolism, immunity, the function of liver, gonad development, the transport capacity of oxygen are changed in different degrees of the two hybrid populations compared with the two inbreed populations. It can be concluded that these changes are due to their different genetic compositions, because they have different parents. The correlation analysis between the hematological indices and growth behavior, stress resistance should be made in order to verify whether these changes have influence on growth behavior, and stress resistance of two hybrid populations and determine the influence degree.

Abstract:In order to find out the main growth traits of Portunus trituberculatus which have influence on body weight, and define degree of correlation among characters， thirty eight fullsib groups and fifteen halfsib groups of Portunus trituberculatus were obtained by directional mating techniques. Full carapace width(X¹),carapace width(X²),carapace length(X³),body height(X⁴),and body weight (Y) of each family on 80 d,100 d and 120 d were measured. The phenotype correlation coefficients and the genetic correlation coefficients were calculated by correlation analysis at the different ages. The first 4 morphometric attributes (X¹-X⁴) were used as independent variables, and body weight (Y) was used as a dependent variable for path analysis. Path coefficients, and determination coefficients were calculated in path analysis. The results showed that the phenotypic correlation of the five traits was between 0.759 and 0.972,and the genetic correlation was between 0.170 and 0.975. All five correlation coefficients achieved very significant difference（P<0.01）levels. The direct effects by the characters have an order at the three stages,80 d: X¹>X²>X³>X⁴,100 d: X¹> X³> X²> X⁴,120 d: X³> X²> X¹> X⁴, among them X¹ weighted the most to the body weight on 80 d and 100 d, X¹ and X³ weighted the most to the body weight on 120 d. Full carapace width which have the largest direct impact on body weight (0.471 9) is the most important indicators for indirect selection of body weight. Multiple regression equation of X¹,X²,X³,X⁴) to body weight at different developmental stages was obtained as: 80 d Y=-54.113+0.491X¹+0.632 X²+0.325 X³+0.451X⁴; 100 d Y=-123.484+0.627 X¹+0.928X²+1.433X³+0.152 X⁴; 120 d Y=-158.239+0.137 X¹+1.410 X²+2.370X³+0.800X⁴.The high compound correlation index(R²=0.960,0.971,0.892) indicated that these growth traits had direct influence on body weight. Efficient scheme of multiple trait selection will be made if we obtain definite quantitative relationship between each two characters．This study provides theoretical evidence and perfect measure target for breeding of Portunus trituberculatus.

Abstract:PCR-DGGE fingerprinting was explored in the present study to analyze intestinal contents of filterfeeding fishes (Hypophthalmichthys molirix and Aristichthys nobilis), which were sampled from the Donghu Lake, Wuhan. Different microbial bands (16S rDNA) and eukaryotic bands (18S rDNA) were detected in all the individuals, and the numbers of 16S rDNA bands were more than that of 18S rDNA bands. Results of the present study suggested that it is possible and useful to apply PCR-DGGE fingerprinting to studying the intestinal contents of filterfeeding fishes. Additionally, PCR-DGGE fingerprinting and sequences analysis of the DGGE bands indicated that intestinal contents composition of silver carp and bighead carp, which had been found to be significantly different in traditional dietary analyses with microscopy identification, was not significantly different. These results suggested that a significant portion of planktonic organisms were fed by the silver carp and bighead carp. In DNA based dietary analyses, multiple samples can be studied simultaneously with a standardized set of procedures, making it practical to perform comparative analyses, and furthermore, this comparison makes it possible to elucidate some objective ecological rules.

Abstract:For the better understanding about the mechanism of lipid metabolism at the molecular level in grass carp Ctenopharyngodon idellas, the partial cDNA sequence of lipoprotein lipase (LPL) gene was cloned (GenBank accession number FJ612596), and its expression in different organs, adipocytes, as well as the influence of fasting and refeeding on it in hepatopancrease were studied by means of semi-quantitative (SQ-RT-PCR . The results showed that the obtained cDNA sequence was 360 bp in length, and the homology is 70%-89% with other species. LPL gene is widely expressed in hepatopancreas, muscle, heart, gill, intrapaneal fat body (IPF)，mature adipocytes and stromal vascular fraction (SVF) cells , with the highest expression level in IPF, lowest one in gill among the organs (P<0.05), and higher level in mature adipocytes, compared to SVF cells (P<0.05). On the other hand, LPL gene expression level significantly increased in hepatopancreas after 48 h fasting and retrieved to the same level as 0 h after 12 h refeeding (P<0.05). It could be concluded that LPL gene is wildely expressed in various organs and at the initiation and final differentiation stage of adipocyte, regulated by the energy expenditure status, which implies its important role in lipid metabolism in grass carp. The further study on the modification mechanism of LPL gene is required in fish.

Abstract:The aim of this study was to assess the effects of adding astaxanthin(100 mg/kg)， canthaxanthin(100 mg/kg) and mixed pigment(50 mg/kg astaxanthin+50 mg/kg canthaxanthin) on muscle pigmentation and total antioxidant capacity of liver in rainbow trout with an initial weight of (56.60±0.63) g. After 60 days feeding, results showed that there were no significant differences of weight gain, feed conversion ratio, muscle proximate composition component , water loss rate and muscle percentage of rainbow trout among groups(P> 0.05). The Salmo Fan score, redness, astaxanthin content of muscle, and carotenoids content of serum in rainbow trout fed diets supplemented with pigments, were higher than that of control group(P< 0.05). SalmoFan score and redness of rainbow trout of astaxanthin group were 26.25 and 18.40, higher than the canthaxanthin group(22.38，14.13) and the mixed pigment group(24.00, 15.70)(P< 0.05). The muscle astaxanthin content of astaxanthin group was 4.75 mg/kg on 30th day and 6.45 mg/kg on 60th day, and both were higher than that of mixed pigment group(3.87 mg/kg, 5.48 mg/kg)(P< 0.05). Fish of astaxanthin group had the highest serum carotenoids content, higher than that of mixed pigment group, the canthaxanthin group. There was no significant difference in total antioxidant capacity of liver among the three pigmentsupplemented groups(2.39 U/mg, 2.26 U/mg, 2.39 U/mg), which was higher than that of control group(2.03 U/mg)(P< 0.05). Results above showed that adding 100 mg/kg astaxanthin, canthaxanthin or mixed pigment(50 mg/kg astaxanthin+50 mg/kg canthaxanthin) in feed could improve the muscle colour and total antioxidant capacity of rainbow trout. Astaxanthin, mixed pigment(50 mg/kg astaxanthin+50 mg/kg canthaxanthin) had better pigmentation than canthaxanthin, and was suggested to be apply in rainbow trout culture.

Abstract:Intersimple sequence repeat (ISSR) analysis was performed in order to evaluate three successively selected populations(F₇/F₈/F₉ of Pujiang No.1 breeding improved variety),two wild populations (Liangzi Lake/Yuni Lake of 2007)and one hatchery population (Base group, offspring of Yuni lake of 1985 ) of blunt snout bream (Megalobrama amblycephala).The sequence analysis of mitochondrial DNA cytchrome gene was also used to evaluate the effectiveness of ISSR analysis．Main results:(1)Among 100 ISSR primers,26 could produce 164 clear and repeatable bands，of which 81 bands appeared to be polymorphic(49.39％). The sequencing analysis of the cyt b gene presents that three selected generations(F₇,F₈,F₉) of Pujiang No.1 possess the same haplotype.(2)There is a high genetic differentiation between wild/base and selected populations. Compared to Yuni Lake and base population, the percentage of polymorphic loci of F9 generation decreased by 32.34% and 30.29% respectively. Genetic distance（0.067 2）,pairwise F values（0.305 72）were the highest between F₉ generation and Yuni lake population. Based on the UPGMA dendrogram：the selected populations(F₇,F₈,F₉ ) are genetically distinct they were placed in a relatively derived position as the sister branch to a branch containing wild and base populations, it also shows the closest relationship between Yuni Lake and base population. (3)The low G_ST value (0.141 7) combined with the high N_m value(3.027 9) contributed to lower degree of genetic divergence among selected populations (F₇,F₈,F₉);The percentage of polymorphic loci/Average gene diversity over loci/Nei's (1973) gene diversity and Shannon genetic diversity show a tendency that it has been decreased generation by generation.(4)The ISSR technique used was confirmed to be more sensitive than cyt b sequence analysis for the study of closely related populations．(5)The authors acknowledged that，through over 20 years and 9 generations selection, compared with the wild population, Pujiang No.1 variety exhibits a significant differentiation from the wild population in genetics, and shows a rather high stability in improved trails, but there is still some distance from the plateau of selection. In the future, based on the monitoring of changes in genetic structure,potential for selection should be tapped further and attention should be paid to the possible mix in genetics, inbreeding and genetic bottleneck.

Abstract:To provide data for immunoglobulin (Ig) phyletic evolution, isotypes classification and vaccine used in Hucho taimen (Pallas) cultivation, cDNA sequence, gene diversity and physicochemical property on Ig VL (variable region of light chain) of Hucho taimen were studied. The cDNA clones of the immunoglobulin light chain variable regions were first time obtained by RTPCR with a pair of specific primers after total RNA was extracted from the spleen of Hucho taimen (Pallas). 51 positive clones were isolated and doublestrand sequenced over the variable regions. 46 complete sequences were obtained without duplicating to determine variety of Ig VL genes families. After comparing with other vertebrate Ig sequences in BLUST, the results show that the 46 sequences obtained are similar to the sequences of rainbow trout Oncorhynchus mykiss Walbaum, no. X68517.1 and no. X68519.1, reported in GenBank, with the similarity rate from the lowest 86.1% (that of P4 and P43) to the highest 99.8% (that of P21 and P38). Jotun Hein method from MegAling of DNAstar soft package was used to analyze amino acid homology of Ig VL and the VL genes were classified into two families. After computing method of variability parameters, it was found that CDRs (complementary determining regions) are the main variable regions, and hyper variability was showed in CDR3. By analysis of CDR3 sequences, a conclusion was drawn that character of rearrangement style of Ig VL of Hucho taimen is that Jκ genes are located at 3′side, and Vκ genes are followed. The rearranging way makes nucleotides link styles variable, and thus causes more diversity of the Ig. The hydropathic analysis showed that hydrophilic amino acids of VL are mainly distributed at forepart of LP, terminal of FR1, the whole FR2, amphisides of FR3 and foreparts of CDR1, CDR2 and CDR3.

Abstract:Spawned yellow catfish (Pseudobagrus fulvidraco Richardson) and their fry usually die of infective diseases. In order to enhance the resistant ability of spawning yellow catfish and their fry against pathogens, it is necessary to illuminate changes of immune parameters of periphery blood in immunized mature yellow catfish. In this study, one hundred and thirty healthy mature yellow catfish were randomly divided into test and control groups. Each individual was intraperitoneally injected with 0.2 mL of bacterial suspension at a concentration of 1.0×10⁸ CFU/mL formalinkilled Aeromonas sobria for the test group, and with 0.2 mL of 0.65% sterile saline solution for the control group. Then, approximate 2 mL of blood was taken from each fish caudal vein at 1, 2, 4, 7, 14, 21, 28, 35 d post injection. Three yellow catfish were sacrificed at each sampling time. The haematological indices, such as leucocyte and erythrocyte number per microlitre, differential leucocyte count (DLC), and serum antibody titer and activity of phagocytes, were investigated for each blood sample of the test and control group. The results were as follows : The leucocyte number per microlitre of test group drastically increased and was significantly (or very significantly) higher than that of control from the 2nd day to 35th day post immunization (Fig. 2). The differential leucocyte count (DLC) of the test group prominently increased and was significantly (or very significantly) higher than that of control for neutrophil and monocyte from day 4 to day 7 (Fig.5 & Fig.6 ), and for lymphocytes from the 4th day to 28th day (Fig. 4). The phagocytic percentage and phagocytic index also gradually increased from day 1 to day 7 and peaked at 7 d, then dropped down to the control level (Tab. 2). However, the test group was significantly lower than the control in erythrocyte number per microlitre between the 7th day and 35th day (Fig. 1), and in DLC of thrombocyte between day 4 and day 28 (Fig. 3). The serum antibody titer gradually increased from day 1 to day 14 and reached the highest value at 14 d, then, slowly decreased from day 14 to day 35, and maintained a higher level between day 14 and day 21 in comparison with the values at other sampling times (Tab. 3). From what has been stated above, the immunized mature yellow catfish were enhanced in their resistant ability against bacterial pathogens by means of increment of nonspecific cellular immunity, such as monocyte and neutrophil, during the early period (from day 1 to day 7) post single dose immunization, and of specific cellular and humoral immunity, such as lymphocytes and antibody, from day 7 to day 35 after the immunization. And the higher antibody titer level could last for a week from day 14 to day 21 post the immunization. Therefore, it is suggested that the mature fish should be immunized with singledose vaccine at day 10-14 before breeding to enhance the resistant ability of spawn yellow catfish against pathogens, and further to reduce mortality rate of the spawned yellow catfish and their fry caused by infective diseases.

Abstract:Knowledge about the cells and organs involved in the fish immune system is important to improve fish immunoprophylaxis. Teleosts lack bone marrow and lymph nodes and the major lymphoid organs are thymus, kidney, spleen and gutassociated lymphoid tissue (GALT). In this paper, monoclonal antibodies (mabs, 2D8 and 1H1) raised against Japanese flounder (Paralichthys olivaceus) serum IgM were applied as probes. Histological, immunocytochemical and immunohistochemical detection of immunoglobulinpositive cells in the peripheral blood cells, kidney, liverpancreas, spleen and gut associated lymphoid tissues of the Japanese flounder were successfully performed. Results showed that immunoglobulinpositive cells were found in all the sampled tissues. Immunoglobulinpositive cells with diffusely stained cytoplasmic were observed in the peripheral blood, but immunoglobulinpositive macrophages were not found. There were many macrophages, granulocytes, lymphocytes and other immunerelated cells in spleen and kidney, immunoglobulinpositive cells were mostly present around the melanomacrophage centers (MMCs) and blood vessels in the spleen; the lymphoid immunoglobulinpositive cells in the kidney were also closely associated with the MMCs and existed in groups or dispersedly. The pancreas was surrounded with liver in flounder and formed liverpancreas, immunoglobulinpositive cells were separately distributed in the hepatic tissue, but not in the pancreas tissue. Additionally, immunoglobulinpositive cells in the gut associated lymphoid tissue were rarely detected in the epithelial layer although many lymphocytes were observed, yet they were frequently present in the lamina propria in groups or separately, presumably as a part of the intestine involved in mucosal immune responses. The thymus of Japanese flounder degenerates with age and no thymus could be found about 12 months old in this study.

Abstract:Crustacean haemocytes play important roles in the host immune response including recognition, phagocytosis, encapsulation melanization, cytotoxicity and cellcell communication. The various types of haemocytes are responsible for a number of defense mechanisms. Thus, the morphology and classification of haemocytes are the basis of crustacean immunology. Generally, classification of the haemocyte types in decapod crustaceans is based mainly on the presence of cytoplasmic granules and relative size of granules. In this paper, Giemsa's staining, hematoxylin and eosin(H.E) staining and electron microscope techniques were used to classify the haemocytes of Procambarus clarkii. And three types of haemocytes, including hyaline cell(HC), semigranular cell(SGC) and granular cell(GC), were observed, accounting for 19.9%，34.1%，46.1% respectively. In addition, the ultrastructures of haemocytes turned out to be most easily recognized under the electron microscope. The hyaline cells were generally the smallest cells with the highest nuclearcytoplasmatic(N/C) ratio, containing few granules, while the semigranulocytes and the granulocytes had increasing amount of granules inside their cells. Afterwards using in vivo and in vitro incubation methods， the haemocyte immunology responses of P. clarkii were investigated, with Sephadex A25 chromatography beads as the antigens. Meanwhile, the cellular recognition mechanism during the immune responses was also preliminarily discussed. When the Sephadex A25 beads were transplanted into the body of P. clarkii for a certain period of time, a large number of haemocytes, which were almost recognized as semigranular cells with slice technique, encapsulated the antigens. It was proved that semigranular cells played a prominent role during the encapsulation process. Therefore, the semigranular cells were the cells active in encapsulation responses. Besides, the results also laid a scientific foundation for the further study of cellular immunological mechanism and antidisease ability of P. clarkii.

Abstract:Arginine kinase (ATP: arginine N-phosphotransferase, AK) in invertebrates catalyzes the reversible phosphorylation of arginine by MgATP to form phosphoarginine and MgADP. The conventional view is that phosphoarginine functions as ATP buffers, permitting maintenance of high ATP values during periods in burst of cellular activity in invertebrates. In this paper, the open reading frame (ORF) was cloned from shrimp, Litopenaeus vannamei, based on the fulllength cDNA sequence that was obtained in our previous study. The ORF encoding 356 amino acids of AK was cloned and inserted to a prokaryotic expression vector pGEX-4T-2. The recombinant protein was expressed as glutathione S-transferase (GST) arginine kinase (GST-AK) fusion protein, which was purified by affinity chromatography using Glutathione Sepharose 4B. The anti-AK polyclonal antibody was prepared by immunization of mice using this purified AK protein. The specific recognition of anti-AK antibody was further verified. The AK protein expression in various tissues was then analyzed using this newly prepared antibody by Western-blotting. The results revealed a strong expression of AK in heart, nerve, hemocyte, stomach and muscle, weak expression in eyestalk, hepatopancrease, gill and skin. The present study may be very useful for studying the function of AK in shrimp.

Abstract:The virus named AVNV (acute viral necrotic virus) can cause the mass mortality in cultured scallops Chlamys farreri. So the research of AVNV vertical transmission is necessary. In the experiment, the study put the parental scallops into the separate breeding waters, then spawning, hatching, and cultivating larvae. According to the artificial spring and autumn seed of scallop indoors, from the forty separate breeding water bodies, 23 parental scallops, 23 eggs, 12 spermoviums, D larvae and shell young, 2 eyebot larvae were obtained. For the qualitative and localized detection, the methods of Indirect ELISA, IFA and Electron microscope were used to detect parental scallops, eggs and the larvae with different development phases. The result shows that the virus can be detected in the gonad of the parental scallops, but it can not be detected in the eggs, spermoviums, D larvae, shell young, and eyebot larvae. This indicates that AVNV can infect the gonad of scallop (Chlamys farreri), but there does not exist the route of vertical transmission sensu stricto. For further study, the research of horizonatal transmission is essential.

Abstract:Agarase plays a key role in degrading many complex polysaccharides.The screening of agardegrading bacteria from marine environment and organisms becomes an important means. A gramnegative bacterial strain, MA-B22 with high agarase activity was isolated from small abalone Haliotis diversicolor. By 16S rRNA analysis, this strain has 99% homology to Tamlana agarivorans sp. nov., it was named Tamlana sp. in this report. Under 2216E medium, the optimal temperature, pH value and culture time are 25 ℃, 6.0 and 60 h respectively. With onefactoratatime method and orthogonal designed experiment, the optimal compositions of the ferment medium were confirmed. It contained 2.5‰ agar, 6.0‰ beef extract based on the salinity of 25. The highest enzyme activity of agarase was detected by DNS method at 1021.79 U/mL, which is 8.82 times higher than before. MAB22 demonstrated highperformance characteristics of enzyme production. This is the first report on the agar degrading Tamlana sp. and optimal cultivation to enhance its enzyme activity. The results provide a basis for constructing engineering bacterium for expression agarase genes and potential probiotics of abalone aquaculture in the future.

Abstract:Based on the data by scientific observer survey in the tropical Central and Eastern Pacific Ocean(05°N-10°S,134° W-173°W) by the tuna longline fishery targeting bigeye tuna during Feb.Nov., 2006, with the total 223 sets, 22 sea turtle bycatch were observed and analysed. The incidental sea turtles were classified as 5 species, namely green sea turtles (Chelonia mydas),loggerhead (Caretta caretta),oliver ridley turtle (Lepidochelys olivacea), hawksbill turtle (Eretmochelys imbricata) and leatherback turtle (Dermochelys coriacea) with total 22 individuals.There were 13 sea turtles mortality after capture. The incidental catch rate is 0.09865 per set and 0.374 0 per thousand hooks. The interaction between fishing gear and sea turtles showed that sea turtle body were hooked with mouth accounting for 41.0%, external parts 13.6%, swallowed 13.6%，foreleg 18.2% and mainline entangement 13.6%. Most sea turtle bycatch occurred north of 04°S and almost all the year round. Analysis was also made to identify the specific hook number for interaction with sea turtles. Discussion was made on factors for affecting catch rate and mortality. Measures were suggested on conservation and bycatch mitigation for sea turtles.

Abstract:Chub mackerel (Scomber japonicus) is one of the important pelagic fishery resources in the China's coastal waters. It is needed to standardize the catch per unit effort (CPUE) in the stock assessment. Many factors including seasonal, regional and marine environmental conditions affect the CPUE. In this paper, generalized linear model (GLM) and generalized additive model (GAM), by which temporal, spatial, environmental, and fisheries vessels variables were chosen for analysis, were used to standardize CPUE of chub mackerel for Chinese large lightingpurse seine fishery in the East China Sea and Yellow Sea from 1998 to 2006, and evaluate impacts of environmental variables on CPUE. Firstly, GLM was applied to evaluate impacts of temporal, spatial, environmental, fisheries operational variables on the CPUE, and the significant factors. Then the significant variables were used in the GAM one by one to select an optimal GAM by using the Akaike Information Criterion (AIC). The derived GAM was used to quantify the effects of temporal, spatial, environmental, and fisheries operational variables on the Chub mackerel catch rates and to derive standardized CPUE. The GLM analysis revealed the importance of eight variables ranked by decreasing magnitude: Year, Fleet, Fleet×Year, Month, Fleet ×Month, Longitude, Latitude and Sea surface temperature. The final GAM including eight significant variables derived from GLM analysis was the optimal model based on AIC and explained 27.78% of the variance in nominal CPUE. GAM analysis indicated that high CPUEs were found in the central East China Sea at sea surface temperatures ranging from 28 to 31 ℃ in summer and in the Yellow Sea at sea surface temperatures from 12 to 16 ℃ in winter. The standardized CPUE tended to decrease from 1998 to 2006, which might result from increased fishing efforts.