Abstract:This study examined the effects of different mode of administration of LHRH-A on spermiation in the red spotted grouper （ Epinephelus akaara ）. Groups of red spotted grouper received either saline injection on 0, 7 day （ C, 50μg?kg^-1 body weight [BW] ） or LHRH-A injection on 0, 7 day（ IN, 50μg?kg^-1 body weight [BW] ） or a slower-releasing implant（ IM, 100μg? kg^-1 body weight [BW] ）. Serum levels of the androgens testosterone （T） and 11-ketotestosteron （11-KT） were significantly affected by the sustained LHRH-A delivery systems, serum T and 11-KT levels increased rapidly after being implanted. These results indicated a close correlation between sustained stimulation of T and 11-KT release, achieved by LHRH-A delivery systems, and long-term enhancement of milt production. Histological evaluation of the testes indicated that, by day 21, control and injection fish testes contained almost exclusively spermatozoa. At the same time, testes from LHRH-A implanted fish still contained large numbers of spermatozoa in the testes, indicating that spermiation was still underway. Over 40 days experimental period a total of milt volume as follows ： controls 〈 injection 〈 implantation. LHRH-A treatments stimulated a significant increase in total milt volume. While milt volumes in injected males returned to control values by day 14, implanting treated with sustained LHRH-A delivery systems maintained significantly elevated milt volumes for 40 days. Sperm motility tended to be positively correlated with milt volume, and there were no significant differences in sperm counts. These data show that sustained administration of LHRH-A significantly increases and prolongs spermiation in the red-spotted grouper without altering sperm concentration or quality.

Abstract:To explore the locations of serotonin （5-HT） and neuropeptide Y （NPY） in the brain of Scylla serrata, immunocytochemical method of Strept Avidin-Biotin-Complex was applied to observe the immunoreaction to two antibodies. Among all the 12 somal clusters and 11 neuropils of the brain in S. serrata, 5-HT-like immunoreactivity occurs in 4 somal clusters and 6 neuropils in the protocerebrum and deutocerebrum; while NPY-like immunoreactivity is present in 7 somal dusters and 3 neuropils in the deutocerebrum and tritocerebrum. The specific distribution patterns of these two immunoreactive substances may thus provide morphological proofs for their different neurophysiological functions.

Abstract:Histological changes of the digestive system and its associated glands were studied in tonguefish Cynoglossus semilaevis from the fast day （ fast day post-hatch dph） until 30 dph. Specimens for this study were hatched from artificially spawned broodstock and maintained in the indoor cement tanks （ 20.0 - 22.0℃ ）. At mouth opening （ 3 dph）, lengthening of the digestive tract, mucosae differentiation and pancreas and liver appearance were the most apparent elements. The yolk was gradually resorbed and disappeared on 5 dph., while the digestive tract was differentiated into five portions： buccal-pharyngeal cavity, esophagus, stomach, anterior and posterior intestine. The larval digestive system was morphologically ready to process external food at this time. During the following period of independent life the most noticeable events occurring were an increase in mucosal folding, cellular differentiation in the luminal epithelia, gut segmentation and looping. Thus, these digestive tract and associated glands became mature gradually and completed the morphological digestive features characteristic with increasing age and feeding activity. Glycogen was then progressively stored in the liver. Gastric glands were observed around 23d ph, which indicates the passage through the iuvenile oeriod.

Abstract:The changes of the structure in oocytes during the oogenesis of Litopenaeus vannamei were studied with transmission electron microscope. The results showed that the structure was simpler and the active metabolism is lower in oogonium. The particles from the nucleus mainly passed through the perinuclear cisternae into the cytoplasm because of fewer nuclearpores. The marked changes occurred in the latter previtellogenic oocytes and the primary vitellogenic oocytes. The nuclear changes consisted of the concavo-convex nuclear membrane, many more nucleoli, numerous nuclearpores and a large amount of nucleolns granular materials penetrating nuclear membrane into cytoplasm. There were various well-developed organelles in the cytoplasm which exhibited higher synthetic activity. The yolk granules formed mainly in active vitellogenesis. At this stage, a lap spherical cortical rods appeared radializedly at the edge of the cytoplasm. Meanwhile active microphagocytosis could be seen along the ooplasma membrane while the vitellin membrane formed on the surface of vitellogenic oocytes. The mature oocytes were fill of big yolk granules and lipid droplets and the number of organelles reduced quickly. The relation between the changes of the structure in oocytes and the vitellogenesis was discussed. The origin and function of the cortical rods were also discussed.

Abstract:Meretrix meretrix Linnaeus is an important cultivated mollusk in north China. As a series of studies, which have been carried out since 2001, the mucous cells in the alimentary tract of M. meretrix were examined with histology and histochemistry methods by the light microscopy. The aim was to offer theoretical evidence for the studies of digestion and the culture of M. meretrix. The distribution of mucous cells in the alimentary tract was revealed by hematoxylin and eosin（ H. E） dyeing method, which indicated that the mucous cells were distributed in all of the digestive organs including labella, mouth, esophagus, stomach, intestines, rectum, etc, and mostly existed in epidermis. Only a small quantity of mucous cells existed in connective tissue of labella. The types and distributions of mucous cells were observed and analyzed also by AB-PAS（alcian blue and periodic acid Schiff＇s reaction, AB pH 2.5） dyeing method. Based on the observations, the mucous cells belonged to four types： type Ⅰ, pure red, PAS positive and AB negative, the cells included neutral mucopolysaccharide; type Ⅱ, pure blue, PAS negative and AB positive, the cells included acid one; type Ⅲ, PAS positive more than AB, the cells included mixed mucopolysaccharide, more neutral than acid, and type Ⅳ, AB positive more than PAS, these cells included mixed mucopolysaccharide, more acid than neutral ones. The statistic results showed that, in different parts, the mucous cells had different types and densities. There was a small quantity of mucous cells in labellurn, and most of them existed in the ruga part. The types of the mucous cells of labellum were types Ⅱ and Ⅳ. There were large quantities of mucous cells in esophagus, which belonged to all four types. There were more mucous cells in stomach than in labellurn, but less than in esophagus. The mucous cells include four types, and the distribution of the mucous cells is dispersed. The mucous cells in intestines increase dramatically. Most mucous cells lie in the rectum, consisting of types Ⅱ, Ⅲ, and Ⅳ. By the studies of AB - PAS （AB, pH1. 0）, it was found that the mucus included neutral mucopolysaccharide, and acid one that included sulphate mucopoiysaccharide and carbonic mucopolysaccharide. The most competent of acid mucopolysaccharide in labella and rectum was carbonic mucopolysaccharide, only a little was sulphate mucopolysaccharide. In addition, the mucous cells had acid phosphoataes （ACP） and alkaline phosphoataes （AKP） activity, but the activity was weak. Our work exhibited that the mucus released from mucous cells had the activity of ACP and AKP.

Abstract:A fragment cDNA of parvalbumin gene has been cloned, which expressed differently between pre-metamorphosis and pro-metamorphosis stages of Japanese flounder, Paralichthys olivaceus. For the requirement of further research on the role of parvalbumin in metamorphosis stage, it is necessary to clone the full-length cDNA of parvalbumin gene. Here we constructed a head tissue full-length cDNA library of pro-metamorphosing flounder （23 dph, day post hatch） using SMARTTM technique. The titer of cDNA library was estimated as 1.2 ? 10^6 PFU?mL^-1, which was about 1000 times coverage of gene expressional profile, and the inserted fragments in the library are about 1000 bp length. Through two-runs anchor-PCR with target gene specific primers and λ TripIEx2 primers, we obtained a full-length cDNA of parvalbumin gene from the cDNA library. The full-length cDNA of parvalbumin gene consisted of 603 bp nucleofides, and encodes 109 amino acids with two EF-hand calcium-binding domains： DQDGSGFIEEEL （52- 64） and DSDGDGKIGVEEF（91 - 103）, and an ATP/GTP-binding site motif A （P-loop）： ACGLAGKS（ 33- 40）. The structure of the sequence shows the parvalbumin is a typical calcium-binding protein. Further analysis with homologue sequence comparison shows the flounder parvalbumin is a conserved protein, which shares 61.5% - 68.8% similarity with other teleosts, such as 61.5% with channel catfish lctalurus punctatus, 68.8 % with zebrafish Danio rerio. The phylogenetic analysis shows the closest relationship existed between parvalbumins of Japanese flounder and cod Theragra chalcogramma. Parvalbumin plays biological role of relaxation of muscle after contraction by soaking free calcium in cell. The recent researches show the parvalbumin gene expressed differentially in GABA neurons. However, it is unclear about the role of parvalbumin in metamorphosing flatfish, including Japanese flounder. The results reported here should be helpful for the lhture work such ~s gene expression location, gene function research.

Abstract:Adhesion gene （ahal）, aerolysin gene （aerA） and cytotonic enterotoxin gene （alt） were considered as major virulence genes of Aeromonas. According to ahal, aerA and alt gene sequences, a multiplex polymerase chain reaction （MPCR） was developed to detect the above virulence genes simultaneously. The ahal, aerA and adt genes were 1087 bp, 721 bp and 480 bp PCR products respectively. Digested with EcoRV, BamnHI and Fbal, the sizes of digestive fragments of each PCR product were consistent with expectation. Using the same pairs, no PCR product was detected from S. aureus, P. putide, V. minicus and unpathogenic Aeromonads. The sensitivity of the MPCR was 100 CFU?mL^-1 of the bacteria. At the same time, multiplex PCR analysis was performed on 15 strains Aeromonas spp. isolated from different aquatic animals in Anhui and the results showed there were 10 strains with virulence genotype alt^＋ ahal^＋ aerA^＋ , 2 strains with virulence genotype alt^＋ ahal^- aerA^- and 1 strain with virulence genotype alt^＋ ahal^＋ aerA^- within 13 strains pathogenic Aeromonas spp. The positive rates of alt, ahal and aerA genes were 100%, 84. 62% and 76.92% respectively. These results indicate that the major virulence genotype of Anhui isolation strains is the high virulence phenotype of alt^＋ ahal^＋ aerA^＋ and the virulence gene alt generally exists in different Aeromonas phenospecies.

Abstract:SRY（sex-determining region of the Y chromosome） gene plays a key role in sex determination in the mammal and some evidences suggest that SRY encodes the TDF （testis determining factor） that is required for male sex determination. Sox genes are closely related to the SRY and code for proteins with a characteristic DNA-binding motif known as SRY-type HMG （high mobility group） box. A large number of Sox genes have been found in a wide variety of species throughout the animal kingdom, from human to Drosophila. Penaeths monodom is an important economical species and its heteromorphic sex chromosomes have not been found. So far, little information is known about P. monodom＇s molecular genetics of sex determination. In this paper, the technique of PCR is used to amplify the Sox genes of P. mondom with a pair of primers which specially amplify the conservation sequences of man HMG-box gene. In addition, the technique of SSCP（single strand conformation polymorphism） is used to analyze the amplification products. The results of PCR and SSCP analysis suggest： 1 ）The lengths of the fragments of PCR products are about 350 bp and 220 bp in both female and male shrimps. 2）There are Sox genes in these shrimps. 3） Introns are also found in Sox genes which is 350 bp. 4） There are different Sox genes in female and male shrimps. And the sex-determining mechanism of prawn is also discussed.

Abstract:Enrichment by magnetic beads is a simple and efficient method for rapid isolation of microsatellite DNA. In this experiment, we isolated microsatellite DNA from grass carp （ Ctenopharyngodon idella ） genome with this method. Grass carp genomic DNA was extracted and digested with restriction enzyme Sau3AI. Fragments of 400 - 900 bp were isolated and purified and then ligated withshort linkers （20 bp）. They were used to create a “whole genome PCR library”. This genomic DNA was hybridized with a biotin-labeled microsatellite probe （CA）15. The hybrid mixture was incubated with magnetic beads coated with streptavidin. After washing to remove the non-SRS fragments, the eluted single-stlanded DNA contains the selected microsatellite DNA. The selected DNAs are then amplified using primers designed complementary to the linkers, cloned into the pGEM - T vector and seq. uenced. In this experiment, we performed the second screening with a radiolabeled （CA）15 probe and obtained 132 positive clones. From these positive clones, we isolated 130 microsatellite sequences. This allowed us to design 83 pairs of primers with the software Primer Premier 5.0.

Abstract:It took 26 days for Pelteobagrus vachelli to grow into juvenile ,after thawing. The larval and juvenile development of P.vachelli were divided into four stages （namely, prelarval, larval, early juvenile and juvenile stages）, and each stage was also divided into 3 periods （amount to 12 periods）. The prelarval period can be divided into adherence, horizontal swimming and initial bait phases. It was necessary to provide suitable adhesive things during the adherence stage. During initial bait phase, its food maily consisted of some small Cladoceras. The larval stage was still a mixtrophic one and it mostly ingested Cladocera besides yolk. After entering the juvenile stage, its feeding characters underwent transitions twice： firstly, its foods changed from zooplankton to zoobenthos; secondly, foods changed from zoobenthos to animal-oriented polyphagia and the juvenile started to ingest artificial intensive feed. According to characters of the larval and juvenile development of P. vacheUi, a series of scientific methods about the rates of hatching and surviving of P.vachelli were suggested in this article.

Abstract:Effects of different water-borne copper（ Cu^2＋ ） （ 0.00, 0.01, 0.10, 1.00, 5.00 mg? L^-1 ） on distribution of copper in the body of Eriocheir sinensis and activities of some main digestive enzymes were studied by Single Gradient Factor Experiments. Among gill, epidermis and hepatopancreas of the animals in the control group without additive Cu^2＋ , average Cu^2＋ was 0.074, 0.036, 0.023 mg?g^-1 wet weight, respectively, which were determined by ICP-AES. When increasing of water-borne Cu^2＋ except for the hepatopancreas and epidermis in tested group with 0.01 mg?L^-1 water-borne Cu^2＋, Cu^2＋ level increased significantly （analyzed by SPSS software） in all organs studied and especially in the hepatopancreas. In the tested group in which water-borne Cu^2＋ was 5.00 mg?L^-1 Cu^2＋ level among the gill, epidermis, and hepatopancreas was 0.307, 0. 191, 0.341 mg?g^-1 wet weight, respectively. Furthermore, in the hepatopancreas the inhibition effect could be noted on activities of some important digestive enzymes, such as the typase, pepsin, amylase and cellulase in different extents during increase of water-borne Cu^2＋ . It is notable that such effect on the amylase was the most obvious even the level of additive Cu^2＋ was only 0.01mg?L^-1 that is the level of the National Water Purity Standard for Fisheries. Comparing with the control group, the activity of the amylase dropped sharply from 305.01 ? 32.23 to 36.81 ? 4.84 U?mg^-1. The maximum inhibition rate of this enzyme could be recorded as high as 89.96% in the tested group with 5.00 mg?L^-1 of water-borne Cu^2＋ . In contrast, the activity of the lipase enzyme was found increasing in all tested groups between 346.15% to 646.15%. The present study suggests that the different functional characteristics of the gill, epidermis and hepatopancreas may contribute to the change of Cu^2＋ distribution pattern among these organs for Eriocheir sinensis when the additive Cu^2＋ level in water was increased. Meanwhile, the hepatopancreas seems to be the main bioac Cu^2＋ mulation site, as well as the metabolic organ for Cu^2 ＋ . Moreover, the variations on the activity of the typase, pepsin, amylase and cellulase enzymes can sensitively indicate the inhibition effect of Cu^2＋ as well as its toxicity.

Abstract:The effect of soybean trypsin inhibitor （STI） on the degradation of silver carp myofibrillar proteins caused by an endogenous myofibril-bound serine proteinase （MBSP） was studied. Soybean trypsin inhibitor was purified to homogeneity and added to myofibril to investigate its effect on preventing myofibrillar protein degradation. In the absence of STI, incubation at 55℃ for 30 min caused the degradation of myosin heavy chain. The degradation of actin and tropomyosin could also be detected after prolonged incubation. Compared with control, in the presence of STI, with final concentration of 0.75μg?mL^-1, proteolysis of myofibrillar proteins was greatly suppressed, the result showed that STI was an effective inhibitor in preventing degradation caused by MBSP. As the integrity of myosin heavy chain （MHC） and actin was the most important factor forming the elasticity of surimi, this result suggested the possibility that STI is applicable in surimi production in order to enhance the elasticity that is the quality of the final products.

Abstract:Sensory analysis and GC/MS were employed to study the effects of two marine flavors on the seafood flavor and sensory characters of the temporarily cultured Palaemon carinicauda, as well as the absorption of the flavors. Two new flavor additives, DBP and TBP, extracted from marine algae, were added into the feedstuff. The flavor contents in P. carinicauda were assayed after feeding with these special additives for 10 d. Volunteers were invited to give sensory analysis to various samples and grade them about their brightness, hue, fragrance, flavor, off-flavor, savor, taste, tissue elasticity and tendemess. And the color, sense, taste, touch characters of P. carinicauda were described and appraised. Results showed that P. carinicauda could absorb a certain of flavor compounds by feed with flavor additives. Two flavors were absorbed at different rates, and TBP was easier to be absorbed with the maximum concentration of 754.5 ng?g^-1 while only 172.2 ng?g^-1 for DBP. The seafood flavor and the comprehensive taste of P. carinicauda were improved by adding proper concentration of TBP into their feedstuff. The best sensory value and seafood flavor were obtained when TBP ranged from 162.6 to 451.8 ng?g^-1.

Abstract:Juvenile yellow catfish Pelteobagrus fulvidraco （Richardson） with 18.0 g in body weight were stocked into 36 net cages of 60 cm ? 60 cm ? 120 cm each and fed semi-purified formulated feed containing 30%, 38% and 46% of dietary protein and supplemented with four levels of ascorbic acid-2-phosphate （AA） （0, 500, 1000, 1500 mg?kg^- 1 diet, ） at 22 ? 3 ℃ for 50 days. The results showed that the fish fed the formulated feed containing 38.0% - 40.0% dietary protein bad the maximal special growth rate （ SGR）, and protein efficiency ratio （PER） and the minimal feed coefficient （ FC）. The catfish had the maximal levels of total plasma protein, γ-globulin, and activities of lysozyme and the minimal activity of superoxide dismutase when fed 847, 850, 994 and 634 AA mg?kg^-1 diet. In the fish fed the formulated feed varying from 30% to 38% in dietary protein, the protein in muscles increased from 60.95% to 69.86%, and the lipid decreased from 29.27% to 21.69%, whereas the fish fed the formulated feed containing 46% dietary protein bad a decrease in protein （64.35 % ） and an increase in lipid （26.06 % ） levels in muscles. There were significantly higher saturated fatty acid levels （SFA） （ P 〈 0.01 ）and monounsaturated fatty acid levels（ P 〈 0.05）in museles in the fish fed the feed containing 38% dietary protein than those in the fish fed the feed containing 30% and 46% dietary protein. There were significantly higher linolenic acid and n - 3 polyunsaturated fatty acid （ n - 3 PUFA） levels in muscles in the fish fed the 38% and 46% （ P 〈 0.01, P 〈 0.05） dietary protein feeds than those in the fish fed the 30% dietary protein feeds. It was found.that there was significantly higher n - 3/n - 6 ratio in the 30% and 46% dietary protein groups than in the 38% dietary protein group. For yellow catfish juveniles, the optimal dietary protein and vitamin C levels have been considered as 35.58 - 40.60% and 600 - 800 mg? kg^-1, respectively.

Abstract:A specific band of Betanodavirus RNA2 was detected from red-spotted grouper（ Epinephelus akaara） larvae by reversetranscription polymerase chain reaction （ RT-PCR）. Under light microscopy, the brain, retina, and spinal cord of affected fish showed conspicuous vacuolation. In the brains, vacuoles were mainly apparent in the telencephalon, the diencephalon and the cerebellum. Affected cells were pyknosis accompanying marked shrinkage and basophilia. Inclusion bodies were usually round and different in size. Transmission electron microscopy revealed affected cells containing dense bodies filled with numerous viral particles in the cytoplasm. Virions were 25 - 28 nm in diameter, icosahedral and non-enveloped. Virions were distributed randomly in the cytoplasm or arranged in crystalline arrays in the dense bodies. The sizes of dense bodies were different. Occasionally, the unit membrane surrounding the larger bodies had ruptured, allowing the viral particles to escape into the cytoplasm.

Abstract:Brine shrimp Artemia is the key animal diet during the course of the penaeid shrimp and crabs＇ larviculture. It is very important for healthy larvae culture to investigate whether it could carry white spot syndrome virus （ WSSV ）. In this study, wild Artemia sp. with eggs and experimentally cultured children Artemia were detected using nested-PCR for the presence of WSSV. Wild adult Artemia sp. were sampled from brine pan from July to October, 2002. 20 control groups were ,set up after ten-days＇ nursery period, that is, one female Artemia with eggs in each 500ml beaker, feeding microalgae. Parents Artemia were taken out after spawning and then frozen at -20℃. About 20 days later, five children ones were taken out and detected by nested-PCR together with the parents, respectively. The results showed that WSSV positive products could be detected in Artemia, not only in wild adult Artemia but also the filial generation, and DNA ,sequence of the amplified product accords with the designed specific amplified one according to sequence analysis using sequence analysis software Bioedit 4.8.8.. The positive rates of wild brine shrimp and the filial generation were 58 % and 43 %, respectively. In addition, there seems to have some relation between parents and filial generation＇ s detecting results of the same genealogies. However, we could not tell whether WSSV could vertically transmit in Artemia or not, and there still needs some work to further verify this. In conclusion, the results showed that brine shrimp Artemia could possibly act as reservoir or mechanical vector for WSSV, thus, Artemia diet during shrimp or crabs＇ larviculture might lead to WSSV infection. Therefore, it is necessary to quarantine the brine shrimp before being fed to shrimp or crabs in order to cultivate healthy larvae.

Abstract:Whitish muscle diseases of freshwater giant prawn, Macrobrachium rosenbergii, is a new epidemic disease in the larvae and post-larvae of M. Rosenbergii in recent years. The clinical sign of the diseam is a whitish or opacity appearance of the muscles in abdomen. Mortalities may reach more than 60%, sometimes even reach 100%. The pathogen of the disease is confirmed as Macrobrachium rosenbergii Nodavirus（MrNV）, an icosahedral and non-enveloped virus sized from 25 - 26 nm with two linear ssRNA sized 3.0 kb and 1.3 kb respectively. MrNV was the first nodavirus founded in crustacean. To develop a rapid method for MrNV detection, monoclonal antibody against MrNV was prepared and used for virus detection. Briefly, mouse myeloma .cells（SP2/0） were fused with spleen cells from BALB/c immunized with purified MrNV from diseased postlarvae（PL）. Positive colonies were screened by indirect ELISA with the plates coated with purified MrNV and cloned 2 - 3 times with limited dilution method. Twelve positive hybridoma cell lines secreting monoclonal antibodies （Mabs） against MrNV were at last obtained and used for ascites preparation. The titres of positive ascites Mabs were ranged from 1 ： 105 to 1 ： 106 by indirect ELISA. These twelve Mabs were isotyped by Southern Biotechnology clonotyping system, and the results showed that six Mabs were IgG1, four IgG2a and two IgG2b, respectively. No cross reactions, were found when the twelve Mabs were used for indirect ELISA with white spot syndrome virus （WSSV） and Taura syndrome virus（TSV）. A triple antibody sandwich enzyme-linked immunosorbent assay（TAS-ELISA） was developed with the plates pre-coated with rabbit polyclonal antibodies against MrNV for virus capturing, followed by MrNV and samples incubaticn, Mabs and goat-anti-mouse-IgG antibodies labeled with HRP. Mab 2B5, characterized as Mab against 43 kD coat protein of MrNV by Western blot, was selected for TAS-ELISA. The results showed that TAS-ELISA can be used successfully for minimum virus detection with the sensitivity of 0.98 ng MrNV, or with the viral titer of 1：40960 when typical diseased PL homogenization supematants were used for TAS-ELISA. TAS-ELISA could be considered as a useful technique for MrNV detection as well as for epidemiological studies for its rapid and inexpensive cost with high specificity and sensitivity.

Abstract:Fluoroquinolones （FQs）are one of the commonest antibiotic groups that have been used for the treatment of bacterial infection. Enrofloxacin（EF） belongs to this group and has been increasingly used in veterinary medicine . A simple and reliable analytical method for this drug is required. The current determination by high performance liquid chromatography （HPLC） is sensitive but labor-intensive, lmmunoassay methods have been successfully developed to rapidly screen EF residues. The synthesis and characteristics of complete antigens of EF were experimented. The results of this study indicated that the antibodies against EF may be used as the basis for an immunoassay to rapidly screen samples for the presence of FQs in tissues or body fluids. EF is a small molecule （MW 〈 1000） that must be conjugated to a cartier to elicit an immune response. Proteins are commonly used carriers, such as bovine serum albumin （BSA）, ovalbumin （OVA）, keyhole limpet hemocyanin （KLH） and Albumin Human （HSA）. Using the free carboxylic acid on EF, it could be linked to protein carriers through the amide linkage. BSA and OVA were used as protein carriers to synthesize EF complete antigens respectively by both carbodiirnide method and active ester method, which were then immunized to BALB/C rats. KLH was coupled to EF by carbodiimide method , the conjugation was used as coating antigen. The effects of coupling between haptens and carriers were investigated by UV spectrum, anti-serum, etc. The UV spectrum showed that the major absorbances of EF were in 208 nm, 271 nm and 334 nm, while protein had its major absorbances at 229 nm and 278 nm ; the EF-protein conjugates had a significant peak at 330 - 335 nm which were the main characteristic of EF but could not be seen in protein, so it could be concluded that the linkage between EF and protein were successfully set up. An indirect enzyme linked immunosorbant assay （ ELISA ） was used to determine the titers of the antiserum, the titers of all the resultant polyclonal antibodies could reach 1：50000. In ELISA procedure, the optimal dilution of coating antigen was determined by the checkerboard test and the optimum condition selected was coating antigen concentration in 10μg?mL^-1 and secondary antibody diluted 1 ： 10000. The haptenprotein ratio was calculated by formula at the range of 1 - 6. It used to believe that the amount of haptens coupled to the protein should be as much as possible. But in theory, the carder protein linked even to one hapten may be enough to produce specific antibody, and in fact many researchers have proved that in some cases overabundance of the haptens with the cartier cannot result in desired responses, because it may prevent the protein from being present at the surface of immunoglobulins on B cells, and then interfere with the immune reaction. The results showed that the UV spectrum of protein-EF could coincide with the results of enzyme immunoassay of anti,rums, so it could be used to indicate the characteristics of the complete antigens simply. During the reaction, amino group on the protein can also be linked with the carboxyl group on itself. To reduce the self-coupling, we added excess ethylenediamine （EDA） first to block the proteins and it could be seen that the characters of the modified proteins did not change from its UV spectrum. Now no relative reports about modified protein could be obtained in China. Also it was found that coupling effects could be obviously improved after the carrier proteins were treated with EDA. But to various carrier proteins, the coupling effects were greatly influenced by different coupling methods. It was better to use active ester method than carbodiimide method when carrier protein was OVA. But when BSA was used as carrier protein, the coupling effects of the carbodiimide method were better.

Abstract:A series of experiments had been carried out to develop and validate the growth kinetics model of specific spoilage organisms for predicting the microbial quality and remaining shelf life of cultured tilapia stored aerobically in cold temperature. The results from microbiological behaviors analysis, organoleptical quality and TVBN evaluation identified Pseudomonas spp. as special spoilage organisms of tilapia stored at 0 - 15℃. At the end of shelf life, the average number of Pseudomonas spp. was 7.70 ? 0.11 log10cfu?g^-1 at temperatures 0,5, 10 and 15℃. The kinetics models of Pseudomonas spp. had been developed and the results showed growth dynamics of this organism under 0 - 15 ℃ could be well described by a Gompertz type model. The temperature had no significant effect on Nmax （maximum cell concentration）, the average value of Nmax was determined to be 8.85 ? 0.18 log10cfu? g^-1 under those four temperatures. The temperature dependence of Pseudomonas spp. kinetic parameters - μmax（ maximum specific growth rate） and Lag （lag phase） were modeled using Belehradek model. The correlation regression showed could finely describe the influence of temperature on growth. Validation of built kinetic models was preferred by comparing with experimental development of Pseudomonas spp. grown on tilapia at temperatures 3℃,8 ℃. Bias and accuracy factors were used as comparison indices and range from 0.97 to 1.01 and from 1.02 to 1.04, respectively. The results derived from validation showed that the developed models could finely predict the growth kinetic of Pseudomonas spp. at 0 - 15 ℃. Basic errors between predicted shelf life and real shelf life were 3.47% and - 7.91% at 3, 8℃ respectively, it suggested microbial growth models built in our research were valuable for rapid and realistic prediction of the microbial quality and remaining shelf life of cultured tilapia stored aerobically from 0 to 15℃.

Abstract:The breaking force of silver carp （ Hypophthalmichthys mollitrix ） and Alaska pollack （ Theragra chalcogramma ）sufimi gel heated by microwave and water-bath was measured respectively. Then the gel-forming rate of different heating methods and the activation energy under microwave were calculated and compared. Energy absorption of the samples heated by different methods was also calculated and compared. The results indicated that, （ 1 ） their gel-forming rate during microwave heating was quicker than that heated by water-hath, and under microwave heating the gel-forming rate of different fish surimi was different. （2） under microwave heating, the activation energy that freshwater fish silver carp needed was larger than the seawater fish Alaska pollack, and the activation energy of their corresponding composite surimi was less than the sunplex surimi. It was closely related with their myofibriUar protein＇s heat stabilities and their content. （3） compared with water-bath heating, the surimi samples absorbed the same or less energy during microwave heating, while the breaking force of the gel was larger instead. Microwave heating showed more advantages.

Abstract:Immunostimulants are valuable for the control of fish diseases. At present, it has already been a main tendency to discover and develop novel irnmunostimulants with characteristics of low toxicity, high efficacy, immediate efficacy and long-term efficacy. In this review, the definition, defense mechanism, categories and application of fish immunostimulants are introduced and fish immunostimulants are classified systematically according to their sources. Moreover, the latest literature about fish immunostimulants in detail were presented and reviewed and three methods for control of fish disease were compared： vaccination, chemotherapy and immunostimulants. Fish immunostimulants were thought to be safer than chemotherapeutics and their range of efficacy was wider than vaccination. They may be able to compensate for these limitations of chemotherapeutics and vaccines, thus they hold tremendous potential for use in fish culture to reduce losses from fish diseases. Meanwhile, concepts of use of fish immunostimulants were presented and principles for use were also noted. For aquaculture, this review provided a scientific reference for control of fish disease by immtmostimulants.

Abstract:Unionidae, one major freshwater family of a Bivalvia with many species, is distributed very extensively. Some of them have high economic value. Due to the deterioration of world environment, its resource is declining continually. The development of eygote of freshwater mussel （Unionidae） undergoes in the gill. When it grows to the stage of glochidia, it must live a parasitic life on fish. Because of this breeding feature, the artificial insemination technology has had no breaking-through in long time. With the rapid development of fresh water pearl culture, it is impending for us to develop new breeding technology, select sceds with good productive traits, protect the resources quality and develop new cultivate technology. This paper synthesized the research achievement for the feature of mussel breeding, the gamete growth and formation, reproduction season and embryonic development, the glochidium formation, structure and abnormal breed, and so on. Finally the future research prospects were described. This review could serve as the reference material for future studying on the freshwater mussel reproduction biology and the artificial propagation technology.

Abstract:During July-November in 2003, pelagic fish survey was conducted by longliner covering areas of 03?S - 17?S , 96?W - 146?W. Sampling gear was longline fishing. With the precaudal length increment, the total gonad weight of male blue shark increases greatly but that of female increases little. The average value of TGSI（total gonadosomatic index） of female amounts to 0.64 but male increases to 0.98. The blue shark is viviparous and the period of parturition appears in Oct. - Nov, The sex ratio of adult blue shark is not fitted to 1 ： 1. Preliminary results show that female matures at total length 183 cm（precaudal length 137cm）. The broods range from 13 to 62 and average 34.29 ? 11.2 （ n = 28 ）. There is no obvious relationship between broods and maternal length. The length at birth arrives at total length 44cm. Based on the observation of egg diameter in the ovary of female blue shark, the maximum of egg diameter is 16- 18 mm occurred in Nov. and at the same time fertilized eggs appear. Based on the clasper length analysis, the smallest male matures at total length 174 cm （precaudal length 131 cm）.

Abstract:This paper focused on the force characteristics of mooring lines of the common used gravity cage and sea station cage under floating status. In this experiment, total four cage models were designed which included two gravity cage models, one sea station cage model and one quasi-sea station cage model. The gravity cages were made of the same materials but with different weighting system configurations. The sea station cage model and the quasi-sea station cage model were of the same diameter but the latter is 1.4 times in cage height to the former one which attributed greatly to the amplification of effective aquaculture volume up to 2.2 times. Four mooring lines were attached to each cage model on one side and the other side fixed on the bottom of the wave-current tank. Several kinds of experiment conditions were set, including pure current conditions, pure wave conditions and combined wave-current conditions. Forces were measured by four transducers attached to the bottom of the mooring lines respectively. Analysis was based on the resultant forces of the two current-ward or wave-ward mooring lines. Results from this study were presented and statically analyzed revealing that the quasi-sea station cage model suffered from greater forces compared with the other two kinds of cage models, which was more apparent under pure current conditions. It was interesting to find that the mass of the weighting system turned out to be relatively small effect on the forces acting on the gravity cage models under most conditions. Under pure wave conditions, several results of the forces acting on the gravity cage with heavier weighting system configuration were even smaller than those with lighter weighting system configuration. Conclusions were drawn that it was feasible to increase the mass of the weighting system properly to reduce the deformation of the netting system since it will not increase the forces acting on the gravity cages apparently. But it should be kept in mind that the operation of the cage will be affected directly by increasing the mass of the weighting system. As to sea station cage model, given the similar maximum aquaculture volume, results showed that the forces acting on it were approximately the same with that of gravity cages under the same conditions. But its huge size would lead to bad operation directly. Thus, when considering the force characteristics, the performance and management of the cages together with the cage prices, the co-authors recommended that gravity cages were more suitable currently as deep-water aquaculture facilities in China.

Abstract:Biofilter technology plays a key role in the production process of recirculating system. The establishment of biofilter＇s full nitrifying function is very important to avoid the aquacultural hazards for producers. There were many reports about the cultivation of the biofilter in the recirculating system. However, almost all of them were carried out in the lab scale and had little meaning to the production scale. This paper compared three methods of cultivating biofilter in the recirculating aquacultural system. The results showed that putting appropriate load fish directly and feed lowly was the best method to establish the nitrification function of the biofilter in the production of the recirculating aquaculture system.

Abstract:Based on the filtration of sand filter bed, a solid and nitrification biofilter of floating plastic bead was designed and tested. In the biofilter, the bead was layered automatically in water according to the size and gravity of plastic bead. With two types of diameter 5 mm gravity 0.94 and diameter 2 mm gravity 0.5 beads, the biofilter performed well in solid separation and ammonia nitrification. The bead filter performances were tested in a recirculating aquaculture system. When pump power was 0.75 kW and flow quantity was 20 m^3?h-1, the recirculating culture system could support 10 m^3 water in culture density of 16 kg?m^-3 Amur sturgeon. Suspended solid filtering rate was 90% and ammonia nitrification capacity was 149 g?m^-3?d^-1 Within water exchanged period of two weeks, the water quality was as following, SS≤ 100 mg?L^-1, Ammonia≤ 1.0 mg?L^- 1, Nitrites≤0.18 mg?L^-1 and Nitrate 24≤mg?L^-1. After 15 days＇ culture, fish survival rate was 99% and growth rate was 50%. The experiment result showed that the bead biofilter could meet the need of recirculating aquaculture system in solid seoaration and ammonia nitrification.