STUDIES ON IMMUNODIAGNOSIS OFHEPATOPANCREATIC PARVO- LIKEVIRUS DISEASE OF THE CHINESEPENAEID, PENAEUS CHINENSISI. PURIFICATION OF THE VIRUS

The viral disease of penaeids is indistinct but highly dangerous because their symptoms are often masked by some secondary ones.In order to gain knowledge on how to prevent the occurrence and spread of this viral disease, diagnostic studies on the early phase of the hepatopancreatic parvo-like viral (HPV)disease of cultured Penaeus chinensis was conducted using immuno-serological techniques. The purification of HPV was successfully done by density gradient ultracentrifugation of cane sugar.     

Studies on immunodiagnosis of hepatopancreatic parvo-like virus diseases ofPenaeus orientalis

This paper describes a rapid procedure for diagnosis of penaeid viral disease using the immune serological method. The SPA (Staphylococcal Protein A) coagglutination test in early diagnosis of penaeid viral disease has yielded satisfactory results. This is the first time SPA is used in China for diagnoses of viral diseases of marine invertebrates, especially penaeid shrimp. The SPA early diagnostic method is characterized by accuracy, rapidity, simplicity and convenience, low cost, high specificity, strong sensitivity and micro-detectability, and easy dissemination and adaptability in prawn farms.     

A study on pathogens of Chinese prawn (Penaeus Chinensis) virus diseases

This pathogenic study shows that the viral diseases of Chinese prawns (Penaeus chinensis, O'sbeck) is due to three kinds of viruses: epithelium envelope baculovirus ofPenaeus chinensis (EEBV-PC, detected by the authors in 1993), infections hypodermal and hematopoietic necrosis virus, and hepatopancreatic parvo-like virus, and that the first two viruses seem to be the main pathogens of the epidemic in the northern regions in 1993.     

Detection of lymphocystis disease virus in Japanese flounder Paralichthys olivaceus and other marine teleosts from northern China

We isolated a strain of lymphocystis disease virus (LCDV) from Japanese flounder (Paralichthys olivaceus) cultured in northern China. Based on published sequences of major capsid protein (MCP) gene of LCDV-cn (GenBank: AF126405), we designed two primer sets P1/P2 and P3/P4. We then used one-step or nested PCR and in-situ hybridization (ISH) to detect LCDV and identify the target tissues or cells in infected Japanese flounder. The PCR products were positive in purified viral supernatant, skin nodules, gut, gill, kidney, spleen, stomach, heart, and liver of Japanese flounder. We compared the DNA sequence with 14 MCP nucleotide sequences from GenBank, including Megalocytivirus (OFIV and RSIV), Iridovirus (CzIV and WIV), Ranavirus (TFV and FV3), and Lymphocystivirus (8 LCDV). Based on the alignment, we confirmed the PCR product was from Lymphocystivirus (GenBank accession number DQ279090 (LCDV-HD)). Using ISH, we noted the presence of LCDV in the skin nodules, gut, gill, spleen, stomach, and heart of spontaneously infected Japanese flounders. We successfully amplified LCDV fragments from Schlegel’s black rockfish (Sebastes schlegeli Higendorf), redwing sea robin (Lepidotrigla microptera Günther) and turbot (Scophthalmus maximus) using the one-step and nested PCR, suggesting the target genes can be widely detected in fish using this method.     

Expression of putative zinc-finger protein lcn61 gene in lymphocystis disease virus China （LCDV-cn） genome

An open reading frame (lcn61) of lymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector. Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene. Supported by High Technology Research and Development Program of China (863 Program, No. 2006AA100309)     

16S rRNA gene phylogenesis of culturable predominant bacteria from diseased Apostichopus japonicus (Holothuroidea, Echinodermata)

Cultured Apostichopus japonicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease, pathogens of which are supposed to be bacteria by most researchers, is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao, China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies, and touchdown PCR was performed to amplify the target sequences. The results suggest that γ- proteobacteria (Alteromonadales and Vibrionales) of CFB group, many strains of which have been also determined as pathogens in other marine species, are the predominant bacterial genera of the diseased Apostichopus japonicus individuals.     

Disease resistance and humoral immunomodulatory effects of vitamin C on grouper,Epinephelus awoara

The disease resistance and humoral immunomodulatory effects of vitamin C administered orally to grouper,Epinephelus awoara maintained on a frozen fish diet supplemented with vitamin C at 500, 1000, 1500 and 2000 mg/kg were investigated. After 20 weeks, the growth rates of the groups with high level of vitamin C apparently increased. The untreated fish had symptoms of vitamin C deficiency. The endogenous liver tissue vitamin C levels were found to reflect well the dietary treatments. After intraperitoneal injection or bath challenge with a virulent strain ofVibrio vulnificus, fish fed with high level vitamin C showed significantly higher survival rate compared with the normal control group. Vaccination with formalin inactivatedV. vulnificus significantly enhanced the specific antibody production in fish treated with vitamin C, and completely protected from strong bacterial challenge the groups fed on fish with vitamin C 1500 and 2000 mg/kg diet. Project 39500113 supported by NSFC and also by Guangdong Science Research Foundation (Grant No. 950711).     

A VIBRIO ANGUILIARUM STRAIN ASSOCIATED WITH SKIN ULCER ON CULTURED FLOUNDER, PARALICHTHYS OLIVACEUS

The characteristics of a bacterium strain M3, isolated from cultured flounderParalichthys olivaceus with remarkable external sign of skin ulcer during an epizootic outbreak, indicated that the bacterium belonged to the speciesVibrio anguillarum. Challenge by I.M. (intramuscular injection), bath, and oral administration with M3 showed that it was highly pathogenic forParalichthys olivacues. The LD₅₀ dose was 5.144×10³ CFU/ per fish infection by I.M. injection. Recovered inoculated bacteria from the surviving fish revealed that the asymptomatic carriers could be a latent contagious source. Study of the effect of bacterial culture CFS (cell-free-supernatant) showed that the exotoxins produced by M3 play an important role in its pathogenicity for flounder. The resistance of M3 to 36 out of 41 antibiotics indicated that the bacterial disease outbreak was mainly attributable to the frequent and excessive use of antimicrobial agents; and that vaccination would be an effective precaution against bacterial disease. Contribution No. 4107 from Institute of Oceanology, Chinese Academy of Science. Contribution No. 270 from Experimental marine Biology Laboratory, CAS. Funded by projects under the Major State Basic Research Development Program, Grant G1999012003.     

Experimental study on the interspecific interactions between the two bloom-forming algal species and the rotifer Brachionus plicatilis

The interspecific interactions between the rotifer Brachionus plicatilis and two harmful algal blooms (HAB) species were investigated experimentally by single culture method. B. plicatilis population and the growth of the two algae were compared at different algal cell densities. The results demonstrated that the B. plicatilis obtained sufficient nutrition from Prorocentrum donghaiense to support net population increase. With exposure to 2.5×10⁴ cells mL⁻¹ of P. donghaiense, the number of B. plicatilis increased faster than it did when exposed to other four algal densities (5, 10, 15 and 20 ×10⁴ cells mL⁻¹), and the increase rate of B. plicatilis population (r) at this algal density was 0.104 ± 0.015 rd⁻¹. Cell densities of P. donghaiense decreased due to the grazing of B. plicatilis. In contrast, Heterosigma akashiwo had an adverse effect on B. plicatilis population and its growth was largely unaffected by rotifer grazing. In this case, B. plicatilis population decreased and H. akashiwo grew at a rate similar to that of the control.     

Two new species of the genusDorylaimopsis Ditlevsen, 1918 (Nematoda: Adenophora,Comesomatidae) from the Bohai Sea,China

Two nematode species of the genusDorylaimopsis (Family Comesometidae) from the Bohai Sea are described.Dorylaimopsis rabalaisi n. sp. similar toD. punctata Ditlevsen, 1918 from which it can be separated by the small size, short and simple spicules without ventral apophyses or joint line.Dorylaimopsis turneri n. sp. differs from the closest speciesDorylaimopsis angelae (Inglis, 1967) by its higher values of de Man ratio ‘a’, much shorter spicules and higher ratio of gubernacular to spicules length. A key to species of the genus based on the key by Jensen (1979) is given. This project was supported by the National Science Foundation, No: 3860804     

Effects of fishes on the growth and survival of Chinese mitten crab,Eriocheir sinensis

This experiment on the effects of fishes (Pelteobagrus fulvidraco, Channa argus, Cyprinus carpio) on growth and survival of juvenile Chinese mitten crab,Eriocheir sinensis was conducted in cages placed in Bao'an Lake. T-tests showed that effects of fishes on the carapace width growth were not significant as compared with the control (no fish). But the effects of these fishes on crab survival were negative. The causes of negative effects of fishes on crab survival were simply discussed on the basis of food habits. This research was financially supported by the Department of Agriculture (96-008-02-03), P. R. China, and Chinese Academy of Sciences (Key Project: KZ951-A1-102).     

A NEW SPECIES OF THE GENUS NEROCILA （ISOPODA： CYMOTHOIDAE） FROM THE EAST CHINA SEA

A new species of cymothoid Isopeda, Nerocila donghaiensis, collected from the body of a sciaenid fish, Johnius sp., is described in the present paper. The new species can be distinguished from the other species of genus Nerocila by its endoped of uropods with deep notch, sharply serrated on the lateral margin and strongly serrated on the medial margin; antennule with 1 st article swollen; dactyli of pereopods Ⅰ, Ⅱ, Ⅳ, Ⅴ swollen.     

Screen and effect analysis of immunostimulants for sea cucumber, <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

Immunostimulants may improve disease resistance of aquaculture animals by promoting the nonspecific immunity response of the organisms. Five types of saccharides, including chitosan, yeast polysaccharide, burdock oligosaccharide, seaweed polysaccharide and lentinus edodes polysaccharide, were screened for potential use as immunostimulants by using spectrophotometry. The saccharides were injected into Apostichopus japonicus, a sea cucumber, and the lysozyme and superoxide dismutase (SOD) activities of the coelomic fluid and epidermal slime were monitored in six consecutive days. The results show that the lysozyme activity of the animal’s coelomic fluid was significantly stimulated on day 2, day 4 and day 6 after the injection of the saccharides (P<0.05). The effects of chitosan and yeast polysaccharide were the most notable. The lysozyme activity of the epidermal slime was significantly increased by chitosana, yeast polysaccharide, seaweed polysaccharide, and burdock oligosaccharide on day 1 and day 2 after the injection (P<0.05). The SOD activity of the coelomic fluid was significantly promoted by the saccharides on day 2 and day 4 post-injection (P<0.05), while the SOD activity of the epidermal slime increased on day 2. These findings indicate that chitosan and yeast polysaccharide are the most effective immunostimulants and potential healthy anti-disease feedstuff for A. japonicus. Supported by the National Key Technology Research and Development Program (863 Program, No. 2006BAD09A06) and the Special Fund of Chinese Central Government for Basic Scientific Research Operations in Commonweal Research Institutes (No. 02-2007B03)     

Fluctuation in <Emphasis Type="Italic">Ommastrephe bartrami</Emphasis> yield in the North Pacific

The fluctuation in Ommastrephe bartrami yield from 1995 to 2001 in the North Pacific was shown obvious, on which this study was conducted using data of sea surface temperature (SST), chlorophyll-a (chl-a) and statistical production. The study shows that, cool water and low food abundance caused by abnormal Kuroshio resulted in the reduction in abundance of O. bartrami, which was worsened by excessive catch and the unawareness to local fishery resources protection. Supported by National Key Project for Basic Research of China (2005CB422300)     

Serum immunoglobulin of the mandarin fish, Siniperca chuatsi with development of polyclonal antibody

Serum immunoglobulin from the mandarin fish, or the so-called Chinese perch,Siniperca chuatsi (Basilewsky), was successfully purified using affinity chromatography. Heavy and light chains were detected on electrophoresis gel, with molecular weights being estimated at 72 and 29 kDa, respectively. The tetrameric IgM ofS. chuatsi was calculated to be 808 kDa. The rabbit polyclonal antisera against the purifed immunoglobulin were developed and tested by Western blot analysis. The antisera reacted strongly with the heavy chains ofS. chuatsi immunoglobulin. Humoral immune responses of the mandarin fish can then be examined using the developed polyclonal antibody. Project No. 30025035 supported by NSFC, State Key Laboratory of Freshwater Ecology and Biotechnology (2000FB02), and the Chinese Academy of Sciences (KZ9511-B1-111).     

The effect of temperature on growth and energy budget of the polychaete, Neanthes japonica Izuka

Growth and energy budget of the polychaete, Neanthes japonica, at various temperatures (17, 20, 23, 26 and 29°) were investigated in this study. The growth, as indicated by final dry weight and specific growth rate (SGR), increased with increasing temperature, with the maximum level at 26°C, and then decreased significantly at 29°C. A similar trend was observed in feeding rate, food conversion efficiency (FCE) and apparent digestive rate (ADR). However, no significant differences were detected in ADR among all the temperature treatments. In the pattern of energy allocation, faeces energy was only a small component of energy budget and had little influence on the proportion of food energy allocated to growth. The metabolic energy accounted for a large portion of energy intake for each temperature treatment. The nitrogen excretion was appreciable with changing temperature. The two expenditure terms (respiration energy and excretion energy) in energy budget were the major factors influencing the proportion of food energy allocated to growth. These results revealed that temperature affected the growth of N. japonica mainly by influencing feeding rate and FCE. In addition, regression equations describing the relationship between feeding rate, faecal production, SGR, FCE and temperature were obtained. The optimum temperatures for feeding rate, FCE and SGR were estimated at 25.01°C, 24.24°C and 24.73°C, respectively, from the regression equations.     

ANALYSIS OF EVOLUFIONARY RELATIONSHIP BETWEEN ASTASIA LONGA AND EUGLENA GRACILIS BY USING RAPD TECHNIQUE AND CLADISTIC ANALYSIS

Although both Astasia longa and Euglena gracdlis belong to different genera, they share many morphological characters except that A. longa has no chloroplast. In the 1940‘ s, on the basis of the finding that in darkness or upon addition of scrne chemicals, E. gmcilis would fade reversibly or irreversibly, scrne scholars hypothesised that A. longa evolved from E. gracilis by losing chloroplast.The authors‘ use of RAPD and cladistic analyses in a study on the evolutionary relationship between A .longa and E. gmcilis showed that the A. longa ‘ s relationship with E. gmcilis was closer than that with other green euglenoids. This proves the hypothesis that A. longa evolved from E. gmcills is reasonable. The results d this study suggest that saprophytic colorless euglenoids were transformed fromgreen euglenoids by losing their choroplasts.     

Effect of dietary phosphorus on the growth and body components of juvenile Synechogobius hasta

The effect of dietary phosphorus on the growth and body components of juvenile Synechogobius hasta was determined. Different percentages of dietary phosphorus (0.63, 0.77, 0.93, 1.06, 1.22 and 1.36) were tested by feeding the fish (body weight, 15.81 g ± 0.32 g; 20 individuals each group; 3 groups each percentage) at a surplus of 5%–10% above satiation for 35 d. Dietary phosphorus did not significantly affect the specific growth rate, feed intake, feed conversion ratio and protein efficiency rate. Nitrogen retention was found to be the highest in fish fed the diet containing 1.06% of phosphorus; however, this was not significantly different from other diets. Fish fed the diet containing 0.93% of phosphorus showed the highest phosphorus retention; similar phosphorus retention rates were found in fish fed the diets containing 0.77% and 1.06% of phosphorus. Fish fed the diet containing the highest percentage of dietary phosphorus were found to contain the least whole body lipid, lower than fish fed other diets (P<0.05). The protein content increased from 18.59% to 19.55% (although not significant) with the decrease of body lipid content (P>0.05). The contents of the whole body ash, whole body phosphorus and vertebrae phosphorus increased with dietary phosphorus percentage up to 1.06 (P<0.05), reaching a plateau after that. Dietary phosphorus did not significantly influence the muscle components (protein, lipid and moisture). Condition factor and hepatosomatic index were the highest in fish fed the diet containing 0.63% of dietary phosphorus; however, this was not significantly different from those of other diets. The second-order polynomial regression of phosphorus retention against dietary phosphorus identified a breakpoint at 0.88% of dietary phosphorus. However, the dietary requirement of phosphorus for maintaining maximum phosphorus storage determined by broken-line analysis of the contents of whole body phosphorus, and ash and vertebrae phosphorus was 1.06% of the diet.     

GENETIC DIVERSITY OF THE WILD AND REARED PSEUDOSCIAENA CROCEA

The genetic diversity of both wild and reared Pseudosciaena crocea (Richardson) col-lected from Guan-Jing-Yang in Ningde, China in May 1999 was investigated by random amplified poly-morphic DNA (RAPD) in the present study. The polymorphism and mean difference of the wild popula-tion as revealed by RAPD were 18.9% and 0.0960 respectively, and those of the reared stocks were rel-atively lower, with 16.7% in polymorphism and 0.0747 in mean difference. The genetic distance be-tween the two stocks was 0.0041. From the comprehensive investigation, the main reasons for the loss of genetic diversity were probably overilshing, small number of parents as broodstocks and the debatable arti-ficial ranching. Results from this study also showed that the large yellow croaker populations distributed along Fujian coastal waters including Guan-Jing-Yang still potentially wide genetic variability. It is sug-gested that genetic management and prevention should be scientifically conducted in order to maintain and improve the genetic diversity of the P. crocea population.