线纹尖塘鳢的形态生物学与核型

This paper reports the morphological character and karyotype of Oxyeleotris lineolatus. Oxyeleotris lineolatus is native in Australia and call ed sleepy cod. It belongs to Oxyeleotris, Eleotridae, Gobioidei, Perciformes in taxonomy. Recently, it was introduced to China and local people were not familiar with it. So we carried out this study. 30 individuals have been observed and some data were recorded. It has a large mouth that is in front and up. The mandible stands out and is longer than the up jaw. There are many rows of thin teeth in up and down jaws. The pelvie fins are located in chest and pectoral fins are large and fanlike. There are two dorsal fins. The tail fin is circular. Gill rakers are sparse and the number of gill rakers is 8-12+3-4. The gas bladder belongs to physoclistaus and its stomach is strong and I-like. The intestine is thick and short and no pyloric caecas. The length of the digestive path is 48.1％-80.3% of the length of body. Its liver has two lobes and the liver weight is 4.1%-7.2% of the body weight. The digestive organs characters are same as the trait of flesheater fish. Its scale belongs to ctenoid scale and its body surface shows several long lines. There is not lateral line in the body. The number of vertebra is 26-27 and it has 10-11 pairs rib. The number of diploid chromosome is 2n=46 and the karyotype formula is 2sm+8st+36t，NF=48. The relative length of chromosome is from 1.37% to 3.48% and it is continuity. No strange size chromosomes relation to sex was observed. The karyotype of Oxyeleotris lineolatus is similar to that of Oxyeletris marmoratus Bleeker from South East Asia and both of them belong to Oxyeleotri. It testifies the correctness of traditional classification on cytology.     

转Hu-IFN-α基因草鱼雌核发育F1的遗传配型分析

The Hu-IFN-α gene, which was transducted into downstream promoter of β-actin gene of common carp (Cyprinus carpio), was recombined by DNA recombination technology. These recombined genes were injected into 1-2 cell fertilized eggs of grass carp (Ctenophatyngodon idellus) by microinjection technology, we gained transgenetic fish by molecular detection methods. In order to analyse the genetic expression of tranHu-IFN-α gene gynogensis F₁, which male individualization were gained by raising methyltestosterone, molecular genetic marker technology was used. In our research, 30 random primers were picked out from 48 and were used into RAPD-PCR, the result indicated that 1 169 clear, steady and repeated DNA finger printing bands were achieved. On the basis of gentic distance matrix among tranHu-IFN-α gene gynogenesis F₁ group, the genetic relationship of gynogenesis F₁ were analysed by UPGMA, the results showed the genetic patterns are close between the 3# male gynogenesis F₁ and the the 23# female of gynogenesis F₁, 5# and 27#, 2# and 28#, 2# and 30#. The data indicated that these group could be served as parent of tranHu-IFN-α grass carp (Ctenophatyngodon idellus) pure line.     

细角螺的繁殖生态条件及繁殖习性

Hemifusus ternatanus(Gmelin), a gastropoda living in the deep water areas of undertide,as well as a delicious seafood, was distributed mainly in Chinese southeast seas and Japanese seas near the coast. In this experiment Hemifusus ternatanus' breeding ecological conditions and its propagation habit were studied in order to provide an experimental foundation for the artificial breeding. The brood stocks of test animals collected from the waters in Taiwan Strait, and then were examinated their growing temperature， salinity, feeding and propagation habit in the laboratory. The re sults showed that the growing temperature for Hemifusus ternatanus ranges from 16 ℃ to 32 ℃, with an optimum temperature between 20 ℃ to 28 ℃ and between 23 ℃ and 28 ℃ for breeding. The optimum growth salinity was 18.3-32.3 though it much adapts to salinity from 13 to 35. They prefer to feed on bivalves particularly those with thin shells and byssusless. Hemifusus ternatanus is dioecious and fertilization finishes inside the body. It was called as egg vesicle procreation that the whole stages of embryo growth were developed within egg vesicle. Larva forms as soon as it leaves egg vesicle, which is called direct occurrence type. The larva became the juvenile after metamorphosis during 20-25 days development. It was first to obtained 233 juveniles with shell height 32-45 mm by an artificial breeding method.     

我国赤潮频发现象分析与海藻栽培生物修复作用

In this paper, the history, main events and present status of red tide (HAB, harmful algal blooms) along China coast in recent years were reviewed and presented. It showed that the HAB's frequency and scale, number of HAB spec ies, percentage of toxic HAB events and the degree of damages to marine environment and economy have sharply increased in China since 1960's. Eutrophication was key factor for high occurrence of red tide. In this paper, main causes of frequent HAB occurrence along China coast was discussed. Many factors might influence the occurrence of red tide, which included weather, climate, coastal current, tidal current, water temperature, salinity, hydrodynamic and nutrient conditions, trace metals and the variation of biological environment. Numerous evidences from all over the world revealed the linkage between the increases in nutrient loading and the occurrences of high biomass blooms. Eutrophication was one of the important causes that involved in high occurrence of HAB. The main sources of nutrients potentially stimulating HABs included terrestrial runoff, aquaculture selfpollution, atmospheric deposition, sea projects and other pollution events in the ocean. Studies showed that the input from land contaminations and the selfpollution of marine aquaculture accelerated eutrophication in coastal waters and were also important impact factors on red tide. Researches suggested that nutrient composition could affect the species composition of phytoplankton as well as the development of some HABs. The changes in nutrient supply ratios, primarily N∶P, often resulted in shifts in red tide species composition. The correlation between cysts and formation of HAB was discussed from the viewpoi nt of transformation of cyst and vegetative cell, the effects of trace elements and other organic substances on the occurrence of HAB were presented also. It indicated that the nutrient control could be an effective way to reduce the risk of red tide occurrence. Seaweed would play an important role for decreasing marine eutrophication. Among the different methods of red tide controlling studied, seaweed biomass has received much attention due to the cost saving, low sensitivity to environmental and impurity factors, the possible contaminant recovery from the biomaterial and its elevated adsorption capacity. Cultivated seaweeds have very high rates of productivity higher than that of seaweed in its natural habits and grow well in water bodies with higher nitrogen and other nutrients. Seaweeds are able to absorb large quantities of nitrogen, phosphorus and carbon dioxide, produce large quanti ties of oxygen, and have excellent effect on decreasing eutrophication. Large amounts of C, N and P are accumulated into seaweed tissues as they accumulate considerable biomass over a period of months or years depending on the cultivation season. When seaweeds are harvested, nutrients are removed from the sea area. An investigation was carried out for inorganic nitrogen and inorganic phosphorus concentration at Lusi Coast, Qidong County, Jiangsu Province in China, where there were about 270 hm2 for Porphyra yezoensis cultivation with eutrophic sea water in recent years. While during Porphyra yezoensis cultivation, from Sep 2003 to May 2004, the concentration of ammonium nitrogen declined form 0.511-0.778 mg·L^-1 to 0.006-0.057 mg·L^-1, nitrite nitrogen concentration declined from 0.010-0.040 mg·L^-1 to 0.001-0.009 mg·L^-1, and nitrate nitrogen concentration declined from 0.466-0.549 mg·L^-1 to 0.286-0.0568 mg·L^-1, the average concentration of inorganic phosphorus declined from 0.024 mg·L^-1 to 0.019 mg·L^-1. Furthermore, during five hours, the concentration of ammonium nitrogen in the seawater declined form 220.88 μmol·L^-1 to 8.59 μmol·L^-1 by cultivated Gracilaria lemanaiformis, and the concentration of ammonium nitrogen declined form 213.84 μmol·L^-1 to zero by cultivated Enteromorpha clathrata. Other bioremediation mechanisms of seaweed inhibiting the red tide microalgae such as nutrients competition and allelopathic effects were also discussed.     

绿色微囊藻等不同食物对大型生长和脂类成分的影响

The growth and reproduction of Daphnia magna fed with Microcystis viridis and Chlorella spp respectively and 20% fish oil + 80% yeast and yeast were studied. The intrinsic rate of natural increase of Daphnia magna (rm) was 0.243 fed with Microcystis viridis, 0.301 with Chlorella spp., 0.244 with 20% fish oil + 80% yeast and 0.193 with yeast. The result showed that：Daphnia magna fed with Microcystis viridis had lower growth rates than that fed with Chlorella spp. an d 20% fish oil + 80% yeast which both had full fatty acids. And the Daphnia magna fed with yeast was the lowest. So the fatty acids composition of diet may affect the growth of Daphnia magna. This paper further examined total lipids and main HUFA (EPA and DHA) compositions of the continuous three generations of Daphnia magna (in order to get rid of t he effects of former diet) fed with different of above diets (except the yeast). The result showed that:the three generations of the D. magna fed with Microcystis viridis had the lowest total lipids and the percentages of the HUFA (EPA, and no DHA) have a significant decrease and get the lowest EPA in the final experiment because of the very low HUFA especially EPA and DHA in Microcystis viridis. So the results indicate that Microcystis viridis with low HUFA made the lower growth rate, spawn ing rate and hatching rate of Daphnia magna.     

鳗源嗜水气单胞菌主要外膜蛋白基因克隆及其表达

A pair of primers were designed according to the published nucleotide sequence of a putative outer membrane protein gene (omp) of Aeromonas hydrophila . With the specific primers, a target fragment about 1.1 kb was amplified from Aeromonas hydrophila ML316 via PCR .The target fragment was inserted into the linearized pGEM-T easy vector. After enzyme restriction and sequencing analysis,the nucleotide data had been further analyzed by DNAman and ClutalW software. The analysis results showed that the cloned DNA fragment had a longest open reading frame (ORF) of 1035 nt,it predicted to be encoded a 344 aa protein with the molecular weight of 36 kD. Hydrophobicity analysis suggested that the protein was highly hydrophilic, especialy at the first 24 aminoacid, this region could function as signal peptide. The homologious comparison proved the cloned gene had 96% homology to the sequence of the omp gene, and the alignment of the amino acid sequence was 98% . The recombinant plasmid was constructed with the target gene and the expressing vector pGEX-4T-1 and then was transformed into E. coli BL21（DE3）by BamH and Sal I .The fusion protein was expressed under the IPTG inducing condition,and exhibited about 62 kD in size,very close to the predicted molecular weight of GSTMOMP, furthermore,the fusion protein was specifically recognized by antiserum which raised against the major outer membrane protein of AHML316. Considering all these together, it proved that the cloned gene represented the major outer membrane protein gene of AHML316, and the expressed gene products shared identical antigenicity with the natural main outer membrane protein,and also provided technical support for developing an advanced gene engineering vaccine against Aeromonas hydrophila.     

鱼类几种新型免疫因子的研究进展

Cytokines are low molecular weight proteins that serve as chemical messengers within the innate and adaptive immune systems. To date, great progresses have been made in fish cytokine researches. A number of cytokine genes have been cloned and sequenced in fish. This review will focus on a number of novel immune-related cytokines including interleukin, interferon, interferon regulatory factors, Myxovirus resistance proteins, transforming growth factor, tumor necrosis factor, chemokines (CC and CXC chemokines), NK dell enhancement factor, MHCⅠ, MHCⅡ and some of their receptors, which have been identified in many fish species recently. Their genes and molecular structures are clarified. These cytokines are evolutionary well conserved. They share high identities at both the nucleotide and amino acid levels with the high vertebrate cytokines, and maintain characteristic structural motifs of those higher vertebrates. The function of some cytokine genes are analyzed in conventional manner by production of recombinant molecules. Several fish cytokines have been identified based on functional similarity to, or cross-reactivity with, mammalian cytokines. Moreover, molecular techniques, such as suppression subtractive hybridization, PCR and cDNA library screening, have recently enabled the identification of fish cytokine genes. Because of fish phylogenetic position and the fact that their immune systems have not been elaborated to the extent seen in mammals, progresses in this field will deepen our understanding of the molecular origins of cytokine genes and extend our knowledge on their mechanisms conferring disease resistance and the recombinant cytokines to control fish diseases.     

Accumulation of waterborne selenium by rainbow trout (Salmo gairdneri), eggs,fry and juveniles

The effect of waterborne selenite levels on selenium accumulated by different developmental stages of rainbow trout (Salmo gairdneri) was studied using⁷⁵SeO ₃ ⁼ as a tracer. All stages readily accumulated selenium at both high and low concentrations, but the rate of accumulation increased as the trout developed from the egg to the juvenile feeding stage. The low rate of selenium accumulation by embryos seemed to be related more to a lack of gills than to the presence of a chorion. The bioconcentration factor (the ratio of tissue-to-water concentrations) declined in all groups with increased waterborne selenium levels; accumulation rates appeared limited by cell permeability. At low levels of waterborne selenium (0.4μg/l), the total accumulated was small relative to existing body burdens and unlikely to contribute significantly to the nutritional requirement for selenium. High levels (45.6μg/l), however, caused considerable selenium accumulation and may be sufficient to overcome the effects of low dietary selenium.     

Insulin-like growth factor I of Japanese eel, Anguilla japonica: cDNA cloning, tissue distribution, and expression after treatment with growth hormone and seawater acclimation

In an attempt to understand growth regulation in the Japanese eel, Anguilla japonica, we cloned insulin-like growth factor-I (IGF-I) cDNAs and examined their mRNA expression in several tissues. Two eel IGF-I (eIGF-I) cDNAs encoding preprohormones, eIGF-I-Ea1and eIGF-I-Ea2, were cloned from the liver by polymerase chain reaction (PCR). The preproIGF-Is were identical in signal peptide and mature IGF-I, but different in the E domain—eIGF-I-Ea2 mRNA was 36 bp longer than eIGF-I-Ea1 mRNA. Eel IGF-I was 83–94% identical with that of teleosts, 71% identical with that of dogfish, 87% identical with that of bullfrog and chicken, and 83% identical with that of humans. In both males and females the highest eIGF-I-Ea1 mRNA levels were observed in the liver, with detectable levels also found in the gills, heart, stomach, spleen, kidney, intestine, swim-bladder, muscle, and gonads. eIGF-I-Ea1 mRNA levels in the liver were higher in females than in males whereas in the intestine they were lower than in males. eIGF-I-Ea2 mRNA was detected in all the tissues examined and at similar levels in males and females. In this experiment higher eIGF-I-Ea1 mRNA levels were observed in the liver of larger glass eels than in those of smaller fish. eIGF-I-Ea2 mRNA levels were also higher in larger eels, although they were lower than IGF-I-Ea1 mRNA levels. Both eIGF-I mRNA levels in liver were positively correlated with the body size of the␣glass eels. Intraperitoneal injection of recombinant eel GH (reGH), 0.25 μg g⁻¹ body weight, into glass eels resulted in a significant increase in both eIGF-I mRNAs in the liver 1 day after injection compared with control fish, but no elevation was observed 2 days after injection. Incubation of liver slices with reGH at concentrations of 10, 100, and 1,000 ng mL⁻¹ for 24 h resulted in a significant concentration-dependent increase in the levels of both eIGF-I mRNAs. Higher levels of eIGF-I-Ea1 and Ea2 mRNA were observed in the gills ofseawater-reared eels than in those of freshwater-reared fish, but no differenceswere observed in the whole kidney. These results suggest that IGF-I is involved in the regulation of somatic growth and also in adaptation of the Japanese eel to seawater.     

Emergence of pathogenic betanodaviruses belonging to the SJNNV genogroup in farmed fish species from the Iberian Peninsula

Betanodaviruses are the causative agents of viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) in cultured marine fish. Based on the RNA2 gene fish nodaviruses have been traditionally classified into four different genotypes and recently a fifth genotype has been proposed. This study presents sequencing data of 24 new nodaviruses obtained from three different fish species: sea bass, Dicentrarchux labrax (L.), sea bream, Sparus aurata L., and Senegalese sole, Solea senegalensis Kaup, cultured in the Iberian Peninsula (Spain and Portugal). Sequence analysis was performed on the T4 region (388 nt) of the coat protein gene. In addition, phylogenetic analysis, according to maximum parsimony and neighbour-joining methods, was performed using these sequences and other nucleotide sequences available in the databases or in the literature. Results obtained indicate that all these new nodaviruses should be classified into the striped jack nervous necrosis virus (SJNNV) genotype. This finding suggests that SJNNV genotype is emerging in the Iberian Peninsula and could easily spread throughout the Mediterranean, representing a serious threat to the fish farming industry.     

Mass mortalities associated with viral nervous necrosis (VNN) disease in two species of hatchery-reared grouper, Epinephelus fuscogutatus and Epinephelus akaara (Temminck & Schlegel)

Mass mortalities of hatchery-reared juvenile groupers have occurred in southern Taiwan. The diseased fish swam in a darting, corkscrew fashion. Light microscopy revealed vacuolation in the brain tissue. Electron microscopy showed numerous non-enveloped, cytoplasmic viral particles (20–25 nm in diameter) in the brain cells, and many virions were enclosed in the membrane-bound organelles of the cells. Two structural proteins of the purified grouper virus, with molecular weights of 44 and 43 kDa, were revealed by SDS-PAGE. Moreover, the results of RT-PCR and nested PCR diagnosis using primers specific to the T2 and T4 target segments of striped jack nervous necrosis virus (SJNNV) RNA2 genes suggest that this virus is a fish nodavirus, and is designated as GNNV 9410 strain (grouper nervous necrosis virus strain 9410). This is the first case report of viral nervous necrosis among marine fish in Taiwan.     

Complete sequencing of Tunisian redspotted grouper nervous necrosis virus betanodavirus capsid gene and RNA‐dependent RNA polymerase gene

Finfish nodaviruses (betanodaviruses) can cause highly destructive infections in numerous species of farmed marine fish larvae and juveniles worldwide. The betanodavirus genome consists of two single‐stranded positive‐sense RNA molecules (RNA1 and RNA2). The virus can be classified into four genotypes based on the partial sequences of the coat protein (CP) gene (T2 and T4 regions). Currently, genomic sequence information for RNA1 regions of RNA2 outside of T2 and T4 is less well documented. This study reports on the characterization of the full RNA2 sequence of a Tunisian betanodavirus with a length of 1433 nt, containing a 339 amino acid open‐reading frame encoding the CP, and typing to the redspotted grouper nervous necrosis virus Ia genotype following phylogenetic analysis. The homology of the capsid protein to other betanodaviruses or alphanodaviruses was compared. In addition, a full length RNA1 sequence of 3104 nt encoding a 982 amino acid RNA‐dependent RNA polymerase was obtained.     

Pathogenicity of the causative agent of viral nervous necrosis disease in striped jack, Pseudocaranx dentex (Bloch & Schneider)

Abstract. The pathogenicity of the agent causing viral nervous necrosis (VNN) of striped jack, Pseudocaranx dentex (Bloch & Schneider), was examined in striped jack and other selected marine fish species. Fish were exposed to purified striped jack nervous necrosis virus (SJNNV) (0·1–100 ng ml⁻¹) or homogenates of diseased striped jack larvae. Striped jack larvae (3·5 and 4·4 mm total length) were susceptible to the virus, but juveniles (78 mm) were not. The viral antigens were detected by indirect ELISA and the characteristic pathological changes, i.e. vacuolation in the retina and brain, were reproduced in the affected larvae. The infection was also established in healthy larvae by cohabitation with the diseased larvae. Larvae of red sea bream, Pagrus major Temminck & Schlegel, yellowtail, Seriola quinqueradiata Temminck & Schlegel, and goldstriped amberjack, Seriola lalandi Valenciennes, were not susceptible to SJNNV.     

鱼类神经坏死病毒研究进展与发展趋势

神经坏死病毒(nervous necrosis virus,NNV)是一种世界范围内流行、严重危害多种海水和淡水鱼类的传染性病原。NNV为单一正链、2节段RNA病毒,基因组由RNA1(3.1 kb)和RNA2(1.4 kb)组成。在病毒复制过程中,会合成亚基因组RNA3。RNA1编码RNA聚合酶。RNA2编码衣壳蛋白,为病毒的唯一结构蛋白。RNA3编码B1和B2两种非结构蛋白。根据病毒衣壳蛋白的基因序列,神经坏死病毒可以分成4种基因型,分别为拟鲹、红鳍东方鲀、条斑星鲽和赤点石斑神经坏死病毒基因型。但是,目前只发现A、B、C三种病毒血清型,A对应拟鲹神经坏死病毒基因型,B对应红鳍东方鲀神经坏死病毒基因型、C对应条斑星鲽神经坏死病毒和赤点石斑神经坏死病毒基因型。病毒存在垂直和水平两种传播途径,而且广泛分布于养殖和野生鱼类中。阻断病毒在野生与养殖鱼类之间的传播和开展新型鱼类疫苗研发是将来研究趋势。     

Susceptibility of cultured juveniles of several marine fish to the sevenband grouper nervous necrosis virus

Piscine nodaviruses (betanodaviruses) have been tentatively divided into four genotypes (SJNNV, RGNNV, TPNNV and BFNNV) and it is suggested that host specificity is different among these genotypes. In the present study, a betanodavirus [sevenband grouper nervous necrosis virus (SGNNV)] belonging to the redspotted grouper nervous necrosis virus (RGNNV) genotype, to which most betanodaviruses from warm water fish are identified, was evaluated for its pathogenicity to hatchery-reared juveniles of several marine fish species. When challenged with the virus by a bath method (10(5.1) TCID50 mL(-1)), sevenband grouper, Epinephelus septemfasciatus, Japanese flounder, Paralichthys olivaceus, and tiger puffer, Takifugu rubripes, displayed behavioural abnormalities and mortalities with distinct histopathological signs of viral nervous necrosis and heavily immunostained cells were observed in the central nervous tissues and retina. Bath-challenged rock fish, Sebastiscus marmoratus, and a hybrid of sevenband grouper and kelp grouper, E. moara, did not display any behavioural abnormality or mortality during the experimental period, although many fish showed slight signs of viral infection in nerve cells. Kelp grouper and red sea bream, Pagrus major, showed no behavioural abnormality, mortality or immunohistopathological changes after the virus challenge. These results are, in part, consistent with the natural host range of RGNNV, indicating the complexity in the host specificity of betanodaviruses.     

Characterization of nodavirus and viral encephalopathy and retinopathy in farmed turbot, Scophthalmus maximus (L.)

An outbreak of nodavirus infection in turbot larvae is described with respect to histopathology, immunohistochemistry, cell culture cultivation, RT-PCR amplification and sequence analysis of the capsid protein gene RNA2. Affected turbot developed classical signs of viral encephalopathy and retinopathy (VER) with abnormal swimming behaviour and high mortality levels. In the acute stage of infection, light microscopy revealed vacuolation of the central nervous system (CNS), with positive immunohistochemical staining for nodavirus. Later in the infection, CNS lesions appeared more chronic and contained clusters of cells immunopositive for nodavirus. Bacterial overgrowth in the intestines of the fish may have provoked or influenced the course of the nodavirus infection. We were unable to propagate the virus in cell culture. While RT-PCR using primers designed to detect Atlantic halibut nodavirus gave negative results, further testing with primers complementary to a more conserved region of RNA2 resulted in amplification of a product of the expected size. The entire RNA2 segment was cloned and sequenced. Sequence alignment showed that the turbot nodavirus (TNV) was different from previously described fish nodaviruses. In addition, phylogenetic analysis based on an 823 nt region of the sequence indicated that TNV clustered outside the four established fish nodavirus genotypes, suggesting a fifth genotype within the betanodaviruses.