短期饥饿胁迫下(鱼免)鱼(Miichthys miiuy)早期生活阶段的生长及消化酶活性研究

采用实验生态学方法,研究了饥饿胁迫对(鱼免)鱼(1—7日龄前期仔鱼,26—35日龄后期仔鱼,42—53日龄稚鱼)的生长、存活及消化酶(淀粉酶、脂肪酶、胰蛋白酶)活性的变化。结果表明,短期饥饿(2天)会显著降低前期仔鱼生长及消化酶活性;在后期仔鱼和稚鱼中,产生这种影响的饥饿时间分别为饥饿后3和6天,表明(鱼免)鱼仔、稚鱼的生长与消化酶活性对饥饿的忍受能力随发育生长而增强。恢复摄食5天后,饥饿4天的后期仔鱼和饥饿6天的稚鱼消化酶活性可达到正常水平,但饥饿6天的后期仔鱼的消化酶活性不能从饥饿损伤中恢复。饥饿4—6天的后期仔鱼和稚鱼的生长在恢复摄食后均未达到正常投喂组个体的生长水平,表明恢复摄食后其生长恢复过程滞后于消化酶活性。(鱼免)鱼仔、稚鱼的生长和消化酶活性可作为评价其遭受的饥饿程度和营养状况的指标。

防治鱼虾病害的新型药物──“病免”

防治鱼虾病害的新型药物──“病免”朱选才，金龙（上海市水产研究所，２００４３３）近几年，全国各地养殖的对虾和淡水鱼类相继流行暴发性病害，给水产养殖业带来了惨重损失。目前，鱼虾病害的种类较多，加之基层养殖单位、养殖专业户普遍缺乏科学的诊断手段，选用水产...

鮸鱼胚胎发育及其仔、稚、幼鱼形态特征和生活习性的初步研究

作者对（鱼免）鱼（Miichthys miiuy）的早期发育（从受精卵到45日龄幼鱼）形态特征及生态习性进行了描述。（鱼免）鱼的受精卵在水温21．4℃～22．O℃，盐度25．7条件下，经过29h30rain仔鱼孵出。从受精卵到仔鱼出膜可分为卵裂期、囊胚期、原肠期、胚体形成期等4个主要时期，然后进入胚后发育阶段。孵出后的3耳龄仔鱼已能在水体中平游，极少数个体可开口撮食轮虫。4日龄仔鱼全部开口摄食，并对光反应敏感，出现明显的集群现象。22日龄的后期仔鱼各鳍鳍条发育形成，鳞片开始出现，进入到稚鱼期。39日龄后，挽鱼全身披满细小的鳞片，全部分布于育苗池的底部，开始进入到幼鱼发育阶段。根据仔、稚、幼鱼形态特征和生活习性对（鱼免）鱼的分类进行了探讨。

鮸鱼亲鱼培育及其人工繁殖的研究

采用自然苗培育成的1龄(鱼免)鱼,通过'春肥、夏育、秋繁、冬保'8字方针培育成亲鱼,性成熟率达到95%.经LHRH-A3 0.8～1.8 μg /kg催产并放人产卵池自行产卵,获产率达到96.83%.获受精卵650万余粒,受精率达到52%.受精后21.8 h孵化出鱼苗,孵化率达到70%.

短期饥饿胁迫对幼鱼的生长、生化组成及其消化酶活力的影响

以(鱼免)(Miichthys miiuy)幼鱼为实验动物,研究短期饥饿对其生长、生化组成及消化酶活力的影响实验.结果表明,随着饥饿时间的延长,(鱼免)幼鱼全长、体质量、粗蛋白、粗脂肪、糖类的含量和能值均逐渐减少.实验期间粗蛋白、粗脂肪、糖类的质量分数和能值的变化与饥饿时间呈负相关关系(P＜0.01);粗蛋白、粗脂肪和糖类的质量分数变化与能值呈正相关关系(P＜0.01);分析认为,蛋白质是影响饥饿期间(鱼免)幼鱼肌肉能值变化的主要含能物质.粗蛋白、粗脂肪和糖类的相对损失率从大到小依次为粗脂肪、糖类、粗蛋白,且粗脂肪降幅高峰的出现时段早于糖类.实验期间蛋白酶呈现出先降低后升高的趋势;脂肪酶和淀粉酶随着饥饿时间的延长而逐渐降低.

大黄鱼和(鱼免)的远缘杂交初步研究

进行了大黄鱼和(鱼免)的远缘杂交试验,结果表明:(鱼免)(♀)×大黄鱼(♂)杂交组受精率为52%,孵化率为71%,但孵化后未能开口摄食,15天内杂交苗逐渐全部死亡。大黄鱼(♀)×(鱼免)(♂)杂交组受精率为75%,孵化率为83%,度过了开口摄食轮虫和开始摄食桡足类2个死亡高峰期,存活率仅为1.2%。并分析了(鱼免)(♀)×大黄鱼(♂)的杂交子代不能存活的原因,以及大黄鱼(♀)×(鱼免)(♂)的杂交子代的遗传本质和成活率低的原因。

IMMUNE FUNCTION OF IMMATURE RAINBOW TROUT ( Oncorhynchus mykiss ) RELATED WITH PHOTOPERIOD

Radioimmunoassay,enzyme linked immunospot assay and enzyme immunoassay were used for the determinations of plasma steroid hormone's level,antibody producing cell's counting and IgM level in this study.The decreased number of antibody producing cells and low IgM levels were observed in sexual immature rainbow trout during the spawning season.These fish were reared under almost constant water temperature and natural photoperiod.Moreover,low IgM level was also observed in immature rainbow trout,which were reared under short photoperiod,and IgM level was not changed by treatment of testosterone.The results suggest that photoperoid may cause the changes in immune competence.It is possible that circadian rhythm accompanied with photoperiod may influence physiological function of fish,so that immune competence is changed.

Ontogeny of Immune Related Organs during Early Development of Carp (Cyprinus Carpio L.)

In this paper, ontogeny of immune-related organs during theearly development of carp was studied by histochemical technique-lcineBlue staining under acid conditions and PAS reaction(AB-PAS staining). Thekidney appeared one day before hatching, spleen, liver and pancreas emergedon the same day of hatching, and thymus was not found until the third day afterhatching. Ontogeny of these immune related organs of the carp in thisresearch is earlier than that reported by Botham.

Identification of zRAP55,a gene preponderantly expressed in StagesⅠandⅡ oocytes of zebrafish

In an in silico search for gonand specific expressed genes, we have identified zRAP55 which is enriched in the ovary of zebrafish . zRAP55 encodes a protein of 382 amino acids with a highly conserved Lsm domain. zRAP55 protein shares more than 56% identities with that of other vertebrate species. RT-PCR results show that it is predominantly expressed in the ovary. In situ hybridization and immunohistochemistry studies reveal that zRAP55 is ubiquitously dispersed throughout the cytoplasm of stages Ⅰ and Ⅱ oocytes, whereas no expression is observed in stages Ⅲ and Ⅳ oocytes. As an RNA associated protein, zRAP55 might function in the control of protein translation at the early stages of oogenesis in zebrafish.

Immune response,stress resistance and bacterial challenge in juvenile rainbow trouts Oncorhynchus mykiss fed diets containing chitosan-oligosaccharides

Effects of dietary supplementation of chitosan-oligosaccharides （COS） on the growth performance, immune response, stress resistance, and disease resistance of juvenile rainbow trout Oncorhynchus mykiss were studied. Four experimental diets containing 0, 20, 40, or 60 mg/kg COS （COSO, COS20, COS40, and COS60, respectively） were fed to juvenile rainbow trout （initial weight = 5.2 ± 0.3 g） for 8 weeks. By the end of the feeding trial, representative groups of fish from each dietary treatment were challenged with stressor （30 see air exposure） and pathogen exposure （intraperitoneal injection with Aeromonas hydrophila ）. Results showed that supplementation of COS in diets did not affect production performance and body composition of rainbow trout. However, fish fed the COS40 diet demonstrated improved phagocytic activities, respiratory burst activities and decreased serum cortisol level. Additionally, survival following A. hydrophila challenge was significant higher among fish fed the COS-supplemented feeds, although there was no difference based on the level of supplementation. The present study suggests that COS can be used as an immuno-stimulant in rainbow trout feeds

Histopathological Observation of Lymphocystis Disease and Lymphocystis Disease Virus (LCDV) Detection in Cultured Diseased Sebastes schlegeli

Lymphocystis nodules occurring in the cultured sting fish Sebastes schlegeli were observed under light and electron microscope. Lymphocystis disease virus (LCDV) in the tissues of diseased fish was detected with indirect immunofluorescence test (IFAT). Results showed that lymphocystis cells had overly irregular nuclei, basophilic intracytoplasmic inclusion bodies with virions budding from the surface, and hyaline capsules outside the cell membrane. Numerous virus particles about 200 nm in diameter scat- tered in the cytoplasm, electron-dense particles 70-80 nm in diameter filled in perinuclear cisterna, and membrane-enveloped parti- cles with electron-dense core of 70-80 nm appeared around cellular nucleus. IFAT using monoclonal antibody against LCDV from Paralichthys olivaceus revealed that specific green fluorescence was present in the cytoplasm of lymphocystis cells, epithelium of stomach, gill lamellae, and muscular fibers under epidermis of S. schlegeli, just as that in the cytoplasm of lymphocystis cells of P. olivaceus, suggesting the presence of LCDV in these tissues.

Simultaneous identification and quantification of tetrodotoxin in fresh pufferfish and pufferfish-based products using immunoaffinity columns and liquid chromatography/quadrupole-linear ion trap mass spectrometry

In this study, we established a comprehensive method for simultaneous identification and quantification of tetrodotoxin （TTX） in fresh pufferfish tissues and pufferfish-based products using liquid chromatography/quadrupole-linear ion trap mass spectrometry （LC-QqLIT-MS）. TTX was extracted by 1% acetic acid-methanol, and most of the lipids were then removed by freezing lipid precipitation, followed by purification and concentration using immunoaffinity columns （IACs）. Matrix effects were substantially reduced due to the high specificity of the IACs, and thus, background interference was avoided. Quantitation analysis was therefore performed using an external calibration curve with standards prepared in mobile phase. The method was evaluated by fortifying samples at 1, 10, and 100 ng/g, respectively, and the recoveries ranged from 75.8%--107%, with a relative standard deviation of less than 15%. The TTX calibration curves were linear over the range of 1-1 000 ~tg/L, with a detection limit of 0.3 ng/g and a quantification limit of 1 ng/g. Using this method, samples can be further analyzed using an information- dependent acquisition （IDA） experiment, in the positive mode, from a single liquid chromatography-tandem mass spectrometry injection, which can provide an extra level of confirmation by matching the full product ion spectra acquired for a standard sample with those from an enhanced product ion （EPI） library. The scheduled multiple reaction monitoring method enabled TTX to be screened for, and TTX was positively identified using the IDA and EPI spectra. This method was successfully applied to analyze a total of 206 samples of fresh pufferfish tissues and pufferfish-based products. The results from this study show that the proposed method can be used to quantify and identify TTX in a single run with excellent sensitivity and reproducibility, and is suitable for the analysis of complex matrix pufferfish samples.

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 of S. 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 of S. chuatsi immunoglobulin. Humoral immune responses of the mandarin fish can then be examined using the developed polyclonal antibody.

Detection of Grass Carp Hemorrhage Virus (GCHV) from Vietnam and Comparison with GCHV Strain from China

Grass carp plays an important role in small-scale aquaculture in Vietnam. However, a severe disease, known in Vietnam as "Red Spot Disease", is causing significant economic loss in grass carp aquaculture. In this study, the tissue samples isolated from the grass carp with Red Spot Disease in Vietnam are investigated and comparied with the control GCHV isolated in China by experimental infection, culture cell infection, serological cross reactivity, and RT-PCR amplification. Infected grass carp exhibits hemorrhage symptoms about 5 days after experimental injection with GCHV-V (Vietnam) strain. The symptoms and lethality induced by the GCHV-V strain are identical to that induced by the Chinese GCHV-9014 strain. The Chinese GCHV-873 strain induces typical cytopathogenic effects in 4 cell lines, such as CIK, CAB, FHM and GCO, from the 6 fish cell lines examined. No cytopathogenic effects are observed in all the 6 examined cell lines, including CAB, FHM, CIK, EPC, CCO and GCO, infected by the GCHV-V strain and GCHV-9014 strain. Immunodiffusion assays demonstrate an obvious cross-reactivity among three GCHV strains. Precipitin lines are clearly observed not only between the anti-GCHV-873 serum and the two strains GCHV-873 and GCHV-9014, but also between the anti-GCHV-873 serum and the GCHV-V strain. GCHV can be detected by immunodiffusion assays after three generations of blind propagations in the cell lines inoculated by GCHV-V strain. This implicates that GCHV-V viruses have been replicated and amplified despite there being no cytopathogenic effects observed in these examined cell lines. Three genome segments of GCHV, including S8, S9 and S10, are amplified by three sets of PCR primers designed according to the segment sequences published recently. The Q8fp and Q8rp primer set specific for genome segment S8 amplifies a 955 bp fragment from the extracted sample of diseased fish with Red Spot Disease, and the fragment size is identical to that amplified by the same primer set from control GCHV-873 strain. Simultaneously, the Q9fp and Q9rp primer set specific for genome segment S9 generates a same 635 bp product, and the Q10fp and Q10rp primer set specific for genome segment S10 produces a same 697 bp fragment from both template samples of diseased fish with Red Spot Disease and control GCHV-873 strain. The RT-PCR amplification and corresponding size comparison data indicate that the three GCHV-V genome segments extracted from the diseased grass carp with Red Spot Disease in Vietnam should be identical to that in control GCHV-873 strain from China. The data confirm that the causative agent of grass carp Red Spot Disease in Vietnam is a virus, and the virus is closely similar to GCHV strain in China.

Genes of the adaptive immune system are expressed early in zebrafish larval development following lipopolysaccharide stimulation

Information regarding immunocompetence of the adaptive immune system （AIS） in zebrafish Danio rerio remains limited. Here, we stimulated an immune response in fish embryos, larvae and adults using lipopolysaccharide （LPS） and measured the upregulation of a number of AIS-related genes （Rag2, AID, TCRAC, IgLC-1, mIg, slg, IgZ and DAB） 3 and 18 h later. We found that all of the genes evaluated were strongly induced following LPS stimulation, with most of them responding at 8 d post fertilization. This confirms that a functional adaptive immune response is present in D. rerio larvae, and provides a window for further functional analyses.

Expression of Outer Capsid Protein VP5 of Grass Carp Reovirus in E.coli and Analysis of its Immunogenicity

Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.

Gene cloning and induced expression pattern of IRF4 and IRF10 in the Asian swamp eel(Monopterus albus)

The Asian swamp eel （Monopterus albus） is one of the most economically important freshwater fish in East Asia, but data on the immune genes of M. albus are scarce compared to other commercially important fish. A better understanding of the eel＇s immune responses may help in developing strategies for disease management, potentially improving yields and mitigating losses. In mammals, interferon regulatory factors （IRFs） play a vital role in both the innate and adaptive immune system; though among teleosts IRF4 and IRFIO have seldom been studied. In this study, we characterized IRF4 and IRFIO from M. albus （malRF4 and malRFlO） and found that malRF4 cDNA consists of 1 716 nucleotides encoding a 451 amino acid （aa） protein, while malRFlO consists of 1 744 nucleotides including an open reading frame （ORF） of 1 236 nt encoding 411 aa. The malRFlO gene was constitutively expressed at high levels in a variety of tissues, while malRF4 showed a very limited expression pattern. Expression of malRF4 and malRFlO in head kidney, and spleen tissues was significantly up-regulated from 12 h to 48 h post-stimulation with polyinosinic： polycytidylic acid （poly I：C）, lipopolysaccharide （LPS） and a common pathogenic bacteria Aeromonas hydrophila. These results suggest that IRF4 and IRF10 play roles in immune responses to both viral and bacterial infections in M. albus.

ANNUAL CHANGES IN PLASMA LEVELS OF CORTISOL AND SEX STEROID HORMONES IN MALE RAINBOW TROUT, ONCORHYNCHUS MYKISS

The profiles of cortisol, testosterone, 11 ketotestosterone and 17α, 20β dihydroxy 4 pregnene 3 one in male rainbow trout reared under constant water temperature and natural photoperiod were determined by radioimmunoassay. Gonads of male rainbow trout reached maturity when the fish were two years old. Changes in the plasma levels of both sex steroid hormones and cortisol were closely related to the GSI. Plasma levels of testosterone, 11 ketotestosterone and 17α, 20β dihydroxy 4 pregnene 3 one showed a clear peak in the annual breeding season, when the GSI reached their maxima. Plasma cortisol levels also showed clearly seasonal changes in both two and three year old fish. The results suggest that the elevated plasma levels of cortisol may not just be due to stresses during the breeding season but have certain physiological functions in the reproduction of rainbow trout.

De novo characterization of Larimichthys crocea transcrip- tome for growth-/immune-related gene identification and massive microsatellite (SSR) marker development

The large yellow croaker, Larimichthys crocea is an important marine fish in China with a high economic value. In the last decade, the stock conservation and aquaculture industry of this species have been facing severe challenges because of wild population collapse and degeneration of important economic traits. However, genes contributing to growth and immunity in L. crocea have not been thoroughly analyzed, and available molecular markers are still not sufficient for genetic resource management and molecular selection. In this work, we sequenced the transcriptome in L. eroeea liver tissue with a Roche 454 sequencing platform and assembled the transcriptome into 93 801 transcripts. Of them, 38 856 transcripts were successfully annotated in nt, nr, Swiss-Prot, InterPro, COG, GO and KEGG databases. Based on the annotation information, 3 165 unigenes related to growth and immunity were identified. Additionally, a total of 6 391 simple sequence repeats （SSRs） were identified from the transcriptome, among which 4 498 SSRs had enough flanking regions to design primers for polymerase chain reactions （PCR）. To access the polymorphism of these markers, 30 primer pairs were randomly selected for PCR amplification and validation in 30 individuals, and 12 primer pairs （40.0%） exhibited obvious length polymorphisms. This work applied RNA-Seq to assemble and analyze a live transeriptome in L. crocea. With gene annotation and sequence information, genes related to growth and immunity were identified and massive SSR markers were developed, providing valuable genetic resources for future gene functional analysis and selective breeding ofL. eroeea.

Characterization and expression of sweetfish （Plecoglossus altivelis） cathepsin D

Cathepsin D （CTSD） is a lysosomal acidic endoproteinase that plays an important role in immune response, In this study, we obtained sweetfish （Plecoglossus altivelis） CTSD （PaCTSD） via de-novo transcriptome sequencing of sweetfish macrophages. The full length cDNA sequence of PaCTSD was 1955 bp encoding a propeptide of 397 amino acids. The deduced protein had a calculated molecular weight of 43.17x 103. Multiple alignment with other known CTSD amino acid sequences revealed amino acid conservation through the teleosts. Phylogenetic tree analysis showed that PaCTSD grouped tightly with other fish CTSD, and was close to that of Atlantic salmon and rainbow trout. Subsequently, PaCTSD was prokaryotically expressed and refolded by the urea gradient method on a nickel-nitrilotriacetic acid column. Enzyme activity analysis showed that PaCTSD exhibited pH-dependent proteolytic activity. Quantitative real-time PCR showed that PaCTSD mRNA was expressed in all detected tissues in healthy sweetfish. The highest expression was observed in the spleen and white blood cells, followed by liver, head-kidney, kidney, intestine, gill, and muscle. After Listonella anguillarum infection, PaCTSD transcripts were up-regulated significantly in liver, spleen, white blood cells, and head-kidney of sweetfish. In summary, PaCTSD has proteolytic activity and is closely involved in the immune response of sweetfish.