养殖大菱鲆溃疡症病原菌的分离鉴定及系统发育分析

2004年夏季山东海阳一带养殖大菱鲆(Scophthalmus maximusL.)暴发溃疡病。症状主要表现为体表病灶部位出血、肌肉溃烂、眼球凸出等,解剖可见鳃丝贫血,肝脏充血,肾脏和胆囊肿大,肠壁充血并呈透明状,从出现发病症状起,大约一周后开始出现死亡。从病鱼体表溃疡部位及内脏分离出优势菌并命名为H1,经人工感染证实H1即为引起本次养殖大菱鲆体表溃疡症的病原菌。对病原菌进行鉴定揭示该菌革兰氏染色阴性,菌体呈短杆状,极生单鞭毛。综合该菌在形态、生理生化、API20NE与API20E自动鉴定结果、16S rDNA同源性等方面的特性,确认H1为哈维氏弧菌(Vibrio harveyi),该菌对呋喃妥因、菌必治等多种抗生素敏感。哈维氏弧菌是海水养殖鱼类的常见致病菌,但作为养殖大菱鲆的病原菌属首次报道。     

不同大菱鲆(Scophthalmus maximus)个体肠道菌群结构差异研究

目的:肠道微生物能够帮助宿主完成多种生理生化功能,对宿主的健康生长也起到非常重要的作用。大菱鲆(Scophthalmus maximus)是我国北方最重要的海水养殖品种之一。然而,细菌性疾病的频发造成大规模的鱼类死亡。此外,许多大菱鲆致病菌也被证实是人类潜在的致病菌。因此鉴定成年养殖大菱鲆肠道中所包含的核心菌群,并筛选可能与大菱鲆或人类健康密切相关的细菌尤为重要。方法:构建三个大菱鲆个体肠道16S rRNA文库,利用限制性片段长度多态性分析(RFLP)及生物信息学技术研究三个大菱鲆个体肠道菌群结构及多样性。结果:共得到758个阳性克隆,16个OTU类型。分类分析显示,变形菌门在大菱鲆肠道中占优势地位。进一步将细菌按属来分类显示,弧菌属所占的比例最高,约为95.3%。韦恩图显示大菱鲆三个个体共有的OTU类型数量为3,分别占三个大菱鲆个体肠道文库中阳性克隆总数的94.9%、96.7%以及93.5%。分类结果显示它们分别属于条件致病菌哈氏弧菌、副溶血弧菌和创伤弧菌。结论:大菱鲆的核心肠道菌群为弧菌属,它的主要成员哈氏弧菌、副溶血弧菌和创伤弧菌对大菱鲆及人类的健康起到关键的作用。在水产养殖过程中,通过监控这些弧菌种类的浓度变化能够及时对养殖环境进行评价,并及时调整温度、水质等因素,这为预防大菱鲆细菌性疾病,保证人类健康提供重要的理论依据。     

Microarray-Based Identification of Differentially Expressed Genes in Families of Turbot (Scophthalmus maximus) After Infection with Viral Haemorrhagic Septicaemia Virus (VHSV)

Viral haemorrhagic septicaemia virus (VHSV) is one of the major threats to the development of the aquaculture industry worldwide. The present study was aimed to identify genes differentially expressed in several turbot (Scophthalmus maximus) families showing different mortality rates after VHSV. The expression analysis was conducted through genome-wide expression profiling with an oligo-microarray in the head kidney. A significant proportion of the variation in the gene expression profiles seemed to be explained by the genetic background, indicating that the mechanisms by which particular species and/or populations can resist a pathogen(s) are complex and multifactorial. Before the experimental infections, fish from resistant families (low mortality rates after VHSV infection) showed high expression of different antimicrobial peptides, suggesting that their pre-immune state may be stronger than fish of susceptible families (high mortality rates after VHSV infection). After infection, fish from both high- and low-mortality families showed an up-modulation of the interferon-induced Mx2 gene, the IL-8 gene and the VHSV-induced protein 5 gene compared with control groups. Low levels of several molecules secreted in the mucus were observed in high-mortality families, but different genes involved in viral entrance into target cells were down-regulated in low-mortality families. Moreover, these families also showed a strong down-modulation of marker genes related to VHSV target organs, including biochemical markers of renal dysfunction and myocardial injury. In general, the expression of different genes involved in the metabolism of sugars, lipids and proteins were decreased in both low- and high-mortality families after infection. The present study serves as an initial screen for genes of interest and provides an extensive overview of the genetic basis underlying the differences between families that are resistant or susceptible to VHSV infection.     

Design and Performance of a Turbot (Scophthalmus maximus) Oligo-microarray Based on ESTs from Immune Tissues

An expressed sequence tag database from immune tissues was used to design the first high-density turbot (Scophthalmus maximus) oligo-microarray with the aim of identifying candidate genes for tolerance to pathogens. Specific oligonucleotides (60 mers) were successfully designed for 2,716 out of 3,482 unique sequences of the database. An Agilent custom oligo-microarray 8 × 15 k (five replicates/gene; eight microarrays/slide) was constructed. The performance of the microarray and the sources of variation along microarray analysis were examined on spleen pools of controls and Aeromonas salmonicida-challenged fish at 3 days postinfection. Only 48 out of 2,716 probes did not show signal of hybridization on the 32 microarrays employed, thus demonstrating the consistency of the bioinformatic applications of our database. An asymmetric hierarchical design was employed to ascertain the noise associated with biological and technical (RNA extraction, labeling, hybridization, slide, and dye bias) factors using 1C and 2C labeling approaches. The high correlation coefficient between replicates at most factors tested demonstrated the high reproducibility of the signal. An analysis of random-effects variance revealed that technical variation was mostly negligible, and biological variation represented the main factor, even using pooled samples. One-color approach performed at least as well as 2C, suggesting their usefulness due to its higher design flexibility and lower cost. A relevant proportion of genes turn out to be differentially labeled depending on fluorophore, which alerts for the likely need of swapping replication in 2C experiments. A set of differentially expressed genes and enriched functions related to immune/defense response were detected at 3 days postchallenging.     

Mapping of DNA Sex-Specific Markers and Genes Related to Sex Differentiation in Turbot (Scophthalmus maximus)

Production of all-female populations in turbot can increase farmer's benefits since sexual dimorphism in growth in this species is among the highest within marine fish, turbot females reaching commercial size 3-6?months earlier than males. Puberty in males occurs earlier than in females, which additionally slows their growth. Thus, elucidating the mechanisms of sex determination and gonad differentiation is a relevant goal for turbot production. A ZZ/ZW sex determination mechanism has been suggested for this species, and four sex-related quantitative trait loci (QTL) were detected, the major one located in linkage group (LG) 5 and the three minor ones in LG6, LG8, and LG21. In the present work, we carried out a linkage analysis for several sex-related markers: (1) three anonymous sex-associated RAPD and (2) several candidate genes related to sex determination and gonad differentiation in other species (Sox3, Sox6, Sox8, Sox9, Sox17, Sox19, Amh, Dmrta2, Cyp19a, Cyp19b). We focused our attention on their co-localization with the major and minor sex-related QTL trying to approach to the master sex-determining gene of this species. Previously described growth-related QTL were also considered since the association observed between growth and sex determination in fish. Amh, Dmrta2, and one RAPD were located in LG5, while Sox9 and Sox17 (LG21), Cyp19b (LG6), and a second RAPD (LG8) co-mapped with suggestive sex-related QTL, thus supporting further analyses on these genes to elucidate the genetic basis of this relevant trait for turbot farming.     

High-Density Genetic Linkage Mapping in Turbot (Scophthalmus maximus L.) Based on SNP Markers and Major Sex- and Growth-Related Regions Detection

This paper describes the development of a high density consensus genetic linkage map of a turbot (Scophthalmus maximus L.) family composed of 149 mapping individuals using Single Nucleotide Polymorphisms (SNP) developed using the restriction-site associated DNA (RAD) sequencing technique with the restriction enzyme, PstI. A total of 6,647 SNPs were assigned to 22 linkage groups, which is equal to the number of chromosome pairs in turbot. For the first time, the average marker interval reached 0.3958 cM, which is equal to approximately 0.1203 Mb of the turbot genome. The observed 99.34% genome coverage indicates that the linkage map was genome-wide. A total of 220 Quantitative Traits Locus (QTLs) associated with two body length traits, two body weight traits in different growth periods and sex determination were detected with an LOD > 5.0 in 12 linkage groups (LGs), which explained the corresponding phenotypic variance (R²), ranging from 14.4–100%. Among them, 175 overlapped with linked SNPs, and the remaining 45 were located in regions between contiguous SNPs. According to the QTLs related to growth trait distribution and the changing of LGs during different growth periods, the growth traits are likely controlled by multi-SNPs distributed on several LGs; the effect of these SNPs changed during different growth periods. Most sex-related QTLs were detected at LG 21 with a linkage span of 70.882 cM. Additionally, a small number of QTLs with high feasibility and a narrow R² distribution were also observed on LG7 and LG14, suggesting that multi LGs or chromosomes might be involved in sex determination. High homology was recorded between LG21 in Cynoglossus semilaevis and turbot. This high-saturated turbot RAD-Seq linkage map is undoubtedly a promising platform for marker assisted selection (MAS) and flatfish genomics research.     

Cryopreservation of turbot (Scophthalmus maximus) spermatozoa

The aim of this study was to develop a method for cryopreserving turbot semen and to compare sperm motility characteristics, metabolic status and fertilization capacity of frozenthawed and fresh semen. The best results were obtained when spermatozoa were diluted at a 1:2 ratio with a modified Mounib extender, supplemented with 10% BSA and 10% DMSO. For freezing sperm samples, straws were placed at 6.5 cm above the surface of liquid nitrogen (LN) and plunged in LN. The straws were thawed in water bath at 30 degrees C for 5 sec. Use of this simple method resulted in a 60 to 80% reactivation rate of the thawed spermatozoa. Although the percentage of motile spermatozoa in the frozen-thawed semen samples was significantly lower than in fresh semen, spermatozoa velocity and respiratory rate remained unchanged. The process of cryopreservation significantly decreased intracellular ATP content. The fertilization rate of frozen-thawed spermatozoa was significantly lower than that of fresh spermatozoa, but it increased with sperm concentration.     

Identification of Microsatellite Repeats in Turbot (Scophthalmus maximus) and Dover Sole (Solea solea) using a RAPD-Based Technique: Characterization of Microsatellite Markers in Dover Sole

We have used a RAPD-based technique to identify several microsatellite repeats in turbot (Scophthalmus maximus) and Dover sole (Solea solea) and report the characterization of six novel polymorphic microsatellite markers for Dover sole. These are the first such markers to be developed for this flatfish species. They exhibit much higher levels of heterozygosity than those previously observed with allozyme loci and should prove useful in addressing population genetic questions as well as more fundamental aquaculture-related questions. Received ; accepted July 13, 1999.     

Seasonal Incidence of Autochthonous Antagonistic Roseobacter spp. and Vibrionaceae Strains in a Turbot Larva (Scophthalmus maximus) Rearing System

Bacteria inhibitory to fish larval pathogenic bacteria were isolated from two turbot larva rearing farms over a 1-year period. Samples were taken from the rearing site, e.g., tank walls, water, and feed for larvae, and bacteria with antagonistic activity against Vibrio anguillarum were isolated using a replica plating assay. Approximately 19,000 colonies were replica plated from marine agar plates, and 341 strains were isolated from colonies causing clearing zones in a layer of V. anguillarum. When tested in a well diffusion agar assay, 173 strains retained the antibacterial activity against V. anguillarum and Vibrio splendidus. Biochemical tests identified 132 strains as Roseobacter spp. and 31 as Vibrionaceae strains. Partial sequencing of the 16S rRNA gene of three strains confirmed the identification as Roseobacter gallaeciensis. Roseobacter spp. were especially isolated in the spring and early summer months. Subtyping of the 132 Roseobacter spp. strains by randomly amplified polymorphic DNA with two primers revealed that the strains formed a very homogeneous group. Hence, it appears that the same subtype was present at both fish farms and persisted during the 1-year survey. This indicates either a common, regular source of the subtype or the possibility that a particular subtype has established itself in some areas of the fish farm. Thirty-one antagonists were identified as Vibrio spp., and 18 of these were V. anguillarum but not serotype O1 or O2. Roseobacter spp. strains were, in particular, isolated from the larval tank walls, and it may be possible to establish an antagonistic, beneficial microflora in the rearing environment of turbot larvae and thereby limit survival of pathogenic bacteria.     

Hemoglobin Genotypes in Turbot (Scophthalmus maximus Rafinesque), Their Oxygen Affinity Properties and Relation With Growth

A growth trial lasting 15 months was conducted with 80 individually tagged turbot at four temperatures (10, 13, 16, and 19°C). Haemoglobins of the individually tagged fish were analysed by agar gel electrophoresis and the fish segregated into three genotypes named; Hb-I(1/1), Hb-I(1/2) and Hb-I(2/2). The genotype specific growth rates of the fish were analysed. In addition, the oxygen affinity properties of the hemoglobin genotypes were studied at 10 and 19°C. The genotype Hb-I(2/2) displayed the overall best growth at 10, 13 and 16°C, but no differences in growth among genotypes were seen at 19°C. Further, Hb-I(2/2) had the highest oxygen affinity at 10 and 19°C, whereas Hb-I(1/1) had the lowest oxygen affinity at both temperatures. In conclusion, the hemoglobin polymorphism in turbot seems to be correlated with physiological performance, and thus useful in future selection programs for turbot as a correlated trait with production characteristics.     

Iron-metabolic function and potential antibacterial role of Hepcidin and its correlated genes (Ferroportin 1 and Transferrin Receptor) in Turbot (Scophthalmus maximus)

Antimicrobial peptide plays an important role in fish immunity. The small molecular antimicrobial peptide Hepcidin in turbot was studied and reported in this paper. The Ferroportin 1 (FPN1) and Transferrin Receptor (TFR) genes, which are related to Hepcidin, were cloned in turbot. The characteristics of Hepcidin and its related genes were studied, including an analysis of the expression patterns and cloning of the Hepcidin promoter, the relationship between Hepcidin and NF-κB and the regulation of iron-metabolism. The results showed that the promoter of SmHepcidin contains the binding sites of NF-κB, and NF-κB may directly or indirectly receive feedback signals from SmHepcidin. In the liver, spleen and kidney, in which there was an increased SmHepcidin expression level, SmFPN1 dramatically decreased and SmTFR was also either decreased or exhibited no obvious change after bacterial/viral infection and an injection of exogenous Hepcidin protein. RNAi experiments in turbot kidney cells confirmed the expression changes of these gene patterns. Furthermore, the administration of exogenous Hepcidin protein, which regulates the level of chelatable iron in cells, further confirmed the function of Hepcidin in iron metabolism. It is speculated that the rapidly increased expression of SmHepcidin may induce changes in the expression of related genes, and that the in vivo chelatable iron concentration which participates in the antibacterial process was also changed when exogenous pathogens are present in turbot. It is suggested that SmHepcidin plays a defensive role against pathogenic infection.     

A microsatellite genetic map of the turbot (Scophthalmus maximus)

A consensus microsatellite-based linkage map of the turbot (Scophthalmus maximus) was constructed from two unrelated families. The mapping panel was derived from a gynogenetic family of 96 haploid embryos and a biparental diploid family of 85 full-sib progeny with known linkage phase. A total of 242 microsatellites were mapped in 26 linkage groups, six markers remaining unlinked. The consensus map length was 1343.2 cM, with an average distance between markers of 6.5 +/- 0.5 cM. Similar length of female and male maps was evidenced. However, the mean recombination at common intervals throughout the genome revealed significant differences between sexes, approximately 1.6 times higher in the female than in the male. The comparison of turbot microsatellite flanking sequences against the Tetraodon nigroviridis genome revealed 55 significant matches, with a mean length of 102 bp and high sequence similarity (81-100%). The comparative mapping revealed significant syntenic regions among fish species. This study represents the first linkage map in the turbot, one of the most important flatfish in European aquaculture. This map will be suitable for QTL identification of productive traits in this species and for further evolutionary studies in fish and vertebrate species.     

Production of Eicosapentaenoic Acid (20:5 n-3) by Vibrio pelagius Isolated from Turbot (Scophthalmus maximus (L.)) Larvae

Fourteen bacterial strains isolated from turbot, Scophthalmus maximus (L.), larvae were screened for eicosapentaenoic acid (20:5 n-3) (EPA) production. Gas chromatography analysis revealed that one bacterial species, Vibrio pelagius, contained a high proportion of EPA in cellular lipid. This finding was confirmed by gas chromatography-mass spectrometry analysis. A higher concentration of EPA was detected when the bacterium was cultured at 4°C.     

Identification of Quantitative Trait Loci Associated with Resistance to Viral Haemorrhagic Septicaemia (VHS) in Turbot (Scophthalmus maximus): A Comparison Between Bacterium,Parasite and Virus Diseases

One of the main objectives of genetic breeding programs in turbot industry is to reduce disease-related mortality. In the present study, a genome scan to detect quantitative trait loci (QTL) affecting resistance and survival to viral haemorrhagic septicaemia (VHS) was carried out. Three full-sib families with approximately 90 individuals each were genotyped and evaluated by linear regression and maximum likelihood approaches. In addition, a comparison between QTL detected for resistance and survival time to other important bacterial and parasite diseases affecting turbot (furunculosis and scuticociliatosis) was also carried out. Finally, the relationship between QTL affecting resistance/survival time to the virus and growth-related QTL was also evaluated. Several genomic regions controlling resistance and survival time to VHS were detected. Also significant associations between the evaluated traits and genotypes at particular markers were identified, explaining up to 14 % of the phenotypic variance. Several genomic regions controlling general and specific resistance to different diseases in turbot were detected. A preliminary gene mining approach identified candidate genes related to general or specific immunity. This information will be valuable to develop marker-assisted selection programs and to discover candidate genes related to disease resistance to improve turbot production.     

盐度对大菱鲆幼鱼鳃Na -K -ATPase活力、血清离子浓度 和激素水平的影响

采用饲养试验方法,研究了平均体质量为(7.16±0.07)g的大菱鲆(Scophthalmus maximus)幼鱼分别在盐度12、18、24、30和36下饲养60 d后,其鳃Na+-K+-ATPase活力、血清离子浓度、生长激素(GH)、皮质醇激素(COR)、特定生长率(SGR)和饲料效率(FCE)的变化。结果表明:幼鱼鳃Na+-K+-ATPase活力、血清Na+浓度均随盐度的升高而上升,分别在3.48~8.30 U/mg和169.99~180.00 mmol/L之间,其中12盐度组最低,36盐度组最高。幼鱼血清中K+和Cl-浓度分别在2.20~3.47 mmol/L和136.67~142.00 mmol/L之间,各盐度组之间差异不显著。幼鱼血清中GH和COR浓度分别在0.41~1.66 ng/ml和35.33~76.41 ng/ml之间;其中GH在36盐度组最高,12盐度组最低,而COR在12盐度组最高,36盐度组最低。幼鱼SGR和FCE分别在(1.45~2.00)%/d和1.12%~1.38%之间,与盐度的相关性不显著,两者均为12盐度组最低。由此可见,盐度变化显著影响大菱鲆幼鱼鳃Na+-K+-ATPase活力、血清Na+浓度和激素含量。本研究对大菱鲆养殖生理生态条件分析具有重要参考意义,研究结果可为大菱鲆养殖的盐度选择提供理论依据。     

Determination of bacteria associated with reared turbot (Scophthalmus maximus) larvae

In order to extend our knowledge of the presence of bacteria in hatcheries and their influence on rearing performance, the aerobic and facultative bacterial flora associated with farmed turbot larvae were studied in relation to the microflora of the water and diets. A settlement of specific groups of bacterial populations was found in the gut of the larvae. A clear succession of bacterial phenotypes was also observed from day 1 to day 90 post-hatching. Oxidative Gram-negative rods were predominant at the initial stages, whereas some phenotypes of Vibrio were frequent at the final stages. A high heterogeneity of Vibrio species was observed in the intermediate period when the highest mortalities of turbot larvae occur.