半滑舌鳎Cs-UBE2D4基因的克隆、表达模式及启动子活性

泛素-蛋白酶体系统作为重要的蛋白质修饰途径,参与多个生物学过程,其中泛素结合酶E2作为关键酶可以决定泛素化对靶蛋白的影响。为了探究泛素结合酶E2在半滑舌鳎雌性性腺发育中的作用,本研究克隆了半滑舌鳎Cs-UBE2D4基因的开放阅读框ORF,检测了该基因在不同组织和不同发育阶段性腺中的表达模式;构建了其启动子报告基因载体(pGL3-Cs-UBE2D4)并进行了启动子活性分析。Cs-UBE2D4开放阅读框为360 bp,编码119个氨基酸,含有保守的泛素结合酶E2结构域。荧光定量PCR结果显示,Cs-UBE2D4在雌鱼的各组织中广泛表达,其中在卵巢和肝脏中表达量较高,而在雄鱼中几乎检测不到表达;在卵巢各发育阶段中,该基因的表达量从90日龄开始上调,6月龄达到峰值,之后开始下调,在1.5龄时表达量最低,在3龄时表达量有所回升,其中6月龄表达量为90日龄表达量的1.97倍。双荧光素酶活性检测结果显示,Cs-UBE2D4启动子具有较强的转录活性。本实验将为进一步开展Cs-UBE2D4基因对半滑舌鳎卵巢发育和性别调控相关机制的研究提供理论依据。     

南极鱼类DNA条形码及分子系统进化研究

本文采用COI基因兼并引物对15种36个南极鱼类DNA进行扩增测序,并结合Gen Bank已有序列进行联配分析,对南极鱼2科22属43种共97条COI基因片段(539 bp)进行序列比较和系统发生关系研究,探索了DNA条形码技术在辅助鱼类物种鉴定和分类中的适应性与可行性。结果分析表明,43种南极鱼科和鳄冰鱼科的鱼类COI基因的平均碱基组成为T:31.9%、G:18.3%、A:22.2%和C:27.6%,具有明显的碱基偏倚性。南极鱼类的种间平均距离为0.157,种内平均遗传距离为0.002,种间平均遗传距离是种内平均距离的79倍;系统分析结果显示,除南极小带腭鱼(Cryodraco antarcticus)和罗斯海小带腭鱼(Cryodraco atkinsoni)外,其余的鱼类皆能够形成独立的分支,且与形态学分支一致。由此可见,DNA条形码对南极鱼亚目鳄冰鱼科和南极鱼科鱼类能够进行有效的物种鉴定,基于COI基因所建的NJ(neighbor-joining)树对物种分类具有较为准确的辨识力。系统发生关系表明,DNA条形码可以对除南极小带腭鱼(Cryodraco antarcticus)和罗斯海小带腭鱼(Cryodraco atkinsoni)外的南极鱼物种进行鉴定,不仅可以作为形态学的辅助手段为南极鱼分类系统的必要补充和佐证,并且可以用于探讨南极鱼类近缘种的系统发育关系。     

Trawl fishing impacts on the status of seabed fauna in diverse regions of the globe

Bottom trawl fishing is a controversial activity. It yields about a quarter of the world's wild seafood, but also has impacts on the marine environment. Recent advances have quantified and improved understanding of large‐scale impacts of trawling on the seabed. However, such information needs to be coupled with distributions of benthic invertebrates (benthos) to assess whether these populations are being sustained under current trawling regimes. This study collated data from 13 diverse regions of the globe spanning four continents. Within each region, we combined trawl intensity distributions and predicted abundance distributions of benthos groups with impact and recovery parameters for taxonomic classes in a risk assessment model to estimate benthos status. The exposure of 220 predicted benthos‐group distributions to trawling intensity (as swept area ratio) ranged between 0% and 210% (mean = 37%) of abundance. However, benthos status, an indicator of the depleted abundance under chronic trawling pressure as a proportion of untrawled state, ranged between 0.86 and 1 (mean = 0.99), with 78% of benthos groups > 0.95. Mean benthos status was lowest in regions of Europe and Africa, and for taxonomic classes Bivalvia and Gastropoda. Our results demonstrate that while spatial overlap studies can help infer general patterns of potential risk, actual risks cannot be evaluated without using an assessment model that incorporates trawl impact and recovery metrics. These quantitative outputs are essential for sustainability assessments, and together with reference points and thresholds, can help managers ensure use of the marine environment is sustainable under the ecosystem approach to management.     

Quantification of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), tissues by qPCR

A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms.     

Dietary calcium/phosphorus ratio influences the efficacy of microbial phytase on growth,mineral digestibility and vertebral mineralization in juvenile tiger puffer,Takifugu rubripes

A 2 × 3 factorial feeding trial was conducted to determine effects of dietary Ca/P ratio and dietary microbial phytase on growth, mineral digestibility and vertebral mineralization in tiger puffer. The treatments consisted of three levels of Ca/P ratios (0.5, 1.0 and 1.5) combined either with phytase (2000 FTU kg^?1 diet) or without supplementation, respectively. The Ca/P ratios were achieved by supplementing calcium at 0, 6 and 12 g kg^?1 combined with the same level of inorganic P at 5 g kg^?1. After a 50‐day feeding trial, puffer fish fed the diet at low Ca/P ratio (0.5) together with phytase had significantly higher growth rate and feed intake (FI) than other groups. Both dietary Ca/P ratio and phytase supplement were independent effects on plasma minerals and alkaline phosphatase. Interactive effect between both dietary treatments was observed on P and Zn contents in vertebrae and whole body. P and Zn digestibilities tended to increase with increased Ca/P ratio from 0.5 to 1.0, especially when phytase was supplemented. In conclusion, fish fed a diet with highest Ca/P ratio (1.5) showed the poorest growth performance and nutrients utilization. Dietary Ca/P ratio of 0.5 (without Ca supplement) with 2000 FTU phytase per kg would be the optimum combination in the diet of tiger puffer.     

Epilithic biofilm as a key factor for small‐scale river fisheries on Caribbean islands

Abstract Numerous freshwater species are consumed or exploited through artisanal fisheries in the rivers of the islands of Guadeloupe, French West Indies. Autochthonous production of organic matter is limited in these flowing rivers and is mainly represented by scarce filamentous green algae and an abundant epilithic biofilm growing on wet stones in the river bed. Stable isotope analysis was used to quantify the relative importance of biofilm and other riverine allochthonous and autochthonous food sources in the diet of tropical shrimps (Palaemonidae, Atyidae and Xiphocarididae) and fishes (Gobiidae and Eleotridae) consumed by the local people. The epilithic biofilm was exploited by most species, constituted an important source of autochthonous carbon and was an important source of organic matter production at the base of freshwater food webs in Caribbean rivers. Biofilm percentages in the diet reached 32% for molluscs, 85% for atyid shrimps, 29% for xiphocaridid shrimps, 14% for palaemonid shrimps and 13% for fish. Assessment of biofilm in nine rivers showed that blue and red cyanobacteria were quantitatively dominant with a moderately rich diatom flora. These results address the interactions between river biofilm and Caribbean freshwater fauna where trophic links between consumers and their potential resources have poorly been documented.     

Growth and production of Pacific ocean perch (Sebastes alutus) in nursery habitats of the Gulf of Alaska

Nursery areas for juvenile fishes are often important for determining recruitment in marine populations by providing habitats that can maximize growth and thereby minimize mortality. Pacific ocean perch (POP, Sebastes alutus) have an extended juvenile period where they inhabit rocky nursery habitats. We examined POP nursery areas to link growth potential to recruitment. Juvenile POP were captured from nursery areas in 2004 and 2008, and estimated growth rates ranged from ?0.19 to 0.60 g day^?1 based on differences in size between June and August. Predicted growth rates from a bioenergetics model ranged from 0.05 to 0.49 g day^?1 and were not significantly different than observed. Substrate preferences and the distribution of their preferred habitats were utilized to predict the extent of juvenile POP nursery habitat in the Gulf of Alaska. Based on densities of fish observed on underwater video transects and the spatial extent of nursery areas, we predicted 278 and 290 million juvenile POP were produced in 2004 and 2008. Growth potential for juvenile POP was reconstructed using the bioenergetics model, spring zooplankton bloom timing and duration and bottom water temperature for 1982–2008. When a single outlying recruitment year in 1986 was removed, growth potential experienced by juvenile POP in nursery areas was significantly correlated to the recruitment time‐series from the stock assessment, explaining ～30% of the variability. This research highlights the potential to predict recruitment using habitat‐based methods and provides a potential mechanism for explaining some of the POP recruitment variability observed for this population.     

Cellular and humoral immune responses of Senegalese sole,Solea senegalensis (Kaup), following challenge with two Photobacterium damselae subsp. piscicida strains from different geographical origins

The present study aimed to investigate leucocyte responses to inflammation as well as some innate immune parameters of Senegalese sole, Solea senegalensis, following challenge with two strains of Photobacterium damselae subsp. piscicida belonging to the European and Japanese clones described for this bacterium. Pathogenicity assays were performed to assess the virulence of each Photobacterium damselae subsp. piscicida strain for sole. Subsequently, fish were intraperitoneally injected with phosphate‐buffered saline (control) or two concentrations (2 × 10² and 2 × 10⁶ CFU mL^?1) of each bacterial strain and sampled after 6 and 24 h. Results showed that the European isolate induces a higher degree of response than the Japanese strain. While blood neutrophilia and monocytosis correlated well with the increase in neutrophil and macrophage numbers in the peritoneal cavity, fish infected with the European isolate presented higher peritoneal cell numbers than fish challenged with the Japanese strain. In addition, alternative complement pathway activity and respiratory burst of head kidney leucocytes increased significantly in fish infected with the European isolate. The enhanced innate immune response displayed by Senegalese sole challenged with the European isolate is probably due to the higher degree of virulence presented by this Photobacterium damselae subsp. piscicida strain.     

Enhancement of innate immune system,survival and yield in Penaeus monodon reared in ponds using Streptococcus phocae PI80

The objective of this study was to evaluate the effect of dietary and water supplementation of probiotic Streptococcus phocae PI80 on growth, immune response and feed utilization of tiger shrimp Penaeus monodon in earthen ponds. The probiotic bacterium S. phocae PI80 was cultured in large fermenter (50 L) by adding additional carbon source in the form of molasses and glucose along with yeast extract as nitrogen source to enrich S. phocae PI80 biomass. This enriched S. phocae PI80 was administered to shrimp in feed (6.5 × 10¹³ bacterial cells mL^?1) as well as in pond water (5 L/pond). Shrimp growth performance was significantly improved (P < 0.05) in 120 days culture when the average body weight of treated molasses + yeast extract (MY) (28.41 ± 0.874 g), glucose + yeast extract (GY) (27.013 ± 0.698 g) was significantly higher than control (23.63 ± 0.684 g). Food conversion ratio FCR was also found to be reduced significantly in ponds treated with probiotics when compared with control pond (1.89 ± 0.09). Vibrio and luminescent bacteria were found to be lower in the treatment receiving MY group, and we hypothesize that this may lead to greater shrimp survival. Furthermore, fermentation product of S. phocae PI80 added to pond water and feed additives enhanced the shrimp immune system. The results indicated that total haemocytes count (THC), phenoloxidase (PO) activity, NBT reductase assay and phagocytic activity significantly increased in shrimps treated with S. phocae PI80. Our study demonstrates that administration of S. phocae PI80 in the water and feed at 6.5 × 10¹³ colony‐forming units (CFU) mL^?1 bacterial cells induce immune modulation and enhances the immune ability of P. monodon in pond‐reared shrimp and increased the shrimp production.