Construction of cytoplasmic molecular markers distinguishing Danio rerio from Gobiocypris rarus at high identity domains based on MP-PCR strategy and Sybr Green I detection

To distinguish the cytoplasm of Danio rerio from that of Gobiocypris rarus, we cloned G. rarus COXI and constructed cytoplasmic molecular markers at the high identity domains of COXI by mutated primer PCR (MP-PCR for short). Then Sybr Green I was used to detect the single amplicon. As a result, we succeeded in getting the cytoplasmic molecular markers, G.M COXI and Z.M COXI, by MP-PCR strategy. They were used to detect the sperm-derived mtDNA in the sexual hybrid embryos (D. rerio female x G. rarus male) before the sphere stage. In the present study, all results demonstrate that MP-PCR approach and Sybr Green I detection are feasible to construct the molecular markers to identify genes that shared high identity.To distinguish the cytoplasm of Danio rerio from that of Gobiocypris rarus, we cloned G. rarus COXI and constructed cytoplasmic molecular markers at the high identity domains of COXI by mutated primer PCR (MP-PCR for short). Then Sybr Green I was used to detect the single amplicon. As a result, we succeeded in getting the cytoplasmic molecular markers, G.M COXI and Z.M COXI, by MP-PCR strategy. They were used to detect the sperm-derived mtDNA in the sexual hybrid embryos (D. rerio female x G. rarus male) before the sphere stage. In the present study, all results demonstrate that MP-PCR approach and Sybr Green I detection are feasible to construct the molecular markers to identify genes that shared high identity

Enzyme-linked immunosorbent assay of changes in serum levels of growth hormone (cGH) in common carps (Cyprinus carpio)

The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1-14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1-6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1-7, 9 and 10) were used in fluorescent staining and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions.The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1-14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1-6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1-7, 9 and 10) were used in fluorescent staining and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions

Site-directed gene integration in transgenic zebrafish mediated by cre recombinase using a combination of mutant Lox sites

With current gene-transfer techniques in fish, insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences, and multiple gene interactions make gene expression experiments difficult to interpret and fish phenotype less predictable. To meet different fish engineering needs, we describe here a gene targeting model in zebrafish. At first, four target zebrafish lines, each harboring a single genomic lox71 target site, were generated by zebrafish transgenesis. The zygotes of transgenic zebrafish lines were coinjected with capped Cre mRNA and a knockin vector pZklox66RFP. Site-specific integration event happened from one target zebrafish line. In this line two integrant zebrafish were obtained from more than 80,000 targeted embryos (integrating efficiency about 10(-4) to 10(-5)) and confirmed to have a sole copy of the integrating DNA at the target genome site. Genomic polymerase chain reaction analysis and DNA sequencing verified the correct gene target events where lox71 and lox66 have accurately recombined into double mutant lox72 and wild-type loxP. Each integrant zebrafish chosen for analysis harbored the transgene rfp at the designated egfp concatenates. Although the Cre-mediated recombination is site specific, it is dependent on a randomly placed target site. That is, a genomic target cannot be preselected for integration based solely on its sequence. Conclusively, an rfp reporter gene was successfully inserted into the egfp target locus of zebrafish genome by Cre-lox-mediated recombination. This site-directed knockin system using the lox71/lox66 combination should be a promising gene-targeting platform serving various purposes in fish genetic engineering

Identification and characterization of a novel gene differentially expressed in zebrafish cross-subfamily cloned embryos

m Background: Cross-species nuclear transfer has been shown to be a potent approach to retain the genetic viability of a certain species near extinction. However, most embryos produced by cross-species nuclear transfer were compromised because that they were unable to develop to later stages. Gene expression analysis of cross-species cloned embryos will yield new insights into the regulatory mechanisms involved in cross-species nuclear transfer and embryonic development. Results: A novel gene, K31, was identified as an up-regulated gene in fish cross-subfamily cloned embryos using SSH approach and RACE method. K31 complete cDNA sequence is 1106 base pairs (bp) in length, with a 342 bp open reading frame (ORF) encoding a putative protein of 113 amino acids (aa). Comparative analysis revealed no homologous known gene in zebrafish and other species database. K31 protein contains a putative transmembrane helix and five putative phosphorylation sites but without a signal peptide. Expression pattern analysis by real time RT-PCR and whole-mount in situ hybridization (WISH) shows that it has the characteristics of constitutively expressed gene. Sub-cellular localization assay shows that K31 protein can not penetrate the nuclei. Interestingly, over-expression of K31 gene can cause lethality in the epithelioma papulosum cyprinid (EPC) cells in cell culture, which gave hint to the inefficient reprogramming events occurred in cloned embryos. Conclusion: Taken together, our findings indicated that K31 gene is a novel gene differentially expressed in fish cross-subfamily cloned embryos and over-expression of K31 gene can cause lethality of cultured fish cells. To our knowledge, this is the first report on the determination of novel genes involved in nucleo-cytoplasmic interaction of fish cross-subfamily cloned embryos.m Background: Cross-species nuclear transfer has been shown to be a potent approach to retain the genetic viability of a certain species near extinction. However, most embryos produced by cross-species nuclear transfer were compromised because that they were unable to develop to later stages. Gene expression analysis of cross-species cloned embryos will yield new insights into the regulatory mechanisms involved in cross-species nuclear transfer and embryonic development

Structure of endemic fish assemblages in the upper Yangtze River basin and population differentiation of an endangered endemic fish (Gobiocypris rarus)

Le fleuve Yangtze (Changjiang en chinois) supporte une très riche biodiversité de poissons, avec environ 400 espèces au total. Assimilée à une barrière eco-fonctionnelle, la partie amont du Yangtze présente des conditions environnementales naturelles exceptionnelles, avec une hétérogeneité bien prononcée des habitats, un système de drainage bien développé, une abondance en ressources en eau and une biodiversité importante. Ainsi, on dénombre 286 espèces de poissons dans la partie amont du Yangtze, dont 124 endémiques à cette région. Cependant, cette biodiversité est fortement menacée, notamment du fait de la surexploitation, de la pollution des eaux, des barrages hydroélectriques, des invasions biologiques et des modifications environnementales. Il est de ce fait impératif de mesurer les influences de ces changements environnementaux sur la ressource piscicole, de telle sorte à accélérer la mise en place de mesures de conservation. En conséquent, cette thèse avait pour objectif d'apporter des éléments visant à favoriser la conservation des espèces endémiques de poisons dans la partie amont du fleuve Yangtze. Dans la première partie de la thèse, l'objectif est d'illustrer les mécanismes de structuration des assemblages de poissons pour améliorer les connaissances des processus écologiques de la partie amont du fleuve Yangtze, i.e. déterminer les patrons spatiaux de distribution des poissons endémiques et les relations avec les variables environnementales (publications P1 & P2). Nous avons pu définir cinq assemblages de poissons endémiques dans le haut Yangtze caractérisés par des différences significatives au niveau de la richesse spécifique. Ces assemblages reflètent le gradient longitudinal de la distribution des espèces et de richesse avec une forte corrélation avec la topographie et la géomorphologie du fleuve Yangtze. Chaque assemblage est caractérisé par des espèces endémiques " indicatrices ". Le patron de distribution des poissons endémiques est significativement corrélé avec les facteurs environnementaux de l'utilisation du sol du bassin versant et des caractéristiques du fleuve. Des modèles mixtes utilisant simultanément des paramètres d'utilisation du sol et des caractéristiques du fleuve permettent d'assurer une meilleure prédiction de l'assemblage des poissons endémiques du Yangtze, comparés à des modèles utilisant séparément les 2 groupes de paramètres environnementaux. Enfin, nous suggérons trois points importants pour la conservation des espèces endémiques de poissons du haut Yangtze: sélection de plusieurs sites protégés de diverses espèces; maintient d'une partie courante naturelle pour chaque cours d'eau parmi les affluents; développement de mise en réserve des affluents. Dans la deuxième partie de la thèse, afin d'obtenir une bonne connaissance d'une espèce de poisson endémique en danger (Gobiocypris rarus) et fournir des instructions pour la conservation des autres espèces du haut Yangtze, nous avons utilisé cette espèce comme exemple d'étude de conservation à l'aide de marqueurs microsatellites et morphologiques (publications P3, P4 et P5). L'habitat typique de G. rarus était caractérisé par une seule population stable qui présentait une taille effective relativement large et aucune évidence de structure cryptique au cours des dix dernières années. Les forces maintenant cette diversité génetique était principalement les fluctuations environnementales et les traits d'histoire de vie propres à G. rarus. A une échelle spatiale plus étendue, nous avons trouvé des patrons significatifs de différentiation entre plusieurs populations de G. rarus, d'un point de vue génétique mais aussi morphologique. Particulièrement, deux clusters génétiques reflétant la structure du réseau hydrographique ont été identifiés. L'étude comparée des patrons d'isolation par la distance nous a permis de conclure que G. rarus était capable de migrer de réseau en réseau davantage via les canaux d'irrigations que via l'embouchure des rivières. Nous avons également mis en évidence un dimorphisme sexuel cryptique (i.e. visible uniquement que quelque trait morphologique continu). Finalement, au vue de cette distribution discontinu (tant d'un point de vue génétique que morphologique) et étant donné les menaces écologiques attendu, nous conseillons que la plupart des populations étudiées soit préservées. Plus particulièrement, les populations T1, T2, Q2, M3 et D2 devraient être prioritaire d'un point de vue de la conservation, avec une gestion de l'habitat et de l'espèce particulièrement forte dans ces localités.The Yangtze River, also called Changjiang, supports rich biodiversity, especially diverse fish fauna, i.e., about 400 fish species and subspecies. As an eco-functional barrier of the Yangtze River, the upper Yangtze River exhibits complicated natural environment, pronounced habitat heterogeneity, well-developed drainage system, abundant water resources and rich biodiversity. There were 286 fish species and subspecies distributing in the upper Yangtze River, among which 124 species were endemic. However, these fish resources are experiencing large threats: overexploitation, water pollution, hydropower projects, invasion by exotic species, and global environmental changes. It is imperative to evaluate the influences of the changes in environmental features on the fish resources and to accelerate the progress of conservation projects. Therefore, two parts of content have been studied in this thesis for the conservation of endemic fishes in the upper Yangtze River. Firstly, for the purpose of providing insight into mechanisms structuring fish assemblages and enhancing knowledge on ecological processes in the upper Yangtze River, spatial pattern of endemic fishes in the upper Yangtze River basin and their relationship with environmental features have been studied in P1 and P2 of this thesis. We identified five endemic fish assemblages in the upper Yangtze River basin. Not only species composition but also endemic species richness varied significantly among these five assemblages. They not only reflect the longitudinal gradient pattern but also are closely correlated with the topography and geomorphology of the Yangtze River. Each endemic fish assemblage has its specific indicator species. The endemic fish distribution patterns are significantly correlated with environmental factors such as land-cover features and river characteristics. The mixed models containing both land-cover features and river characteristics are more effective than any individual one in explaining complex endemic fish distribution patterns in the upper Yangtze River basin. Finally, we suggested that three key points for the conservation of endemic fishes in the upper Yangtze River basin should be paid more attention: selection of several protected sites aiming at various species; maintenance of at least one flowing reach in each river; developing the conservation of tributaries. Secondly, in order to obtain enough background of an endangered endemic fish (Gobiocypris rarus) and provide guidelines for other species conservation in the upper Yangtze River basin, the author used this Chinese rare minnow (G. rarus) as an example to do the studies of conservation biology using microsatellite markers and morphological traits in P3, P4 and P5 of this thesis. The type locality of G. rarus held a single stable and healthy population with a relatively large Ne and no cryptic structure during nearly ten years. The forces maintaining their genetic diversity were mainly from environmental fluctuations and life history traits. In addition, there were significant differentiations among wild populations of G. rarus not only in genetic markers but also in morphometric traits. Two obvious genetic clusters were revealed among wild populations of G. rarus, reflecting out water system structure to some extent. An isolation-by-riparian-distance pattern was identified, indicating that G. rarus might migrate through some man-made channels of hydropower projects but not through the mouth of the Minjiang and Tuojiang Rivers to exchange genes. Sexual dimorphism existed in morphometric traits of G. rarus wild populations. Finally, in the view of discontinuous distribution, significant genetic and quantitative differentiation of wild populations, and large threats from human activities, all the studied populations should be protected. Especially, populations T1, T2, Q2, M3 and D2 should be in prior conservation, and a habitat and species management area should be established in its type locality (viz. population D2)

Sustainability in China: Bridging Global Knowledge with Local Action

China’s road to sustainability has attracted global attention. Since the “Reform & Opening Up” policy, China’s rapid pace of both urbanization and industrialization has made its being the second largest economy but meantime a heavy environmental price has been paid over the past few decades for addressing the economic developmental target. Today, as the biggest developing country, China needs to take more responsibilities for constructing its local ecological-civilization society as well as for addressing the global challenges such as climate change, resources scary and human beings well-fare; therefore, we need to have deeper understandings into China’s way to sustainability at very different levels, both spatially and structurally, concerns ranging from generating sustainable household livelihoods to global climate change, from developing technological applications to generate institutional changes. In this spirit, this publication, “Sustainability in China: Bridging Global Knowledge with Local Action” aims to investigate the intended and spontaneous issues concerning China’s road to sustainability in a combined top-down and bottom-up manner, linking international knowledge to local-based studies

Ranaviruses: Lethal Pathogens of Ectothermic Vertebrates

Animal Ecology; Environmental Health; Fish & Wildlife Biology & Management; Virology; Freshwater & Marine Ecology; Conservation Biology/Ecolog

Gut Microbiota and Energy Homeostasis in Fish

The microorganisms within the intestinal tract (termed gut microbiota) have been shown to interact with the gut-brain axis, a bidirectional communication system between the gut and the brain mediated by hormonal, immune, and neural signals. Through these interactions, the microbiota might affect behaviors, including feeding behavior, digestive/absorptive processes (e.g., by modulating intestinal motility and the intestinal barrier), metabolism, as well as the immune response, with repercussions on the energy homeostasis and health of the host. To date, research in this field has mostly focused on mammals. Studies on non-mammalian models such as fish may provide novel insights into the specific mechanisms involved in the microbiota-brain-gut axis. This review describes our current knowledge on the possible effects of microbiota on feeding, digestive processes, growth, and energy homeostasis in fish, with emphasis on the influence of brain and gut hormones, environmental factors, and inter-specific differences