Project description:This project aims to study the role played by small non-coding RNAs in the response of aquatic organisms to the presence of micropollutants in the environment. MiRNA were purified from Eels (Anguilla anguilla) sampled from two sites along the Gironde aquatic system with contrasted pollution profiles.
Project description:Since the early 1980s, the population of European eels (Anguilla anguilla) has dramatically declined. Nowadays, the European eel is listed on the red list of threatened species (IUCN Red List) and is considered as critically endangered of extinction. Pollution is one of the explanations of the collapse of this species. Among their possible effects, pollutants gradually accumulated in eels during their somatic growth phase (yellow eel stage) would be remobilized during their reproductive migration leading to potential toxic events in gonads. The aim of this study was to investigate the potential effect of pollution on the gonad development of wild female silver eels. Female silver eels from two sites with differing contamination levels were artificially matured. Transcriptomic analyses by means of a 1000 candidate gene cDNA microarray were performed on gonads after 11 weeks of maturation. The results showed that the transcription levels of several genes that were associated to the gonadosomatic index (GSI) were involved in mitotic cell division but also in spermatogenesis. Genes associated to pollution were mainly involved in the mechanisms of protection against oxidative stress, in DNA repair, in the purinergic signaling pathway and in steroidogenesis, suggesting an impairment of gonad development in eels from the polluted site. This was in agreement with the fact that eels from the reference site showed a higher gonad growth in comparison to contaminated fish.
Project description:In spite of many decades of research, the spawning migration of the European eel Anguilla anguilla from the European coast to the Sargasso Sea remains a mystery. In particular, the role of the swimbladder as a buoyancy regulating structure is not yet understood. In this study, we exercised silver eels in a swim tunnel under elevated hydrostatic pressure. The transcriptome of gas gland tissue of these exercised eels was then compared to the known transcriptome of not exercised (control) silver eel gas gland cells. Due to the high infection rate of the eel population with the swimbladder parasite Anguillicola crassus, the comparison also included an exercised group of silver eels with a heavily damaged swimbladder, and we compared the previously published transcriptome of not exercised silver eels with a highly damaged swimbladder with the exercised group of silver eels with a heavily damaged swimbladder. The comparisons of unexercised (control) silver eels with exercised silver eels with functional swimbladder (EF), as well as with exercised silver eels with damaged swimbladder(ED), both showed a significant elevation in transcripts related to glycolytic enzymes. This could also be observed within the comparison of unexercised silver eels with a highly infected swimbladder with exercised eels with a damaged swimbladder (DED). In contrast to EF, in ED a significant elevation in transcript numbers of mitochondrial NADH dehydrogenase was observed. While in EF the transcriptional changes suggested that acid production and secretion was enhanced, in ED these changes appeared to be related to thickened tissue and thus elevated diffusion distances. The remarkable number of differentially expressed transcripts coding for proteins connected to cAMP-dependent signaling pathways indicated that metabolic control in gas gland cells includes cAMP-dependent pathways. In contrast to ED, in EF significant transcriptional changes could be related to the reconstruction of the extracellular matrix, while in ED tissue repair and inflammation was more pronounced. Surprisingly, in exercised eels hypoxia inducible transcription factor expression was elevated. In EF, a large number of genes related to the circadian clock were transcriptionally modified, which may be connected to the circadian vertical migrations observed during the spawning migration.
Project description:No studies systematically compared time-resolved and multi-tissue innate immune responses for European eels (Anguilla anguilla) with Aeromonas hydrophila infection through a high throughput method. Here, we challenged European eels with A. hydrophila (infected) or PBS (control). A low fatality rate (16.1%) was observed for infected group at 2 days post-infection (dpi). Then we examined transcriptional profiles of intestines, livers and spleens from 12 European eels with/without the infection of A. hydrophila at 6, 18 and 36 hours post-infection (hpi). The results showed that the most differentially expressed genes (DEGs) were found in the spleens at 6 hpi (7569 DEGs), followed by the intestines at 6 hpi (3129 DEGs) and the livers at 18 hpi (2722 DEGs). Only a few DEGs were found in three tissues at 36 hpi. The comparisons for DEGs numbers and KEGG enrichments suggested a consistent time-course immune responses among these three tissues. A similar enrichment of PRRs-related pathways including TLRs, NLRs and RLRs was revealed in the livers and spleens, but not in the intestines. Among immune-related DEGs, 62 paralogues pairs were found, and the expression trends of most paralogues pairs were consistent. Some paralogues of immune-related DEGs had unique domains. Furthermore, a larger clusters representing the similar tissue were revealed for the intestines and spleens. The distinct cytokine signal transduction and interaction existed between the livers and spleens. Altogether, the present study provides time-resolved and multi-tissue transcriptome characteristics of A. anguilla in response to A. hydrophila infection.
Project description:Ecotoxicogenomic studies face the problem of the lack of gene sequence information available for toxicologically relevant non-model pollution sentinel species. In this sense, next generation sequencing technologies allow obtaining deep de novo sequence information cost-effectively. We have thus employed the platform GS-FLX of Roche technologies, in order to sequence the multitissue transcriptome of a fish species under severe population decline, the European eel (Anguilla anguilla), using normalized cDNA (Evrogen). The European eel could be successfully employed in pollution biomonitoring studies as it is a euryhaline teleost that is quite resistant to chemical exposure. We have completed a one whole plate sequencing-run using the Titanium kit of Roche from which a total of 7.8x105 reads were obtained with a length average of 303.53 bp. Annotated genes, for instance, could be further assessed as biomarkers of exposure to specific chemical compounds in marine, estuarine and river waters. Designed tool will be thus useful, in the study of: a.- the mode of action of chemical compounds in active monitoring studies using caged eels, b.- the physiological processes that are specific to freshwater eels such as the one of sexual development and reproduction, c.- reasons that could explain the disappearance of the species from European waters. In the first context, a caging experiment was performed to measure the putative effects of a paper industry effluent on eel hepatic transcription levels. For this purpose, eels were caged upstream and downstream to the SMURFFIT-KAPPA paper industry in Iurreta (Biscay, Basque Country). Thus, hepatic transcription profiles reflect the stress levels suffered by eels. The experiment contains a time serial sampling: T0 = before sampling; T1 = after 3 days of exposure; T2=after 15 days of exposure. In addition, caged eels were placed in 2 sites, Upstream (Up) and Downstream (Do) of the hotspot. 6 samples were dissected per sampling group (5 groups= ToUP; T1UP; T1Do; T2Up; T2Do), 30 samples in total
Project description:Ecotoxicogenomic studies face the problem of the lack of gene sequence information available for toxicologically relevant non-model pollution sentinel species. In this sense, next generation sequencing technologies allow obtaining deep de novo sequence information cost-effectively. We have thus employed the platform GS-FLX of Roche technologies, in order to sequence the multitissue transcriptome of a fish species under severe population decline, the European eel (Anguilla anguilla), using normalized cDNA (Evrogen). The European eel could be successfully employed in pollution biomonitoring studies as it is a euryhaline teleost that is quite resistant to chemical exposure. We have completed a one whole plate sequencing-run using the Titanium kit of Roche from which a total of 7.8x105 reads were obtained with a length average of 303.53 bp. Annotated genes, for instance, could be further assessed as biomarkers of exposure to specific chemical compounds in marine, estuarine and river waters. Designed tool will be thus useful, in the study of: a.- the mode of action of chemical compounds in active monitoring studies using caged eels, b.- the physiological processes that are specific to freshwater eels such as the one of sexual development and reproduction, c.- reasons that could explain the disappearance of the species from European waters. In the first context, a caging experiment was performed to measure the putative effects of a paper industry effluent on eel hepatic transcription levels. For this purpose, eels were caged upstream and downstream to the SMURFFIT-KAPPA paper industry in Iurreta (Biscay, Basque Country). Thus, hepatic transcription profiles reflect the stress levels suffered by eels.
Project description:Nanometric revolution is underway, promising technical innovations in a wide range of applications, leading to a potential boost in environmental discharges. Nanoparticle propensity to be transferred throughout trophic chains and to generate toxicity was mainly assessed in primary consumers while a lack of knowledge for higher trophic levels persists. This study focused on a predatory fish, the European eel Anguilla anguilla exposed to gold nanoparticles (AuNP, 10 nm, PEG-coated) for 21 days at three concentration levels in food: 0 (NP0), 1 (NP1) and 10 (NP10) mg Au.kg-1 . Transfer was assessed by gold quantification in eel tissues and transcriptomic responses in the liver and brain were revealed by a high-throughput RNA-sequencing approach. Eels fed at NP10 presented an erratic feeding behaviour while gold quantification only indicated transfer to intestine and kidney of NP1 exposed eels. RNA-Sequencing was performed in NP0 and NP1 eels. A total of 258 genes and 156 genes were significantly differentially transcribed in response to AuNP trophic exposure in the liver and brain, respectively. Enrichment analysis highlighted modifications in the immune system-related processes in the liver. In addition, results pointed out a shared response of both organs regarding 13 genes, most of them being involved in immune functions. This finding may shed light into the mode of action and toxicity of AuNP in fish.
Project description:Gene expression analyses have been performed on brain, liver and muscle of eels (Anguilla anguilla) sampled in three different segment of the Canal des Etangs ( artificial canal linking Arcachon Basin and Lacanau Lake). The present study relies on intraspecific differences of glass eels encountered below and above the water dams. Eels were collected using electric fishing under similar climatic and hydrological conditions. Individuals were sampled below the obstacle, close to the fishway entry in segment 1 (Pas du Bouc; +44° 50' 27.95, -1° 9' 8.09) and 2 (Langouarde, +44° 51' 32.92, -1° 9' 5.03). Fish from the segment 3 (Joncru; +44° 52' 57.13, -1° 8' 11.70) were sampled on the fishway, as water depth before the obstacle, approximately 2 meters, precluded the use of electro-fishing technique. Ten individuals were selected from each site according to their body size (between 83 and 155 mm) and health status (no externally visible pathogens) criteria. Internal parasites such as Anguilicola crassus were taken into account. Sampled and selected fish were immediately sacrified by decapitation and the whole brain, liver and muscle tissue (posterior bottom body part) were dissected and stored at -80°C in separate tubes with RNAlater buffer (Quiagen) for gene expression analysis. Microarray analysis was conducted using an European eel-specific array consisting of a total of 14,913 probes based on a large collection of high-throughput transcriptomic sequences (Pujolar et al. 2012). Probe sequences and further details on the microarray platform can be found on the GEO database under accession number GPL15124. A comparative analysis of gene expression was conducted between eels sampled in differents segments of Canal des Etangs. Total RNAs were extracted from the whole liver,muscle and brain using the Absolutely RNA RT-PCR Miniprep kit (Agilent) according to the manufacturer’s instructions. Three independent RNA pools, each consisting of three individuals RNA samples, were prepared for each sampling site and tissues. Due to technical problems, it was no possible to perform microarray analyses in muscle of eels sampled in forebay 3. Accordingly, DNA microarray analysis has been performed in a total of 24 pools. RNAs’ quality was evaluated by electrophoresis on a 1% agarose gel and their concentrations were determined by spectrophotometry. Total RNA was stored in RNAse free water at -80° C. Sample labelling and hybridization were conducted following the details in Pujolar et al. (2012). Hybridized slides were scanned at 5 µm resolution using an Agilent DNA microarray scanner. Slides were scanned at two different sensitivity levels (XDR Hi 100% and XDR Lo 10%) and the two linked images generated were analysed together. Data were extracted and background subtracted using the standard procedure in Agilent Feature Extraction (FE) software v. 9.5.1. Data were normalized separately for each tissue using a quantile normalization procedure using R (http://www.r-project.org).
Project description:Gene expression analyses have been performed on brain, liver and muscle of eels (Anguilla anguilla) sampled in three different segment of the Canal des Etangs ( artificial canal linking Arcachon Basin and Lacanau Lake). The present study relies on intraspecific differences of glass eels encountered below and above the water dams. Eels were collected using electric fishing under similar climatic and hydrological conditions. Individuals were sampled below the obstacle, close to the fishway entry in segment 1 (Pas du Bouc; +44° 50' 27.95, -1° 9' 8.09) and 2 (Langouarde, +44° 51' 32.92, -1° 9' 5.03). Fish from the segment 3 (Joncru; +44° 52' 57.13, -1° 8' 11.70) were sampled on the fishway, as water depth before the obstacle, approximately 2 meters, precluded the use of electro-fishing technique. Ten individuals were selected from each site according to their body size (between 83 and 155 mm) and health status (no externally visible pathogens) criteria. Internal parasites such as Anguilicola crassus were taken into account. Sampled and selected fish were immediately sacrified by decapitation and the whole brain, liver and muscle tissue (posterior bottom body part) were dissected and stored at -80°C in separate tubes with RNAlater buffer (Quiagen) for gene expression analysis. Microarray analysis was conducted using an European eel-specific array consisting of a total of 14,913 probes based on a large collection of high-throughput transcriptomic sequences (Pujolar et al. 2012). Probe sequences and further details on the microarray platform can be found on the GEO database under accession number GPL15124.