Project description:Gene expression analyses have been performed on brain tissue of sexually mature and immature males using microarrays. 60 eels were transferred to two independent temperature controlled recirculation water units connected to two 500 L cylindro-conical tanks (30 fish per tank) where the fish were acclimated to seawater (35 PSU salinity) over a 2 week period.Eel males in one of the seawater units were injected intramuscularly every week over a 140 day period with 2000 IU hCG/kg (human chorionic gonadotropin, Sigma–Aldrich Chemical) dissolved in 0.9% saline to induce sexual maturation. Eel males in the other recirculation unit were injected weekly over the same period with 0.9% NaCl (vehicle).At the end of the experiment, eels were anesthetized in a solution of 0.1mg/L tricaine methanesulfonate (MS-222, Sigma Aldrich) and blood samples collected into heparinized syringes. Tissues (brain, including the olfactory bulbs, telencephalon, diencephalon and mesencephalon, and gonads) were collected from sexually immature (n=12) and sexually mature males (n=12).RNA was extracted from brain tissue of a total of 24 (12 mature and 12 immature) males. For each individual, RNA was extracted separately for olfactory bulb, telencephalon and diencephalon. Because RNA concentrations of olfactory bulb, telencephalon and diencephalon were below the amount needed for microarray analysis, a separate experiment for each part of the brain was not possible. Therefore, we combined equal concentrations of olfactory bulb, telencephalon and diencephalon as a single brain sample for each individual.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. Data was normalized using a quantile normalization procedure using R (http://www.r-project.org) A comparative analysis of gene expression was conducted between sexually mature and immature males. 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. Hybridization success was evaluated using flag values, excluding those intensities not equal to 1.

Project description:Gene expression analyses have been performed on liver tissue of sexually mature and immature males using microarrays. 60 eels were transferred to two independent temperature controlled recirculation water units connected to two 500 L cylindro-conical tanks (30 fish per tank) where the fish were acclimated to seawater (35 PSU salinity) over a 2 week period.Eel males in one of the seawater units were injected intramuscularly every week over a 140 day period with 2000 IU hCG/kg (human chorionic gonadotropin, Sigma–Aldrich Chemical) dissolved in 0.9% saline to induce sexual maturation. Eel males in the other recirculation unit were injected weekly over the same period with 0.9% NaCl (vehicle).At the end of the experiment, eels were anesthetized in a solution of 0.1mg/L tricaine methanesulfonate (MS-222, Sigma Aldrich) and blood samples collected into heparinized syringes. Tissues (brain, liver and gonads) were collected from sexually immature (n=12) and sexually mature males (n=12).RNeasy Mini Kit (Qiagen) was used to extract RNA from the livers. Tissue samples were pooled and, therefore, each of the biological replicates (n= 4 sexually immature, n=4 sexually mature) contains tissue from three fish. Total RNA concentration was determined using the Nanodrop ND-100 spectrophotometer (NanoDrop Technologies) and sample integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). 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. Data was normalized using a quantile normalization procedure using R (http://www.r-project.org) A comparative analysis of gene expression was conducted between sexually mature and immature males. 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. Hybridization success was evaluated using flag values, excluding those intensities not equal to 1.

Project description:Many enigmas surround different aspects of freshwater eel biology and life cycle. In the same way different hypothesis about why eels are disappearing from European continental waters have been proposed. One such proposal defends that poor fat accumulation of eels, due to pollution in continental waters, might be stopping eels from reproductive migration to the Sargasso Sea. Thus, habitat deterioration could be blamed for the decline in the catchment potential of the European rivers. In this context, and with the aim to study the mode of action of environmentally relevant chemicals in eels, the multi tissue transcriptome was sequenced (454 Titanium Roche) in order to design a high density custom oligonucleotide microarray (eArray, Agilent). To validate this tool a laboratory experiment was carried to analyze the gene trasncrition profiles related to chemical compounds released from pulp and paper mills; 100 μg/L mercury and 150 μg/L β-sitosterol (only Hg data is presented here). 20 yellow European eel (Anguilla anguilla) elvers (11.7±5.35 g) obtained from a local farm (Acuivas SL, Usurbil, Gipuzkoa) were exposed for 3, 6 and 9 days. Pyrosequencing allowed the design and construction of a 60K microarray platform containing 3923 gene signatures identified through BlastN analysis and 7212 sequences annotated through BlastX coupled to Blast2Go analysis. Additional 226 sequences were incorporated from NCBI databases and 3551 from the information available in EeelBase in 2011. Two probes were generated per sequence and they were spotted twice in the array. Hepatic gene expression profiling of the exposed eels indicated that Hg significantly down-regulated (LIMMA, adj. p<0.005) only gene signatures related to selenoprotein W-1 (SeW), something typically described in mammals exposed to methyl-mercury. Increasing the adj. p value to <0.05, 116 genes were significantly regulated (38 down-regulated and 79 up-regulated). Among them, we found additional selenoproteins such as ROS metabolism related genes; glutathione peroxidases (gpx1 & gpx4b) and thioredoxine which were up-regulated. In addition, complement system genes (C3 & C4b) were also up-regulated. Studying enriched Go pathways (p<0.005) and in relation with lipid homeostasis we observed that the following pathways were enriched after exposure to Hg: fatty acids degradation and metabolism of arachidonic acid, linoleic acid, ether lipids, alpha linolenic acid, and glycerophospholipids. In addition, among the top 10 significantly enriched KEGG pathways, p53 signalling, apoptosis, and MAPK signalling were present, suggesting possible effects on cell cycle regulation. In summary, transcriptome pyrosequencing and subsequently designed microarray provided the molecular tools to successfully study the gene transcription profiles of toxic chemical compounds such as Hg in European eel tissues. In addition to study the molecular modes of action of specific chemical compounds, the developed gene expression microarray will be useful in active monitoring of the quality of freshwater environments using caged sentinel eels. This study was funded by Basque Government (SAIOTEK S-PE09UN32; Consolidated Research Groups IT810-13) and UPV/EHU (UFI 11/37). Technical and human support provided by SGIker (UPV/EHU) is acknowledged.

Project description:Many enigmas surround different aspects of freshwater eel biology and life cycle. In the same way different hypothesis about why eels are disappearing from European continental waters have been proposed. One such proposal defends that poor fat accumulation of eels, due to pollution in continental waters, might be stopping eels from reproductive migration to the Sargasso Sea. Thus, habitat deterioration could be blamed for the decline in the catchment potential of the European rivers. In this context, and with the aim to study the mode of action of environmentally relevant chemicals in eels, the multi tissue transcriptome was sequenced (454 Titanium Roche) in order to design a high density custom oligonucleotide microarray (eArray, Agilent). To validate this tool a laboratory experiment was carried to analyze the gene trasncrition profiles related to chemical compounds released from pulp and paper mills; 100 Î¼g/L mercury and 150 Î¼g/L Î²-sitosterol (only Hg data is presented here). 20 yellow European eel (Anguilla anguilla) elvers (11.7Â±5.35 g) obtained from a local farm (Acuivas SL, Usurbil, Gipuzkoa) were exposed for 3, 6 and 9 days. Pyrosequencing allowed the design and construction of a 60K microarray platform containing 3923 gene signatures identified through BlastN analysis and 7212 sequences annotated through BlastX coupled to Blast2Go analysis. Additional 226 sequences were incorporated from NCBI databases and 3551 from the information available in EeelBase in 2011. Two probes were generated per sequence and they were spotted twice in the array. Hepatic gene expression profiling of the exposed eels indicated that Hg significantly down-regulated (LIMMA, adj. p<0.005) only gene signatures related to selenoprotein W-1 (SeW), something typically described in mammals exposed to methyl-mercury. Increasing the adj. p value to <0.05, 116 genes were significantly regulated (38 down-regulated and 79 up-regulated). Among them, we found additional selenoproteins such as ROS metabolism related genes; glutathione peroxidases (gpx1 & gpx4b) and thioredoxine which were up-regulated. In addition, complement system genes (C3 & C4b) were also up-regulated. Studying enriched Go pathways (p<0.005) and in relation with lipid homeostasis we observed that the following pathways were enriched after exposure to Hg: fatty acids degradation and metabolism of arachidonic acid, linoleic acid, ether lipids, alpha linolenic acid, and glycerophospholipids. In addition, among the top 10 significantly enriched KEGG pathways, p53 signalling, apoptosis, and MAPK signalling were present, suggesting possible effects on cell cycle regulation. In summary, transcriptome pyrosequencing and subsequently designed microarray provided the molecular tools to successfully study the gene transcription profiles of toxic chemical compounds such as Hg in European eel tissues. In addition to study the molecular modes of action of specific chemical compounds, the developed gene expression microarray will be useful in active monitoring of the quality of freshwater environments using caged sentinel eels. This study was funded by Basque Government (SAIOTEK S-PE09UN32; Consolidated Research Groups IT810-13) and UPV/EHU (UFI 11/37). Technical and human support provided by SGIker (UPV/EHU) is acknowledged. An exposure laboratory experiment was performed in the âUr Biologia eta Experimentazio Zerbitzuaâ (UBEZ) of the University of the Basque Country (UPV/EHU) A total of 84 eels, 20.23 Â± 2.84 cm in length, were acclimatized to laboratory conditions for 2 weeks in a 10 L water glass tanks under controlled conditions: 12 h light/dark cycle at 18oC, conductivity 600 Î¼S. During this period animals were fed with commercial flakes. Ammonium, nitrites and nitrates kits (Sera GmbH) were used to systematically asses the levels of nitrogenous compounds which were maintained at 0-0.5mg/l; 0-0.5 mg/l, and 5-10 mg/l, respectively. When highest concentrations within those ranges were achieved seawater was partially replaced. A day before exposure experiment, animals were placed in 4 aquaria with 8 L of water at 600 Î¼S. In each aquarium 21 eels were randomly distributed and exposed to: metal-mercury (Hg) (Fluka-Sigma-Aldrich) at a concentration of 100Î¼g/L; and to a phitosterol, Î²-sitosterol (Sigma) at a concentration of 150 Î¼g/ L. The last compound needed ethanol (0.01%) as vehicle. In addition to an ethanol control group, a water control (600Î¼S) without contaminant was also prepared. Water used during the experiment was filtered at 0.5mm and treated with UV light. Toxic metabolites were measured everyday during experiment using ammonium, nitrites and nitrates test (Sera GmbH), and water was partially replaced every day. Eels were fed everyday with 10mg of commercial food per eel. After 3, 6 and 9 days exposure (T1, T2 and T3 respectively) 7 eels were anaesthetized with 3-aminobenzoate methylsulfonate salt (Sigma-Aldrich). Size and weight were measured for FultonÂ´s conditioning index assessments, and liver was dissected and frozen with RNA laterÂ® in liquid nitrogen for molecular studies.

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Changes in brain gene expression profiles during sexual maturation of European eel (Anguilla anguilla) males.

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