Project description:Dietary immunostimulants are considered as an effective tool for enhancing the immune status of cultured organisms and improving their general welfare. Immunostimulants are chemical substances which activate transiently elements of the immune response and may potentially render animals more resistant to infectious diseases. They also reduce the risk of disease outbreaks, if administrated prior to situations known to result in stress and impaired general performance (e.g. handling, change of temperature and environment, weaning to inert diets) or prior to expected increase in exposure to pathogenic microorganisms and parasites. In this study, we have evaluated the effect of dietary LPS derived from Pantoea agglomerans (LPSp, SOMACY SL100, MACROPHI Inc., Japan) in Anguilla anguilla elvers considering the general condition, growth and immune status of the animals. Elvers (1.7 Â± 0.2 g) obtained from local fishermen (Delta del Ebro, Spain) were fed at different doses of c (0, 20 and 40 Î¼g LPSp kg BW-1 day-1) for a period of 70 days (4 replicates per dietary group). LPSp was added to the diet (52% protein, 24% fat, 9% ash) by top coating the pellets with the lyophilized powder of LPSp dissolved in 2% fish oil (diets manufactured by ALLERQUA, Denmark). The trial was conducted in 100 L tanks (40 L functional volume) connected to a recirculation unit (IRTAmarÂ®) and water quality was as follows: temperature, 21 Â± 0.5 ÂºC; pH, 7.5 Â± 0.1; photoperiod, 12 L:12 D and oxygen at saturation levels. At the end of the trial, fish were measured for growth (BW), size dispersion, histological organization of the intestinal mucosa and the transcriptomic responses of the spleen were evaluated by means of a 60K (15000 gene signatures) custom oligonucleotide microarray (Agilent Technologies Inc.). No significant differences in BW nor survival were observed among groups regardless of the dose of LPSp (BW: 3.9 Â± 0.3 g, survival: 98-99%). Micorarray analysis (LIMMA F<0.05; adj. p value < 0.005) resulted in 81 significantly up-regulated genes and only 3 down-regulated genes, in the highest LPSp dose. The lowest dose of LPSp up-regulated 26 genes and down-regulated 50. Both doses resulted in regulation of immune related genes, such as, sensors for detection of viral and bacterial products, innate antiviral response genes, cytokines, adipocytokines, and their regulatory nuclear factors. The pathway enrichment analysis (FatiGO; p < 0.005) of up-regulated genes (40 Î¼g LPSp kg BW-1 day-1) indicated among the most significantly regulated KEGG pathways: cytokine-cytokine receptor interaction, Jak-STAT signalling, natural killer cell mediated cytotoxicity, Toll-like, RIG-I-like and NOD-like receptor signalling and Wnt, Notch and mTOR signalling. In conclusion, LPSp diet highly regulates spleen transcription levels, mainly enhancing the immune response capacity of the immunostimulated elvers. This study was funded by the CENIT-ACUISOST project (CDTI, Spain), IRTA, Basque Government (S-PC08UN03; IT810-13) And UPV/EHU (UFI 11/37). Technical and human support provided by SGIker (UPV/EHU) is acknowledged. Authors are also in debt to G. MaciÃ (IRTA-SCR) for fish care and maintenance during the trial. 21 samples are analyzed, 7 pools of 3 spleens in each experimental group,Control group; Diet of 20ug/kg and 40ug/kg

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.

Dataset Information

Transcriptomic responses of Anguilla anguilla elvers fed dietary LPS

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