Winter and fall gene expression in rainbow smelt liver
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ABSTRACT:
ORGANISM(S): Osmerus mordax Salmo salar
PROVIDER: GSE17961 | GEO | 2010/01/31
SECONDARY ACCESSION(S): PRJNA119927
REPOSITORIES: GEO
Similar Datasets
Project description:Rainbow smelt (Osmerus mordax) are freeze-resistant fish that accumulate glycerol and produce an antifreeze protein during winter. Quantitative reverse transcription-PCR (qPCR) and subtractive hybridization studies have revealed a few genes in smelt liver to be differentially regulated in winter in comparison with the fall when water temperatures are warmer. In order to further define the suite of processes that are regulated seasonally, we undertook a large-scale analysis of gene expression by hybridization of smelt cDNA to the salmonid 16K cGRASP cDNA microarray. These microarray experiments were conducted as a focused sieving exercise, which identified informative genes for further study in the microarray samples and over a seasonal sampling series using quantitative reverse-transcription PCR. Total RNA was obtained from livers of 3 male fish sampled on October 20th and aliquots were pooled to contain equimolar concentrations of each RNA preparation. Total RNA was prepared from 3 male fish on January 3rd and processed in the same manner. Fluorescently labeled cDNA preparations were made using the fall (October) and winter (January) pools and mixed before hybridization to the microarray. The cDNAs were hybridized to three separate microarrays in order to generate technical replicates. Differential expression was assessed in order to select genes for further study.
2010-05-15 | E-GEOD-17961 | biostudies-arrayexpress
Project description:The rainbow smelt (Osmerus mordax, Mitchill, 1814) is an anadromous teleost that overwinters in the estuaries and inshore waters along the North American Atlantic coastline. In the winter months, smelt avoid freezing in subzero temperatures by the production of antifreeze proteins and high levels of glycerol. Glycerol production (glyceroneogenesis) occurs in the liver via a branch point in glycolysis and gluconeogenesis and is directly activated by low temperature. In these studies, hepatocytes were isolated from the liver of individual warm fish and incubated at either a warm (8ºC; non-glycerol accumulating) or cold (0.4ºC; glycerol accumulating) temperature over a 72 h time course. Functional genomic techniques were used to identify and validate hepatic transcripts that were differentially expressed between the warm and cold cells. Reciprocal suppression subtractive hybridization (SSH) cDNA libraries enriched for cold-responsive liver transcripts were constructed at the 72 h incubation time. Microarray analyses using the consortium for Genomic Research on All Salmonids Project (cGRASP) 16K (salmonid) cDNA array were performed at the 24, 48 and 72 h incubation times. For quantitative reverse transcription – polymerase chain reaction (QPCR) studies, we focused specifically on the non-colligative [type II antifreeze protein (AFPII)] and colligative (glycerol accumulation) freeze prevention strategies. AFPII (SSH identified) and 21 transcripts (SSH and/or microarray identified or selected by the authors based on a conceptual link to glycerol production) involved in the metabolism of glycolytic (glycogen, glucose) and gluconeogenic (amino acids) sources of glycerol and of lipids with a glycerol backbone (triglyceride, phosphoplipid) were analyzed using QPCR.
2010-08-11 | GSE23516 | GEO
Project description:The rainbow smelt (Osmerus mordax, Mitchill, 1814) is an anadromous teleost that overwinters in the estuaries and inshore waters along the North American Atlantic coastline. In the winter months, smelt avoid freezing in subzero temperatures by the production of antifreeze proteins and high levels of glycerol. Glycerol production (glyceroneogenesis) occurs in the liver via a branch point in glycolysis and gluconeogenesis and is directly activated by low temperature. In these studies, hepatocytes were isolated from the liver of individual warm fish and incubated at either a warm (8ºC; non-glycerol accumulating) or cold (0.4ºC; glycerol accumulating) temperature over a 72 h time course. Functional genomic techniques were used to identify and validate hepatic transcripts that were differentially expressed between the warm and cold cells. Reciprocal suppression subtractive hybridization (SSH) cDNA libraries enriched for cold-responsive liver transcripts were constructed at the 72 h incubation time. Microarray analyses using the consortium for Genomic Research on All Salmonids Project (cGRASP) 16K (salmonid) cDNA array were performed at the 24, 48 and 72 h incubation times. For quantitative reverse transcription – polymerase chain reaction (QPCR) studies, we focused specifically on the non-colligative [type II antifreeze protein (AFPII)] and colligative (glycerol accumulation) freeze prevention strategies. AFPII (SSH identified) and 21 transcripts (SSH and/or microarray identified or selected by the authors based on a conceptual link to glycerol production) involved in the metabolism of glycolytic (glycogen, glucose) and gluconeogenic (amino acids) sources of glycerol and of lipids with a glycerol backbone (triglyceride, phosphoplipid) were analyzed using QPCR. Rainbow smelt were collected by seine netting from Mount Arlington Heights, Placentia Bay, Newfoundland in late October 2007 and transported to the Ocean Sciences Centre, Memorial University of Newfoundland. The fish were held in a 3000 L indoor free-flowing seawater tank maintained at 8°C to 10°C and followed a natural photoperiod with fluorescent lights set by an outdoor photocell. Hepatocyte isolations were performed for individual male fish (i.e. no pooling of cells from different fish) from Nov. 27th to Dec.17th, 2007. Hepatocytes were not pooled as individual smelt produce different amounts of glycerol in response to cold temperature challenge. Each liver was perfused with medium containing collagenase and yielded approximately 300 million cells per individual. Initial or “pre-incubation” samples were collected and the remaining cell suspension was divided into 20 ml glass scintillation vials (6 x106 cells per vial) containing 2 ml of incubation medium and incubated at either a warm (8ºC; non-glycerol accumulating) or cold (0.4ºC; glycerol accumulating) temperature. Duplicate vials from each temperature were sampled at 24, 48 and 72 h incubation times for RNA isolation. RNA isolated from the hepatocytes of the 9 fish with the highest levels of glycerol production at 0.4°C were used to generate 2 mRNA pools (a “cold” pool and a “warm” pool) for each incubation time (24, 48 and 72 h). To summarize, the “cold” pools contained cells from 9 individual fish incubated at 0.4°C for either 24, 48 or 72 h and which exhibited the highest increase in glycerol levels at 72 h relative to pre-incubation levels, and the “warm” pools contained cells from these same 9 individual fish but incubated at 8°C for either 24, 48 or 72 h and therefore exhibited no increase in glycerol levels at 72 h relative to pre-incubation levels. Comparisons were made for the cold compared to warm pools at the 24, 48 and 72 h time points. For each time point, technical quadruplicate slides including two dye swaps were run per comparison. Incubation time: 24 h: Cold_24h_1, Cold_24h_2, Cold_24h_3, Cold_24h_4 Incubation time: 48 h: Cold_48h_1, Cold_48h_2, Cold_48h_3, Cold_48h_4 Incubation time: 72 h: Cold_72h_1, Cold_72h_2, Cold_72h_3, Cold_72h_4 Technical replicate: Cold_24h_1, Cold_24h_2, Cold_24h_3, Cold_24h_4 Technical replicate: Cold_48h_1, Cold_48h_2, Cold_48h_3, Cold_48h_4 Technical replicate: Cold_72h_1, Cold_72h_2, Cold_72h_3, Cold_72h_4
2010-08-11 | E-GEOD-23516 | biostudies-arrayexpress
Project description:The delta smelt (Hypomesus transpacificus) is an endangered pelagic fish species endemic to the Sacramento-San Joaquin estuary, California, and considered an indicator of ecosystem health. The experimental combination of molecular biomarkers with higher level condition indicators may allow for interpretation of responses in a functional context that can be used to predict detrimental outcomes caused by contaminant exposure. Copper is a contaminant of concern in Californian waterways that may affect the development and survival of this endangered species. We have developed and applied a delta smelt microarray in order to screen for probable candidate molecular biomarkers that may be used in monitoring programs. Functional classifications of microarray responses are presented along with quantitative Polymerase Chain Reaction (qPCR) assessments measuring effects upon neuromuscular, digestive and immune responses in delta smelt exposed to copper. Differences in sensitivity were measured between juvenile and larval delta smelt (LC¬5096h= 25.2 and 80.4 ?g/L Cu2+ respectively). Swimming velocity declined with higher exposure concentrations in a dose-dependent manner, though were not statistically significant to controls. Genes encoding for aspartoacylase (ASPA), hemopexin, alpha-actin and calcium regulation proteins were significantly affected by exposure and were functionally interpreted with measured swimming responses. Effects on digestion were measured by upregulation of chitinase and downregulation of amylase, whilst downregulation of tumor necrosis factor indicated a probable compromised immune system.
2010-08-18 | GSE23238 | GEO
Project description:The delta smelt (Hypomesus transpacificus) is a pelagic fish species endemic to the Sacramento-San Joaquin Estuary in Northern California, listed as endangered under both the USA Federal and Californian State Endangered Species Acts and acts as an indicator of ecosystem health in its habitat range. Interrogative tools are required to successfully monitor effects of contaminants upon the delta smelt, and to research potential causes of population decline in this species. We used microarray technology to investigate genome-wide effects in 57-day old larvae after a 4-day exposure to ammonia; one of multiple contaminants arising from wastewater treatment plants and agricultural runoff. Genomic assessments were carried out between larvae exposed to 10 mg/L total ammonium; the lowest observed effect concentration (LOEC), and controls. Microarray assessments were conducted on larvae exposed for 4-days to 10 mg/L (nominal) ammonium chloride and controls. Assessments were carried out in quadruplicate, using 5 fish per treatment. RNA was extracted from frozen whole, individual organisms, using Trizol Reagent (Invitrogen) as per manufacturer's guidelines. Total RNA from 5 fish was pooled per treatment and cDNA was synthesized from a total of 500ng total RNA, amplified using a SuperScripttm Indirect RNA Amplification System (Invitrogen). Resulting cDNA was labeled with Alexa fluor dyes (Invitrogen) as per manufacturer’s instructions. Two color microarray assessments were carried out on quadruplicate treatments, using 1µg of amplified cDNA for each control vs exposed sample, incorporating dye swaps for each (total 8 samples). Microarray hybridizations were performed using an automated Tecan HS4800 hybridization station. Slides were scanned using a GenePix 4000B scanner (Axon Instruments). Data was analyzed using LIMMA GUI (Linear model for microarray analysis graphical user interface) (Smyth, 2005), written in the R-programming language available through Bioconductor http://www.Bioconductor.org. Data was normalized within using print-tip Lowess and between arrays applying average intensity quantile (Aquantile) normalization methods with background correction (Smyth, 2005). A linear model fit was computed using the duplicates on the arrays and the least-squares method, with Benjamin Hochberg false discovery rate adjustment.
2011-05-05 | E-GEOD-29089 | biostudies-arrayexpress
Project description:The delta smelt (Hypomesus transpacificus) is a pelagic fish species endemic to the Sacramento-San Joaquin Estuary in Northern California, listed as endangered under both the USA Federal and Californian State Endangered Species Acts and acts as an indicator of ecosystem health in its habitat range. Interrogative tools are required to successfully monitor effects of contaminants upon the delta smelt, and to research potential causes of population decline in this species. We used microarray technology to investigate genome-wide effects in 57-day old larvae after a 4-day exposure to ammonia; one of multiple contaminants arising from wastewater treatment plants and agricultural runoff. Genomic assessments were carried out between larvae exposed to 10 mg/L total ammonium; the lowest observed effect concentration (LOEC), and controls.
2011-05-05 | GSE29089 | GEO
Project description:The delta smelt (Hypomesus transpacificus) is an endangered pelagic fish species endemic to the Sacramento-San Joaquin estuary, California, and considered an indicator of ecosystem health. The experimental combination of molecular biomarkers with higher level condition indicators may allow for interpretation of responses in a functional context that can be used to predict detrimental outcomes caused by contaminant exposure. Copper is a contaminant of concern in Californian waterways that may affect the development and survival of this endangered species. We have developed and applied a delta smelt microarray in order to screen for probable candidate molecular biomarkers that may be used in monitoring programs. Functional classifications of microarray responses are presented along with quantitative Polymerase Chain Reaction (qPCR) assessments measuring effects upon neuromuscular, digestive and immune responses in delta smelt exposed to copper. Differences in sensitivity were measured between juvenile and larval delta smelt (LC¬5096h= 25.2 and 80.4 ?g/L Cu2+ respectively). Swimming velocity declined with higher exposure concentrations in a dose-dependent manner, though were not statistically significant to controls. Genes encoding for aspartoacylase (ASPA), hemopexin, alpha-actin and calcium regulation proteins were significantly affected by exposure and were functionally interpreted with measured swimming responses. Effects on digestion were measured by upregulation of chitinase and downregulation of amylase, whilst downregulation of tumor necrosis factor indicated a probable compromised immune system. We utilized 3 replicates for exposed samples and 3 replicates for controls, both of which were hybridized to a reference pool. Each replicate contained 4 pooled larval delta smelt. The Reference pool consisted of delta smelt samples from this experiment and a number of prior tests. No dye swaps were performed due to limited material. Supplementary file linked below reports candidate differentially expressed cDNAs. Data represented as filtered, normalized, Log-2 Alexa (547/555) ratios.
2010-08-18 | E-GEOD-23238 | biostudies-arrayexpress
Project description:Research on the effects of contaminants on fishes is often conducted on well-studied model test species, whose responses may be different than those of species of conservation concern. We used an oligonucleotide microarray to examine the effects of permethrin, a widely used pyrethroid pesticide, on a critically endangered fish species endemic to Northern California, the delta smelt (Hypomesus transpacificus). These results demonstrate the effects of a widely used pesticide on a sensitive fish species at concentrations below those that affect model test species.
2015-09-01 | GSE67521 | GEO
Project description:Research on the effects of contaminants on fishes is often conducted on well-studied model test species, whose responses may be different than those of species of conservation concern. We used an oligonucleotide microarray to examine the effects of permethrin, a widely used pyrethroid pesticide, on a critically endangered fish species endemic to Northern California, the delta smelt (Hypomesus transpacificus). These results demonstrate the effects of a widely used pesticide on a sensitive fish species at concentrations below those that affect model test species. Twenty samples were run on twenty arrays, there were four replicates for each of four permethrin exposure concentrations and a control group.
2015-09-01 | E-GEOD-67521 | biostudies-arrayexpress
Project description:Winter and fall gene expression in rainbow smelt liver
| PRJNA119927 | ENA