Unknown,Transcriptomics,Genomics,Proteomics

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Ammonia exposures on the endangered delta smelt

ABSTRACT: 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.

ORGANISM(S): Hypomesus transpacificus

SUBMITTER: Richard Connon

PROVIDER: E-GEOD-29089 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 47-day old larvae after a 7-day exposure to ambient water samples from the Sacramento River at a monitoring field station (Hood) situated 8 miles downstream of the Sacramento regional Wastewater Treatment Plant. Genomic assessments were carried out on surviving organisms and contrasted to laboratory controls. Microarray assessments were conducted on larvae exposed for 7-days to Sacramento River water collected at Hood and pooled laboratory controls. Assessments were carried out in quadruplicate, using 3 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 2ug total RNA, amplified using a SuperScripttm Indirect RNA Amplification System (Invitrogen). Resulting aRNA was labeled with Alexa fluor dyes (Invitrogen) as per manufacturerM-bM-^@M-^Ys instructions. Two color microarray assessments were carried out on quadruplicate treatments, using 5M-BM-5g of aRNA for pooled controls vs exposed sample, (total 4 samples). Microarray hybridizations were performed manually, and incubated in a waterbath at 42C for 16 hours. 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.

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.

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Ammonia exposures on the endangered delta smelt

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