Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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A Transcriptomic Analysis of Land Use Impacts on the Oyster, Crassostrea virginica, in the South Atlantic Bight.

ABSTRACT: Increasing utilization and human population density in the coastal zone is widely believed to place increasing stresses on the resident biota, but confirmation of this belief is somewhat lacking. While we have solid evidence that highly disturbed estuarine systems have dramatic changes in the resident biota (black and white if you will), we lack tools that distinguish the shades of grey. In part this lack of ability to distinguish shades of grey stems from the analytical tools that have been applied to studies of estuarine systems and perhaps more important is the insensitivity of the biological end points that we have used to assess these impacts. In this paper we will present data on the phenotypic adjustments as measured by transcriptomic signatures of a resilient organism (oysters) to land use practices in the surrounding watershed using advanced machine learning algorithms. We will demonstrate that such an approach can reveal subtle and meaningful shifts in oyster gene expression in response to land use. Further, the data shows that gill tissues are far more responsive and provide superior discrimination of land use classes than hepatopancreas and that transcript encoding proteins involved in energy productions, protein synthesis and basic metabolism are more robust indicators of land use than classic biomarkers such as metallothioneins, GST and cytochrome P450. Keywords: Comparative genomics, ecogenomics. Tissue differences, impacts of land use and contaminants on gene expression. Oysters were collected from 11 tidal creeks in Georgia, South Carolina and North Carolina at sites variously impacted by human development. A total of 267 individuals were examined for gene expression profiles in gill and hepatopancreas tissues for a total of 534 arrays. The data were filtered though standard tools and ultimately analyzed using advance machine learning techniques.

ORGANISM(S): Crassostrea virginica

SUBMITTER: G Warr

PROVIDER: E-GEOD-14793 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Marine intertidal organisms commonly face hypoxic stress during low tide emersion; moreover, eutrophic conditions and sediment nearness could lead to hypoxic phenomena; it is indeed important to understand the molecular processes involved in the response to hypoxia. In this study the molecular response of the Pacific oyster Crassostrea gigas to prolonged hypoxia (2 mg O2 L-1 for 20 d) was investigated under experimental conditions. A transcriptomic approach was employed using a cDNA microarray of 9058 C. gigas clones to highlight the genetic expression patterns of the Pacific oyster under hypoxic conditions. Lines of oysters resistant (R) and susceptible (S) to summer mortality were used in this study. This is the first study employing microarrays to characterize the genetic markers and metabolic pathways responding to hypoxic stress in C. gigas. For the microarray analysis oysters were sampled at days 0, 2, 10, and 20 after the beginning of hypoxia. On each date, the digestive gland from 6 oysters was dissected, pooled, and stored at -80°C in Extract-All Reagent (Eurobio) at a concentration of 1 mL/50 mg tissue until total RNA was extracted. For each condition (hypoxia and normoxia) and oyster line (R and S), 4 pools were sampled (biological replicates) for a total of 56 pools during the 4 days of sampling. Furthermore, the entire tissue from 10 wild oysters was collected, pooled, and homogenized in Extract-all Reagent (Eurobio) to constitute a single total RNA sample to use as a reference in all slide hybridizations and Real-Time PCR analyses. Five ?g of total RNA were directly labeled by reverse transcription using the Direct ChipShot Direct Labeling and Clean-Up Kit (Promega). The samples were labeled with Cyanine-5 (Cy5). The reference samples were labeled with Cyanine-3 (Cy3) in separate tubes following the same protocol. The Cy3-labeled cDNAs were pooled and re-divided to obtain a homogeneous reference sample. cDNA samples and references were evaporated in a SpeedVac and mixed into a single pool in equimolar amounts with the Chip Hybe hybridization buffer (Ventana Discovery). Hybridization was performed using a Ventana automatic hybridization station (Ventana Discovery).

Project description:Summer mortality of the Pacific oyster Crassostrea gigas is the result of a complex interaction between oysters, their environment and pathogens. Heredity appears to be a major factor determining the sensitivity of oysters to summer mortality, allowing resistant (R) and susceptible (S) lines to be produced. We conducted genome-wide expression profiling of R and S gonads during the 3-month period preceding a summer mortality event using a 9K cDNA microarray that we designed. This transcriptional analysis provides new indications to define markers for Quantitative Trait Loci searches and functional studies, and evaluates the potential role of each gene in the resistance to summer mortality For microarray analysis, R and S oysters were sampled four times (dates 1 to 4: May 9, May 25, June 6, and June 20, respectively). On each date, 3 replicates of 8 oysters were sampled from each line (R and S) and the gonads prepared for total RNA extraction. Furthermore, the entire tissues of 10 wild oysters were collected, pooled and homogenized to constitute a single total RNA sample for use as a reference in all slide hybridizations and RT-PCR analysis. For microarray hybridizations, 5µg of total RNA were directly labeled by reverse transcription and then purified using the Direct ShipShot Labeling kit (Promega). This reaction was performed for each of the 24 gonad samples, with Cy5 (red) incorporation. The reference sample was Cy3-labeled (green) in 24 separate tubes following the same protocol. The 24 Cy3-labeled cDNAs were next pooled, and then divided once more into 24 samples to obtain a homogeneous reference. Equimolar amounts of cDNA samples and cDNA reference labeled with Cy5 and Cy3, respectively, were SpeedVac evaporated and mixed into a single pool with the hybridization buffer (ChipHyb™ hybridization buffer, Ventana Discovery, Tucson, AZ, USA). They were then co-hybridized on the same microarray slide, in a Ventana hybridization station (Ventana Discovery, Tucson, AZ, USA). The data submitted here correspond to the mean of the three replicates for each line and each date, representing 8 samples : gonad_resistant_date1 gonad_sensitive_date1 gonad_resistant_date2 gonad_sensitive_date2 gonad_resistant_date3 gonad_sensitive_date3 gonad_resistant_date4 gonad_sensitive_date4

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A Transcriptomic Analysis of Land Use Impacts on the Oyster, Crassostrea virginica, in the South Atlantic Bight.

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