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Transcriptomic response of the Pacific oyster Crassostrea gigas to hypoxia

ABSTRACT: 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.

ORGANISM(S): Crassostrea gigas

PROVIDER: GSE23883 | GEO | 2010/09/01

SECONDARY ACCESSION(S): PRJNA130497

REPOSITORIES: GEO

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Project description:Intermittent hypoxia is a common stressor in estuarine and coastal habitats due to the diurnal and tidal cycles of oxygen availability. Oxygen deficiency results in energy stress by limiting aerobic ATP production, while reoxygenation may lead to oxidative injury due to the excessive production of reactive oxygen species (ROS) in mitochondria. Mitochondria are a key intracellular target of hypoxia-reoxygenation (H/R) stress due to their central role in ATP production, ROS generation and stress signaling. Marine intertidal bivalves such as the Pacific oyster Crassostrea gigas are adapted to frequent H/R cycles in the intertidal zone and maintain mitochondrial integrity and aerobic function despite frequent oxygen fluctuations. To gain insight into the molecular responses of mitochondria of the hypoxia-tolerant Pacific oyster to H/R stress, we studied changes in oxidative phosphorylation (OXPHOS) capacity, proton leak and activity of the electron transport system enzymes (ETS) in gill mitochondria of C. gigas. We furthermore investigated shifts in mitochondrial proteome and phosphoproteome after 24 h of hypoxia and 1 h of post-hypoxic recovery. Oyster mitochondria maintained normal ETS and OXPHOS capacity during H/R stress, despite a slight but significant decline in cytochrome c oxidase (Complex IV) activity after reoxygenation. Fifty total proteins and 36 phosphoproteins were differentially abundant in mitochondria of H/R exposed oysters compared with the controls. Rearrangements of the mitochondrial (phospho)proteome during H/R stress involved upregulation of mitochondrial ETS (most notably Complexes I and IV) and iron-binding proteins (frataxin and ferrochelatase) as well as suppression of the metabolic pathways that channel electrons to ubiquinone, possibly as a mechanism to limit ROS production due to the iron overload and reverse flow of electrons through ETS. H/R stress also led to upregulation of a mitophagic activator serine/threonine protein phosphatase PGAM5 and dephosphorylation of metalloendopeptidase OMA1 indicating stimulation of mitochondrial quality control mechanisms during reoxygenation. Changes in the overall abundance and phosphorylation levels of several key proteins involved in the mitochondrial protein homeostasis (including subunits of the mitochondrial ribosome, mitochondrial inner membrane translocase and elongation factor G) are consistent with the suppression of the protein synthesis during hypoxia, likely as an energy-saving mechanism. Overall, our study indicates that shifts in the mitochondrial (phospho-)proteome play an important role in responses of oysters to H/R stress and might complement adaptations of anaerobic metabolism and metabolic rate depression in ensuring hypoxic survival and rapid recovery in this hypoxia-tolerant intertidal species.

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).

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Transcriptomic response of the Pacific oyster Crassostrea gigas to hypoxia

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