Response of the Pacific oyster Crassostrea gigas to hypoxia exposure under experimental conditions

Abstract

In the last few decades, marine hypoxia has become one of the major ecological concerns in the world, because of the increase of excessive anthropogenic input of nutrients and organic matter into coastal seawater [1]. Benthic communities are the most sensitive parts of the coastal ecosystem to eutrophication and resulting hypoxia [2]. High production in stratified waters results from nutrient enrichment and can cause hypoxic or anoxic bottom waters because of the subsequent deposition of algal biomass [3]. Marine organisms are directly affected by hypoxia at various levels of organization and behavioural, biochemical and physiological responses to limited availability of oxygen have been well studied in fish and marine invertebrates [4]. Most of the invertebrate species that inhabit the intertidal zone, and especially sedentary ones, have developed mechanisms for surviving twicedaily oxygen deprivation at low tide. Depression of metabolic rate can be considered as one of the most important adaptations for hypoxia endurance [5,6]. Many marine molluscs do indeed show reversible protein phosphorylation to limit the activity of many enzymes and functional proteins during anoxia [5,7]. The same response to hypoxia has already been The molecular response to hypoxia stress in aquatic invertebrates remains relatively unknown. In this study, we investigated the response of the Pacific oyster Crassostrea gigas to hypoxia under experimental conditions and focused on the analysis of the differential expression patterns of specific genes associated with hypoxia response. A suppression subtractive hybridization method was used to identify specific hypoxia up-and downregulated genes, in gills, mantle and digestive gland, after 7-10 days and 24 days of exposure. This method revealed 616 different sequences corresponding to 12 major physiological functions. The expression of eight potentially regulated genes was analysed by RT-PCR in different tissues at different sampling times over the time course of hypoxia. These genes are implicated in different physiological pathways such as respiration (carbonic anhydrase), carbohydrate metabolism (glycogen phosphorylase), lipid metabolism (delta-9 desaturase), oxidative metabolism and the immune system (glutathione peroxidase), protein regulation (BTF3, transcription factor), nucleic acid regulation (myc homologue), metal sequestration (putative metallothionein) and stress response (heat shock protein 70). Stress proteins (metallothioneins and heat shock proteins) were also quantified. This study contributes to the characterization of many potential genetic markers that could be used in future environmental monitoring, and could lead to explore new mechanisms of stress tolerance in marine mollusc species.Abbreviations GPx, glutathione peroxidase; HIF-1, hypoxia-inducible factor-1; HSP, heat shock protein; MT, metallothionein; SSH, suppression subtractive hybridization.

Cloning of cytoplasmic heat shock protein 90 (FcHSP90) from Fenneropenaeus chinensis and its expression response to heat shock and hypoxia

“…This difference might depend on the different roles that these two tissues play during hypoxia stress. Similar results have been reported in C. gigas that hypoxia causes the variation in levels of heat shock proteins including HSP70 and HSP25, and different expression profiles were obtained for HSP70 in different tissues after hypoxia stress (David et al 2005). HSP90 can modulate the stabilization of hypoxia-inducible factor-1α (HIF-1α) and subsequently change the conformation of the heterodimeric complex comprised of HIF-1α and aryl hydrocarbon receptor nuclear translocator to regulate the gene expression for cytoprotection under hypoxia (Ali et al 1998;Hur et al 2002;Isaacs et al 2002Isaacs et al , 2004.…”

“…In a previous report of hypoxia experiments in C. gigas, the expression levels of several genes including carbonic anhydrase, glutathione peroxidase, metallothionein, HSP70, etc. varied greatly in the control group after 3 days (David et al 2005). Due to the sensitivity of FcHSP90 expression in the shrimp response to heat shock and hypoxia, FcHSP90 might play very important roles to allow shrimp to cope with environmental stresses.…”

Intertidal oysters reach their physiological limit in a future high-CO2 world

“…The findings of this and previous studies (Widdows and Shick, 1985;Lesser, 2016) in relation to changing metabolisms of intertidal bivalves when transplanted, suggest that oysters are highly plastic (Collicutt and Hochachka, 1977;Greenway and Storey, 1999;Hamdoun et al, 2003;Ernande et al, 2004;David et al, 2005;Zhang et al, 2012). Plastic responses allow for an immediate response to cope with, and potentially overcome, a stressor (West-Eberhard, 1989).…”

“…When emersed, marine bivalves close their shell, which restricts gas exchange and can result in internal hypercapnia and extracellular acidosis (Burnett, 1988;Truchot, 1990). When hypercapnic, normal physiological processes of bivalves are impeded, and metabolism is slowed until normocapnia returns (Burnett, 1988;Greenway and Storey, 1999;David et al, 2005). Intertidal emersion also reduces the time available to sessile organisms for feeding, waste excretion and other vital processes (Truchot, 1990).…”

Assessment of housekeeping genes as internal references in quantitative expression analysis during early development of oyster

“…In previous research, although their expression stability was not validated, several housekeeping genes of C. gigas have been used as internal references; they include α subunit of elongation factor 1 (elf1α) (Fabioux et al, 2004;Badariotti et al, 2007;Gonzalez et al, 2007), actin (Zhang et al, 2011), glyceraldehyde-3-phosphate dehydrogenase (gapdh) (Badariotti et al, 2007;Herpin et al, 2007), 18s rRNA (Meistertzheim et al, 2007) and 28s rRNA (David et al, 2005). However, it is inappropriate to simply assume a gene to be stably expressed without validation, especially given the complex early development of C. gigas.…”