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The Pacific oyster Crassostrea gigas is a sessile bivalve mollusc whose homeostasis relies, at least partially, upon cells circulating in hemolymph and referred to as hemocytes. Oyster’s hemocytes have been reported to produce reactive oxygen species (ROS), even in absence of stimulation. Although ROS production in bivalve molluscs is mostly studied for its defence involvement, ROS may also be involved in cellular and tissue homeostasis. ROS sources have not yet been described in oyster hemocytes. The objective of the present work was to characterize the ROS sources in unstimulated hemocytes. We studied the effects of chemical inhibitors on the ROS production and the mitochondrial membrane potential (Δψm) of hemocytes. First, this work confirmed the specificity of JC-10 probe to measure Δψm in oyster hemocytes, without being affected by ΔpH, as reported in mammalian cells. Second, results show that ROS production in unstimulated hemocytes does not originate from cytoplasmic NADPH-oxidase, nitric oxide synthase or myeloperoxidase, but from mitochondria. In contrast to mammalian cells, incubation of hemocytes with rotenone (complex I inhibitor) had no effect on ROS production. Incubation with antimycin A (complex III inhibitor) resulted in a dose-dependent ROS production decrease while an over-production is usually reported in vertebrates. In hemocytes of C. gigas, the production of ROS seems similarly dependent on both Δψm and ΔpH. These findings point out differences between mammalian models and bivalve cells, which warrant further investigation about the fine characterization of the electron transfer chain and the respective involvement of mitochondrial complexes in ROS production in hemocytes of bivalve molluscs.

Atmospheric carbon dioxide concentrations have been rising during the past century, leading to ocean acidification (OA). Coastal and estuarine habitats experience annual pH variability that vastly exceeds the magnitude of long-term projections in open ocean regions. Eastern oyster (Crassostrea virginica) reproduction season coincides with periods of low pH occurrence in estuaries, thus we investigated effects of moderate (pH 7.5, pCO2 2260 µatm) and severe OA (pH 7.1, pCO2 5584 µatm; and 6.7, pCO2 18480 µatm) on oyster gametogenesis, fertilization, and early larval development successes. Exposure at severe OA during gametogenesis caused disruption in oyster reproduction. Oogenesis appeared to be more sensitive compared to spermatogenesis. However, Eastern oyster reproduction was resilient to moderate OA projected for the near-future. In the context of projected climate change exacerbating seasonal acidification, OA of coastal habitats could represent a significant bottleneck for oyster reproduction which may have profound negative implications for coastal ecosystems reliant on this keystone species.