Global climate change is rapidly altering coastal marine ecosystems important for food production. A comprehensive understanding of how organisms will respond to these complex environmental changes can come only from observing and studying species within their natural environment. To this end, the effects of environmental drivers — pH, dissolved oxygen content, salinity, and temperature — on Pacific oyster (Crassostrea gigas) physiology were evaluated in an outplant experiment. Sibling juvenile oysters were outplanted to eelgrass and unvegetated habitat at five different estuarine sites within the Acidification Nearshore Monitoring Network in Washington State, USA to evaluate how regional environmental drivers influence molecular physiology. Within each site, we also determined if eelgrass presence that buffered pH conditions changed the oysters’ expressed proteome. A novel, two-step, gel-free proteomic approach was used to identify differences in protein abundance in C. gigas ctenidia tissue after a 29 day outplant by 1) identifying proteins in a data independent acquisition survey step and 2) comparing relative quantities of targeted environmental response proteins using selected reaction monitoring. While there was no difference in protein abundance detected between habitats or among sites within Puget Sound, C. gigas outplanted at Willapa Bay had significantly higher abundances of antioxidant enzymes and molecular chaperones. Environmental factors at Willapa Bay, such as higher average temperature, may have driven this protein abundance pattern. These findings generate a suite of new hypotheses for lab and field experiments to compare the effects of regional conditions on physiological responses of marine invertebrates.

Pacific oyster ctendia was obtained after a 29 day outplant. Peptides were analyzed on an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) using Data Independent Acquisition Mass Spectrometry (DIA). DIA analyses were completed as a comprehensive, non-random analytical method for detecting peptide ions present within a sample to create a peptide library. The peptide library was then leveraged to develop a targeted proteomics assay for quantification (see Selected Reaction Monitoring Assay).

For the DIA analysis, ctenidia was used from one oyster from each unique site-habitat combination (10 samples total). SRM analysis was completed using 5 ctenidia samples from each site-habitat combination.