SUMMARYClimate change is affecting speciesʼ physiology, pushing environmental tolerance limits and shifting distribution ranges. In addition to temperature and ocean acidification, increasing levels of hyposaline stress due to extreme precipitation events and freshwater runoff may be driving some of the reported recent range shifts in marine organisms. Using two-dimensional gel electrophoresis and tandem mass spectrometry, we characterized the proteomic responses of the cold-adapted blue mussel Mytilus trossulus, a native to the Pacific coast of North America, and the warm-adapted M. galloprovincialis, a Mediterranean invader that has replaced the native from the southern part of its range, but may be limited from expanding north due to hyposaline stress. After exposing laboratory-acclimated mussels for 4h to two different experimental treatments of hyposaline conditions and one control treatment (24.5, 29.8 and 35.0psu, respectively) followed by a 0 and 24h recovery at ambient salinity (35psu), we detected changes in the abundance of molecular chaperones of the endoplasmic reticulum (ER), indicating protein unfolding, during stress exposure. Other common responses included changes in small GTPases of the Ras superfamily during recovery, which suggests a role for vesicle transport, and cytoskeletal adjustments associated with cell volume, as indicated by cytoskeletal elements such as actin, tubulin, intermediate filaments and several actin-binding regulatory proteins. Changes of proteins involved in energy metabolism and scavenging of reactive oxygen species suggest a reduction in overall energy metabolism during recovery. Principal component analyses of protein abundances suggest that M. trossulus is able to respond to a greater hyposaline challenge (24.5psu) than M. galloprovincialis (29.8psu), as shown by changing abundances of proteins involved in protein chaperoning, vesicle transport, cytoskeletal adjustments by actin-regulatory proteins, energy metabolism and oxidative stress. While proteins involved in energy metabolism were lower in M. trossulus during recovery from hyposaline stress, M. galloprovincialis showed higher abundances of those proteins at 29.8psu, suggesting an energetic constraint in the invader but not the native congener. Both species showed lower levels of oxidative stress proteins during recovery. In addition, oxidative stress proteins associated with protein synthesis and folding in the ER showed lower levels during recovery in M. galloprovincialis, in parallel with ER chaperones, indicating a reduction in protein synthesis. These differences may enable the native M. trossulus to cope with greater hyposaline stress in the northern part of its range, as well as to outcompete M. galloprovincialis in the southern part of M. trossulusʼ range, thereby preventing M. galloprovincialis from expanding further north. Supplementary material available online at

ACUTE NITRATE EXPOSURE CAUSES A PROTEOMIC RESPONSE TO REACTIVE NITROGEN SPECIES IN THE PACIFIC OYSTER, CRASSOSTREA GIGAS Lauren R. HittNitrate is the most common ionic form of nitrogen in aquatic ecosystems. Although nitrate is known to affect ecosystems at high levels through eutrophication, hypoxia and loss of biodiversity, it is considered to be physiologically inert to the individual aquatic organism. To test the physiological effects of nitrate on aquatic life, we exposed gill tissue of the Pacific oyster, Crassostrea gigas, to nitrate and characterized changes in protein expression, using a gel-based proteomics approach. Of the 642 protein spots detected, we found that 24 proteins (15 identified) changed expression in response to a 6-hour exposure to nitrate concentrations ranging from 0-73 mg/L, values that characterize highly contaminated surface and ground waters. Proteins changing expression included the oxidative stress proteins thioredoxin and cavortin (a member of the superoxide dismutase family) as well as proteins that are involved in G-protein signaling (Rho-GDI, ADP-ribosylation factor, G-protein ß-subunit), protein homeostasis (heat shock protein 70, prohibitin, calreticulin, and proteasome α-type 4 subunit), glycolysis (enolase), transport of hydrophobic molecules (lipocalin) and cytoskeletal arrangements (intermediate filaments and a gelsolin-like adseverin). The most parsimonious explanation for these changes in protein expression assumes that C. gigas reduces nitrate to nitrite and nitric oxide, which reacts with superoxide anions to form the very reactive peroxynitrite. We propose that part of the cellular response to reactive nitrogen species,phagocytic hemocytes inhibit the production of reactive oxygen species, potentially compromising the immune response of oysters to invading pathogens. v ACKNOWLEDGEMENTS

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