SUMMARYThe Mediterranean blue mussel, Mytilus galloprovincialis, an invasive species in California, has displaced the more heat-sensitive native congener, Mytilus trossulus, from its former southern range, possibly due to climate change. By comparing the response of their proteomes to acute heat stress we sought to identify responses common to both species as well as differences that account for greater heat tolerance in the invasive. Mussels were acclimated to 13°C for four weeks and exposed to acute heat stress (24°C, 28°C and 32°C) for 1h and returned to 13°C to recover for 24h. Using two-dimensional gel electrophoresis and tandem mass spectrometry we identified 47 and 61 distinct proteins that changed abundance in M. galloprovincialis and M. trossulus, respectively. The onset temperatures of greater abundance of some members of the heat shock protein (Hsp) 70 and small Hsp families were lower in M. trossulus. The abundance of proteasome subunits was lower in M. galloprovincialis but greater in M. trossulus in response to heat. Levels of several NADH-metabolizing proteins, possibly linked to the generation of reactive oxygen species (ROS), were lower at 32°C in the cold-adapted M. trossulus whereas proteins generating NADPH, important in ROS defense, were higher in both species. The abundance of oxidative stress proteins was lower at 32°C in M. trossulus only, indicating that its ability to combat heat-induced oxidative stress is limited to lower temperatures. Levels of NADdependent deacetylase (sirtuin 5), which are correlated with lifespan, were lower in M. trossulus in response to heat stress. In summary, the expression patterns of proteins involved in molecular chaperoning, proteolysis, energy metabolism, oxidative damage, cytoskeleton and deacetylation revealed a common loci of heat stress in both mussels but also showed a lower sensitivity to high-temperature damage in the warm-adapted M. galloprovincialis, which is consistent with its expanding range in warmer waters. Supplementary material available online at

SUMMARYEstuaries are characterized by extreme fluctuations in CO 2 levels due to bouts of CO 2 production by the resident biota that exceed its capacity of CO 2 consumption and/or the rates of gas exchange with the atmosphere and open ocean waters. Elevated partial pressures of CO 2 (P CO2; i.e. environmental hypercapnia) decrease the pH of estuarine waters and, ultimately, extracellular and intracellular pH levels of estuarine organisms such as mollusks that have limited capacity for pH regulation. We analyzed proteomic changes associated with exposure to elevated P CO2 in the mantle tissue of eastern oysters (Crassostrea virginica) after 2weeks of exposure to control (~39Pa P CO2 ) and hypercapnic (~357Pa P CO2 ) conditions using two-dimensional gel electrophoresis and tandem mass spectrometry. Exposure to high P CO2 resulted in a significant proteome shift in the mantle tissue, with 12% of proteins (54 out of 456) differentially expressed under the high P CO2 compared with control conditions. Of the 54 differentially expressed proteins, we were able to identify 17. Among the identified proteins, two main functional categories were upregulated in response to hypercapnia: those associated with the cytoskeleton (e.g. several actin isoforms) and those associated with oxidative stress (e.g. superoxide dismutase and several peroxiredoxins as well as the thioredoxin-related nucleoredoxin). This indicates that exposure to high P CO2 (~357Pa) induces oxidative stress and suggests that the cytoskeleton is a major target of oxidative stress. We discuss how elevated CO 2 levels may cause oxidative stress by increasing the production of reactive oxygen species (ROS) either indirectly by lowering organismal pH, which may enhance the Fenton reaction, and/or directly by CO 2 interacting with other ROS to form more free radicals. Although estuarine species are already exposed to higher and more variable levels of CO 2 than other marine species, climate change may further increase the extremes and thereby cause greater levels of oxidative stress.

SUMMARYThe ability to acclimate to variable environmental conditions affects the biogeographic range of species, their success at colonizing new habitats, and their likelihood of surviving rapid anthropogenic climate change. Here we compared responses to temperature acclimation (4weeks at 7, 13 and 19°C) in gill tissue of the warm-adapted intertidal blue mussel Mytilus galloprovincialis, an invasive species in the northeastern Pacific, and the cold-adapted M. trossulus, the native congener in the region, to better understand the physiological differences underlying the ongoing competition. Using two-dimensional gel electrophoresis and tandem mass spectrometry, we showed that warm acclimation caused changes in cytoskeletal composition and proteins of energy metabolism in both species, consistent with increasing rates of filtration and respiration due to increased ciliary activity. During cold acclimation, changes in cytoskeletal proteins were accompanied by increasing abundances of oxidative stress proteins and molecular chaperones, possibly because of the increased production of aldehydes as indicated by the upregulation of aldehyde dehydrogenase. The cold-adapted M. trossulus showed increased abundances of molecular chaperones at 19°C, but M. galloprovincialis did not, suggesting that the two species differ in their long-term upper thermal limits. In contrast, the warm-adapted M. galloprovincialis showed a stronger response to cold acclimation than M. trossulus, including changes in abundance in more proteins and differing protein expression profiles between 7 and 13°C, a pattern absent in M. trossulus. In general, increasing levels of oxidative stress proteins inversely correlate with modifications in Krebs cycle and electron transport chain proteins, indicating a trade-off between oxidative stress resistance and energy production. Overall, our results help explain why M. galloprovincialis has replaced M. trossulus in southern California over the last century, but also suggest that M. trossulus may maintain a competitive advantage at colder temperatures. Anthropogenic global warming may reinforce the advantage M. galloprovincialis has over M. trossulus in the warmer parts of the latterʼs historical range. Supplementary material available online at

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

The blue mussels and are competing species with biogeographical ranges set in part by environmental exposure to heat and hyposalinity. The underlying cellular mechanisms influencing interspecific differences in stress tolerance are unknown, but are believed to be under regulation by sirtuins, nicotinamide adenine dinucleotide (NAD)-dependent deacylases that play a critical role in the cellular stress response. A comparison of the proteomic responses of and to an acute heat shock in the presence and absence of the sirtuin inhibitor suramin (SIRT1, 2 and 5) showed that sirtuins affected molecular chaperones, oxidative stress proteins, metabolic enzymes, cytoskeletal and signaling proteins more in the heat-sensitive than in the heat-tolerant Interactions between sirtuin inhibition and changes in the abundance of proteins of β-oxidation and oxidative stress in suggest a greater role of sirtuins in shifting metabolism to reduce the production of reactive oxygen species near thermal limits. Furthermore, RNA-binding proteins initiating and inhibiting translation were affected by suramin in and , respectively. Western blot analysis showed that the levels of mitochondrial sirtuin 5 (SIRT5) were generally three times higher and increased with acute heat stress in response to sirtuin inhibition in but not in , suggesting a possible feedback response in the former species and a greater reliance on SIRT5 for its stress response. Our findings suggest that SIRT5 plays an important role in setting interspecific differences in stress tolerance in by affecting the stress proteome.