Metabolic cost of thermoregulation decreases after the molt in developing Weddell seal pups.

Allocation of energy to thermoregulation greatly contributes to the metabolic cost of endothermy, especially in extreme ambient conditions. Weddell seal (Leptonychotes weddellii) pups born in Antarctica must survive both on ice and in water, two environments with very different thermal conductivities. This disparity likely requires pups to allocate additional energy toward thermoregulation rather than growth or development of swimming capabilities required for independent foraging. We measured longitudinal changes in resting metabolic rate (RMR) for Weddell seal pups (n=8) in air and water from one to seven weeks of age, using open-flow respirometry. Concurrently, we collected molt, morphometric and dive behavior data. Absolute metabolic rate (MR) in air followed the expected allometric relationship with mass. Absolute MR in water was not allometric with mass, despite a 3-fold increase in mass between one and seven weeks of age. Developmental stage (or molting stage), rather than calendar age, determined when pups were thermally capable of being in the water. We consistently observed post-molt pups had lower RMR in air and water (6.67±1.4 and 7.90±2.38 ml O2 min-1 kg-1, respectively) than pre-molt (air: 9.37±2.42 ml O2 min-1 kg-1, water: 13.40±3.46 ml O2 min-1 kg-1) and molting pups (air: 8.45±2.05 ml O2 min-1 kg-1, water: 10.4±1.63 ml O2 min-1 kg-1). RMR in air and water were equivalent only for post-molt pups. Despite the increased energy cost, molting pups spent three times longer in the water than other pups. These results support the idea of an energetic trade-off during early development; pups expend more energy for thermoregulation in water, yet gain experience needed for independence.

Circadian and circatidal rhythms of protein abundance in the California mussel (Mytilus californianus).

Coastal habitats fluctuate with the 12.4 h tidal and 24 h light/dark cycle to predictably alter conditions such as air exposure, temperature, and food availability. Intertidal sessile bivalves exhibit behavioural and physiological adjustments to minimize the challenges of this environment. We investigated a high-resolution time course of the changes in protein abundance in the gill tissue of the intertidal mussel Mytilus californianus in a simulated tidal environment of 12:12 h light:dark cycles and a matching 6:6 h high:low tide cycle within each 12 h period. Approximately 38% of detected proteins showed significant rhythms in their abundances, with diversity in the phases of rhythmic isoforms. The circadian rhythm was dominant in protein abundance changes, particularly with oxidative metabolism. A tidal cycle elicited changes within functional groups, including in cytoskeletal proteins, chaperones, and oxidative stress proteins. In addition to protein abundance changes, we found the possibility for post-translational modifications driving rhythms, including methylation, mitochondrial peptide processing (proteolysis), and acylation. Dynamic changes in the proteome across functional categories demonstrate the importance of the tidal environment in entraining cellular processes, confirming that differential expression studies should not assume a static baseline of cellular conditions in intertidal organisms.

Dynamic regulation of coral energy metabolism throughout the diel cycle.

Coral reefs are naturally exposed to daily and seasonal variations in environmental oxygen levels, which can be exacerbated in intensity and duration by anthropogenic activities. However, coral's diel oxygen dynamics and fermentative pathways remain poorly understood. Here, continuous oxygen microelectrode recordings in the coral diffusive boundary layer revealed hyperoxia during daytime and hypoxia at nighttime resulting from net photosynthesis and net respiration, respectively. The activities of the metabolic enzymes citrate synthase (CS), malate dehydrogenase, and strombine dehydrogenase remained constant throughout the day/night cycle, suggesting that energy metabolism was regulated through adjustments in metabolite fluxes and not through changes in enzyme abundance. Liquid chromatography-mass spectrometry analyses identified strombine as coral's main fermentative end product. Strombine levels peaked as oxygen became depleted at dusk, indicating increased fermentation rates at the onset of nightly hypoxia, and again at dawn as photosynthesis restored oxygen and photosynthate supply. When these peaks were excluded from the analyses, average strombine levels during the day were nearly double those at night, indicating sifnificant fermentation rates even during aerobic conditions. These results highlight the dynamic changes in oxygen levels in the coral diffusive boundary layer, and the importance of fermentative metabolism for coral biology.

Multiple stressor responses are regulated by sirtuins in Mytilus congeners.

Understanding physiological tolerances of marine organisms to environmental stress is key to predicting species susceptability under climate change. Along the Pacific Coast of the U.S.A. intertidal mussel congeners (genus Mytilis) vary in their physiological stress tolerances, with the invasive M. galloprovincialis being heat tolerant but vulnerable to hyposalinity while the native M. trossulus is vulnerable to heat stress and tolerant of hyposalinity. Sirtuins, a family of NAD+-dependent deacylases, may influence the environmental stressor tolerances in these mussel congeners. The purpose of our study was to determine the mechanism by which sirtuins may confer differential stress responses in the two mussel congeners. Mussels (N = 6 per species) were acclimated to laboratory conditions in tidal simulators and exposed to sirtuin inhibitors (suramin and nicotinamide). Following inhibition, mussels were exposed to hyposalinity stress (29 ppt) for 6 h followed by aerial heat stress (32 °C) for 6 h after which mussel gill was dissected for proteomic analysis. During sirtuin inhibition we found a reduction of cellular stress response (CSR) proteins (molecular chaperones, antioxidants), which are key to maintaining cellular homeostasis. Moreover, we found differential stress responses between the two species under aerial heat combined with hyposalinity exposure. Three-way interactions (aerial heat, hyposalinity and sirtuin inhibition combined) showed complex interactive effects with sirtuins as potential modulators. Thus, our study suggests that sirtuins are contributing to the species-specific CSR in Mytilus and our multiple-stressor approach provides information used to make predictions regarding climate change effects on these competing species.

Cadmium-Related Effects on Cellular Immunity Comprises Altered Metabolism in Earthworm Coelomocytes.

The heavy metal cadmium (Cd) is known to modulate the immune system, challenging soil-dwelling organisms where environmental Cd pollution is high. Since earthworms lack adaptive immunity, we determined Cd-related effects on coelomocytes, the cellular part of innate immunity, which is also the site of detoxification processes. A proteomics approach revealed a set of immunity-related proteins as well as gene products involved in energy metabolism changing in earthworms in response to Cd exposure. Based on these results, we conducted extracellular flux measurements of oxygen and acidification to reveal the effect of Cd on coelomocyte metabolism. We observed a significantly changing oxygen consumption rate, extracellular acidification, as well as metabolic potential, which can be defined as the response to an induced energy demand. Acute changes in intracellular calcium levels were also observed, indicating impaired coelomocyte activation. Lysosomes, the cell protein recycling center, and mitochondrial parameters did not change. Taken together, we were able to characterize coelomocyte metabolism to reveal a potential link to an impaired immune system upon Cd exposure.

Ocean Acidification and Coastal Marine Invertebrates: Tracking CO2 Effects from Seawater to the Cell.

In the last few decades, numerous studies have investigated the impacts of simulated ocean acidification on marine species and communities, particularly those inhabiting dynamic coastal systems. Despite these research efforts, there are many gaps in our understanding, particularly with respect to physiological mechanisms that lead to pathologies. In this review, we trace how carbonate system disturbances propagate from the coastal environment into marine invertebrates and highlight mechanistic links between these disturbances and organism function. We also point toward several processes related to basic invertebrate biology that are severely understudied and prevent an accurate understanding of how carbonate system dynamics influence organismic homeostasis and fitness-related traits. We recommend that significant research effort be directed to studying cellular phenotypes of invertebrates acclimated or adapted to elevated seawater pCO2 using biochemical and physiological methods.

Sirtuins as regulators of the cellular stress response and metabolism in marine ectotherms.

The effects of climate change are altering the environmental landscape of marine habitats and exposing organisms to stressful conditions that may exceed their tolerance limits. Marine intertidal organisms are well adapted to fluctuating environments by adjusting energy metabolism and inducing the cellular stress response (CSR). Recent studies have shown that food availability can influence stress tolerance of marine ectotherms where a well-fed organism is more "robust" and more likely to survive a stressor than an animal under a low-food regime. We propose that the link between food availability and stress tolerance in marine ectotherms may be regulated by sirtuins, NAD+-dependent deacylases. In model organisms sirtuins act as energy sensors and are active under calorie restricted states where they target and regulate cellular metabolism, minimize oxidative stress, and influence the CSR. However, we know little regarding sirtuins in marine ectotherms. Herein we review the current literature on sirtuins in marine ectotherms including marine teleosts, limpets, and mussels. We show that the role of sirtuins in marine ectotherms is conserved from model organisms in regulating the CSR and energy, but the direct connection to NAD+ status under fed and starved conditions requires more attention. Although there is a beginning foundation of research regarding sirtuins in marine organisms, it is limited and would benefit from targeted studies investigating sirtuin activity in various tissues and animals under multiple stressors, NAD+/NADH levels under various fed states, and by using known sirtuin inhibitors and activators to elucidate the potential targets of sirtuins in marine animals.

Proteomic changes across a natural temperature gradient in a marine gastropod.

Responses of marine ectotherms to variable environmental temperature often entails maintanence of cellular homeostasis and physiological function through temperature compensation and physiological changes. We investigated the physiological response to thermal stress by examining proteomic changes in the marine kelp forest gastropod and emerging fisheries species Kellet's whelk (Kelletia kelletii) across a naturally-existing thermal gradient that ranges from a warmer-water site inside the species' native range and extends to the northern, cold-water edge of the range. We hypothesized that abundance of cellular stress response and energy metabolism proteins would increase with decreasing temperature in support of cold-compensation. Our exploratory proteomic analysis of whelk gill tissue (N = 6 whelks) from each of the four California Channel Island sites revealed protein abundance changes related to the cytoskeleton, energy metabolism/oxidative stress, and cell signaling. The changes did not correlate consistently with temperature. Nonetheless, whelks from the coldest island site showed increased abundance of energy metabolism and oxidative stress proteins, possibly suggesting oxidative damage of lipid membranes that is ameliorated by antioxidants and may aid in their cold stress response. Similarly, our exploratory analysis revealed abundances of cell signaling proteins that were higher at the coldest site compared to the warmest site, possibly indicating an importance for cell signaling regulation in relatively cooler environments. This study provides protein targets for future studies related to thermal effects in marine animals and may contribute to understanding the physiological response of marine organisms to future ocean conditions.

Phytochrome A Regulates Carbon Flux in Dark Grown Tomato Seedlings.

Phytochromes comprise a small family of photoreceptors with which plants gather environmental information that they use to make developmental decisions, from germination to photomorphogenesis to fruit development. Most phytochromes are activated by red light and de-activated by far-red light, but phytochrome A (phyA) is responsive to both and plays an important role during the well-studied transition of seedlings from dark to light growth. The role of phytochromes during skotomorphogenesis (dark development) prior to reaching light, however, has received considerably less attention although previous studies have suggested that phytochrome must play a role even in the dark. We profiled proteomic and transcriptomic seedling responses in tomato during the transition from dark to light growth and found that phyA participates in the regulation of carbon flux through major primary metabolic pathways, such as glycolysis, beta-oxidation, and the tricarboxylic acid (TCA) cycle. Additionally, phyA is involved in the attenuation of root growth soon after reaching light, possibly via control of sucrose allocation throughout the seedling by fine-tuning the expression levels of several sucrose transporters of the SWEET gene family even before the seedling reaches the light. Presumably, by participating in the control of major metabolic pathways, phyA sets the stage for photomorphogenesis for the dark grown seedling in anticipation of light.

Proteomic analysis of the crustacean molting gland (Y-organ) over the course of the molt cycle.

Molting in crustaceans is a highly complex physiological process involving regulation by two paired endocrine glands, the X-organ/sinus gland complex (XO/SG) and the Y-organ (YO). The XO/SG complex is responsible for making molt-inhibiting hormone, which negatively regulates synthesis of molting hormones, ecdysteroids, by the YO. In this study, changes in protein abundance in the YO were characterized over the course of a molt cycle induced by multiple leg autotomy in the blackback land crab, Gecarcinus lateralis. In all, 457 distinct protein spots were detected using two-dimensional gel electrophoresis, of which 230 (50%) changed significantly in abundance over the course of the molt cycle. Protein abundance differed most notably between intermolt and the three premolt stages, indicative of a biological 'on-off' switch. Changes in hemolymph proteins were correlated with stage-specific processes of sclerotization and melanization that facilitate cuticle hardening and support immune reactions. An abundance of cytoskeletal proteins were identified, which corresponded with glandular hypertrophy associated with synthesis and secretion of ecdysteroids. Many proteins involved in energetic pathways including glycolysis, the citric acid cycle, amino acid metabolism, and one­carbon metabolism changed in abundance in response to increasing energy demands and the requirement for precursors of macromolecular synthesis. Several proteins involved in immune, proteostasis, and oxidative stress responses were correlated with the dynamic and demanding cellular changes associated with ecdysteroidogenesis. These changes in diverse physiological pathways represent the complexity involved with molecular regulation of the YO in decapod crustaceans.

Sirtuins regulate proteomic responses near thermal tolerance limits in the blue mussels Mytilus galloprovincialis and Mytilus trossulus.

The blue mussels Mytilus galloprovincialis and M. trossulus 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 M. galloprovincialis and M. trossulus 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 M. trossulus than in the heat-tolerant M. galloprovincialis Interactions between sirtuin inhibition and changes in the abundance of proteins of ß-oxidation and oxidative stress in M. trossulus 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 M. galloprovincialis and M. trossulus, 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 M. trossulus but not in M. galloprovincialis, 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 Mytilus by affecting the stress proteome.

Time course of lead induced proteomic changes in gill of the Antarctic limpet Nacella Concinna (Gastropoda: Patellidae).

The effect of increasing levels of metals from anthropogenic sources on Antarctic invertebrates is poorly understood. Here we exposed limpets (Nacella concinna) to 0, 0.12 and 0.25 µg L− 1 lead for 12, 24, 48 and 168 h. We subsequently quantified the changes in protein abundance from gill, using 2D gel electrophoresis and mass spectrometry. We identified several antioxidant proteins, including the metal binding Mn-superoxide dismutase and ferritin, increasing abundances early on. Chaperones involved in the redox-dependent maturation of proteins in the endoplasmic reticulum (ER) showed higher abundance with lead at 48 h. Lead also increased the abundance of Zn-binding carbonic anhydrase at 12 h, suggesting a challenge to acid-base balance. Metabolic proteins increased abundance at 168 h, suggesting a greater ATP demand during prolonged exposure. Changes in abundance of the small G-protein cdc42, a signaling protein modifying cytoskeleton, increased early and subsequently reversed during prolonged exposure, possibly leading to the modification of thick filament structure and function. We hypothesize that the replacement of metals initially affected antioxidant proteins and increased the production of reactive oxygen species. This disrupted the redox-sensitive maturation of proteins in the ER and caused increased ATP demand later on, accompanied by changes in cytoskeleton. SIGNIFICANCE: Proteomic analysis of gill tissue in Antarctic limpets exposed to different concentrations of lead (Pb) over a 168 h time period showed that proteomic changes vary with time. These changes included an increase in the demand of scavenging reactive oxygen species, acid-base balance and a challenge to protein homeostasis in the endoplasmic reticulum early on and subsequently an increase in energy metabolism, cellular signaling, and cytoskeletal modifications. Based on this time course, we hypothesize that the main mode of action of lead is a replacement of metal-cofactors of key enzymes involved in the scavenging of reactive oxygen species and the regulation of acid-base balance.

Proteomic responses to environmentally induced oxidative stress.

Environmental (acute and chronic temperature, osmotic, hypoxic and pH) stress challenges the cellular redox balance and can lead to the increased production of reactive oxygen species (ROS). This review provides an overview of the reactions producing and scavenging ROS in the mitochondria, endoplasmic reticulum (ER) and peroxisome. It then compares these reactions with the findings of a number of studies investigating the proteomic responses of marine organisms to environmentally induced oxidative stress. These responses indicate that the thioredoxin-peroxiredoxin system is possibly more frequently recruited to scavenge H2O2 than the glutathione system. Isoforms of superoxide dismutase (SOD) are not ubiquitously induced in parallel, suggesting that SOD scavenging activity is sometimes sufficient. The glutathione system plays an important role in some organisms and probably also contributes to protecting protein thiols during environmental stress. Synthesis pathways of cysteine and selenocysteine, building blocks for glutathione and glutathione peroxidase, also play an important role in scavenging ROS during stress. The increased abundance of glutaredoxin and DyP-type peroxidase suggests a need for regulating the deglutathionylation of proteins and scavenging of peroxynitrite. Reducing equivalents for these scavenging reactions are generated by proteins of the pentose phosphate pathway and by NADP-dependent isocitrate dehydrogenase. Furthermore, proteins representing reactions of the tricarboxylic acid cycle and the electron transport system generating NADH and ROS, including those of complex I, II and III, are frequently reduced in abundance with stress. Protein maturation in the ER likely represents another source of ROS during environmental stress, as indicated by simultaneous changes in ER chaperones and antioxidant proteins. Although there are still too few proteomic analyses of non-model organisms exposed to environmental stress for a general pattern to emerge, hyposaline and low pH stress show different responses from temperature and hypoxic stress. Furthermore, comparisons of closely related congeners differing in stress tolerance start to provide insights into biochemical processes contributing to adaptive differences, but more of these comparisons are needed to draw general conclusions. To fully take advantage of a systems approach, studies with longer time courses, including several tissues and more species comparisons are needed.

Proteomic analysis of cardiac response to thermal acclimation in the eurythermal goby fish Gillichthys mirabilis.

Cardiac function is thought to play a central role in determining thermal optima and tolerance limits in teleost fishes. Investigating proteomic responses to temperature in cardiac tissues may provide insights into mechanisms supporting the thermal plasticity of cardiac function. Here, we utilized a global proteomic analysis to investigate changes in cardiac protein abundance in response to temperature acclimation (transfer from 13°C to 9, 19 and 26°C) in a eurythermal goby, Gillichthys mirabilis. Proteomic data revealed 122 differentially expressed proteins across acclimation groups, 37 of which were identified using tandem mass-spectrometry. These 37 proteins are involved in energy metabolism, mitochondrial regulation, iron homeostasis, cytoprotection against hypoxia, and cytoskeletal organization. Compared with the 9 and 26°C groups, proteins involved in energy metabolism increased in 19°C-acclimated fish, indicating an overall increase in the capacity for ATP production. Creatine kinase abundance increased in 9°C-acclimated fish, suggesting an important role for the phosphocreatine energy shuttle in cold-acclimated hearts. Both 9 and 26°C fish also increased abundance of hexosaminidase, a protein directly involved in post-hypoxia stress cytoprotection of cardiac tissues. Cytoskeletal restructuring appears to occur in all acclimation groups; however, the most prominent effect was detected in 26°C-acclimated fish, which exhibited significantly increased actin levels. Overall, proteomic analysis of cardiac tissue suggests that the capacity to adjust ATP-generating processes is crucial to the thermal plasticity of cardiac function. Furthermore, G. mirabilis may optimize cellular functions at temperatures near 19°C, which lies within the species' preferred temperature range.

The proteomic response of cheliped myofibril tissue in the eurythermal porcelain crab Petrolisthes cinctipes to heat shock following acclimation to daily temperature fluctuations.

The porcelain crab Petrolisthes cinctipes lives under rocks and in mussel beds in the mid-intertidal zone where it experiences immersion during high tide and saturating humid conditions in air during low tide, which can increase habitat temperature by up to 20°C. To identify the biochemical changes affected by increasing temperature fluctuations and subsequent heat shock, we acclimated P. cinctipes for 30 days to one of three temperature regimes: (1) constant 10°C, (2) daily temperature fluctuations between 10 and 20°C (5 h up-ramp to 20°C, 1 h down-ramp to 10°C) and (3) 10-30°C (up-ramp to 30°C). After acclimation, animals were exposed to either 10°C or a 30°C heat shock to analyze the proteomic changes in claw muscle tissue. Following acclimation to 10-30°C (measured at 10°C), enolase and ATP synthase increased in abundance. Following heat shock, isoforms of arginine kinase and glycolytic enzymes such as aldolase, triose phosphate isomerase and glyceraldehyde 3-phosphate dehydrogenase increased across all acclimation regimes. Full-length isoforms of hemocyanin increased abundance following acclimation to 10-30°C, but hemocyanin fragments increased after heat shock following constant 10°C and fluctuating 10-20°C, possibly playing a role as antimicrobial peptides. Following constant 10°C and fluctuating 10-20°C, paramyosin and myosin heavy chain type-B increased in abundance, respectively, whereas myosin light and heavy chain decreased with heat shock. Actin-binding proteins, which stabilize actin filaments (filamin and tropomyosin), increased during heat shock following 10-30°C; however, actin severing and depolymerization proteins (gelsolin and cofilin) increased during heat shock following 10-20°C, possibly promoting muscle fiber restructuring. RAF kinase inhibitor protein and prostaglandin reductase increased during heat shock following constant 10°C and fluctuating 10-20°C, possibly inhibiting an immune response during heat shock. The results suggest that ATP supply, muscle fiber restructuring and immune responses are all affected by temperature fluctuations and subsequent acute heat shock in muscle tissue. Furthermore, although heat shock after acclimation to constant 10°C and fluctuating 10-30°C showed the greatest effects on the proteome, moderately fluctuating temperatures (10-20°C) broadened the temperature range over which claw muscle was able to respond to an acute heat shock with limited changes in the muscle proteome.

Proteomics to study adaptations in marine organisms to environmental stress.

Comparisons of proteomic responses of closely related congeners and populations have shown which cellular processes are critical to adapt to environmental stress. For example, several proteomic species comparisons showed that increasing abundances of oxidative stress proteins indicate that reactive oxygen species (ROS) represent a ubiquitous signal and possible co-stressor of warm and cold temperature, acute hyposaline and low pH stress, possibly causing a shift from pro-oxidant NADH-producing to anti-oxidant NADPH-producing and -consuming metabolic pathways. Changes in cytoskeletal and actin-binding proteins in response to several stressors, including ROS, suggest that both are important structural and functional elements in responding to stress. Disruption of protein homeostasis, e.g., increased abundance of molecular chaperones, was severe in response to acute heat stress, inducing proteolysis, but was also observed in response to chronic heat and cold stress and was concentrated to the endoplasmic reticulum during hyposaline stress. Small GTPases affecting vesicle formation and transport, Ca(2+)-signaling and ion transport responded to salinity stress in species- and population-specific ways. Aerobic energy metabolism was in general down-regulated in response to temperature, hypoxia, hyposalinity and low pH stress, but other metabolic pathways were activated to respond to increased oxidative stress or to switch metabolic fuels. Thus, comparative proteomics is a powerful approach to identify functionally adaptive variation. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Introduction to the symposium "Comparative proteomics of environmental and pollution stress".

The study of the proteome in response to environmental change is beginning to generate a number of new hypotheses about how organisms respond and adapt to a variety of stressors. The contributions to this symposium highlight how comparisons at the levels of species, populations, and tissues provide exciting new perspectives on the diversity of biochemical responses involved in the tolerance of stress. Despite limited genomic information, a number of studies of nonmodel organisms provide insights that are only accessible through a systems approach like proteomics. The realization that these systemic responses differ among closely related species, populations, and tissues illustrates the potential importance of the proteome to an organism's evolutionary response to a rapidly changing environment. Changes in an organism's proteome may occur as early as during the first stages of development and continue through acclimatization of the adult and adaptation of the following generations. A proteomic approach can also demonstrate how pollutants have systemic effects that may be counter-intuitive to expectations, emphasizing how isolating a single mode of action for a pollutant, e.g., xeno-androgen, is often inadequate. To continue with the progress made, we need a critical evaluation of the experimental designs used in proteomics studies, a reevaluation of some of the statistical analyses, and new technical advances in order to identify a greater number of proteins. The contributions to the current symposium offer the novice a starting point for assessing the potential of proteomics to generate novel hypotheses about how organisms interact with their environment.

Environmental proteomics of the mussel Mytilus: implications for tolerance to stress and change in limits of biogeographic ranges in response to climate change.

Climate change will affect temperature extremes and averages, and hyposaline conditions in coastal areas due to extreme precipitation events and oceanic pH. How climate change will push species close to, or beyond, their physiological tolerance limits as well as change the limits of their biogeographic ranges can probably be investigated best in species that have already responded to climate change and whose distribution ranges are currently in flux. Blue mussels provide such a study system, with the invading warm-adapted Mediterranean Mytilus galloprovincialis having replaced the native more cold-adapted Mytilus trossulus from the southern part of its range in southern California over the past century, possibly due to climate change. However, freshwater input may prevent the latter species from expanding further north. We used a proteomics approach to characterize the responses of the two congeners to acute heat stress, chronic thermal acclimation, and hyposaline stress. In addition, we investigated the proteomic changes in response to decreasing seawater pH in another bivalve, the eastern oyster Crassostrea virginica. The results suggest that reactive oxygen species (ROS) are a common costressor during environmental stress, including oceanic acidification, and possibly cause modifications of cytoskeletal elements. All stressors disrupted protein homeostasis, indicated by the induction of molecular chaperones and, in the case of acute heat stress, proteasome isoforms, possibly due both to protein denaturation directly by the stressor and to the production of ROS. Acute stress by heat and hyposalinity changed several small G-proteins implicated in cytoskeletal modifications and vesicular transport, respectively. Changes in abundance of proteins involved in energy metabolism and ROS scavenging further suggest a possible trade-off during acute and chronic stress from heat and cold between ROS-generating NADH-producing pathways and ROS-scavenging NADPH-producing pathways, especially through the reaction of NADPH-dependent isocitrate dehydrogenase and the pentose-phosphate pathway. Some of the proteomic changes may not constitute de novo protein synthesis but rather shifts in abundance of isoforms differing in posttranslational modifications, specifically acetylation by a NAD-dependent deacetylase (sirtuin). Interspecific differences suggest that these processes set physiological tolerance limits and thereby contribute to recent biogeographic shifts in range, possibly caused by climate change.

Proteomics of hyposaline stress in blue mussel congeners (genus Mytilus): implications for biogeographic range limits in response to climate change.

Climate change is affecting species' 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. trossulus' range, thereby preventing M. galloprovincialis from expanding further north.