Lars Tomanek

Variation in the heat shock response and its implication for predicting the effect of global climate change on species' biogeographical distribution ranges and metabolic costs

SUMMARY The preferential synthesis of heat shock proteins (Hsps) in response to thermal stress [the heat shock response (HSR)] has been shown to vary in species that occupy different thermal environments. A survey of case studies of aquatic (mostly marine) organisms occupying stable thermal environments at all latitudes, from polar to tropical, shows that they do not in general respond to heat stress with an inducible HSR. Organisms that occupy highly variable thermal environments (variations up to >20°C), like the intertidal zone, induce the HSR frequently and within the range of body temperatures they normally experience, suggesting that the response is part of their biochemical strategy to occupy this thermal niche. The highest temperatures at which these organisms can synthesize Hsps are only a few degrees Celsius higher than the highest body temperatures they experience. Thus, they live close to their thermal limits and any further increase in temperature is probably going to push them beyond those limits. In comparison, organisms occupying moderately variable thermal environments (<10°C), like the subtidal zone, activate the HSR at temperatures above those they normally experience in their habitats. They have a wider temperature range above their body temperature range over which they can synthesize Hsps. Contrary to our expectations, species from highly (in comparison with moderately) variable thermal environments have a limited acclimatory plasticity. Due to this variation in the HSR, species from stable and highly variable environments are likely to be more affected by climate change than species from moderately variable environments.

The proteomic response of the mussel congeners Mytilus galloprovincialis and M. trossulus to acute heat stress: implications for thermal tolerance limits and metabolic costs of thermal stress

SUMMARY The 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 NAD-dependent 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.

Proteomic response to elevated PCO2 level in eastern oysters, Crassostrea virginica: evidence for oxidative stress.

SUMMARY Estuaries are characterized by extreme fluctuations in CO2 levels due to bouts of CO2 production by the resident biota that exceed its capacity of CO2 consumption and/or the rates of gas exchange with the atmosphere and open ocean waters. Elevated partial pressures of CO2 (PCO2; 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 PCO2 in the mantle tissue of eastern oysters (Crassostrea virginica) after 2 weeks of exposure to control (∼39 Pa PCO2) and hypercapnic (∼357 Pa PCO2) conditions using two-dimensional gel electrophoresis and tandem mass spectrometry. Exposure to high PCO2 resulted in a significant proteome shift in the mantle tissue, with 12% of proteins (54 out of 456) differentially expressed under the high PCO2 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 PCO2 (∼357 Pa) induces oxidative stress and suggests that the cytoskeleton is a major target of oxidative stress. We discuss how elevated CO2 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 CO2 interacting with other ROS to form more free radicals. Although estuarine species are already exposed to higher and more variable levels of CO2 than other marine species, climate change may further increase the extremes and thereby cause greater levels of oxidative stress.

Time Course and Magnitude of Synthesis of Heat‐Shock Proteins in Congeneric Marine Snails (Genus Tegula) from Different Tidal Heights

The time course and magnitude of the heat‐shock response in relation to severity of thermal stress are important, yet poorly understood, aspects of thermotolerance. We examined patterns of protein synthesis in congeneric marine snails (genus Tegula) that occur at different heights along the subtidal to intertidal gradient after a thermal exposure (30°C for 2.5 h, followed by 50 h recovery at 13°C) that induced the heat‐shock response. We monitored the kinetics and magnitudes of protein synthesis by quantifying incorporation of 35S‐labeled methionine and cysteine into newly synthesized proteins and observed synthesis of putative heat‐shock proteins (hsps) of size classes 90, 77, 70, and 38 kDa. In the low‐ to mid‐intertidal species, Tegula funebralis, whose body temperature frequently exceeds 30°C during emersion, synthesis of hsps commenced immediately after heat stress, reached maximal levels 1–3 h into recovery, and returned to prestress levels by 6 h, except for hsp90 (14 h). In contrast, in the low‐intertidal to subtidal species, Tegula brunnea, for which 2.5 h at 30°C represents a near lethal heat stress, synthesis of hsps commenced 2–14 h after heat stress; reached maximal levels after 15–30 h, which exceeded magnitudes of synthesis in T. funebralis; and returned to prestress levels in the case of hsp90 (50 h) and hsp77 (30 h) but not in the case of hsp70 and hsp38. Exposures to 30°C under aerial (emersion) and aquatic (immersion) conditions resulted in differences in hsp synthesis in T. brunnea but not in T. funebralis. The different time courses and magnitudes of hsp synthesis in these congeners suggest that the vertical limits of their distributions may be set in part by thermal stress.

Physiological Ecology of Rocky Intertidal Organisms: A Synergy of Concepts

Abstract The rocky intertidal zone is among the most physically harsh environments on earth. Marine invertebrates and algae living in this habitat are alternatively pounded by waves and exposed to thermal extremes during low tide periods (Denny and Wethey, 2001). Additionally, they must deal with strong selective pressures related to predation and competition for space (Connell, 1961). As a result, the steep physical gradient and spatially condensed community has made the rocky intertidal zone an ideal “natural laboratory” to study the coupled role of physical and biological factors in determining the abundance and distribution of organisms in nature (Connell, 1961; Paine, 1966, 1994).

The Importance of Physiological Limits in Determining Biogeographical Range Shifts due to Global Climate Change: The Heat‐Shock Response

Physiological processes that set an organism’s thermal limits are in part determining recent shifts in biogeographic distribution ranges due to global climate change. Several characteristics of the heat‐shock response (HSR), such as the onset, maximal, and upper limit of heat‐shock protein (Hsp) synthesis, contribute to setting the acute upper thermal limits of most organisms. Aquatic animals from stable, moderately variable, or highly variable thermal environments differ in their HSR. Some animals living in extremely stable thermal environments lack the response altogether. In contrast, rocky intertidal animals that experience highly variable thermal conditions start synthesizing Hsps, that is, the onset of synthesis, below the highest temperatures that they experience. Thus, these organisms experience thermal conditions in their environment that are close to the upper thermal limits in which they can defend themselves against cellular thermal insults by employing the HSR. Subtidal animals are characterized by moderately variable thermal environments, and their cells start synthesizing Hsps above the highest temperatures that they experience. The upper thermal limits against which they can defend themselves are thus much higher than the highest body temperatures they currently experience. Furthermore, the ability to acclimate to changing thermal conditions seems greatest among animals from moderately variable environments and limited in animals from stable and highly variable environments. Thus, these findings suggest that organisms with the narrowest (stenothermal) and the widest (highly eurythermal) temperature tolerance ranges live closest to their thermal limits and have a limited ability to acclimate, suggesting that they will be most affected by global climate change.

Heat-Shock Protein 70 (Hsp70) as a Biochemical Stress Indicator: an Experimental Field Test in Two Congeneric Intertidal Gastropods (Genus: Tegula)

Although previous studies have demonstrated that heat-shock protein 70 (Hsp70) can be induced by environmental stress, little is known about natural variation in this response over short time scales. We examined how Hsp70 levels varied over days to weeks in two intertidal snail species of the genus Tegula. Sampling was conducted both under naturally changing environmental conditions and in different vertical zones on a rocky shore. The subtidal to low-intertidal T. brunnea was transplanted into shaded and unshaded mid-intertidal cages to assess temporal variation in Hsps under conditions of increased stress. For comparison, the low to mid-intertidal T. funebralis was transplanted into mid-intertidal cages, within this species’ natural zone of occurrence. Snails were sampled every 3 to 4 days for one month, and endogenous levels of two Hsp70-kDa family members (Hsp72 and Hsp74) were quantified using solid-phase immunochemistry. Following periods of midday low tides, levels of Hsps increased greatly in transplanted T. brunnea but not in T. funebralis. Levels of Hsps increased less in T. brunnea transplanted to shaded cages than to unshaded cages, suggesting that prolonged emersion and reduction in feeding time per se are factors that are only mildly stressful. Upregulated levels of Hsps returned to base levels within days. In unmanipulated snails collected from their natural zones, Hsp levels showed little change with thermal variation, indicating that these species did not experience thermally stressful conditions during this study. However, under common conditions in the mid-intertidal zone, Hsp70 levels reflected the different thermal sensitivities of the physiological systems of these two species.

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.

Proteomic responses of blue mussel (Mytilus) congeners to temperature acclimation

SUMMARY The 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 latter’s historical range.

Two-Dimensional Gel Analysis of the Heat-Shock Response in Marine Snails (genus Tegula ): Interspecific Variation in Protein Expression and Acclimation Ability

SUMMARY The degree to which temperature acclimation modifies the acute synthesis of the entire heat-shock protein (Hsp) complement is still unknown, but it may constitute an important mechanism for understanding the differences in acclimation ability among closely related ectothermic species that occupy widely varying thermal environments. In general, eurythermal (heat-tolerant) species modify physiological function in response to an increase in acclimation temperature to a greater extent than stenothermal (heat-sensitive) species. In the present work I used 35S-labelled amino acids and two-dimensional gel electrophoresis to test this assumption for how acclimation affects acute Hsp expression (referred to as phenotypic plasticity) in two heat-sensitive, low-intertidal to subtidal zone turban snails, Tegula brunnea and T. montereyi, in comparison to a heat-tolerant, mid- to low-intertidal zone congener, T. funebralis. I was able (i) to detect the synthesis of over 30 proteins in gill tissue, primarily in the 70 kDa range, in response to an increase in temperature (13°C, 24°C, 27°C and 30°C), (ii) to assess the effect of acclimation (13°C vs 22°C) on acute Hsp synthesis, and (iii) to compare this effect among the three Tegula congeners. After increasing acclimation temperature from 13°C to 22°C, synthesis of the most highly expressed Hsps decreased more in T. brunnea and T. montereyi than in T. funebralis. Two highly expressed proteins of molecular mass 71 and 74 kDa, however, were also synthesized constitutively at 13°C and changed with increasing acclimation temperature in all three species. Although similar in phenotypic plasticity, T. brunnea and T. montereyi synthesized either a 76 or a 72 kDa cluster of proteins, respectively, and differed in how acclimation affected the acute synthesis of several 77 kDa proteins. Thus, in Tegula, the effect of acclimation on Hsp expression is (i) Hsp-specific, (ii) dependent on a proteins expression pattern (constitutive and inducible vs only inducible), (iii) and is actually limited in the more eurythermal mid- to low-intertidal congener. These results contradict the general assumption that greater heat tolerance correlates with an increased ability to modify physiological function in response to acclimation.