Thermal performance curves (TPCs), which quantify how an ectotherm's body temperature (T b ) affects its performance or fitness, are often used in an attempt to predict organismal responses to climate change. Here, we examine the key -but often biologically unreasonable -assumptions underlying this approach; for example, that physiology and thermal regimes are invariant over ontogeny, space and time, and also that TPCs are independent of previously experienced T b. We show how a critical consideration of these assumptions can lead to biologically useful hypotheses and experimental designs. For example, rather than assuming that TPCs are fixed during ontogeny, one can measure TPCs for each major life stage and incorporate these into stage-specific ecological models to reveal the life stage most likely to be vulnerable to climate change. Our overall goal is to explicitly examine the assumptions underlying the integration of TPCs with T b , to develop a framework within which empiricists can place their work within these limitations, and to facilitate the application of thermal physiology to understanding the biological implications of climate change.

Sensitivity to temperature helps determine the success of organisms in all habitats, and is caused by the susceptibility of biochemical processes, including enzyme function, to temperature change. A series of studies using two structurally and catalytically related enzymes, A 4 -lactate dehydrogenase (A 4 -LDH) and cytosolic malate dehydrogenase (cMDH) have been especially valuable in determining the functional attributes of enzymes most sensitive to temperature, and identifying amino acid substitutions that lead to changes in those attributes. The results of these efforts indicate that ligand binding affinity and catalytic rate are key targets during temperature adaptation: ligand affinity decreases during cold adaptation to allow more rapid catalysis. Structural changes causing these functional shifts often comprise only a single amino acid substitution in an enzyme subunit containing approximately 330 residues; they occur on the surface of the protein in or near regions of the enzyme that move during catalysis, but not in the active site; and they decrease stability in cold-adapted orthologs by altering intra-molecular hydrogen bonding patterns or interactions with the solvent. Despite these structure-function insights, we currently are unable to predict a priori how a particular substitution alters enzyme function in relation to temperature. A predictive ability of this nature might allow a proteome-wide survey of adaptation to temperature and reveal what fraction of the proteome may need to adapt to temperature changes of the order predicted by global warming models. Approaches employing algorithms that calculate changes in protein stability in response to a mutation have the potential to help predict temperature adaptation in enzymes; however, using examples of temperatureadaptive mutations in A 4 -LDH and cMDH, we find that the algorithms we tested currently lack the sensitivity to detect the small changes in flexibility that are central to enzyme adaptation to temperature.

Orthologous proteins of species adapted to different temperatures exhibit differences in stability and function that are interpreted to reflect adaptive variation in structural "flexibility." However, quantifying flexibility and comparing flexibility across proteins has remained a challenge. To address this issue, we examined temperature effects on cytosolic malate dehydrogenase (cMDH) orthologs from differently thermally adapted congeners of five genera of marine molluscs whose field body temperatures span a range of ∼60 °C. We describe consistent patterns of convergent evolution in adaptation of function [temperature effects on of cofactor (NADH)] and structural stability (rate of heat denaturation of activity). To determine how these differences depend on flexibilities of overall structure and of regions known to be important in binding and catalysis, we performed molecular dynamics simulation (MDS) analyses. MDS analyses revealed a significant negative correlation between adaptation temperature and heat-induced increase of backbone atom movements [root mean square deviation (rmsd) of main-chain atoms]. Root mean square fluctuations (RMSFs) of movement by individual amino acid residues varied across the sequence in a qualitatively similar pattern among orthologs. Regions of sequence involved in ligand binding and catalysis-termed mobile regions 1 and 2 (MR1 and MR2), respectively-showed the largest values for RMSF. Heat-induced changes in RMSF values across the sequence and, importantly, in MR1 and MR2 were greatest in cold-adapted species. MDS methods are shown to provide powerful tools for examining adaptation of enzymes by providing a quantitative index of protein flexibility and identifying sequence regions where adaptive change in flexibility occurs.

Limpets of the genus Lottia occupy a broad vertical distribution on wave-exposed rocky shores, a range that encompasses gradients in the frequency and severity of thermal and desiccation stress brought on by aerial emersion. Using western blot analysis of levels of heat-shock protein 70 (Hsp70), we examined the heat-shock responses of four Lottia congeners: Lottia scabra and L. austrodigitalis, which occur in the high-intertidal zone, and L. pelta and L. scutum, which are restricted to the low- and mid-intertidal zones. Our results suggest distinct strategies of Hsp70 expression in limpets occupying different heights and orientations in the rocky intertidal zone. In freshly field-collected animals and in specimens acclimated at ambient temperature ( approximately 14 degrees C) for 14 days, the two high-intertidal species had higher constitutive levels of Hsp70 than the low- and mid-intertidal species. During aerial exposure to high temperatures, the two low-shore species and L. austrodigitalis exhibited an onset of Hsp70 expression at 28 degrees C; no induction of Hsp70 occurred in L. scabra. Our findings suggest that high-intertidal congeners of Lottia employ a "preparative defense" strategy involving maintenance of high constitutive levels of Hsp70 in their cells as a mechanism for protection against periods of extreme and unpredictable heat stress.

SUMMARY Supplementary material available online at

Biogeographic distributions are driven by cumulative effects of smaller scale processes. Thus, vulnerability of animals to thermal stress is the result of physiological sensitivities to body temperature (T b ), microclimatic conditions, and behavioural thermoregulation. To understand interactions among these variables, we analysed the thermal tolerances of three species of intertidal snails from different latitudes along the Chinese coast, and estimated potential T b in different microhabitats at each site. We then empirically determined the temperatures at which heart rate decreased sharply with rising temperature (Arrhenius breakpoint temperature, ABT) and at which it fell to zero (flat line temperature, FLT) to calculate thermal safety margins (TSM). Regular exceedance of FLT in sun-exposed microhabitats, a lethal effect, was predicted for only one mid-latitude site. However, ABTs of some individuals were exceeded at sun-exposed microhabitats in most sites, suggesting physiological impairment for snails with poor behavioural thermoregulation and revealing inter-individual variations (physiological polymorphism) of thermal limits. An autocorrelation analysis of T b showed that predictability of extreme temperatures was lowest at the hottest sites, indicating that the effectiveness of behavioural thermoregulation is potentially lowest at these sites. These results illustrate the critical roles of mechanistic studies at small spatial scales when predicting effects of climate change.

SUMMARYContemporary theory for thermal adaptation of ectothermic metazoans focuses on the maximization of energy gain and performance (locomotion and foraging). Little consideration is given to the selection for mechanisms that minimize resting energy loss in organisms whose energy gain is severely constrained. We tested a hypothetical framework for thermal performance of locomotor activity (a proxy for energy gain) and resting metabolism (a proxy for energy loss) in energetically compromised snails in the littoral fringe zone, comparing this with existing theory. In contrast to theory, the thermal ranges and optima for locomotor performance and metabolic performance of Echinolittorina malaccana are mismatched, and energy gain is only possible at relatively cool temperatures. To overcome thermal and temporal constraints on energy gain while experiencing high body temperatures (23-50°C), these snails depress resting metabolism between 35 and 46°C (thermally insensitive zone). The resulting bimodal relationship for metabolism against temperature contrasts with the unimodal or exponential relationships of most ectotherms. Elevation of metabolism above the breakpoint temperature for thermal insensitivity (46°C) coincides with the induction of a heat shock response, and has implications for energy expenditure and natural selection. Time-dependent mortality is initiated at this breakpoint temperature, suggesting a threshold above which the rate of energy demand exceeds the capacity for cellular energy generation (rate of ATP turnover). Mortality in a thermal range that elevates rather than limits aerobic metabolism contrasts with the hypothesis that cellular oxygen deficiency underlies temperature-related mortality. The findings of this study point to the need to incorporate aspects of resting metabolism and energy conservation into theories of thermal adaptation.

Fish and other marine life are affected by ocean weather: drastic variations in temperature, pH, oxygen and salinity that are in turn influenced by climate change. ALEX MUSTARD/NPL 1 6 A U G U S T 2 0 1 8 | V O L 5 6 0 | N A T U R E | 2 9 9 COMMENT © 2 0 1 8 S p r i n g e r N a t u r e L i m i t e d . A l l r i g h t s r e s e r v e d . A full list of author affiliations accompanies this Comment online (see go.nature.com/2m8acbj).A Weddell seal equipped with a sensor for measuring ocean conductivity, temperature and depth.