Thermal adaptation of mRNA secondary structure: stability versus lability.

Macromolecular function commonly involves rapidly reversible alterations in three-dimensional structure (conformation). To allow these essential conformational changes, macromolecules must possess higher order structures that are appropriately balanced between rigidity and flexibility. Because of the low stabilization free energies (marginal stabilities) of macromolecule conformations, temperature changes have strong effects on conformation and, thereby, on function. As is well known for proteins, during evolution, temperature-adaptive changes in sequence foster retention of optimal marginal stability at a species' normal physiological temperatures. Here, we extend this type of analysis to messenger RNAs (mRNAs), a class of macromolecules for which the stability-lability balance has not been elucidated. We employ in silico methods to determine secondary structures and estimate changes in free energy of folding (ΔGfold) for 25 orthologous mRNAs that encode the enzyme cytosolic malate dehydrogenase in marine mollusks with adaptation temperatures spanning an almost 60 °C range. The change in free energy that occurs during formation of the ensemble of mRNA secondary structures is significantly correlated with adaptation temperature: ΔGfold values are all negative and their absolute values increase with adaptation temperature. A principal mechanism underlying these adaptations is a significant increase in synonymous guanine + cytosine substitutions with increasing temperature. These findings open up an avenue of exploration in molecular evolution and raise interesting questions about the interaction between temperature-adaptive changes in mRNA sequence and in the proteins they encode.

Roles of multi-level temperature-adaptive responses and microhabitat variation in establishing distributions of intertidal species.

How intertidal species survive their harsh environment and how best to evaluate and forecast range shifts in species distribution are two important and closely related questions for intertidal ecologists and global change biologists. Adaptive variation in responses of organisms to environmental change across all levels of biological organization - from behavior to molecular systems - is of key importance in setting distribution patterns, yet studies often neglect the interactions of diverse types of biological variation (e.g. differences in thermal optima owing to genetic and acclimation-induced effects) with environmental variation, notably at the scale of microhabitats. Intertidal species have to cope with extreme and frequently changing thermal stress, and have shown high variation in thermal sensitivities and adaptive responses at different levels of biological organization. Here, I review the physiological and biochemical adaptations of intertidal species to environmental temperature on multiple spatial and temporal scales. With fine-scale datasets for the thermal limits of individuals and for environmental temperature variation at the microhabitat scale, we can map the thermal sensitivity for each individual in different microhabitats, and then scale up the thermal sensitivity analysis to the population level and, finally, to the species level by incorporating physiological traits into species distribution models. These more refined mechanistic models that include consideration of physiological variations have higher predictive power than models that neglect these variations, and they will be crucial to answering the questions posed above concerning adaptive mechanisms and the roles they play in governing distribution patterns in a rapidly changing world.

Exploring the adaptability of the secondary structure of mRNA to temperature in intertidal snails based on SHAPE experiments.

RNA-based thermal regulation is an important strategy for organisms to cope with temperature changes. Inhabiting the intertidal rocky shore, a key interface of the ocean, atmosphere and terrestrial environments, intertidal species have developed variable thermal adaptation mechanisms; however, adaptions at the RNA level remain largely uninvestigated. To examine the relationship between mRNA structural stability and species distribution, in the present study, the secondary structure of cytosolic malate dehydrogenase (cMDH) mRNA of Echinolittorina malaccana, Echinolittorina radiata and Littorina brevicula was determined using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), and the change in folding free energy of formation (ΔGfold) was calculated. The results showed that ΔGfold increased as the temperature increased. The difference in ΔGfold (ΔΔGfold) between two specific temperatures (25 versus 0°C, 37 versus 0°C and 57 versus 0°C) differed among the three species, and the ΔΔGfold value of E. malaccana was significantly lower than those of E. radiata and L. brevicula. The number of stems of cMDH mRNA of the snails decreased with increasing temperature, and the breakpoint temperature of E. malaccana was the highest among these. The number of loops was also reduced with increasing temperature, while the length of the loop structure increased accordingly. Consequently, these structural changes can potentially affect the translational efficiency of mRNA. These results imply that there were interspecific differences in the thermal stability of RNA secondary structures in intertidal snails, and these differences may be related to snail distribution.

Surviving hot summer: Roles of phenotypic plasticity of intertidal mobile species considering microhabitat environmental heterogeneity.

For species inhabiting warming and variable thermal environment, coordinated changes in heat tolerance to temperature fluctuations, which largely depend on phenotypic plasticity, are pivotal in buffering high temperatures. Determining the roles of phenotypic plasticity in wild populations and common garden experiments help us understand how organisms survive hot summer and the warming world. We thus monitored the operative temperature of the intertidal limpets Cellana toreuma in both emergent rock and tidal pool microhabitats from June to October 2021, determined the variations of upper thermal limits of short-term acclimated and long-term acclimated limpets from different microhabitats (emergent rock and tidal pool), and further calculated the relationship between the upper thermal limits and acclimation capacity. Our results indicated that living on the emergent rock, limpets encountered more extreme events in summer. For the short-term acclimated samples, limpets on the emergent rock exhibited obvious variations of sublethal thermal limit (i.e., Arrhenius Break Point of cardiac performance, ABT) during summer months, however, this variation of ABT was absent in the limpets in the tidal pool. After the laboratory long-term acclimation, the ABTs and FLTs (Flat Line Temperature of cardiac performance, as an indicator of lethal temperature) of limpets both on the rock and in the tidal pool increased significantly in October, implying the potential existence of selection during the hot summer. Our results further showed that environmental temperature was an important driver of phenotypic plasticity. This study highlighted the changes in the thermal tolerance of intertidal limpets during summer in different microhabitats.

Demographic, physiological and genetic factors linked to the poleward range expansion of the snail Nerita yoldii along the shoreline of China.

Species range shift is one of the most significant consequences of climate change in the Anthropocene. A comprehensive study, including demographic, physiological and genetic factors linked to poleward range expansion, is crucial for understanding how the expanding population occupies the new habitat. In the present study, we investigated the demographic, physiological and genetic features of the intertidal gastropod Nerita yoldii, which has extended its northern limit by ~200 km over the former biogeographical break of the Yangtze River Estuary in recent decades. Data from neutral single nucleotide polymorphisms (SNPs) showed that the new marginal populations formed a distinct cluster established by a few founders. Demographic modelling analysis revealed that the new marginal populations experienced a strong genetic bottleneck followed by recent demographic expansion. Successful expansion that overcame the founder effect might be attributed to its high capacity for rapid population growth and multiple introductions. According to the non-neutral SNPs under diversifying selection, there were high levels of heterozygosity in the new marginal populations, which might be beneficial for adapting to the novel thermal conditions. The common garden experiment showed that the new marginal populations have evolved divergent transcriptomic and physiological responses to heat stress, allowing them to occupy and survive in the novel environment. Lower transcriptional plasticity was observed in the new marginal populations. These results suggest a new biogeographical pattern of N. yoldii has formed with the occurrence of demographic, physiological and genetic changes, and emphasize the roles of adaptation of marginal populations during range expansion.

Multiple genetic sources facilitate the northward range expansion of an intertidal oyster along China's coast.

Coastal artificial structures on the former mudflats provide available habitats for the rocky intertidal species which can establish new populations in these emerging habitats over their former distribution range limits. As a former southern species, the oyster Crassostrea sikamea has become a pioneer and rapidly invaded the artificial shorelines in northern China. We used a seascape genomics approach to investigate the population structure and genetic sources of C. sikamea on the coastal artificial structures, which is crucial for understanding the genetic mechanisms driving species distribution range expansion and invasion pathway of intertidal species. Five C. sikamea populations, including two artificial substrate populations (WGZ and ZAP), one oyster reef population (LS), and two natural rocky shore populations (ZS and XM), were measured using single nucleotide polymorphism (SNPs) obtained from double digest restriction-site associated DNA sequencing (ddRAD-Seq). Redundancy analyses (RDA) were implemented for investigating the relationship between local temperature variables and the temperature adaptability of C. sikamea. Genetic diversity, direction and strength of gene flow, and population structure all revealed that the LS and ZS populations were the genetic sources for the oyster populations on the emerging northern coastal artificial structures. Results of RDA showed that there were different adaptive potentials for northern and southern populations to local temperature variables and the oyster reef population which frequently suffers from heat stress owned high heat adaptability. The ZS population as a genetic source nearby the Yangtze River estuary provided mass larvae for the northern populations, and the other genetic source, the heat-tolerant LS population, in the oyster reef played an important role in the post-settlement success by providing preadapted genotypes. These results highlight the importance of multiple sources with divergent adaptative capabilities for biological invasion, and also emphasize the importance of the oyster reef in coastal biodiversity and conservation.

Comparing mutagenesis and simulations as tools for identifying functionally important sequence changes for protein thermal adaptation.

Comparative studies of orthologous proteins of species evolved at different temperatures have revealed consistent patterns of temperature-related variation in thermal stabilities of structure and function. However, the precise mechanisms by which interspecific variations in sequence foster these adaptive changes remain largely unknown. Here, we compare orthologs of cytosolic malate dehydrogenase (cMDH) from marine molluscs adapted to temperatures ranging from -1.9 °C (Antarctica) to ∼55 °C (South China coast) and show how amino acid usage in different regions of the enzyme (surface, intermediate depth, and protein core) varies with adaptation temperature. This eukaryotic enzyme follows some but not all of the rules established in comparisons of archaeal and bacterial proteins. To link the effects of specific amino acid substitutions with adaptive variations in enzyme thermal stability, we combined site-directed mutagenesis (SDM) and in vitro protein experimentation with in silico mutagenesis using molecular dynamics simulation (MDS) techniques. SDM and MDS methods generally but not invariably yielded common effects on protein stability. MDS analysis is shown to provide insights into how specific amino acid substitutions affect the conformational flexibilities of mobile regions (MRs) of the enzyme that are essential for binding and catalysis. Whereas these substitutions invariably lie outside of the MRs, they effectively transmit their flexibility-modulating effects to the MRs through linked interactions among surface residues. This discovery illustrates that regions of the protein surface lying outside of the site of catalysis can help establish an enzyme's thermal responses and foster evolutionary adaptation of function.

Aerobic scope in fishes with different lifestyles and across habitats: Trade-offs among hypoxia tolerance, swimming performance and digestion.

Exercise and aerobic scope in fishes have attracted scientists' attention for several decades. While it has been suggested that aerobic scope may limit behavioral expression and tolerance to environmental stressors in fishes, the exact importance of aerobic scope in an ecological context remains poorly understood. In this review, we examine the ecological relevance of aerobic scope by reconsidering and reanalyzing the existing literature on Chinese freshwater fishes across a wide-range of habitats and lifestyles. The available evidence suggests that natural selection in fast-flowing aquatic habitats may favor species with a high aerobic scope and anaerobic capacity for locomotion, whereas in relatively slow-flowing habitats, hypoxia tolerance may be favored at the cost of reduced locomotor capacity. In addition, while physical activity can usually cause fishes from fast-flowing habitats to reach their aerobic metabolic ceiling (i.e., maximum metabolic rate), possibly due to selection pressure on locomotion, most species from slow-flowing habitats can only reach their metabolic ceiling during digestion, either alone or in combination with physical activity. Overall, we suggest that fish exhibit a continuum of metabolic types, from a 'visceral metabolic type' with a higher digestive performance to a 'locomotion metabolic type' which appears to have reduced capacity for digestion but enhanced locomotor performance. Generally, locomotor-type species can either satisfy the demands of their high swimming capacity with a high oxygen uptake capacity or sacrifice digestion while swimming. In contrast, most visceral-type species show a pronounced decrease in swimming performance while digesting, probably owing to conflicts within their aerobic scope. In conclusion, the ecological relevance of aerobic scope and the consequent effects on other physiological functions are closely related to habitat and the lifestyle of a given species. These results suggest that swimming performance, digestion and hypoxia tolerance might coevolve due to dependence on metabolic traits such as aerobic scope.

Physiological determinants of biogeography: The importance of metabolic depression to heat tolerance.

A quantitative understanding of physiological thermal responses is vital for forecasting species distributional shifts in response to climate change. Many studies have focused on metabolic rate as a global metric for analyzing the sublethal effects of changing environments on physiology. Thermal performance curves (TPCs) have been suggested as a viable analytical framework, but standard TPCs may not fully capture physiological responses, due in part to failure to consider the process of metabolic depression. We derived a model based on the nonlinear regression of biological temperature-dependent rate processes and built a heart rate data set for 26 species of intertidal molluscs distributed from 33°S to ~40°N. We then calculated physiological thermal performance limits with continuous heating using T 1 / 2 H , the temperature at which heart rate is decreased to 50% of the maximal rate, as a more realistic measure of upper thermal limits. Results indicate that heat-induced metabolic depression of cardiac performance is a common adaptive response that allows tolerance of harsh environments. Furthermore, our model accounted for the high inter-individual variability in the shape of cardiac TPCs. We then used these TPCs to calculate physiological thermal safety margins (pTSM), the difference between the maximal operative temperature (95th percentile of field temperatures) and T 1 / 2 H of each individual. Using pTSMs, we developed a physiological species distribution model (pSDM) to forecast future geographic distributions. pSDM results indicate that climate-induced species range shifts are potentially less severe than predicted by a simple correlative SDM. Species with metabolic depression below the optimum temperature will be more thermal resistant at their warm trailing edges. High intraspecific variability further suggests that models based on species-level vulnerability to environmental change may be problematic. This multi-scale, mechanistic understanding that incorporates metabolic depression and inter-individual variability in thermal response enables better predictions about the relationship between thermal stress and species distributions.

Membrane lipid metabolism, heat shock response and energy costs mediate the interaction between acclimatization and heat-hardening response in the razor clam Sinonovacula constricta.

Thermal plasticity on different time scales, including acclimation/acclimatization and heat-hardening response - a rapid adjustment for thermal tolerance after non-lethal thermal stress, can interact to improve the resilience of organisms to thermal stress. However, little is known about physiological mechanisms mediating this interaction. To investigate the underpinnings of heat-hardening responses after acclimatization in warm seasons, we measured thermal tolerance plasticity, and compared transcriptomic and metabolomic changes after heat hardening at 33 or 37°C followed by recovery of 3 or 24 h in an intertidal bivalve Sinonovacula constricta. Clams showed explicit heat-hardening responses after acclimatization in a warm season. The higher inducing temperature (37°C) caused less effective heat-hardening effects than the inducing temperature that was closer to the seasonal maximum temperature (33°C). Metabolomic analysis highlighted the elevated content of glycerophospholipids in all heat-hardened clams, which may help to maintain the structure and function of the membrane. Heat shock proteins (HSPs) tended to be upregulated after heat hardening at 37°C but not at 33°C, indicating that there was no complete dependency of heat-hardening effects on upregulated HSPs. Enhanced energy metabolism and decreased energy reserves were observed after heat hardening at 37°C, suggesting more energy costs during exposure to a higher inducing temperature, which may restrict heat-hardening effects. These results highlight the mediating role of membrane lipid metabolism, heat shock responses and energy costs in the interaction between heat-hardening response and seasonal acclimatization, and contribute to the mechanistic understanding of evolutionary change and thermal plasticity during global climate change.

Temperature adaptations of the thermophilic snail Echinolittorina malaccana: insights from metabolomic analysis.

The periwinkle snail Echinolittorina malaccana, for which the upper lethal temperature is near 55°C, is one of the most heat-tolerant eukaryotes known. We conducted a multi-level investigation - including cardiac physiology, enzyme activity, and targeted and untargeted metabolomic analyses - that elucidated a spectrum of adaptations to extreme heat in this organism. All systems examined showed heat intensity-dependent responses. Under moderate heat stress (37-45°C), the snail depressed cardiac activity and entered a state of metabolic depression. The global metabolomic and enzymatic analyses revealed production of metabolites characteristic of oxygen-independent pathways of ATP generation (lactate and succinate) in the depressed metabolic state, which suggests that anaerobic metabolism was the main energy supply pathway under heat stress (37-52°C). The metabolomic analyses also revealed alterations in glycerophospholipid metabolism under extreme heat stress (52°C), which likely reflected adaptive changes to maintain membrane structure. Small-molecular-mass organic osmolytes (glycine betaine, choline and carnitine) showed complex changes in concentration that were consistent with a role of these protein-stabilizing solutes in protection of the proteome under heat stress. This thermophilic species can thus deploy a wide array of adaptive strategies to acclimatize to extremely high temperatures.

Genomic and physiological mechanisms underlying skin plasticity during water to air transition in an amphibious fish.

The terrestrial radiation of vertebrates required changes in skin that resolved the dual demands of maintaining a mechanical and physiological barrier while also facilitating ion and gas transport. Using the amphibious killifish Kryptolebias marmoratus, we found that transcriptional regulation of skin morphogenesis was quickly activated upon air exposure (1 h). Rapid regulation of cell-cell adhesion complexes and pathways that regulate stratum corneum formation was consistent with barrier function and mechanical reinforcement. Unique blood vessel architecture and regulation of angiogenesis likely supported cutaneous respiration. Differences in ionoregulatory transcripts and ionocyte morphology were correlated with differences in salinity acclimation and resilience to air exposure. Evolutionary analyses reinforced the adaptive importance of these mechanisms. We conclude that rapid plasticity of barrier, respiratory and ionoregulatory functions in skin evolved to support the amphibious lifestyle of K. marmoratus; similar processes may have facilitated the terrestrial radiation of other contemporary and ancient fishes.

Structural flexibility and protein adaptation to temperature: Molecular dynamics analysis of malate dehydrogenases of marine molluscs.

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 KM 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.

Timing Metabolic Depression: Predicting Thermal Stress in Extreme Intertidal Environments.

AbstractAnticipatory changes in organismal responses, triggered by reliable environmental cues for future conditions, are key to species' persistence in temporally variable environments. Such responses were tested by measuring the physiological performance of a tropical high-shore oyster in tandem with the temporal predictability of environmental temperature. Heart rate of the oyster increased with environmental temperatures until body temperature reached ∼37°C, when a substantial depression occurred (∼60%) before recovery between ∼42° and 47°C, after which cardiac function collapsed. The sequential increase, depression, and recovery in cardiac performance aligned with temporal patterns in rock surface temperatures, where the risk of reaching temperatures close to the oysters' lethal limit accelerates if the rock heats up beyond ∼37°C, coinciding closely with the body temperature at which the oysters initiate metabolic depression. The increase in body temperature over a critical threshold serves as an early-warning cue to initiate anticipatory shifts in physiology and energy conservation before severe thermal stress occurs on the shore. Cross-correlating the onset of physiological mechanisms and temporal structures in environmental temperatures, therefore, reveals the potential role of reliable real-time environmental cues for future conditions in driving the evolution of anticipatory responses.

Help, there are 'omics' in my comparative physiology!

'Omics' methods, such as transcriptomics, proteomics, lipidomics or metabolomics, yield simultaneous measurements of many related molecules in a sample. These approaches have opened new opportunities to generate and test hypotheses about the mechanisms underlying biochemical and physiological phenotypes. In this Commentary, we discuss general approaches and considerations for successfully integrating omics into comparative physiology. The choice of omics approach will be guided by the availability of existing resources and the time scale of the process being studied. We discuss the use of whole-organism extracts (common in omics experiments on small invertebrates) because such an approach may mask underlying physiological mechanisms, and we consider the advantages and disadvantages of pooling samples within biological replicates. These methods can bring analytical challenges, so we describe the most easily analyzed omics experimental designs. We address the propensity of omics studies to digress into 'fishing expeditions' and show how omics can be used within the hypothetico-deductive framework. With this Commentary, we hope to provide a roadmap that will help newcomers approach omics in comparative physiology while avoiding some of the potential pitfalls, which include ambiguous experiments, long lists of candidate molecules and vague conclusions.

Phenotypic flexibility in respiratory traits is associated with improved aerial respiration in an amphibious fish out of water.

Amphibious fishes have evolved multiple adaptive strategies for respiring out of water, but there has been less focus on reversible plasticity. We tested the hypothesis that when amphibious fishes leave water, enhanced respiratory performance on land is the result of rapid functional phenotypic flexibility of respiratory traits. We acclimated four isogenic strains of Kryptolebias marmoratus to air for 0, 1, 3 or 7 days. We compared respiratory performance out of water with traits linked to the O2 cascade. Aerial O2 consumption rate was measured over a step-wise decrease in O2 levels. There were significant differences between strains, but time out of water had the largest impact on measured parameters. Kryptolebiasmarmoratus had improved respiratory performance [lower aerial critical oxygen tension (Pcrit), higher regulation index (RI)] after only 1 day of air exposure, and these changes were strongly associated with the change in hematocrit and dorsal cutaneous angiogenesis. Additionally, we found that 1 h of air exposure induced the expression of four angiogenesis-associated genes - vegfa, angpt2, pecam-1 and efna1 - in the skin. After 7 days in air, respiratory traits were not significantly linked to the variation in either aerial Pcrit or RI. Overall, our data indicate that there are two phases involved in the enhancement of aerial respiration: an initial rapid response (1 day) and a delayed response (7 days). We found evidence for the hypothesis that respiratory performance on land in amphibious fishes is the result of rapid flexibility in both O2 uptake and O2 carrying capacity.

Pathological evaluation of neoadjuvant chemotherapy in advanced gastric cancer.

BACKGROUND: Although pathological evaluation has been considered an effective evaluation method, some problems still exist in practice. Therefore, we explored whether there are more reasonable and practical pathological evaluation criteria for neoadjuvant chemotherapy in patients with advanced gastric cancer. Here, we aim to determine pathological judgment criteria for neoadjuvant chemotherapy in patients with advanced gastric cancer. METHODS: Eighty-seven patients with cT2-4 or cN+ were enrolled in this study. Pathological factors for overall survival (OS) were investigated using univariate and multivariate analyses, and the pathological criteria for neoadjuvant chemotherapy were then determined. RESULTS: A total of 87 patients underwent 3-4 cycles of neoadjuvant chemotherapy, with 67 (77.0%), 15 (17.2%), and 5 (5.8%) receiving Folfox6, Xelox, and SOX regimens, respectively. All patients showed different levels of graded histological regression (GHR) of the primary tumor, with a ≥ 50% regression rate of 50.6%. The univariate analysis showed that GHR ≥ 50% (p = 0.022), 66.7% (p = 0.013), and 90% (p = 0.028) were significantly correlated with OS. The multivariate analysis demonstrated that ypTNM (II/III) stage was significantly associated with OS compared with ypTNM (0+I) stage [HR = 3.553, 95% CI 1.886-6.617; HR = 3.576, 95% CI 1.908-6.703, respectively] and that the Lauren classification of diffuse type was also an independent risk factor for OS compared with the intestinal type (HR = 3.843, 95% CI 1.443-10.237). CONCLUSIONS: The Lauren classification and ypTNM stage after neoadjuvant chemotherapy are independent prognostic factors in advanced gastric cancer. A GHR ≥ 50%/< 50% can be used as the primary criterion for advanced gastric cancer after neoadjuvant chemotherapy to determine postoperative adjuvant chemotherapy regimens.

Substantial heat tolerance acclimation capacity in tropical thermophilic snails, but to what benefit?

The theory for thermal acclimation of ectotherms suggests that (1) heat tolerance is traded off for thermal acclimation in thermophilic species and that (2) plasticity is constrained in tropically distributed ectotherms, which commonly experience relatively thermally stable environments. We observed substantial heat tolerance plasticity in a test of this theory using tropical, thermophilic marine intertidal snails that inhabit extremely hot and highly variable thermal environments. The implication of these results is that plasticity selection is largely driven by habitat temperature conditions irrespective of basal heat tolerance or latitude. However, heat tolerance of field-fresh snails was comparable with that of laboratory warm-acclimated snails, suggesting that snails in the field may often be unable to improve heat hardening with further environmental warming. These findings suggest that field referencing is crucial to using laboratory-measured acclimation capacity when inferring climate warming vulnerability in ectotherms, and overall they question how well current thermal biology theory predicts the outcomes of global change in intertidal environments.

Untangling the roles of microclimate, behaviour and physiological polymorphism in governing vulnerability of intertidal snails to heat stress.

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 (Tb), 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 Tb 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 Tb 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.