From molecules to morphology: How food supply influences the larvae of sea urchins across all levels of biological organization.

An important goal of many studies in molecular ecology is to utilize molecular tools to elucidate how critical traits like metabolism and growth are affected by environmental stressors and how organisms offset these stresses by adaptive molecular-level responses. Stress from food deprivation may be critical for early developmental stages that require a continued supply of substrates for energy metabolism and growth if development is to be completed. In a 'From the Cover' article in this issue of Molecular Ecology, Li et al. (2023) examined the effects of withholding food (unicellular algae) on 10 traits of larvae of the purple sea urchin (Strongylocentrotus purpuratus), ranging from the molecular level (gene expression) to morphology. Overall, this study sheds new light on the plasticity of larval development and the tight linkages that exist among traits as they respond to changes in food availability. Importantly, shifts in the sources of food utilized under different dietary treatments show the plasticity of these larvae to alter reliance on endogenous energy stores and dissolved organic matter (DOM) as algae deprivation continues. The effects of global change on the amounts and phenology of productivity in the seas make this type of integrated, multi-level analysis an important tool for predicting the future states of marine ecosystems.

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.

Tissue-specific temperature dependence of RNA editing levels in zebrafish.

BACKGROUND: RNA editing by adenosine deaminase acting on RNA (ADAR) occurs in all metazoans and fulfils several functions. Here, we examined effects of acclimation temperature (27 °C, 18 °C,13 °C) on editing patterns in six tissues of zebrafish (Danio rerio). RESULTS: Sites and total amounts of editing differed among tissues. Brain showed the highest levels, followed by gill and skin. In these highly edited tissues, decreases in temperatures led to large increases in total amounts of editing and changes in specific edited sites. Gene ontology analysis showed both similarities (e.g., endoplasmic reticulum stress response) and differences in editing among tissues. The majority of edited sites were in transcripts of transposable elements and the 3'UTR regions of protein coding genes. By experimental validation, translation efficiency was directly related to extent of editing of the 3'UTR region of an mRNA. CONCLUSIONS: RNA editing increases 3'UTR polymorphism and affects efficiency of translation. Such editing may lead to temperature-adaptive changes in the proteome through altering relative amounts of synthesis of different proteins.

Turning a page: remaining a top competitor in an evolving publication ecosystem.

Journal of Experimental Biology (JEB) is celebrating its first 100 years this year. My own relationship with the journal spans over six decades and encompasses a variety of roles: reader, author, Editor (1995-2000), Editorial Advisory Board member (2000 to present) and Director on the board of its publisher, The Company of Biologists (2003-2009). I was therefore delighted when the journal Editors asked me to write a Perspective to reflect on how the journal and the publishing environment in which it competes have evolved over this long period, and to peek into my crystal ball and comment on what the future might hold for the journal and the primary fields it covers: comparative-environmental-evolutionary physiology, neuroethology and biomechanics.

Oxidative stress effects are not correlated with differences in heat tolerance among congeners of Mytilus.

The physiological mechanisms that limit thermal tolerance are broadly relevant to comparative biology and global change. Species differences in macromolecular stability play important roles in evolved patterns of heat tolerance, but other mechanisms such as oxidative stress have also been hypothesized to contribute. For example, mussels in the genus Mytilus exhibit evolved physiological differences at several levels of organization that have been linked with interspecific differences in whole-organism heat tolerance. Both omics and behavioral studies suggested that variation in resistance to oxidative stress plays a role in these differences. Functional data are needed to test this hypothesis. Here, we compared three Mytilus congeners to examine whether susceptibility to oxidative stress contributes to acute heat tolerance. We assayed the activity of two antioxidant enzymes (catalase, superoxide dismutase), as well as levels of oxidative damage to lipids, DNA and individual proteins (using gel-based proteomics methods). In addition, we assessed these oxidative stress responses after repeated episodes of heat stress experienced in air or while immersed in seawater, given that survival and competitive outcomes between Mytilus congeners differ in these two contexts. The results are generally inconsistent with patterns that would be expected if oxidative stress contributes to thermal sensitivity. Rather, the more heat-tolerant congeners suffer comparable or even elevated levels of oxidative damage. As predicted, different treatment contexts led to distinct changes in proteome-wide abundance patterns and, to a lesser extent, protein carbonylation profiles. Overall, the results question the relevance of oxidative damage as a mediator of heat tolerance in this genus.

Effects of heat acclimation on cardiac function in the intertidal mussel Mytilus californianus: can laboratory-based indices predict survival in the field?

Thermal performance curves are commonly used to investigate the effects of heat acclimation on thermal tolerance and physiological performance. However, recent work indicates that the metrics of these curves heavily depend on experimental design and may be poor predictors of animal survival during heat events in the field. In intertidal mussels, cardiac thermal performance (CTP) tests have been widely used as indicators of animals' acclimation or acclimatization state, providing two indices of thermal responses: critical temperature (Tcrit; the temperature above which heart rate abruptly declines) and flatline temperature (Tflat; the temperature where heart rate ceases). Despite the wide use of CTP tests, it remains largely unknown how Tcrit and Tflat change within a single individual after heat acclimation, and whether changes in these indices can predict altered survival in the field. Here, we addressed these issues by evaluating changes in CTP indices in the same individuals before and after heat acclimation. For control mussels, merely reaching Tcrit was not lethal, whereas remaining at Tcrit for ≥10 min was lethal. Heat acclimation significantly increased Tcrit only in mussels with an initially low Tcrit (<35°C), but improved their survival time above Tcrit by 20 min on average. Tflat increased by ∼1.6°C with heat acclimation, but it is unlikely that increased Tflat improves survival in the field. In summary, Tcrit and Tflat per se may fall short of providing quantitative indices of thermal tolerance in mussels; instead, a combination of Tcrit and tolerance time at temperatures ≥Tcrit better defines changes in thermal tolerance with heat acclimation.

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.

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.

A single heat-stress bout induces rapid and prolonged heat acclimation in the California mussel, Mytilus californianus.

Climate change is not only causing steady increases in average global temperatures but also increasing the frequency with which extreme heating events occur. These extreme events may be pivotal in determining the ability of organisms to persist in their current habitats. Thus, it is important to understand how quickly an organism's heat tolerance can be gained and lost relative to the frequency with which extreme heating events occur in the field. We show that the California mussel, Mytilus californianus-a sessile intertidal species that experiences extreme temperature fluctuations and cannot behaviourally thermoregulate-can quickly (in 24-48 h) acquire improved heat tolerance after exposure to a single sublethal heat-stress bout (2 h at 30 or 35°C) and then maintain this improved tolerance for up to three weeks without further exposure to elevated temperatures. This adaptive response improved survival rates by approximately 75% under extreme heat-stress bouts (2 h at 40°C). To interpret these laboratory findings in an ecological context, we evaluated 4 years of mussel body temperatures recorded in the field. The majority (approx. 64%) of consecutive heat-stress bouts were separated by 24-48 h, but several consecutive heat bouts were separated by as much as 22 days. Thus, the ability of M. californianus to maintain improved heat tolerance for up to three weeks after a single sublethal heat-stress bout significantly improves their probability of survival, as approximately 33% of consecutive heat events are separated by 3-22 days. As a sessile animal, mussels likely evolved the capability to rapidly gain and slowly lose heat tolerance to survive the intermittent, and often unpredictable, heat events in the intertidal zone. This adaptive strategy will likely prove beneficial under the extreme heat events predicted with climate change.

Mussel acclimatization to high, variable temperatures is lost slowly upon transfer to benign conditions.

Climate change is increasing the temperature variability animals face, and thermal acclimatization allows animals to adjust adaptively to this variability. Although the rate of heat acclimatization has received some study, little is known about how long these adaptive changes remain without continuing exposure to heat stress. This study explored the rate at which field acclimatization states are lost when temperature variability is minimized during constant submersion. California mussels (Mytilus californianus) with different acclimatization states were collected from high- and low-zone sites (∼12 versus ∼5°C daily temperature ranges, respectively) and then kept submerged at 15°C for 8 weeks. Each week, the cardiac thermal performance of mussels was measured as a metric of acclimatization state: critical (Tcrit) and flatline (Tflat) temperatures were recorded. Over 8 weeks of constant submersion, the mean Tcrit of high-zone mussels decreased by 1.07°C from baseline, but low-zone mussels' mean Tcrit was unchanged. High- and low-zone mussels' mean maximum heart rate (HR) and resting HR decreased ∼12 and 35%, respectively. Tflat was unchanged in both groups. These data suggest that Tcrit and HR are more physiologically plastic in response to the narrowing of an animal's daily temperature range than Tflat is, and that an animal's prior acclimatization state (high versus low) influences the acclimatory capacity of Tcrit Approximately 2 months were required for the cardiac thermal performance of the high-zone mussels to reach that of the low-zone mussels, suggesting that acclimatization to high and variable temperatures may persist long enough to enable these animals to cope with intermittent bouts of heat stress.

Establishing typical values for hemocyte mortality in individual California mussels, Mytilus californianus.

Hemocytes are immune cells in the hemolymph of invertebrates that play multiple roles in response to stressors; hemocyte mortality can thus serve as an indicator of overall animal health. However, previous research has often analyzed hemolymph samples pooled from several individuals, which precludes tracking individual responses to stressors over time. The ability to track individuals is important, however, because large inter-individual variation in response to stressors can confound the interpretation of pooled samples. Here, we describe protocols for analysis of inter- and intra-individual variability in hemocyte mortality across repeated hemolymph samples of California mussels, Mytilus californianus, free from typical abiotic stressors. To assess individual variability in hemocyte mortality with serial sampling, we created four groups of 15 mussels each that were repeatedly sampled four times: at baseline (time zero) and three subsequent times separated by either 24, 48, 72, or 168 h. Hemocyte mortality was assessed by fluorescence-activated cell sorting (FACS) of cells stained with propidium iodide. Our study demonstrates that hemolymph can be repeatedly sampled from individual mussels without mortality; however, there is substantial inter- and intra-individual variability in hemocyte mortality through time that is partially dependent on the sampling interval. Across repeated samples, individual mussels' hemocyte mortality had, on average, a range of ~6% and a standard deviation of ~3%, which was minimized with sampling periods ≥72 h apart. Due to this intra-individual variability, obtaining ≥2 samples from a specimen will more accurately establish an individual's baseline. Pooled-sample means were similar to individual-sample means; however, pooled samples masked the individual variation in each group. Overall, these data lay the foundation for future work exploring individual mussels' temporal responses to various stressors on a cellular level.

Impact of heating rate on cardiac thermal tolerance in the California mussel, Mytilus californianus.

Intertidal communities of wave-swept rocky shores have served as a powerful model system for experiments in ecology, and mussels (the dominant competitor for space in the mid-intertidal zone) play a central role in determining community structure in this physically stressful habitat. Consequently, the ability to account for mussels' physiological responses to thermal stress affects ecologists' capacity to predict the impacts of a warming climate on this ecosystem. Here, we examined the effect of heating rate on cardiac thermal tolerance in the ribbed mussel, Mytilus californianus, comparing populations from high and low sites in the intertidal zone where emersion duration leads to different mean daily heating rates. Two temperature-related cardiac variables were examined: (1) the critical temperature (Tcrit) at which heart rate (HR) precipitously declines, and (2) flatline temperature (FLT) where HR reaches zero. Mussels were heated in air at slow, moderate and fast rates, and HR was measured via an infrared sensor affixed to the shell. Faster heating rates significantly increased Tcrit in high- but not low-zone mussels, and Tcrit was higher in high- versus low-zone mussels, especially at the fastest heating rate. By contrast, FLT did not differ between zones, and was minimally affected by heating rate. As heating rate significantly impacted high- but not low-zone mussels' cardiac thermal tolerance, realistic zone-specific heating rates must be used in laboratory tests if those tests are to provide accurate information for ecological models attempting to predict the effects of increasing temperature on intertidal communities.

RNA thermosensors: how might animals exploit their regulatory potential?

The secondary and tertiary orders of RNA structure are crucial for a suite of RNA-related functions, including regulation of translation, gene expression and RNA turnover. The temperature sensitivity of RNA secondary and tertiary structures is exploited by bacteria to fabricate RNA thermosensing systems that allow a rapid adaptive response to temperature change. RNA thermometers (RNATs) present in non-coding regions of certain mRNAs of pathogenic bacteria enable rapid upregulation of translation of virulence proteins when the temperature of the bacterium rises after entering a mammalian host. Rapid upregulation of translation of bacterial heat-shock proteins likewise is governed in part by RNATs. Turnover of mRNA may be regulated by temperature-sensitive RNA structures. Whereas the roles of temperature-sensitive RNA structures similar to RNATs in Eukarya and Archaea are largely unknown, there would appear to be a potential for all taxa to adaptively regulate their thermal physiology through exploitation of RNA-based thermosensory responses akin to those of bacteria. In animals, these responses might include regulation of translation of stress-induced proteins, alternative splicing of messenger RNA precursors, differential expression of allelic proteins, modulation of activities of small non-coding RNAs, regulation of mRNA turnover and control of RNA editing. New methods for predicting, detecting and experimentally modifying RNA secondary structure offer promising windows into these fascinating aspects of RNA biochemistry. Elucidating whether animals too have exploited the types of RNA thermosensing tools that are used so effectively by bacteria seems likely to provide exciting new insights into the mechanisms of evolutionary adaptation and acclimatization to temperature.

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.

Heat-resistant cytosolic malate dehydrogenases (cMDHs) of thermophilic intertidal snails (genus Echinolittorina): protein underpinnings of tolerance to body temperatures reaching 55°C.

Snails of the genus Echinolittorina are among the most heat-tolerant animals; they experience average body temperatures near 41-44°C in summer and withstand temperatures up to at least 55°C. Here, we demonstrate that heat stability of function (indexed by the Michaelis-Menten constant of the cofactor NADH, KMNADH) and structure (indexed by rate of denaturation) of cytosolic malate dehydrogenases (cMDHs) of two congeners (E. malaccana and E. radiata) exceeds values previously found for orthologs of this protein from less thermophilic species. The ortholog of E. malaccana is more heat stable than that of E. radiata, in keeping with the congeners' thermal environments. Only two inter-congener differences in amino acid sequence in these 332 residue proteins were identified. In both cases (positions 48 and 114), a glycine in the E. malaccana ortholog is replaced by a serine in the E. radiata protein. To explore the relationship between structure and function and to characterize how amino acid substitutions alter stability of different regions of the enzyme, we used molecular dynamics simulation methods. These computational methods allow determination of thermal effects on fine-scale movements of protein components, for example, by estimating the root mean square deviation in atom position over time and the root mean square fluctuation for individual residues. The minor changes in amino acid sequence favor temperature-adaptive change in flexibility of regions in and around the active sites. Interspecific differences in effects of temperature on fine-scale protein movements are consistent with the differences in thermal effects on binding and rates of heat denaturation.

Thermal history and gape of individual Mytilus californianus correlate with oxidative damage and thermoprotective osmolytes.

The ability of animals to cope with environmental stress depends - in part - on past experience, yet knowledge of the factors influencing an individual's physiology in nature remains underdeveloped. We used an individual monitoring system to record body temperature and valve gaping behavior of rocky intertidal zone mussels (Mytilus californianus). Thirty individuals were selected from two mussel beds (wave-exposed and wave-protected) that differ in thermal regime. Instrumented mussels were deployed at two intertidal heights (near the lower and upper edges of the mussel zone) and in a continuously submerged tidepool. Following a 23-day monitoring period, measures of oxidative damage to DNA and lipids, antioxidant capacities (catalase activity and peroxyl radical scavenging) and tissue contents of organic osmolytes were obtained from gill tissue of each individual. Univariate and multivariate analyses indicated that inter-individual variation in cumulative thermal stress is a predominant driver of physiological variation. Thermal history over the outplant period was positively correlated with oxidative DNA damage. Thermal history was also positively correlated with tissue contents of taurine, a thermoprotectant osmolyte, and with activity of the antioxidant enzyme catalase. Origin site differences, possibly indicative of developmental plasticity, were only significant for catalase activity. Gaping behavior was positively correlated with tissue contents of two osmolytes. Overall, these results are some of the first to clearly demonstrate relationships between inter-individual variation in recent experience in the field and inter-individual physiological variation, in this case within mussel beds. Such micro-scale, environmentally mediated physiological differences should be considered in attempts to forecast biological responses to a changing environment.

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.

Adaptations of protein structure and function to temperature: there is more than one way to 'skin a cat'.

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, A4-lactate dehydrogenase (A4-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 temperature-adaptive mutations in A4-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.

Master of all trades: thermal acclimation and adaptation of cardiac function in a broadly distributed marine invasive species, the European green crab, Carcinus maenas.

As global warming accelerates, there is increasing concern about how ecosystems may change as a result of species loss and replacement. Here, we examined the thermal physiology of the European green crab (Carcinus maenas Linnaeus 1758), a globally invasive species, along three parallel thermal gradients in its native and invasive ranges. At each site, we assessed cardiac physiology to determine heat and cold tolerance and acclimatory plasticity. We found that, overall, the species is highly tolerant of both heat and cold, and that it survives higher temperatures than co-occurring native marine crustaceans. Further, we found that both heat and cold tolerance are plastic in response to short-term acclimation (18-31 days at either 5 or 25°C). Comparing patterns within ranges, we found latitudinal gradients in thermal tolerance in the native European range and in the invasive range in eastern North America. This pattern is strongest in the native range, and likely evolved there. Because of a complicated invasion history, the latitudinal pattern in the eastern North American invasive range may be due either to rapid adaptation post-invasion or to adaptive differences between the ancestral populations that founded the invasion. Overall, the broad thermal tolerance ranges of green crabs, which may facilitate invasion of novel habitats, derive from high inherent eurythermality and acclimatory plasticity and potentially adaptive differentiation among populations. The highly flexible physiology that results from these capacities may represent the hallmark of a successful invasive species, and may provide a model for success in a changing world.

Functional determinants of temperature adaptation in enzymes of cold- versus warm-adapted mussels (Genus Mytilus).

Temperature is a strong selective force on the evolution of proteins due to its effects on higher orders of protein structure and, thereby, on critical protein functions like ligand binding and catalysis. Comparisons among orthologous proteins from differently thermally adapted species show consistent patterns of adaptive variation in function, but few studies have examined functional adaptation among multiple structural families of proteins. Thus, with our present state of knowledge, it is difficult to predict what fraction of the proteome will exhibit adaptive variation in the face of temperature increases of a few to several degrees Celsius, that is, temperature increases of the magnitude predicted by models of global warming. Here, we compared orthologous enzymes of the warm-adapted Mediterranean mussel Mytilus galloprovincialis and the cold-adapted Mytilus trossulus, a native of the North Pacific Ocean, species whose physiologies exhibit significantly different responses to temperature. We measured the effects of temperature on the kinetics (Michaelis-Menten constant-K(m)) of five enzymes that are important for ATP generation and that represent distinct protein structural families. Among phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (GTP) (PEPCK), and isocitrate dehydrogenase (NADP) (IDH), only IDH orthologs showed significantly different thermal responses of K(m) between the two species. The K(m) of isocitrate of M. galloprovincialis-IDH was intrinsically lower and more thermally stable than that of M. trossulus-IDH and thus had higher substrate affinity at high temperatures. Two amino acid substitutions account for the functional differences between IDH orthologs, one of which allows for more hydrogen bonds to form near the mobile region of the active site in M. galloprovincialis-IDH. Taken together, our findings cast light on the targets of adaptive evolution in the context of climate change; only a minority of proteins might adapt to small changes in temperature, and these adaptations may involve only small changes in sequence.