George N. Somero

The physiology of climate change: how potentials for acclimatization and genetic adaptation will determine ‘winners’ and ‘losers’

SUMMARY Physiological studies can help predict effects of climate change through determining which species currently live closest to their upper thermal tolerance limits, which physiological systems set these limits, and how species differ in acclimatization capacities for modifying their thermal tolerances. Reductionist studies at the molecular level can contribute to this analysis by revealing how much change in sequence is needed to adapt proteins to warmer temperatures — thus providing insights into potential rates of adaptive evolution — and determining how the contents of genomes — protein-coding genes and gene regulatory mechanisms — influence capacities for adapting to acute and long-term increases in temperature. Studies of congeneric invertebrates from thermally stressful rocky intertidal habitats have shown that warm-adapted congeners are most susceptible to local extinctions because their acute upper thermal limits (LT50 values) lie near current thermal maxima and their abilities to increase thermal tolerance through acclimation are limited. Collapse of cardiac function may underlie acute and longer-term thermal limits. Local extinctions from heat death may be offset by in-migration of genetically warm-adapted conspecifics from mid-latitude ‘hot spots’, where midday low tides in summer select for heat tolerance. A single amino acid replacement is sufficient to adapt a protein to a new thermal range. More challenging to adaptive evolution are lesions in genomes of stenotherms like Antarctic marine ectotherms, which have lost protein-coding genes and gene regulatory mechanisms needed for coping with rising temperature. These extreme stenotherms, along with warm-adapted eurytherms living near their thermal limits, may be the major ‘losers’ from climate change.

Thermal Physiology and Vertical Zonation of Intertidal Animals: Optima, Limits, and Costs of Living

Abstract Temperatures pervasive effects on physiological systems are reflected in the suite of temperature-adaptive differences observed among species from different thermal niches, such as species with different vertical distributions (zonations) along the subtidal to intertidal gradient. Among the physiological traits that exhibit adaptive variation related to vertical zonation are whole organism thermal tolerance, heart function, mitochondrial respiration, membrane static order (fluidity), action potential generation, protein synthesis, heat-shock protein expression, and protein thermal stability. For some, but not all, of these thermally sensitive traits acclimatization leads to adaptive shifts in thermal optima and limits. The costs associated with repairing thermal damage and adapting systems through acclimatization may contribute importantly to energy budgets. These costs arise from such sources as: (i) activation and operation of the heat-shock response, (ii) replacement of denatured proteins that have been removed through proteolysis, (iii) restructuring of cellular membranes (“homeoviscous” adaptation), and (iv) pervasive shifts in gene expression (as gauged by using DNA microarray techniques). The vertical zonation observed in rocky intertidal habitats thus may reflect two distinct yet closely related aspects of thermal physiology: (i) intrinsic interspecific differences in temperature sensitivities of physiological systems, which establish thermal optima and tolerance limits for species; and (ii) ‘cost of living’ considerations arising from sub-lethal perturbation of these physiological systems, which may establish an energetics-based limitation to the maximal height at which a species can occur. Quantifying the energetic costs arising from heat stress represents an important challenge for future investigations.

Linking biogeography to physiology: Evolutionary and acclimatory adjustments of thermal limits

Temperature-adaptive physiological variation plays important roles in latitudinal biogeographic patterning and in setting vertical distributions along subtidal-to-intertidal gradients in coastal marine ecosystems. Comparisons of congeneric marine invertebrates reveal that the most warm-adapted species may live closer to their thermal tolerance limits and have lower abilities to increase heat tolerance through acclimation than more cold-adapted species. In crabs and snails, heart function may be of critical importance in establishing thermal tolerance limits. Temperature-mediated shifts in gene expression may be critical in thermal acclimation. Transcriptional changes, monitored using cDNA microarrays, have been shown to differ between steady-state thermal acclimation and diurnal temperature cycling in a eurythermal teleost fish (Austrofundulus limnaeus). In stenothermal Antarctic notothenioid fish, losses in capacity for temperature-mediated gene expression, including the absence of a heat-shock response, may reduce the abilities of these species to acclimate to increased temperatures. Differences among species in thermal tolerance limits and in the capacities to adjust these limits may determine how organisms are affected by climate change.

Temperature Tolerance of Some Antarctic Fishes

Three species of Antarctic fishes which live in constantly near-freezing waters have a markedly low upper-lethal temperature of 6� C ; this is the lowest upper-lethal temperature reported for any organism. The fishes survive supercooling to —2.5�C. Data on brain metabolism in vitro support the hypothesis that the central nervous system is a primary site of thermal injury.

A Comparative Analysis of the Upper Thermal Tolerance Limits of Eastern Pacific Porcelain Crabs, Genus Petrolisthes: Influences of Latitude, Vertical Zonation, Acclimation, and Phylogeny

Marine intertidal organisms are subjected to a variety of abiotic stresses, including aerial exposure and wide ranges of temperature. Intertidal species generally have higher thermal tolerance limits than do subtidal species, and tropical species have higher thermal tolerance limits than do temperate species. The adaptive significance of upper thermal tolerance limits of intertidal organisms, however, has not been examined within a comparative context. Here, we present a comparative analysis of the adaptive significance of upper thermal tolerance limits in 20 congeneric species of porcelain crabs, genus Petrolisthes, from intertidal and subtidal habitats throughout the eastern Pacific. Upper thermal tolerance limits are positively correlated with surface water temperatures and with maximal microhabitat temperatures. Analysis of phylogenetically independent contrasts (from a phylogenetic tree on the basis of the 16s rDNA gene sequence) suggests that upper thermal tolerance limits have evolved in response to maximal microhabitat temperatures. Upper thermal tolerance limits increased during thermal acclimation at elevated temperatures, the amount of increase being greater for subtidal than for intertidal species. This result suggests that the upper thermal tolerance limits of some intertidal species may be near current habitat temperature maxima, and global warming thus may affect the distribution limits of intertidal species to a greater extent than for subtidal species.

A Violation of the Metabolism-Size Scaling Paradigm: Activities of Glycolytic Enzymes in Muscle Increase in Larger-Size Fish

The activities (units per gram wet weight of tissue) of two glycolytic enzymes (lactate dehydrogenase [LDH] and pyruvate kinase [PK]) and two citric acid cycle enzymes (citrate synthase [CS] and malate dehydrogenase [MDH]) were measured in epaxial white muscle of different-sized specimens of 13 teleost species. For CS, an enzyme associated with aerobic metabolism, the units of activity per gram muscle decreased with increasing body size, following the scaling pattern characteristically observed for aerobic respiration as a function of body size. The two anaerobically poised enzymes, LDH and PK, exhibited significantly higher activity per gram muscle in larger specimens. The dramatic increases in LDH and PK activities, both in terms of units per gram muscle and units of muscle activity per gram of total fish mass, were analyzed from the standpoint of power requirements for burst-swimming performance, since glycolytic capacity may determine a fishs ability to swim at high relative (body lengths per second) velocities. The scaling of muscle power required for maintenance of identical burst-swimming abilities (in body lengths per second) in large and small individuals of a species, based on calculations of drag scaling with body size and relative swimming velocity, agrees closely with the observed scaling of LDH and PK activity in white epaxial muscle. Brain enzymic activities were examined in three species to determine whether enzyme scaling with body size also occurred in a nonlocomotory tissue. The LDH and PK activities were virtually constant over a wide range of body size in all three species; CS activity exhibited a scaling with body size similar to the relationship found for white muscle CS activity and similar to the relationship expected between respiratory rate and body size. These different enzymic activity scaling patterns for aerobically and anaerobically poised enzymes are discussed in terms of the body-sizedependent aerobic. swimming abilities and body-size-independent anaerobic (burst) swimming abilities of fish. The ecological significance of maintaining size-independent burst-swimming ability, as measured in body lengths per second, is also considered. The contrasting aerobic and anaerobic enzyme scaling patterns found in this study indicate that the metabolic scaling paradigm observed in numerous studies of respiratory metabolism may not be applicable to anaerobic metabolism since different constraints on metabolic rate may apply under aerobic and anaerobic conditions. Limitations on aerobic metabolism may derive from surface-volume relationships and on oxygen transport capacities, i.e., on intertissue relationships. In contrast, limitations on short-term anaerobic metabolism, as used in burst swimming, may involve only intratissue factors, notably glycogen concentration and the activities of glycolytic enzymes like PK and LDH that are instrumental in supplying ATP and in regenerating NAD⁺.

Heat-Shock Protein Expression in Mytilus californianus: Acclimatization (Seasonal and Tidal-Height Comparisons) and Acclimation Effects

Heat-shock protein (hsp) expression was examined in gill of field-acclimatized and laboratory-acclimated mussels (Mytilus californianus) from the Oregon coast. Endogenous levels of heat-shock proteins in the 70-kDa class (hsp70 isoforms) and profiles of induction temperature for newly synthesized hsp 70 were measured in freshly field-collected specimens as functions of location height in the intertidal and season, and in mussels after 7 weeks of laboratory thermal acclimation. There were significant differences in endogenous levels of hsp70 as functions of season and collection height. Strong induction of new hsp70 synthesis occurred at body temperatures within the range measured in field specimens. Profiles of hsp70 thermal induction varied significantly with season, but not with height of collection. In contrast to the large differences in hsp70 expression between winter- and summer-acclimatized mussels, no differences related to temperature occurred in the differently acclimated mussels. The differences found between the effects of field acclimatization and laboratory thermal acclimation suggest that the stress response is modulated by environmental factors in addition to body temperature. Thus, caution is required in extrapolating from laboratory acclimation studies to acclimatization effects in field populations. The seasonal and tidal-height variations in the heat-shock response are discussed in the context of energy costs of protein turnover.

Buffering capacity of vertebrate muscle: Correlations with potentials for anaerobic function

SummaryBuffering capacities (β), measured in slykes (μmoles of base required to titrate the pH of one gram wet weight of muscle by one pH unit, over the pH range of pH 6 to pH 7) due to non-bicarbonate buffers were measured in locomotory muscles from a variety of terrestrial and marine mammals and teleost fishes (Tables 1 and 2). The highest buffering capacities were found in muscles capable of either intense, burst glycolytic function or prolonged, low-level anaerobic function. Marine mammals had higher muscle buffering capacity on the average than terrestrial mammals. Among the fishes studied, warm-bodied species had the greatest β values of all animals examined (Table 2). Deep-sea fishes and shallow-living fishes with sluggish locomotory abilities had low β values. Fish white muscle displayed higher buffering capacity than red muscle (Fig. 1; Table 2), in keeping with the more aerobic poise and higher capillary density of the latter type of muscle. Strong correlations were found between (1) β and muscle myoglobin concentrations in the mammalian species (Table 1; Fig. 2), and (2) β and muscle lactate dehydrogenase (LDH) activities in both the mammals and the fishes (Tables 1 and 2; Fig. 3). No correlation was found between β and the activity of a citric acid cycle indicator enzyme, citrate synthase, in the mammalian species. While strongly correlated with buffering capacity, the amounts of myoglobin and LDH in a muscle are not the principal determinants of β. The results indicate that muscle intracellular buffering capacity is especially critical in locomotory muscles which must function under conditions (burst locomotion and prolonged, low-level anaerobic function) where circulatory perfusion is inadequate to rapidly remove the acidic end-products such as lactic acid that are produced by anaerobic glycolysis. In this respect, the locomotory muscle of diving mammals and the white skeletal muscles of teleost fishes face a common acid-base regulatory problem and utilize a common biochemical strategy to resolve it.

Thermal limits and adaptation in marine Antarctic ectotherms: an integrative view

A cause and effect understanding of thermal limitation and adaptation at various levels of biological organization is crucial in the elaboration of how the Antarctic climate has shaped the functional properties of extant Antarctic fauna. At the same time, this understanding requires an integrative view of how the various levels of biological organization may be intertwined. At all levels analysed, the functional specialization to permanently low temperatures implies reduced tolerance of high temperatures, as a trade-off. Maintenance of membrane fluidity, enzyme kinetic properties (Km and kcat) and protein structural flexibility in the cold supports metabolic flux and regulation as well as cellular functioning overall. Gene expression patterns and, even more so, loss of genetic information, especially for myoglobin (Mb) and haemoglobin (Hb) in notothenioid fishes, reflect the specialization of Antarctic organisms to a narrow range of low temperatures. The loss of Mb and Hb in icefish, together with enhanced lipid membrane densities (e.g. higher concentrations of mitochondria), becomes explicable by the exploitation of high oxygen solubility at low metabolic rates in the cold, where an enhanced fraction of oxygen supply occurs through diffusive oxygen flux. Conversely, limited oxygen supply to tissues upon warming is an early cause of functional limitation. Low standard metabolic rates may be linked to extreme stenothermy. The evolutionary forces causing low metabolic rates as a uniform character of life in Antarctic ectothermal animals may be linked to the requirement for high energetic efficiency as required to support higher organismic functioning in the cold. This requirement may result from partial compensation for the thermal limitation of growth, while other functions like hatching, development, reproduction and ageing are largely delayed. As a perspective, the integrative approach suggests that the patterns of oxygen- and capacity-limited thermal tolerance are linked, on one hand, with the capacity and design of molecules and membranes, and, on the other hand, with life-history consequences and lifestyles typically seen in the permanent cold. Future research needs to address the detailed aspects of these interrelationships.

The cellular response to heat stress in the goby Gillichthys mirabilis: a cDNA microarray and protein-level analysis.

SUMMARY The cellular response to stress relies on the rapid induction of genes encoding proteins involved in preventing and repairing macromolecular damage incurred as a consequence of environmental insult. To increase our understanding of the scope of this response, a cDNA microarray, consisting of 9207 cDNA clones, was used to monitor gene expression changes in the gill and white muscle tissues of a eurythermic fish, Gillichthys mirabilis (Gobiidae) exposed to ecologically relevant heat stress. In each tissue, the induction or repression of over 200 genes was observed. These genes are associated with numerous biological processes, including the maintenance of protein homeostasis, cell cycle control, cytoskeletal reorganization, metabolic regulation and signal transduction, among many others. In both tissues, the molecular chaperones, certain transcription factors and a set of additional genes with various functions were induced in a similar manner; however, the majority of genes displayed tissue-specific responses. In gill, thermal stress induced the expression of the major structural components of the cytoskeleton, whereas these same genes did not respond to heat in muscle. In muscle, many genes involved in promoting cell growth and proliferation were repressed, perhaps to conserve energy for repair and replacement of damaged macromolecules, but a similar repression was not observed in the gill. Many of the observed changes in gene expression were similar to those described in model species whereas many others were unexpected. Measurements of the concentrations of the protein products of selected genes revealed that in each case an induction in mRNA synthesis correlated with an increase in protein production, though the timing and magnitude of the increase in protein was not consistently predicted by mRNA concentration, an important consideration in assessing the condition of the stressed cell using transcriptomic analysis.