Hypoxia is important in both biomedical and environmental contexts and necessitates rapid adaptive changes in metabolic organization. Mammals, as air breathers, have a limited capacity to withstand ...sustained exposure to hypoxia. By contrast, some aquatic animals, such as certain fishes, are routinely exposed and resistant to severe environmental hypoxia. Understanding the changes in gene expression in fishes exposed to hypoxic stress could reveal novel mechanisms of tolerance that may shed new light on hypoxia and ischemia in higher vertebrates. Using cDNA microarrays, we have studied gene expression in a hypoxia-tolerant burrow-dwelling goby fish, Gillichthys mirabilis. We show that a coherent picture of a complex transcriptional response can be generated for a nonmodel organism for which sequence data were unavailable. We demonstrate that: (i) although certain shifts in gene expression mirror changes in mammals, novel genes are differentially expressed in fish; and (ii) tissue-specific patterns of expression reflect the different metabolic roles of tissues during hypoxia.
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 activities 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.
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 (ΔG
) 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: ΔG
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.
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).
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.
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.
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,
, 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 (
) 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
in high- but not low-zone mussels, and
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.
The capacities of eurythermal ectotherms to withstand wide ranges of temperature are based, in part, on abilities to modulate gene expression as body temperature changes, notably genes encoding ...proteins of the cellular stress response. Here, using a complementary DNA microarray, we investigated the sequence in which cellular stress response-linked genes are expressed during acute heat stress, to elucidate how severity of stress affects the categories of genes changing expression. We also studied how prior acclimation history affected gene expression in response to acute heat stress. Eurythermal goby fish (Gillichthys mirabilis) were acclimated to 9 ± 0.5, 19 ± 0.5, and 28 ± 0.5°C for 1 mo. Then fish were given an acute heat ramp (4°C/h), and gill tissues were sampled every +4°C to monitor gene expression. The average onset temperature for a significant change in expression during acute stress increased by ∼2°C for each ∼10°C increase in acclimation temperature. For some genes, warm acclimation appeared to obviate the need for expression change until the most extreme temperatures were reached. Sequential expression of different categories of genes reflected severity of stress. Regardless of acclimation temperature, the gene encoding heat shock protein 70 (HSP70) was upregulated strongly during mild stress; the gene encoding the proteolytic protein ubiquitin (UBIQ) was upregulated at slightly higher temperatures; and a gene encoding a protein involved in cell cycle arrest and apoptosis, cyclin-dependent kinase inhibitor 1B (CDKN1B), was upregulated only under extreme stress. The tiered, stress level-related expression patterns and the effects of acclimation on induction temperature yield new insights into the fundamental mechanisms of eurythermy.
An insufficient supply of oxygen under thermal stress is thought to define thermal optima and tolerance limits in teleost fish. When under thermal stress, cardiac function plays a crucial role in ...sustaining adequate oxygen supply for respiring tissues. Thus, adaptive phenotypic plasticity of cardiac performance may be critical for modifying thermal limits during temperature acclimation. Here we investigated effects of temperature acclimation on oxygen consumption, cardiac function and blood oxygen carrying capacity of a eurythermal goby fish, Gillichthys mirabilis, acclimated to 9, 19 and 26°C for 4 weeks. Acclimation did not alter resting metabolic rates or heart rates; no compensation of rates was observed at acclimation temperatures. However, under an acute heat ramp, warm-acclimated fish exhibited greater heat tolerance (CTmax=33.3, 37.1 and 38.9°C for 9°C-, 19°C- and 26°C-acclimated fish, respectively) and higher cardiac arrhythmia temperatures compared with 9°C-acclimated fish. Heart rates measured under an acute heat stress every week during 28 days of acclimation suggested that both maximum heart rates and temperature at onset of maximum heart rates changed over time with acclimation. Hemoglobin levels increased with acclimation temperature, from 35 g l(-1) in 9°C-acclimated fish to 60-80 g l(-1) in 19°C- and 26°C-acclimated fish. Oxygen consumption rates during recovery from acute heat stress showed post-stress elevation in 26°C-acclimated fish. These data, coupled with elevated resting metabolic rates and heart rates at warm temperatures, suggest a high energetic cost associated with warm acclimation in G. mirabilis. Furthermore, acclimatory capacity appears to be optimized at 19°C, a temperature shown by behavioral studies to be close to the species' preferred temperature.
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 (
) 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 (
) and flatline (
) temperatures were recorded. Over 8 weeks of constant submersion, the mean
of high-zone mussels decreased by 1.07°C from baseline, but low-zone mussels' mean
was unchanged. High- and low-zone mussels' mean maximum heart rate (HR) and resting HR decreased ∼12 and 35%, respectively.
was unchanged in both groups. These data suggest that
and HR are more physiologically plastic in response to the narrowing of an animal's daily temperature range than
is, and that an animal's prior acclimatization state (high versus low) influences the acclimatory capacity of
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.
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.
Coping with environmental stress may involve combinations of behavioral and physiological responses. We examined potential interactions between adult mussels' simple behavioral repertoire - ...opening/closing of the shell valves - and thermal stress physiology in common-gardened individuals of three Mytilus congeners found on the West Coast of North America: two native species (M. californianus and M. trossulus) and one invasive species from the Mediterranean (M. galloprovincialis). We first continuously monitored valve behavior over three consecutive days on which body temperatures were gradually increased, either in air or in seawater. A temperature threshold effect was evident between 25 and 33°C in several behavioral measures. Mussels tended to spend much less time with the valves in a sealed position following exposure to 33°C body temperature, especially when exposed in air. This behavior could not be explained by decreases in adductor muscle glycogen (stores of this metabolic fuel actually increased in some scenarios), impacts of forced valve sealing on long-term survival (none observed in a second experiment), or loss of contractile function in the adductor muscles (individuals exhibited as many or more valve adduction movements following elevated body temperature compared with controls). We hypothesize that this reduced propensity to seal the valves following thermal extremes represents avoidance of hypoxia-reoxygenation cycles and concomitant oxidative stress. We further conjecture that prolonged valve gaping following episodes of elevated body temperature may have important ecological consequences by affecting species interactions. We then examined survival over a 90 day period following exposure to elevated body temperature and/or emersion, observing ongoing mortality throughout this monitoring period. Survival varied significantly among species (M. trossulus had the lowest survival) and among experimental contexts (survival was lowest after experiencing elevated body temperature in seawater). Surprisingly, we observed no cumulative impact on survival of 3 days relative to 1 day of exposure to elevated body temperature. The delayed mortality and context-specific outcomes we observed have important implications for the design of future experiments and for interpretation of field distribution patterns of these species. Ultimately, variation in the catalog of physiological and behavioral capacities among closely related or sympatric species is likely to complicate prediction of the ecological consequences of global change and species invasions.
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