Owing to its unique combination of extreme physical severity and exceptional biological diversity, the intertidal zone of rocky shores has long served as a model system for development of ecological theories and experimental tests of their predictions. This narrow interface between marine and terrestrial environments is a natural microcosm that presents organisms with rapid, extensive and often unpredictable variation in hydrodynamic forces, temperature, pH, oxygen availability and salinity. As ecologists struggle to predict the physical environment's effects on future community structure and function, it is essential to understand the physiological interactions among these factors and their role in ecology. Thus, the dynamic and heterogeneous nature of wave-swept rocky coasts provides an opportunity to exploit shoreline microcosms as a bellwether of climate change. Here, we review aspects of the intertidal environment that distinguish it from other microcosms and explore the nature of three smaller, embedded microcosms-tidepools, splashpools and the internal microcosms of individual organisms. Each of these systems comes with a distinct suite of physiological challenges and experimental potential. The variety of environmental interactions embodied by these microcosms positions rocky shores to continue serving as a model system for investigating environmental physiology, community ecology and the interplay between the two. This article is part of the theme issue 'Life in natural microcosms'.

Non-additive interactions between environmental stressors, where biological responses to simultaneous stressors do not equal the sum of individual-stressor responses, commonly occur across organisms and environments. To enable predictions of organismal resilience to shifting patterns of environmental stressors, it is important to both identify these interactions and document the mechanisms underlying them. The supratidal copepod Tigriopus californicus demonstrates a non-additive, antagonistic pattern of increased heat tolerance when simultaneously exposed to high salinities. We investigated salinity's chronic and acute effects on heat tolerance in a northern population and quantified responses in protein abundance to hypersalinity and heat stress, both alone and in combination. The overall proteomic response to multiple stressors was non-additive and largely reflected that to high temperature. However, 42% of multi-stressor proteins were absent from either single-stressor response; we refer to these proteins that are only differentially abundant in the multi-stressor scenario as "emergent". Our results suggest that the increased heat tolerance of T. californicus conferred by hypersalinity may be driven by a combination of these emergent proteins, several proteins induced by hypersalinity in both single- and multi-stressor conditions that may contribute to cross-tolerance, and four proteins with additive abundance patterns (including a small heat shock protein). These candidate proteins play putative roles in several relevant processes including the heat shock response, protein folding, and regulation of metabolism and oxidative stress responses. Our results connect to prior whole-organism findings and highlight promising pathways for future investigation in the context of heat tolerance and multi-stressor interactions.

Epigenetic modifications to DNA are proposed to underpin plastic responses to environmental change, and the manner in which DNA methylation contributes to plasticity likely differs among tissues. However, few studies have investigated tissue-specific DNA methylation responses to ecologically relevant environmental stressors in natural settings. Here, we used reduced representation bisulfite sequencing on foot and gill to examine the influence of in situ microhabitats on DNA methylation in juvenile California mussels ( Mytilus californianus ), a foundation species with widespread dispersal and little evidence of genetic population structure. We examined mussels from a one-month reciprocal transplant experiment between a cool, wave-exposed and a warm, wave-protected microhabitat. These manipulations, which were previously shown to alter juvenile mussels’ heat tolerance, led to significant and highly tissue-specific changes in CpG methylation, including within a number of genes with roles in stress response pathways. Differentially methylated genes were involved in processes including heat shock response, proteolysis, DNA repair, and temperature sensing. In gill, differentially methylated CpGs were more likely to occur in introns relative to other inter- and intragenic features. This study expands on previous research that examined environmentally driven shifts in DNA methylation by documenting plastic and tissue-specific changes in DNA methylation between microhabitats in a natural setting.

In the Anthropocene, species are increasingly faced with multiple stressors that are more severe and less predictable than before. While multiple stressors often interact to affect organisms negatively, sometimes these interactions can be beneficial, enhancing resilience through cross-protection. Cross-protection interactions occur when exposure to one stressor, such as elevated temperature, enhances an organism's tolerance to a different stressor, like hypoxia, through shared protective mechanisms or signaling pathways. Understanding the potential for cross-protection to combat rapid and diverse environmental change is crucial for conservation, as it potentially alters the predicted consequences of such change. Here, we outline 10 key considerations for investigating cross-protection in a conservation context. These considerations include the importance of stressor intensity and timing, recognizing species-specific and sex-specific responses, and embracing temporal variability in environmental stressors. Additionally, predictions will depend upon uncovering the underlying mechanisms of cross-protection by integrating emerging approaches like omics and meta-analyses. By better understanding-and in some cases explicitly leveraging-cross-protective interactions, conservation practitioners may be able to develop more effective management plans to enhance species resilience, potentially mitigating the immediate effects of emerging stressors. These insights are vital for guiding future research directions and informing conservation policies and management practices to preserve biodiversity in the Anthropocene.

In natural environments, two or more abiotic parameters often vary simultaneously, and interactions between co-varying parameters frequently result in unpredictable, non-additive biological responses. To better understand the mechanisms and consequences of interactions between multiple stressors, it is important to study their effects on not only fitness (survival and reproduction) but also performance and intermediary physiological processes. The splash-pool copepod Tigriopus californicus tolerates extremely variable abiotic conditions and exhibits a non-additive, antagonistic interaction resulting in higher survival when simultaneously exposed to high salinity and acute heat stress. Here, we investigated the response of T. californicus in activity and oxygen consumption under simultaneous manipulation of salinity and temperature to identify whether this interaction also arises in these sublethal measures of performance. Oxygen consumption and activity rates decreased with increasing assay salinity. Oxygen consumption also sharply increased in response to acute transfer to lower salinities, an effect that was absent upon transfer to higher salinities. Elevated temperature led to reduced rates of activity overall, resulting in no discernible impact of increased temperature on routine metabolic rates. This suggests that swimming activity has a non-negligible effect on the metabolic rates of copepods and must be accounted for in metabolic studies. Temperature also interacted with assay salinity to affect activity, and with acclimation salinity to affect routine metabolic rates upon acute salinity transfer, implying that the sublethal impacts of these co-varying factors are also not predictable from experiments that study them in isolation.

Stenothermal Antarctic notothenioid fishes are noteworthy for their history of isolation in extreme cold and their corresponding lack of the canonical heat shock response. Despite extensive transcriptomic studies, the mechanistic basis for stenothermy has not been fully elucidated. Given that the proteome better represents an organism's physiology, the possibility exists that some aspects of stenothermy arise posttranscriptionally. Here, Antarctic emerald rockcod (Trematomus bernacchii) were sampled after exposure to chronic and/or acute high temperatures, followed by a thorough assessment of proteomic responses in the brain, gill, and kidney. Few cellular stress response proteins were induced, and overall responses were modest in terms of the numbers of differentially expressed proteins and their fold changes. Inconsistencies in protein induction across treatments and tissues are suggestive of dysregulation, rather than an adaptive response. Changes in regulation of the translational machinery in Antarctic notothenioids could explain these patterns. Some components of translational regulatory pathways are highly conserved [e.g., Ser-52, eukaryotic translation initiation factor 2α (eIF2α)], but other proteins comprising the cellular "integrated stress response," specifically, the eIF2α kinases general control nonderepressible 2 (GCN2) and PKR-like endoplasmic reticulum kinase (PERK), may have evolved along different trajectories in Antarctic fishes. Taken together, these observations suggest a novel hypothesis for stenothermy and the absence of a coordinated cellular stress response in Antarctic fishes.NEW & NOTEWORTHY Antarctic fishes have some of the lowest known heat tolerances among vertebrates, but the molecular mechanisms underlying this pattern are not fully understood. By combining detailed analyses of protein expression patterns in several tissues under various heat treatments with a broader evolutionary perspective, this study offers a novel hypothesis to explain the narrow range of temperature tolerance in this extraordinary group of fishes.

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.

ABSTRACT Organismal responses to stressful environments are influenced by numerous transcript- and protein-level mechanisms, and the relationships between expression changes at these levels are not always straightforward. Here, we used paired transcriptomic and proteomic datasets from two previous studies from gill of the California mussel, Mytilus californianus, to explore how simultaneous transcript and protein abundance patterns may diverge under different environmental scenarios. Field-acclimatized mussels were sampled from two disparate intertidal sites; individuals from one site were subjected to three further treatments (common garden, low-intertidal or high-intertidal outplant) that vary in temperature and feeding time. Assessing 1519 genes shared between the two datasets revealed that both transcript and protein expression patterns differentiated the treatments at a global level, despite numerous underlying discrepancies. There were far more instances of differential expression between treatments in transcript only (1451) or protein only (226) than of the two levels shifting expression concordantly (68 instances). Upregulated expression of cilium-associated transcripts (likely related to feeding) was associated with relatively benign field treatments. In the most stressful treatment, transcripts, but not proteins, for several molecular chaperones (including heat shock proteins and endoplasmic reticulum chaperones) were more abundant, consistent with a threshold model for induction of translation of constitutively available mRNAs. Overall, these results suggest that the relative importance of transcript- and protein-level regulation (translation and/or turnover) differs among cellular functions and across specific microhabitats or environmental contexts. Furthermore, the degree of concordance between transcript and protein expression can vary across benign versus acutely stressful environmental conditions.

To better understand life in the sea, marine scientists must first quantify how individual organisms experience their environment, and then describe how organismal performance depends on that experience. In this review, we first explore marine environmental variation from the perspective of pelagic organisms, the most abundant life forms in the ocean. Generation time, the ability to move relative to the surrounding water (even slowly), and the presence of environmental gradients at all spatial scales play dominant roles in determining the variation experienced by individuals, but this variation remains difficult to quantify. We then use this insight to critically examine current understanding of the environmental physiology of pelagic marine organisms. Physiologists have begun to grapple with the complexity presented by environmental variation, and promising frameworks exist for predicting and/or interpreting the consequences for physiological performance. However, new technology needs to be developed and much difficult empirical work remains, especially in quantifying response times to environmental variation and the interactions among multiple covarying factors. We call on the field of global-change biology to undertake these important challenges.

The environment can alter the magnitude of phenotypic variation among individuals, potentially influencing evolutionary trajectories. However, environmental influences on variation are complex and remain understudied. Populations in heterogeneous environments might exhibit more variation, the amount of variation could differ between benign and stressful conditions, and/or variation might manifest in different ways among stages of the gene-to-protein expression cascade or among physiological functions. Here, we explore these three issues by quantifying patterns of inter-individual variation in both transcript and protein expression levels among California mussels, Mytilus californianus Conrad. Mussels were exposed to five ecologically relevant treatments that varied in the mean and interindividual heterogeneity of body temperature. To target a diverse set of physiological functions, we assessed variation within 19 expression subnetworks, including canonical stress-response pathways and empirically derived coexpression clusters that represent a diffuse set of cellular processes. Variation in expression was particularly pronounced in the treatments with high mean and heterogeneous body temperatures. However, with few exceptions, environment-dependent shifts of variation in the transcriptome were not reflected in the proteome. A metric of phenotypic integration provided evidence for a greater degree of constraint on relative expression levels (i.e., stronger correlation) within expression subnetworks in benign, homogeneous environments. Our results suggest that environments that are more stressful on average - and which also tend to be more heterogeneous - can relax these expression constraints and reduce phenotypic integration within biochemical subnetworks. Context-dependent "unmasking" of functional variation may contribute to interindividual differences in physiological phenotype and performance in stressful environments.

Synopsis Accurate forecasting of organismal responses to climate change requires a deep mechanistic understanding of how physiology responds to present-day variation in the physical environment. However, the road to physiological enlightenment is fraught with complications: predictable environmental fluctuations of any single factor are often accompanied by substantial stochastic variation and rare extreme events, and several factors may interact to affect physiology. Lacking sufficient knowledge of temporal patterns of co-variation in multiple environmental stressors, biologists struggle to design and implement realistic and relevant laboratory experiments. In this study, we directly address these issues, using measurements of the thermal tolerance of freshly collected animals and long-term field records of environmental conditions to explore how the splash-pool copepod Tigriopus californicus adjusts its physiology as its environment changes. Salinity and daily maximum temperature—two dominant environmental stressors experienced by T. californicus—are extraordinarily variable and unpredictable more than 2–3 days in advance. However, they substantially co-vary such that when temperature is high salinity is also likely to be high. Copepods appear to take advantage of this correlation: median lethal temperature of field-collected copepods increases by 7.5°C over a roughly 120 parts-per-thousand range of ambient salinity. Complementary laboratory experiments show that exposure to a single sublethal thermal event or to an abrupt shift in salinity also elicits rapid augmentation of heat tolerance via physiological plasticity, although the effect of salinity dwarfs that of temperature. These results suggest that T. californicus’s physiology keeps pace with the rapid, unpredictable fluctuations of its hypervariable physical environment by responding to the cues provided by recent sublethal stress and, more importantly, by leveraging the mechanistic cross-talk between responses to salinity and heat stress.

Shifting climate patterns may impose novel combinations of abiotic conditions on animals, yet understanding of the present-day interactive effects of multiple stressors remains under-developed. We tested the oxygen and capacity limited thermal tolerance (OCLTT) hypothesis and quantified environmental preference of the copepod Tigriopus californicus, which inhabits rocky-shore splashpools where diel fluctuations of temperature and dissolved oxygen (DO) are substantial. Egg-mass bearing females were exposed to a 5 h heat ramp to peak temperatures of 34.1–38.0 °C crossed with each of four oxygen levels: 22, 30, 100 and 250% saturation (4.7–5.3, 5.3–6.4, 21.2–21.3, and 50.7–53.3 kPa). Survival decreased at higher temperatures but was independent of DO. The behavioral preference of females was quantified in seven combinations of gradients of both temperature (11–37 °C) and oxygen saturation (17–206% or 3.6–43.6 kPa). Females avoided high temperatures regardless of DO levels. This pattern was more pronounced when low DO coincided with high temperature. In uniform temperature treatments, the distribution shifted toward high DO levels, especially in uniform high temperature, confirming that Tigriopus can sense environmental pO2. These results question the ecological relevance of OCLTT for Tigriopus and raise the possibility of microhabitat selection being used within splashpool environments to avoid physiologically stressful combinations of conditions.

Accelerating shifts in global climate have focused the attention of ecologists and physiologists on extreme environmental events. However, the dynamic process of physiological acclimatization complicates study of these events' consequences. Depending on the range of plasticity and the amplitude and speed of environmental variation, physiology can be either in tune with the surroundings or dangerously out of synch. We implement a modified quantitative approach to identifying extreme events in environmental records, proposing that organisms are stressed by deviations of the environment from the current level of acclimatization, rather than by the environment's absolute state. This approach facilitates an unambiguous null model for the consequences of environmental variation, identifying a unique subset of events as ‘extremes’. Specifically, it allows one to examine how both the temporal extent (the acclimatization window) and type of an environmental signal affect the magnitude and timing of extreme environmental events. For example, if physiology responds to the moving average of past conditions, a longer acclimatization window generally results in greater imposed stress. If instead physiology responds to historical maxima, longer acclimatization windows reduce imposed stress, albeit perhaps at greater constitutive cost. This approach should be further informed and tested with empirical experiments addressing the history-dependent nature of acclimatization.

The rocky intertidal zone is a dynamic and stressful habitat, dominated by strong vertical gradients of emersion time. Marine organisms that settle high on the shore are exposed to greater temperature stress, increased desiccation risk, and reduced access to their suspended food compared with their low-shore neighbors. The ribbed mussel Mytilus californianus is one such species that occupies a broad vertical intertidal range. The present study asked whether high-shore mussels might conserve energy by employing larger, energetically cheaper adductor muscle fibers. This conjecture is based on the optimal fiber size hypothesis, which posits in part that the reduction in surface area (i.e., cell membrane area) to volume ratio of large-diameter muscle fibers also reduces the ATP costs of maintaining resting ion gradients across the sarcolemmal membrane. The evidence suggests that high-shore mussels collected in Pacific Grove, California, USA, in 2015 do have fibers with larger cross-sectional areas when controlling for body mass. This morphological trait is paired with a lower mass-specific activity of the aerobic enzyme citrate synthase in adductor muscle. A common-garden treatment revealed that enzyme activity, but not muscle fiber area, changed relatively quickly (4weeks). Previous studies have supported the optimal fiber size hypothesis by documenting hypertrophic growth of muscle fibers during ontogeny, but few studies have examined whether fiber sizes in ectotherms might be adjusted based on an individual's environmental context. Although a definitive link to organismal costs remains to be demonstrated, this mechanism may mitigate some of the costs for organisms living in energy-constrained habitats.

The interaction of ocean conditions and weather with small-scale physical features of a habitat can have profound effects on the experiences of individual organisms. On topographically complex shorelines, and particularly within dense aggregations of organisms such as mussel beds, a mosaic of environmental conditions can develop, and the resulting variation in conditions within the aggregation could drastically alter the performance of neighboring individuals. Using a suite of sensors mounted to individual Mytilus californianus mussels over two summer field deployments, we have characterized the temperature variation and valve gaping behavior differences found at two spatial scales: within a group separated by centimeters, and between groups of mussels located at the upper and lower extents of the natural mussel zone separated by meters. While temperature conditions near the lower edge of the mussel bed were generally more benign, temperature extremes were similar at both heights in the bed, and variation in body temperature among neighbors increased as the daily mean temperature increased. These patterns were similar across years despite a 3.8 °C difference in mean air and seawater temperatures between years. Gaping behavior was also highly variable among individuals, though that variability diminished at the high end of the mussel bed where the total time mussels spent submerged was much more constrained. These data indicate that an individual mussel's physiological status and past history can be drastically different than those of its nearby neighbors, complicating our ability to characterize representative conditions within a habitat. These observations also provide for the possibility that the impacts of future climate change will be highly specific to certain individuals based on their relative exposure or protection within the mosaic. To address such possibilities, future work must examine the correlation between genotypic and physiological traits that determine performance and individuals' unique experiences in their disparate micro-environments.

Greater understanding of physiological responses to climate change demands deeper comprehension of the causes and consequences of physiological variation. Increasingly, population trait means are being deconstructed into variable signals at the level of individuals. We advocate for greater consideration of such inter-individual physiological variation and how it both depends on and interacts with environmental variability. First, we review several studies on the intertidal mussel Mytilus californianus to illustrate how the magnitude of inter-individual variation may depend on the environmental context analyzed (i.e., is the mean condition benign or stressful?) and/or on the specific physiological metric investigated. Stressful conditions may reveal or mask variation in disparate ways at different levels of analysis (e.g., transcriptome vs. proteome), but we often lack crucial information regarding the relationships among these different physiological metrics and their consequences for fitness. We then reanalyze several published datasets to ask whether individuals employ divergent strategies over time in response to acute heat stress; such time-dependence would further complicate interpretation of physiological variation. However, definitive conclusions are precluded by limited sample sizes and short timescales in extant datasets. A key remaining challenge is to extend these analytical frameworks to longer periods over which individuals in a population experience repeated, but spatially variable, episodic stress events. We conclude that variation at multiple levels of analysis should be investigated over longer periods and, where possible, within individuals (or genotypes) experiencing repeated environmental challenges. Although difficult in practice, such studies will facilitate improved understanding of potential population-level physiological responses to climate change.