To investigate the role of gene expression in adaptation of marine ectotherms to different temperatures, we examined the transcriptome-wide thermal stress response in geographically separated populations of the intertidal snail Chlorostoma funebralis. Snails from two southern (heat tolerant) and two northern (heat sensitive) populations were acclimated to a common thermal environment, exposed to an environmentally relevant thermal stress and analysed using RNA-seq. Pooling across all populations revealed 306 genes with differential expression between control and heat-stressed samples, including 163 significantly upregulated and 143 significantly downregulated genes. When considered separately, regional differences in response were widely apparent. Heat shock proteins (Hsps) were upregulated in both regions, but the magnitude of response was significantly greater in northern populations for most Hsp70s, while the southern populations showed greater upregulation for approximately half of the Hsp40s. Of 177 stress-responsive genes in northern populations, 55 responded to heat stress only in northern populations. Several molecular chaperones and antioxidant genes that were not differentially expressed in southern populations showed higher expression under control conditions compared with northern populations. This suggests that evolution of elevated expression of these genes under benign conditions preadapts the southern populations to frequent heat stress and contributes to their higher thermal tolerance. These results indicate that evolution has resulted in different transcriptome responses across populations, including upregulation of genes in response to stress and preadaptation of genes in anticipation of stress (based on evolutionary history of frequent heat exposure). The relative importance of the two mechanisms differs among gene families and among populations.

The balance between natural selection, gene flow and genetic drift is difficult to resolve in marine invertebrates with extensive dispersal and fluctuating population sizes. The intertidal snail Chlorostoma funebralis has planktonic larvae and previous work using mtDNA polymorphism reported no genetic population structure. Nevertheless, recent studies have documented differences in thermal tolerance and transcriptomic responses to heat stress between northern and southern California, USA, populations. To gain insight into the dynamics influencing adaptive divergence, we used double-digest restriction site-associated DNA (ddRAD) sequencing to identify 1861 genomewide, quality-filtered single-nucleotide polymorphism (SNP) loci for C. funebralis collected from three northern and three southern California sites (15 individuals per population). Considering all SNPs, there was no evidence for genetic differentiation among populations or regions (average FST  = 0.0042). However, outlier tests revealed 34 loci putatively under divergent selection between northern and southern populations, and structure and SNP tree analyses based on these outliers show clear genetic differentiation between geographic regions. Three of these outliers are known or hypothesized to be involved in stress granule formation, a response to environmental stress such as heat. Combined with previous work that found thermally tolerant southern populations show high baseline expression of stress response genes, these results further suggest that thermal stress is a strong selective pressure across C. funebralis populations. Overall, this study increases our understanding of the factors constraining local adaptation in marine organisms, while suggesting that ecologically driven, strong differentiation can occur at relevant loci in a species with planktonic larvae.

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 (). 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.

The location and abundance of fish eggs provide information concerning the timing and location of spawning activities and can provide fishery-independent estimates of spawning biomass. However, the full value of egg and larval surveys is severely restricted because many species' eggs and larvae are morphologically similar, making species-level identification difficult. Recent efforts have shown that nearly all species of fish may be identified by mitochondrial DNA (mtDNA) sequences (e.g. via 'DNA barcoding'). By taking advantage of a DNA barcode database, we have developed oligonucleotide probes for 23 marine fish species that produce pelagic eggs commonly found in California waters. Probes were coupled to fluorescent microspheres to create a suspension bead array. Biotin-labelled primers were used to amplify the mitochondrial cytochrome oxidase subunit I (COI) and 16S ribosomal rRNA genes from individual fish eggs. The amplicons were then hybridized to the bead array, and after the addition of a reporter fluorophore, samples were analysed by flow cytometry with Luminex 100 instrumentation. Probes specifically targeted eggs that are abundant and/or from morphologically indistinguishable species pairs. Results showed that the 33 different probes designed for this study accurately identified all samples when PCR was successful. Suspension bead arrays have a number of benefits over other methods of molecular identification; these arrays permit high multiplexing, simple addition of new probes, high throughput and lower cost than DNA sequencing. The increasing availability of DNA barcode data for numerous fish faunas worldwide suggests that bead arrays could be developed and widely used for fish egg, larval and tissue identifications.

Southern California (USA) populations of the intertidal marine snailChlorostoma (formerly Tegula) funebralis generally occupy warmer climates and are exposed to high air temperatures during low tides more often than northern California populations. Available genetic data suggest there is extensive gene flow across a broad range of C. funebralis populations, so it is unclear if populations can adapt to differences in local environments. To test for population-specific responses to heat stress, three phenotypic assays were performed on three northern and on three southern populations of C. funebralis, after acclimation to common-garden conditions in the laboratory.Thermal drop-down, heat stress mortality, and heat stress reattachment assays were designed to evaluate ecologically relevant phenotypic responses to heat stress; these assays assessed tolerance during, mortality following, and speed of recovery following heat stress. The latter two tests indicate that southern populations consistently suffer significantly lower mortality and recover significantly more quickly following heat stress compared to northern populations. Hierarchical cluster analysis of stress response data clearly identified northern California and southern California regional groupings of populations. Thus, these results indicate that southern populations have higher tolerance to heat stress than northern populations and suggest that adaptation to local environmental differences can evolve despite moderate potential for larval dispersal in this species. Accounting for intraspecific population variation in thermal tolerance may provide important insights for predicting how species distributions will respond to global warming.al., 2010) and that adaptive differentiation often occurs in species with planktonic dispersal (Sanford and Kelly, 2010), local adaptation in the sea remains understudied. As rates of environmental change are accelerating

Complex life cycles characterized by uncertainty at transitions between larval/juvenile and adult environments could favour irreversible physiological plasticity at such transitions. To assess whether thermal tolerance of intertidal mussels () adjusts to post-settlement environmental conditions, we collected juveniles from their thermally buffered microhabitat from high- and low-shore locations at cool (wave-exposed) and warm (wave-protected) sites. Juveniles were transplanted to unsheltered cages at the two low sites or placed in a common garden. Juveniles transplanted to the warm site for one month in summer had higher thermal tolerance, regardless of origin site. By contrast, common-garden juveniles from all sites had lower tolerance indistinguishable from exposed site transplants. After six months in the field plus a common garden period, there was a trend for higher thermal tolerance at the protected site, while reduced thermal tolerance at both sites indicated seasonal acclimatization. Thermal tolerance and growth rate were inversely related after one but not six months; protected-site transplants were more tolerant but grew more slowly. In contrast to juveniles, adults from low-shore exposed and protected sites retained differences in thermal tolerance after common garden treatment in summer. Both irreversible and reversible forms of plasticity must be considered in organismal responses to changing environments.

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 interindividual 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.

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