Lani U Gleason

In this era of climate change there is an urgent need to better understand the mechanisms that allow organisms to thrive vs. fail in thermally stressful environments. In particular, there is growing evidence that the “holobiont” (host animal + microbiome community of bacteria, fungi, and archaea that live in an organism) affects how organisms respond to environmental stressors such as temperature and thus should be studied further. Rocky intertidal species such as Tegula snails are ideal organisms for these types of studies because closely related species exhibit variability in heat tolerance. Here, we assess potential microbiome bacterial contributions to thermal tolerance in Tegula eiseni, Tegula funebralis , and Tegula gallina that co-occur in southern California but occupy different intertidal heights that vary in thermal stress exposure. 16S sequencing of the V4 region of individuals of each species exposed to control conditions (ambient temperature = 15°C) or a single short duration 5.5-hour heat stress (maximum temperature = 34°C) revealed distinct bacterial communities across species. Moreover, unique bacterial genera of the microbiome were significantly enriched (more abundant) in each Tegula species. Lutimonas, Polaribacter , and the exopolysaccharide (EPS)-producing bacteria Pelagicoccus were most abundant in T. gallina , the species that occupies the highest intertidal heights and thus experiences heat stress most frequently. These results suggest that microbiome-derived metabolites such as EPS could be contributing to the higher thermal tolerance of T. gallina. Overall, this study demonstrates that the bacterial microbiome should be considered when examining mechanisms of thermal tolerance in marine invertebrates.

Climate change-induced population declines of highly abundant intertidal invertebrates will affect ecosystem function and stability, but it is not fully understood which species are most vulnerable to these declines. Rocky intertidal Tegula snails live at different tidal heights and exhibit variability in heat tolerance, but the temperatures these different species experience in the field, and thus which species are most susceptible to climate change, remains unknown. Here, we use HOBO data loggers to record field temperatures in the unique thermal habitats of Tegula eiseni, Tegula funebralis, and Tegula gallina in San Diego, California. Determining the maximum temperatures each Tegula species is exposed to in the field will ultimately inform conservation efforts by identifying which Tegula species are most threatened by heat stress.

Although most marine invertebrates are experiencing multiple environmental stressors simultaneously, the transcriptome-wide gene expression responses to multiple stressors remain understudied. We used RNA-sequencing to assess the transcriptomic responses to heat stress, starvation, and heat stress plus starvation in the red abalone Haliotis rufescens. Results indicate that the response to each stressor is distinct and is characterized by unique gene functions. The heat stress plus starvation treatment produced the largest transcriptomic response, including a significant upregulation of genes involved in translation. Overall, this study highlights the importance of multi-stressor experiments that reflect the complex modalities of climate change.

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 Conrad 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 vs. acutely stressful environmental conditions.

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 inter-individual 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 co-expression 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 inter-individual differences in physiological phenotype and performance in stressful environments.

RNA-sequencing has provided a new way to study transcriptomic population differences of non-model organisms such as marine invertebrates. Importantly, population transcriptomics can provide insight regarding the genetic mechanisms that allow some populations to tolerate various environmental stressors in this era of climate change. Such studies can help us predict future population dynamics and species survival for marine invertebrates of ecological and economic value. Here, I review practical considerations for marine invertebrate researchers using RNA-sequencing, recent applications of population transcriptomics in marine invertebrates, current limitations of this approach, and future areas of study.Recent marine invertebrate studies have examined population-specific responses to abiotic stressors including temperature, salinity, pH, and water quality. Populations tolerant of environmental stressors generally show high constitutive gene expression before stress exposure and a muted overall transcriptional response following stress exposure. Evidence for molecular signatures of selection on several genes including ATP synthase and aquaporins has been identified in marine invertebrate populations exposed to broad temperature regimes. At present, the genetic mechanisms underlying individual variation within a population and the transcriptome response to multiple environmental stressors remain poorly understood.Complete assessment of marine invertebrate population responses to climate change will require pairing RNA-sequencing approaches with complementary phenotype studies. Improvements in transcriptome annotation and future work investigating gene expression regulation will lead to an increased understanding of the genes responsible for population-level tolerance to environmental stressors.

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 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.

Southern California (USA) populations of the intertidal snail
occupy warmer climates than northern California populations, and southern populations are more thermally tolerant and have unique transcriptomic responses to heat stress compared to northern populations. To investigate how climate affects body temperature patterns for
, iButton temperature loggers encased in empty
shells (robosnails) were deployed at 3 northern and 3 southern California sites for 1.5 mo in the late summer and early fall of 2014, typically when maximum annual temperatures are reached. Measurements revealed that southern, thermally tolerant populations experienced higher average daily maximum and absolute maximum temperatures than northern, less tolerant populations, and that robosnails in southern, but not northern, California exceeded temperatures that cause 100% mortality. Similarly, the probability of a site reaching 27°C, the temperature that induces the heat shock response in
, was 3 times higher at the southern compared to the northern sites. To determine whether these exposures to stressful temperatures are related to gene expression differences, we then tested for a correlation between the probability of reaching 27°C and the constitutive (non-induced) expression of genes previously implicated as pre-adapted in southern California populations. We identified 222 genes (including 14 involved in ubiquitin protein degradation, a response to heat stress) with a significant correlation. The results demonstrate how combining
temperature and transcriptome data can increase our understanding of thermal adaptation and better inform predictions regarding the impact of future climate change.