Effects of Intertidal Position on Metabolism and Behavior in the Acorn Barnacle, Balanus glandula.

The intertidal zone is characterized by persistent, tidally-driven fluctuations in both abiotic (e.g., temperature, oxygen, and salinity) and biotic (e.g., food availability and predation) factors, which make this a physiologically challenging habitat for resident organisms. The relative magnitude and degree of variability of environmental stress differ between intertidal zones, with the most extreme physiological stress often being experienced by organisms in the high intertidal. Given that so many of the constantly shifting parameters in this habitat are primary drivers of metabolic rate (e.g., temperature, [O2], and food availability), we hypothesized that sessile conspecifics residing in different tidal zones would exhibit distinct "metabolic phenotypes," a term we use to collectively describe the organisms' baseline metabolic performance and capacity. To investigate this hypothesis, we collected acorn barnacles (Balanus glandula) from low, mid, and high intertidal positions in San Luis Obispo Bay, CA, and measured a suite of biochemical (whole-animal citrate synthase (CS) and lactate dehydrogenase (LDH) activity, and aerial [D-lactate]), physiological (O2 consumption rates), morphological (body size), and behavioral (e.g., cirri beat frequency and percentage of time operculum open) indices of metabolism. We found tidal zone-dependent differences in B. glandula metabolism that primarily related to anaerobic capacity, cirral activity patterns, and body size. Barnacles from the low intertidal tended to have a greater capacity for anaerobic metabolism (i.e., increased LDH activity and increased baseline [D-lactate]), have reduced cirral beating activity-and presumably reduced feeding-when submerged, and be smaller in size compared to conspecifics in the high intertidal. We did not, however, see any D-lactate accumulation in barnacles from any tidal height throughout 96 h of air exposure. This trend indicates that the enhanced capacity of low intertidal barnacles for anaerobic metabolism may have evolved to support metabolism during more prolonged episodes of emersion or during events other than emersion (e.g., coastal hypoxia and predation). There were also no significant differences in CS activity or baseline O2 consumption rates (in air or seawater at 14°C) across tidal heights, which imply that aerobic metabolic capacity may not be as sensitive to tidal position as anaerobic processes. Understanding how individuals occupying different shore heights differ in their metabolic capacity becomes increasingly interesting in the context of global climate change, given that the intertidal zone is predicted to experience even greater extremes in abiotic stress.

Physiological responses to heat stress in an invasive mussel Mytilus galloprovincialis depend on tidal habitat.

Mussels are ecologically important organisms that can survive in subtidal and intertidal zones where they experience thermal stress. We know little about how mussels from different tidal habitats respond to thermal stress. We used the mussel Mytilus galloprovincialis from separate subtidal and intertidal populations to test whether heart rate and indicators of potential aerobic (citrate synthase activity) and anaerobic (cytosolic malate dehydrogenase activity) metabolic capacity are affected by increased temperatures while exposed to air or submerged in water. Subtidal mussels were affected by warming when submerged in water (decreased heart rate) but showed no effect in air. In contrast, intertidal mussels were affected by exposure to air (increased anaerobic capacity) but not by warming. Overall, physiological responses of mussels to thermal stress were dependent on their tidal habitat. These results highlight the importance of considering the natural habitat of mussels when assessing their responses to environmental challenges.

Characterization of thermally sensitive miRNAs reveals a central role of the FoxO signaling pathway in regulating the cellular stress response of an extreme stenotherm, Trematomus bernacchii.

Despite the lack of an inducible heat shock response (HSR), the Antarctic notothenioid fish, Trematomus bernacchii, has retained a level of physiological plasticity that can at least partially compensate for the effects of acute heat stress. Over the last decade, both physiological and transcriptomic studies have signaled these fish can mitigate the effects of acute heat stress by employing other aspects of the cellular stress response (CSR) that help confer thermotolerance as well as drive homeostatic mechanisms during long-term thermal acclimations. However, the regulatory mechanisms that determine temperature-induced changes in gene expression remain largely unexplored in this species. Therefore, this study utilized next generation sequencing coupled with an in silico approach to explore the regulatory role of microRNAs in governing the transcriptomic level response observed in this Antarctic notothenioid with respect to the CSR. Using RNAseq, we characterized the expression of 125 distinct miRNA orthologues in T. bernacchii gill tissue. Additionally, we identified 12 miRNAs that appear to be thermally responsive based on differential expression (DE) analyses performed between fish acclimated to control (-1.5 °C) and an acute heat stress (+4 °C). We further characterized the functional role of these DE miRNAs using bioinformatics pipelines to identify putative gene targets of the DE miRNAs and subsequent gene set enrichment analyses, which together suggest these miRNAs are involved in regulating diverse aspects of the CSR in T. bernacchii.

Subcellular localization of dinoflagellate polyketide synthases and fatty acid synthase activity.

Dinoflagellates are prolific producers of polyketide secondary metabolites. Dinoflagellate polyketide synthases (PKSs) have sequence similarity to Type I PKSs, megasynthases that encode all catalytic domains on a single polypeptide. However, in dinoflagellate PKSs identified to date, each catalytic domain resides on a separate transcript, suggesting multiprotein complexes similar to Type II PKSs. Here, we provide evidence through coimmunoprecipitation that single-domain ketosynthase and ketoreductase proteins interact, suggesting a predicted multiprotein complex. In Karenia brevis (C.C. Davis) Gert Hansen & Ø. Moestrup, previously observed chloroplast localization of PKSs suggested that brevetoxin biosynthesis may take place in the chloroplast. Here, we report that PKSs are present in both cytosol and chloroplast. Furthermore, brevetoxin is not present in isolated chloroplasts, raising the question of what chloroplast-localized PKS enzymes might be doing. Antibodies to K. brevis PKSs recognize cytosolic and chloroplast proteins in Ostreopsis cf. ovata Fukuyo, and Coolia monotis Meunier, which produce different suites of polyketide toxins, suggesting that these PKSs may share common pathways. Since PKSs are closely related to fatty acid synthases (FAS), we sought to determine if fatty acid biosynthesis colocalizes with either chloroplast or cytosolic PKSs. [(3) H]acetate labeling showed fatty acids are synthesized in the cytosol, with little incorporation in chloroplasts, consistent with a Type I FAS system. However, although 29 sequences in a K. brevis expressed sequence tag database have similarity (BLASTx e-value <10(-10) ) to PKSs, no transcripts for either Type I (cytosolic) or Type II (chloroplast) FAS are present. Further characterization of the FAS complexes may help to elucidate the functions of the PKS enzymes identified in dinoflagellates.

High latitude fish in a high CO2 world: Synergistic effects of elevated temperature and carbon dioxide on the metabolic rates of Antarctic notothenioids.

Although the physiological response of teleost fishes to increased temperature has been well documented, there is only a small body of literature that examines the effects of ocean acidification on fish under ecologically relevant scenarios. Furthermore, little data exists which examines the possible synergistic effects of increased sea surface temperatures and pCO(2) levels, although it is well established that both will co-committedly change in the coming centuries. In this study we examined the effects of increased temperature, increased pCO(2), and a combination of these treatments on the resting metabolic rate (RMR) of four species of notothenioid fish, Trematomus bernacchii, T. hansoni, T. newnesi, and Pagothenia borchgrevinki, acclimated to treatment conditions for 7, 14 or 28days. While most species appear capable of rapidly acclimating to increased pCO(2), temperature continues to impact RMRs for up to 28days. One species in particular, T. newnesi, displayed no acclimatory response to any of the treatments regardless of acclimation time and may have a reduced capacity to respond to environmental change. Furthermore, we present evidence that temperature and pCO(2) act synergistically to further elevate the RMR and slow acclimation when compared to temperature or pCO(2) increases alone.

Effects of temperature change on mussel, Mytilus.

An increasing body of research has demonstrated the often idiosyncratic responses of organisms to climate-related factors, such as increases in air, sea and land surface temperatures, especially when coupled with non-climatic stressors. This argues that sweeping generalizations about the likely impacts of climate change on organisms and ecosystems are likely less valuable than process-based explorations that focus on key species and ecosystems. Mussels in the genus Mytilus have been studied for centuries, and much is known of their physiology and ecology. Like other intertidal organisms, these animals may serve as early indicators of climate change impacts. As structuring species, their survival has cascading impacts on many other species, making them ecologically important, in addition to their economic value as a food source. Here, we briefly review the categories of information available on the effects of temperature change on mussels within this genus. Although a considerable body of information exists about the genus in general, knowledge gaps still exist, specifically in our ability to predict how specific populations are likely to respond to the effects of multiple stressors, both climate and non-climate related, and how these changes are likely to result in ecosystem-level responses. Whereas this genus provides an excellent model for exploring the effects of climate change on natural and human-managed ecosystems, much work remains if we are to make predictions of likely impacts of environmental change on scales that are relevant to climate adaptation.

Physiological tolerances across latitudes: thermal sensitivity of larval marine snails (Nucella spp.).

A critical step in understanding how temperature will affect biodiversity in coastal ecosystems is to gain insight into how the tolerances, and ultimately survival, of early life history stages will influence the distribution and abundance of adults. We assessed the thermal tolerance of encapsulated veliger-stage larvae of a common dogwhelk, Nucella ostrina, that occur in the rocky intertidal zone on the west coast of North America. Results showed that veligers collected from northern latitudes in Washington State were less tolerant of heat stress than those from central sites in California. For all sites, we found there to be a subtle difference between the temperatures at which veligers first began to die compared to when veligers reached 100% mortality. On a biogeographic scale, the LT50 temperatures, a measure of larval sensitivity, for N. ostrina veligers displayed a strong latitudinal trend. These findings provide a conservative measurement of the upper thermal limits of encapsulated veligers while illustrating how these early life history stages could be physiologically compromised under future climate warming scenarios.

Constitutive roles for inducible genes: evidence for the alteration in expression of the inducible hsp70 gene in Antarctic notothenioid fishes.

Previous research on the Antarctic notothenioid fish Trematomus bernacchii demonstrated the loss of the heat shock response (HSR), a classical cellular defense mechanism against thermal stress, characterized by the rapid synthesis of heat shock proteins (Hsps). In the current study, we examined potential mechanisms for the apparent loss of the HSR in Antarctic notothenioids and, in addition, compared expression patterns of two genes from the 70-kDa Hsp family (hsc71 and hsp70) in tissues from T. bernacchii to expression patterns in tissues of two closely related temperate notothenioid fishes from New Zealand, Bovichtus variegatus and Notothenia angustata. The results showed that transcript for both the constitutive and inducible genes in the Hsp70 gene family were expressed in detectable levels in all three species. However, only the cold-temperate New Zealand fishes displayed the ability to upregulate the inducible transcript, hsp70. Although hsp70 was present in detectable levels in several tissues of the Antarctic notothen T. bernacchii, in vitro thermal stresses failed to produce a significant increase in mRNA levels. In all species, the expression of the constitutive transcript hsc71 was variable and nonresponsive to temperature increases, even at temperatures as high as 10 degrees C above the ecologically relevant range for the species under study. Field-collected tissues from T. bernacchii (sampled immediately after capture) indicated that hsp70 mRNA was expressed at high levels in field-acclimatized fishes. Thus upregulation of molecular chaperones suggested that low-temperature stress may be significantly denaturing to cellular proteins in Antarctic fish, an observation that was supported by elevated levels of ubiquitin-conjugated protein.

The molecular chaperone Hsc70 from a eurythermal marine goby exhibits temperature insensitivity during luciferase refolding assays.

The role and function of molecular chaperones has been widely studied in model systems (e.g. yeast, Escherichia coli and cultured mammalian cells), however, comparatively little is known about the function of molecular chaperones in eurythermal ectotherms. To investigate the thermal sensitivity of molecular chaperone function in non-model ectotherms, we examined the in vitro activity of Hsc70, a constitutively expressed member of the 70-kDa heat-shock protein gene family, purified from white muscle of the eurythermal marine goby Gillichthys mirabilis. The activity of G. mirabilis Hsc70 was assessed with an in vitro refolding assay where the percent refolding of thermally denatured luciferase was monitored using a luminometer. Assays were conducted from 10-40 degrees C, a range of temperatures that is ecologically relevant for this estuarine species. The results showed that isolated Hsc70 displayed chaperone characteristics in vitro, and was relatively thermally insensitive across the range of experimental temperatures. In addition, the thermal stability of the luciferase refolding capacity of Hsc70 was relatively stable, with refolding activity occurring as high as 50 degrees C. Overall, Hsc70 from G. mirabilis displayed thermal properties in vitro that suggest that the molecular chaperone is capable of binding and chaperoning proteins at temperatures that the goby encounters in nature.

Molecular chaperones in ectothermic marine animals: biochemical function and gene expression.

The intertidal zone has historically functioned as an important natural laboratory for testing ideas about how physical factors such as temperature influence organismal physiology and in turn influence the distribution patterns of organisms. Key to our understanding of how the physical environment helps structure organismal distribution is the identification of physiological processes that have ecological relevance. We have focused on biochemical- and molecular-level physiology that would contribute to thermal tolerance and maintenance of a functional intracellular protein pool in the face of extreme and fluctuating environmental temperatures. Past research has addressed processes central to protein homeostasis (e.g., protein ubiquitination) and the molecular ecology of molecular chaperones, a.k.a. heat shock proteins (Hsps), in ectothermic animals. In this presentation, we focus on two new developments regarding the biology of heat shock proteins as molecular chaperones in intertidal organisms. First, we present data on the functional characteristics of the transcriptional factor, HSF1 and discuss how these data relate to the plasticity of Hsp gene expression observed in intertidal organisms in nature. Second, we present data on the biochemical function of heat shock proteins purified from our non-model study organisms and discuss the temperature relationships of these molecules as they assist in protein folding in situ.