W. Wesley Dowd

Proteomic and physiological responses of leopard sharks (Triakis semifasciata) to salinity change

SUMMARY Partially euryhaline elasmobranchs may tolerate physiologically challenging, variable salinity conditions in estuaries as a trade-off to reduce predation risk or to gain access to abundant food resources. To further understand these trade-offs and to evaluate the underlying mechanisms, we examined the responses of juvenile leopard sharks to salinity changes using a suite of measurements at multiple organizational levels: gill and rectal gland proteomes (using 2-D gel electrophoresis and tandem mass spectrometry), tissue biochemistry (Na+/K+-ATPase, caspase 3/7 and chymotrypsin-like proteasome activities), organismal physiology (hematology, plasma composition, muscle moisture) and individual behavior. Our proteomics results reveal coordinated molecular responses to low salinity – several of which are common to both rectal gland and gill – including changes in amino acid and inositol (i.e. osmolyte) metabolism, energy metabolism and proteins related to transcription, translation and protein degradation. Overall, leopard sharks employ a strategy of maintaining plasma urea, ion concentrations and Na+/K+-ATPase activities in the short-term, possibly because they rarely spend extended periods in low salinity conditions in the wild, but the sharks osmoconform to the surrounding conditions by 3 weeks. We found no evidence of apoptosis at the time points tested, while both tissues exhibited proteomic changes related to the cytoskeleton, suggesting that leopard sharks remodel existing osmoregulatory epithelial cells and activate physiological acclimatory responses to solve the problems posed by low salinity exposure. The behavioral measurements reveal increased activity in the lowest salinity in the short-term, while activity decreased in the lowest salinity in the long-term. Our data suggest that physiological/behavioral trade-offs are involved in using estuarine habitats, and pathway modeling implicates tumor necrosis factor α (TNFα) as a key node of the elasmobranch hyposmotic response network.

Biological Impacts of Thermal Extremes: Mechanisms and Costs of Functional Responses Matter

Thermal performance curves enable physiological constraints to be incorporated in predictions of biological responses to shifts in mean temperature. But do thermal performance curves adequately capture the biological impacts of thermal extremes? Organisms incur physiological damage during exposure to extremes, and also mount active compensatory responses leading to acclimatization, both of which alter thermal performance curves and determine the impact that current and future extremes have on organismal performance and fitness. Thus, these sub-lethal responses to extreme temperatures potentially shape evolution of thermal performance curves. We applied a quantitative genetic model and found that beneficial acclimatization and cumulative damage alter the extent to which thermal performance curves evolve in response to thermal extremes. The impacts of extremes on the evolution of thermal performance curves are reduced if extremes cause substantial mortality or otherwise reduce fitness differences among individuals. Further empirical research will be required to understand how responses to extremes aggregate through time and vary across life stages and processes. Such research will enable incorporating passive and active responses to sub-lethal stress when predicting the impacts of thermal extremes.

Biophysics, environmental stochasticity, and the evolution of thermal safety margins in intertidal limpets.

Summary As the air temperature of the Earth rises, ecological relationships within a community might shift, in part due to differences in the thermal physiology of species. Prediction of these shifts – an urgent task for ecologists – will be complicated if thermal tolerance itself can rapidly evolve. Here, we employ a mechanistic approach to predict the potential for rapid evolution of thermal tolerance in the intertidal limpet Lottia gigantea. Using biophysical principles to predict body temperature as a function of the state of the environment, and an environmental bootstrap procedure to predict how the environment fluctuates through time, we create hypothetical time-series of limpet body temperatures, which are in turn used as a test platform for a mechanistic evolutionary model of thermal tolerance. Our simulations suggest that environmentally driven stochastic variation of L. gigantea body temperature results in rapid evolution of a substantial ‘safety margin’: the average lethal limit is 5–7°C above the average annual maximum temperature. This predicted safety margin approximately matches that found in nature, and once established is sufficient, in our simulations, to allow some limpet populations to survive a drastic, century-long increase in air temperature. By contrast, in the absence of environmental stochasticity, the safety margin is dramatically reduced. We suggest that the risk of exceeding the safety margin, rather than the absolute value of the safety margin, plays an underappreciated role in the evolution of thermal tolerance. Our predictions are based on a simple, hypothetical, allelic model that connects genetics to thermal physiology. To move beyond this simple model – and thereby potentially to predict differential evolution among populations and among species – will require significant advances in our ability to translate the details of thermal histories into physiological and population-genetic consequences.

Natural feeding influences protein expression in the dogfish shark rectal gland: A proteomic analysis.

The rectal gland is the principal salt-secreting organ in elasmobranchs, yet its functional response to normal physiological variation (e.g., due to feeding, stress) has only recently been examined. To complement studies on acid-base, digestive, and osmoregulatory physiology in response to natural feeding, we investigated protein-level responses in the rectal gland of spiny dogfish (Squalus acanthias) 6 h, 20 h, and 5 days (reference control) after a meal. Our objective was to identify proteins involved in regulation of osmoregulatory and metabolic processes in response to feeding. Proteins were separated by two-dimensional gel electrophoresis, and protein spots that were significantly up- or down-regulated >2 fold (i.e., abundance increased more than 100% or decreased more than 50%) were detected using gel image analysis software. Of 684 proteins analyzed on 2D gels, 16 proteins changed significantly 6 h after feeding vs. 5 day controls (5 decreased; 11 increased), and 12 proteins changed >2 fold 20 h after feeding vs. 5 day controls (2 decreased; 10 increased). Thirteen of these proteins were identified using mass spectrometry and classified into functional pathways using the PANTHER bioinformatics database. Rectal gland proteins that were regulated following feeding fell into three main categories: cytoskeletal/muscular (e.g., tropomyosin alpha chain, transgelin), energy metabolism (e.g., malate dehydrogenase, ATP synthase), and nucleotide metabolism (nucleoside diphosphate kinase). The data also revealed that previously documented increases in the activity of isocitrate dehydrogenase after feeding are at least partially due to increased abundance of a cytosolic, NADP-dependent isoform of this enzyme. One of the primary components of the rectal glands response to feeding appears to be maintenance of the cellular supply of energy, which would be necessary to fuel increased activities of enzymes involved in salt secretion and oxidative metabolism in the rectal gland following a meal.

Thermal history and gape of individual Mytilus californianus correlate with oxidative damage and thermoprotective osmolytes

ABSTRACT The ability of animals to cope with environmental stress depends – in part – on past experience, yet knowledge of the factors influencing an individuals 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. Highlighted Article: Variation in thermal experience and behavior contributes to inter-individual physiological variation within intertidal mussel beds, providing valuable insight into physiological plasticity and variable costs of defending against environmental stress.

Plasticity of thermal tolerance and its relationship with growth rate in juvenile mussels ( Mytilus californianus )

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 (Mytilus californianus) 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.

Small Non-coding RNA Expression and Vertebrate Anoxia Tolerance

Background: Extreme anoxia tolerance requires a metabolic depression whose modulation could involve small non-coding RNAs (small ncRNAs), which are specific, rapid, and reversible regulators of gene expression. A previous study of small ncRNA expression in embryos of the annual killifish Austrofundulus limnaeus, the most anoxia-tolerant vertebrate known, revealed a specific expression pattern of small ncRNAs that could play important roles in anoxia tolerance. Here, we conduct a comparative study on the presence and expression of small ncRNAs in the most anoxia-tolerant representatives of several major vertebrate lineages, to investigate the evolution of and mechanisms supporting extreme anoxia tolerance. The epaulette shark (Hemiscyllium ocellatum), crucian carp (Carassius carassius), western painted turtle (Chrysemys picta bellii), and leopard frog (Rana pipiens) were exposed to anoxia and recovery, and small ncRNAs were sequenced from the brain (one of the most anoxia-sensitive tissues) prior to, during, and following exposure to anoxia. Results: Small ncRNA profiles were broadly conserved among species under normoxic conditions, and these expression patterns were largely conserved during exposure to anoxia. In contrast, differentially expressed genes are mostly unique to each species, suggesting that each species may have evolved distinct small ncRNA expression patterns in response to anoxia. Mitochondria-derived small ncRNAs (mitosRNAs) which have a robust response to anoxia in A. limnaeus embryos, were identified in the other anoxia tolerant vertebrates here but did not display a similarly robust response to anoxia. Conclusion: These findings support an overall stabilization of the small ncRNA transcriptome during exposure to anoxic insults, but also suggest that multiple small ncRNA expression pathways may support anoxia tolerance, as no conserved small ncRNA response was identified among the anoxia-tolerant vertebrates studied. This may reflect divergent strategies to achieve the same endpoint: anoxia tolerance. However, it may also indicate that there are multiple cellular pathways that can trigger the same cellular and physiological survival processes, including hypometabolism.