Patricia M. Schulte

Heat shock protein genes and their functional significance in fish

Despite decades of intensive investigation, important questions remain regarding the functional, ecological, and evolutionary roles of heat shock proteins. In this paper, we discuss the utility of fish as a model system to address these questions, and review the relevant studies of heat shock protein genes and the regulation of their expression in fish. Although molecular studies of the heat shock proteins in fish are still in their early descriptive phase, data are rapidly being collected. More is known about the biotic and abiotic factors regulating heat shock proteins. We briefly review these studies and focus on the role of heat shock proteins in development, their regulation by the endocrine system, and their importance in fish in nature. Functional genomics approaches will provide the tools necessary to gain a comprehensive understanding of the significance of heat shock proteins in the cellular stress response, in the physiological processes at higher levels of organization, and in the whole animal in its natural environment.

Intraspecific variation in thermal tolerance and heat shock protein gene expression in common killifish, Fundulus heteroclitus

SUMMARY Populations of common killifish, Fundulus heteroclitus, are distributed along the Atlantic coast of North America through a steep latitudinal thermal gradient. We examined intraspecific variation in whole-animal thermal tolerance and its relationship to the heat shock response in killifish from the northern and southern extremes of the species range. Critical thermal maxima were significantly higher in southern than in northern fish by ∼1.5°C at a wide range of acclimation temperatures (from 2-34°C), and critical thermal minima differed by ∼1.5°C at acclimation temperatures above 22°C, converging on the freezing point of brackish water at lower acclimation temperatures. To determine whether these differences in whole-organism thermal tolerance were reflected in differences in either the sequence or regulation of the heat shock protein genes (hsps) we obtained complete cDNA sequences for hsc70, hsp70-1 and hsp70-2, and partial sequences of hsp90α and hsp90β. There were no fixed differences in amino acid sequence between populations in either hsp70-1 or hsp70-2, and only a single conservative substitution between populations in hsc70. By contrast, there were significant differences between populations in the expression of many, but not all, of these genes. Both northern and southern killifish significantly increased hsp70-2 levels above control values (Ton) at a heat shock temperature of 33°C, but the magnitude of this induction was greater in northern fish, suggesting that northern fish may be more susceptible to thermal damage than are southern fish. In contrast, hsp70-1 mRNA levels increased gradually and to the same extent in response to heat shock in both populations. Hsc70 mRNA levels were significantly elevated by heat shock in southern fish, but not in northern fish. Similarly, the more thermotolerant southern killifish had a Ton for hsp90α of 30°C, 2°C lower than that of northern fish. This observation combined with the ability of southern killifish to upregulate hsc70 in response to heat shock suggests a possible role for these hsps in whole-organism differences in thermal tolerance. These data highlight the importance of considering the complexity of the heat shock response across multiple isoforms when attempting to make linkages to whole-organism traits such as thermal tolerance.

Na+/K+-ATPase alpha-isoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer.

SUMMARY We identified five Na+/K+-ATPase α-isoforms in rainbow trout and characterized their expression pattern in gills following seawater transfer. Three of these isoforms were closely related to other vertebrate α1 isoforms (designated α1a, α1b and α1c), one isoform was closely related to α2 isoforms (designated α2) and the fifth was closely related to α3 isoforms (designated α3). Na+/K+-ATPase α1c- and α3-isoforms were present in all tissues examined, while all others had tissue specific distributions. Four Na+/K+-ATPase α-isoforms were expressed in trout gills (α1a, α1b, α1c and α3). Na+/K+-ATPase α1c- and α3-isoforms were expressed at low levels in freshwater trout gills and their expression pattern did not change following transfer to 40% or 80% seawater. Na+/K+-ATPase α1a and α1b were differentially expressed following seawater transfer. Transfer from freshwater to 40% and 80% seawater decreased gill Na+/K+-ATPaseα 1a mRNA, while transfer from freshwater to 80% seawater caused a transient increase in Na+/K+-ATPase α1b mRNA. These changes in isoform distribution were accompanied by an increase in gill Na+/K+-ATPase enzyme activity by 10 days after transfer to 80% seawater, though no significant change occurred following transfer to 40% seawater. Isoform switching in trout gills following salinity transfer suggests that the Na+/K+-ATPase α1a- andα 1b-isoforms play different roles in freshwater and seawater acclimation, and that assays of Na+/K+-ATPase enzyme activity may not provide a complete picture of the role of this protein in seawater transfer.

Macrophysiology: A Conceptual Reunification

Widespread recognition of the importance of biological studies at large spatial and temporal scales, particularly in the face of many of the most pressing issues facing humanity, has fueled the argument that there is a need to reinvigorate such studies in physiological ecology through the establishment of a macrophysiology. Following a period when the fields of ecology and physiological ecology had been regarded as largely synonymous, studies of this kind were relatively commonplace in the first half of the twentieth century. However, such large‐scale work subsequently became rather scarce as physiological studies concentrated on the biochemical and molecular mechanisms underlying the capacities and tolerances of species. In some sense, macrophysiology is thus an attempt at a conceptual reunification. In this article, we provide a conceptual framework for the continued development of macrophysiology. We subdivide this framework into three major components: the establishment of macrophysiological patterns, determining the form of those patterns (the very general ways in which they are shaped), and understanding the mechanisms that give rise to them. We suggest ways in which each of these components could be developed usefully.

Thermal Performance Curves, Phenotypic Plasticity, and the Time Scales of Temperature Exposure

Thermal performance curves (TPCs) describe the effects of temperature on biological rate processes. Here, we use examples from our work on common killifish (Fundulus heteroclitus) to illustrate some important conceptual issues relating to TPCs in the context of using these curves to predict the responses of organisms to climate change. Phenotypic plasticity has the capacity to alter the shape and position of the TPCs for acute exposures, but these changes can be obscured when rate processes are measured only following chronic exposures. For example, the acute TPC for mitochondrial respiration in killifish is exponential in shape, but this shape changes with acclimation. If respiration rate is measured only at the acclimation temperature, the TPC is linear, concealing the underlying mechanistic complexity at an acute time scale. These issues are particularly problematic when attempting to use TPCs to predict the responses of organisms to temperature change in natural environments. Many TPCs are generated using laboratory exposures to constant temperatures, but temperature fluctuates in the natural environment, and the mechanisms influencing performance at acute and chronic time scales, and the responses of the performance traits at these time scales may be quite different. Unfortunately, our current understanding of the mechanisms underlying the responses of organisms to temperature change is incomplete, particularly with respect to integrating from processes occurring at the level of single proteins up to whole-organism functions across different time scales, which is a challenge for the development of strongly grounded mechanistic models of responses to global climate change.

Reciprocal expression of gill Na+/K+-ATPase alpha-subunit isoforms alpha1a and alpha1b during seawater acclimation of three salmonid fishes that vary in their salinity tolerance.

SUMMARY The upregulation of gill Na+/K+-ATPase activity is considered critical for the successful acclimation of salmonid fishes to seawater. The present study examines the mRNA expression of two recently discovered α-subunit isoforms of Na+/K+-ATPase (α1a and α1b) in gill during the seawater acclimation of three species of anadromous salmonids, which vary in their salinity tolerance. Levels of these Na+/K+-ATPase isoforms were compared with Na+/K+-ATPase activity and protein abundance and related to the seawater tolerance of each species. Atlantic salmon (Salmo salar) quickly regulated plasma Na+, Cl– and osmolality levels within 10 days of seawater exposure, whereas rainbow trout (Oncorhynchus mykiss) and Arctic char (Salvelinus alpinus) struggled to ionoregulate, and experienced greater perturbations in plasma ion levels for a longer period of time. In all three species, mRNA levels for theα 1a isoform quickly decreased following seawater exposure whereasα 1b levels increased significantly. All three species displayed similar increases in gill Na+/K+-ATPase activity during seawater acclimation, with levels rising after 10 and 30 days. Freshwater Atlantic salmon gill Na+/K+-ATPase activity and protein content was threefold higher than those of Arctic char and rainbow trout, which may explain their superior seawater tolerance. The role of the α1b isoform may be of particular importance during seawater acclimation of salmonid fishes. The reciprocal expression of Na+/K+-ATPase isoforms α1a and α1b during seawater acclimation suggests they may have different roles in the gills of freshwater and marine fishes; ion uptake in freshwater fish and ion secretion in marine fishes.

Linking genotypes to phenotypes and fitness: how mechanistic biology can inform molecular ecology

The accessibility of new genomic resources, high‐throughput molecular technologies and analytical approaches such as genome scans have made finding genes contributing to fitness variation in natural populations an increasingly feasible task. Once candidate genes are identified, we argue that it is necessary to take a mechanistic approach and work up through the levels of biological organization to fully understand the impacts of genetic variation at these candidate genes. We demonstrate how this approach provides testable hypotheses about the causal links among levels of biological organization, and assists in designing relevant experiments to test the effects of genetic variation on phenotype, whole‐organism performance capabilities and fitness. We review some of the research programs that have incorporated mechanistic approaches when examining naturally occurring genetic and phenotypic variation and use these examples to highlight the value of developing a comprehensive understanding of the relationship between genotype and fitness. We give suggestions to guide future research aimed at uncovering and understanding the genetic basis of adaptation and argue that further integration of mechanistic approaches will help molecular ecologists better understand the evolution of natural populations.

Gene expression after freshwater transfer in gills and opercular epithelia of killifish: insight into divergent mechanisms of ion transport

SUMMARY We have explored the molecular basis for differences in physiological function between the gills and opercular epithelium of the euryhaline killifish Fundulus heteroclitus. These tissues are functionally similar in seawater, but in freshwater the gills actively absorb Na+ but not Cl–, whereas the opercular epithelium actively absorbs Cl– but not Na+. These differences in freshwater physiology are likely due to differences in absolute levels of gene expression (measured using real-time PCR), as several proteins important for Na+ transport, namely Na+,H+-exchanger 2 (NHE2), carbonic anhydrase 2 (CA2), Na+,HCO3–cotransporter 1, and V-type H+-ATPase, were expressed at 3- to over 30-fold higher absolute levels in the gills. In gills, transfer from 10% seawater to freshwater increased the activity of Na+,K+-ATPase by twofold (from 12 h to 7 days), increased the expression of NHE2 (at 12 h) and CA2 (from 12 h to 7 days), and decreased the expression of NHE3 (from 12 h to 3 days). In opercular epithelium, NHE2 was not expressed; furthermore, Na+,K+-ATPase activity was unchanged after transfer to freshwater, CA2 mRNA levels decreased, and NHE3 levels increased. Consistent with their functional similarities in seawater, killifish gills and opercular epithelium expressed Na+,K+-ATPase α1a, Na+,K+,2Cl–cotransporter 1 (NKCC1), cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel and the signalling protein 14-3-3a at similar absolute levels. Furthermore, NKCC1 and CFTR were suppressed equally in each tissue after freshwater transfer, and 14-3-3a mRNA increased in both. These results provide insight into the mechanisms of ion transport by killifish gills and opercular epithelia, and demonstrate a potential molecular basis for the differences in physiological function between these two organs.

Evolutionary adaptations of gene structure and expression in natural populations in relation to a changing environment: A multidisciplinary approach to address the million‐year saga of a small fish

We have used an experimentally based strategy to address molecular mechanisms underlying adaptation in Fundulus heteroclitus. In an attempt to falsify the hypothesis that selection is a major driving force in the maintenance of genetic diversity, we employed a multidisciplinary approach including allelic isozyme and mtDNA phylogeography, kinetic analyses of allelic isozymes, analysis of variation in coding and regulatory DNA sequences, metabolic biochemistry, organismal physiology, and selection experiments. Observed differences in gene structure and expression led us to make testable predictions about differences in metabolic flux, whole organism performance, and differential survival between allotypes. We have shown that variation in the lactate dehydrogenase-B (Ldh-B) protein results in differences in physiological function and is correlated with differences in survival at high temperatures. Recent work has investigated the role of variation in Ldh-B expression. There are differences in the levels of Ldh-B protein, mRNA, and transcription rate. We have addressed the mechanisms responsible for differences in transcription rate by a combination of sequence comparison, DNase I footprinting, and functional analyses both in vitro and in vivo. We have shown that variation in the regulatory sequence of Ldh-B is responsible for the differences in transcription rate between populations and that the patterns of variation are inconsistent with a neutral model of molecular evolution. This functional differentiation, coupled with departures from neutral expectations, suggests that natural selection has acted on the regulation of Ldh-B. This article illustrates the value of a multidisciplinary approach in addressing problems in gene structure, expression, and evolutionary adaptation.

Molecular Evolution of Cytochrome c Oxidase Underlies High-Altitude Adaptation in the Bar-Headed Goose

Bar-headed geese (Anser indicus) fly at up to 9,000 m elevation during their migration over the Himalayas, sustaining high metabolic rates in the severe hypoxia at these altitudes. We investigated the evolution of cardiac energy metabolism and O(2) transport in this species to better understand the molecular and physiological mechanisms of high-altitude adaptation. Compared with low-altitude geese (pink-footed geese and barnacle geese), bar-headed geese had larger lungs and higher capillary densities in the left ventricle of the heart, both of which should improve O(2) diffusion during hypoxia. Although myoglobin abundance and the activities of many metabolic enzymes (carnitine palmitoyltransferase, citrate synthase, 3-hydroxyacyl-coA dehydrogenase, lactate dehydrogenase, and pyruvate kinase) showed only minor variation between species, bar-headed geese had a striking alteration in the kinetics of cytochrome c oxidase (COX), the heteromeric enzyme that catalyzes O(2) reduction in oxidative phosphorylation. This was reflected by a lower maximum catalytic activity and a higher affinity for reduced cytochrome c. There were small differences between species in messenger RNA and protein expression of COX subunits 3 and 4, but these were inconsistent with the divergence in enzyme kinetics. However, the COX3 gene of bar-headed geese contained a nonsynonymous substitution at a site that is otherwise conserved across vertebrates and resulted in a major functional change of amino acid class (Trp-116 → Arg). This mutation was predicted by structural modeling to alter the interaction between COX3 and COX1. Adaptations in mitochondrial enzyme kinetics and O(2) transport capacity may therefore contribute to the exceptional ability of bar-headed geese to fly high.