Responses to environmental stressors in an estuarine fish: Interacting stressors and the impacts of local adaptation

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Journal of Thermal Biology 32 (2007) 152–161 www.elsevier.com/locate/jtherbio

Responses to environmental stressors in an estuarine fish: Interacting stressors and the impacts of local adaptation Patricia M. Schulte Department of Zoology, The University of British Columbia, 6270 University Boulevard, Vancouver, BC, Canada V6T 1Z4

Abstract Common killifish, Fundulus heteroclitus, are found in marshes and estuaries along the Atlantic coast of North America from Newfoundland to Florida. Although these habitats are highly productive, they are also characterized by variation in a number of abiotic stressors, including temperature, salinity, oxygen, and anthropogenic toxicants, which vary substantially in both space and time. In order to survive in these habitats, killifish must be able to cope with these stressors, both individually and in combination. There is substantial evidence to suggest that populations of F. heteroclitus have undergone local adaptation to multiple abiotic stressors, including temperature, salinity, and toxicants, but most studies have examined the effects of single stressors in isolation. Here I review some of the studies on local adaptation in F. heteroclitus, focusing on the molecular basis of local adaptation to abiotic stressors, and the acute responses to these stressors both singly and in combination. This work demonstrates that there are substantial interactions between the responses to both natural and anthropogenic stressors at the cellular level. r 2007 Elsevier Ltd. All rights reserved. Keywords: Mummichogs; Temperature; Salinity; Toxicants; Cortisol; Adaptation; Fish; Gene expression

1. Introduction Coastal aquatic environments such as estuaries, marshes, and tidal creeks are characterized by the presence of multiple interacting abiotic stressors, including changes in temperature, oxygen concentration and environmental salinity (Bianchi, 2006). Over the last 100 years, these habitats have also undergone a remarkable degree of anthropogenic environmental change, subjecting the organisms that live in these already challenging habitats to an increasingly complex pattern of environmental stressors. Both global and local anthropogenic factors are having and will continue to have substantial impacts on these habitats. Marshes and estuaries are expected to be particularly strongly affected by global warming and associated sea level increases (Scavia et al., 2002), and many estuaries have also been highly modified as a result of factors acting at local scales (Nichols et al., 1986) because Tel.: +1 604 822 4276; fax: +1 604 822 2416.

E-mail address: [email protected]. 0306-4565/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2007.01.012

of the high human population in coastal areas around the world (Vitousek et al., 1997). For example, land-use practices and the resulting introduction of nutrients into estuaries and coastal environments has caused increased frequency of harmful algal blooms, and episodes of prolonged aquatic hypoxia (Diaz and Rosenberg, 1995; Conley et al., 2002; Gray et al., 2002; Rabalais et al., 2002). Industrialization along coastlines has also resulted in substantial inputs of toxic substances into estuarine and coastal habitats. As a result, in order to survive, organisms in these habitats must cope with a complex set of interacting stressors, both natural and anthropogenic, which vary in both space and time. As elucidated in a recent United States Environmental Protection Agency Report (US EPA, 2004), one of the critical obstacles in developing a risk assessment framework to guide environmental legislation is to understand the ways in which multiple interacting stressors affect organisms, populations, and ecosystems. This problem is particularly challenging because different stressors may affect related physiological and biochemical pathways, or

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invoke overlapping cellular responses (NRC, 2000). As a result, there is ample opportunity for additive, synergistic, or antagonistic effects and crosstalk among the cellular and organismal responses to these stressors. In this review I summarize some of our recent work on the mechanisms via which fish respond to interacting abiotic stressors through both the organismal and cellular stress responses, and the relationships between these two types of response across a variety of temporal scales.

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local adaptation and their impacts on the ability of these populations to tolerate other stressors either singly or in combination has seldom been investigated. Here I summarize some of the evidence for local adaptation to temperature, salinity, and anthropogenic toxicants in F. heteroclitus, and outline the results of some preliminary investigations into the effects of interacting stressors in this species. 3. Local adaptation in F. heteroclitus: thermal tolerance

2. The model system The small teleost fish, Fundulus heteroclitus (also known as the common killifish or the mummichog) has been widely used as a model in which to address questions in ecotoxicology and environmental physiology (Powers et al.,1993). F. heteroclitus is a member of the family Fundulidae, a relatively large group of cyprinodontiform fishes that live in fresh, brackish, and coastal marine waters in Central and North America. The genus Fundulus is thought to be subtropical in origin, and F. heteroclitus is one of the few species in this genus that penetrates north into Canada (Lee et al., 1980). Indeed, F. heteroclitus are extremely abundant throughout their distribution range, which stretches along the Atlantic coast of North America from Nova Scotia to Florida. F. heteroclitus have strong site fidelity, often having home ranges of less than 30 m (Brown and Chapman, 1991), and mark-recapture studies indicate that these fish seldom disperse more than 1 km even in relatively highflow environments such as tidal creeks (Lotrich, 1975; Sweeney et al., 1998). In addition, F. heteroclitus lays adherent eggs on vegetation or empty mussel shells, and lacks a pelagic larval stage (Scott and Crossman, 1973; Taylor, 1999), suggesting limited dispersal possibility for juveniles. As a result, there is likely to be limited gene flow between geographically distinct populations. Limited gene flow could result in genetic divergence, even among local populations, and particularly at the extremes of the species range. In fact, studies of genetic variation in F. heteroclitus suggest that there are two genetic groupings within this species (a northern and a southern form), with a zone of admixture between them in intermediate latitudes, centered around the Hudson River (Bernardi et al., 1993; Adams et al., 2006). The northern and southern killifish are sufficiently distinct that they are sometimes considered to be subspecies: Fundulus heteroclitus macrolepidotus (the northern form) and Fundulus heteroclitus heteroclitus (the southern form) (Morin and Able, 1983). There is substantial evidence to suggest that these subspecies have diverged both morphologically and physiologically (Morin and Able, 1983; Powers et al., 1993; Scott et al., 2004). In addition, local adaptation in response to toxicant exposure has been demonstrated on a much smaller geographic scale, at least in the southern subspecies (Nacci et al., 1999; Cohen, 2002; Roark et al., 2005). However, the interactions among the processes involved in

Populations of F. heteroclitus are exposed to a wide range of environmental temperatures, and this variation is expressed at a variety of temporal and spatial scales (Fig. 1). At a latitudinal scale, there is a steep thermal gradient along the Atlantic coast such that there is almost a 1 1C change in mean annual temperature with every degree change in latitude (Fig. 1A). At the southern end of the species distribution, summer temperatures often approach 35 1C, whereas northern fish seldom experience temperatures above 25 1C. In contrast, extensive ice formation is the norm in winter at the northern end of the species range, while southern populations are unlikely ever to encounter ice. In addition to this latitudinal variation, there is substantial seasonal variation within both the northern and southern habitats (Fig. 1B), with mean winter temperatures being more than 10 1C lower than mean summer temperatures (3 versus 15 1C for northern populations and 15 versus 28 1C for southern populations). Because of the tidal influence in many of these habitats, there is also the potential for large acute temperature changes. For example, Fig. 1C illustrates that habitat temperature can change by more than 10 1C within a few hours in some areas. Previous studies have suggested that northern and southern populations of F. heteroclitus might have undergone local adaptation to their respective habitat temperatures (reviewed in Schulte, 2001). Indeed, data from a comprehensive examination of the whole-organism thermal tolerance of individuals from several populations at the northern and southern extremes of the species range is consistent with this hypothesis (Fangue et al., 2006). Thermal tolerance in fish is often assessed using critical thermal methodology. The critical thermal maximum (CTMax) and critical thermal minimum (CTMin) are defined as the upper and lower temperatures, respectively, at which fish lose the ability to escape conditions that will ultimately lead to death (Beitinger et al., 2000). In the laboratory, CTMax and CTMin are usually estimated using the temperature at which loss of equilibrium occurs following gradual heating or cooling as an empirical endpoint. Southern F. heteroclitus have consistently higher CTMax and CTMin than do northern fish, with a difference between populations on the order of 1.5 1C (Fangue et al., 2006). This phenotypic difference is consistent with the predictions of local adaptation of these

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Temperature (οC)

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Fig. 1. Environmental temperatures in Atlantic Coast Estuaries. (A) Mean monthly temperatures with latitude along the Atlantic coast. (B) Mean monthly, monthly maximum, and monthly minimum temperature for two locations on the Atlantic Coast. Open symbols and gray line indicate temperatures in Matanzas, Florida. Solid symbols and black line indicate temperatures in Wells, ME. Triangles indicate monthly maximum temperature, squares represent monthly minimum temperature. Monthly average temperature is indicated by a solid line with no symbols. Data are for 2001. (C) An example of temperature variation within a day in a Wells, ME estuary. Data are for July 5, 2001. All data were collected as part of the National Estuarine Research Reserve System-wide Monitoring Program (National Oceanographic and Atmospheric Administration, NOAA).

populations to their respective habitat temperatures, but since these studies were performed on wild-caught fish that had been laboratory acclimated to a common temperature, the possibility of irreversible effects of differing developmental temperatures cannot be eliminated. There is also a very large effect of acclimation temperature on both CTMax and CTMin, with both of these parameters increasing by more than 10 1C between acclimation temperatures of 3 and 35 1C (Fangue et al., 2006). However, a consistent difference between populations in both CTMax and CTMin was present regardless of acclimation temperature (with the exception of CTMin at acclimation temperatures below 10 1C, which converged on

the freezing point of brackish water in both populations). This strongly suggests either a genetic or irreversible phenotypic difference in thermal tolerance between the two populations. The heat shock proteins (HSPs) are logical candidate gene to examine in the search for biochemical mechanisms underlying the observed differences in thermal tolerance among populations of F. heteroclitus (Basu et al., 2002; Hoffmann et al., 2003). As molecular chaperones, HSPs interact with proteins that are in their non-native conformation and prevent these denatured proteins from interacting inappropriately with one another (for reviews see Lindquist, 1986; Hightower, 1991; Morimoto, 1998). HSPs are encoded by multiple genes and are assigned to families based on sequence similarity and molecular weight. HSP families may contain both constitutive and inducible members, such as the inducible hsp70 genes, and the constitutively expressed hsc70 genes (the heat shock cognates). There is ample evidence to suggest that various aspects of the heat shock response, such as the temperature at which HSPs are induced, are correlated with habitat temperature among species (Feder and Hofmann, 1999; Tomanek and Somero, 1999; Buckley et al., 2001, Somero, 2005; Tomanek, 2005). The role of HSPs in thermal adaptation among populations of F. heteroclitus, however, is far from clear. The coding sequences of the hsp70 and hsc70 genes do not differ between populations, suggesting local thermal adaptation in this species did not proceed via changes in the efficiency of heat shock protein function (Fangue et al., 2006). There are, however, differences between populations in a number of aspects of the response of these genes to heat shock (such as the onset temperature for gene expression [Ton], and the extent of the increase in mRNA in response to a common heat shock protocol). But there is no consistent pattern of induction across genes, so that each cDNA exhibits a unique pattern of differences between populations in its expression profile (Fangue et al., 2006). Particularly interestingly, F. heteroclitus expresses two hsp70 genes that are extremely similar in coding sequence, but are still easily distinguishable at the cDNA level, and clearly within the hsp70 subfamily rather than the related hsc70s (Fangue et al., 2006). These two closely related hsp70 gene products display entirely different expression profiles. The hsp70-1 show a pattern of gradual induction with increasing temperature and no clear differences in expression between populations, while hsp70-2 shows a clear induction temperature threshold. Although Ton for hsp70-2 did not differ between populations, northern fish had a significantly greater magnitude of induction than did southern fish at all heat shock temperatures (Fangue et al., 2006). This difference in the magnitude of expression between populations is consistent with the hypothesis that fish from the northern populations experience a greater protein denaturing stress at the cellular level in response to any given heat shock temperature, resulting a in larger hsp70 response (as assessed at the

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transcript level). However, there was no difference in the magnitude of the response of the inducible Hsp90a between populations, which is somewhat inconsistent with this simple hypothesis of greater susceptibility to protein denaturing stress in northern populations. The hsp70 and hsp90 responses likely have somewhat different targets, which could account for this difference in expression pattern (Young et al., 2003), but this hypothesis remains to be tested. The observed variation in expression patterns among the HSP genes hints at the complexity of the cellular stress response in this extremely thermally tolerant fish. In addition to HSPs, there have been several suggestions that differences in organismal thermal tolerance may be related to metabolic differences among species (see for example Portner et al., 2006). Indeed, much of the work that has addressed potential biochemical mechanisms underlying differences in thermal tolerance in F. heteroclitus has concentrated on differences in the biochemical pathways that mediate energy supply, as a result of early work indicating strong associations between allozyme variants at a number of metabolically important loci and differences in organismal performance, developmental rate and survival between populations at either temperature extreme, (DiMichele and Powers, 1982a, b, 1991; DiMichele et al., 1991; Paynter et al., 1991). LDH catalyzes the reversible interconversion of lactate to pyruvate at the terminal step of ‘‘anaerobic glycolysis’’, and thus plays an essential role in maintaining the energy balance of the cell. There are differences in both the coding sequence and the regulation of the Ldh-B gene northern and southern populations of F. heteroclitus (Crawford and Powers, 1989, 1992; Bernardi et al., 1993; Pierce and Crawford, 1997). To test whether differences in the sequence of the promoter of the Ldh-B gene could be responsible for the differences in transcription rate, we first cloned and sequenced the promoter from a sample of individuals across the species range (Schulte et al., 1997). Interestingly, the sequence variation in the promoter was non-randomly distributed, and the pattern of variation was not consistent with a simple neutral model of molecular evolution, suggesting the possibility of adaptively significant variation in this non-coding region of the genome. Using a combination of in vitro assays and transgenic technologies, we have shown that the observed differences in transcription rate between populations can be explained by functionally important differences in the sequence of the Ldh-B promoter between the populations (Schulte et al., 1997, 2000, reviewed in Schulte, 2001). Comprehensive deletion and mutagenesis experiments implicated a possible stress-responsive element approximately 500 bp upstream of the transcription start site of this gene. A consensus glucocorticoid responsive element is present in this region of the Ldh-B promoter in fish of the southern genotype, but is mutated by several base pairs in fish of the northern genotype (Schulte et al., 2000). To test the hypothesis that a stress responsive element is present in the Ldh-B promoter

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of southern but not northern fish, we exposed fish to a repeated netting stress, which is known to increase glucocorticoid levels in F. heteroclitus (Leach and Taylor, 1980). Exposure to this stressor resulted in increased LDHB specific activity in southern but not northern fish, consistent with the presence of a stress-responsive repressor element in the Ldh-B gene of southern fish (Schulte et al., 2000). We then directly tested the hypothesis that a glucocorticoid responsive element in the Ldh-B promoter was responsible for differences in promoter activity using fish that had been rendered transiently transgenic with a luciferase reporter gene under the control of the Ldh-B promoter from either northern or southern fish. Regardless of the genetic background of the transgenic fish, luciferase activity was low but stress-responsive in fish injected with a southern construct and high and non-stress responsive in fish injected with a northern construct (Schulte et al., 2000). Together these data strongly suggest that differences in the stress-responsiveness of the Ldh-B promoter may underlie the putatively adaptive differences between populations in LDH-B activity observed in liver and heart. To determine whether this difference in stress-responsiveness was present at higher levels of organization, we examined the behavior of the remainder of the genes in the glycolytic pathway. Only three of the 10 other glycolytic genes were stress responsive, but that all of these genes were stress responsive in fish from the southern population but not in fish from the northern population. Two of these genes (glucokinase; Picard and Schulte, 2004, and phosphofructokinase; DeKoning et al., 2004) are known to play important roles in the regulation of glycolysis (Hochachka and Somero, 2002) suggesting that there may be a fundamental difference in the metabolic response to stress by northern and southern populations of F. heteroclitus. Indeed, preliminary evidence indicates that the regulation of the entire stress-hormone axis may differ between populations, because we observed that fish from a southern population have higher levels of glucorticoid stress hormones following exposure to a common stressor or following injection of a standardized dose of cortisol than do fish from a northern population (DeKoning et al., 2004). There is substantial evidence for interactions between the glucocorticoid-mediated organismal stress response and the expression of HSPs during the cellular stress response. In fish, chronic glucocortoid treatment is known to decrease the induction of hsp70 in response to heat shock in trout liver (Basu et al., 2001) and attenuate heat shock-induced hsp90 mRNA accumulation in trout hepatocytes in primary culture (Sathiyaa et al., 2001). Taken together, these data suggest the possibility that differences in the organismal stress response between populations of F. heteroclitus may be related to differences in the cellular stress response and thermal tolerance between populations.

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4. Local adaptation in F. heteroclitus: salinity tolerance Glucocorticoids participate in the regulation of many physiological functions including glucose metabolism and gluconeogenesis, proteolysis, and lipid metabolism. In addition, in fish the glucocorticoids are involved in the response of fish to salinity transfer (Evans et al., 2005). Several lines of evidence suggest that northern and southern populations of F. heteroclitus differ in their response to salinity transfer, particularly with respect to their tolerance to freshwater. Northern populations of F. heteroclitus have higher fertilization success and larval survival in freshwater than do southern populations (Able and Palmer, 1988). In addition, in areas such as the Chesapeake Bay the southern genotype dominates at the coast, but the proportion of northern genotypes increases in freshwater habitats, even at latitudes and temperatures that are typical for the southern subspecies (Powers et al., 1993). Consistent with these observations, we have recently shown that there are substantial differences in adult mortality following acute freshwater transfer between northern and southern populations. Fish from a southern population experienced up to 20% mortality following freshwater transfer, while fish from a northern population experienced no significant mortality (Scott et al., 2004). At the level of the whole organism, this difference in mortality was associated with differences in the ability of these fish to maintain ion homeostasis following freshwater transfer. Southern fish were less able to regulate plasma Na+, and levels were decreased for a longer period following transfer in surviving southern individuals than in northern fish. Similarly, plasma Cl was maintained in northern fish following freshwater transfer, but declined rapidly and remained low in southern fish throughout the duration of the experiment (Scott et al., 2004). At the level of the gill, there appear to be substantial differences in the cellular responses to freshwater transfer. For example, southern fish demonstrated a smaller degree of activation of the Na+/K+-ATPase (at the mRNA and protein levels) than did northern fish, which might account for differences in the ability to regulate plasma Na+ levels. There was also a marked difference in the flux rate of chloride across the gills between populations. Northern fish were able to essentially eliminate Cl loss following freshwater transfer, whereas flux remained high in southern fish. These differences in flux can be accounted for by differences in the paracellular permeability of the gills between populations (Scott et al., 2004). Electron microscopic studies of the gill indicate that, unlike northern fish, southern fish are unable to undergo a complete transformation from a ‘‘seawater-type’’ gill morphology to a ‘‘freshwater-type’’ gill morphology (Scott et al., 2004). The process of acclimation to freshwater appears to be mediated, in part, by cortisol in F. heteroclitus (Scott et al., 2005). Given that we have shown that northern and southern fish differ in their cortisol dynamics (DeKoning et al., 2004), it is possible that differences in the cellular

stress response may influence the ability of these fish to colonize freshwater habitats. 5. Local adaptation in F. heteroclitus: anthropogenic stressors In addition to coping with a wide range of natural abiotic stressors, some populations of F. heteroclitus are also exposed to anthropogenic toxicants in their environment. For example, in the heavily dioxin and PCB contaminated New Bedford Harbor, these fish are found in waters where PCB sediment levels are 4 orders of magnitude higher than sediment guidelines correlated with adverse biological effects. This population of fish is genetically distinct from reference populations at noncontaminated sites, as detected using polymorphism at a highly variable region of the major histocompatibility loci (MHC) (Cohen, 2002), although this differentiation could not be detected at other alleles (Roark et al., 2005). Consistent with the hypothesis of local adaptation to toxicant exposure, fish from highly contaminated sites in New Bedford Harbor have been shown to be more resistant to the toxic effects of PCBs than are fish from reference sites (Nacci et al., 1999). Similarly, fish from contaminated sites in the Elizabeth River (Virginia) are less sensitive to toxicant exposure than are fish from reference sites (Van Veld and Westbrook, 1995; Mulvey et al., 2003). This difference has been attributed to lack of induction of cytochrome P4501A (CYP1A) by toxicants that are potent inducers of this gene in fish from reference populations (see Meyer et al., 2002 for review). However, in this case the differences do not persist after several generations of laboratory rearing, suggesting that non-genetic factors are responsible for the phenotypic variation among the populations (Meyer et al., 2002). Although F. heteroclitus at contaminated sites exhibit greater resistance to the effects of environmental contaminants than do fish from reference sites, even fish from reference sites are able to tolerate toxicant exposures that would be lethal to more sensitive fish species. In addition, many estuarine sites are contaminated with low levels of anthropogenic pollutants, which opens the question of how these anthropogenic stressors interact with the stressors naturally present in the highly variable estuarine environment to affect physiological processes in F. heteroclitus. 6. Interacting stressors: cellular mechanisms Although a true science of ‘‘predictive toxicology’’ is more a goal than a reality (Maggioli et al., 2006), the last two decades have seen an explosion of data that provide insights into the mechanisms of action of a variety of toxicants, and the nature of the cellular response to these stressors (Nendza and Wenzel, 2006). As a result, it is becoming possible to predict a priori, based on the mechanism of action of a toxicant, situations in which toxicants could interfere with the responses to natural

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abiotic stressors at a cellular level (Lydy et al., 2004; Munns, 2006). Using this approach, we have shown that two toxicants (arsenic and polychlorinated biphenyls), which are encountered by F. heteroclitus in their natural environment, have the potential to interfere with the normal responses to environmental hypoxia and behavioral stress, respectively (Kraemer and Schulte, 2004; Bears et al., 2006). This suggests that these toxicants could potentially have adverse effects on fish populations even at very low levels of environmental contamination. During summer months in temperate regions, when temperatures and salinities are highest, it is not uncommon for estuaries to show diel variations in dissolved oxygen, often producing hypoxic waters at night (Cochran and Burnett, 1996). As is expected for a species living in such a dynamic oxygen environment, F. heteroclitus are extremely hypoxia tolerant, and are able to survive in waters where the dissolved oxygen levels are as low as 1.5–2.5 ppm (Greaney et al., 1980). At a cellular level, one of the classic responses to hypoxia is to increase the transcription of the genes involved in glycolysis. This pathway is responsible for cellular energy production when oxygen is limiting, and the genes coding for glycolytic enzymes are known to be upregulated during hypoxic exposure in mammals (Semenza, 2000). This response provides a degree of protection against short-term hypoxia. A similar protective response may be present in F. heteroclitus, since lactate dehydrogenase activity has been shown to increase in response to hypoxia in this species (Greaney et al., 1980). PCB and dioxin exposure also affect the regulation of the enzymes of central metabolism in mammals. In particular, dioxin exposure may down-regulate the enzymes of gluconeogenesis (Weber et al., 1991). However, the effects of dioxins and PCBs on the regulation of glycolytic enzymes are less well characterized. In general, only moderate effects have been detected, although significant PCB

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induction of lactate dehydrogenase has been reported (Buu-Hoi et al., 1972). Because of their ability to survive stressors that are lethal to more sensitive fish species, F. heteroclitus provides a unique model in which to study the interactive effects of hypoxia and PCB exposure. Gene regulation in response to hypoxia and PCB exposure occurs via two distinct, but interrelated, biochemical pathways (Fig. 2; see Wenger et al., 2005; Mimura and Fujii-Kuriyama, 2003 for reviews) initiated by activation of (1) the aryl hydrocarbon receptor (AHR) in response to PCB or dioxin exposure and (2) the hypoxia inducible factor (HIF-1a) in response to hypoxic stress. When activated by dioxins or hypoxia the AHR or HIF-1a, respectively are released from their interactions with a heat shock protein complex and heterodimerize with the protein aryl hydrocarbon receptor nuclear translocator (ARNT). This heterodimer (Ahr/ARNT or HIF/ARNT) acts as a transcription factor to regulate gene expression by binding to either a xenobiotic responsive element (XRE) or hypoxia responsive element (HRE) located adjacent to regulated genes. ARNT plays a prominent role in specifying the DNA binding activity of the heterodimer. As a result, HREs and XREs share a common core sequence recognized by ARNT (CGTG). The overlap between the two transcription factor pathways suggests the possibility of cross-talk between oxygen and dioxin signal transduction pathways (Gassmann et al., 1997). We have shown that hypoxia results in coordinate upregulation of glycolytic genes, and that prior PCB exposure prevents this increase (Kraemer and Schulte, 2004). Enzyme activities in fish exposed to both stressors was intermediate between that of fish exposed to either hypoxia or PCBs independently, and not significantly different from the value in normoxic control fish. This result is consistent with the hypothesis that there is cross talk between the two pathways, possibly via competition for ARNT. Previous studies that have

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Fig. 2. An outline of the pathways involved in transcriptional regulation in response to hypoxia and aryl hydrocarbons. PCBs bind to the aryl hydrocarbon receptor (AHR), causing it to dissociate from its chaperone comple. Hypoxia results in the stabilization of HIF1a, disrupting its interaction with chaperones and causing its concentration to increase. The AHR and HIF1a then bind to the aryl hydrocarbon nuclear translocator (ARNT), move to the nucleus and interact with either a xenobiotic responsive element (XRE) or a hypoxia responsive element (HRE) adjacent to target genes, regulating their transcription.

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investigated the possibility of cross-talk between arylhydrocarbon and hypoxic induction pathways have yielded conflicting results, with some studies indicating that hypoxia exposure inhibits the aryl-hydrocarbon response (Gradin et al., 1996; Gassmann et al., 1997; Chan et al., 1999; Kim and Sheen, 2000; Prasch et al., 2004), and others demonstrating reciprocal repression (Nie et al., 2001). As was the case with the interacting responses to arsenic and behavioral stress, the complexity of the responses to combined exposure to hypoxia and PCBs suggests that there is substantial gene, tissue and species-specificity. In addition, dose and relative timing of exposure likely plays a role in the observed variation among studies. However, our results in F. heteroclitus clearly indicate the possibility for potentially adverse interactions between these two stressors. Increases in glycolysis in response to hypoxia are a highly conserved protective cellular response to the restrictions in aerobic energy supply caused by hypoxia. Inhibition of this response by prior PCB exposure in F. heteroclitus presents a potential hazard to these fish. In fact, in preliminary experiments we observed that fish could not survive combined exposure to these stressors at levels that were non-lethal when the stressors were tested independently. These data demonstrate the importance of considering interacting stressors, and the mechanisms of the biological responses to these stressors when developing predictions regarding the responses of organisms and populations to anthropogenic environmental change. In addition to contamination with organic pollutants such as PCBs and dioxins, estuarine habitats are often contaminated with heavy metals as a result of anthropogenic processes such as wood preservation, mining, electronics manufacturing, and agriculture (Sanders et al., 1994; Reimer et al., 2002). One such heavy metal, arsenic, is a well known poison that is lethal to most animals at high doses, but chronic exposure to much lower doses (ca. 5–10 ppb, an environmentally relevant range) is also deleterious, being associated with carcinogenesis, damage to organs, diabetes, and cardiovascular disease (Hughes, 2002). When arsenic levels increase substantially in aquatic habitats, fish die-offs and compromised health have been reported (Gilderhus, 1966; Sorenson et al., 1980; Reimer et al., 2002; Larsen and Francesconi, 2003; Liao et al., 2003). The toxic mechanisms of arsenic exposure at high doses are fairly well understood, and involve processes such as the inhibition of both substrate level and oxidative phosphorylation, and the inhibition of enzymes such as pyruvate dehydrogenase (see Hughes, 2002 for review). However, fish chronically exposed to much lower doses of arsenic display a variety of physiological effects including reduced ability to cope with changes in abiotic factors such as salinity and temperature (Holland et al., 1964; Sorenson et al., 1980; Nichols et al., 1984; Kotsanis and IliopoulouGeorgudaki, 1999; Pedlar and Klaverkamp, 2002). The mechanisms underlying these chronic effects are not well understood, but these processes are directly or indirectly mediated by the cortisol-mediated stress response in fish,

suggesting the possibility that arsenic might interfere in some way with these pathways. Cortisol mediates physiological responses by binding to the glucocorticoid receptor (GR). Cortisol-bound GR then undergoes nuclear translocation and acts as a transcription factor, by binding to glucocorticoid responsive elements (GREs) associated with appropriate target genes (Schoneveld et al., 2004). Evidence from a variety of model systems suggests that arsenic (at environmentally relevant levels) may interfere with GR-mediated transcriptional regulation (Simons et al., 1990; Hamilton et al., 1998; Kaltreider et al., 2001). The amino acid sequence of the DNA binding domain of the GR is thought to be the primary target for arsenic effects (Bodwell et al., 2004). There are some differences between the DNA binding domains of fish and mammals (Ducouret et al., 1995; Takeo et al., 1996) that cause fish GR to bind more loosely to GREs, which could render GR-mediated transcription even more susceptible to interference by arsenic in fish. To test the hypothesis that arsenic exposure interferes with glucorticoid-regulated gene expression in F. heteroclitus, we chronically exposed fish from the southern population to environmentally relevant doses of arsenic, and then measured the stress-mediated gene expression of the Ldh-B and phosphoenolpyruvate carboxykinase (Pepck) genes (Bears et al., 2006), which we had previously demonstrated to be stress responsive in this population (Schulte et al., 2000; Picard and Schulte, 2004). Arsenic exposure inhibited the stress-induced induction of Ldh-B (at both the protein and mRNA levels), but did not affect the induction of Pepck (Bears et al., 2006). These data provide qualified support to the hypothesis that low-dose arsenic mediates some of its toxic action via modification of the normal organismal stress response. However, other studies have failed to show effects of arsenic on cortisolmediated gene expression of the cystic fibrosis transmembrane receptor (CFTR gene) in F. heteroclitus (Shaw et al., 2006). The differences in the effects of arsenic on the transcriptional regulation of a variety of cortisol-regulated genes hints at the complexity both of the cellular response to glucocorticoids and of the effects of arsenic on these processes. Whether these effects of arsenic on the stress response are sufficient to result in increased mortality in response to arsenic exposure in F. heteroclitus is currently unknown. 7. Conclusions Organismal responses to environmental stressors can occur on a variety of time scales. Over many generations, populations may adapt to tolerate a stressor that would prove lethal or highly deleterious to the parent population. Within an organism’s lifetime, many species are capable of undergoing acclimatory changes involving changes in gene expression or alterations in membrane properties. Indeed, organisms even have a variety of mechanisms for coping with stressors on very acute time scales. Little is known,

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however, about the interactions among mechanisms operating at these various temporal and spatial scales. In addition, because of the nature of the responses to environmental stressors, there is ample opportunity for cross talk among responses pathways, potentially resulting in synergistic, additive, or antagonistic effects depending upon the combination of stressors. Investigations into the effects of combinations of stressors investigations are currently in their infancy (Foran and Ferenc, 1999), but it is clear that an understanding of the interacting effects of multiple stressors and how this affects and is affected by local adaptation of a species will be a critical component in the development of rational environmental policies.

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