Microevolution and Ecotoxicology of Metals in Invertebrates

Environ. Sci. Technol. 2007, 41, 1085-1096

Microevolution and Ecotoxicology of Metals in Invertebrates A . J O H N M O R G A N , * ,† P E T E R K I L L E , † A N D STEPHEN R. STU ¨ R Z E N B A U M †,‡ Cardiff School of Biosciences, Cardiff University, Cardiff, UK, and School of Biomedical and Health Sciences, Department of Biochemistry, Pharmaceutical Sciences Research Division, King’s College London, London, UK

Risk assessment of metal-contaminated habitats based on responses in the field is complicated by the evolution of local, metal-resistant ecotypes. The unpredictability of occurrence of genetically determined adaptive traits, in terms of site-specific geochemistry, a population’s inferred exposure history, and in the physiology of resistance, exacerbates the problem. Micro-evolutionary events warrant the attention of ecotoxicologists because they undermine the application of the bedrock of toxicology, the doseresponse curve, to in situ field assessments. Here we survey the evidence for the existence of genetically differentiated, metal-resistant, invertebrate populations; we also describe some of the molecular mechanisms underpinning the adaptations. Quantitative changes in tissue-metal partitioning, and in the molecular and cellular responses (biomarkers) to alterations in internal bioreactive metal pools, are widely accepted as indicators of toxicity and/or exposure in free-living organisms. Both can be modulated by resistance. The understanding that all genomes are intrinsically flexible, with subtle sequence changes in promoter regions or epigenetic adjustments conferring significant phenotypic consequences, is deemed highly relevant. Equally relevant is the systems biology insight that genes and proteins are woven into networks. We advocate that biomarker studies should work toward assimilating and exploiting these biological realities through monitoring the activities of suites of genes (transcriptomics) and their expressed products (proteomics), as well as profiling the metabolite signatures of individuals (metabolomics) and by using neutral genetic markers to genotype populations. Ecotoxicology requires robust tools that recognize the imprint of evolution on the constitution of field populations, as well as sufficient mechanistic understanding of the moleculargenetic observations to interpret them in meaningful environmental diagnostic ways.

Introduction “Most or perhaps all the variations from the typical form of a species must have some definite effect, however slight, on the habits or capacities of the individuals.” Alfred Russel Wallace (1858). On the Tendency of Varieties to Depart Indefinitely from the Original Type * Corresponding author phone: +44 2920875872; fax: +44 29874305; e-mail: [email protected]. † Cardiff University. ‡ King’s College London. 10.1021/es061992x CCC: $37.00 Published on Web 01/20/2007

 2007 American Chemical Society

“...natural selection is daily and hourly scrutinizing ...every variation even the slightest.... Each slight variation, if useful, is preserved.” Charles Darwin (1859). The Origin of Species by Means of Natural Selection “Whatever does not kill me makes me stronger.” Friedrich Nietzsche (1888). Twilight of the Idols Ecotoxicology is frequently concerned with assessing the relatively short-term effects of stressors (1) on ecological or environmental health. In practice the subject of study is the functional performance of individual organisms because ecological units (populations and communities) are comprised of individuals. Thus, ecotoxicologists often measure the phenotypic consequences of the interaction of potential toxicants on the genotype of a test organism. In the cases of laboratory tests, what are measured are alterations in biochemical, physiological, morphological, or behavioral traits during the more-or-less recent (exposure) history of the individual. This is significantly different from observations on field populations exposed to long-term pollution stress, even though the same traits may be measured by identical techniques. The phenotype of a field population may reflect both its multi-generation exposure history and its genetic background. Natural selection acts on phenotypic variations between individuals in a population. The function of genes is to ensure that traits favored by selection are inherited by the offspring of individuals possessing what Darwin referred to as useful variations. In this a posteriori fashion (2) a population may evolve through a combination of individual elimination and differential survival into a local adapted population with the capacity to tolerate, for example, elevated concentrations of chemical contaminants. There are a few general points worth noting here. First, the presence of a population of reproducing individuals of a given species at a chronically polluted site does not justify the immediate inference that it is adapted. Lagisz et al. (3), for example, were unable to find any differences in metal uptake and excretion rates, or in respiratory rates, in F1-generation carabid beetles (Pterostichus oblongopunctatus) bred from parents originating from clean and metal-contaminated sites, respectively. It is also possible that, where a population chronically exposed to elevated metal loads does not display constitutive adaptation, its individual members possess sufficient phenotypic plasticity to enable them to make the necessary biochemical or physiological adjustments within their individual life-histories to ensure survival at least until the successful completion of a minimum of one reproductive cycle. Alternatively, the population living in a contaminated zone may be sustained by continuous recruitment of reproductively active, nonadapted, individuals from the adjacent ‘normal’ surroundings. Immigration of juveniles, coupled with heterogeneous VOL. 41, NO. 4, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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metal distribution, has been offered as an explanation of earthworm colonization of a Cu-contaminated site (4). Second, an adapted population may be genetically differentiated from a population of non-adapted con-specifics if directional selection pressure in the polluted zone is heavy, even if there is no geographical barrier preventing gene-flow between them. Third, an adapted population may be genetically and phenotypically heterogeneous, especially if the selection pressure in the polluted zone is low or moderate: tolerant genotypes may be intermixed with relatively sensitive genotypes in both polluted and unpolluted field sites. The latter scenario, providing evidence of genetic erosion, was observed by Lopes et al. (5) in field populations of Daphnia longispina, where a stressed population did not contain the most Cu-sensitive lineages but the reference population did contain the most Cu-tolerant lineages. Ecotoxicologists are periodically urged to conduct tests in the field. Indeed, the expanding interest in biomarkers is partly a response to these calls. Measuring functional parameters in field populations does, however, present a number of difficulties. For example, the parameter may be modulated by intrinsic biotic (seasonal-linked variations in the reproductive cycle; population density effects; developmental and age-related parameters) and extrinsic abiotic factors (spatial heterogeneity in the habitat; climatic variations; food availability and quality). These types of “background noise” are difficult to factor into risk assessments, but at least their presence is widely recognized. Dealing with the potential confounding effects of local microevolutionary events is a much thornier matter. For example, Luoma and Rainbow (6), in an insightful review, listed four interacting factors that influence metal bioaccumulation by animals: (i) metal specificity; (ii) environmental influences; (iii) route of exposure; (iv) species-specific characteristics. Note that the possible influence of intra-specific genetic differentiation on bioaccumulation and ensuing toxic responses is not included. Aims. A central tenet of evolutionary ecology may be presented in the form of a familiar relationship: Phenotype ) Genotype × Environment (7-9). In ecotoxicology the effect of the environment on a genotype is measured as a phenotypic parameter. In classical toxicology and in laboratory-based ecotoxicology the acquisition of dose-response curves implies genotypic constancy. But populations are not genetically homogeneous across their distribution ranges (10); thus, they can be subject locally to evolutionary processes and modified dose-response relationships (1013) (Figure 1). Moreover, two or more (hypothetical) populations of a species that are exposed to identical stressful conditions at different localities across the distribution range will not necessarily both/all evolve identical, or indeed any, locally adapted ecotypes (14-16). Evolution is contingent on genetic variability and environmental conditions. Fieldbased ecotoxicology, however, seeks easily interpretable dose (“environment”) versus response (“phenotype”) relationships analogous to those regularly found in laboratory exposures with clonal, often parthenogenetic, organisms (17). The main purpose of this review is, therefore, heuristic: to explore how evolutionary events that have modified genetic constitution, toxicant sequestration and partitioning, gene expression efficiency, and toxicant susceptibility can profoundly influence here-and-now pollution bio-monitoring and ecological risk assessments. Our examples will be confined to invertebrates and their evolved responses to metal pollution, and will focus on molecular-genetic mechanistic pathways.

2. Adapted Populations: Evidence and Mechanisms This is not the place to present an exhaustive review of genetically based metal resistance in invertebrates. It is worth 1086

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FIGURE 1. Hypothetical dose-response curves and performance indicators in field populations. Panel A provides survival-dose curves for the laboratory-reared F2-generation offspring of six fieldsampled populations exposed to a range of metal concentrations under laboratory conditions; populations 1, 2, 3, and 5 have, for present purposes, very similar LC50 values, although their slopes differ, indicating a progressive intra-population decrease in the variability of sensitivities. Populations 4 and 6 have significantly higher LC50 values than the others, indicating that both are metalresistant ecotypes. Inter-population variations in susceptibility revealed in laboratory tests can be reflected in measured performance indicators and biomarkers in the geographically isolated source populations (Panels B and C). The curves in both cases plot the expected relationships if the six populations were genotypically similar (and the convenient stepwise differences in pollution intensity across the sites is not subject to site-specific moderating or accentuating edaphic variables), with a decrease in demographic parameters reciprocated by the curve for a general stress-responsive biomarker (Tier 1) such as hsp70. Note, however, that populations 4 and 6 (metal-resistant) are outliers (they do not occupy the “expected” 4′ and 6′ positions - note the “dashed boxes”)seven though both inhabit polluted sites, with population 6 experiencing the most severe pollution intensity. It is inappropriate to speculate how the two populations have adapted to the elevated metal loads in their native environments (see Figure 3), but it is appropriate to grasp the risk assessment implications of such local adaptations: for instance, if the demographics or hsp70 expression level of field-sampled individuals from population 6 were to be measured in “isolation” (i.e., without independent knowledge that the population is genetically differentiatedsupper panel), then a reasonable diagnostic conclusion would rank the risk status of sites 6 and 3 at similar levels, and not appreciably different from reference site 1. [Note: (i) the population at site 5 is not an outliers as indicated in the text, the evolution of metal-resistance is not merely a function of the intensity of pollution, but is contingent on local biotic and abiotic variables; (ii) although the Figure is presented as a set of hypothetical curves, it is noteworthy that similar hsp70 response patterns with identical interpretations have been described in terrestrial isopods inhabiting a metal pollution-gradient (13).

TABLE 1. Two Generic Mechanisms Whereby Organisms Cope with Excessive Metal Burdens in Their Native Habitats (after ref 25) acclimation

adaptation

tolerancea

resistanceb

stress responses during an individual’s lifetime

stress responses evolve during multi-generational exposure histories responses are often constitutive heritable, genetically-determined, adaptations resulting from directional selection genetically differentiated populations are “ecotypes” adapted organisms may have lower fitness than non-adapted organisms in “clean” environmentssthe cost of tolerance removal of stress may result in population reverting to the non-adapted state if the adaptation has a fitness cost

responses are induced rather than being “fixed” or constitutive phenotypic plasticitysmorphology, biochemistry, behavior phenotypic plasticity is genetically determined offspring raised in clean environments may be acclimated if stressor (e.g., metal) is transferred from mother to embryo traits may be lost if stressor is withdrawn

a Tolerance for the purposes of this article is considered synonymous with “phenotypic plasticity”, “acclimation”, and “inducible tolerance”. According to The Shorter Oxford English Dictionary (SOED) one definition of tolerance is “the power, constitutional or acquired, of enduring large doses of active drugs, or of resisting the action of poison”. b Resistance is considered synonymous with “adaptation”, “constitutive tolerance”, and “reduced susceptibility”. The SOED defines resistance variously: (i) the act, on the part of persons, of resisting, opposing, or withstanding; (ii) opposition of one material thing to another material thing, force etc. There are somesunhelpfulsoverlaps in the dictionary definitions. [There are also some unhelpful overlaps in the biological connotations of the terms: the mechanistic bases (i.e., phenotypic expressions) of resistance and tolerance may be very similar and dynamic; both the width of an organism’s homeostatic window (the capacity to adjust or to acclimate), or its locally evolved adaptation, are genetically determined. Indeed, phenotypic plasticity itself evolves like other quantitative traits (26, 27). Tolerance implies enduring or “putting up with (the challenges of a stressor)”, with a tendency to revert toward normality in the absence of the stressor, i.e., a switching on (induction) followed by a switching off of a response within the homeostatic range. Resistance implies a directional act and commitment; resistant organisms are genetically differentiated from con-specifics inhabiting unstressed sites. In the absence of robust evidence confirming genetic differentiation (local adaptation or a resistant ecotype) in a given population it may be prudent to use the looser term “tolerance”. This is our practice in this review.]

noting, however, that although there are many observations suggestive of tolerance in diverse aquatic and terrestrial taxa (18-20), close scrutiny indicates that many are inconclusive because they were performed directly on adults collected from the field, and not on their F1 or (preferably, to counteract the effects of the transmission of stress-inducing toxicants, or stress-responsive proteins or mRNAs, into the ooplasm of first-generation zygotes from adapted mothersssee refs 19, 21, 22) later-generation offspring raised under noncontaminated conditions. In other words, the heritability of tolerance traits is a prerequisite to distinguish constitutive adaptation from the physiological adjustment (“acclimation” or “phenotypic plasticity”; see refs 23-27; Table 1) that occurs during an individual’s exposure history to stressors. Sound evidence has been presented for Cu-tolerance in the marine polychaete Nereis diversicolor (28), and for Cdtolerance in the freshwater oligochaete Limnodrilus hoffmeisteri (29), the obligate parthenogenetic earthworm Dendrobaena octaedra (30), the freshwater dipteran larva Chironomous riparius (31), and the terrestrial collembolan Orchesella cincta (16, 32), for example. It has been suggested (33, 34) that metal-resistant populations are more common in terrestrial than in aquatic organisms. The explanation offered is that metal exposures are relatively constant, and organism dispersal and consequential gene-flow rates are relatively low in contaminated terrestrial habitats. However, this ecosystem dichotomy is not borne out in the “at least eighteen species, most (of which are) invertebrates” that van Straalen and Roelofs (35) consider to exhibit metal resistance. It is very significant that adaptation in a field population of C. riparius inhabiting a metal-contaminated river was highly unstable over a 5 month period, probably reflecting both seasonal fluxes in toxicant concentrations (i.e., in the intensity of the selective pressure) and gene flow from unpolluted regions (36). Analogous episodic gene pool variations have not to our knowledge been observed in any metal-resistant terrestrial population. The ligand affinity of a metal influences the biochemical responses of an organism. This is an important consideration when attempting to identify adapted populations based on molecular-genetic and biochemical biomarkers on the one hand, and the interpretation of laboratory-exposure toxicity

tests on the other. For instance, an ecotype with an evolved capacity behaviorally to avoid a metal pollutant in its spatially heterogeneous native habitat (e.g., the freshwater pulmonate snail Physella columbiana (37)) will have a component of its resistance armory short-circuited during toxicant exposures under uniform laboratory conditions. Ecotoxicology can also be mired by the notion that an organism has a mechanism of evolved tolerance rather than a web of integrated strategies and functional pathways. Let us explore this point by examining a familiar detoxification system. Metallothioneins are sulfur-donating, cysteine-rich, peptides that are frequently associated with tolerance to “soft acid” metals such as Cd (35). Metallothionein-gene amplifications have been detected in Cd-resistant natural populations of Drosophila melanogaster (35, 38, 39). Callaghan and Denny (40), however, showed that an ATP-dependent membrane protein, p-glycoprotein, promotes Cd efflux and plays a significant role in reducing Cd toxicity in an artificially selected laboratory “strain” of D. melanogaster. One question that arises is whether the upregulated Cd-efflux and Cdsequestration are mutually exclusive, ecotype-specific, tolerance mechanisms or coexistent, synergistic mechanisms in some or all Cd-tolerant Drosophila populations. The distinction is not trivial. Upregulated Cd efflux is, in effect, a biochemical equivalent of avoidance and is synonymous with reduced Cd toxico-availability; it may lead to lower Cdinduced metallothionein expression than predicted from combining site-specific Cd concentrations and known metallothionein Cd response levels in non-adapted animals. In other words, one protective pathway can in principle cooperate with, or serve as a functional alternative to, another, thus confounding easily interpretable dose-response relationships. A survey of the expressed sequence tag libraries for L. rubellus (www.earthworms.org) reveals that this species has two genes, hemerythrin and phytochelatin synthase, whose products are known to be intimately involved in the sequestration of Cd in annelid worms (41, 42), nematodes (43), and plants (44), respectively. Moreover, L. rubellus has homologues (www.earthworms.org) of two mammalian RNAbinding proteins, T-cell internal antigen-1 (TIA-1) and TIA1-related protein (TIAR), that play crucial roles in mRNA VOL. 41, NO. 4, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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we do advocate a more holistic approach to the mechanistic study of metal resistance. Developing a biomarker assay based on one molecular pathway is futile if the measured parameter is not induced or suppressed by an environmental toxicant because the inducer/stressor has been circumvented by the organism (e.g., the potential toxicant is behaviorally avoided, or rendered unavailable by export pumps), or its intensity has been functionally “suppressed” (e.g., it is trafficked and sequestered in other, perhaps unsuspected, molecular pathways).

3. Bioaccumulation and Bioreactivity in Metal-Tolerant Ecotypes FIGURE 2. Cadmium trafficking pathways in a hypothetical invertebrate cell, but based on transcriptomic and bioinformatic observations on earthworms (www.earthworms.org). The toxicity of Cd is regulated by a series of independent and inter-dependent pathways. Uptake is thought to be via either calcium and/or zinc channels. Once inside the cell a plethora of metal chaperones combine to minimize the risk of cadmium interfering with cellular processes. These chaperones include the gene-encoded proteins metallothionein, myohemerythrin, and Cd-binding protein (Cd-BP or MP II), as well as the enzymically synthesized (i.e., posttranscriptionally regulated) peptides and phytochelatins (PC). Either associated with these chaperones, or via a proton antiporter, cadmium is packaged into intracellular vesicles and subsequently excreted from the cell. [Note: (i) many details and connections are unresolved, but it is evident that there exists a complex network of metal trafficking pathways in a “typical cell”ssee Figure 7; (ii) descriptions of TIA-1, TIAR, and stress granules are given in the text.] stabilization, segregation, and subsequent translatability during stress (45). Whether or not these systems contribute to Cd resistance is unknown, but their until-recently unsuspected presence raises the possibility that metallothioneins are but components of a multi-factorial network of Cd-protective mechanisms in earthworms (Figure 2). Fourth, putative Cd-adapted earthworm populations may have evolved enhanced metallothionein expression neither by amplifying the gene(s) nor through modified coding regions, but by mutating and increasing the efficiency of metallothionein promoter regions. This is the hypothesis formulated by Van Straalen and Roelofs (35), and recently confirmed by Roelofs et al. (46a), to explain the 2- to 3-fold overexpression of metallothionein in a Cd-resistant population of the soil-dwelling springtail Orchesella cincta compared with controls exposed experimentally to the metal (34). Roelofs et al. also suggested that Cd-induced up-regulation of metallothionein may be functionally linked to the enhancement of a Cd excretion pathway. Not remote in terms of phenotypic outcome from the ability to elevate transcriptional efficiency are evolved adaptations in a pathway’s kinetics. Mouneyrac et al. (46b) found that a population of polychaete worms, Hediste (previously Nereis) diversicolor from a heavily metal-contaminated sediment had lower concentrations of sulfur-containing metal-binding molecules, including metallothionein, than a reference population inhabiting unpolluted sediment. The authors suggested that the turnover of sequestering metals in the adapted population is high, with the metals efficiently delivered to and immobilized within intracellular storage organelles. Clearly, a “snapshot” of metallothionein content in metal-adapted Hediste would not correlate with environmental contamination levels or accumulated body burdens. Van Straalen and Roelofs (35) implied that research into the role of metallothionein in Cd resistance has hitherto been too narrow in scope. We do not dissent from this view, but 1088

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Ehlers and Luthy (47), in their summary of a National Research Council report (2002), concluded that a better understanding of contaminant bioavailability would improve soil and sediment risk assessment and remediation. In essence this statement is an application of a central paradigm in pharmacology and ecotoxicology: the effect evoked by a chemical reflects its concentration at the receptor. An ecotoxicological derivative of the paradigm is the critical body residue (CBR) notion, where CBR is defined as “the threshold concentration of a substance in an organism that marks the transition between no effect and adverse effect” (48). The relationship between CBR and total accumulated body burden is relatively difficult to predict for metals, mainly because of the species-specific variety of accumulation patterns and the metal-specific modes of toxicity (49). Thus, species- and metal-specific understanding of the connection between bioaccumulation and toxicity is a matter of priority in predictive ecotoxicology (see refs 6, 50-53). Vijver et al. (48) formulated a plausible hypothesis: “(the) differences between species in critical body concentrations of metals reflect different internal compartmentalization strategies and that the fraction causing toxic effects may be similar in all species”. The hypothesis implicitly invites conjecture about the effects of intra-specific adaptation to long-term field exposures on the tripartite relationship between metal burden, bio-reactive metal fraction, and metal toxicity within an organism (Figure 3). We are unaware of any evidence either refuting or supporting the notion that a given metal’s CBR value is similar in adapted and non-adapted populations. There is, however, ample evidence that resistant ecotypes are relatively insensitive to the metals to which they have multi-generational histories of exposure (see above). There are two generic functional strategies for achieving this. The first reduces net tissue metal accumulation, the other increases the proportion of the body burden accumulated in non-bioreactive states. Limiting the accumulated-metal burden in metal-laden environments can be genetically determined by downregulating cation influx pumps. The principle is exemplified by the repression of high-affinity phosphate pumps in arsenate-resistant plants (54), but similar tolerance strategies appear not to have been described in invertebrates. The alternative strategy of reducing body metal burdens through up-regulating excretory or periodic-shedding mechanisms is well-known in invertebrates (32, 55, 56), with Cd tolerance in a number of different taxa aided by efflux pumps similar to multidrug resistance p-glycoproteins (40, 57, 58) being a particularly well described mechanism. However, increased bio-immobilization capacity may be the most common strategy. It is exemplified by the enhanced ability of L. hoffmeisteri from a long-term polluted site to sequester Cd in insoluble intracellular, metal-rich, granules (59, 60); in contrast, worms from a clean reference site appeared to rely mainly on metallothionein induction to protect against shortterm Cd exposure. Pre-induction effects notwithstanding, the phenomenon may also be exemplified by the higher

Cadmium mobility from nonadapted worms was approximately 4-fold higher than that from the adapted worms (60). Analogous, albeit inter- rather than intra-specific, observations were made with another shrimp species fed two bivalve species with contrasting Cd and Zn partitioning patterns (62). It seems clear that heritable changes in tissue metal compartmentation can potentially confound intersite comparisons reliant on straightforward whole organism metal bioaccumulation data. Measuring metal effects rather than metal burdens might alleviate the problem.

4. Bioreactive Metal Pools and Sublethal Toxicity (Biomarkers)

FIGURE 3. Conceptual relationships between total metal body burdens (solid lines) and bioreactive metal fractions (broken lines), respectively, and environmental metal concentrations in a nonadapted genotype (Panel A) and two adapted, metal-resistant genotypes characterized by different metal handling physiologies: enhanced metal-sequestration efficiency (Panel B); enhanced metal exclusion/excretion (Panel C). The area between the solid and broken lines in each panel represents the “detoxified” (alternatively, the “sequestered” or “immobilized”) metal pools; genotypes with the physiological features depicted in (Panel B) have the highest detoxification capacity and efficiency because they suppress expansion of the bio-reactive pool relative to increase in total body burden. CBR is the critical body concentration above which an organism displays adverse, metal-mediated, effects; by definition, it is the metal concentration in the bioreactive pool that mediates direct toxicosis. It is hypothesized that the CBR value is constant across all genotypes within a species, i.e., it is independent of local physiological strategies or adaptations. Note that the two adapted genotypes exceed the CBR at higher environmental metal concentrations than their non-adapted counterpart, but achieve resistance by different physiological strategies. [See ref 22 for an example of an attempt to relate tissue metal fractionation to toxic effects.] proportion of the Cd body burden sequestered by metallothionein-like proteins (contributing significantly to the “detoxified” subcellular metal pool) in caddisflies with a metal exposure history compared with con-specifics from a clean site (61). It is evident that resistant ecotypes can have different sub-cellular metal partitioning profiles compared with nontolerants, although Vijvier et al. (53) did not observe such an interpopulation difference in the earthworm Aporrectodea caliginosa. On the whole, resistance is characterized by relatively low bioreactive non-essential-metal pools per unit “total” body burden, thus reducing the likelihood of vulnerable biochemical pathways encountering >CBRmetalx. This is a conclusion drawn from an experimental study of the trophic transference of Cd from metal adapted or non-adapted prey (L. hoffmeisteri) to a predatory shrimp (Palaemonetes pugio).

Biomarkers are used in environmental diagnostics as early and predictive molecular-genetic, biochemical, cellular, or physiological responses to sublethal toxicant stress (63, 64). Their potential value in ecotoxicology, though recently questioned in the context of environmental risk assessments (65), rests on their perceived sensitivity, early responsiveness, relative ease of quantitative measurement, and the variety of functional pathways and organizational levels that they report on (Figure 4). There are two major types of biomarkers: markers of exposure and of effect (66). Biomarkers of exposure report on the environmental availability of a metal. Metallothionein is a typical example. But we saw earlier (Section 2) that the level of metallothionein protein in Cd tolerant individuals can be lower than that in a reference population exposed to background Cd concentrations (46). In this particular instance, where it was suggested that the tolerant animals have a much higher metallothionein turnover, measuring metallothionein-gene transcription, 35S-cysteine kinetics, or the volume fraction of the insoluble granules into which metallothionein chaperones Cd, would probably serve as better biomarkers of exposure. The higher metallothionein m-RNA levels recorded in a laboratory-raised metal-resistant “strain” of the collembolan Orchesella cincta compared with a reference strain (34) tends to confirm this view. The stress-inducible heat shock protein, hsp70, (67) is a widely used biomarker of effect (64). However, a number of studies have shown that natural selection favors diminished expression of hsp70 in natural populations of Drosophila buzzatii exposed to long-term thermal stress (68), and in three species of terrestrial arthropods (64) and an orthopteran insect Tetrix tenuicornis (69) chronically exposed to metal pollutants. Interpreting these findings is difficult, but in the case of metals it is conceivable that the transporting and sequestration mechanisms in adapted ecotypes are sufficiently effective to minimize metal “spillover”, and the consequent hsp-inducing molecular damage. In practical ecotoxicity terms, these manifestations of local microevolutionary events add to the raft of difficulties identified (70, 71) in the use of hsp70 expression as a reliable, easy to evaluate, biomarker of general stress. This is not to say that hsp70 expression does not faithfully reflect the degree of stress experienced by individual organisms in particular polluted habitats; indeed, it probably does. The difficulty is that the level of stress reported by biomarkers of effect can be less a consequence of the site-specific contamination level to which the monitored individuals are exposed than it is a consequence of the combined exposure and microevolutionary histories of the given population. The notion that “hidden variation” can be uncovered by the application of stress has a long history (15, 72), although the mechanism underpinning it proved elusive. The recently described buffering roles of heat-shock proteins, but in particular hsp90, during early development has potentially profound evolutionary (73-75) and ecotoxicological implications. Seminal laboratory observations on Arabidopsis VOL. 41, NO. 4, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Biomarkers at the lowest biological levels of organization. Monitoring chemical-induced departures from the norm in genetic activities is perceived to be the acme of the biomarker approach. Genetic modifications are the source of the cascade of an organism’s responses to altered environmental conditions, a cascade that reverberates “upward” through individual survival strategies and reproductive output to, ultimately, ecological-level consequences. But, the hierarchal complexities at the target gene and its expressed product level invites caution. Gene transcription (Panel A) is subject to a number of up-regulating and down-regulating genetic and epi-genetic elements which may (i) provide the functional basis for an ecotype’s resistance to a chemical stressor without requiring alterations in a gene’s coding regions; or (ii) override or buffer the otherwise negative effects of a toxic metal on a particular gene’s activity. In analogous fashion, but mediated by different molecular players, translation efficiency (Panel B) and protein activity (Panel C) are also subject to up- and down-regulatory influences. Transcription, translation, and protein activity are connected links in a chain, so is it more effective to employ early or late components as biomarkers? To illustrate the dilemma, let us assume that it is the level of activity of an effector (an enzyme, say) that is the most relevant index of toxicity (at this low molecular-genetic level under consideration). It is, however, feasible, that the enzyme’s activity has equilibrated within the normal range despite a major toxicant-evoked enhancement in its degradation and inactivation; this could be achieved by positive feedback on transcriptome production or on the efficiency of translation. Abbreviations: CDS, coding sequence; Co-A/R, co-activator/repressor; EBS, enhancer binding site; EN, enhancer; M, modification; Me, methylation; S, mRNA stabilizing factor; TF, transcription factor; TFBS, transcription factor binding site; TM, translational mask; Ub, ubiquitin. thaliana (76) and Drosophila (77, 78) have shown that genomic modifications, that if unchecked during development yield significant phenotypic variations, can be silenced by the reparative functions of molecular chaperones belonging to the heat shock super-family. Such effectively unexpressed mutations, the majority of which are potentially lethal, can accumulate under non-stressful conditions. They may be termed “latent” or “cryptic mutations” because they are revealed when, say, hsp90 is challenged with an additional burden of damaged proteins caused by physical or chemical stressors (78) (Figure 5). Populations may, as a result of buffering, be more genetically variable than trait variation indicates under neutral or favorable environmental conditions. The corollary is that should conditions deteriorate some individuals may possess previously unsuspected heritable traits that are by chance adaptive, thus promoting rapid micro-evolutionary response to spatially or temporally abrupt environmental challenges (78). We are unaware of any concrete examples of this potentially important phenomenon in any invertebrate population, but the possibility of its existence should be recognized. Fluctuating asymmetry (FA), or developmental instability, has been advocated by some (e.g., 79), but not by others (e.g., 80), as an indicator of population stress. Part of the problem with FA stems from the differential responses of individual traits to stress: some traits are relatively impervious to stressors, others are more susceptible. Another problem is that even relatively unstable traits in a species do not deviate consistently from the “norm” in different populations inhabiting polluted habitats. For these reasons a number of 1090

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authors recommend integrating FA data from several traits rather than relying on one (81). Hendrickx et al. (82) adopted the multiple-trait approach to test the hypothesis that FA is positively related to stress and negatively related to fitness (egg mass and egg size) in female wolf spiders Pirata piraticus at six sites with different degrees of metal pollution. The authors found that spiders exposed to long-term pollution had adapted to site-specific stress through reproductive output alterations, but these bioindicators of stress did not correlate with increased FA, indicating that developmentally unstable individuals were selected against. Recent work on Drosophila (83) similarly shows that hsp90 is an effective genetic buffer but is an ineffective buffer against stochastic disturbances in developmental symmetry. Both studies provide salutary examples of how natural selection during a population’s protracted history of exposure to a stressful environment can dislocate inter-site dose-response relationships (Figure 1). Calow (84) stated that the phenomenon of compensatory adjustments, or homeostasis, raises serious difficulties in the diagnosis of environmental stress, not least because they can result in a lack of concordance between molecular/ cellular changes and organism-level performance indicators. The difficulty is acute if the purpose of a study is to predict population-level consequences from biomarker perturbations, but remains a concern if the purpose is merely to detect a stress-mediated response. Take for instance a stressor that selectively inhibits a house-keeping enzyme, but where the receptor organism responds by up-regulating transcription of the enzyme-encoding gene to yield a “normal” number

FIGURE 5. Highly schematized attempt to illustrate the principles of the release of latent or cryptic mutations by stressful conditions experienced during an organism’s early development. Under unstressed “normal” environmental conditions, the misfolded products (U1, 3) of basal genetic mutations may be refolded by heat shock protein 90 (hsp90; U2) so that the proteins have normal activities (U3) and, thus, the mutations have no apparent phenotypic consequences. Under stressful conditions, there is an additional mutation burden above the basal level, yielding new cohorts of misfolded proteins (S1, gray triangle); a finite quantity of hsp90 can only repair a limited proportion of the damaged proteins (S2), such that some of the previously silent basal mutations are revealed in the phenotype through their unbuffered products (S3, 3). The “new” phenotypes are subject to natural selection and may perchance be adaptive under prevailing stressful conditions. [O, normally folded proteins; b, repaired proteins].

FIGURE 6. Schemes and illustrative examples of alternative resistance pathways that confound the exploitation of a single molecular biomarker to indicate metal exposure. Many key detoxification proteins have multiple isoforms providing alternative responses leading to a similar functional outcome, a situation observed for most class I Metallothioneins (MTs) and illustrated by mtl-1 and mtl-2 in C. elegans (87) (Panel A). Parallel pathways employing discrete detoxification mechanisms (incorporating single or multiple proteins) provide a potential overlap of response, a situation illustrated by the exploitation of Class I MTs or phytochelatins (Class III MTs) by cadmium-exposed plants (88) (Panel B). Resistance may be achieved through reciprocal responses; for example, Zn balance may be maintained in the presence of excess Zn by either up-regulating export or down-regulating import (89) (Panel C). The majority of resistance mechanisms are multistep processes incorporating linear (Panel D) or split pathways (Panel F). Additional complexity often originates from linked pathways associated with responses to alternative factors (Panel E). Well characterized examples of the last three schemes (Panels D, E, F) are the Cu-challenge response pathways (90). [The abbreviated notations (Atx 1, Ccc 2, Cup 1, Fre 1, Zip 1, Znt 1) are defined and described in the primary sources given in this caption.]

of functionally active enzyme molecules. If enzyme activity is the stress-responsive biomarker being measured it would be reasonable, but erroneous, to conclude that the organism was not suffering a significant deleterious effect. If, on the other hand, the biomarker measured is a transcriptome then stress might be inferred because of the departure of the measured parameter from the unstressed condition. The phenomenon of pathway redundancy, so well characterized in yeast (85) but likely to be widely dispersed in all living systems, is both a fail-safe source of functional robustness (86) and another potential confounder of the biomarker approach (Figure 6: (87-90)). Stress-induced responses can involve down- as well as up-regulation of particular pathways through the suppression of the translational apparatus involving the sequestration of poly(A)+ mRNA in so-called stress granules (91, 92) (see Figure 2). Stress-induced transcripts such as hsp70 are, however, specifically excluded from the granules, and are preferentially translated to promote cellular survival (45). Translation of aggregated mRNAs is reinitiated by the dissolution of the granules when the stress abates. This intricate and yet economical molecular machine has hitherto been overlooked in the context of environmental diagnostics and local evolution to metal-stressed “natural” environments. Stress

granules have been shown to form in cultured vertebrate cells under cadmium or arsenite exposures (93-96). Heat shock proteins, as we have already seen (Section 2), are ubiquitous stress-alleviating molecules, but the overexpression of hsp70, for example, is not always beneficial. The copy number of hsp70 in Drosophila is considered to be a compromise between positive selection for its anti-stress chaperone roles and negative selection against its deleterious effects on demographic parameters (67). This notion is supported by the observation that female Drosophila with extra transgenic copies of hsp70 display reduced fertility when exposed to heat shock (97). Moreover, hsp70-deleted Drosophila display protracted heat response coupled with an extended developmental delay after exposure to sublethal heat shock (98). Whether the negative effects of stress are mediated through the hsp70-implicated redirection if not stasis of housekeeping gene translation, involving a dynamic equilibrium between stress granules and polysomes (99), is an attractive hypothesis worth exploring. It is tempting to hypothesize that the lower than expected hsp70 contents of certain arthropod field populations chronically exposed to metals (64, 69) indicates that a component of the local resistance is a means of releasing “normal” functions, including growth and/or reproductive pathways, from the VOL. 41, NO. 4, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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suppressive web of hsp70 influences. Perhaps in this way highly adapted populations become sustainable under constant more-or-less severe metal-exposures conditions.

5. Cost of Resistance Animals exposed to chemical stressors must mobilize defensive and repair processes if they are to survive. These processes are energy-demanding and can have negative fitness consequences (100). Two hypotheses describe how a cost of metal resistance might be manifested (101-103): (a) the tradeoff hypothesissfueling adaptive metal-responsive traits at the expense of resource expenditures on growth and reproduction reduces individual fitness in unpolluted environments; (b) the metal requirement hypothesissresistance based on less efficient metal bioavailability can evoke micronutrient deficiency syndromes in nutrient-poor polluted environments. Studies on plants indicate that metalresistant ecotypes are not always less fit than their nonresistant counterparts on unpolluted soils (101, 104). Intraspecific cost differences ranging from low to none have been observed among resistant populations of Daphnia magna (105). There are at least two possible reasons why the cost of tolerance is negligible in some populations. Both hinge on the local absence of resistant genotypes, characterized by phenotypic plasticity predominating as an evolutionary alternative to local adaptation (106-108). First, a polluted habitat, such as an abandoned metal mine site comprised of a spatially heterogeneous mosaic of contaminated and uncontaminated patches, may favor highly plastic genotypes rather than “committed” resistant genotypes adapted to the hostile pockets. Second, plastic genotypes may be favored in habitats, notably freshwater ones, where the intensity of stress is temporally variable. Some laboratory selection (109, 110) and field observations (111) do show that metal resistance in vertebrates and invertebrates can incur significant fitness penalties. The rapid change after environmental cleanup in the genetic constitution of a previously Cd-resistant benthic worm (112) illustrates how costly resistance can be if the selection pressure is intense. The energy/nutrient budget or allocation model has been useful for conceptualizing how one advantage might be achieved at the expense of another (100). But it is a flawed model, because there is a lack of strong evidence that detoxification imposes a direct energetic burden (103), and it fails to furnish mechanistic explanations underpinning the tradeoff in species- and site-specific instances. However, it is probable that the trade-offs are consequences of the pleiotropic effects of the major genes conferring metal resistance (103).

6. Synthesis and Prospects It is not inevitable that a polluted site supports an adapted population. For example, a recent study on a reference and five tributyltin-exposed natural populations of the dogwhelk, Nucella lapillus, showed that there were no significant differences in genetic diversity and long term adaptive potential among the populations (113). A number of conditions should normally be satisfied before metal pollution results in the site-specific evolution of resistance: (i) a stress intensity exceeding the capacity of the local species to prevail by plastic responses alone; (ii) a sufficiently long populationexposure history relative to the generation period; (iii) the availability of resistant genotypes, albeit at low frequency, in the nonpolluted region contiguous with the polluted site; and (iv) limited mobility and gene flow (Figure 7). An overriding requirement, of course, is the presence of appropriate genetic variability for selection. The post-genomic era is providing new insights into how adaptive variability can be generated in response to environmental perturbations 1092

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FIGURE 7. Schematic representation of predicted genetic diversity trends along a gradient radiating from a point metal-pollution source. Selection pressure will tend to intensify (with accompanying decrease in genetic diversity) in the vicinity of the source, while gene flow (and genetic diversity) will tend to increase with distance away from the source. High genetic diversity is inevitably accompanied by relatively low genetic differentiation (i.e., the probability of the occurrence of metal-resistant ecotypes or “outlier” populations) away from the point source. Note that, unlike the geographically isolated populations depicted in Figure 1 (e.g., a series of mine-associated soils), serial sampling stations on pointsource gradients comprise a spatial continuum. [This figure is the product of personal communication with Professor M.W. Bruford.] leading to stress in receptor organisms. Accumulated “cryptic or latent mutations”, normally silenced by hsp90 interventions but articulated by stress overload during early development (76-78) (see Section 4), present one source of genestructure variations that, if by chance are adaptive in the new stressful environment, have the potential to engender rapid evolution of a resistant ecotype. The activities of transposable elements may create novel genetic variability favoring rapid evolution in response to long-term stress (114). Another molecular-genetic mechanism that may also lead to fast adaptation to stress-induced environmental changes in prokaryotes and eukaryotes involve satellite repeat sequences (115, 116) and microsatellites or simple sequence repeats (SSRs) (117). There is accumulating evidence that repeated sequences in invertebrates as well as vertebrates are transcribed under physiological and pathological stress. Although the function of most transcripts is presently unknown, some serve as precursors of interfering RNAs (RNAi) (116) which favor the production of new phenotypes (118). As an illustrative example, it has been hypothesized that sat III transcripts have two possible functions in heatstressed HeLa cells: (i) they protect a vulnerable genomic domain against damage; and, (ii) they protect the transcription machinery and gene-splicing activities under stress (115). Recently, it has become evident that SSRs are nonrandomly distributed across protein coding regions, untranslated regions, and introns, and that they lead to phenotypic change through, where coding regions are involved, the loss or gain of gene function via frame-shift mutation or expanded toxic mRNA (117). The phenotype could also be modified by alterations in SSR copy number (117). The role of RNA in the evolution of metal-resistant populations is likely to be a fruitful area of future research. The following quotations from Herbert (118) give a flavor of the fundamental importance of the field, and serve as a prompt to evolutionary geneticists and ecotoxicologists: “We find that non-protein coding RNAs are central to the translation of coding RNAs and to the coregulation of other cellular events. Through combinatorial processes that lead to new coRNAs, these events can vary over time. New RNA spaces can be explored in search of those that create selective advantage for their host.” Epigenetics has been defined as “heritable modification of gene expression without change in its nucleotide sequence, in which the modification is attributable to patterns of DNA methylation and/or histone modification associated with the gene” (119). Methylation is often associated with repeat sequences and can be triggered by environmental stress; they lead to gene silencing and inexorably alter phenotypes

(119, 120). Finnegan (120) stated that if the randomly formed epialleles following stress exposure are subjected to natural selection this could lead to rapid local adaptation without, by definition, accompanying changes in genomic sequence. It has also been stated that silent sequence mutations (effectively “cryptic mutations” sensu (78)) could accumulate in methylated alleles, and then be uncovered by demethylation (121). Such epigenetic events have not to our knowledge been identified in any metal-resistant invertebrate population, but they are surely worth seeking? Indeed there are a number of techniques available for examining genespecific methylation (e.g., 122), including high-throughput methods (e.g., 123), although none are straightforward (119). It is germane and encouraging that demethylation, for example, causes the activation of methylated metallothionein I gene promoter in murine lymphosarcoma cells (124). The hypermethylation and demethylation of such genetic targets provides a plausible subtle mechanism for tweaking the expression of a protein with known metal protective function in a range of plant and animal taxa but without altering the genetic architecture. There is little doubt that techniques need to be deployed in the context of field-based ecotoxicology to screen for genetic differentiation among populations. A number of genetic tools, including polymorphic microsatellite (e.g., 125), amplified fragment length polymorphism (AFLP) (e.g., 16), and mitochondrial DNA (mtDNA) (e.g., 16) markers, are available for genotyping invertebrate populations. These tools have a considerable utility for genetic profiling along pollution gradients radiating from point sources, and for comparing genetic diversity on either side of discrete pollution boundaries. They can also perform the crucial task of screening for cryptic species, because genetic inhomogeneities of this type may: (i) be linked with differences in susceptibility or resistance to certain environmental contaminants, thus confounding site-to-site biomonitoring studies (126); or (ii) indicate that the observed erosion of genetic diversity within a polluted site is not a loss of susceptible genotypes within a species, but a loss of morphologically indistinguishable species from an unsuspected cryptic species complex (127). These techniques can also address a range of ecotoxicological questions on non-model organisms (12), but go beyond the description of modes of toxicant action (128) to confront the fundamental issues enunciated by Bickham et al. (129): “Because population genetic changes are expected to be independent of the mechanisms of toxicity, and yet highly sensitive transgenerational effects, we propose that they represent the ultimate biomarker of effect. This is because such genetic changes, especially the loss of genetic variability, might be permanent .....Whereas population numbers can potentially recover to pre-bottleneck levels as a result of adaptation to the polluted environment or disappearance of the stressor, genetic diversity will only recover if the population survives for a very long time (assuming the absence of gene flow from other populations). This contrasts with other biomarkers of effect, which represent somatic effects on individuals, not permanent effects in populations.” In conclusion, ecotoxicologists may engage the confounding realities of evolutionary events on the predictability, or otherwise, of the responses of their subject organisms to chemical stressors in one of three ways. First, by sidestepping the local evolutionary issue and working with “standard” genotypes exposed to field-sampled media under controlled conditions. If the chosen strain is a relatively sensitive one, such pragmatism yields the conservative rather than permissive outcomes that regulators favor because the sampling of structurally complex environmental media, such as soils and sediments, inevitably alter their chemical fidelities thus reversing the processes of geochemical “aging” and increasing toxic potential. Second, by simultaneously assessing effect

biomarkers in resident and transplanted individuals at field sites after the latter have equilibrated with local geochemical variables. Third, ecotoxicologists assessing the health of freeliving field populations using transcriptomic, proteomic, or metabolomic technologies (130) might prudently discard the reductionist “gene for” tendency in favor of a systems perspective where phenotypes emanate from the geneprotein interactions that Noble (86) described as complex, modular, gene-protein networks. Such networks are, of course, heritable products of evolution. Describing and interpreting toxicant-mediated changes in gene-protein network patterns, in ways that inform both an understanding of physiological functions and risk assessment decisionmaking, are conspicuous ecotoxicological challenges, not least because of the poor representation of fully annotated sequence data in those non-model species of invertebrates most frequently used in ecotoxicology. These challenges are evaded but not fully resolved unless they acknowledge and assimilate the micro-evolutionary dimension.

Acknowledgments Authors A.J.M. and P.K. are joint first authors of this manuscript. We thank the Natural Environment Research Council (NERC) for their generous financial support. This review was written during the tenure of a NERC grant (GST/ 02/1782) under the Environmental Genomics thematic programme. We also thank Dr. M.S. Davies and four anonymous reviewers for their perspicacious comments on an early version of the manuscript.

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Received for review August 18, 2006. Revised manuscript received December 5, 2006. Accepted December 8, 2006. ES061992X