Predator- and competitor-induced responses in amphibian populations that evolved different levels of pesticide tolerance.

Exposure to agrochemicals can drive rapid phenotypic and genetic changes in exposed populations. For instance, amphibian populations living far from agriculture (a proxy for agrochemical exposure) exhibit low pesticide tolerance, but they can be induced to possess high tolerance following a sublethal pesticide exposure. In contrast, amphibian populations close to agriculture exhibit high, constitutive tolerance to pesticides. A recent study has demonstrated that induced pesticide tolerance appears to have arisen from plastic responses to predator cues. As a result, we might expect that selection for constitutive pesticide tolerance in populations near agriculture (i.e., genetic assimilation) will lead to the evolution of constitutive responses to natural stressors. Using 15 wood frog (Rana sylvatica) populations from across an agricultural gradient, we conducted an outdoor mesocosm experiment to examine morphological (mass, body length, and tail depth) and behavioral responses (number of tadpoles observed and overall activity) of tadpoles exposed to three stressor environments (no-stressor, competitors, or predator cues). We discovered widespread differences in tadpole traits among populations and stressor environments, but no population-by-environment interaction. Subsequent linear models revealed that population distance to agriculture (DTA) was occasionally correlated with tadpole traits in a given environment and with magnitudes of plasticity, but none of the correlations were significant after Bonferroni adjustment. The magnitudes of predator and competitor plasticity were never correlated with the magnitude of pesticide-induced plasticity that we documented in a companion study. These results suggest that while predator-induced plasticity appears to have laid the foundation for the evolution of pesticide-induced plasticity and its subsequent genetic assimilation, inspection of population-level differences in plastic responses show that the evolution of pesticide-induced plasticity has not had a reciprocal effect on the evolved plastic responses to natural stressors.

Local adaptation of the MHC class IIß gene in populations of wood frogs (Lithobates sylvaticus) correlates with proximity to agriculture.

Major histocompatibility complex (MHC) genes code for membrane-embedded proteins that are involved in parasite/pathogen recognition. The link between the MHC and immunity makes these genes important genetic markers to evaluate in systems where infectious disease is associated with population declines. As human impacts on wildlife populations continue to increase, it is also essential to evaluate the role of MHC and immunity in the context of anthropogenic change. Amphibians are an ideal model to test the role of the MHC in infectious disease resistance, as parasites and anthropogenic disturbances currently threaten populations worldwide. We characterized the diversity of MHC class IIß peptide binding region alleles, 13 microsatellite loci, and population-level trematode resistance in 14 populations of wood frogs (Lithobates sylvaticus) in northwestern Pennsylvania with varying geographic distances to agriculture. To assess local adaptation in the MHC IIß, we compared genetic differentiation of MHC IIß and microsatellite markers (FST). We also tested for an effect of isolation by distance on genetic differentiation of MHC IIß and microsatellite markers. In addition, we evaluated whether population-level MHC IIß diversity and common allele frequencies correlate with distance to agriculture and trematode resistance. We found no evidence for genetic structure based on microsatellite analysis nor an effect of isolation by distance on neutral and immunogenetic markers. However, we did detect structure based on the MHC IIß locus, suggesting that it is under local selection. The MHC IIß allele Lisy-DAB*1 was more common in populations living near agricultural sites. Populations with higher MHC IIß diversity showed increased resistance to trematodes. Our results suggest that wood frog populations experience immunogenetic differences at a small scale. In addition, agriculture may disturb natural associations between hosts and parasites through its influence on immunocompetence, underscoring the importance of examining the effects of environmental context on host-parasite interactions.

Evolved pesticide tolerance influences susceptibility to parasites in amphibians.

Because ecosystems throughout the globe are contaminated with pesticides, there is a need to understand how natural populations cope with pesticides and the implications for ecological interactions. From an evolutionary perspective, there is evidence that pesticide tolerance can be achieved via two mechanisms: selection for constitutive tolerance over multiple generations or by inducing tolerance within a single generation via phenotypic plasticity. While both mechanisms can allow organisms to persist in contaminated environments, they might result in different performance trade-offs including population susceptibility to parasites. We have identified 15 wood frog populations that exist along a gradient from close to agriculture and high, constitutive pesticide tolerance to far from agriculture and inducible pesticide tolerance. Using these populations, we investigated the relationship between evolutionary responses to the common insecticide carbaryl and host susceptibility to the trematode Echinoparyphium lineage 3 and ranavirus using laboratory exposure assays. For Echinoparyphium, we discovered that wood frog populations living closer to agriculture with high, constitutive tolerance experienced lower loads than populations living far from agriculture with inducible pesticide tolerance. For ranavirus, we found no relationship between the mechanism of evolved pesticide tolerance and survival, but populations living closer to agriculture with high, constitutive tolerance experienced higher viral loads than populations far from agriculture with inducible tolerance. Land use and mechanisms of evolved pesticide tolerance were associated with susceptibility to parasites, but the direction of the relationship is dependent on the type of parasite, underscoring the complexity between land use and disease outcomes. Collectively, our results demonstrate that evolved pesticide tolerance can indirectly influence host-parasite interactions and underscores the importance of including evolutionary processes in ecotoxicological studies.

Effect of Simultaneous Amphibian Exposure to Pesticides and an Emerging Fungal Pathogen, Batrachochytrium dendrobatidis.

Amphibian declines have been linked to numerous factors, including pesticide use and the fungal pathogen Batrachochytrium dendrobatidis (Bd). Moreover, research has suggested a link between amphibian sensitivity to Bd and pesticide exposure. We simultaneously exposed postmetamorphic American toads (Anaxyrus americanus), western toads (A. boreas), spring peepers (Pseudacris crucifer), Pacific treefrogs (P. regilla), leopard frogs (Lithobates pipiens), and Cascades frogs (Rana cascadae) to a factorial combination of two pathogen treatments (Bd+, Bd-) and four pesticide treatments (control, ethanol vehicle, herbicide mixture, and insecticide mixture) for 14 d to quantify survival and infection load. We found no interactive effects of pesticides and Bd on anuran survival and no effects of pesticides on infection load. Mortality following Bd exposure increased in spring peepers and American toads and was dependent upon snout-vent length in western toads, American toads, and Pacific treefrogs. Previous studies reported effects of early sublethal pesticide exposure on amphibian Bd sensitivity and infection load at later life stages, but we found simultaneous exposure to sublethal pesticide concentrations and Bd had no such effect on postmetamorphic juvenile anurans. Future research investigating complex interactions between pesticides and Bd should employ a variety of pesticide formulations and Bd strains and follow the effects of exposure throughout ontogeny.

Sex-specific nutrient use and preferential allocation of resources to a sexually selected trait in Hyalella amphipods.

Although sexually dimorphic traits are often well studied, we know little about sex-specific resource use strategies that should underlie such dimorphism. We measured sex-specific responses in acquisition and assimilation of two fundamental resources, carbon (C) and phosphorus (P) in juvenile and mature Hyalella amphipods given low and high supplies of inorganic phosphate, analogous to oligotrophic and eutrophic conditions, respectively. Additionally, we quantified allocation of resources to sexual traits in males. Dual radiotracer ((14)C and (33)P) assays revealed substantial age- and sex-specific differences in acquisition and assimilation. Furthermore, a phenotypic manipulation experiment revealed that amphipods fed low-P food allocated more C to all traits than those fed high-P food. Importantly, we found that amphipods preferentially allocated more C to the development of a sexually selected trait (the posterior gnathopod), compared with a serially homologous trait (the fifth pereopod) not under sexual selection. Substantial differences in how the sexes use fundamental resources, and the impact of altered nutrient supply on such differences, illuminate sexual dimorphism at the lowest level of biological organization. Such information will be important in understanding how sex- and age-specific life history demands influence nutrient processing in a biosphere characterized by rapidly changing alterations to biogeochemical cycles.

Evolved pesticide tolerance in amphibians: Predicting mechanisms based on pesticide novelty and mode of action.

We examined 10 wood frog populations distributed along an agricultural gradient for their tolerance to six pesticides (carbaryl, malathion, cypermethrin, permethrin, imidacloprid, and thiamethoxam) that differed in date of first registration (pesticide novelty) and mode-of-action (MOA). Our goals were to assess whether: 1) tolerance was correlated with distance to agriculture for each pesticide, 2) pesticide novelty predicted the likelihood of evolved tolerance, and 3) populations display cross-tolerance between pesticides that share and differ in MOA. Wood frog populations located close to agriculture were more tolerant to carbaryl and malathion than populations far from agriculture. Moreover, the strength of the relationship between distance to agriculture and tolerance was stronger for older pesticides compared to newer pesticides. Finally, we found evidence for cross-tolerance between carbaryl and malathion (two pesticides that share MOA). This study provides one of the most comprehensive approaches for understanding patterns of evolved tolerance in non-pest species.

The contribution of phenotypic plasticity to the evolution of insecticide tolerance in amphibian populations.

Understanding population responses to rapid environmental changes caused by anthropogenic activities, such as pesticides, is a research frontier. Genetic assimilation (GA), a process initiated by phenotypic plasticity, is one mechanism potentially influencing evolutionary responses to novel environments. While theoretical and laboratory research suggests that GA has the potential to influence evolutionary trajectories, few studies have assessed its role in the evolution of wild populations experiencing novel environments. Using the insecticide, carbaryl, and 15 wood frog populations distributed across an agricultural gradient, we tested whether GA contributed to the evolution of pesticide tolerance. First, we investigated the evidence for evolved tolerance to carbaryl and discovered that population-level patterns of tolerance were consistent with evolutionary responses to pesticides; wood frog populations living closer to agriculture were more tolerant than populations living far from agriculture. Next, we tested the potential role of GA in the evolution of pesticide tolerance by assessing whether patterns of tolerance were consistent with theoretical predictions. We found that populations close to agriculture displayed constitutive tolerance to carbaryl whereas populations far from agriculture had low naïve tolerance but high magnitudes of induced tolerance. These results suggest GA could play a role in evolutionary responses to novel environments in nature.

Differences in sensitivity to the fungal pathogen Batrachochytrium dendrobatidis among amphibian populations.

Contributing to the worldwide biodiversity crisis are emerging infectious diseases, which can lead to extirpations and extinctions of hosts. For example, the infectious fungal pathogen Batrachochytrium dendrobatidis (Bd) is associated with worldwide amphibian population declines and extinctions. Sensitivity to Bd varies with species, season, and life stage. However, there is little information on whether sensitivity to Bd differs among populations, which is essential for understanding Bd-infection dynamics and for formulating conservation strategies. We experimentally investigated intraspecific differences in host sensitivity to Bd across 10 populations of wood frogs (Lithobates sylvaticus) raised from eggs to metamorphosis. We exposed the post-metamorphic wood frogs to Bd and monitored survival for 30 days under controlled laboratory conditions. Populations differed in overall survival and mortality rate. Infection load also differed among populations but was not correlated with population differences in risk of mortality. Such population-level variation in sensitivity to Bd may result in reservoir populations that may be a source for the transmission of Bd to other sensitive populations or species. Alternatively, remnant populations that are less sensitive to Bd could serve as sources for recolonization after epidemic events.

An empirical test of stable species coexistence in an amphipod species complex.

The ability of phenotypically similar species to coexist at local scales is paradoxical given that species that closely resemble each other should compete strongly for resources and thus be subject to competitive exclusion. Although theory has identified the key requirements for species to stably coexist, empirical tests of coexistence have rarely been conducted. We explored a key requirement for species to stably coexist: a species can invade a community when it is initially rare. We also assessed whether primary productivity (manipulated using phosphorus availability) affected invasion success by increasing the amount of resources available. Using two mesocosm experiments and an assemblage of phenotypically similar amphipod species in the genus Hyalella, we found no evidence for invasion success among the three Hyalella species. Further, patterns of species exclusions differed among the species, which suggests that one species is an especially poor competitor. Finally, these patterns were consistent regardless of whether mesocosms were fertilized with low or high levels of phosphorus. Our results, suggest that species differences in resource competition and predator avoidance ability found in previous studies using these Hyalella species may not be sufficient to allow for coexistence. Moreover, our study demonstrates the importance of using a variety of empirical approaches to test species coexistence theory.

Carotenoids and amphibians: effects on life history and susceptibility to the infectious pathogen, Batrachochytrium dendrobatidis.

Carotenoids are considered beneficial nutrients because they provide increased immune capacity. Although carotenoid research has been conducted in many vertebrates, little research has been done in amphibians, a group that is experiencing global population declines from numerous causes, including disease. We raised two amphibian species through metamorphosis on three carotenoid diets to quantify the effects on life-history traits and post-metamorphic susceptibility to a fungal pathogen (Batrachochytrium dendrobatidis; Bd). Increased carotenoids had no effect on survival to metamorphosis in gray treefrogs (Hyla versicolor) but caused lower survival to metamorphosis in wood frogs [Lithobates sylvaticus (Rana sylvatica)]. Increased carotenoids caused both species to experience slower development and growth. When exposed to Bd after metamorphosis, wood frogs experienced high mortality, and the carotenoid diets had no mitigating effects. Gray treefrogs were less susceptible to Bd, which prevented an assessment of whether carotenoids could mitigate the effects of Bd. Moreover, carotenoids had no effect on pathogen load. As one of only a few studies examining the effects of carotenoids on amphibians and the first to examine potential interactions with Bd, our results suggest that carotenoids do not always serve amphibians in the many positive ways that have become the paradigm in other vertebrates.

Testing the ecological consequences of evolutionary change using elements.

Understanding the ecological consequences of evolutionary change is a central challenge in contemporary biology. We propose a framework based on the ˜25 elements represented in biology, which can serve as a conduit for a general exploration of poorly understood evolution-to-ecology links. In this framework, known as ecological stoichiometry, the quantity of elements in the inorganic realm is a fundamental environment, while the flow of elements from the abiotic to the biotic realm is due to the action of genomes, with the unused elements excreted back into the inorganic realm affecting ecological processes at higher levels of organization. Ecological stoichiometry purposefully assumes distinct elemental composition of species, enabling powerful predictions about the ecological functions of species. However, this assumption results in a simplified view of the evolutionary mechanisms underlying diversification in the elemental composition of species. Recent research indicates substantial intraspecific variation in elemental composition and associated ecological functions such as nutrient excretion. We posit that attention to intraspecific variation in elemental composition will facilitate a synthesis of stoichiometric information in light of population genetics theory for a rigorous exploration of the ecological consequences of evolutionary change.

Proximity to agriculture is correlated with pesticide tolerance: evidence for the evolution of amphibian resistance to modern pesticides.

Anthropogenic environmental change is a powerful and ubiquitous evolutionary force, so it is critical that we determine the extent to which organisms can evolve in response to anthropogenic environmental change and whether these evolutionary responses have associated costs. This issue is particularly relevant for species of conservation concern including many amphibians, which are experiencing global declines from many causes including widespread exposure to agrochemicals. We used a laboratory toxicity experiment to assess variation in sensitivity to two pesticides among wood frog (Lithobates sylvaticus) populations and a mesocosm experiment to ascertain whether resistance to pesticides is associated with decreased performance when animals experience competition and fear of predation. We discovered that wood frog populations closer to agriculture were more resistant to a common insecticide (chlorpyrifos), but not to a common herbicide (Roundup). We also found no evidence that this resistance carried a performance cost when facing competition and the fear of predation. To our knowledge, this is the first study demonstrating that organophosphate insecticide (the most commonly applied class of insecticides in the world) resistance increases with agricultural land use in an amphibian, which is consistent with an evolutionary response to agrochemicals.

Eutrophication and predation risk interact to affect sexual trait expression and mating success.

Sexual traits are especially sensitive to low food resources. Other environmental parameters (e.g., predation) should also affect sexual trait expression by favoring investment in viability traits rather than sexual traits. We know surprisingly little about how predators alter investment in sexual traits, or how predator and resource environments interact to affect sexual trait investment. We explored how increasing phosphorous (P) availability, at a level mimicking cultural eutrophication, affects the development of sexual, nonsexual, and viability traits of amphipods in the presence and absence of predators. Sexual traits and growth were hypersensitive to low P compared to nonsexual traits. However, a key sexual trait responded to low P only when predator cues were absent. Furthermore, investment trade-offs between sexual traits and growth only occurred when P was low. The phenotypic changes caused by predator cues and increased P availability resulted in higher male mating success. Thus, eutrophication not only affects sexual trait expression but also masks the trade-off between traits with similar P demand. Sensitivity of sexually selected traits to changes in P, combined with the important roles these traits play in determining fitness and driving speciation, suggests that human-induced environmental change can greatly alter the evolutionary trajectories of populations.

Condition dependence of a sexually selected trait in a crustacean species complex: importance of the ecological context.

The genic capture model offers a promising solution to the lek paradox. Heightened condition dependency of sexually selected traits is a prerequisite of this model. Condition dependency is empirically inferred by the sensitivity of traits to stressors. The magnitude of ecological stress (e.g., competition and predation) experienced by populations varies considerably. Thus, condition dependence should manifest more in populations experiencing higher levels of stress. We experimentally assessed the sensitivity of a sexually selected trait (posterior gnathopod) to food resource stress in an amphipod species. We found that gnathopod size variation was 59% higher under food stress, with no corresponding effect on nonsexually selected traits. In addition, we assessed levels of gnathopod variation and the allometry of gnathopods for males sampled from natural populations for two amphipod species that experience different levels of stress (driven by contrasting size-selective predation and associated life-history trade-offs). Populations that experience higher resource stress had both steeper allometries and greater gnathopod size variation. These results suggest that the magnitude of ecological stress experienced by natural populations strongly impacts condition dependency of sexually selected traits, and could play an important role in shaping trait variation and thus the opportunity for sexual selection.

No evidence that a common pesticide impairs female mate choice in a freshwater amphipod.

Sublethal concentrations of pesticides may drastically alter the evolutionary trajectories of populations by interfering with mating behaviors. We used a median lethal concentration (LC50) experiment to test the sensitivity of three amphipod (Hyalella sp.) classes (females, small males, and large males) to five concentrations [10.4, 1.52, 0.16, 0.05, none detected (ND) microg/L] of the most commonly applied pesticide (malathion) in the United States. We then tested the sensitivity of female mate choice to sublethal concentrations (0.55, 0.38, and 0.05 microg/L) of malathion using a dichotomous choice test. Mortality was relatively high at low concentrations for all three amphipod classes with LC50s ranging from 0.06 microg/L in females to 0.19 microg/L in small males. There was overlap in the 95% CI of these estimates across amphipod classes suggesting no class-specific differences in sensitivity to malathion. We found no evidence that malathion interferes with female choice in this species, suggesting that mating behavior may not always be a good indicator of sublethal pesticide contamination.

Direct and indirect fitness consequences of female choice in a crustacean.

Understanding the evolution and maintenance of female mate choice requires information on both the benefits (the sum of direct and indirect benefits) and costs of selective mating. In this study, I assessed the fitness consequences of female mate choice in a freshwater crustacean. In Hyalella amphipods, males attempt to form precopulatory pairs with females. Large males, bearing large posterior gnathopods, tend to be over-represented in precopulatory pairs. I show that females receive both direct (reduced risk of predation while paired) and indirect (sexy sons) benefits from mating with these males. Furthermore, the behavioral mechanisms used to filter male phenotypes carry no detectable energetic cost for females. Thus, females that choose males with successful phenotypes are expected to have higher Darwinian fitness than females that mate at random. This study shows that direct and indirect selection act together to favor large male size, which explains the sexual size dimorphism and size-based mating biases observed in this species.

Niche diversity in crustacean cryptic species: complementarity in spatial distribution and predation risk.

Recent genetic studies indicate that species with very close phenotypic similarity ("cryptic species") are a common feature of nature, and that such cryptic species often coexist in communities. Because traditional views of species coexistence demand that species differ in phenotype to coexist stably, the existence of sympatric cryptic species appears to challenge traditional perspectives of coexistence. We evaluated niche diversity in three recently discovered species of Hyalella amphipods that occur sympatrically in lakes and share close phenotypic similarity. We found that, in some cases, these species exhibited strong complementary spatial distributions within the littoral zone of lakes, both across a distance-from-shore gradient, and a vertical depth gradient. Additionally, we compared fish stomach contents with habitat samples and found that species differed in their vulnerability to predation from sunfish (Lepomis spp.). Complementarity among species across axes of spatial distribution and predation risk, two important niche components, suggests that species with close phenotypic similarity may differ appreciably along ecologically relevant axes. Our results, considered in the light of previous studies, suggest a community structured by predator-mediated coexistence or sequential dominance across environmental gradients in the littoral zone.