Current water quality guidelines across North America and Europe do not protect lakes from salinization.

Human-induced salinization caused by the use of road deicing salts, agricultural practices, mining operations, and climate change is a major threat to the biodiversity and functioning of freshwater ecosystems. Yet, it is unclear if freshwater ecosystems are protected from salinization by current water quality guidelines. Leveraging an experimental network of land-based and in-lake mesocosms across North America and Europe, we tested how salinization-indicated as elevated chloride (Cl-) concentration-will affect lake food webs and if two of the lowest Cl- thresholds found globally are sufficient to protect these food webs. Our results indicated that salinization will cause substantial zooplankton mortality at the lowest Cl- thresholds established in Canada (120 mg Cl-/L) and the United States (230 mg Cl-/L) and throughout Europe where Cl- thresholds are generally higher. For instance, at 73% of our study sites, Cl- concentrations that caused a ≥50% reduction in cladoceran abundance were at or below Cl- thresholds in Canada, in the United States, and throughout Europe. Similar trends occurred for copepod and rotifer zooplankton. The loss of zooplankton triggered a cascading effect causing an increase in phytoplankton biomass at 47% of study sites. Such changes in lake food webs could alter nutrient cycling and water clarity and trigger declines in fish production. Current Cl- thresholds across North America and Europe clearly do not adequately protect lake food webs. Water quality guidelines should be developed where they do not exist, and there is an urgent need to reassess existing guidelines to protect lake ecosystems from human-induced salinization.

Effects of amphibian genetic diversity on ecological communities.

The amount of genetic diversity within a population can affect ecological processes at population, community, and ecosystem levels. However, the magnitude, consistency, and scope of these effects are largely unknown. To investigate these issues, we conducted two experiments manipulating the amount of genetic diversity and environmental factors in larval amphibians. The first experiment manipulated wood frog genetic diversity, the presence or absence of caged predators, and competition from leopard frogs to test whether these factors affected survival, growth, and morphology of wood frogs and leopard frogs. The second experiment manipulated wood frog genetic diversity, the presence or absence of uncaged predators, and resource abundance to test whether these factors affected wood frog traits (survival, morphology, growth, development, and behavior) and other components of the ecological community (zooplankton abundance, phytoplankton, periphyton, and bacterial community structure). Genetic diversity did not affect wood frog survival, growth, and development in either experiment. However, genetic diversity did affect the mean morphology of wood frog tadpoles in the first experiment and the abundance and distribution of zooplankton in the second experiment. It did not affect phytoplankton abundance, periphyton abundance, or bacterial community structure. While effect sizes (Cohen's d) of genetic diversity were approximately half those of environment treatments, the greatest effect sizes were for interaction effects between genetic diversity and environment. Our results indicate that genetic diversity can have a large effect on ecological processes, but the direction of those effects is highly dependent upon environmental conditions, and not easily predicted from simple measures of traits.

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.

Timing and order of exposure to two echinostome species affect patterns of infection in larval amphibians.

The study of priority effects with respect to coinfections is still in its infancy. Moreover, existing coinfection studies typically focus on infection outcomes associated with exposure to distinct sets of parasite species, despite that functionally and morphologically similar parasite species commonly coexist in nature. Therefore, it is important to understand how interactions between similar parasites influence infection outcomes. Surveys at seven ponds in northwest Pennsylvania found that multiple species of echinostomes commonly co-occur. Using a larval anuran host (Rana pipiens) and the two most commonly identified echinostome species from our field surveys (Echinostoma trivolvis and Echinoparyphium lineage 3), we examined how species composition and timing of exposure affect patterns of infection. When tadpoles were exposed to both parasites simultaneously, infection loads were higher than when exposed to Echinoparyphium alone but similar to being exposed to Echinostoma alone. When tadpoles were sequentially exposed to the parasite species, tadpoles first exposed to Echinoparyphium had 23% lower infection loads than tadpoles first exposed to Echinostoma. These findings demonstrate that exposure timing and order, even with similar parasites, can influence coinfection outcomes, and emphasize the importance of using molecular methods to identify parasites for ecological studies.

Range position and climate sensitivity: The structure of among-population demographic responses to climatic variation.

Species' distributions will respond to climate change based on the relationship between local demographic processes and climate and how this relationship varies based on range position. A rarely tested demographic prediction is that populations at the extremes of a species' climate envelope (e.g., populations in areas with the highest mean annual temperature) will be most sensitive to local shifts in climate (i.e., warming). We tested this prediction using a dynamic species distribution model linking demographic rates to variation in temperature and precipitation for wood frogs (Lithobates sylvaticus) in North America. Using long-term monitoring data from 746 populations in 27 study areas, we determined how climatic variation affected population growth rates and how these relationships varied with respect to long-term climate. Some models supported the predicted pattern, with negative effects of extreme summer temperatures in hotter areas and positive effects on recruitment for summer water availability in drier areas. We also found evidence of interacting temperature and precipitation influencing population size, such as extreme heat having less of a negative effect in wetter areas. Other results were contrary to predictions, such as positive effects of summer water availability in wetter parts of the range and positive responses to winter warming especially in milder areas. In general, we found wood frogs were more sensitive to changes in temperature or temperature interacting with precipitation than to changes in precipitation alone. Our results suggest that sensitivity to changes in climate cannot be predicted simply by knowing locations within the species' climate envelope. Many climate processes did not affect population growth rates in the predicted direction based on range position. Processes such as species-interactions, local adaptation, and interactions with the physical landscape likely affect the responses we observed. Our work highlights the need to measure demographic responses to changing climate.

A pesticide paradox: fungicides indirectly increase fungal infections.

There are many examples where the use of chemicals have had profound unintended consequences, such as fertilizers reducing crop yields (paradox of enrichment) and insecticides increasing insect pests (by reducing natural biocontrol). Recently, the application of agrochemicals, such as agricultural disinfectants and fungicides, has been explored as an approach to curb the pathogenic fungus, Batrachochytrium dendrobatidis (Bd), which is associated with worldwide amphibian declines. However, the long-term, net effects of early-life exposure to these chemicals on amphibian disease risk have not been thoroughly investigated. Using a combination of laboratory experiments and analysis of data from the literature, we explored the effects of fungicide exposure on Bd infections in two frog species. Extremely low concentrations of the fungicides azoxystrobin, chlorothalonil, and mancozeb were directly toxic to Bd in culture. However, estimated environmental concentrations of the fungicides did not reduce Bd on Cuban tree frog (Osteopilus septentrionalis) tadpoles exposed simultaneously to any of these fungicides and Bd, and fungicide exposure actually increased Bd-induced mortality. Additionally, exposure to any of these fungicides as tadpoles resulted in higher Bd abundance and greater Bd-induced mortality when challenged with Bd post-metamorphosis, an average of 71 d after their last fungicide exposure. Analysis of data from the literature revealed that previous exposure to the fungicide itraconazole, which is commonly used to clear Bd infections, made the critically endangered booroolong frog (Litoria booroolongensis) more susceptible to Bd. Finally, a field survey revealed that Bd prevalence was positively associated with concentrations of fungicides in ponds. Although fungicides show promise for controlling Bd, these results suggest that, if fungicides do not completely eliminate Bd or if Bd recolonizes, exposure to fungicides has the potential to do more harm than good. To ensure that fungicide applications have the intended consequence of curbing amphibian declines, researchers must identify which fungicides do not compromise the pathogen resistance mechanisms of amphibians.

Salinization triggers a trophic cascade in experimental freshwater communities with varying food-chain length.

The application of road deicing salts in northern regions worldwide is changing the chemical environment of freshwater ecosystems. Chloride levels in many lakes, streams, and wetlands exceed the chronic and acute thresholds established by the United States and Canada for the protection of freshwater biota. Few studies have identified the impacts of deicing salts in stream and wetland communities and none have examined impacts in lake communities. We tested how relevant concentrations of road salt (15, 100, 250, 500, and 1000 mg Cl- /L) interacted with experimental communities containing two or three trophic levels (i.e., no fish vs. predatory fish). We hypothesized that road salt and fish would have a negative synergistic effect on zooplankton, which would then induce a trophic cascade. We tested this hypothesis in outdoor mesocosms containing filamentous algae, periphyton, phytoplankton, zooplankton, several macroinvertebrate species, and fish. We found that the presence of fish and high salt had a negative synergistic effect on the zooplankton community, which in turn caused an increase in phytoplankton. Contributing to the magnitude of this trophic cascade was a direct positive effect of high salinity on phytoplankton abundance. Cascading effects were limited with respect to impacts on the benthic food web. Periphyton and snail grazers were unaffected by the salt-induced trophic cascade, but the biomass of filamentous algae decreased as a result of competition with phytoplankton for light or nutrients. We also found direct negative effects of high salinity on the biomass of filamentous algae and amphipods (Hyalella azteca) and the mortality of banded mystery snails (Viviparus georgianus) and fingernail clams (Sphaerium simile). Clam mortality was dependent on the presence of fish, suggesting a non-consumptive interactive effect with salt. Our results indicate that globally increasing concentrations of road salt can alter community structure via both direct and indirect effects.

Inducible Tolerance to Agrochemicals Was Paved by Evolutionary Responses to Predators.

Recent research has reported increased tolerance to agrochemicals in target and nontarget organisms following acute physiological changes induced through phenotypic plasticity. Moreover, the most inducible populations are those from more pristine locations, far from agrochemical use. We asked why do populations with no known history of pesticide exposure have the ability to induce adaptive responses to novel agrochemicals? We hypothesized that increased pesticide tolerance results from a generalized stressor response in organisms, and would be induced following sublethal exposure to natural and anthropogenic stressors. We exposed larval wood frogs (Lithobates sylvaticus) to one of seven natural or anthropogenic stressors (predator cue (Anax spp.), 0.5 or 1.0 mg carbaryl/L, road salt (200 or 1000 mg Cl-/L), ethanol-vehicle control, or no-stressor control) and subsequently tested their tolerance to a lethal carbaryl concentration using time-to-death assays. We observed induced carbaryl tolerance in tadpoles exposed to 0.5 mg/L carbaryl and also in tadpoles exposed to predator cues. Our results suggest that the ability to induce pesticide tolerance likely arose through evolved antipredator responses. Given that antipredator responses are widespread among species, many animals might possess inducible pesticide tolerance, buffering them from agrochemical exposure.

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.

A salty landscape of fear: responses of fish and zooplankton to freshwater salinization and predatory stress.

Predator-prey relationships are altered by anthropogenic contaminants. Road salt is a widespread contaminant among freshwater ecosystems, yet a relatively understudied subject in community ecology. Unknown is whether road salt salinization interacts with predatory stress to influence the growth, behavior, or reproduction of freshwater organisms. Using rainbow trout (Oncorhynchus mykiss) and zooplankton (Daphnia pulex), we exposed them to variable levels of road salt (NaCl) crossed with the presence or absence of alarm cues or kairomones. Alarm cue reduced trout activity and aggression and increased shoaling behavior. Road salt reduced trout growth in the high compared to moderate salt concentration, but neither concentration was different from the control. There was no interaction between alarm cues and salt for trout. Road salt and predatory stress had an additive effect on Daphnia abundance. Predatory stress decreased Daphnia abundance by 11%. Compared to the control, salt decreased Daphnia abundance by 40% in 860 mg Cl-/L and 79% in 1300 mg Cl-/L, and by the final day abundance was reduced by 85% in 1300 mg Cl-/L. Road salt and predatory stress had an interactive effect on Daphnia reproduction. Predatory stress in control water and moderate salt levels (230 mg Cl-/L) increased sexual reproduction of Daphnia, but these responses disappeared at high salt concentrations. Thus, road salt could limit reproductive adaptations to natural and anthropogenic stressors in Daphnia. Our results indicate road salt salinization could alter zooplankton population dynamics directly and by interacting with predatory stress, which might affect energy flow through freshwater food webs.

Litter chemistry and chemical diversity drive ecosystem processes in forest ponds.

Research suggests that a positive relationship exists between diversity and ecological function, yet the multi-trophic effects of biodiversity remain poorly understood. The resource complementarity hypothesis suggests that increasing the trait diversity of resources provides a more complete diet for consumers, elevating consumer feeding rates. Whereas previous tests of this mechanism have measured trait diversity as the variation of single traits or the richness of functional groups, we employed a multivariate trait index to manipulate the chemical diversity of temperate tree litter species in outdoor pond mesocosms. We inoculated outdoor mesocosms with diverse and multi-trophic communities of microbial and macro-consumer species that rely on leaf litter for energy and nutrients. Litter was provided at three levels of chemical trait diversity, a constant level of species richness, and an equal representation of all litter species. Over three months, we measured more than 65 responses, and assessed the effects of litter chemical diversity and chemical trait means (i.e., community-weighted means). We found that litter chemical diversity positively correlated with decomposition rate of leaf litter, but had no effect on biomass or density of producers and consumers. However, the pond communities often responded to chemical trait means, particularly those related to nutrients, structure, and defense. Our results suggest that resource complementarity does have some effect on the release of energy and nutrients from decomposing substrates in forest ponds, but does not have multi-trophic effects. Our results further suggest that loss of tree biodiversity could affect forest ecosystem functionality, and particularly the processes occurring in and around ponds and wetlands.

Wetland defense: naturally occurring pesticide resistance in zooplankton populations protects the stability of aquatic communities.

Anthropogenic stressors are ubiquitous and have been implicated in worldwide declines of terrestrial and aquatic species. Pesticides are one such stressor that can have profound effects on aquatic communities by directly affecting sensitive species and indirectly affecting other species via trophic cascades, which can alter ecosystem function. However, there is growing evidence that non-target species can evolve increased resistance. When such species are important drivers of the food web, then evolved resistance should help buffer communities from the effects of pesticides. To examine this possibility, we cultured four populations of the common zooplankton Daphnia pulex that we previously demonstrated were either sensitive or resistant to a common insecticide (i.e., chlorpyrifos) due to their proximity to agriculture. Using outdoor mesocosms that contained identical aquatic communities of phytoplankton, periphyton, and leopard frog tadpoles (Lithobates pipiens), we manipulated four D. pulex populations and four insecticide concentrations. As we monitored the communities for nearly 3 months, we found that the insecticide caused direct mortality of D. pulex in communities containing sensitive populations, and this led to a bloom of phytoplankton. In contrast, the insecticide caused much less direct mortality in communities containing resistant D. pulex populations, and the trophic cascade was prevented under low to moderate insecticide concentrations. Across all insecticide treatments, survivorship of leopard frogs was approximately 72 % in communities with resistant D. pulex but only 35 % in communities with sensitive D. pulex. To our knowledge, this is one of the first studies to use naturally occurring population variation in insecticide resistance to show that the evolution of pesticide resistance in zooplankton can mitigate the effects of insecticide-induced trophic cascades, and that this outcome can have far-reaching community effects.

Dissecting the smell of fear from conspecific and heterospecific prey: investigating the processes that induce anti-predator defenses.

Prey use chemical cues from predation events to obtain information about predation risk to alter their phenotypes. Though we know how many prey respond to predators, we still have a poor understanding of the processes and chemical cues involved during a predation event. We examined how gray treefrog tadpoles (Hyla versicolor) altered their behavior and morphology when raised with cues from different stages of predator attack, predators fed different amounts of prey, and predators consuming different combinations of treefrog tadpoles or snails (Helisoma trivolvis). We found that starved predators and predators fed snails induced no defensive responses whereas tadpoles exposed to a predator consuming gray treefrogs induced greater hiding, lower activity, and relatively deeper tails. We also found that the tadpoles did not respond to crushed, chewed, or digested conspecifics, but they did respond to consumed (i.e., chewed plus digested) conspecifics. When we increased the treefrog biomass consumed by predators, tadpoles frequently increased their defenses when only tadpoles were consumed and always increased their defenses when the total diet biomass was held constant via the inclusion of snails. When predators experienced temporal variation in diet composition, including cues from snails to cause additional digestive cues or chemical noise, there was no effect on tadpole phenotypes. Our results suggest that amphibian prey rely on cues from both chewing and digestion of conspecifics and that the presence of cues from digested heterospecifics play little or no role in adding chemical noise or increased digestive enzymes and by-products that could potentially interfere with induced defenses.

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.

Exposure to sublethal concentrations of a pesticide or predator cues induces changes in brain architecture in larval amphibians.

Naturally occurring environmental factors shape developmental trajectories to produce variable phenotypes. Such developmental phenotypic plasticity can have important effects on fitness, and has been demonstrated for numerous behavioral and morphological traits. However, surprisingly few studies have examined developmental plasticity of the nervous system in response to naturally occurring environmental variation, despite accumulating evidence for neuroplasticity in a variety of organisms. Here, we asked whether the brain is developmentally plastic by exposing larval amphibians to natural and anthropogenic factors. Leopard frog tadpoles were exposed to predator cues, reduced food availability, or sublethal concentrations of the pesticide chlorpyrifos in semi-natural enclosures. Mass, growth, survival, activity, larval period, external morphology, brain mass, and brain morphology were measured in tadpoles and after metamorphosis. Tadpoles in the experimental treatments had lower masses than controls, although developmental rates and survival were similar. Tadpoles exposed to predator cues or a high dose of chlorpyrifos had altered body shapes compared to controls. In addition, brains from tadpoles exposed to predator cues or a low dose of chlorpyrifos were narrower and shorter in several dimensions compared to control tadpoles and tadpoles with low food availability. Interestingly, the changes in brain morphology present at the tadpole stage did not persist in the metamorphs. Our results show that brain morphology is a developmentally plastic trait that is responsive to ecologically relevant natural and anthropogenic factors. Whether these effects on brain morphology are linked to performance or fitness is unknown.

Leaf litter resource quality induces morphological changes in wood frog (Lithobates sylvaticus) metamorphs.

For organisms that exhibit complex life cycles, resource conditions experienced by individuals before metamorphosis can strongly affect phenotypes later in life. Such resource-induced effects are known to arise from variation in resource quantity, yet little is known regarding effects stemming from variation in resource quality (e.g., chemistry). For larval anurans, we hypothesized that variation in resource quality will induce a gradient of effects on metamorph morphology. We conducted an outdoor mesocosm experiment in which we manipulated resource quality by rearing larval wood frogs (Lithobates sylvaticus) under 11 leaf litter treatments. The litter species represented plant species found in open- and closed-canopy wetlands and included many plant species of current conservation concern (e.g., green ash, common reed). Consistent with our hypothesis, we found a gradient of responses for nearly all mass-adjusted morphological dimensions. Hindlimb dimensions and gut mass were positively associated with litter nutrient content and decomposition rate. In contrast, forelimb length and head width were positively associated with concentrations of phenolic acids and dissolved organic carbon. Limb lengths and widths were positively related with the duration of larval period, and we discuss possible hormonal mechanisms underlying this relationship. There were very few, broad differences in morphological traits of metamorphs between open- and closed-canopy litter species or between litter and no-litter treatments. This suggests that the effects of litter on metamorph morphology are litter species-specific, indicating that the effects of changing plant community structure in and around wetlands will largely depend on plant species composition.

Evolutionary change in continuous reaction norms.

Understanding the evolution of reaction norms remains a major challenge in ecology and evolution. Investigating evolutionary divergence in reaction norm shapes between populations and closely related species is one approach to providing insights. Here we use a meta-analytic approach to compare divergence in reaction norms of closely related species or populations of animals and plants across types of traits and environments. We quantified mean-standardized differences in overall trait means (Offset) and reaction norm shape (including both Slope and Curvature). These analyses revealed that differences in shape (Slope and Curvature together) were generally greater than differences in Offset. Additionally, differences in Curvature were generally greater than differences in Slope. The type of taxon contrast (species vs. population), trait, organism, and the type and novelty of environments all contributed to the best-fitting models, especially for Offset, Curvature, and the total differences (Total) between reaction norms. Congeneric species had greater differences in reaction norms than populations, and novel environmental conditions increased the differences in reaction norms between populations or species. These results show that evolutionary divergence of curvature is common and should be considered an important aspect of plasticity, together with slope. Biological details about traits and environments, including cryptic variation expressed in novel environmental conditions, may be critical to understanding how reaction norms evolve in novel and rapidly changing environments.

Interactive effects of competition and predator cues on immune responses of leopard frogs at metamorphosis.

Recent hypotheses suggest that immunosuppression, resulting from altered environmental conditions, may contribute to the increased incidence of amphibian disease around the world. Antimicrobial peptides (AMPs) in amphibian skin are an important innate immune defense against fungal, viral and bacterial pathogens. Their release is tightly coupled with release of the stress hormone noradrenaline (norepinephrine). During metamorphosis, AMPs may constitute the primary immune response in the skin of some species because acquired immune functions are temporarily suppressed in order to prevent autoimmunity against new adult antigens. Suppression of AMPs during this transitional stage may impact disease rates. We exposed leopard frog tadpoles (Lithobates pipiens) to a factorial combination of competitor and caged-predator environments and measured their development, growth and production of hydrophobic skin peptides after metamorphosis. In the absence of predator cues, or if the exposure to predator cues was late in ontogeny, competition caused more than a 250% increase in mass-standardized hydrophobic skin peptides. Predator cues caused a decrease in mass-standardized hydrophobic skin peptides when the exposure was late in ontogeny under low competition, but otherwise had no effect. Liquid chromatography tandem mass spectrometry of the skin peptides showed that they include six AMPs in the brevinin and temporin families and at least three of these peptides are previously uncharacterized. Both of these peptide families have previously been shown to inhibit harmful microbes including Batrachochytrium dendrobatidis, the fungal pathogen associated with global amphibian declines. Our study shows that amphibians may be able to adjust their skin peptide defenses in response to stressors that are experienced early in ontogeny and that these effects extend through an important life-history transition.

Induced tolerance from a sublethal insecticide leads to cross-tolerance to other insecticides.

As global pesticide use increases, the ability to rapidly respond to pesticides by increasing tolerance has important implications for the persistence of nontarget organisms. A recent study of larval amphibians discovered that increased tolerance can be induced by an early exposure to low concentrations of a pesticide. Since natural systems are often exposed to a variety of pesticides that vary in mode of action, we need to know whether the induction of increased tolerance to one pesticide confers increased tolerance to other pesticides. Using larval wood frogs (Lithobates sylvaticus), we investigated whether induction of increased tolerance to the insecticide carbaryl (AChE-inhibitor) can induce increased tolerance to other insecticides that have the same mode of action (chlorpyrifos, malathion) or a different mode of action (Na(+)channel-interfering insecticides; permethrin, cypermethrin). We found that embryonic exposure to sublethal concentrations of carbaryl induced higher tolerance to carbaryl and increased cross-tolerance to malathion and cypermethrin but not to chlorpyrifos or permethrin. In one case, the embryonic exposure to carbaryl induced tolerance in a nonlinear pattern (hormesis). These results demonstrate that that the newly discovered phenomenon of induced tolerance also provides induced cross-tolerance that is not restricted to pesticides with the same mode of action.