Phenotypically plastic responses to freshwater salinization in larval amphibians: Induced tolerance and induced sensitivity.

Contamination of aquatic ecosystems is pervasive around the world and there has been a growing interest in understanding the ecological and evolutionary impacts. For contaminants such as pesticides, researchers are discovering widespread evolution of increased tolerance in target and non-target species and the role of phenotypic plasticity in facilitating this evolution. In contrast, we know much less about the evolution of tolerance in response to the increasing problem of freshwater salinization. In amphibians, recent studies have discovered that some populations from ponds with high salt pollution (from deicing road salts) have evolved higher tolerance. In this study, we examined whether populations of wood frog tadpoles (Rana sylvatica) possess rapid, inducible tolerance to salinity in a manner similar to their inducible tolerance to pesticides. Using newly hatched tadpoles from nine populations, we discovered that eight of the populations were able to alter their tolerance to salt. However, seven of the eight inducible populations experienced a higher sensitivity to salt while the eighth population experienced a higher tolerance to salt. Such inducible responses likely reflect the interplay of salt dynamics in the ponds, combined with the available genetic variation and selection intensity of each pond. This appears to be the first example of inducible salt tolerance in any animal and future studies should examine the generality of the response and how it may affect the evolution of tolerance to the global issue of freshwater salinization.

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.

Timing and frequency of sublethal exposure modifies the induction and retention of increased insecticide tolerance in wood frogs (Lithobates sylvaticus).

Although the paradigm for increased tolerance to pesticides has been by selection on constitutive (naïve) traits, recent research has shown it can also occur through phenotypic plasticity. However, the time period in which induction can occur, the duration of induced tolerance, and the influence of multiple induction events remain unknown. We hypothesized that the induction of increased pesticide tolerance is limited to early sensitive periods, the magnitude of induced tolerance depends on the number of exposures, and the retention of induced tolerance depends on the time elapsed after an exposure and the number of exposures. To test these hypotheses, we exposed wood frog tadpoles to either a no-carbaryl control (water) or 0.5 mg/L carbaryl at 4 time periods, and later tested their tolerance to carbaryl using time-to-death assays. We discovered that tadpoles induced increased tolerance early and midway but not late in our experiment and their constitutive tolerance increased with age. We found no difference in the magnitude of induced tolerance after a single or 2 exposures. Finally, induced pesticide tolerance was reversed within 6 d but was retained only when tadpoles experienced all 4 consecutive exposures. Phenotypic plasticity provides an immediate response for sensitive amphibian larvae to early pesticide exposures and reduces phenotypic mismatches in aquatic environments contaminated by agrochemicals. Environ Toxicol Chem 2018;37:2188-2197. © 2018 SETAC.

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.

Rapid evolution of tolerance to road salt in zooplankton.

Organisms around the globe are experiencing novel environments created by human activities. One such disturbance of growing concern is the salinization of freshwater habitats from the application of road deicing salts, which creates salinity levels not experienced within the recent evolutionary history of most freshwater organisms. Moreover, salinization can induce trophic cascades and alter the structure of freshwater communities, but knowledge is still scarce about the ability of freshwater organisms to adapt to elevated salinity. We examined if a common zooplankton of freshwater lakes (Daphnia pulex) could evolve a tolerance to the most commonly used road deicing salt (sodium chloride, NaCl). Using a mesocosm experiment, we exposed freshwater communities containing Daphnia to five levels of NaCl (15, 100, 200, 500, and 1000 mg Cl- L-1). After 2.5 months, we collected Daphnia from each mesocosm and raised them in the lab for three generations under low salt conditions (15 mg Cl- L-1). We then conducted a time-to-death experiment with varying concentrations of NaCl (30, 1300, 1500, 1700, 1900 mg Cl- L-1) to test for evolved tolerance. All Daphnia populations exhibited high survival when subsequently exposed to the lowest salt concentration (30 mg Cl- L-1). At the intermediate concentration (1300 mg Cl- L-1), however, populations previously exposed to elevated concentrations (i.e.100-1000 mg Cl- L-1) had higher survival than populations previously exposed to natural background levels (15 mg Cl- L-1). All populations survived poorly when subsequently exposed to the highest concentrations (1500, 1700, and 1900 mg Cl- L-1). Our results show that the evolution of tolerance to moderate levels of salt can occur within 2.5 months, or 5-10 generations, in Daphnia. Given the importance of Daphnia in freshwater food webs, such evolved tolerance might allow Daphnia to buffer food webs from the impacts of freshwater salinization.

Investigation of road salts and biotic stressors on freshwater wetland communities.

The application of road deicing salts has led to the salinization of freshwater ecosystems in northern regions worldwide. Increased chloride concentrations in lakes, streams, ponds, and wetlands may negatively affect freshwater biota, potentially threatening ecosystem services. In an effort to reduce the effects of road salt, operators have increased the use of salt alternatives, yet we lack an understanding of how these deicers affect aquatic communities. We examined the direct and indirect effects of the most commonly used road salt (NaCl) and a proprietary salt mixture (NaCl, KCl, MgCl2), at three environmentally relevant concentrations (150, 470, and 780 mg Cl-/L) on freshwater wetland communities in combination with one of three biotic stressors (control, predator cues, and competitors). The communities contained periphyton, phytoplankton, zooplankton, and two tadpole species (American toads, Anaxyrus americanus; wood frogs, Lithobates sylvaticus). Overall, we found the two road salts did not interact with the natural stressors. Both salts decreased pH and reduced zooplankton abundance. The strong decrease in zooplankton abundance in the highest NaCl concentration caused a trophic cascade that resulted in increased phytoplankton abundance. The highest NaCl concentration also reduced toad activity. For the biotic stressors, predatory stress decreased whereas competitive stress increased the activity of both tadpole species. Wood frog survival, time to metamorphosis, and mass at metamorphosis all decreased under competitive stress whereas toad time to metamorphosis increased and mass at metamorphosis decreased. Road salts and biotic stressors can both affect freshwater communities, but their effects are not interactive.

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.

Combined effects of road salt and an insecticide on wetland communities.

As the numbers of chemical contaminants in freshwater ecosystems increase, it is important to understand whether contaminants interact in ecologically important ways. The present study investigated the independent and interactive effects of 2 contaminants that frequently co-occur in freshwater environments among higher latitudes, including a commonly applied insecticide (carbaryl) and road salt (NaCl). The hypothesis was that the addition of either contaminant would result in a decline in zooplankton, an algal bloom, and the subsequent decline of both periphyton and periphyton consumers. Another hypothesis was that combining the contaminants would result in synergistic effects on community responses. Outdoor mesocosms were used with communities that included phytoplankton, periphyton, zooplankton, amphipods, clams, snails, and tadpoles. Communities were exposed to 4 environmentally relevant concentrations of salt (27 mg Cl- L-1 , 77 mg Cl- L-1 , 277 mg Cl- L-1 , and 727 mg Cl- L-1 ) fully crossed with 4 carbaryl treatments (ethanol, 0 µg L-1 , 5 µg L-1 , and 50 µg L-1 ) over 57 d. Contaminants induced declines in rotifer and cladoceran zooplankton, but only carbaryl induced an algal bloom. Consumers exhibited both positive and negative responses to contaminants, which were likely the result of both indirect community interactions and direct toxicity. In contrast to the hypothesis, no synergistic effects were found, although copepod densities declined when high concentrations of both chemicals were combined. The results suggest that low concentrations of salt and carbaryl are likely to have mostly independent effects on aquatic communities. Environ Toxicol Chem 2017;36:771-779. © 2016 SETAC.

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.

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.

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.