Jessica Hua

Pesticide tolerance in amphibians: induced tolerance in susceptible populations, constitutive tolerance in tolerant populations.

The role of plasticity in shaping adaptations is important to understanding the expression of traits within individuals and the evolution of populations. With increasing human impacts on the environment, one challenge is to consider how plasticity shapes responses to anthropogenic stressors such as contaminants. To our knowledge, only one study (using mosquitoes) has considered the possibility of induced insecticide tolerance. Using populations of wood frogs (Lithobates sylvaticus) located close to and far from agricultural fields, we discovered that exposing some populations of embryos and hatchlings to sublethal concentrations of the insecticide carbaryl induced higher tolerance to a subsequent lethal concentration later in life. Interestingly, the inducible populations were located >800 m from agricultural areas and were the most susceptible to the insecticide. In contrast, the noninducible populations were located close to agricultural areas and were the least susceptible. We also found that sublethal concentrations of carbaryl induced higher tadpole AChE concentrations in several cases. This is the first study to demonstrate inducible tolerance in a vertebrate species and the pattern of inducible and constitutive tolerance among populations suggests the process of genetic assimilation.

Experimental evidence for American bullfrog (Lithobates catesbeianus) susceptibility to chytrid fungus (Batrachochytrium dendrobatidis).

The emerging fungal pathogen, Batrachochytrium dendrobatidis (Bd), has been associated with global amphibian population declines and extinctions. American bullfrogs (Lithobates catesbeianus) are widely reported to be a tolerant host and a carrier of Bd that spreads the pathogen to less tolerant hosts. Here, we examined whether bullfrogs raised from eggs to metamorphosis in outdoor mesocosms were susceptible to Bd. We experimentally exposed metamorphic juveniles to Bd in the laboratory and compared mortality rates of pathogen-exposed animals to controls (non-exposed) in two separate experiments; one using a Bd strain isolated from a Western toad and another using a strain isolated from an American bullfrog. We wanted to examine whether metamorphic bullfrogs were susceptible to either of these strains. We show that bullfrogs were susceptible to one strain of Bd and not the other. In both experiments, infection load detected in the skin decreased over time, suggesting that metamorphic bullfrogs from some populations may be inefficient long-term carriers of Bd.

Cross-tolerance in amphibians: wood frog mortality when exposed to three insecticides with a common mode of action.

Insecticide tolerance and cross-tolerance in nontarget organisms is often overlooked despite its potential to buffer natural systems from anthropogenic influence. We exposed wood frog tadpoles from 15 populations to three acetylcholine esterase-inhibiting insecticides and found widespread variation in insecticide tolerance and evidence for cross-tolerance to these insecticides. Our results demonstrate that amphibian populations with tolerance to one pesticide may be tolerant to many other pesticides.

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.

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.

Effects of clothianidin on aquatic communities: Evaluating the impacts of lethal and sublethal exposure to neonicotinoids

The widespread usage of neonicotinoid insecticides has sparked concern over their effects on non-target organisms. While research has largely focused on terrestrial systems, the low soil binding and high water solubility of neonicotinoids, paired with their extensive use on the landscape, puts aquatic environments at high risk for contamination via runoff events. We assessed the potential threat of these compounds to wetland communities using a combination of field surveys and experimental exposures including concentrations that are representative of what invertebrates experience in the field. In laboratory toxicity experiments, LC50 values ranged from 0.002 ppm to 1.2 ppm for aquatic invertebrates exposed to clothianidin. However, freshwater snails and amphibian larvae showed high tolerance to the chemical with no mortality observed at the highest dissolvable concentration of the insecticide. We also observed behavioral effects of clothianidin. Water bugs, Belostoma flumineum, displayed a dose-dependent reduction in feeding rate following exposure to clothianidin. Similarly, crayfish, Orconectes propinquus, exhibited reduced responsiveness to stimulus with increasing clothianidin concentration. Using a semi-natural mesocosm experiment, we manipulated clothianidin concentration (0.6, 5, and 352 ppb) and the presence of predatory invertebrates to explore community-level effects. We observed high invertebrate predator mortality with increases in clothianidin concentration. With increased predator mortality, prey survival increased by 50% at the highest clothianidin concentration. Thus, clothianidin contamination can result in a top-down trophic cascade in a community dominated by invertebrate predators. In our Indiana field study, we detected clothianidin (max = 176 ppb), imidacloprid (max = 141 ppb), and acetamiprid (max = 7 ppb) in soil samples. In water samples, we detected clothianidin (max = 0.67 ppb), imidacloprid (max = 0.18 ppb), and thiamethoxam (max = 2,568 ppb). Neonicotinoids were detected in >56% of soil samples and >90% of the water samples, which reflects a growing understanding that neonicotinoids are ubiquitous environmental contaminants. Collectively, our results underscore the need for additional research into the effects of neonicotinoids on aquatic communities and ecosystems.

Effects of Pesticide Mixtures on Host-Pathogen Dynamics of the Amphibian Chytrid Fungus.

Anthropogenic and natural stressors often interact to affect organisms. Amphibian populations are undergoing unprecedented declines and extinctions with pesticides and emerging infectious diseases implicated as causal factors. Although these factors often co-occur, their effects on amphibians are usually examined in isolation. We hypothesized that exposure of larval and metamorphic amphibians to ecologically relevant concentrations of pesticide mixtures would increase their post-metamorphic susceptibility to the fungus Batrachochytrium dendrobatidis (Bd), a pathogen that has contributed to amphibian population declines worldwide. We exposed five anuran species (Pacific treefrog, Pseudacris regilla; spring peeper, Pseudacris crucifer; Cascades frog, Rana cascadae; northern leopard frog, Lithobates pipiens; and western toad, Anaxyrus boreas) from three families to mixtures of four common insecticides (chlorpyrifos, carbaryl, permethrin, and endosulfan) or herbicides (glyphosate, acetochlor, atrazine, and 2,4-D) or a control treatment, either as tadpoles or as newly metamorphic individuals (metamorphs). Subsequently, we exposed animals to Bd or a control inoculate after metamorphosis and compared survival and Bd load. Bd exposure significantly increased mortality in Pacific treefrogs, spring peepers, and western toads, but not in Cascades frogs or northern leopard frogs. However, the effects of pesticide exposure on mortality were negligible, regardless of the timing of exposure. Bd load varied considerably across species; Pacific treefrogs, spring peepers, and western toads had the highest loads, whereas Cascades frogs and northern leopard frogs had the lowest loads. The influence of pesticide exposure on Bd load depended on the amphibian species, timing of pesticide exposure, and the particular pesticide treatment. Our results suggest that exposure to realistic pesticide concentrations has minimal effects on Bd-induced mortality, but can alter Bd load. This result could have broad implications for risk assessment of amphibians; the outcome of exposure to multiple stressors may be unpredictable and can differ between species and life stages.

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.

Linking Ecology and Epidemiology to Understand Predictors of Multi-Host Responses to an Emerging Pathogen, the Amphibian Chytrid Fungus

Variation in host responses to pathogens can have cascading effects on populations and communities when some individuals or groups of individuals display disproportionate vulnerability to infection or differ in their competence to transmit infection. The fungal pathogen, Batrachochytrium dendrobatidis (Bd) has been detected in almost 700 different amphibian species and is implicated in numerous global amphibian population declines. Identifying key hosts in the amphibian-Bd system–those who are at greatest risk or who pose the greatest risk for others–is challenging due in part to many extrinsic environmental factors driving spatiotemporal Bd distribution and context-dependent host responses to Bd in the wild. One way to improve predictive risk models and generate testable mechanistic hypotheses about vulnerability is to complement what we know about the spatial epidemiology of Bd with data collected through comparative experimental studies. We used standardized pathogen challenges to quantify amphibian survival and infection trajectories across 20 post-metamorphic North American species raised from eggs. We then incorporated trait-based models to investigate the predictive power of phylogenetic history, habitat use, and ecological and life history traits in explaining responses to Bd. True frogs (Ranidae) displayed the lowest infection intensities, whereas toads (Bufonidae) generally displayed the greatest levels of mortality after Bd exposure. Affiliation with ephemeral aquatic habitat and breadth of habitat use were strong predictors of vulnerability to and intensity of infection and several other traits including body size, lifespan, age at sexual maturity, and geographic range also appeared in top models explaining host responses to Bd. Several of the species examined are highly understudied with respect to Bd such that this study represents the first experimental susceptibility data. Combining insights gained from experimental studies with observations of landscape-level disease prevalence may help explain current and predict future pathogen dynamics in the Bd system.