Exposure to Amphibian Chytrid Fungus Alters Terrestrial Growth and Feeding Rate in Metamorphic Anurans

Infectious diseases are increasing in frequency and severity for wildlife, livestock, and humans (Cunningham et al., 2017) and are responsible for putting hundreds of species of plants and animals at risk for population declines or extinction (Smith et al., 2006; Scheele et al., 2019; Lambert et al., 2020). Wildlife diseases alter population dynamics through increased host mortality, as well as sublethal changes in host condition, such as reduced growth or fecundity, and behavioral changes (including altered breeding, anti-predator, and feeding behaviors; Marra et al., 2004; Parris et al., 2006; Tompkins et al., 2011; Gahl et al., 2011). Yet, how species are influenced by the sublethal effects of infectious diseases is poorly understood, despite their capacity to influence population and community interactions.

Amphibians represent 75% of species for which infectious disease is hypothesized to be a contributing factor in population declines and are the most at-risk vertebrate group (Smith et al., 2006; Scheele et al., 2019; Lambert et al., 2020). The emerging amphibian pathogen, Batrachochytrium dendrobatidis (Bd), is linked to global declines and extinctions of amphibians and impacts the host in a multitude of ways, including sublethal changes in behavior (Daszak et al., 2003; Parris and Beaudoin, 2004; Retallick and Miera, 2007; Doody et al., 2019). In populations where anurans do not appear to be experiencing high levels of mortality associated with Bd, sublethal responses may still have population-level effects. Reduced anuran body size in response to Bd exposure could influence both overwintering survival and fecundity (Fitzpatrick, 1976; Garner et al., 2011; Rumschlag and Boone, 2018; Wetsch et al., 2022). While overwintering, amphibians rely on energy reserves to survive near-freezing temperatures (Fitzpatrick, 1976; Kristin and Gvozdik, 2014); a reduction in these energy reserves could put individuals at greater risk of mortality (Rumschlag and Boone, 2018). For most amphibian species, body mass positively correlates with breeding success and fecundity, such that if infection leads to smaller body size, individuals may experience an overall loss in fitness (Kuramoto, 1978).

Bd exposure and infection can result in reduced growth (Caseltine et al., 2016; Burrow et al., 2017; Wetsch et al., 2022), which could be a consequence of increased metabolic costs associated with disease development or from altered feeding behaviors (Gahl et al., 2011; Wise et al., 2014; Rumschlag and Boone, 2015; Grogan et al., 2018). Although few studies have examined the effect of this pathogen on foraging behavior in anurans post metamorphosis, some evidence suggests infection with Bd reduces live food consumption in some anuran species (Searle et al., 2011). Regardless of the mechanism, the impact of reduced growth has long-lasting implications for individual fitness and requires further study. As such, it is critical to evaluate the drivers associated with reduced terrestrial growth in species that frequently contract Bd and experience sublethal effects.

The central objective of this study was to investigate whether terrestrial feeding behavior is affected by exposure to Bd, particularly as feeding behavior may pertain to decreased terrestrial growth of infected individuals. Furthermore, this study aimed to evaluate the effect of prey refugia on these behaviors because in previous studies live prey concentrated in refugia (McQuigg, pers. obs.) potentially increasing effort and energetic costs of feeding behavior. To achieve this goal, we evaluated the feeding efficiency of anurans exposed or not to Bd in two species, American Toads (Anaxyrus americanus) and Northern Leopard Frogs (Lithobates pipiens), when their prey did or did not have access to refugia. American Toads, which emerge at a relatively small body size at metamorphosis, are susceptible to Bd in laboratory studies, exhibiting high levels of mortality and reduced terrestrial growth (Wise et al., 2014; Burrow et al., 2017). Northern Leopard Frogs, which are relatively large at metamorphosis, show a range of responses to this pathogen, from increased skin shedding without decreased growth or survival (Paetow et al., 2012), to Bd-attributed terrestrial growth reduction (Caseltine et al., 2016). We predicted that Bd-exposed amphibians would 1) grow less than unexposed amphibians, 2) be less efficient at obtaining prey in general, and 3) be less efficient at capturing prey when prey refugia (i.e., cover objects) are available.

We collected nine partial Northern Leopard Frog egg masses from Talawanda High School Restoration Wetland in Butler County, Ohio on 21 March 2017. A minimum of 12 partial egg strands from American Toads were collected from Rush Run Wildlife Area in Preble County, Ohio on 6 April 2017. We housed eggs in a temperature-controlled room at 23°C with a 12 : 12 h light : dark cycle in single-species aquaria until hatching. Upon hatching, we fed tadpoles ground TetraMin tropical fish flakes (Tetra Holding) ad libitum until they were free-swimming (Gosner stage 25; Gosner, 1960). Tadpoles from all aquaria were mixed within species and transferred to outdoor mesocosm ponds. We stocked six mesocosms with 1,000 L water (18 Feb 2017), 1 kg leaf litter (19 Feb 2017), inoculations of plankton (20, 22, 24 Feb 2017), and 30 tadpoles from each species for a total of 60 (Northern Leopard Frogs: 30 March 2017, American Toads: 13 April 2017).

We reared tadpoles in mesocosms until metamorphosis, defined as emergence of at least one forelimb (Gosner stage 42; Gosner, 1960). Mass and time to metamorphosis was measured for each individual upon tail resorption and 40 total individuals of each species were randomly assigned to treatments and placed directly into individual terrestrial rearing conditions. Metamorphs were individually housed in 2-L terraria beakers (193 mm tall × 131 mm diameter) stocked with ∼3 cm pea gravel, ∼6 cm topsoil, and a 60 mm diameter circular water dish; we covered each beaker with a fiberglass screen lid to prevent escape. Each day, we determined survival and sprayed terraria with dechlorinated water. Terraria were housed in a temperature-controlled room at 23°C with a 14 : 10 h light : dark cycle for the duration of the study.

Prior to feeding trials, metamorphs were fed live crickets dusted in calcium and vitamin D₃ powder (ZooMed Repti Calcium) equivalent to 10% of the average mass per species, rounding up to the nearest cricket, three times weekly. American Toads received 3.2 mm crickets and Northern Leopard Frogs received 6.4 mm crickets for the duration of the experiment, because of differences in metamorph size. Feeding regime varied during feeding trials as described below. Anurans were weighed once weekly for the duration of the experiment, which began with Bd exposure and addition of refugia on 22 June 2017 (experimental day 0). In this study we manipulated Bd exposure and presence of refugia; to summarize, we had 2 Bd exposures (present or absent) × 2 prey refugia (present or absent) × 10 replicates = 40 individuals per species.

Approximately 1 wk after metamorphosis, on 22 June 2017, we recorded mass for all individuals (Table 1) and determined via ANOVA that there was no statistically significant difference between the starting mass of individuals assigned to each treatment (American Toads: P = 0.662; Northern Leopard Frogs: P = 0.433). We exposed 20 metamorphs of each species to a solution with a total of 1 × 10⁵Bd zoospores of isolate JSOH 01 (Toledo, Ohio, USA, received from J. Longcore; Longcore et al., 1999), made by flooding inoculated 10% tryptone agarose plates with dechlorinated water for 30 min, and 20 metamorphs of each species were exposed to a Bd-free solution made by flooding uninoculated agarose plates with dechlorinated water for 30 min. Anurans were housed for 12 h in ventilated petri dishes with 1 ml of zoospore solution containing 1 × 10⁵ or zero zoospores, and 7 ml of dechlorinated water using techniques described in Rumschlag and Boone (2018).

We conducted short- and long-term feeding trials to assess the effect of Bd exposure and prey refugia on multiple aspects of metamorph feeding behavior. Short-term feeding trials were conducted to assess immediate changes in prey consumption, and long-term feeding trials were conducted in terraria to assess changes in feeding behavior and prey consumption over time. All individuals in the study were used in the short-term feeding trial on day 8 and then placed in terraria for the long-term feeding trial.

Short-term feeding trials occurred over a 12-h period 8 d post exposure. We placed individuals in a ventilated plastic container (30 × 21 × 27 cm) lined with moistened white paper towels. For trials with prey refugia, a 60-mm plastic petri dish was elevated on 12 mm tall cardstock supports. At 1100, we added 26 3.2-mm crickets to each container containing an American Toad and 18 6.4-mm crickets to each container containing a Northern Leopard Frog. Crickets equated to 30% of the average mass for each species. After 12 h (2300), we removed anurans and counted the number of crickets remaining in each container to determine how many crickets were consumed by each animal.

Long-term feeding trials occurred over a 5-wk period beginning 11 d post exposure in beaker terraria. For trials lacking a cover object for prey, a 60-mm petri dish containing water was inserted in the soil so that the top of the dish was flush with the soil surface, prohibiting crickets from retreating beneath the dish. For trials containing a cover object, a 60-mm petri dish containing water was placed on top of the soil elevated by four rocks, ∼1 cm tall, with the ability for crickets to retreat below the petri dish.

Crickets equivalent to 15% of the mean mass of each frog species, a quantity chosen to ensure frogs were not food limited, were counted and added to individual terraria three times weekly for the duration of the study (14 feedings total). At each feeding, we recorded all remaining live crickets in each terraria and deducted that from the number added at feeding. Two days following the final feeding, we counted all remaining crickets in each terraria. Throughout the experiment, cricket size was maintained at 3.2 mm for American Toads and 6.4 mm for Northern Leopard Frogs and number of crickets was scaled with mass. This ensured that crickets never exceeded the gape limit of the smallest anurans in the study.

All data analyses were conducted in R version 3.6.1. All mass and long-term feeding data were analyzed by species using a repeated measures ANOVA model with animal ID as a random factor (R Core Team, 2015). Three unexposed Northern Leopard Frogs from the no-prey-refugia treatment died prior to the end of the experiment and were excluded from mass and long-term feeding analyses. Because all mass data were nonnormally distributed, they were log transformed to achieve normality. To analyze the effect of treatment on mass over time for each species, prey refugia presence, Bd exposure status, and their interaction were evaluated with a repeated-measure ANOVA. Long-term feeding was analyzed using an ANOVA model with proportion of crickets consumed as the response variable, and time, prey refugia presence, Bd exposure status, and all interactions therein as predictors. Prior to analysis, all long-term feeding data were transformed using a constant deduction from the number of crickets consumed (used to calculate proportion consumed) equivalent to one half of one cricket for the largest number of crickets fed. For American Toads, this equated to a standard deduction of 0.015 and for Northern Leopard Frogs the standard deduction was 0.02. This deduction was used to induce greater variance in the data by eliminating 100% proportions. Following this deduction, data were logit transformed following the suggestions of Warton and Hui (2011). The number of crickets consumed in the short-term feeding trials was analyzed using a Generalized Linear Model (GLM) Poisson regression with a log link function. We report all means ± SE.

All American Toads survived for the duration of the study. Three unexposed Northern Leopard Frogs from the no-prey-refugia treatment died prior to the end of the experiment; all others survived. American Toads and Northern Leopard Frogs exposed to Bd were smaller (American Toads: mean = 1.45 g ± 0.05 SE; Northern Leopard Frogs: 4.30 ± 0.10 g) than unexposed individuals (American Toads: 1.78 ± 0.06 g; Northern Leopard Frogs: 4.64 ± 0.15 g) by the end of the study, an effect that emerged over time for both species (Figure 1; Table 2). The effect of Bd on mass of American Toads was also affected by presence of prey refugia, whereby the largest toads by the end of the study were not exposed to Bd and had no refugia present (1.9 ± 0.30 g). In contrast, the presence of prey refugia did not significantly affect Northern Leopard Frog mass (Table 2).

In short-term feeding trials, American Toads ate 51.1 ± 2.2% of crickets and Northern Leopard Frogs ate 85.6 ± 2.3%. Neither American Toad (GLM with Poisson distribution: Z = −1.234, P = 0.217) nor Northern Leopard Frog (GLM with Poisson distribution: Z = −1.428, P = 0.153) feeding behavior was affected by exposure to Bd. Short-term feeding was also not affected by prey refugia presence (American Toad GLM with Poisson distribution: Z = 0.000, P = 1.000; Northern Leopard Frog GLM with Poisson distribution: Z = −1.143, P = 0.253) or an interaction between prey refugia and Bd exposure in either species (American Toad GLM with Poisson distribution: Z = 0.323, P = 0.746; Northern Leopard Frog GLM with Poisson distribution: Z = 1.420, P = 0.156).

In long-term feeding trials, American Toads were more strongly affected by treatment combinations than Northern Leopard Frogs. Overall, American Toad feeding behavior was affected by the interaction of prey refugia and Bd that was consistent over time (Table 3). Generally, toads without refugia ate the most crickets (Bd, no refugia: 98.0 ± 0.7%; no Bd, no refugia: 98.8 ± 0.4%), and when refugia were present, toads that were not exposed to Bd ate the fewest crickets overall (Bd, refugia: 95.5 ± 0.6%; no Bd, no refugia: 90.2 ± 0.9%; Fig. 2A; Table 3). In Northern Leopard Frogs, prey refugia presence had an effect on proportion of crickets consumed (no refugia: 99.9 ± 0.6%, refugia 99.4 ± 2.7%), but Bd exposure and time had no effect (Fig. 2B; Table 3). This effect, however, should be interpreted with caution because there was very little variation in the data and nearly 100% of crickets were consistently consumed by Northern Leopard Frogs regardless of treatment.

Many amphibian populations co-exist with Bd without apparent population declines (Lam et al., 2010). Yet, knowledge of how sublethal infections may influence individual responses and fitness remains limited. Sublethal effects include reduced growth (e.g., Caseltine et al., 2016; Burrow et al., 2017; Wetsch et al., 2022), which may have population-level consequences resulting in reduced fecundity and/or overwinter survival for temperate and arctic amphibians. Although we did not confirm infection using pathogen diagnostics (thus rendering the infection status of animals unknown), our study indicates exposure to Bd reduced terrestrial growth in Northern Leopard Frogs and American Toads. These sublethal effects occurred across all Bd-exposed treatments even though feeding effort was equal to or greater than feeding effort in unexposed anurans. Toads in particular were more likely to increase feeding activity when they were exposed to Bd while Northern Leopard Frog feeding did not change, yet in both cases Bd-exposed frogs were the smallest by the end of the study. Our results suggest that for both American Toads and Northern Leopard Frogs, Bd's negative impact on growth is likely a result of the high metabolic costs of disease.

Some studies suggest that exposure to pathogens can result in changes in feeding behavior of animals by causing morphological changes that influence time and efficiency of feeding, by changing activity levels of hosts, or by altering metabolic needs of hosts (Hart, 1990; Venesky et al., 2009). The present study was designed to evaluate if feeding activity was reduced by Bd exposure, which would have resulted in reduced growth and fewer crickets eaten, or by increased metabolic demands, which would have resulted in reduced growth and more or equal crickets eaten. Among juvenile and adult anurans, exposure to Bd can result in a skin infection triggering an immune response, including the release of skin antimicrobial peptides and other innate responses (Woodhams et al., 2007), which would increase metabolic costs of infection. The extent of these responses varies among species and is still the subject of much research, but while many immune responses in ectothermic animals are thermally dependent, they are still metabolically costly and confer added energy demands in exposed individuals (Moretti et al., 2019).

American Toads and Northern Leopard Frogs exposed to Bd experienced decreased terrestrial growth over the duration of this experiment, similar to other recent studies (Gahl et al., 2011; Wise et al., 2014; Casteltine et al., 2016). The results of the current study suggest the growth differential, at least in American Toads and likely in Northern Leopard Frogs, is the result of increased metabolic needs, rather than a decrease in feeding rate. Unexposed individuals that faced the challenge of finding prey when a prey refuge was present ate less than other groups. In contrast, exposed individuals were smaller on average than all unexposed individuals, despite Bd-exposed animals with or without prey refugia eating more prey than unexposed animals with prey refugia. This suggests that exposed American Toads experienced heightened metabolic needs, for which they attempted to compensate by obtaining food even when prey could retreat. Northern Leopard Frogs, however, ate less when refugia were present with no differences among Bd treatments; yet, only Bd exposure influenced Northern Leopard Frog mass. This result indicated that while apparent appetite or feeding success did not change with pathogen exposure, growth was negatively impacted by Bd, suggesting a metabolic cost of pathogen exposure.

We did not find any effects of Bd exposure or presence of prey refugia on short-term feeding in either species. This is consistent with other feeding trials that observe feeding over a single feeding event (Webber et al., 2010) and suggests that observing feeding over multiple feeding events or longer periods of time may be more informative than single, short trials. In our short-term trial, which took place over 12 h, American Toads ate 51.1 ± 2.2% of crickets and Northern Leopard Frogs consumed 85.6 ± 2.3%, but during the long-term feeding trial they consumed 95.6 ± 0.3% and 99.7 ± 0.08% on average between feeding dates, respectively. This suggests that both species, but American Toads in particular, may feed over longer periods of time and our short-term trial was not long enough to observe effects in feeding behavior.

Exposure to pathogens can result in a number of responses from hosts across endo- and ectothermic taxa, including metabolically costly immune responses, often resulting in trade-offs between pathogen resistance and life history end points such as growth and fecundity (Bonneaud et al., 2003; Romanyukha et al., 2006; Agugliaro et al., 2019). A recent study found that 28% of adult American Toads surveyed were positive for Bd infection (Rumschlag and Boone, 2020), which suggests that populations may be at heightened risk of indirect mortality or decreased fecundity associated with heightened metabolic needs. In our study, exposed American Toads exhibited increased foraging effort over unexposed individuals, which may put them at greater risk of indirect mortality through desiccation or predation. Foraging behavior is driven by a cost-benefit analysis including factors like predation risk, risk of encountering or staying in an unfavorable microhabitat, food availability, nutritional needs, and energy density of food (Brown and Kotler, 2004; Zollner and Lima, 2004; Verdolin, 2006). These foraging trade-offs may be even more complex because many animals change foraging behavior when faced with unfavorable microhabitats, such as areas where predators or environmental variables increase chances of mortality, thereby obtaining less energy than is necessary to meet their metabolic needs (Brown and Kolter, 2004; Zollner and Lima, 2004; Verdolin, 2006). Furthermore, simply being in motion, as is necessary to forage for food, makes frogs more likely to be captured by predators (Heinen and Hammond, 1997) and has metabolic costs. These combined factors suggest that changes in feeding behavior such as those observed here may result in an increased risk of mortality over time.

Reduced access to or availability of prey could disproportionately affect pathogen-exposed individuals, which must obtain more prey to meet metabolic needs. Many invertebrate populations, which act as prey for amphibians, are experiencing population declines (Thomas, 2005). Changes in prey abundance in the wild paired with potential changes in metabolic demand for exposed individuals could exacerbate the growth effects we found, making exposed individuals more vulnerable to reduced fecundity or death in terrestrial life stages, including during overwintering (Fitzpatrick, 1976; Garner et al., 2011; Rumschlag and Boone, 2018; Wetsch et al., 2022).

The effects of Bd in regions where it is present in amphibian populations yet not associated with population declines are poorly understood because most research focuses on decline-prone species and geographic regions. This study illustrates the complexity of interactions between Bd, its hosts, and host prey, and suggests that changes in prey availability could have profound impacts on individuals exposed to Bd. In amphibians with sublethal responses to Bd during terrestrial life stages, abundance of suitable invertebrate prey could be associated with growth and, ultimately, survival and fecundity. This effect is particularly poignant as more invertebrate populations are experiencing population declines that could impact their availability to amphibian predators (Thomas, 2005). Evidence that Bd can increase metabolic costs, even when foraging increases, suggests that the sublethal impacts of pathogen exposure could have serious negative consequences for infected individuals and populations, particularly when exposure occurs early in development.

We thank the Society for the Study of Amphibians and Reptiles and the Miami University Undergraduate Summer Scholars program for funding this project. We thank past and present members of the Boone Amphibian Conservation Lab, M. Strasburg, M. Murphy, C. Dvorsky, O. Wetsch, A. Saul, and F. Lopez, for assistance in the field and lab. We acknowledge J. Fruth and A. Rypstra of the Miami University Ecology Research Center for services and assistance during larval rearing and the Miami University Department of Statistics for statistical analysis advice. This research was conducted with collection permission from the Ohio Division of Wildlife (permit 20-177) and the Miami University Institutional Animal Care and Use Committee approval (IACUC 827).