Recovery and resiliency of skin microbial communities on the southern leopard frog (Lithobates sphenocephalus) following two biotic disturbances.

BACKGROUND: Microorganisms have intimate functional relationships with invertebrate and vertebrate taxa, with the potential to drastically impact health outcomes. Perturbations that affect microbial communities residing on animals can lead to dysbiosis, a change in the functional relationship, often associated with disease. Batrachochytrium dendrobatidis (Bd), a fungal pathogen of amphibians, has been responsible for catastrophic amphibian population declines around the globe. Amphibians harbor a diverse cutaneous microbiome, including some members which are known to be antagonistic to Bd (anti-Bd). Anti-Bd microorganisms facilitate the ability of some frog populations to persist in the presence of Bd, where other populations that lack anti-Bd microorganisms have declined. Research suggests disease-antagonistic properties of the microbiome may be a function of microbial community interactions, rather than individual bacterial species. Conservation efforts have identified amphibian-associated bacteria that exhibit anti-fungal properties for use as 'probiotics' on susceptible amphibian populations. Probiotic application, usually with a single bacterial species, may benefit from a greater understanding of amphibian species-specific microbiome responses to disturbances (e.g. dysbiosis vs. recovery). We assessed microbiome responses to two microbial disturbance events over multiple time points. RESULTS: Exposing Lithobates sphenocephalus (southern leopard frog) adults to the biopesticidal bacteria Bacillus thuringiensis, followed by exposure to the fungal pathogen Bd, did not have long term impacts on the microbiome. After initial shifts, microbial communities recovered and returned to a state that resembled pre-disturbance. CONCLUSIONS: Our results indicate microbial communities on L. sphenocephalus are robust and resistant to permanent shifts from some disturbances. This resiliency of microbial communities may explain why L. sphenocephalus is not experiencing the population declines from Bd that impacts many other species. Conservation efforts may benefit from studies outlining amphibian species-specific microbiome responses to disturbances (e.g. dysbiosis vs. recovery). If microbial communities on a threatened amphibian species are unlikely to recover following a disturbance, additional measures may be implemented to ameliorate the impacts of physical and chemical stressors on host-associated microbial communities.

The effects of a fungicide and chytrid fungus on anuran larvae in aquatic mesocosms.

The amphibian disease chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), has been linked to significant amphibian declines over the past three decades. The most severe effects of the pathogen have been primarily observed in relatively pristine areas that are not affected by many anthropogenic factors.One hypothesis concerning improved amphibian persistence with Bd in disturbed landscapes is that contaminants may abate the effects of Bd on amphibians. Recent laboratory studies have shown that pesticides, specifically the fungicide thiophanate-methyl (TM), can kill Bd outside of hosts and clear Bd infections within hosts. Using aquatic mesocosms, we tested the hypothesis that TM (0.43 mg/L) would alter growth and development of Lithobates sphenocephalus (southern leopard frog) tadpoles and Bd-infection loads in infected individuals. We hypothesized that the scope of such alterations and infection clearing would be affected by aquatic community variables, specifically zooplankton. TM altered zooplankton diversity (reduced cladoceran and increased copepod and ostracod abundances) and caused mortality to all tadpoles in TM-exposed tanks. In TM-free tanks, Bd-exposed tadpoles in high-density treatments metamorphosed smaller than Bd-unexposed, effects that were reversed in low-density treatments. Our study demonstrates the potential adverse effects of a fungicide and Bd on tadpoles and aquatic systems.

Batrachochytrium dendrobatidis exposure effects on foraging efficiencies and body size in anuran tadpoles.

Chytridiomycosis, the amphibian disease caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), is fatal to adults of many species. Bd is largely sublethal to amphibian larvae; however, it is known to reduce larval (i.e. tadpole) growth rates, with possible long-term effects on population dynamics and fitness. We conducted an experiment to test how Bd altered southern leopard frog Lithobates sphenocephalus tadpole mouthpart damage, percentage of food ingested, and subsequent body size. We examined our results using path analyses. We hypothesized that Bd would increase mouthpart damage, causing less food to be ingested, and ultimately reduce body size. In our model, both Bd exposure and increased mouthpart damage significantly reduced food ingested and subsequent body size. However, our study provides evidence against the long-standing hypothesis of mouthpart damage as a pathway for Bd-induced reductions in larval group. Here we provide evidence for reduced foraging efficiency (percentage of food ingested) as a mechanism for Bd-induced reductions in body size. This work highlights the importance of studying the sublethal effects of Bd on larval amphibians.

Development of antimicrobial peptide defenses of southern leopard frogs, Rana sphenocephala, against the pathogenic chytrid fungus, Batrachochytrium dendrobatidis.

Amphibian species face the growing threat of extinction due to the emerging fungal pathogen Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis. Antimicrobial peptides (AMPs) produced in granular glands of the skin are an important defense against this pathogen. Little is known about the ontogeny of AMP production or the impact of AMPs on potentially beneficial symbiotic skin bacteria. We show here that Rana (Lithobates) sphenocephala produces a mixture of four AMPs with activity against B. dendrobatidis, and we report the minimum inhibitory concentration (MIC) of synthesized replicates of these four AMPs tested against B. dendrobatidis. Using mass spectrometry and protein quantification assays, we observed that R. sphenocephala does not secrete a mature suite of AMPs until approximately 12 weeks post-metamorphosis, and geographically disparate populations produce a different suite of peptides. Use of norepinephrine to induce maximal secretion significantly reduced levels of culturable skin bacteria.

A stable niche assumption-free test of ecological divergence.

Understanding the impact of geological events on diversification processes is central to evolutionary ecology. The recent amalgamation between ecological niche models (ENMs) and phylogenetic analyses has been used to estimate historical ranges of modern lineages by projecting current ecological niches of organisms onto paleoclimatic reconstructions. A critical assumption underlying this approach is that niches are stable over time. Using Notophthalmus viridescens (eastern newt), in which four ecologically diverged subspecies are recognized, we introduce an analytical framework free from the niche stability assumption to examine how refugial retreat and subsequent postglacial expansion have affected intraspecific ecological divergence. We found that the current subspecies designation was not congruent with the phylogenetic lineages. Thus, we examined ecological niche overlap between the refugial and modern populations, in both subspecies and lineage, by creating ENMs independently for modern and estimated last glacial maximum (LGM) newt populations, extracting bioclimate variables by randomly generated points, and conducting principal component analyses. Our analyses consistently showed that when tested as a hypothesis, rather than used as an assumption, the niches of N. viridescens lineages have been unstable since the LGM (both subspecies and lineages). There was greater ecological niche differentiation among the subspecies than the modern phylogenetic lineages, suggesting that the subspecies, rather than the phylogenetic lineages, is the unit of the current ecological divergence. The present study found little evidence that the LGM refugial retreat caused the currently observed ecological divergence and suggests that ecological divergence has occurred during postglacial expansion to the current distribution ranges.

The interactive effects of chytrid fungus, pesticides, and exposure timing on gray treefrog (Hyla versicolor) larvae.

Aquatic organisms are often exposed to a wide variety of perturbations in nature, including pathogens and chemical contaminants. Despite the co-occurrence of these 2 stressors, few studies have examined the effects of chemical contaminants on host-pathogen dynamics. The authors tested the individual and combined effects on gray treefrog (Hyla versicolor) tadpoles of 2 commonly used pesticides (Roundup® and Sevin®) and the pathogenic fungus Batrachochytrium dendrobatidis (Bd). A fully factorial design was used, and tadpoles were exposed to Bd, Roundup, or Sevin alone, or a combination of Bd and either pesticide at 3 points during larval development (early, mid, late). It was predicted that pesticides would mediate the effect of Bd on tadpoles and reduce the likelihood of negative consequences of infection and that timing of exposure would influence these effects. Tadpoles exposed to Bd at the mid point experienced higher survival through metamorphosis than those exposed to Bd at the early or late points, while tadpoles exposed to Sevin at the early point experienced reduced survival compared with those exposed to Roundup or no-pesticide control at the same exposure point. Roundup ameliorated the effects of Bd on survival compared with tadpoles exposed to Bd alone, while there was no interactive effect of Sevin on survival. In addition, Sevin reduced mass of new metamorphs compared with Roundup and reduced snout-vent length compared with all other treatments. The present study supports the hypothesis that pesticides can mitigate the effects of Bd on amphibian hosts and that such effects may depend on the timing of exposure.

Mouthparts of southern leopard frog, Lithobates sphenocephalus, tadpoles not affected by exposure to a formulation of glyphosate.

This study evaluated the impacts of Roundup® on tadpole mouthpart damage as a mechanism for reduced growth and developmental rates in Lithobates sphenocephalus (Southern leopard frog) tadpoles. We did not find evidence that Roundup(®) damages larval mouthparts, nor was there a significant relationship between mouthpart damage and either body condition or developmental rate. However, the highest concentration of Roundup® significantly stunted development compared to all other treatments. Although we observed a significant effect of Roundup® on developmental rate, we conclude that mouthpart damage is likely not a mechanism for this life history response.

Re-isolating Batrachochytrium dendrobatidis from an amphibian host increases pathogenicity in a subsequent exposure.

Controlled exposure experiments can be very informative, however, they are based on the assumption that pathogens maintained on artificial media under long-term storage retain the infective and pathogenic properties of the reproducing pathogen as it occurs in a host. We observed that JEL284, an in vitro cultured and maintained isolate of Batrachochytrium dendrobatidis (Bd), was becoming less infectious with successive uses. We hypothesized that passing an isolate propagated on artificial media through an amphibian host would make the isolate more infectious and pathogenic in subsequent exposures. To test our hypothesis, we used two discreet steps, a reisolation step (step 1) and a comparative exposure step (step 2). In step 1, we exposed eastern spadefoot toads, Scaphiopus holbrooki, to JEL284 and JEL197, another isolate that had been maintained in vitro for over six years. We then re-isolated JEL284 only from a successful infection and named this new isolate JEL284(FMBa). JEL197 did not infect any amphibians and, thus, did not proceed to step 2. In step 2, we compared infectivity and pathogenicity (mortality and survival time) of JEL284 and JEL284(FMBa) by exposing 54 naïve S. holbrooki to three treatments (JEL284, JEL284(FMBa), and negative control) with 18 individuals per group. We found that JEL284(FMBa) caused higher mortality and decreased survival time in infected individuals when compared to JEL284 and negative controls. Thus, our data show that pathogenicity of Bd can decrease when cultured successively in media only and can be partially restored by passage through an amphibian host. Therefore, we have demonstrated that pathogenicity shifts can occur rapidly in this pathogen. Given the potential for shifts in pathogenicity demonstrated here, we suspect Bd to have similar potential in natural populations. We suggest that, when possible, the use of freshly isolated or cryopreserved Bd would improve the quality of controlled exposure experiments using this pathogen.

Optimal digestion theory does not predict the effect of pathogens on intestinal plasticity.

One prediction of optimal digestion theory is that organisms will increase the relative length of their digestive tracts when food resources become limited. We used theory of optimal digestion to test whether tadpoles can adjust the relative length of their intestines when challenged with the fungal pathogen Batrachochytrium dendrobatidis (Bd). The degree of tadpole mouthpart damage, a symptom of Bd infections that reduces food consumption, was associated positively with the length of tadpole intestines relative to their body size, consistent with optimal digestion theory. After controlling for mouthpart damage, tadpoles exposed to Bd had shorter intestines relative to their body size, opposite to the predictions of optimal digestion theory. One explanation of why tadpoles with higher Bd loads have shorter relative intestinal lengths is that they divert energy from maintaining intestinal and overall growth towards anti-parasite defences.

Previous exposure of predatory fish to a pesticide alters palatability of larval amphibian prey.

Habitat preferences of organisms are reliant on a variety of factors. For amphibians specifically, preferences can depend on factors such as food availability, water quality, and the presence of potential predators. Because some amphibians breed in permanent bodies of water (e.g., ponds), the threat of predation (e.g., from fish) is constant. Thus, some amphibians are unpalatable to many predators, allowing them to coexist in the same habitats. However, the addition of anthropogenic stressors (i.e., pesticides) may alter the perceived palatability of prey items to predators. The authors tested the hypothesis that bluegill fish (Lepomis macrochirus), previously exposed to the pesticide carbaryl, would consume more unpalatable prey (Fowler's toad [Anaxyrus fowleri] tadpoles) than unexposed predators. Carbaryl is a pesticide that attacks the nervous system and is linked to taste sense in organisms. Moreover, the authors conducted an identical test using palatable prey (gray treefrog [Hyla versicolor] tadpoles) and predicted that no change in preference would be observed. In support of the primary hypothesis, bluegill exposed to the highest concentration of carbaryl consumed more A. fowleri tadpoles compared with those exposed to carbaryl at the lowest concentration or water control. Moreover, an effect of carbaryl on predation success on H. versicolor tadpoles was not observed. The present study shows that an anthropogenic stressor (carbaryl) can alter the perceived palatability of noxious prey to fish predators, potentially altering predator-prey relationships in natural settings.

Unlikely remedy: fungicide clears infection from pathogenic fungus in larval southern leopard frogs (Lithobates sphenocephalus).

Amphibians are often exposed to a wide variety of perturbations. Two of these, pesticides and pathogens, are linked to declines in both amphibian health and population viability. Many studies have examined the separate effects of such perturbations; however, few have examined the effects of simultaneous exposure of both to amphibians. In this study, we exposed larval southern leopard frog tadpoles (Lithobates sphenocephalus) to the chytrid fungus Batrachochytrium dendrobatidis and the fungicide thiophanate-methyl (TM) at 0.6 mg/L under laboratory conditions. The experiment was continued until all larvae completed metamorphosis or died. Overall, TM facilitated increases in tadpole mass and length. Additionally, individuals exposed to both TM and Bd were heavier and larger, compared to all other treatments. TM also cleared Bd in infected larvae. We conclude that TM affects larval anurans to facilitate growth and development while clearing Bd infection. Our findings highlight the need for more research into multiple perturbations, specifically pesticides and disease, to further promote amphibian heath.

The impact of pesticides on the pathogen Batrachochytrium dendrobatidis independent of potential hosts.

Amphibians around the world are experiencing the greatest organismal decline in recent history. Xenobiotics, such as pesticides, and pathogenic biotic perturbations, including the fungus Batrachochytrium dendrobatidis (Bd), have played major roles in amphibian decreases. We conducted laboratory culture studies to determine the effects of three pesticides {carbaryl, glyphosate, and thiophanate-methyl [TM; Topsin-M(R) (Cerexagri-Nisso LLC)]} on Bd zoospore production and zoosporangia growth. We applied Bd to pesticides mixed in an agar culture to simulate pathogen introduction to a system with pre-existing pesticides (Bd addition). Alternatively, pesticides were applied to pre-established Bd to simulate pesticide introduction after Bd establishment (pesticide addition). We then measured Bd zoosporangia and zoospore production. All pesticides significantly inhibited zoospore production; however, glyphosate and TM were more effective at doing so than carbaryl. In addition, only carbaryl and glyphosate inhibited zoosporangia production. Our data suggest that carbaryl and glyphosate are equally effective at inhibiting both zoosporangia and zoospore production; however, TM is selectively toxic to zoospores but not zoosporangia. One possible explanation for this observation could be that TM is toxic to zoospores but not the protective zoosporangia. In the case of pesticides applied to established Bd cultures, all pesticides caused significant mortality in both zoosporangia and zoospores, and no differences were found among pesticides. We conclude that examining pesticide and pathogen interactions independent of hosts provides mechanistic understanding of such interactions before and after host infection or contamination.

Dietary protein restriction impairs growth, immunity, and disease resistance in southern leopard frog tadpoles.

The immune system is a necessary, but potentially costly, defense against infectious diseases. When nutrition is limited, immune activity may consume a significant amount of an organism's energy budget. Levels of dietary protein affect immune system function; high levels can enhance disease resistance. We exposed southern leopard frog [Lithobates sphenocephalus (=Rana sphenocephala)] tadpoles to high and low protein diets crossed with the presence or absence of the pathogenic amphibian chytrid fungus (Batrachochytrium dendrobatidis; Bd) and quantified: (1) tadpole resistance to Bd; (2) tadpole skin-swelling in response to phytohaemagglutinin (PHA) injection (a measure of the T cell-mediated response of the immune system); (3) bacterial killing ability (BKA) of tadpole blood (a measure of the complement-mediated cytotoxicity of the innate immune system); and (4) tadpole growth and development. Tadpoles raised on a low-protein diet were smaller and less developed than tadpoles on a high-protein diet. When controlled for developmental stage, tadpoles raised on a low-protein diet had reduced PHA and BKA responses relative to tadpoles on a high-protein diet, but these immune responses were independent of Bd exposure. High dietary protein significantly increased resistance to Bd. Our results support the general hypothesis that host condition can strongly affect disease resistance; in particular, fluctuations in dietary protein availability may change how diseases affect populations in the field.

Can differences in host behavior drive patterns of disease prevalence in tadpoles?

Differences in host behavior and resistance to disease can influence the outcome of host-pathogen interactions. We capitalized on the variation in aggregation behavior of Fowler's toads (Anaxyrus [ = Bufo] fowleri) and grey treefrogs (Hyla versicolor) tadpoles and tested for differences in transmission of Batrachochytrium dendrobatidis (Bd) and host-specific fitness consequences (i.e., life history traits that imply fitness) of infection in single-species amphibian mesocosms. On average, A. fowleri mesocosms supported higher Bd prevalences and infection intensities relative to H. versicolor mesocosms. Higher Bd prevalence in A. fowleri mesocosms may result, in part, from higher intraspecific transmission due to the aggregation of tadpoles raised in Bd treatments. We also found that, independent of species, tadpoles raised in the presence of Bd were smaller and less developed than tadpoles raised in disease-free conditions. Our results indicate that aggregation behavior might increase Bd prevalence and that A. fowleri tadpoles carry heavier infections relative to H. versicolor tadpoles. However, our results demonstrate that Bd appears to negatively impact larval growth and developmental rates of A. fowleri and H. versicolor similarly, even in the absence of high Bd prevalence.

Fungal pathogen changes the feeding kinematics of larval anurans.

Pathogens can alter host life-history traits by affecting host feeding activities. In anuran tadpoles, keratinized mouthparts (teeth and jaw sheaths) are essential for feeding. Batrachochytrium dendrobatidis ( Bd ) is a pathogenic fungus of amphibians that can infect these mouthparts and reduce tadpole survival. However, the precise way that Bd-induced changes in tadpole mouthparts impact tadpole feeding is unknown. We use high-speed (500 frames/sec) videography to study how Bd-induced mouthpart deformities affect the feeding kinematics of Fowler's toad (Anaxyrus [= Bufo] fowleri ) and grey tree frog (Hyla chrysoscelis) tadpoles. We tested for species-specific patterns of Bd-induced mouthpart deformities to assess how deformations to specific areas of tadpole mouthparts alter feeding kinematics. The teeth of tadpoles from the Bd-exposed treatment slipped off of surfaces on which tadpoles graze and were in contact with an algal-covered substratum for a shorter duration in each gape cycle compared to teeth of control tadpoles. We also found that the jaw sheaths had significantly more deformations than labial teeth; however, how this relates to feeding kinematics is unclear. Our data show explicitly how Bd infection reduces foraging efficiency of anuran tadpoles by altering feeding kinematics and elucidate a mechanistic link between the pathogen infection and reduced host fitness.

How does a change in labial tooth row number affect feeding kinematics and foraging performance of a ranid tadpole (Lithobates sphenocephalus)?

Recent studies have explored feeding kinematics in tadpoles with intact labial teeth; however, it is unknown how missing teeth impacts foraging. We explored the impact of missing labial teeth on the feeding mechanics and foraging performance of Southern leopard frog (Lithobates sphenocephalus [= Rana sphenocephala]) tadpoles by controlling the pattern of labial tooth loss; that is, by surgically removing one row of labial teeth. We then used high-speed (500 frames/second) videography to test the hypothesis that tooth loss reduces the time that tadpoles attach to and graze upon an algal-covered substrate. We next conducted trials of foraging efficiency and foraging activity to test the hypothesis that tadpoles with fewer teeth forage less effectively than control tadpoles. The teeth of tadpoles from the surgery treatment slipped while closing and were in contact with an algal-covered substrate for a shorter duration compared to control tadpoles. Surprisingly, tadpoles with missing labial teeth obtained similar amounts of food and were as active as tadpoles with intact mouthparts. However, tadpoles with missing teeth completed about 25% more gape cycles per unit time than control tadpoles. Our data suggest that tadpoles with missing teeth compensate for inferior feeding kinematics during mouth closing in each gape cycle by increasing the number of gape cycles per unit time.

Impacts of Batrachochytrium dendrobatidis infection on tadpole foraging performance.

Pathogen-induced modifications in host behavior, including alterations in foraging behavior or foraging efficiency, can compromise host fitness by reducing growth and development. Chytridiomycosis is an infectious disease of amphibians caused by the fungus Batrachochytrium dendrobatidis (Bd), and it has played an important role in the worldwide decline of amphibians. In larval anurans, Bd infections commonly result in reduced developmental rates, however, the mechanism(s) responsible are untested. We conducted laboratory experiments to test whether Bd infections reduced foraging performance of Grey Treefrog (Hyla chrysoscelis) and Fowler's Toad (Anaxyrus [= Bufo] fowleri) tadpoles. In the first experiment, we observed foraging behavior of Bd-infected and uninfected tadpoles to test for differences in foraging activity. In a second experiment, we tested for differences in the ingestion rates of tadpoles by examining the amount of food in their alimentary track after a 3-hour foraging period. We hypothesized that Bd-infected tadpoles would forage less often and less efficiently than uninfected tadpoles. As predicted, Bd-infected larvae forage less often and were less efficient at obtaining food than uninfected larvae. Our results show that Bd infections reduce foraging efficiency in Anaxyrus and Hyla tadpoles, and that Bd differentially affects foraging behavior in these species. Thus, our results provide a potential mechanism of decreased developmental rates of Bd-infected tadpoles.

Life cycle polyphenism as a factor affecting ecological divergence within Notophthalmus viridescens.

Polyphenism, which allows a single genotype to express multiple discrete phenotypes in response to environmental cues, is an adaptive trait in heterogeneous environments. Pond hydroperiod is an important ecological parameter affecting amphibian life history, and variation in local pond hydrology has been hypothesized to play a role in species divergence via changes in polyphenism. The eastern newt (Notophthalmus viridescens) expresses life cycle polyphenism. Larvae develop along three possible pathways: metamorphosis to aquatic lunged adult via a terrestrial juvenile (eft) stage, metamorphosis directly to an aquatic lunged adult, or maturation directly to an aquatic gilled adult without metamorphosis (i.e., paedomorphosis). Subspecies of N. viridescens vary in their polyphenic patterns, suggesting possible adaptation to different environments. However, no studies have experimentally tested how genetic and environmental components contribute to the observed differences among subspecies and whether such differences may facilitate divergence. We tested whether adaptation to local pond hydrology via polyphenic changes existed among subspecies by rearing larvae of three subspecies (N. v. dorsalis, N. v. louisianensis, and N. v. viridescens) along three hydroperiod regimes (short, long, and constant) in outdoor artificial ponds. We found that larval N. v. viridescens obligately metamorphosed to efts under all hydroperiods, whereas N. v. dorsalis and N. v. louisianensis exhibited plasticity: larvae metamorphosed to efts under drying conditions but metamorphosed directly to aquatic adults or became paedomorphic in constant water. Also, N. v. viridescens metamorphosed to efts faster and at a smaller body size than the other two subspecies. These data suggest that subspecies of N. viridescens are adapted to different pond hydroperiods, supporting the potential for polyphenism to facilitate divergence. Canalizing selection for certain alternative phenotypes within a single species in which other populations remain plastic may play an important role in the initiation of ecological divergence.

Phylogenetic concordance analysis shows an emerging pathogen is novel and endemic.

Distinguishing whether pathogens are novel or endemic is critical for controlling emerging infectious diseases, an increasing threat to wildlife and human health. To test the endemic vs. novel pathogen hypothesis, we present a unique analysis of intraspecific host-pathogen phylogenetic concordance of tiger salamanders and an emerging Ranavirus throughout Western North America. There is significant non-concordance of host and virus gene trees, suggesting pathogen novelty. However, non-concordance has likely resulted from virus introductions by human movement of infected salamanders. When human-associated viral introductions are excluded, host and virus gene trees are identical, strongly supporting coevolution and endemism. A laboratory experiment showed an introduced virus strain is significantly more virulent than endemic strains, likely due to artificial selection for high virulence. Thus, our analysis of intraspecific phylogenetic concordance revealed that human introduction of viruses is the mechanism underlying tree non-concordance and possibly disease emergence via artificial selection.

Chytridiomycosis impacts predator-prey interactions in larval amphibian communities.

Despite ecologists increasingly recognizing pathogens as playing significant roles in community dynamics, few experimental studies have quantified patterns of disease impacts on natural systems. Amphibians are experiencing population declines, and a fungal pathogen ( Batrachochytrium dendrobatidis; Chytridiomycota) is a suspected causal agent in many declines. We studied the effects of a pathogenic fungus on community interactions between the gray treefrog, Hyla chrysoscelis, and eastern newts, Notophthalmus viridescens. Recent studies have characterized chytridiomycosis as an emerging infectious disease, whose suspected rapid range expansion and widespread occurrence pose a significant risk for amphibian populations worldwide. We reared larvae in outdoor polyethylene experimental tanks and tested the effects of initial larval density, predator presence, and fungal exposure on Hyla recruitment and predator-prey interactions between Hyla and Notophthalmus. Newts reduced treefrog survival, and high intraspecific density decreased metamorphic body mass independent of B. dendrobatidis. The presence of fungi reduced treefrog body mass at metamorphosis by 34%, but had no significant main effect on survival or larval period length. B. dendrobatidis differentially affected larval development in the presence of predators; Hyla developed slower when reared with the pathogen, but only when newts were present. This significant predator-by-pathogen interaction suggests that the impact of chytridiomycosis on larval amphibians may be exacerbated in complex communities. Our data suggest that B. dendrobatidis effects on host life history may be complex and indirect. Direct measurements of the community-level effects of pathogens offer an important opportunity to understand a significant threat to global biodiversity-declining amphibian populations.