Effect of Simultaneous Amphibian Exposure to Pesticides and an Emerging Fungal Pathogen, Batrachochytrium dendrobatidis.

Amphibian declines have been linked to numerous factors, including pesticide use and the fungal pathogen Batrachochytrium dendrobatidis (Bd). Moreover, research has suggested a link between amphibian sensitivity to Bd and pesticide exposure. We simultaneously exposed postmetamorphic American toads (Anaxyrus americanus), western toads (A. boreas), spring peepers (Pseudacris crucifer), Pacific treefrogs (P. regilla), leopard frogs (Lithobates pipiens), and Cascades frogs (Rana cascadae) to a factorial combination of two pathogen treatments (Bd+, Bd-) and four pesticide treatments (control, ethanol vehicle, herbicide mixture, and insecticide mixture) for 14 d to quantify survival and infection load. We found no interactive effects of pesticides and Bd on anuran survival and no effects of pesticides on infection load. Mortality following Bd exposure increased in spring peepers and American toads and was dependent upon snout-vent length in western toads, American toads, and Pacific treefrogs. Previous studies reported effects of early sublethal pesticide exposure on amphibian Bd sensitivity and infection load at later life stages, but we found simultaneous exposure to sublethal pesticide concentrations and Bd had no such effect on postmetamorphic juvenile anurans. Future research investigating complex interactions between pesticides and Bd should employ a variety of pesticide formulations and Bd strains and follow the effects of exposure throughout ontogeny.

Virulence variation among strains of the emerging infectious fungus Batrachochytrium dendrobatidis (Bd) in multiple amphibian host species.

Emerging infectious diseases have been documented in numerous plant and animal populations. The infectious disease amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is associated with global amphibian population declines. While much Bd-amphibian research has centered on response variation in hosts, a paucity of information exists on how variation in the pathogen, such as strain differences, affects infection dynamics. To examine how different Bd strains may differentially impact multiple hosts, we conducted laboratory experiments to measure 2 infection outcomes, viz. host survival and pathogen load, in 3 amphibian host species (Pacific treefrog, western toad, and Cascades frog) after exposure to 3 different Bd strains (an additional fourth Bd strain was tested in toads only). Our results confirm that the infection response differs among host species. Western toads experienced significant mortality, but Pacific treefrogs and Cascades frogs did not. Interestingly, our experiment also captured strain-dependent virulence variation but only in 1 host species, the western toad. Increased mortality was observed in 2 of the 4 Bd strains tested in this host species. Toads were also the only host species found to have variable pathogen load dependent on strain type; individuals exposed to the Panama strain harbored significantly higher loads compared to all other strains. These findings underscore the dynamic nature of Bd infection, showing that virulence can vary contingent on host and strain type. We highlight the importance of both host- and pathogen-dependent factors in determining overall infection virulence and show the need for in vivo testing to fully assess pathogenicity.

Complex interactive effects of water mold, herbicide, and the fungus Batrachochytrium dendrobatidis on Pacific treefrog Hyliola regilla hosts.

Infectious diseases pose a serious threat to global biodiversity. However, their ecological impacts are not independent of environmental conditions. For example, the pathogenic fungus Batrachochytrium dendrobatidis (Bd), which has contributed to population declines and extinctions in many amphibian species, interacts with several environmental factors to influence its hosts, but potential interactions with other pathogens and environmental contaminants are understudied. We examined the combined effects of Bd, a water mold (Achlya sp.), and the herbicide Roundup® Regular (hereafter, Roundup®) on larval Pacific treefrog Hyliola regilla hosts. We employed a 2 wk, fully factorial laboratory experiment with 3 ecologically realistic levels (0, 1, and 2 mg l-1 of active ingredient) of field-formulated Roundup®, 2 Achlya treatments (present and absent), and 2 Bd treatments (present and absent). Our results were consistent with sublethal interactive effects involving all 3 experimental factors. When Roundup® was absent, the proportion of Bd-exposed larvae infected with Bd was elevated in the presence of Achlya, consistent with Achlya acting as a synergistic cofactor that facilitated the establishment of Bd infection. However, this Achlya effect became nonsignificant at 1 mg l-1 of the active ingredient of Roundup® and disappeared at the highest Roundup® concentration. In addition, Roundup® decreased Bd loads among Bd-exposed larvae. Our study suggests complex interactive effects of a water mold and a contaminant on Bd infection in amphibian hosts. Achlya and Roundup® were both correlated with altered patterns of Bd infection, but in different ways, and Roundup® appeared to remove the influence of Achlya on Bd.

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.

Trophic dynamics in an aquatic community: interactions among primary producers, grazers, and a pathogenic fungus.

Free-living stages of parasites are consumed by a variety of predators, which might have important consequences for predators, parasites, and hosts. For example, zooplankton prey on the infectious stage of the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), a pathogen responsible for amphibian population declines and extinctions worldwide. Predation on parasites is predicted to influence community structure and function, and affect disease risk, but relatively few studies have explored its consequences empirically. We investigated interactions among Rana cascadae tadpoles, zooplankton, and Bd in a fully factorial experiment in outdoor mesocosms. We measured growth, development, survival, and infection of amphibians and took weekly measurements of the abundance of zooplankton, phytoplankton (suspended algae), and periphyton (attached algae). We hypothesized that zooplankton might have positive indirect effects on tadpoles by consuming Bd zoospores and by consuming phytoplankton, thus reducing the shading of a major tadpole resource, periphyton. We also hypothesized that zooplankton would have negative effects on tadpoles, mediated by competition for algal resources. Mixed-effects models, repeated-measures ANOVAs, and a structural equation model revealed that zooplankton significantly reduced phytoplankton but had no detectable effects on Bd or periphyton. Hence, the indirect positive effects of zooplankton on tadpoles were negligible when compared to the indirect negative effect mediated by competition for phytoplankton. We conclude that examination of host-pathogen dynamics within a community context may be necessary to elucidate complex community dynamics.

Shifty salamanders: transient trophic polymorphism and cannibalism within natural populations of larval ambystomatid salamanders.

INTRODUCTION: Many species of ambystomatid salamanders are dependent upon highly variable temporary wetlands for larval development. High larval densities may prompt the expression of a distinct head morphology that may facilitate cannibalism. However, few studies have characterized structural cannibalism within natural populations of larval salamanders. In this study we used two species of larval salamanders, long-toed (Ambystoma macrodactylum) and ringed salamanders (A. annulatum). Head morphometrics and stable isotopic values of carbon (δ(13)C) and nitrogen (δ(15)N) were used to identify the presence or absence of structural cannibalism. Weather conditions were also analyzed as a potential factor associated with the expression of cannibalistic morphology. RESULTS: Populations of salamander larvae did not consistently exhibit cannibalistic morphologies throughout collection periods. Larval long-toed salamanders exhibited trophic polymorphisms when relatively lower precipitation amounts were observed. Larval ringed salamanders were observed to be cannibalistic but did not exhibit polymorphisms in this study. CONCLUSIONS: Structural cannibalism may be transient in both species; however in long-toed salamanders this morphology is necessary for cannibalism. Ringed salamanders can be cannibalistic without morphological adaptations; however the cannibal morph may prolong the viable time period for cannibalism. Additionally, weather conditions may alter pond hydroperiod, subsequently influencing head morphology and cannibalism.

A dilution effect in the emerging amphibian pathogen Batrachochytrium dendrobatidis.

Global declines in biodiversity are altering disease dynamics in complex and multifaceted ways. Changes in biodiversity can have several outcomes on disease risk, including dilution and amplification effects, both of which can have a profound influence on the effects of disease in a community. The dilution effect occurs when biodiversity and disease risk are inversely related, whereas the amplification effect is a positive relationship between biodiversity and disease risk. We tested these effects with an emerging fungal pathogen of amphibians, Batrachochytrium dendrobatidis (Bd), which is responsible for catastrophic amphibian population declines and extinctions worldwide. Despite the rapid and continued spread of Bd, the influence of host diversity on Bd dynamics remains unknown. We experimentally manipulated host diversity and density in the presence of Bd and found a dilution effect where increased species richness reduced disease risk, even when accounting for changes in density. These results demonstrate the general importance of incorporating community structure into studies of disease dynamics and have implications for the effects of Bd in ecosystems that differ in biodiversity.

Larval exposure to predator cues alters immune function and response to a fungal pathogen in post-metamorphic wood frogs.

For the past several decades, amphibian populations have been decreasing around the globe at an unprecedented rate. Batrachochytrium dendrobatidis (Bd), the fungal pathogen that causes chytridiomycosis in amphibians, is contributing to amphibian declines. Natural and anthropogenic environmental factors are hypothesized to contribute to these declines by reducing the immunocompetence of amphibian hosts, making them more susceptible to infection. Antimicrobial peptides (AMPs) produced in the granular glands of a frog's skin are thought to be a key defense against Bd infection. These peptides may be a critical immune defense during metamorphosis because many acquired immune functions are suppressed during this time. To test if stressors alter AMP production and survival of frogs exposed to Bd, we exposed wood frog (Lithobates sylvaticus) tadpoles to the presence or absence of dragonfly predator cues crossed with a single exposure to three nominal concentrations of the insecticide malathion (0, 10, or 100 parts per billion [ppb]). We then exposed a subset of post-metamorphic frogs to the presence or absence of Bd zoospores and measured frog survival. Although predator cues and malathion had no effect on survival or size at metamorphosis, predator cues increased the time to metamorphosis by 1.5 days and caused a trend of a 20% decrease in hydrophobic skin peptides. Despite this decrease in peptides determined shortly after metamorphosis, previous exposure to predator cues increased survival in both Bd-exposed and unexposed frogs several weeks after metamorphosis. These results suggest that exposing tadpoles to predator cues confers fitness benefits later in life.

Experimental evidence that host species composition alters host-pathogen dynamics in a ranavirus-amphibian assemblage.

Losses in biodiversity can alter disease risk through changes in host species composition. Host species vary in pathogen susceptibility and competence, yet how changes in diversity alter host-pathogen dynamics remains unclear in many systems, particularly with respect to generalist pathogens. Amphibians are experiencing worldwide population declines linked to generalist pathogens, such as ranavirus, and thus represent an ideal group to investigate how host species composition affects disease risk. We conducted experiments in which amphibian larvae of three native species (Pacific tree frogs, Pseudacris regilla; Cascades frogs, Rana cascadae; and Western toads, Anaxyrus boreas) were exposed to ranavirus individually (in the laboratory) or as assemblages (in outdoor mesocosms). In a laboratory experiment, we observed low survival and high viral loads in P. regilla compared to the other species, suggesting that this species was highly susceptible to the pathogen. In the mesocosm experiment, we observed 41% A. boreas mortality when alone and 98% mortality when maintained with P. regilla and R. cascadae. Our results suggest that the presence of highly susceptible species can alter disease dynamics across multiple species, potentially increasing infection risk and mortality in co-occurring species.

Corrigendum: Global Patterns of the Fungal Pathogen Batrachochytrium dendrobatidis Support Conservation Urgency.

[This corrects the article DOI: 10.3389/fvets.2021.685877.].

Regional decline of an iconic amphibian associated with elevation, land-use change, and invasive species.

Ecological theory predicts that species with restricted geographic ranges will have the highest probability of extinction, but species with extensive distributions and high population densities can also exhibit widespread population losses. In the western United States populations of northern leopard frogs (Lithobates pipiens)-historically one of the most widespread frogs in North America-have declined dramatically in abundance and geographic distribution. To assess the status of leopard frogs in Colorado and evaluate causes of decline, we coupled statewide surveys of 196 historically occupied sites with intensive sampling of 274 wetlands stratified by land use. We used an information-theoretic approach to evaluate the contributions of factors at multiple spatial extents in explaining the contemporary distribution of leopard frogs. Our results indicate leopard frogs have declined in Colorado, but this decline was regionally variable. The lowest proportion of occupied wetlands occurred in eastern Colorado (2-28%), coincident with urban development and colonization by non-native bullfrogs (Lithobates catesbeianus). Variables at several spatial extents explained observed leopard frog distributional patterns. In low-elevation wetlands introduced fishes, bullfrogs, and urbanization or suburbanization associated negatively with leopard frog occurrence, whereas wetland area was positively associated with occurrence. Leopard frogs were more abundant and widespread west of the Continental Divide, where urban development and bullfrog abundance were low. Although the pathogenic chytrid Batrachochytrium dendrobatidis (Bd) was not selected in our best-supported models, the nearly complete extirpation of leopard frogs from montane wetlands could reflect the individual or interactive effects of Bd and climate patterns. Our results highlight the importance of considering multiple, competing hypotheses to explain species declines, particularly when implicated factors operate at different spatial extents.

Individual and combined effects of multiple pathogens on Pacific treefrogs.

In nature, individual hosts often encounter multiple pathogens simultaneously, which can lead to additive, antagonistic, or synergistic effects on hosts. Synergistic effects on infection prevalence or severity could greatly affect host populations. However, ecologists and managers often overlook the influence of pathogen combinations on hosts. This is especially true in amphibian conservation, even though multiple pathogens coexist within amphibian populations, and several pathogens have been implicated in amphibian population declines and extinctions. Using an amphibian host, Pseudacris regilla (Pacific treefrog), we experimentally investigated interactive effects among three pathogens: the trematode Ribeiroia sp. (hereafter, Ribeiroia), the fungus Batrachochytrium dendrobatidis (hereafter, BD), and the water mold Achlya flagellata. We detected no effects of A. flagellata, but did find effects of Ribeiroia and BD that varied depending on context. Low doses of Ribeiroia caused relatively few malformations, while higher Ribeiroia doses caused numerous deformities dominated by missing and reduced limbs and limb elements. Exposure to low doses of BD accelerated larval host development, despite there being no detectable BD infections, while exposure to higher BD doses caused infection but did not alter developmental rate. Hosts exposed to both Ribeiroia and BD exhibited the highest mortality, although overall evidence of interactive effects of multiple pathogens was limited. We suggest further research on the influence of multi-pathogen assemblages on amphibians, particularly under a variety of ecological conditions and with a wider diversity of hosts and pathogens.

Direct and Latent Effects of Pathogen Exposure Across Native and Invasive Amphibian Life Stages.

Emerging infectious diseases are one of the multiple factors contributing to the current "biodiversity crisis". As part of the worldwide biodiversity crisis, amphibian populations are declining globally. Chytridiomycosis, an emerging infectious disease, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is a major cause of amphibian population declines. This fungus primarily affects keratinized structures in larval, juvenile, and adult amphibians as well as heart function. However, we know little about how Bd can impact embryos as well as potential latent effects of Bd exposure over ontogeny. Using two different Bd strains and multiple exposure times, we examined the effects of Bd exposure in Pacific chorus frog (Pseudacris regilla), Western toad (Anaxyrus boreas) and American bullfrog (Lithobates catesbeianus) life stages. Using a factorial experimental design, embryos of these three species were exposed to Bd at early and late embryonic stages, with some individuals re-exposed after hatching. Embryonic Bd exposure resulted in differential survival as a function of host species, Bd strain and timing of exposure. P. regilla experienced embryonic mortality when exposed during later developmental stages to one Bd strain. There were no differences across the treatments in embryonic mortality of A. boreas and embryonic mortality of L. catesbeianus occurred in all Bd exposure treatments. We detected latent effects in A. boreas and L. catesbeianus larvae, as mortality increased when individuals had been exposed to any of the Bd strains during the embryonic stage. We also detected direct effects on larval mortality in all three anuran species as a function of Bd strain, and when individuals were double exposed (late in the embryonic stage and again as larvae). Our results suggest that exposure to Bd can directly affect embryo survival and has direct and latent effects on larvae survival of both native and invasive species. However, these impacts were highly context dependent, with timing of exposure and Bd strain influencing the severity of the effects.

Global Patterns of the Fungal Pathogen Batrachochytrium dendrobatidis Support Conservation Urgency.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) is a skin pathogen that can cause the emerging infectious disease chytridiomycosis in susceptible species. It has been considered one of the most severe threats to amphibian biodiversity. We aimed to provide an updated compilation of global Bd occurrences by host taxon and geography, and with the larger global Bd dataset we reanalyzed Bd associations with environmental metrics at the world and regional scales. We also compared our Bd data compilation with a recent independent assessment to provide a more comprehensive count of species and countries with Bd occurrences. Bd has been detected in 1,375 of 2,525 (55%) species sampled, more than doubling known species infections since 2013. Bd occurrence is known from 93 of 134 (69%) countries at this writing; this compares to known occurrences in 56 of 82 (68%) countries in 2013. Climate-niche space is highly associated with Bd detection, with different climate metrics emerging as key predictors of Bd occurrence at regional scales; this warrants further assessment relative to climate-change projections. The accretion of Bd occurrence reports points to the common aims of worldwide investigators to understand the conservation concerns for amphibian biodiversity in the face of potential disease threat. Renewed calls for better mitigation of amphibian disease threats resonate across continents with amphibians, especially outside Asia. As Bd appears to be able to infect about half of amphibian taxa and sites, there is considerable room for biosecurity actions to forestall its spread using both bottom-up community-run efforts and top-down national-to-international policies. Conservation safeguards for sensitive species and biodiversity refugia are continuing priorities.

Projected climate impacts for the amphibians of the Western hemisphere.

Given their physiological requirements, limited dispersal abilities, and hydrologically sensitive habitats, amphibians are likely to be highly sensitive to future climatic changes. We used three approaches to map areas in the western hemisphere where amphibians are particularly likely to be affected by climate change. First, we used bioclimatic models to project potential climate-driven shifts in the distribution of 413 amphibian species based on 20 climate simulations for 2071-2100. We summarized these projections to produce estimates of species turnover. Second, we mapped the distribution of 1099 species with restricted geographic ranges. Finally, using the 20 future climate-change simulations, we mapped areas that were consistently projected to receive less seasonal precipitation in the coming century and thus were likely to have altered microclimates and local hydrologies. Species turnover was projected to be highest in the Andes Mountains and parts of Central America and Mexico, where, on average, turnover rates exceeded 60% under the lower of two emissions scenarios. Many of the restricted-range species not included in our range-shift analyses were concentrated in parts of the Andes and Central America and in Brazil's Atlantic Forest. Much of Central America, southwestern North America, and parts of South America were consistently projected to experience decreased precipitation by the end of the century. Combining the results of the three analyses highlighted several areas in which amphibians are likely to be significantly affected by climate change for multiple reasons. Portions of southern Central America were simultaneously projected to experience high species turnover, have many additional restricted-range species, and were consistently projected to receive less precipitation. Together, our three analyses form one potential assessment of the geographic vulnerability of amphibians to climate change and as such provide broad-scale guidance for directing conservation efforts.

Experimental examination of the effects of ultraviolet-B radiation in combination with other stressors on frog larvae.

Ultraviolet-B radiation (UVB) is a ubiquitous stressor with negative effects on many aquatic organisms. In amphibians, ambient levels of UVB can result in impaired growth, slowed development, malformations, altered behavior and mortality. UVB can also interact with other environmental stressors to amplify these negative effects on individuals. In outdoor mesocosm and laboratory experiments we studied potential synergistic effects of UVB, a pathogenic fungus, Batrachochytrium dendrobatidis (Bd), and varying temperatures on larval Cascades frogs (Rana cascadae). First, we compared survivorship, growth and development in two mesocosm experiments with UVB- and Bd-exposure treatments. We then investigated the effects of UVB on larvae in the laboratory under two temperature regimes, monitoring survival and behavior. We found reduced survival of R. cascadae larvae with exposure to UVB radiation in all experiments. In the mesocosm experiments, growth and development were not affected in either treatment, and no effect of Bd was found. In the laboratory experiment, larvae exposed to UVB demonstrated decreased activity levels. We also found a trend towards reduced survival when UVB and cold temperatures were combined. Our results show that amphibian larvae can suffer both lethal and sublethal effects when exposed to UVB radiation.

Reproductive characteristics of American bullfrogs (Lithobates catesbeianus) in their invasive range of the Pacific Northwest, USA.

Invasive species pose a major threat to global biodiversity. The effects of invasive species can be strongly influenced and potentially mediated by their reproductive characteristics, such as fecundity, egg production, and duration and number of reproductive events. Selection for smaller body size at first reproduction can also play a role in their establishment, facilitating colonization and spread. The American bullfrog, native to the eastern U.S. (Lithobates catesbeianus), is a species that has invaded more than 40 countries across 4 continents. This species has become especially prevalent in the western United States since its introduction in the early 1900s. This study characterized reproductive characteristics of bullfrogs with emphasis on the minimum size at which males and females reach sexual maturity in the Willamette Valley, Oregon, USA invasion range. We collected and dissected 121 individuals in 2013 and 2017, quantifying characteristics of sexual maturity including snout-vent length, total length, sex, tympanum diameter, presence of distended oviducts or eggs for females, and testes length and sperm activity in males. Our results showed that the minimum reproductive size of both males and females was smaller relative to bullfrogs in their native range as well as in populations across their invasive range. Reduction in size at reproductive maturity is likely impacting the invasive success of American bullfrogs and this study gives us insight on management actions to control the invasion. Applying this insight, managers can adjust their definition of reproductively active adults, increasing the target population of culling and other control methods.

Projected climate-induced faunal change in the Western Hemisphere.

Climate change is predicted to be one of the greatest drivers of ecological change in the coming century. Increases in temperature over the last century have clearly been linked to shifts in species distributions. Given the magnitude of projected future climatic changes, we can expect even larger range shifts in the coming century. These changes will, in turn, alter ecological communities and the functioning of ecosystems. Despite the seriousness of predicted climate change, the uncertainty in climate-change projections makes it difficult for conservation managers and planners to proactively respond to climate stresses. To address one aspect of this uncertainty, we identified predictions of faunal change for which a high level of consensus was exhibited by different climate models. Specifically, we assessed the potential effects of 30 coupled atmosphere-ocean general circulation model (AOGCM) future-climate simulations on the geographic ranges of 2954 species of birds, mammals, and amphibians in the Western Hemisphere. Eighty percent of the climate projections based on a relatively low greenhouse-gas emissions scenario result in the local loss of at least 10% of the vertebrate fauna over much of North and South America. The largest changes in fauna are predicted for the tundra, Central America, and the Andes Mountains where, assuming no dispersal constraints, specific areas are likely to experience over 90% turnover, so that faunal distributions in the future will bear little resemblance to those of today.

Effects of the pathogenic water mold Saprolegnia ferax on survival of amphibian larvae.

Infectious diseases are a significant threat to worldwide biodiversity. Amphibian declines, a significant part of current biodiversity losses, are in many cases associated with infectious disease. Water molds are one group of pathogens affecting amphibians on a worldwide basis. Although water molds have been studied extensively for their effects on host embryos, little information is available about how they affect post-embryonic amphibians. We tested the effects of one species of water mold, Saprolegnia ferax, in a comparative study of larvae of 4 amphibian species: Pseudacris regilla (Pacific treefrog), Rana cascadae (Cascades frog), Ambystoma macrodactylum (long-toed salamander), and R. aurora (red-legged frog). S. ferax can kill amphibians at the embryonic and juvenile life history stages, depending on the amphibian species. In the present study, a 1 wk exposure to S. ferax killed P. regilla larvae and a 2 wk exposure killed R. aurora larvae. Larvae of the other host species were unaffected after 1 wk of exposure to S. ferax. Our results suggest that S. ferax can kill amphibian larvae and further suggest that evaluation of how pathogens affect amphibians at the population level requires investigation at various life stages.