Broad host susceptibility of North American amphibian species to Batrachochytrium salamandrivorans suggests high invasion potential and biodiversity risk.

Batrachochytrium salamandrivorans (Bsal) is a fungal pathogen of amphibians that is emerging in Europe and could be introduced to North America through international trade or other pathways. To evaluate the risk of Bsal invasion to amphibian biodiversity, we performed dose-response experiments on 35 North American species from 10 families, including larvae from five species. We discovered that Bsal caused infection in 74% and mortality in 35% of species tested. Both salamanders and frogs became infected and developed Bsal chytridiomycosis. Based on our host susceptibility results, environmental suitability conditions for Bsal, and geographic ranges of salamanders in the United States, predicted biodiversity loss is expected to be greatest in the Appalachian Region and along the West Coast. Indices of infection and disease susceptibility suggest that North American amphibian species span a spectrum of vulnerability to Bsal chytridiomycosis and most amphibian communities will include an assemblage of resistant, carrier, and amplification species. Predicted salamander losses could exceed 80 species in the United States and 140 species in North America.

Isolation and maintenance of Batrachochytrium salamandrivorans cultures.

Discovered in 2013, the chytrid fungus Batrachochytrium salamandrivorans (Bsal) is an emerging amphibian pathogen that causes ulcerative skin lesions and multifocal erosion. A closely related pathogen, B. dendrobatidis (Bd), has devastated amphibian populations worldwide, suggesting that Bsal poses a significant threat to global salamander biodiversity. To expedite research into this emerging threat, we seek to standardize protocols across the field so that results of laboratory studies are reproducible and comparable. We have collated data and experience from multiple labs to standardize culturing practices of Bsal. Here we outline common culture practices including a medium for standardized Bsal growth, standard culturing protocols, and a method for isolating Bsal from infected tissue.

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.

Community ecology theory predicts the effects of agrochemical mixtures on aquatic biodiversity and ecosystem properties.

Ecosystems are often exposed to mixtures of chemical contaminants, but the scientific community lacks a theoretical framework to predict the effects of mixtures on biodiversity and ecosystem properties. We conducted a freshwater mesocosm experiment to examine the effects of pairwise agrochemical mixtures [fertiliser, herbicide (atrazine), insecticide (malathion) and fungicide (chlorothalonil)] on 24 species- and seven ecosystem-level responses. As postulated, the responses of biodiversity and ecosystem properties to agrochemicals alone and in mixtures was predictable by integrating information on each functional group's (1) sensitivity to the chemicals (direct effects), (2) reproductive rates (recovery rates), (3) interaction strength with other functional groups (indirect effects) and (4) links to ecosystem properties. These results show that community ecology theory holds promise for predicting the effects of contaminant mixtures on biodiversity and ecosystem services and yields recommendations on which types of agrochemicals to apply together and separately to reduce their impacts on aquatic ecosystems.

Nonmonotonic and monotonic effects of pesticides on the pathogenic fungus Batrachochytrium dendrobatidis in culture and on tadpoles.

Pesticides and the pathogenic fungus Batrachochytrium dendrobatidis (Bd) co-occur and are implicated in the global decline of amphibians, a highly threatened taxon. Here, we investigated the effects of ecologically relevant concentrations of chlorothalonil and atrazine, two of the most commonly used, immunomodulatory pesticides in the United States, on tadpole (Osteopilus septentrionalis) survival and Bd growth. Tadpole survival was unaffected by the pesticides but was reduced by Bd. Atrazine monotonically (i.e., consistently increasing or decreasing) reduced Bd in culture and on tadpoles, and every concentration tested (0.0106-106 µg/L) significantly reduced Bd growth compared to controls. Chlorothalonil had a nonmonotonic (i.e., nonlinear) effect on Bd growth both in culture and on tadpoles, where low (0.0176-1.76 µg/L) and high (32-176 µg/L) concentrations inhibited Bd growth significantly more than did intermediate concentrations (8.2-17.6 µg/L) and controls. To our knowledge, this is one of only a handful of studies to document a nonmonotonic dose response of a nonvertebrate (Bd) to a pesticide. Although both pesticides reduced Bd growth on frogs, neither cleared the infection entirely, and because we know little about the long-term effects of the pesticides on hosts, we do not recommend using these chemicals to control Bd.

Fungicide-induced declines of freshwater biodiversity modify ecosystem functions and services.

Although studies on biodiversity and ecosystem function are often framed within the context of anthropogenic change, a central question that remains is how important are direct vs. indirect (via changes in biodiversity) effects of anthropogenic stressors on ecosystem functions in multitrophic-level communities. Here, we quantify the effects of the fungicide chlorothalonil on 34 species-, 2 community- and 11 ecosystem-level responses in a multitrophic-level system. At ecologically relevant concentrations, chlorothalonil increased mortality of amphibians, gastropods, zooplankton, algae and a macrophyte (reducing taxonomic richness), reduced decomposition and water clarity and elevated dissolved oxygen and net primary productivity. These ecosystem effects were indirect and predictable based on changes in taxonomic richness. A path analysis suggests that chlorothalonil-induced reductions in biodiversity and top-down and bottom-up effects facilitated algal blooms that shifted ecosystem functions. This work emphasises the need to re-evaluate the safety of chlorothalonil and to further link anthropogenic-induced changes in biodiversity to altered ecosystem functions.

The fungicide chlorothalonil is nonlinearly associated with corticosterone levels, immunity, and mortality in amphibians.

BACKGROUND: Contaminants have been implicated in declines of amphibians, a taxon with vital systems similar to those of humans. However, many chemicals have not been thoroughly tested on amphibians or do not directly kill them. OBJECTIVE: Our goal in this study was to quantify amphibian responses to chlorothalonil, the most commonly used synthetic fungicide in the United States. METHODS: We reared Rana sphenocephala (southern leopard frog) and Osteopilus septentrionalis (Cuban treefrog) in outdoor mesocosms with or without 1 time (1×) and 2 times (2×) the expected environmental concentration (EEC) of chlorothalonil (~ 164 µg/L). We also conducted two dose-response experiments on O. septentrionalis, Hyla squirella (squirrel treefrog), Hyla cinerea (green treefrog), and R. sphenocephala and evaluated the effects of chlorothalonil on the stress hormone corticosterone. RESULTS: For both species in the mesocosm experiment, the 1× and 2× EEC treatments were associated with > 87% and 100% mortality, respectively. In the laboratory experiments, the approximate EEC caused 100% mortality of all species within 24 hr; 82 µg/L killed 100% of R. sphenocephala, and 0.0164 µg/L caused significant tadpole mortality of R. sphenocephala and H. cinerea. Three species showed a nonmonotonic dose response, with low and high concentrations causing significantly greater mortality than did intermediate concentrations or control treatments. For O. septentrionalis, corticosterone exhibited a similar nonmonotonic dose response and chlorothalonil concentration was inversely associated with liver tissue and immune cell densities (< 16.4 µg/L). CONCLUSIONS: Chlorothalonil killed nearly every amphibian at the approximate EEC; at concentrations to which humans are commonly exposed, it increased mortality and was associated with elevated corticosterone levels and changes in immune cells. Future studies should directly quantify the effects of chlorothalonil on amphibian populations and human health.

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.

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.

Evaluating the links between climate, disease spread, and amphibian declines.

Human alteration of the environment has arguably propelled the Earth into its sixth mass extinction event and amphibians, the most threatened of all vertebrate taxa, are at the forefront. Many of the worldwide amphibian declines have been caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd), and two contrasting hypotheses have been proposed to explain these declines. Positive correlations between global warming and Bd-related declines sparked the chytrid-thermal-optimum hypothesis, which proposes that global warming increased cloud cover in warm years that drove the convergence of daytime and nighttime temperatures toward the thermal optimum for Bd growth. In contrast, the spatiotemporal-spread hypothesis states that Bd-related declines are caused by the introduction and spread of Bd, independent of climate change. We provide a rigorous test of these hypotheses by evaluating (i) whether cloud cover, temperature convergence, and predicted temperature-dependent Bd growth are significant positive predictors of amphibian extinctions in the genus Atelopus and (ii) whether spatial structure in the timing of these extinctions can be detected without making assumptions about the location, timing, or number of Bd emergences. We show that there is spatial structure to the timing of Atelopus spp. extinctions but that the cause of this structure remains equivocal, emphasizing the need for further molecular characterization of Bd. We also show that the reported positive multi-decade correlation between Atelopus spp. extinctions and mean tropical air temperature in the previous year is indeed robust, but the evidence that it is causal is weak because numerous other variables, including regional banana and beer production, were better predictors of these extinctions. Finally, almost all of our findings were opposite to the predictions of the chytrid-thermal-optimum hypothesis. Although climate change is likely to play an important role in worldwide amphibian declines, more convincing evidence is needed of a causal link.

Adding infection to injury: synergistic effects of predation and parasitism on amphibian malformations.

We explored the importance of interactions between parasite infection and predation in driving an emerging phenomenon of conservation importance: amphibian limb malformations. We suggest that injury resulting from intraspecific predation in combination with trematode infection contributes to the frequency and severity of malformations in salamanders. By integrating field surveys and experiments, we evaluated the individual and combined effects of conspecific attack and parasite (Ribeiroia ondatrae) infection on limb development of long-toed salamanders (Ambystoma macrodactylum). In the absence of Ribeiroia, abnormalities involved missing digits, feet, or limbs and were similar to those produced by cannibalistic attack in experimental trials. At field sites that supported Ribeiroia, malformations were dominated by extra limbs and digits. Correspondingly, laboratory exposure of larval salamanders to Ribeiroia cercariae over a 30-day period induced high frequencies of malformations, including extra digits, extra limbs, cutaneous fusion, and micromelia. However, salamander limbs exposed to both injury and infection exhibited 3-5 times more abnormalities than those exposed to either factor alone. Infection also caused significant delays in limb regeneration and time-to-metamorphosis. Taken together, these results help to explain malformation patterns observed in natural salamander populations while emphasizing the importance of interactions between parasitism and predation in driving disease.

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

We tested for a synergism between nitrate and Saprolegnia, a pathogenic water mold, using larvae of 3 amphibian species: Ambystoma gracile (northwestern salamander), Hyla regilla (Pacific treefrog) and Rana aurora (red-legged frog). Each species was tested separately, using a 3 x 2 fully factorial experiment with 3 nitrate treatments (none, low and high) and 2 Saprolegnia treatments (Saprolegnia and control). Survival of H. regilla was not affected significantly by either experimental factor. In contrast, survival of R. aurora was affected by a less-than-additive interaction between Saprolegnia and nitrate. Survival of R. aurora was significantly lower in the Saprolegnia compared to the control treatment when nitrate was not added, but there was no significant difference in survival between Saprolegnia and control treatments in the low and high nitrate treatments, consistent with increased nitrate preventing Saprolegnia from causing mortality of R. aurora. Survival of A. gracile followed a similar pattern, but the difference between Saprolegnia and control treatments when nitrate was not added was not significant, nor was the nitrate x Saprolegnia interaction. Our study suggests that Saprolegnia can cause mortality in amphibian larvae, that there are interspecific differences in susceptibility and that the effects of Saprolegnia on amphibians are context-dependent.