Nearly two decades ago, the global biodiversity crisis was catapulted to the front pages of newspapers with the recognition of worldwide amphibian declines. Amphibians earned their appellation, 'canaries in a coal mine', because of apparent high sensitivity to human-mediated environmental change. The most frequently cited causes for high susceptibility include permeable skin, a dual aquatic-terrestrial life cycle and a relatively rudimentary immune system. While some researchers have questioned the basis for the canary assertion, there has been no systematic evaluation of amphibian sensitivity to environmental challenges relative to other taxa. Here, we apply a database representing thousands of toxicity tests to compare the responses of amphibians relative to that of other taxonomic groups. The use of standardized methods combined with large numbers of identical challenges enables a particularly powerful test of relative effect size. Overall, we found that amphibians only exhibit moderate relative responses to water-borne toxins. Our findings imply that, as far as chemical contaminants are concerned, amphibians are not particularly sensitive and might more aptly be described as 'miners in a coal mine'. To the extent that amphibian declines have been mediated by chemical contaminants, our findings suggest that population losses and extinctions may have already occurred in a variety of taxa much more sensitive than amphibians.

Dispersal and exposure to amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd) is not confined to the aquatic habitat, but little is known about pathways that facilitate exposure to wild terrestrial amphibians that do not typically enter bodies of water. We explored the possible spread of Bd from an aquatic reservoir to terrestrial substrates by the emergence of recently metamorphosed infected amphibians and potential deposition of Bd-positive residue on riparian vegetation in Cusuco National Park, Honduras (CNP). Amphibians and their respective leaf perches were both sampled for Bd presence and the pathogen was detected on 76.1% (35/46) of leaves where a Bd-positive frog had rested. Although the viability of Bd detected on these leaves cannot be discerned from our quantitative PCR results, the cool air temperature, closed canopy, and high humidity of this cloud forest environment in CNP is expected to encourage pathogen persistence. High prevalence of infection (88.5%) detected in the recently metamorphosed amphibians and frequent shedding of Bd-positive residue on foliage demonstrates a pathway of Bd dispersal between aquatic and terrestrial habitats. This pathway provides the opportunity for environmental transmission of Bd among and between amphibian species without direct physical contact or exposure to an aquatic habitat.

Summary1. Batrachochytrium dendrobatidis is a chytrid fungal pathogen driving many amphibian species extinct. For some species, the only place they can be found is in natural history museum collections. Harnessing molecular tools to uncover this pathogen's date of arrival and movement throughout a region and through time will prove useful to broader B. dendrobatidis research. However, it has remained difficult to access B. dendrobatidis DNA from within preserved host tissues, especially specimens that were originally fixed in formalin. 2. Herein, I describe two methods to extract and detect, via qPCR, B. dendrobatidis DNA from herpetological natural history collections. Both extraction methods use a DNA-binding matrix, either a magnetic bead resin or a silica membrane, that retains and separates the extracted DNA from potentially qPCR-inhibiting contaminates. Nine positive control specimens enabled initial method optimizations and coincidently limited empirical comparisons between each extraction method. A museum study involving 164 formalin-fixed amphibians from Connecticut, either having been originally fixed in the 1960s or 2000s, were swabbed and samples extracted by one of the two presented methods. 3. Each method successfully extracted B. dendrobatidis DNA; of the 164 specimens tested, 37 were found B. dendrobatidis-positive, including six specimens (representing three caudate species) from 1968. In addition, results show that an infected individual stored in a jar of multiple specimens does not always contaminate the other specimens with B. dendrobatidis. 4. These results suggest that qPCR methods are well suited to assess B. dendrobatidis presence but to not assign zoospore loads in preserved specimens.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) is an emerging infectious fungal pathogen of amphibians and is linked to global population declines. Until now, there has only been 1 survey for the fungus in the northeastern USA, which focused primarily on northern New England. We tested for Bd in a large number of samples (916 individuals from 116 sites) collected throughout the state of Connecticut, representing 18 native amphibian species. In addition, 239 preserved wood frog Lithobates sylvaticus tadpoles from throughout the state were screened for the fungus. Bd presence was assessed in both the fresh field swabs and the preserved samples using a sensitive quantitative PCR assay. Our contemporary survey found widespread Bd prevalence throughout Connecticut, occurring in 14 species and in 28% of all sampled animals. No preserved L. sylvaticus specimens tested positive for the fungus. Two common species, bullfrogs R. catesbeiana and green frogs R. clamitans had particularly high infection rates (0.21−0.39 and 0.33−0.42, respectively), and given their wide distribution throughout the state, we suggest they may serve as sentinels for Bd occurrence in this region. Further analyses found that several other factors increase the likelihood of infection, including life stage, host sex, and host family. Within sites, ponds with ranids, especially green frogs, increased the likelihood of Bd prevalence. By studying Bd in populations not facing mass declines, the results from this study are an important contribution to our understanding of how some amphibian species and populations remain infected yet exhibit no signs of chytridiomycosis even when Bd is widely distributed.

Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80–90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum specimens should ensure that the techniques they are using are known to provide high-quality throughput DNA for later analysis.