Are novel or locally adapted pathogens more devastating and why? Resolving opposing hypotheses.

There is a rich literature highlighting that pathogens are generally better adapted to infect local than novel hosts, and a separate seemingly contradictory literature indicating that novel pathogens pose the greatest threat to biodiversity and public health. Here, using Batrachochytrium dendrobatidis, the fungus associated with worldwide amphibian declines, we test the hypothesis that there is enough variance in "novel" (quantified by geographic and phylogenetic distance) host-pathogen outcomes to pose substantial risk of pathogen introductions despite local adaptation being common. Our continental-scale common garden experiment and global-scale meta-analysis demonstrate that local amphibian-fungal interactions result in higher pathogen prevalence, pathogen growth, and host mortality, but novel interactions led to variable consequences with especially virulent host-pathogen combinations still occurring. Thus, while most pathogen introductions are benign, enough variance exists in novel host-pathogen outcomes that moving organisms around the planet greatly increases the chance of pathogen introductions causing profound harm.

Cost-Effective DNA Extraction for DNA Barcoding Diverse Biological Samples.

DNA barcoding employs standard molecular techniques (e.g., DNA extraction, PCR, and Sanger sequencing) to taxonomically identify biological samples. While DNA barcoding is a useful experimental workflow for in-class active learning exercises, extracting DNA from diverse sample types in a time and cost-effective manner can be challenging in a classroom setting. Here, we provide two time and cost-effective methods that have been used by novice students to successfully extract DNA from a variety of animal, fungal, algal, and plant tissues for DNA barcoding.

Metabolite compositions on skins of eastern hellbenders Cryptobranchus alleganiensis alleganiensis differ with location and captivity.

Eastern hellbenders Cryptobranchus alleganiensis alleganiensis, large aquatic salamanders, are declining over most of their range. The amphibian-killing fungus Batrachochytrium dendrobatidis (Bd) has contributed to global amphibian declines and has been detected on eastern hellbenders, but infection intensities were lower than those of species that are more susceptible to Bd. The factors limiting Bd on hellbenders may include antifungal metabolites produced by their skin microbiota. We used a metabolite fingerprinting technique to noninvasively identify the presence, but not identity, of metabolites associated with eastern hellbenders. We surveyed the skin of wild eastern hellbenders to test whether the composition and richness (i.e. number of metabolites) of their metabolites are explained by Bd status or location. Furthermore, we surveyed for metabolites on captive eastern hellbenders to test whether metabolite compositions were different between captive and wild eastern hellbenders. Bd detection was not associated with either metabolite richness or composition. Both metabolite composition and richness differed significantly on hellbenders from different locations (i.e. states). For metabolite composition, there was a statistical interaction between location and Bd status. Metabolite richness was greater on captive eastern hellbenders compared to wild hellbenders, and metabolite compositions differed between wild and captive eastern hellbenders. The methods we employed to detect metabolite profiles effectively grouped individuals by location even though metabolite composition and richness have high levels of intraspecific variation. Understanding the drivers and functional consequences of assemblages of skin metabolites on amphibian health will be an important step toward understanding the mechanisms that result in disease vulnerability.

Phylogenetic patterns of trait and trait plasticity evolution: Insights from amphibian embryos.

Environmental variation favors the evolution of phenotypic plasticity. For many species, we understand the costs and benefits of different phenotypes, but we lack a broad understanding of how plastic traits evolve across large clades. Using identical experiments conducted across North America, we examined prey responses to predator cues. We quantified five life-history traits and the magnitude of their plasticity for 23 amphibian species/populations (spanning three families and five genera) when exposed to no cues, crushed-egg cues, and predatory crayfish cues. Embryonic responses varied considerably among species and phylogenetic signal was common among the traits, whereas phylogenetic signal was rare for trait plasticities. Among trait-evolution models, the Ornstein-Uhlenbeck (OU) model provided the best fit or was essentially tied with Brownian motion. Using the best fitting model, evolutionary rates for plasticities were higher than traits for three life-history traits and lower for two. These data suggest that the evolution of life-history traits in amphibian embryos is more constrained by a species' position in the phylogeny than is the evolution of life history plasticities. The fact that an OU model of trait evolution was often a good fit to patterns of trait variation may indicate adaptive optima for traits and their plasticities.

Pathogenic chytrid fungus Batrachochytrium dendrobatidis, but not B. salamandrivorans, detected on eastern hellbenders.

Recent worldwide declines and extinctions of amphibian populations have been attributed to chytridiomycosis, a disease caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd). Until recently, Bd was thought to be the only Batrachochytrium species that infects amphibians; however a newly described species, Batrachochytrium salamandrivorans (Bs), is linked to die-offs in European fire salamanders (Salamandra salamandra). Little is known about the distribution, host range, or origin of Bs. In this study, we surveyed populations of an aquatic salamander that is declining in the United States, the eastern hellbender (Cryptobranchus alleganiensis alleganiensis), for the presence of Bs and Bd. Skin swabs were collected from a total of 91 individuals in New York, Pennsylvania, Ohio, and Virginia, and tested for both pathogens using duplex qPCR. Bs was not detected in any samples, suggesting it was not present in these hellbender populations (0% prevalence, 95% confidence intervals of 0.0-0.04). Bd was found on 22 hellbenders (24% prevalence, 95% confidence intervals of 0.16 ≤ 0.24 ≤ 0.34), representing all four states. All positive samples had low loads of Bd zoospores (12.7 ± 4.9 S.E.M. genome equivalents) compared to other Bd susceptible species. More research is needed to determine the impact of Batrachochytrium infection on hellbender fitness and population viability. In particular, understanding how hellbenders limit Bd infection intensity in an aquatic environment may yield important insights for amphibian conservation. This study is among the first to evaluate the distribution of Bs in the United States, and is consistent with another, which failed to detect Bs in the U.S. Knowledge about the distribution, host-range, and origin of Bs may help control the spread of this pathogen, especially to regions of high salamander diversity, such as the eastern United States.

Evaluation of a filtration-based method for detecting Batrachochytrium dendrobatidis in natural bodies of water.

Infectious diseases are emerging as a significant threat to wildlife. The resulting increased effort to monitor wildlife diseases is driving the development of innovative pathogen monitoring techniques, including many polymerase chain reaction (PCR)-based diagnostics. Despite the utility of these PCR-based techniques, there is still much to be learned about their ability to accurately detect target pathogens in nature. We assessed the diagnostic sensitivity of a PCR-based water filtration technique to detect the directly transmitted aquatic fungal pathogen Batrachochytrium dendrobatidis (Bd) by comparing the results of 4 repeated filter sampling events from 20 ponds to those of skin swabs from ca. 60 boreal chorus frogs Pseudacris maculata from each pond. Filters failed to detect Bd in 31 to 77% of the swab-positive ponds, depending on the time of sampling. However, after 3 repeated sampling events, filtration of small volumes of water (ca. 600 ml) correctly identified 94% of the ponds that tested Bd positive with swabbing, with the highest rates of detection occurring after breeding but before larvae reached metamorphosis. Our results are a case study demonstrating the importance of timing and resampling for the detection of an aquatic microbial pathogen, Bd, from water. This will be a useful technique for monitoring Bd, but additional data are needed to test the degree to which our findings are species or population specific. Future studies need to examine the sensitivity of this technique in other habitats and species that host Bd. These studies will aid in the development of cost-effective monitoring regimes for Bd and potentially other aquatic pathogens.