Panzootic chytrid fungus exploits diverse amphibian host environments through plastic infection strategies.

While some pathogens are limited to single species, others can colonize many hosts, likely contributing to the emergence of novel disease outbreaks. Despite this biodiversity threat, traits associated with host niche expansions are not well understood in multihost pathogens. Here, we aimed to uncover functional machinery driving multihost invasion by focusing on Batrachochytrium dendrobatidis (Bd), a pathogen that infects the skin of hundreds of amphibians worldwide. We performed a meta-analysis of Bd gene expression using data from published infection experiments and newly generated profiles. We analysed Bd transcriptomic landscapes across the skin of 14 host species, reconstructed Bd isolates phylogenetic relationships, and inferred the origin and evolutionary history of differentially expressed genes under a phylogenetic framework comprising other 12 zoosporic fungi. Bd displayed plastic infection strategies when challenged by hosts with different disease susceptibility. Our analyses identified sets of differentially expressed genes under host environments with similar infection outcome. We stressed nutritional immunity and gene silencing as important processes required to overcome challenging skin environments in less susceptible hosts. Overall, Bd genes expressed during amphibian skin exploitation have arisen mainly via gene duplications with great family expansions, increasing the gene copy events previously described for this fungal species. Finally, we provide a comprehensive gene data set that can be used to further examine eco-evolutionary hypotheses for this host-pathogen system. Our study supports the idea that host environments exert contrasting selective pressures, such that gene expression plasticity could be one of the evolutionary keys leading to the success of multihost pathogens.

Detection of pathogenic Batrachochytrium dendrobatidis using water filtration, animal and bait testing.

The pathogen Batrachochytrium dendrobatidis (Bd) can be challenging to detect at endangered amphibian reintroduction sites. Pre-release Bd detection can be confounded by imperfect animal sampling and the absence of animals. In Study 1, we used historical Bd-positive sites, to concurrently evaluate water filtrates and mouth bar (tadpoles) or skin swab (caudates) samples for Bd using molecular beacon realtime PCR. In Study 2, during a natural outbreak, we used PCR to detect Bd from zoospore-attracting keratin baits (three avian, three snake species). In Study 1, no captured animals (n=116) exhibited clinical signs, although 10.6% were positive, representing three of seven species sampled. In contrast, 5.4% of water filters (n=56) were Bd-positive. In Study 2, after short incubation times, a single duck down feather tested Bd-positive. In conclusion, Bd was detected in asymptomatic amphibians and water filtrate at two sites, and from water only, at two other sites. With continued refinement, semi-quantitative Bd water filtrate screening could better define zoospore-specific disease risk, allowing better characterization of the free-living phase of the organism's life cycle. Finally, these results suggest wild aquatic birds (e.g., waterfowl) should be systematically explored as a means of Bd spread. Since large numbers of aquatic birds migrate, even low Bd transfer rates could be a significant means for disease dissemination.

Comparison of methods for detection of chytrid fungus (Batrachochytrium dendrobatidis) in bullfrog tadpole mouthparts.

We previously reported that the tadpole of bullfrog (Lithobates catesbeiana) is a useful model for the field surveillance of the Batrachochytrium dendrobatidis (Bd) distribution. In the present study, we compared Bd detection rates in swab-scraped and resected mouthpart samples, using nested polymerase chain reaction (PCR). The resulting detection rates for swab-scraped and resected specimens were 67 and 65%, respectively, with no significant difference. Furthermore, we performed a histopathological examination for Bd distribution in the mouthparts; we found that Bd infection occurred in the tip and basement of the jaw sheaths and tooth rows. We recommend using swab-scraped samples for Bd detection. Moreover, careful attention should be paid to scraping the tip and basement of the jaw sheaths and the entire oral cavity to reduce the rates of false-negative results on nested PCR of the mouthparts of bullfrog tadpoles.

Four new species of terrestrial-breeding frogs of the genus Phrynopus (Anura: Terrarana: Craugastoridae) from Río Abiseo National Park, Peru.

We describe four new species of terrestrial-breeding frogs belonging to the genus Phrynopus from specimens collected on the eastern slopes of the Cordillera Oriental (2800-3850 m) near and within Río Abiseo National Park, Provincia Mariscal Cáceres, Departments of San Martín and La Libertad, northeastern Peru. All four species lack a visible tympanum and inhabit the upper ridges and slopes within or adjacent to the Park. Phrynopus anancites sp. nov. and P. capitalis sp. nov. inhabit the wet montane grasslands on the upper ridges and valleys from 3600 to 3850 m. Phrynopus anancites (SVL = 25.3 mm) has coarsely aerolated skin and olive green coloration and has small vomerine teeth, while P. capitalis (female SVL = 35.6 mm) is characterized by a large head, short limbs, and distinctive dorsal pattern. Phrynopus dumicola sp. nov. (female SVL = 25.3 mm) has a short head and dark colored body with granular skin on the flanks, and is known only from forest patches along the treeline from 3225 to 3550 m, whereas P. personatus sp. nov. (female SVL = 28.2 mm) has a dark facemask and bright yellow groin spots (possibly aposematic), and inhabits a narrow band of continuous tropical montane rain forest from 2890 to 3110 m. We report infection with Batrachochytrium dendrobatidis from one specimen of P. dumicola collected in July of 1988. With the addition of these four new species, Phrynopus now includes 32 nominal species.

Body length of Hylodes cf. ornatus and Lithobates catesbeianus tadpoles, depigmentation of mouthparts, and presence of Batrachochytrium dendrobatidis are related / Tamanho do corpo, despigmentação das partes bucais e presença de Batrachochytrium dendrobatidis estão relacionados em Hylodes cf. ornatus e Lithobates catesbeianus

A fungal pathogen Batrachochytrium dendrobatidis (Bd), which can cause morbidity and death of anurans, has affected amphibian populations on a worldwide basis. Availability of pure cultures of Bd isolates is essential for experimental studies to understand the ecology of this pathogen. We evaluated the relationships of body length of Hylodes cf. ornatus and Lithobates catesbeianus tadpoles to depigmentation of mouthparts and determined if dekeratinization indicated an infection by Batrachochytrium dendrobatidis. A strong association existed for both species, one from South America (Brazil: São Paulo) and one from North America (USA: Maine). We believe it prudent not to kill adult amphibians if avoidable, thus obtaining tissue for isolating Bd from tadpoles is reasonable because infected specimens of some species can be selectively collected based on depigmentation of mouthparts.

O fungo patógeno Batrachochytrium dendrobatidis (Bd) é apontado como o causador de morbidade e morte em anuros, e tem afetado populações de anfíbios em uma base mundial. Avaliar culturas puras de isolados de Bd é essencial para estudos experimentais para o entendimento da ecologia desse patógeno. Avaliou-se a relação entre o comprimento do corpo em girinos de Hylodes cf. ornatus e Lithobates catesbeianus com a despigmentação das peças bucais, para verificar se a desqueratinização indica uma infecção por Batrachochytrium dendrobatidis. Uma forte associação existe para ambas as espécies, uma da América do Sul (Brasil: São Paulo) e uma da América do Norte (USA: Maine). Acredita-se ser prudente este uso, para evitar a morte de anfíbios adultos; dessa forma, obter tecidos para isolar o Bd de girinos é razoável, porque espécimes infectados podem ser coletados seletivamente com base na despigmentação do aparelho bucal.