External Reinfection of a Fungal Pathogen Does not Contribute to Pathogen Growth.

Chytridiomycosis is an emerging infectious disease of amphibians caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which has led to devastating declines in amphibian populations worldwide. Current theory predicts that Bd infections are maintained through both reproduction on the host's skin and reinfection from sources outside of the host. To investigate the importance of external reinfection on pathogen burden, we infected captive-bred individuals of the highly susceptible Panamanian Golden Frog, Atelopus glyphus, and wild-caught glass frogs, Espadarana prosoblepon, with Bd. We housed the animals in one of three treatments: individually, in heterospecific pairs, and in conspecific pairs. For 8 weeks, we measured the Bd load and shedding rate of all frogs. We found that Atelopus had high rates of increase in both Bd load and shedding rate, but pathogen growth rates did not differ among treatments. The infection intensity of Espadarana co-housed with Atelopus was indistinguishable from those housed singly and those in conspecific pairs, despite being exposed to a large external source of Bd zoospores. Our results indicate that Bd load in both species is driven by pathogen replication within an individual, with reinfection from outside the host contributing little to the amplification of host fungal load.

Prodigiosin, Violacein, and Volatile Organic Compounds Produced by Widespread Cutaneous Bacteria of Amphibians Can Inhibit Two Batrachochytrium Fungal Pathogens.

Symbiotic bacteria can produce secondary metabolites and volatile compounds that contribute to amphibian skin defense. Some of these symbionts have been used as probiotics to treat or prevent the emerging disease chytridiomycosis. We examined 20 amphibian cutaneous bacteria for the production of prodigiosin or violacein, brightly colored defense compounds that pigment the bacteria and have characteristic spectroscopic properties making them readily detectable, and evaluated the antifungal activity of these compounds. We detected violacein from all six isolates of Janthinobacterium lividum on frogs from the USA, Switzerland, and on captive frogs originally from Panama. We detected prodigiosin from five isolates of Serratia plymuthica or S. marcescens, but not from four isolates of S. fonticola or S. liquefaciens. All J. lividum isolates produced violacein when visibly purple, while prodigiosin was only detected on visibly red Serratia isolates. When applied to cultures of chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), prodigiosin caused significant growth inhibition, with minimal inhibitory concentrations (MIC) of 10 and 50 µM, respectively. Violacein showed a MIC of 15 µM against both fungi and was slightly more active against Bsal than Bd at lower concentrations. Although neither violacein nor prodigiosin showed aerosol activity and is not considered a volatile organic compound (VOC), J. lividum and several Serratia isolates did produce antifungal VOCs. White Serratia isolates with undetectable prodigiosin levels could still inhibit Bd growth indicating additional antifungal compounds in their chemical arsenals. Similarly, J. lividum can produce antifungal compounds such as indole-3-carboxaldehyde in addition to violacein, and isolates are not always purple, or turn purple under certain growth conditions. When Serratia isolates were grown in the presence of cell-free supernatant (CFS) from the fungi, CFS from Bd inhibited growth of the prodigiosin-producing isolates, perhaps indicative of an evolutionary arms race; Bsal CFS did not inhibit bacterial growth. In contrast, growth of one J. lividum isolate was facilitated by CFS from both fungi. Isolates that grow and continue to produce antifungal compounds in the presence of pathogens may represent promising probiotics for amphibians infected or at risk of chytridiomycosis. In a global analysis, 89% of tested Serratia isolates and 82% of J. lividum isolates were capable of inhibiting Bd and these have been reported from anurans and caudates from five continents, indicating their widespread distribution and potential for host benefit.

Epizootic to enzootic transition of a fungal disease in tropical Andean frogs: Are surviving species still susceptible?

The fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis, has been linked to catastrophic amphibian declines throughout the world. Amphibians differ in their vulnerability to chytridiomycosis; some species experience epizootics followed by collapse while others exhibit stable host/pathogen dynamics where most amphibian hosts survive in the presence of Bd (e.g., in the enzootic state). Little is known about the factors that drive the transition between the two disease states within a community, or whether populations of species that survived the initial epizootic are stable, yet this information is essential for conservation and theory. Our study focuses on a diverse Peruvian amphibian community that experienced a Bd-caused collapse. We explore host/Bd dynamics of eight surviving species a decade after the mass extinction by using population level disease metrics and Bd-susceptibility trials. We found that three of the eight species continue to be susceptible to Bd, and that their populations are declining. Only one species is growing in numbers and it was non-susceptible in our trials. Our study suggests that some species remain vulnerable to Bd and exhibit ongoing population declines in enzootic systems where Bd-host dynamics are assumed to be stable.

Phylogenetic distribution of symbiotic bacteria from Panamanian amphibians that inhibit growth of the lethal fungal pathogen Batrachochytrium dendrobatidis.

The introduction of next-generation sequencing has allowed for greater understanding of community composition of symbiotic microbial communities. However, determining the function of individual members of these microbial communities still largely relies on culture-based methods. Here, we present results on the phylogenetic distribution of a defensive functional trait of cultured symbiotic bacteria associated with amphibians. Amphibians are host to a diverse community of cutaneous bacteria and some of these bacteria protect their host from the lethal fungal pathogen Batrachochytrium dendrobatidis (Bd) by secreting antifungal metabolites. We cultured over 450 bacterial isolates from the skins of Panamanian amphibian species and tested their interactions with Bd using an in vitro challenge assay. For a subset of isolates, we also completed coculture experiments and found that culturing isolates with Bd had no effect on inhibitory properties of the bacteria, but it significantly decreased metabolite secretion. In challenge assays, approximately 75% of the bacterial isolates inhibited Bd to some extent and these inhibitory isolates were widely distributed among all bacterial phyla. Although there was no clear phylogenetic signal of inhibition, three genera, Stenotrophomonas, Aeromonas and Pseudomonas, had a high proportion of inhibitory isolates (100%, 77% and 73%, respectively). Overall, our results demonstrate that antifungal properties are phylogenetically widespread in symbiotic microbial communities of Panamanian amphibians and that some functional redundancy for fungal inhibition occurs in these communities. We hope that these findings contribute to the discovery and development of probiotics for amphibians that can mitigate the threat of chytridiomycosis.

Survey of helminths, ectoparasites, and chytrid fungus of an introduced population of cane toads, Rhinella marina (Anura: Bufonidae), from Grenada, West Indies.

One hundred specimens of Rhinella marina , (Anura: Bufonidae) collected in St. George's parish, Grenada, from September 2010 to August 2011, were examined for the presence of ectoparasites and helminths. Ninety-five (95%) were parasitized by 1 or more parasite species. Nine species of parasites were found: 1 digenean, 2 acanthocephalans, 4 nematodes, 1 arthropod and 1 pentastome. The endoparasites represented 98.9% of the total number of parasite specimens collected. Grenada represents a new locality record for Mesocoelium monas, Raillietiella frenatus, Pseudoacanthacephalus sp., Aplectana sp., Physocephalus sp., Acanthacephala cystacanth, and Physalopteridae larvae. The digenean M. monas occurred with the highest prevalence of 82%, contrasting many studies of R. marina where nematodes dominate the parasite infracommunity. Female toads were found to have a significantly higher prevalence of Amblyomma dissimile than male toads. Only 2 parasites exhibited a significant difference between wet and dry season with Parapharyngodon grenadensis prevalence highest in the wet season and A. dissimile prevalence highest during the dry season. Additionally, A. dissimile was significantly more abundant during the dry season.

Thermal physiology, disease, and amphibian declines on the eastern slopes of the Andes.

Rising temperatures, a widespread consequence of climate change, have been implicated in enigmatic amphibian declines from habitats with little apparent human impact. The pathogenic fungus Batrachochytrium dendrobatidis (Bd), now widespread in Neotropical mountains, may act in synergy with climate change causing collapse in thermally stressed hosts. We measured the thermal tolerance of frogs along a wide elevational gradient in the Tropical Andes, where frog populations have collapsed. We used the difference between critical thermal maximum and the temperature a frog experiences in nature as a measure of tolerance to high temperatures. Temperature tolerance increased as elevation increased, suggesting that frogs at higher elevations may be less sensitive to rising temperatures. We tested the alternative pathogen optimal growth hypothesis that prevalence of the pathogen should decrease as temperatures fall outside the optimal range of pathogen growth. Our infection-prevalence data supported the pathogen optimal growth hypothesis because we found that prevalence of Bd increased when host temperatures matched its optimal growth range. These findings suggest that rising temperatures may not be the driver of amphibian declines in the eastern slopes of the Andes. Zoonotic outbreaks of Bd are the most parsimonious hypothesis to explain the collapse of montane amphibian faunas; but our results also reveal that lowland tropical amphibians, despite being shielded from Bd by higher temperatures, are vulnerable to climate-warming stress.

Surviving chytridiomycosis: differential anti-Batrachochytrium dendrobatidis activity in bacterial isolates from three lowland species of Atelopus.

In the Neotropics, almost every species of the stream-dwelling harlequin toads (genus Atelopus) have experienced catastrophic declines. The persistence of lowland species of Atelopus could be explained by the lower growth rate of Batrachochytrium dendrobatidis (Bd) at temperatures above 25 °C. We tested the complementary hypothesis that the toads' skin bacterial microbiota acts as a protective barrier against the pathogen, perhaps delaying or impeding the symptomatic phase of chytridiomycosis. We isolated 148 cultivable bacterial strains from three lowland Atelopus species and quantified the anti-Bd activity through antagonism assays. Twenty-six percent (38 strains representing 12 species) of the bacteria inhibited Bd growth and just two of them were shared among the toad species sampled in different localities. Interestingly, the strongest anti-Bd activity was measured in bacteria isolated from A. elegans, the only species that tested positive for the pathogen. The cutaneous bacterial microbiota is thus likely a fitness-enhancing trait that may (adaptation) or not (exaptation) have appeared because of natural selection mediated by chytridiomycosis. Our findings reveal bacterial strains for development of local probiotic treatments against chytridiomycosis and also shed light on the mechanisms behind the frog-bacteria-pathogen interaction.

How does chytrid infection vary among habitats? The case of Litoria wilcoxii (Anura, Hylidae) in SE Queensland, Australia.

Most analyses dealing with the geographical distribution of the chytrid fungus (Batrachochytrium dendrobatidis) have been performed on large geographical scales and data on more localized distribution of the chytrid within catchments are scarce. In this study, we compare the prevalence and intensity of infection of chytrid within and outside rainforest habitats at five independent catchments in southeast Queensland. In each catchment, we sampled adult Litoria wilcoxii along two transects on the same stream: one in forested areas, and the other in open nearby farmland. We analyzed swabs using quantitative PCR techniques. Male frogs were in higher densities in open habitats compared with the nearby forested areas. Infected male frogs were found in all catchments surveyed; however, prevalence of B. dendrobatidis in adult males was higher in the forested habitats than in the open habitats in four of the catchments. There was no significant difference in intensity of infection between forested and open habitats. For adult females and juveniles, sample sizes were not high enough for comparisons. Our results suggest that habitat influences chytrid prevalence and open areas may provide refuge from chytrid-induced population declines.

Developmental regulation of expression of the regulatory subunit of the cAMP-dependent protein kinase of Blastocladiella emersonii.

A monospecific polyclonal antiserum to the regulatory subunit (R) of the cAMP-dependent protein kinase of Blastocladiella emersonii has been developed by immunization with purified regulatory subunit. In Western blots, the antiserum displays high affinity and specificity for the intact R monomer of Mr = 58,000, as well as for its proteolytic products of Mr = 43,000 and Mr = 36,000, even though the antiserum has been raised against the Mr = 43,000 fragment. Western blots of cell extracts prepared at different times during the life cycle of the fungus indicate that the increase in cAMP-binding activity occurring during sporulation, as well as its decrease during germination, are associated with the accumulation of the regulatory subunit during sporulation and its disappearance during germination, respectively. Pulse labeling with [35S]methionine and immunoprecipitation indicate that the accumulation of R is due to its increased synthesis during sporulation. Two-dimensional gel electrophoresis of affinity purified cell extracts obtained after [35S]methionine pulse labeling during sporulation confirms de novo synthesis of R during this stage and furthermore shows that the protein is rapidly phosphorylated after its synthesis. In vitro translation studies using RNA isolated from different stages of the life cycle followed by immunoprecipitation have shown that the time course of expression of the mRNA coding for the regulatory subunit parallels the rate of its synthesis in vivo.