Population genetics of the frog-killing fungus Batrachochytrium dendrobatidis

Global amphibian decline by chytridiomycosis is a major environmental disaster that has been attributed to either recent fungal spread or environmental change that promotes disease. Here, we present a population genetic comparison of Batrachochytrium dendrobatidis isolates from an intensively studied region of frog decline, the Sierra Nevada of California. In support of a novel pathogen, we find low diversity, no amphibian-host specificity, little correlation between fungal genotype and geography, local frog extirpation by a single fungal genotype, and evidence of human-assisted fungus migration. In support of endemism, at a local scale, we find some diverse, recombining populations. Therefore neither epidemic spread nor endemism alone explains this particular amphibian decline. Recombination raises the possibility of resistant sporangia and a mechanism for rapid spread as well as persistence that could greatly complicate global control of the pathogen.

Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay

Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.

Interaction between breeding habitat and elevation affects prevalence but not infection intensity of Batrachochytrium dendrobatidis in Brazilian anuran assemblages

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Batrachochytrium dendrobatidis in amphibians confiscated from illegal wildlife trade and used in an ex situ breeding program in Brazil

This paper describes an outbreak of chytridiomycosis affecting a group of Dendrobates tinctorius, a Neotropical anuran species, confiscated from the illegal wildlife trade and housed in a private zoo in Brazil as part of an ex situ breeding program. We examined histological sections of the skin of 30 D. tinctorius and 20 Adelphobates galactonotus individuals. Twenty D. tinctorius (66.7%) and none of the A. galactonotus were positive for Batrachochytrium dendrobatidis (Bd). Multiple development stages of Bd infection were observed. The reasons for the interspecific difference in the rate of infection could not be determined, and further studies are advised. Because the examined population consisted of confiscated frogs, detailed epidemiological aspects could not be investigated, and the source of the fungus remains uncertain. The existence of ex situ amphibian populations is important for protecting species at higher risk in the wild, and ex situ amphibian conservation and breeding programs in Brazil may be established using confiscated frogs as founders. However, this paper alerts these programs to the urgency of strict quarantine procedures to prevent the introduction of potential pathogens, particularly Bd, into ex situ conservation programs.

Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay

Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.

Promoting Amphibian Conservation through the College Classroom: Detection of Batrachochytrium dendrobatidis among Local Amphibians

Many global amphibian declines have been linked to the fungal pathogen Batrachochytrium dendrobatidis (Bd). The knowledge on Bd distribution provides a fundamental basis for amphibian conservation planning. Yet, such Bd distribution information is currently insufficient, in particular at a regional scale. The college classroom provides an excellent opportunity to expand the knowledge of Bd distribution. Here we provide an example of such research projects to detect Bd prevalence among local amphibians in a college course setting and present the results of work conducted in central Pennsylvania, USA. We collected toe clips and conducted PCR assays of six species, Plethodon cinereus, Desmognathus fuscus, Notophthalmus viridescens, Lithobates catesbeianus, L. clamitans, and L. sylvaticus (59 individuals). Four groups of students independently conducted entire projects, orally presented their findings, and submitted manuscripts to the professor at the end of the semester. This example demonstrates that it is feasible for an undergraduate class to complete a Bd-detection project within a single semester. Such a project not only contributes to Bd research but also promotes conservation education among students through hands-on research experiences. We found Bd infection in only one sample of N. viridescens, but no sign of infection in the rest of the samples. As a relatively high prevalence of Bd has been reported in surrounding areas, our results suggest spatial heterogeneity in Bd occurrence at a regional scale and thus, the need for continued efforts to monitor Bd prevalence.

Accumulation of the pathogenic fungus Batrachochytrium dendrobatidis on the regressing tail of midwife toads Alytes obstetricans undergoing metamorphosis

Batrachochytrium dendrobatidis (Bd) is a fungus infecting the skin of amphibians. On metamorphosing animals the infection is difficult to detect because of the limited information concerning the location of Bd on the animals during this stage. Histological investigation revealed that Bd accumulated on the reabsorbing tail of metamorphosing animals. This observation may facilitate the detection of Bd in metamorphosing amphibians.

Tadpole mouthpart depigmentation as an accurate indicator of chytridiomycosis, an emerging disease of amphibians

Chytridiomycosis is an emerging infectious disease of amphibians caused by the fungal pathogen Batrachochytrium dendrobatidis, and its role in causing population declines and species extinctions worldwide has created an urgent need for methods to detect it. Several reports indicate that in anurans chytridiomycosis can cause the depigmentation of tadpole tnouthparts, but the accuracy of using depigmentation to determine disease status remains uncertain. Our objective was to determine for the Mountain Yellow-legged Frog (Rana muscosa) whether visual inspections of the extent of tadpole mouthpart depigmentation could be used to accurately categorize individual tadpoles or R. muscosa populations as B. dendrobatidis-positive or negative. This was accomplished by assessing the degree of mouthpart depigmentation in tadpoles of known disease status (based on PCR assays). The depigmentation of R. muscosa tadpole mouthparts was associated with the presence of B. dendrobatidis, and this association was particularly strong for upper jaw sheaths. Using a rule that classifies tadpoles with upper jaw sheaths that are 100% pigmented as uninfected and those with jaw sheaths that are <100% pigmented as infected resulted in the infection status of 86% of the tadpoles being correctly classified. By applying this rule to jaw sheath pigmentation scores averaged across all tadpoles inspected per site, we were able to correctly categorize the infection status of 92% of the study populations. Similar research on additional anurans is critically needed to determine how broadly applicable our results for R. muscosa are to other species.

Rapid Global Expansion of the Fungal Disease Chytridiomycosis into Declining and Healthy Amphibian Populations.

The fungal disease chytridiomycosis, caused by Batrachochytrium dendrobatidis, is enigmatic because it occurs globally in both declining and apparently healthy (non-declining) amphibian populations. This distribution has fueled debate concerning whether, in sites where it has recently been found, the pathogen was introduced or is endemic. In this study, we addressed the molecular population genetics of a global collection of fungal strains from both declining and healthy amphibian populations using DNA sequence variation from 17 nuclear loci and a large fragment from the mitochondrial genome. We found a low rate of DNA polymorphism, with only two sequence alleles detected at each locus, but a high diversity of diploid genotypes. Half of the loci displayed an excess of heterozygous genotypes, consistent with a primarily clonal mode of reproduction. Despite the absence of obvious sex, genotypic diversity was high (44 unique genotypes out of 59 strains). We provide evidence that the observed genotypic variation can be generated by loss of heterozygosity through mitotic recombination. One strain isolated from a bullfrog possessed as much allelic diversity as the entire global sample, suggesting the current epidemic can be traced back to the outbreak of a single clonal lineage. These data are consistent with the current chytridiomycosis epidemic resulting from a novel pathogen undergoing a rapid and recent range expansion. The widespread occurrence of the same lineage in both healthy and declining populations suggests that the outcome of the disease is contingent on environmental factors and host resistance.

Conservation, duplication, and loss of the Tor signaling pathway in the fungal kingdom.

BACKGROUND: The nutrient-sensing Tor pathway governs cell growth and is conserved in nearly all eukaryotic organisms from unicellular yeasts to multicellular organisms, including humans. Tor is the target of the immunosuppressive drug rapamycin, which in complex with the prolyl isomerase FKBP12 inhibits Tor functions. Rapamycin is a gold standard drug for organ transplant recipients that was approved by the FDA in 1999 and is finding additional clinical indications as a chemotherapeutic and antiproliferative agent. Capitalizing on the plethora of recently sequenced genomes we have conducted comparative genomic studies to annotate the Tor pathway throughout the fungal kingdom and related unicellular opisthokonts, including Monosiga brevicollis, Salpingoeca rosetta, and Capsaspora owczarzaki. RESULTS: Interestingly, the Tor signaling cascade is absent in three microsporidian species with available genome sequences, the only known instance of a eukaryotic group lacking this conserved pathway. The microsporidia are obligate intracellular pathogens with highly reduced genomes, and we hypothesize that they lost the Tor pathway as they adapted and streamlined their genomes for intracellular growth in a nutrient-rich environment. Two TOR paralogs are present in several fungal species as a result of either a whole genome duplication or independent gene/segmental duplication events. One such event was identified in the amphibian pathogen Batrachochytrium dendrobatidis, a chytrid responsible for worldwide global amphibian declines and extinctions. CONCLUSIONS: The repeated independent duplications of the TOR gene in the fungal kingdom might reflect selective pressure acting upon this kinase that populates two proteinaceous complexes with different cellular roles. These comparative genomic analyses illustrate the evolutionary trajectory of a central nutrient-sensing cascade that enables diverse eukaryotic organisms to respond to their natural environments.

Do green and golden bell frogs (Litoria aurea) occupy habitats with fungicidal properties?

The Green and Golden Bell frog Litoria aureo is in major decline in Australia, where its distribution is now confined mainly to the east coast of New South Wales (NSW). Infection by the newly emerged amphibian fungal pathogen Batrachochytrium dendrobatidis has been identified as one of the main threats affecting L. aurea. Surprisingly, some of the sites in NSW sustaining the largest populations of this species are industrial and urban habitats that are often disturbed and polluted, which could protect L. aurea from chytrid infection if pollution had fungicidal capacity.The aim of this study was to characterise the trace metal concentration of several L. aurea breeding sites in the Sydney and IIlawarra regions of NSW and to evaluate the fungicidal efficacy of the main "'ace metals identified. Selected L. aurea sites were sampled throughout the breeding season (September to February) to establish the concentration of trace metals in both surface sediment and waters. Physico-chemical parameters including pH and salinity were also measured. Of the trace metals identified, copper and zinc were consistently elevated across sites. Over 50% of sites exceeded the National Sediment Quality Guideline for both copper and zinc concentration, and over 90% of sites exceeded the National Water Quality Guideline for these metals. Consequently, we evaluated their effect on the growth and survival of a laboratory culture of B. dendrobatidis,These tests were performed in media containing dissolved metal concentrations of 0.02 - 0.65 mgL^-1 Cu and 0.24 - 5.0 mgL^-1 Zn. Growth rates were inferred by total fungal density in liquid culture (based on spectral absorbance measurements), final dry weight, and the density of zoospores in fungal cultures grown for 28 days. Both copper and zinc were found to reduce the growth and proliferation of B. dendrobatidis, but in a non-linear manner. This suggests that L. aurea may be gaining some protection from B. dendrobatidis infection at several of the sites examined.

Long-term endemism of two highly divergent lineages of the amphibian-killing fungus in the Atlantic Forest of Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Partitioning the net effect of host diversity on an emerging amphibian pathogen

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)