Luisa Ribeiro

and 4 more

The international amphibian trade raises concerns regarding its impact on native populations due to the potential introduction of different lineages of the chytrid fungus (Batrachochytrium dendrobatidis or Bd) into new environments. Current diagnostic methods, particularly qPCR, lack the capability to differentiate between these lineages, necessitating more accurate genotyping approaches. Here we propose the application of TaqMan SNP genotyping assays to discriminate Bd genotypes (Bd-GPL, Bd-ASIA2/Bd-BRAZIL [hereafter Bd-BRAZIL], and Hybrid) in samples from Brazilian bullfrog farms. We collected samples, including skin swabs, tadpole mouthparts, and pure Bd cultures, from bullfrog farms across Brazil. Employing two assays utilizing both qPCR and dPCR, we identified genotype presence and analyzed the relationship between Bd load and genotype determination. The genotyping assay achieved a 56.6 % success rate, notably improving with higher Bd loads, reaching 81.8 % for loads over 1,000 genomic equivalents. Culture samples achieved a 100 % success rate. We identified all Bd genotypes in the bullfrog farms, highlighting the issue of coinfections and hybrids in densely populated farms. We hereby present an efficient method for discriminating Bd genotypes, applicable to both pure cultures and field samples with low Bd loads. We emphasize the need for advanced discriminatory methods and comprehensive genetic studies, particularly regarding national regulations governing breeding sites and the global amphibian trade. Our research underscores the feasibility and significance of the proposed method and advocates for further investigations into infection dynamics by different Bd lineages to inform amphibian conservation efforts and trade regulatory policies.

Anat Belasen

and 4 more

Anat Belasen

and 5 more

Habitat fragmentation and infectious disease threaten amphibians globally, but little is known about how these two threats interact. In this study, we examined the effects of Brazilian Atlantic Forest habitat fragmentation on frog genetic diversity at an immune locus known to affect disease susceptibility in amphibians, the MHC IIB locus. We used a custom high-throughput assay to sequence the MHC IIB locus across six focal frog species in two regions of the Atlantic Forest. We also used a molecular assay to quantify infections by the fungal pathogen Batrachochytrium dendrobatidis (Bd). We found that habitat fragmentation is associated with genetic erosion at the MHC IIB locus, and that this erosion is most severe in frog species restricted to intact forests. Significant Bd infections were recovered only in one Atlantic Forest region, potentially due to the relatively higher elevation. In this region, forest specialists showed an increase in both Bd prevalence and loads in fragmented habitats. We also found that reduced population-level MHC IIB diversity was associated with increased Bd infection risk. On the individual-level, MHC IIB heterozygotes (by allelic genotype as well as supertype) exhibited a reduced risk of Bd infection. Our results suggest that habitat fragmentation increases infection susceptibility in amphibians, mediated at least in part through loss of immunogenetic diversity. Our findings have implications for the conservation of fragmented populations in the face of emerging infectious diseases.