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.

International socioeconomic relationships form the background that underlies the history of invasive species. Species with economic value, such as the North American bullfrog (Lithobates catesbeianus), are more likely to become internationally distributed and to be subsequently introduced to non-native areas and, consequently, become more difficult to control. Using population genetics methods, we investigated the invasion pathways, the connectivity among clusters in different countries and the native population of origin of globally introduced bullfrog populations. Throughout the analysis of seven microsatellite nuclear loci, one fragment of the mitochondrial cytochrome b locus, and historical information, four main lineages were identified and analyzed along with previous findings. This species’ capability to colonize several countries from few starting lineages highlights the necessity to control new propagule pressure to ensure successful management programs, as high inbreeding and bottleneck effect seem not to diminish the invasive success of this species. There is a consensus between markers that most areas of South America belong to the same genetic population while populations in Asia have a more complex history of introduction.

The host-associated microbiome plays a significant role in health. However, the roles of factors such as host genetics and microbial interactions in determining microbiome diversity remain unclear. We examined these factors using amplicon-based sequencing of 175 Thoropa taophora frog skin swabs collected from a naturally fragmented landscape in southeastern Brazil. Specifically, we examined (1) the effects of geography and host genetics on microbiome diversity and structure; (2) the structure of microbial eukaryotic and bacterial co-occurrence networks; and (3) co-occurrence between microeukaryotes with bacterial OTUs known to affect growth of the fungal frog pathogen Batrachochytrium dendrobatidis (including anti-Bd bacteria commonly referred to as “antifungal”). Microbiome structure correlated with geographic distance, and microbiome diversity varied with both overall host genetic diversity and diversity at the frog MHC IIB immunity locus. Our network analysis showed the highest connectivity when both eukaryotes and bacteria were included, implying that ecological interactions occur among Domains. Lastly, anti-Bd bacteria did not demonstrate broad negative co-occurrence with fungal OTUs in the microbiome, indicating that these bacteria are unlikely to be broadly antifungal. Our findings emphasize the importance of considering both Domains in microbiome research, and suggest that probiotic strategies for amphibian disease management should be considered with caution.

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.