Monitoring the Prevalence and Distribution of Chytrid Fungus ( <i>Batrachochytrium dendrobatidis</i> ) in the Kihansi Spray Toad ( <i>Nectophrynoides asperginis</i> ) Population in the Kihansi Gorge Spray Wetlands, Tanzania

Chytridiomycosis, caused by the fungus <i>Batrachochytrium dendrobatidis</i> (<i>Bd</i>), is fatal to some amphibian species, whereas others can carry the pathogen without developing disease. Among the vulnerable species is the Kihansi Spray Toad, <i>Nectophrynoides asperginis</i>, endemic to the Kihansi Gorge spray wetlands in Tanzania's Udzungwa Mountains. By 2009, chytridiomycosis had driven <i>N. asperginis</i> to extinction in the wild, though it survives in captivity. Re-introduction efforts have faced difficulties, underlining the importance of understanding <i>Bd's</i> prevalence in the wild to guide re-introduction programs. Twenty years after <i>Bd</i> was first detected in Tanzania's Kihansi Gorge spray wetlands, we re-evaluated its prevalence and examined whether the pathogen was responsible for the 98% mortality observed among the 1000 captive-bred <i>N. asperginis</i> released there in February 2022. In December 2022, time-constrained surveys were conducted across three spray wetlands covering <i>N. asperginis</i> habitat. Amphibians were skin-swabbed following a protocol developed by the University of California, resulting in two sets of 44 samples from seven species. These samples were analyzed for <i>Bd</i> presence using conventional and real-time quantitative polymerase chain reaction, followed by nucleotide sequencing of PCR products. <i>Bd</i> was detected in 32% of samples, representing four out of the seven species: <i>Arthroleptides yakusini</i> (14%), <i>N. asperginis</i> (9%), <i>Ptychadena anchietae</i> (7%), and <i>Hyperolius substriatus</i> (2%). Whereas <i>N. asperginis</i> was severely affected, the other species did not show signs of the disease. The other three species, namely, <i>Hyperolius mitchelli, Afrixalus fornasinii</i>, <i>and Arthroleptis xenodactyloides</i>, were not detected with <i>Bd.</i> A lineage-specific qPCR diagnostic test confirmed <i>Bd</i>-CAPE as the cause of the mass deaths of the released toads. The continuing presence of <i>Bd</i>-CAPE in the spray wetlands remains a barrier to the successful re-introduction of <i>N. asperginis</i>, necessitating further experimentation to develop strategies for coexistence.