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

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