Batrachochytrium dendrobatidis detection by Real-Time and Endpoint PCR in two species of frogs that inhabit in the Quitos Metropolitan Guangüiltagua Park. The First Confirmed Report of Chytridiomycosis in an Urban Park located in Ecuador
Alexander Genoy-PuertoEl Gaff
David A. Narváez-Narváez, Alejandro Cabrera-Andrade, Andrés Merino-Viteri, César Paz-y-Miño, Germán Burgos, Alexander Genoy-Puerto
Batrachochytrium dendrobatidis (Bd) is one of the principal causes of amphibian declination worldwide. A previous sample (not reported) determined the presence of Bd in tadpoles of Gastrotheca riobambae that inhabits in ponds of Quitos Metropolitan Guangüiltagua Park (Ecuador). This study sought to determine whether the tadpoles are still infected, and principally determine the presence of chytridiomycosis in another frog that also inhabits the park, Pristimantis unistrigatus. We used two types of PCR's molecular techniques (real-time and end-point) for the diagnosis of Bd, in order to establish their effectiveness and differences in detection of fungal DNA. To compare between invasive and non-invasive techniques, only tadpoles were euthanized with an overdose of MS-222 to obtain their mouth tissue. Finally, environmental variables were evaluated (iButton® temperature loggers DS1922L). For a period of one-year samples were taken (through swabs) of the skin of P. unistrigatus and mouthparts of tadpoles. Standard curves were generated from different plasmids concentrations to determine the quantity of zoospores/µL. We found that the two species were infected. From 382 individuals of P. unistrigatus, 57 were positive for Bd (prevalence of 15%). Likewise, between 135 individuals of G. riobambae, 53 (39% prevalence) were positive. The individual with the largest amount of zoospores had 7556 zoospores/µL and an infection level of 459 zoospores/mm in the population of P. unistrigatus studied. The two types of sample (tissue and swabs) from mouthparts showed differences in the zoospores/µL loads. We found a correlation of the maximum mean monthly temperature in both, the pond and the environment, with the levels of Bd infection and disease prevalence of the two species. The technique of RT-PCR showed a different diagnostic sensitivity of PCR. Possibly individuals of P. unistigatus are somehow in contact with the fungus through water from the pond or juveniles and adults infected of G. riobambae.