Apparent Batrachochytrium dendrobatidis in the Endangered Gastrotheca chrysosticta (Anura: Hemiphractidae) in Argentina

Abstract

After 25 years without detection in Argentina, Baritu?s Marsupial Frog (Gastrotheca chrysosticta) was rediscovered in the wild in 2018. Currently listed as an Endangered species on both the IUCN and Argentinean Red Lists, this marsupial frog faces habitat loss as the main threat. The recent confirmation of Batrachochytrium dendrobatidis (Bd) infection of the closely related La Banderita Marsupial Frog (Gastrotheca gracilis) suggests that southern marsupial frogs could be susceptible to infection as well. Herein, we evaluate the presence of Bd in the only known rediscovered population of G. chrysosticta. On 27 February 2019, we collected swab samples (DELTA LAB-300201 swabs) from the oral disc of 10 tadpoles (Gosner?s stages 35?41) captured in a temporary spring at Baritú National Park, Province of Salta, Argentina (22.56301°S, 64.75219°W; 1468 m asl). Tadpoles were immediately released after swabbing. We stored the swabs in labeled refrigerated cryogenic vials until the laboratory processing. We extracted DNA using the Qiagen DNeasy Blood & Tissue commercial kit, and quantified Bd-DNA following the protocol proposed by Boyle et al. (2004). We conducted a real-time amplification (qPCR) using a StepOnePlus thermal cycler (Applied Biosystems). We performed thermal profiling of the reaction at 95°C for 20 s, then 50 cycles (90°C for 1 s, followed by 60°C for 20 s). Each plate included a negative control (UltraPure? DNase/RNase-free distilled water) and a standard curve from 0.01?1000 zoospore genome equivalents as a posi tive control for the qPCR. We ran all samples in duplicate, and we used StepOne v2.3 software (Applied Biosystems) to estimate Bd DNA loads in zoospores equivalents from the amplification AMPHIBIAN AND REPTILE DISEASES curves. We considered a Bd-positive sample if Bd DNA was amplified in at least one sample per animal. We detected Bd in one sample. However, less than one zoospore equivalent was detected. This raises uncertainty about whether this is an accurate result due to uncertainties due to the low sample size of 10 animals swabbed relative to host and pathogen population densities , and inherent uncertainties in diagnostic procedures. Additional sampling is warranted to confirm the finding. Nevertheless, this result appears to represent the first record of Bd in the endangered G. chrysosticta and the second record of Bd in the Yungas Andean forest. The presence of Bd would represent a new threat to the conservation of the Endangered G. chrysosticta, The amphibian population decline and the local extinction of several species were associated with the spread of Bd in the Southern Andes. This could be the case for the three southernmost marsupial frog species, which have suffered substantial population declines since the 1990s (i.e., G. christinani, chrysosticta, and G. gracilis;). A Bd monitoring program is warranted to assess the Bd impact on the amphibian populations in the Yungas Andean forest