Dual Detection of the Chytrid Fungi Batrachochytrium spp. with an Enhanced Environmental DNA Approach

Abstract

Environmental DNA (eDNA) is becoming an indispensable tool in biodiversity monitoring, including the monitoring of invasive species and pathogens. Aquatic chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal) are major threats to amphibians. However, the use of eDNA for detecting these pathogens has not yet become widespread, due to technological and economic obstacles. Using the enhanced eDNA approach (a simple and cheap sampling protocol) and the universally accepted qPCR assay, we confirmed the presence of Bsal and Bd in previously identified sites in Spain, including four sites that were new for Bsal. The new approach was successfully tested in laboratory conditions using manufactured gene fragments (gBlocks) of the targeted DNA sequence. A comparison of storage methods showed that samples kept in ethanol had the best DNA yield. Our results showed that the number of DNA copies in the Internal Transcribed Spacer region was 120 copies per Bsal cell. Eradication of emerging diseases requires quick and cost-effective solutions. We therefore performed cost-efficiency analyses of standard animal swabbing, a previous eDNA approach, and our own approach. The procedure presented here was evaluated as the most cost-efficient. Our findings will help to disseminate information about efforts to prevent the spread of chytrid fungi.

Batrachochytrium salamandrivorans Threat to the Iberian Urodele Hotspot

“…None of the 1395 samples, collected between 2015 and 2021, were positive for Bsal ( Figure 1 ). An intensive sampling effort was conducted in northern Spain (Asturias and southwestern Galicia) due to recent reports of Bsal- positive qPCRs from Asturias and its neighbor Cantabria [ 13 , 14 ]. However, Bsal was not detected in any of the 326 samples collected in this region.…”

“…The first report [ 14 ] mentions five positive skin swabs in palmate newts ( Lissotriton helveticus ). This study was followed by a second study sampling water bodies for eDNA, which yielded a high proportion of qPCR positive samples [ 13 ]. Despite our intensive sampling effort, we did not succeed in confirming these results in this region.…”

“…Between 2015–2021, we opportunistically sampled 66 populations of 10 Iberian urodele species, with a total of 1395 individuals sampled ( Table S1 ). Recent reports of Bsal -positive qPCRs from Asturias [ 13 , 14 ] resulted in more intensive sampling efforts in this region, where we resampled two localities previously reported as positive. The vast majority of samples derived from surveys of live urodeles, with 21 samples from dead animals.…”

“…For all species, five or six adult specimens were inoculated with 10 3 zoospores of the Bsal type strain AMFP13/1 using an established protocol [ 11 ]. In addition, given recent positive qPCR results from the Palmate newt ( Lissotriton helveticus ) in northern Spain [ 13 , 14 ], we also infected 10 captive bred palmate newts with two additional Bsal isolates (isolates AMFP14/1 and AMFP15/1; 5 animals per isolate) to study infection dynamics in this species. An earlier infection trial has indicated that L. helveticus has low susceptibly to Bsal infection [ 11 ].…”

“…In 2018, a disjunct Bsal outbreak was detected in the Iberian Peninsula in northeastern Spain, nearby to Europe’s most threatened newt species, the Montseny brook newt ( Calotriton arnoldi ) [ 12 ]. Two other recent reports have reported positive qPCR results in north central Spain [ 13 , 14 ]. With seven genera, ten species and a high number of small-range endemic lineages, the Iberian Peninsula is a hotspot of diversification of the family Salamandridae.…”