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Background: Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are two pathogenic fungi that are a significant threat to amphibian communities worldwide. European populations are strongly impacted and the monitoring of the presence and spread of these pathogens is crucial for efficient decision-making in conservation management., Results: Here we proposed an environmental DNA (eDNA) monitoring of these two pathogenic agents through droplet digital PCR (ddPCR) based on water samples from 24 ponds in Luxembourg. In addition, amphibians were swabbed in eight of the targeted ponds in order to compare the two approaches at site-level detection. This study allowed the development of a new method taking below-Limit of Detection (LOD) results into account thanks to the statistical comparison of the frequencies of false positives in no template controls (NTC) and below-LOD results in technical replicates. In the eDNA-based approach, the use of this method led to an increase in Bd and Bsal detection of 28 and 50% respectively. In swabbing, this resulted in 8% more positive results for Bd. In some samples, the use of technical replicates allowed to recover above-LOD signals and increase Bd detection by 35 and 33% respectively for eDNA and swabbing, and Bsal detection by 25% for eDNA., Conclusions: These results confirmed the usefulness of technical replicates to overcome high levels of stochasticity in very low concentration samples even for a highly sensitive technique such as ddPCR. In addition, it showed that below-LOD signals could be consistently recovered and the corresponding amplification events assigned either to positive or negative detection via the method developed here. This methodology might be particularly worth pursuing in pathogenic agents' detection as false negatives could have important adverse consequences. In total, 15 ponds were found positive for Bd and four for Bsal. This study reports the first record of Bsal in Luxembourg., (© 2023. The Author(s).)

Background: Important information on movement pathways and introduction routes of invasive parasites can be obtained by comparing the genetic makeup of an invader with its spatial genetic structure in other distribution areas. Sometimes, the population genetic structure of the host might be more informative than that of the parasite itself, and it is important to collect tissue samples of both host and parasite. However, host tissue samples are frequently not available for analysis. We aimed to test whether it is possible to generate reliable microsatellite profiles of host individuals by amplifying DNA extracted from a nematode parasite, using the raccoon (Procyon lotor) and the raccoon roundworm (Baylisascaris procyonis) as a test case., Methods: Between 2020 and 2021, we collected tissue as well as a single roundworm each from 12 raccoons from central Germany. Both the raccoon and the roundworm DNA extracts were genotyped using 17 raccoon-specific microsatellite loci. For each roundworm DNA extract, we performed at least eight amplification reactions per microsatellite locus., Results: We extracted amplifiable raccoon DNA from all 12 roundworms. We obtained at least two amplification products for 186 of the 204 possible genotypes. Altogether 1077 of the 1106 genotypes (97.4%) matched the host-DNA derived reference genotypes and thus did not contain genotyping errors. Nine of the 12 roundworm-derived genetic profiles matched the reference profiles from the raccoon hosts, with one additional genetic profile containing genotyping errors at a single locus. The remaining two genetic profiles were deemed unsuitable for downstream analysis because of genotyping errors and/or a high proportion of missing data., Conclusions: We showed that reliable microsatellite-based genetic profiles of host individuals can be obtained by amplifying DNA extracted from a parasitic nematode. Specifically, the approach can be applied to reconstruct invasion pathways of roundworms when samples of the raccoon hosts are lacking. Further research should assess whether this method can be replicated in smaller species of parasitic nematodes and other phyla of parasites more generally., (© 2023. The Author(s).)