Opening Pandora's box: First insights into the genetic diversity of Aphanomyces astaci in Switzerland.

Aphanomyces astaci, the etiological agent of crayfish plague, varies in virulence depending on their genetic strains. Five genotype groups (A-E) are well characterized and, with exception of genotype group A, cause significant mortality in European freshwater crayfish species. In this study, we aimed to investigate the presence and diversity of A. astaci genotype groups in Switzerland by adapting the qPCR assays (Simplex-qPCR and Duplex-qPCR) published by Di Domenico et al. (2021) to a Pentaplex-qPCR assay. The Pentaplex-qPCR was compared to Simplex-qPCR, Duplex-qPCR and Triplex-qPCR assays using artificial DNA fragments, culture and tissue samples. We performed the Pentaplex-qPCR on DNA extracted from formalin-fixed, paraffin-embedded tissue samples (1991-2020) and fresh tissue samples (2020-2024) of crayfish collected in Switzerland. Crayfish were mainly native crayfish species undergoing a crayfish plague outbreak and few invasive crayfish tested positive for A. astaci. The intra-assay repeatability and inter-assay reproducibility of the qPCR was assessed. The Pentaplex-qPCR assay was not inferior to the other qPCR assays (Simplex-qPCR, Duplex-qPCR, Triplex-qPCR) and reliably detected genotype groups A, B, D, and E in 31 of 45 crayfish populations (68.9%) across Switzerland. Most positive samples were linked to crayfish plague outbreaks in European crayfish species, except for four populations, where genotype groups B and D were found in North American signal crayfish (Pacifastacus leniusculus) and Louisiana red swamp crayfish (Procambarus clarkii), respectively. Genotype group C was not detected in any of the samples. Genotype group occurrence and heterogeneity were highest in the Rhine basin, particularly between 2016 and 2020, where groups A, B, D, and E were all detected. In the Ticino basin, genotype group diversity was high and genotype groups B, D and E were detected, while in the Rhone basin, only genotype group B was found. Genotype group D, typically associated with Louisiana red swamp crayfish, was identified in the Rhine basin in 1991 and suspected in 1994, respectively four and one year before the first known detection of its carrier species. Genotype group A was detected once in 2017 in the Rhine basin, causing limited mortality in a European crayfish population. The use of archived samples revealed a long history of A. astaci presence, dating back to 1991, providing valuable insights into the spatiotemporal dynamics of crayfish plague. Identifying genotype groups helps clarify potential routes of introduction for both pathogen genotype groups and carrier host species. Furthermore, it facilitates tracking outbreaks and identifying possible primary sources of spread. Future studies are needed to deepen our understanding of the pathogen's genetic variability in relation to virulence differences and spread.