Time capsules of infection: Updating the distribution of Aphanomyces astaci in Croatia using archived crayfish specimens

The crayfish plague pathogen Aphanomyces astaci Schikora, 1906 is one of the most damaging invasive pathogens affecting European freshwater ecosystems, causing declines in native crayfish populations. In Croatia, crayfish plague was first reported in the late 19 th century and was subsequently molecularly confirmed in 2016. However, its national distribution remains incompletely resolved because the initial molecular survey covered only a limited number of localities. To obtain an initial country-scale distribution of A. astaci , ethanol-preserved specimens of all seven crayfish species present in the country, collected across Croatia between 1998 and 2025, were analyzed. Tissue samples were screened for A. astaci using quantitative PCR (qPCR), and positive results were confirmed by Sanger sequencing. Genotype groups were assessed using pentaplex qPCR, with microsatellite genotyping and mitochondrial haplotyping applied to samples with sufficient pathogen loads. Of the 238 samples, 91 (38.2%) were A. astaci -positive. Pathogen presence was confirmed in both the Black Sea and Adriatic Sea drainages and across all investigated host species, extending its known distribution beyond previously surveyed localities. The integration of different genotyping methods yielded 11 interpretable consensus genotype profiles, including genotype groups A and B, SSR-UP-like, and a putatively novel genotype profile. However, additional genotypes may have remained undetected, for instance, in positive samples with low pathogen loads from Faxonius limosus , an invasive host widespread in Croatia and a typical carrier of genotype group E. Overall, the study shows that A. astaci is a widespread, long-established, and genetically diverse pathogen in Croatia, highlighting the need for coordinated, genotype-resolved national monitoring focused on invasive crayfish reservoirs and threatened indigenous populations.