Caterina Francesconi

• European crayfish species are considered keystone in freshwater ecosystems. As such, their conservation is of paramount importance to prevent biodiversity decline and loss of ecosystem function. Unfortunately, today, European crayfish species are among the most threatened crayfish species worldwide. An especially relevant threat is represented by the invasive pathogen Aphanomyces astaci. This oomycete, native of North America, has been one of the main causes of crayfish population declines across Europe since its first introduction 150 years ago, to the point of causing the local extinction of many populations. Over the years, several introductions of A. astaci strains into Europe took place through translocation of infected North American crayfish, and were followed by mass mortalities across European crayfish populations. However, in the past 20 years, more and more reports emerged of European crayfish populations surviving A. astaci infections or being latently infected with the pathogen. The survival of infected crayfish can be ascribed to both increased resistance of some crayfish populations and decreased virulence of some A. astaci strains. As the relationship between host and pathogen in Europe is changing, it is imperative to gain insights on what shapes these changes to understand the implications for the long-term coexistence of crayfish and A. astaci in Europe. With this thesis, I focused on the virulence of A. astaci, looking for mechanisms, patterns and determinants underlying the pathogen’s virulence variability. In particular, by characterising the virulence of several A. astaci strains, I identified two possible different mechanisms of loss of virulence. I revealed that A. astaci’s virulence variability is not linked to variation of in vitro growth and sporulation, traits classically associated with a pathogen’s virulence. Based on these results, I suggest that the pathogen’s virulence determinants are likely its “virulence effectors”, of which A. astaci genome is enriched. Additionally, with the present work I provided transcriptomic evidence of coevolution between A. astaci and European crayfish. I showed that the haplogroups based on the canonical mitochondrial markers, often used to assess A. astaci’s virulence to inform management actions, do not differ for some of their characterising phenotypical traits, including virulence. Finally, after experimental characterisation of virulence and assessment of its likely phenotypical determinants, i.e., sporulation and growth, the next and more comprehensive step to study the pathogen’s virulence is through genomic approaches. To this aim, I provided key data for future comparative genomic studies, i.e., highly complete genome assemblies based on Nanopore (3) and Illumina reads (11). These data can be exploited in several ways, from building a pangenome of the species to a genome-wide association study (GWAS), that can offer a much deeper understanding of A. astaci’s virulence and adaptability. In particular, the identification of the loci associated with virulence through a GWAS has the potential to be revolutionary for the management of A. astaci, as it can become the basis to create a genomic tool to quickly and accurately assess the virulence of newly introduced strains, directing management actions towards the more dangerous strains.