Improved method for genotyping the causative agent of crayfish plague (Aphanomyces astaci) based on mitochondrial DNA

Abstract

AbstractAphanomyces astacicauses crayfish plague, which is a devastating disease of European freshwater crayfish. The likely first introduction ofA. astaciinto Europe was in the mid-19th century in Italy, presumably with the introduction of North American crayfish. These crayfish can carryA. astaciin their cuticle as a benign infection.Aphanomyces astacirapidly spread across Europe causing the decline of the highly susceptible indigenous crayfish species. Random amplified polymorphic DNA-PCR analysis ofA. astacipure cultures characterized five genotype groups (A, B, C, D and E). CurrentA. astacigenotyping techniques (microsatellites and genotype-specific regions, both targeting nuclear DNA) can be applied directly to DNA extracted from infected cuticles but require high infection levels. Therefore, they are not suitable for genotyping benign infections in North American crayfish (carriers). In the present study, we combine bioinformatics and molecular biology techniques to developA. astacigenotyping molecular markers that target the mitochondrial DNA, increasing the sensitivity of the genotyping tools. The assays were validated on DNA extracts ofA. astacipure cultures, crayfish tissue extractions from crayfish plague outbreaks and tissue extractions from North American carriers. We demonstrate the presence ofA. astacigenotype groups A and B in UK waters.