Targeted eDNA Survey Enables Consistent Detection of the Invasive Blue Crab in Trans‐Border Adriatic Coastal‐Marine Ecosystems

Abstract

ABSTRACT The invasive blue crab Callinectes sapidus poses a significant ecological and economic threat to Mediterranean marine ecosystems, highlighting the need for sensitive and reliable tools for its early detection. We developed and validated a species‐specific environmental DNA (eDNA) assay designed for sensitive detection of C. sapidus . Species‐specific primers targeting a 464 bp fragment of the mitochondrial COI gene were designed, and their specificity was confirmed through in silico analyses using Primer‐BLAST and Sanger sequencing, and their effectiveness was validated via high‐throughput sequencing (HTS) of environmental DNA (eDNA) samples. The assay based on eDNA proved to be reliable and robust in confirming the presence of blue crab DNA in five cross‐border Adriatic pilot areas impacted by this invasive species. In addition, the dominant OTU was consistently present in all sites, while haplotype network analysis using ASVs revealed a shared central haplotype across Adriatic populations, supporting their genetic cohesiveness and a common invasion history. These results emphasize the importance of molecular tools for tracking invasion dynamics and reinforce the potential for uniform genetic profiles to facilitate management efforts. Overall, our findings demonstrate that targeted eDNA approaches are essential for effective early detection and containment strategies in marine ecosystems threatened by biological invasions, and that the blue crab in the Adriatic Sea constitutes a potential genetically cohesive invasive entity.