Haplosporidium pinnae Detection from the Faeces of Pinna nobilis: A Quick and Noninvasive Tool to Monitor the Presence of Pathogen in Early-Stage or during Fan Mussel Mass Mortalities

Due to the increasing mass mortality of Pinna nobilis, mainly caused by the protozoan Haplosporidium pinnae along the Mediterranean Sea, it is necessary to develop rapid and effective methods to detect the pathogen. The present study describes the development and validation of a species-specific assay based on hydrolysis probe chemistry to detect H. pinnae DNA from faeces and pseudofaeces of P. nobilis. During a study campaign in the Gulf of Trieste (Italy) in the spring and summer of 2022, 18 samples (10 faeces and 8 pseudofaeces) were collected. DNA was isolated from all samples and the presence of H. pinnae was tested by amplifying a small portion of 18S rDNA using qPCR. The newly developed assay detected positive H. pinnae in the faeces of the fan mussel in the spring, while no evidence of an outbreak of H. pinnae was found in the summer. In addition, the method proved to be noninvasive and can be used to monitor suspected H. pinnae infections in the early stages when bivalves are still vital. Furthermore, fecal analysis allows the monitoring of P. nobilis without dissecting tissues. The presented assay can also be used to routinely monitor the progress of mass mortalities caused by H. pinnae and to screen for the pathogen in live fan mussels and other environmental matrices, such as water, sediment, and faeces from other species that can host the protozoan.