Development of two quantitative real-time PCRs for the detection of Perkinsus beihaiensis and P. marinus

Oyster production is an expanding aquaculture activity worldwide. Protozoan parasites such as Perkinsus spp. affects bivalve populations in the Americas, Europe, and Asia, causing mass mortality in both cultured and wild populations. Their impact contributed to the depletion of natural resources, resulting in significant economic losses. A recent concern is the rapid spread of Perkinsus into regions far from their natural geographical areas, posing a threat to native mollusk populations. Since 2008, Perkinsus spp. has been detected among bivalves from Brazil. Two species (P. olsenii and P. chesapeaki) were sporadically observed. In contrast, two others (P. marinus and P. beihaiensis) are frequently detected in oyster populations, reaching high infection intensities and prevalences depending on the populations and species (Crassostrea gasar, C. rhizophorae, and Magallana gigas). Diagnosis based on molecular techniques, such as qPCR, has been developed to detect protozoan parasites, including Perkinsus spp.. However, the qPCRs developed for the diagnosis of P. marinus and P. beihaiensis have limitations; because they were not designed to distinguish among the seven species within the genus, they are neither fully specific nor validated. Considering the spread of Perkinsus spp. over the continents, prompted by several factors (environmental or anthropogenic), there is an urgent need to accurately detect Perkinsus species infecting hosts in areas of co-occurrence or coinfection. In the present study, we developed two species-specific qPCRs for the detection of these protozoan parasites P. marinus and P. beihaiensis that are spreading worldwide.