Establishment and characteristics of Perkinsus beihaiensis in vitro isolates from Mediterranean mussels Mytilus galloprovincialis

Two monoclonal in vitro isolates of Perkinsus beihaiensis were established from tissues of infected Mediterranean mussels Mytilus galloprovincialis, using a lipid concentrate and yeast extract medium (LpcYM) to induce enlarged prezoospoangia from histozoic trophozoites, and Perkinsus broth medium (PBM) to support subsequent zoosporulation and schizogonic proliferation from those cells. Zoospores of the isolates developed into uninucleate signet-ring trophozoites followed by schizonts typical of Perkinsus spp., but an additional type of unknown botryoidal cell clusters appeared and increased in density. These botryoidal cell clusters had second-generation daughter schizonts within which further internal cells were produced. Although botryoidal cell clusters in primary cultures rarely proliferated in PBM, cells contained by botryoidal clusters enlarged as prezoosporangia in LpcYM, and a large number of proliferative stages (i.e. trophozoites and schizonts typical for Perkinsus spp.) were obtained from these prezoosporangia in PBM. At lower cell density, trophozoites transformed into the botryoidal cell clusters in PBM, and the number of cells increased little, while trophozoites rapidly increased by typical schizogony at higher density in PBM. Based on these observations, we determined an optimal cell density for continuous culture of P. beihaiensis isolates, confirmed their ability to proliferate and survive after cryopreservation, and describe an experimental protocol to culture this most recently described species in the genus Perkinsus.