Identification of glycine betaine as a host-derived molecule required for the vegetative proliferation of the protozoan parasite Perkinsus olseni.

Perkinsus olseni is an industrially significant protozoan parasite of Manila clam, Ruditapes philippinarum. So far, various media, based on Dulbecco's Modified Eagle Medium and Ham's F-12 nutrient mixture with supplementation of fetal bovine serum (FBS), have been developed to proliferate the parasitizing trophozoite stage of P. olseni. The present study showed that P. olseni did not proliferate in FBS-deficient Perkinsus broth medium (PBMΔF), but proliferated well in PBMΔF supplemented with tissue extract of host Manila clams, indicating that FBS and Manila clam tissue contained molecule(s) required for P. olseni proliferation. Preliminary characterization suggested that the host-derived molecule(s) was a heat-stable molecule(s) with a molecular weight of less than 3 kDa, and finally a single molecule required for the proliferation was purified by high-performance liquid chromatography processes. High-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance analyses identified this molecule as glycine betaine (=trimethylglycine), and the requirement of this molecule for P. olsseni proliferation was confirmed by an assay using chemically synthesized, standard glycine betaine. Although glycine betaine was required for the proliferation of all examined Perkinsus species, supplementation of glycine betaine precursors, such as choline and betaine aldehyde, enhanced the proliferation of 4 Perkinsus species (P. marinus, P. chesapeaki, P. mediterraneus and P. honshuensis), but not of 2 others (P. olseni and P. beihaiensis). Thus, it was concluded that the ability to biosynthesise glycine betaine from its precursors varied among Perkinsus species, and that P. olseni and P. beihaiensis lack the ability required to biosynthesize glycine betaine for proliferation.

Supplementation with lipids enhances zoosporulation of Perkinsus species.

The protozoan parasite Perkinsus olseni is a major pathogen of marine mollusks. A large number of zoospores (the most infective stage) is required to experimentally examine the transmission and invasion processes of this parasite, but the conventional zoosporulation method in seawater is inefficient. A recent study found that P. olseni zoosporulation was highly enhanced in a nutrient-rich medium which was developed for trophozoite proliferation in Perkinsus. Here, we examined the effect of each medium component on P. olseni zoosporulation and found that lipids enhanced zoosporulation. Moreover, zoospores failed to develop into trophozoites in seawater supplemented with lipids, indicating that this zoosporulation method using seawater supplemented with lipids is ideal for preparing a large number of P. olseni zoospores for various biological assays. Lipid supplementation also significantly enhanced zoosporulation in P. honshuensis, P. chesapeaki, and P. marinus, although the enhancing effect of the lipid supplementation varied by species. Considering the limited availability of lipids in the sea water column, our findings suggest that Perkinsus zoosporulation likely occurs mainly in the vicinity of hosts (not in the sea water column), such as in the mantle cavity or on the body surface of mollusk hosts.