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

is an industrially significant protozoan parasite of Manila clam, . 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 . The present study showed that 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 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 proliferation was confirmed by an assay using chemically synthesized, standard glycine betaine. Although glycine betaine was required for the proliferation of all examined species, supplementation of glycine betaine precursors, such as choline and betaine aldehyde, enhanced the proliferation of 4 species ( and ), but not of 2 others ( and ). Thus, it was concluded that the ability to biosynthesise glycine betaine from its precursors varied among species, and that and lack the ability required to biosynthesize glycine betaine for proliferation.