title: An Agar-Based Method for Plating Marine Protozoan Parasites of the Genus Perkinsus.
abstract: The genus Perkinsus includes protozoan parasites of mollusks responsible for losses in the aquaculture industry and hampering the recovery of natural shellfish beds worldwide, and they are a key taxon for understanding intracellular parasitism adaptations. The ability to propagate the parasite in liquid media, in the absence of the host, has been crucial for improving understanding of its biology; however, alternative techniques to grow the parasite are needed to explore other basic aspects of the Perkinsus spp. biology. We optimized a DME: Ham's F12-5% FBS- containing solid agar medium for plating Perkinsus marinus. This solid medium supported trophozoite propagation both by binary fission and schizogony. Colonies were visible to the naked eye 17 days after plating. We tested the suitability of this method for several applications, including the following: 1) Subcloning P. marinus isolates: single discrete P. marinus colonies were obtained from DME: Ham's F12-5% FBS- 0.75% agar plates, which could be further propagated in liquid medium; 2) Subcloning engineered Perkinsus mediterraneus MOE[MOE]: GFP by streaking cultures on plates; 3) Chemical susceptibility: Infusing the DME: Ham's F12-5% FBS- 0.75% agar plates with triclosan resulted in inhibition of the parasite propagation in a dose-dependent manner. Altogether, our plating method has the potential for becoming a key tool for investigating diverse aspects of Perkinsus spp. biology, developing new molecular tools, and for biotechnological applications.
authors.affiliation: Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, United States of America.|Research Experiences for Undergraduates (REU) NSF Program - 2015 - Bigelow Laboratory for Ocean Sciences, Boothbay, Maine, United States of America.
authors.forename: Emma R
authors.initials: ER
authors.lastname: Cold
authors.affiliation: Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, United States of America.|Université de Toulon, Toulon, France.
authors.forename: Nastasia J
authors.initials: NJ
authors.lastname: Freyria
authors.affiliation: Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, United States of America.
authors.forename: Joaquín
authors.initials: J
authors.lastname: Martínez Martínez
authors.affiliation: Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, United States of America.
authors.forename: José A
authors.initials: JA
authors.lastname: Fernández Robledo
mesh_terms: D000362:Agar; D056893:Alveolata / Q000254:growth & development*; D000818:Animals; D059001:Aquatic Organisms / Q000254:growth & development*; D003470:Culture Media; D056890:Eukaryota / Q000254:growth & development; D008974:Mollusca / Q000469:parasitology; D010271:Parasites / Q000254:growth & development*; D012758:Shellfish / Q000469:parasitology; D053058:Trophozoites / Q000254:growth & development
chemical_list: D003470:Culture Media; D000362:Agar