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.
Highlights
Protozoan parasites significantly affect wild and farmed mollusk species around the world (OIE; http://www.oie.int/; Aquatic Animal Health Code, Section 11: Diseases of Mollusks)
Cultures of the wild-type P. marinus ATCC PRA-240 and P. mediterraneus ATCC PRA-238 [25] were maintained in Dulbecco’s modified Eagle medium (DME): Ham's F12 (1:2) supplemented with 5% fetal bovine serum (FBS) in 25 cm2 (5–8 ml) polystyrene canted neck cell culture flasks with vent caps (Corning1, Corning, NY) at 26–28°C in a microbiology incubator as reported elsewhere [26]
The stock solution was prepared in 100% ethanol and diluted in the DME: Ham’s F12–5% FBS (2x) prior to mixing with agar medium to yield final concentrations of 50, 100, and 200 μM; final concentration of ethanol on the plates was 1% or less, a concentration known to have no negative effects on Perkinsus spp. viability [27, 28]
Summary
Protozoan parasites significantly affect wild and farmed mollusk species around the world (OIE; http://www.oie.int/; Aquatic Animal Health Code, Section 11: Diseases of Mollusks). The ability to grow cells onto solid media plates can facilitate subcloning and may become crucial for selecting Perkinsus spp. transfectants once specific resistance cassettes become available.
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