An Agar-Based Method for Plating Marine Protozoan Parasites of the Genus Perkinsus

PLoS One. 2016 May 5;11(5):e0155015. doi: 10.1371/journal.pone.0155015. eCollection 2016.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agar
  • Alveolata / growth & development*
  • Animals
  • Aquatic Organisms / growth & development*
  • Culture Media
  • Eukaryota / growth & development
  • Mollusca / parasitology
  • Parasites / growth & development*
  • Shellfish / parasitology
  • Trophozoites / growth & development

Substances

  • Culture Media
  • Agar

Grants and funding

This study was supported by institutional funds from Bigelow Laboratory for Ocean Sciences, supported by the National Science Foundation grants OCE0755142 (REU Program), EAGER IOS1550399, and by Gordon and Betty Moore Foundation (GBMF4962). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.