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.

Toward establishing model organisms for marine protists: Successful transfection protocols for Parabodo caudatus (Kinetoplastida: Excavata)

“…and Symbiodinium microadriaticum (Te et al, 1998;De Riso et al, 2009;van Ooijen et al, 2012). Transfection of two dinoflagellates has been successful using silicon carbide whiskers to deliver plasmid constructs (Te et al, 1998), and transfection of Perkinsus marinus, a marine protist parasite, has been achieved using electroporation (Fernandez-Robledo et al, 2008;Cold et al, 2016). Recent gene editing protocols have been established for few diatoms (Hopes et al, 2016;Liu et al, 2016), and progress toward useful forward genetics approaches for choanoflagellates have been established (Hoffmeyer and Burkhardt, 2016).…”

Transient Expression of Plasmodium berghei MSP8 and HAP2 in the Marine Protozoan Parasite Perkinsus marinus

“…The selection of transfectants with the new plasmid containing the resistance cassette will also benefit from the recently developed methodology for plating Perkinsus spp. in solid medium (Ferna´ndez Robledo et al, 2008;Cold et al, 2016). The difference in GC content between P. marinus and P. berghei genome (47.4% vs. 23.7%) (Nikbakht et al, 2014) did not hinder the expression of the selected P. berghei genes in P. marinus.…”

“…The availability of culturing methodologies for Perkinsus spp. enabled a variety of studies addressing diverse aspects of Perkinsus biology and the development of a transfection system (Ferna´ndez Robledo et al, 2008;Cold et al, 2016). On the other hand, the Perkinsozoa is one of the earliest diverging groups in the lineage leading to dinoflagellates, branching off close to the ancestor from which the Ciliata, Dinozoa, Chromera, and Apicomplexa originated (Leander and Keeling, 2003;Saldarriaga et al, 2003;Gile et al, 2006;Moore et al, 2008).…”

“…In this study, we improved the original plasmid vector used to develop the transfection system in Perkinsus spp. (Ferna´ndez Robledo et al, 2008;Cold et al, 2016) to include a multiple cloning site (MCS) and the sequences coding for enterokinase and a His-tag. Two partial Plasmodium berghei genes, which are currently recognized as good candidates for a malaria vaccine, were cloned into new plasmid vector and used to transfect P. marinus trophozoites.…”

“…The vector used to clone P. berghei HAP2 and MSP8 genes was derived from the original plasmid used to develop the transfection methodology for Perkinsus spp. (Ferna´ndez Robledo et al, 2008;Cold et al, 2016). The newly improved plasmid p[MOE]:GFP uses the genome sequences and untranslated regions (UTRs) of MOE, a highly expressed gene in P. marinus trophozoites with no homologues so far in GenBanke other than Perkinsus spp.…”