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

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 li...

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Autores principales: Cold, Emma R., Freyria, Nastasia J., Martínez Martínez, Joaquín, Fernández Robledo, José A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4858233/
https://www.ncbi.nlm.nih.gov/pubmed/27149378
http://dx.doi.org/10.1371/journal.pone.0155015
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author Cold, Emma R.
Freyria, Nastasia J.
Martínez Martínez, Joaquín
Fernández Robledo, José A.
author_facet Cold, Emma R.
Freyria, Nastasia J.
Martínez Martínez, Joaquín
Fernández Robledo, José A.
author_sort Cold, Emma R.
collection PubMed
description 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.
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spelling pubmed-48582332016-05-13 An Agar-Based Method for Plating Marine Protozoan Parasites of the Genus Perkinsus Cold, Emma R. Freyria, Nastasia J. Martínez Martínez, Joaquín Fernández Robledo, José A. PLoS One Research Article 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. Public Library of Science 2016-05-05 /pmc/articles/PMC4858233/ /pubmed/27149378 http://dx.doi.org/10.1371/journal.pone.0155015 Text en © 2016 Cold et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Cold, Emma R.
Freyria, Nastasia J.
Martínez Martínez, Joaquín
Fernández Robledo, José A.
An Agar-Based Method for Plating Marine Protozoan Parasites of the Genus Perkinsus
title An Agar-Based Method for Plating Marine Protozoan Parasites of the Genus Perkinsus
title_full An Agar-Based Method for Plating Marine Protozoan Parasites of the Genus Perkinsus
title_fullStr An Agar-Based Method for Plating Marine Protozoan Parasites of the Genus Perkinsus
title_full_unstemmed An Agar-Based Method for Plating Marine Protozoan Parasites of the Genus Perkinsus
title_short An Agar-Based Method for Plating Marine Protozoan Parasites of the Genus Perkinsus
title_sort agar-based method for plating marine protozoan parasites of the genus perkinsus
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4858233/
https://www.ncbi.nlm.nih.gov/pubmed/27149378
http://dx.doi.org/10.1371/journal.pone.0155015
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