Cold Emma R, Freyria Nastasia J, Martínez Martínez Joaquín, Fernández Robledo José A
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.
PLoS One. 2016 May 5;11(5):e0155015. doi: 10.1371/journal.pone.0155015. eCollection 2016.
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.
派金虫属包括软体动物的原生动物寄生虫，这些寄生虫会给水产养殖业造成损失，并阻碍全球天然贝类养殖场的恢复，它们是理解细胞内寄生适应的关键分类群。在没有宿主的情况下，在液体培养基中繁殖这种寄生虫的能力对于增进对其生物学特性的了解至关重要；然而，需要其他培养该寄生虫的技术来探索派金虫属生物学的其他基本方面。我们优化了一种含有DME：Ham's F12 - 5%胎牛血清的固体琼脂培养基来培养海派金虫。这种固体培养基支持滋养体通过二分裂和裂体生殖进行繁殖。接种后17天，菌落肉眼可见。我们测试了该方法在几种应用中的适用性，包括：1）亚克隆海派金虫分离株：从DME：Ham's F12 - 5%胎牛血清 - 0.75%琼脂平板上获得单个离散的海派金虫菌落，这些菌落可在液体培养基中进一步繁殖；2）通过在平板上划线培养对工程化的地中海派金虫MOE[MOE]：绿色荧光蛋白进行亚克隆；3）化学敏感性：向DME：Ham's F12 - 5%胎牛血清 - 0.75%琼脂平板中加入三氯生会导致寄生虫繁殖受到剂量依赖性抑制。总之，我们的平板培养方法有潜力成为研究派金虫属生物学各个方面、开发新的分子工具以及用于生物技术应用的关键工具。
