McMullen John G, Stock S Patricia
School of Animal and Comparative Biomedical Sciences, University of Arizona.
Department of Entomology, University of Arizona;
J Vis Exp. 2014 Sep 22(91):52096. doi: 10.3791/52096.
Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the family Enterobacteriaceae. Xenorhabdus bacteria are associated with steinernematids nematodes while Photorhabdus are symbionts of heterorhabditids. Together nematodes and bacteria form a potent insecticidal complex that kills a wide range of insect species in an intimate and specific partnership. Herein, we demonstrate in vivo and in vitro techniques commonly used in the rearing of these nematodes under laboratory conditions. Furthermore, these techniques represent key steps for the successful establishment of EPN cultures and also form the basis for other bioassays that utilize these organisms for research. The production of aposymbiotic (symbiont-free) nematodes is often critical for an in-depth and multifaceted approach to the study of symbiosis. This protocol does not require the addition of antibiotics and can be accomplished in a short amount of time with standard laboratory equipment. Nematodes produced in this manner are relatively robust, although their survivorship in storage may vary depending on the species used. The techniques detailed in this presentation correspond to those described by various authors and refined by P. Stock's Laboratory, University of Arizona (Tucson, AZ, USA). These techniques are distinct from the body of techniques that are used in the mass production of these organisms for pest management purposes.
昆虫病原线虫（EPN）（斯氏线虫科和异小杆线虫科）与肠杆菌科中的革兰氏阴性γ-变形菌有着共生关系。嗜线虫致病杆菌属细菌与斯氏线虫科线虫相关联，而发光杆菌属则是异小杆线虫科的共生菌。线虫和细菌共同形成了一种高效的杀虫复合体，以一种紧密且特定的共生关系杀死多种昆虫物种。在此，我们展示了在实验室条件下饲养这些线虫时常用的体内和体外技术。此外，这些技术是成功建立昆虫病原线虫培养物的关键步骤，也是利用这些生物进行研究的其他生物测定的基础。无共生体线虫的生产对于深入、多方面地研究共生关系通常至关重要。本方案无需添加抗生素，使用标准实验室设备即可在短时间内完成。以这种方式生产的线虫相对强健，不过它们在储存中的存活率可能因所使用的物种而异。本报告中详细介绍的技术与各位作者所描述并经美国亚利桑那大学（图森，亚利桑那州）P. 斯托克实验室完善的技术相对应。这些技术与用于大规模生产这些生物以进行害虫管理的技术体系不同。
