Siva-Jothy Jonathon A, Prakash Arun, Vasanthakrishnan Radhakrishnan B, Monteith Katy M, Vale Pedro F
Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh.
IGDR - CNRS UMR 6290.
J Vis Exp. 2018 May 31(135):57676. doi: 10.3791/57676.
The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.
果蝇（黑腹果蝇）是感染与固有免疫领域中最完善的模式系统之一。尽管大多数研究工作聚焦于全身性感染，但最近人们对肠道针对病原体的免疫能力机制的兴趣有所增加，而这需要通过口服感染果蝇的方法来实现。在此，我们介绍一种将单个果蝇经口暴露于机会性细菌病原体（铜绿假单胞菌）和黑腹果蝇天然细菌病原体（嗜虫假单胞菌）的实验方案。该方案的目的是提供一种可靠的方法，使雄性和雌性果蝇接触这些病原体。我们给出了具有代表性的结果，展示了生存表型、微生物载量和细菌排泄情况，这对于研究病原体传播中的异质性具有重要意义。最后，我们证实Dcy突变体（肠道上皮中缺乏保护性围食膜）和Relish突变体（缺乏功能性免疫缺陷（IMD）途径）对经口细菌感染的易感性增加。因此，本方案描述了一种利用经口感染途径感染果蝇的可靠方法，该方法可扩展用于研究肠道感染结果和细菌传播中多种遗传和环境变异来源。
