Oral Bacterial Infection and Shedding in <i>Drosophila Melanogaster</i>

International audienceThe 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

Extensive de novo mutation rate variation between individuals and across the genome of <i>Chlamydomonas reinhardtii</i>

Describing the process of spontaneous mutation is fundamental for understanding the genetic basis of disease, the threat posed by declining population size in conservation biology, and much of evolutionary biology. Directly studying spontaneous mutation has been difficult, however, because new mutations are rare. Mutation accumulation (MA) experiments overcome this by allowing mutations to build up over many generations in the near absence of natural selection. Here, we sequenced the genomes of 85 MA lines derived from six genetically diverse strains of the green alga Chlamydomonas reinhardtii. We identified 6843 new mutations, more than any other study of spontaneous mutation. We observed sevenfold variation in the mutation rate among strains and that mutator genotypes arose, increasing the mutation rate approximately eightfold in some replicates. We also found evidence for fine-scale heterogeneity in the mutation rate, with certain sequence motifs mutating at much higher rates, and clusters of multiple mutations occurring at closely linked sites. There was little evidence, however, for mutation rate heterogeneity between chromosomes or over large genomic regions of 200 kbp. We generated a predictive model of the mutability of sites based on their genomic properties, including local GC content, gene expression level, and local sequence context. Our model accurately predicted the average mutation rate and natural levels of genetic diversity of sites across the genome. Notably, trinucleotides vary 17-fold in rate between the most and least mutable sites. Our results uncover a rich heterogeneity in the process of spontaneous mutation both among individuals and across the genome

The route of infection determines Wolbachia antibacterial protection in Drosophila

International audienceBacterial symbionts are widespread among metazoans and provide a range of beneficial functions. -mediated protection against viral infection has been extensively demonstrated in In mosquitoes that are artificially transinfected with (wMel), protection from both viral and bacterial infections has been demonstrated. However, no evidence for -mediated antibacterial protection has been demonstrated in to date. Here, we show that the route of infection is key for -mediated antibacterial protection. carrying showed reduced mortality during enteric-but not systemic-infection with the opportunist pathogen -mediated protection was more pronounced in male flies and is associated with increased early expression of the antimicrobial peptide , and also increased expression of a reactive oxygen species detoxification gene (). These results highlight that the route of infection is important for symbiont-mediated protection from infection, that can protect hosts by eliciting a combination of resistance and disease tolerance mechanisms, and that these effects are sexually dimorphic. We discuss the importance of using ecologically relevant routes of infection to gain a better understanding of symbiont-mediated protection

Data from: The route of infection determines Wolbachia antibacterial protection in Drosophila

Bacterial symbionts are widespread among metazoans and provide a range of beneficial functions. Wolbachia-mediated protection against viral infection has been extensively demonstrated in Drosophila. In mosquitoes that are artificially transinfected with Drosophila melanogaster Wolbachia (wMel), protection from both viral and bacterial infections has been demonstrated. However, no evidence for Wolbachia-mediated antibacterial protection has been demonstrated in Drosophila to date. Here, we show that the route of infection is key for Wolbachia-mediated antibacterial protection. Drosophila melanogaster carrying Wolbachia showed reduced mortality during enteric—but not systemic—infection with the opportunist pathogen Pseudomonas aeruginosa. Wolbachia-mediated protection was more pronounced in male flies and is associated with increased early expression of the antimicrobial peptide Attacin A, and also increased expression of a reactive oxygen species detoxification gene (Gst D8). These results highlight that the route of infection is important for symbiont-mediated protection from infection, that Wolbachia can protect hosts by eliciting a combination of resistance and disease tolerance mechanisms, and that these effects are sexually dimorphic. We discuss the importance of using ecologically relevant routes of infection to gain a better understanding of symbiont-mediated protection

Supplementary material from The route of infection determines <i>Wolbachia</i> antibacterial protection in <i>Drosophila</i>

Bacterial symbionts are widespread among metazoans and provide a range of beneficial functions. <i>Wolbachia</i>-mediated protection against viral infection has been extensively demonstrated in <i>Drosophila.</i> In mosquitoes that are artificially transinfected with <i>Drosophila melanogaster Wolbachia</i> (wMel), protection from both viral and bacterial infections has been demonstrated. However, no evidence for <i>Wolbachia</i>-mediated antibacterial protection has been demonstrated in <i>Drosophila</i> to date. Here, we show that the route of infection is key for <i>Wolbachia</i>-mediated antibacterial protection. <i>Drosophila melanogaster</i> carrying <i>Wolbachia</i> showed reduced mortality during enteric—but not systemic—infection with the opportunist pathogen <i>Pseudomonas aeruginosa</i>. <i>Wolbachia</i>-mediated protection was more pronounced in male flies and is associated with increased early expression of the antimicrobial peptide <i>Attacin A</i>, and also increased expression of a reactive oxygen species detoxification gene (<i>Gst D8</i>). These results highlight that the route of infection is important for symbiont-mediated protection from infection, that <i>Wolbachia</i> can protect hosts by eliciting a combination of resistance and disease tolerance mechanisms and that these effects are sexually dimorphic. We discuss the importance of using ecologically relevant routes of infection to gain a better understanding of symbiont-mediated protection