Established cotton stainer gut bacterial mutualists evade regulation by host antimicrobial peptides

Symbioses with microorganisms are ubiquitous in nature and confer important ecological traits on animal hosts, but also require control mechanisms to ensure homeostasis of the symbiotic interaction. In addition to protecting hosts against pathogens, animal immune systems recognize, respond to and regulate mutualists. The gut bacterial symbionts of the cotton stainer bug, Dysdercus fasciatus, elicit an immune response characterized by the upregulation of C-type lysozyme and the antimicrobial peptide pyrrhocoricin in bugs with their native gut microbiota compared to dysbiotic insects. In this study, we investigated the impact of the elicited antimicrobial immune response on the established cotton stainer gut bacterial symbiont populations. To this end, we used RNAi to knock down immune genes hypothesized to regulate the symbionts and subsequently measured the effect of this silencing on host fitness and on the abundance of the major gut bacterial symbionts. Despite successful downregulation of target genes by both ingestion and injection of dsRNA, silencing of immune genes neither had an effect on host fitness nor on the qualitative and quantitative composition of established gut bacterial symbionts, indicating that the host immune responses are not actively involved in the regulation of the nutritional and defensive gut bacterial mutualists. These results suggest that close associations of bacterial symbionts with their hosts can result in the evolution of mechanisms ensuring that symbionts remain insensitive to host immunological responses, which may be important for the evolutionary stability of animal-microbe symbiotic associations. Importance Animal immune systems are central for the protection of hosts against enemies by preventing or eliminating successful infections. However, in the presence of beneficial bacterial mutualists, the immune system must strike a balance of not killing the beneficial symbionts while at the same time preventing enemy attacks. Here, using the cotton stainer bug, we reveal that its long-term associated bacterial symbionts are insensitive to the host9s immune effectors, suggesting adaptation to the host9s defenses, thereby strengthening the stability of the symbiotic relationship. The ability of the symbionts to elicit host immune responses but remain insensitive themselves may be a mechanism by which the symbionts prime hosts to fight future pathogenic infections.