Identifying bacterial symbionts of the kissing bug Rhodnius prolixus

Triatomine kissing bugs vector the parasite Trypanosoma cruzi, the causative agent of Chagas’ disease, which affects millions of people throughout the Americas. Vector capacity varies among the over 100 species of Triatominae, but all are obligately and exclusively hematophagous, requiring a blood meal from a vertebrate host to successfully molt and produce eggs. Like other exclusively hematophagous insects, kissing bugs are known to harbor bacteria that are essential for their development. Unlike essential bacterial symbionts of other exclusively hematophagous insects, the symbionts of kissing bugs reside extracellularly in the gut and are acquired from the nymph’s environment, often via coprophagy of other triatomine’s feces. Rhodococcus rhodnii, an Actinobacteria, has been demonstrated to function as symbionts of Rhodnius prolixus, Triatoma infestans, and other triatomines, yet is not universally present in microbiome surveys of wild triatomines. We developed methods to produce axenic, bacteria free, R. prolixus nymphs, which we can then artificially infect with bacteria via an inoculated blood meal. We examined several species of Rhodococcus, as well as Escherichia coli, for their ability to function as symbionts of R. prolixus. We found that R. rhodnii-infected insects developed faster and had increased survival relative to insects harboring other bacteria. Bacterial titer varied significantly among the species tested, and that the immune response of R. prolixus differed depending on the bacteria found in the gut. These results suggest that while many bacteria can persist in the gut of triatomines, only certain bacteria are functional symbionts.