Wolbachia genomics support a tripartite nutritional symbiosis in blood-sucking Triatomine bugs

The nutritional symbiosis promoted by bacteria is a key determinant for adaptation and evolution of many insect lineages. A complex form of nutritional mutualism that arose in blood-sucking insects critically depends on diverse bacterial symbionts that supplement the diet of their nutrient-poor hosts with B vitamins. For instance, the triatomine bug Rhodnius prolixus, one of the main vectors of the Chagas disease in humans, is known to maintain a nutritional symbiosis with the gut symbionts Rhodococcus rhodnii. In this study, we show that Wolbachia symbionts are also widely distributed in the Rhodnius genus. We have screened a large set of Rhodnius blood-sucking bugs samples belonging to 17 different species and to the three phylogenetic groups, prolixus, pallescens and pictipes. We assembled 13 genomes of Wolbachia infecting eight Rhodnius species from prolixus and pictipes groups. We demonstrate that these Wolbachia belong to supergroup F and are closely related to Wolbachia infecting the bedbug Cimex lectularius (wCle). Although bedbugs and triatomines are very distantly related hemipteran bugs, the genomes of their respective Wolbachia were highly similar, suggesting recent horizontal host switches. We also show that Rhodnius Wolbachia genomes infecting the prolixus group encode intact biotin operon, the hallmark of nutritional symbiosis in bedbugs. This operon is lacking from all the other Wolbachia infecting R. pictipes. Finally, host genome analyses provide evidence of massive Wolbachia-to-Rhodnius gene transfers in almost samples, providing footprints of past infections that support a widespread and probably ancient symbiotic association between Wolbachia and triatomine bugs. Our results suggest that both Wolbachia and R. rhodnii gut symbionts and their Rhodnius host maintain a highly prevalent symbiotic relationship, in which the vertically-inherited Wolbachia has the metabolic potantial to ensure or complement, the nutritional mutualism provided by the gut symbionts. Specific loss of the biotin operon in some symbiont genomes suggests that the boundaries between obligatory mutualism, facultative mutualism and parasitism in Wolbachia are transient and fluid, supporting a dynamic process of transition and reversion from one state to another.