Pigmentation in insects has been linked to mate selection and predator evasion, thus representing an important aspect for natural selection. Insect body color is classically associated to the activity of tyrosine pathway enzymes, and eye color to pigment synthesis through the tryptophan and guanine pathways, and their transport by ABC proteins. Among the hemiptera, the genetic basis for pigmentation in kissing bugs such as Rhodnius prolixus, that transmit Chagas disease to humans, has not been addressed. Here we report the functional analysis of R. prolixus eye and cuticle pigmentation genes. Consistent with data for most insect clades, we show that knockdown for yellow results in a yellow cuticle, while scarlet and cinnabar knockdowns display red eyes as well as cuticle phenotypes. In addition, tyrosine pathway aaNATpreto knockdown resulted in a striking dark cuticle that displays no color pattern or UV reflectance. In contrast, knockdown of ebony and tan, that encode NBAD branch tyrosine pathway enzymes, did not generate the expected dark and light brown phenotypes, respectively, as reported for other insects. We hypothesize that R. prolixus, which requires tyrosine pathway enzymes for detoxification from the blood diet, evolved an unusual strategy for cuticle pigmentation based on the preferential use of a color erasing function of the aaNATpreto tyrosine pathway branch. We also show that genes classically involved in the generation and transport of eye pigments regulate red body color in R. prolixus. This is the first systematic approach to identify the genes responsible for the generation of color in a blood-feeding hemiptera, providing potential visible markers for future transgenesis.

The kissing bug Rhodnius prolixus is a major vector for Chagas disease in the Americas, and also considered as the primary model for functional studies.Prospective transgenic approaches and genome editing strategies hold great promise for controlling insect populations as well as disease propagation. In this context, identifying visible genetic markers for transgenic methodologies is of paramount importance to advance the field. Here we have identified and analyzed the function of putative cuticle and eye color genes by investigating the effect of gene knockdown on fertility, viability, and the generation of visible phenotypes. Synthesis of the dark, yellow and tan pigments present in the cuticle of most insects depends on the function of key genes encoding enzymes in the tyrosine pathway. Knockdown of the R. prolixus yellow and aaNAT/pro orthologs produces striking alterations in cuticle color.Surprisingly, knockdown of ebony does not generate visible phenotypes. Since loss of ebony function results in a dark cuticle in several insect orders, we conclude that R. prolixus evolved alternative strategies for cuticle coloration, possibly including the loss of a pigmentation function for an entire branch of the tyrosine pathway. Knockdown of the scarlet and brown genes -encoding ABC transporters -alters cuticle and eye pigmentation, implying that the transport of pigment into proper organelles is an important process both for cuticle and eye coloration in this species. Therefore, this analysis identifies for the first time potential visible markers for transgenesis in a hemipteran vector for a debilitating human disease. Author SummaryThe hemipteran Rhodnius prolixus -also known as a kissing bug -is a main vector transmitting the parasite Trypanosoma cruzi, the causative agent of Chagas disease, a debilitating infection estimated to affect more than 6 million people in Central and South America. In order to limit disease spread, an important measure is insect vector control. However, kissing bugs -like other insects -develop resistance to insecticides.Alternative strategies based on transgenesis and the recently developed CRISPRbased genome edition hold great promise to control vector population or generate parasite-resistant insects. For these approaches to be feasible in R. prolixus, it is critical to identify visible phenotypic markers. Here we identify and describe several genes controlling cuticle and eye pigmentation that are well-suited putative landing sites for transformation strategies. Among these, loss-of-function mutations in the ABC transporter encoding scarlet and the tyrosine pathway enzyme encoding aaNAT/pro generate striking and easily visible phenotypes. Importantly, the knockdown of these genes does not affect insect viability and fertility under laboratory conditions. Our results suggest that R. prolixus has developed alternative strategies for cuticle coloration involving the loss of an entire branch of tanning loci, while the other branch producing cuticle patterns by generating non-pigmented a...