Pigmentation loci as markers for genome editing in the Chagas disease vector Rhodnius prolixus

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.