Pigmentation loci as markers for genome editing in the Chagas disease vector Rhodnius prolixus
Authors: Berni M. , Bressan D. , Simão Y. , Julio A. , Oliveira P. L. , Pane A. , Araujo H. 
Published: April 2020

The kissing bugRhodnius prolixusis 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 theR. prolixus yellowandaaNAT/proorthologs produces striking alterations in cuticle color. Surprisingly, knockdown ofebonydoes not generate visible phenotypes. Since loss ofebonyfunction results in a dark cuticle in several insect orders, we conclude thatR. prolixusevolved alternative strategies for cuticle coloration, possibly including the loss of a pigmentation function for an entire branch of the tyrosine pathway. Knockdown of thescarletandbrowngenes - 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 Summary

The hemipteranRhodnius prolixus- also known as a kissing bug - is a main vector transmitting the parasiteTrypanosoma 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 CRISPR- based genome edition hold great promise to control vector population or generate parasite-resistant insects. For these approaches to be feasible inR. 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 encodingscarletand the tyrosine pathway enzyme encodingaaNAT/progenerate striking and easily visible phenotypes. Importantly, the knockdown of these genes does not affect insect viability and fertility under laboratory conditions. Our results suggest thatR. prolixushas 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 areas has gained critical importance.