Testing for off-target impacts of gene modification in Lygus

Lygus bugs are among the costliest pests of cotton on a per acreage basis, requiring numerous pesticide applications and accounting for 30% of all losses. Increasing resistance to insecticides requires development of alternative control approaches. Use of CRISPR/Cas9 to create targeted mutations has potential to overcome many of the limitations to manipulating and destabilizing pest populations in a way that reduces management costs, risks, and effort. Facilitating the deployment of this gene editing technology is incorporation of mutations that produce externally evident phenotypic markers, such as changes to cuticle pigmentation. However, such modification can unintentionally impact the behavior and/or physiology of mutant strains, preventing integration into local populations. In L. hesperus, knockout of aralkylamine N-acetyltransferase (aaNAT) causes body surfaces to turn black, an easily discerned external marker. However, potential negative consequences include reduced structural integrity of the cuticle that enhances susceptibility to dehydration or limits mobility, reduced competitiveness during mate selection, reduced crypsis in their normal green habitat, and behavioral changes arising from circulating neurotransmitter levels. In this study, we examined many of the potential pitfalls to identify unintended consequences of silencing aaNAT. Overall, we find that the negative consequences are negligible, and the mutant phenotype appears to be suitable for use in developing new control strategies for L. hesperus that are based on gene manipulation.