RNAi-Mediated Manipulation of Cuticle Coloration Genes in Lygus hesperus Knight (Hemiptera: Miridae)

SIMPLE SUMMARY: Insect cuticle coloration results from pigments accumulating in a species-specific pattern. Numerous genes are involved in regulating pigmentation, and their manipulation can be used to create externally visible markers of successful gene editing. To clarify the roles for many of these genes and examine their suitability as markers in Lygus hesperus Knight (western tarnished plant bug), we screened existing gene expression data for sequences similar to those with known functions in pigmentation. We identified six genes (aaNAT, black, ebony, pale, tan, and yellow), with two variants for black and found that expression varied for each by developmental stage, adult age, body part, and sex. Silencing the expression of each gene produced varied effects in adults, ranging from the non-detectable (black 1, yellow), to moderate decreases (pale, tan) and increases (black 2, ebony) in darkness, to extremely dark and pervasive pigmentation (aaNAT). Based solely on its expression profile and easily observed color changes, aaNAT appears to be the best marker for tracking transgenic L. hesperus. ABSTRACT: Cuticle coloration in insects is a consequence of the accumulation of pigments in a species-specific pattern. Numerous genes are involved in regulating the underlying processes of melanization and sclerotization, and their manipulation can be used to create externally visible markers of successful gene editing. To clarify the roles for many of these genes and examine their suitability as phenotypic markers in Lygus hesperus Knight (western tarnished plant bug), transcriptomic data were screened for sequences exhibiting homology with the Drosophila melanogaster proteins. Complete open reading frames encoding putative homologs for six genes (aaNAT, black, ebony, pale, tan, and yellow) were identified, with two variants for black. Sequence and phylogenetic analyses supported preliminary annotations as cuticle pigmentation genes. In accord with observable difference in color patterning, expression varied for each gene by developmental stage, adult age, body part, and sex. Knockdown by injection of dsRNA for each gene produced varied effects in adults, ranging from the non-detectable (black 1, yellow), to moderate decreases (pale, tan) and increases (black 2, ebony) in darkness, to extreme melanization (aaNAT). Based solely on its expression profile and highly visible phenotype, aaNAT appears to be the best marker for tracking transgenic L. hesperus.