Doublesex is essential for masculinization but not feminization in Lygus hesperus

In most holometabolous insects, sex differentiation occurs via a hierarchical cascade of transcription factors, with doublesex (dsx) regulating genes that control sex-specific traits. Although less is known in hemimetabolous insects, early evidence suggests that substantial differences exist from more evolutionarily advanced insects. Here, we identified and characterized dsx in Lygus hesperus (western tarnished plant bug), a hemipteran pest of many agricultural crops in western North America. The full-length transcript for L. hesperus dsx (Lhdsx) and several variants encode proteins with conserved DNA binding and oligomerization domains. Transcript profiling revealed that Lhdsx is ubiquitously expressed, likely undergoes alternative pre-mRNA splicing, and, unlike several model insects, is sex-biased rather than sex-specific. Embryonic RNA interference (RNAi) of Lhdsx only impacted sex development in adult males, which lacked both internal reproductive organs and external genitalia. No discernible impacts on adult female development or reproductivity were observed. RNAi knockdown of Lhdsx in nymphs likewise only affected adult males, which lacked the characteristic dimorphic coloration but had dramatically elevated vitellogenin transcripts. Gene knockout of Lhdsx by CRISPR/Cas9 editing yielded only females in G₀ and strongly biased heterozygous G₁ offspring to females with the few surviving males showing severely impaired genital development. These results indicate that L. hesperus male development requires Lhdsx, whereas female development proceeds via a basal pathway that functions independently of dsx. A fundamental understanding of sex differentiation in L. hesperus could be important for future gene-based management strategies of this important agricultural pest.