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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 G 0 and strongly biased heterozygous G 1 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. [Display omitted] • Lygus hesperus has multiple doublesex transcripts that exhibit sex-biased expression. • RNAi knockdown of Lygus hesperus doublesex impedes male sexual development but has no effect on female development. • Following CRISPR/Cas9-mediated doublesex knockout, the sex ratio of the resulting progeny is heavily biased towards females. [ABSTRACT FROM AUTHOR]

[Display omitted] • Lygus hesperus has 7 β-tubulin genes. • β-tubulin2 is predominantly expressed in the testes. • Knockdown or knockout of β-tubulin2 reduces both spermatozoa abundance and quality, which limits fertilization and the subsequent egg hatch. Lygus hesperus Knight is an important insect pest of crops across western North America, with field management heavily reliant on the use of chemical insecticides. Because of the evolution of resistance to these insecticides, effective and environmentally benign pest management strategies are needed. Traditional sterile insect technique (SIT) has been successfully employed to manage or eradicate some insect pests but involves introducing irradiated insects with random mutations into field populations. New genetically-driven SIT techniques are a safer alternative, causing fixed mutations that manipulate individual genes in target pests to produce sterile individuals for release. Here, we identified seven β-tubulin coding genes from L. hesperus and show that Lhβtub2 is critical in male sperm production and fertility. Lhβtub2 is expressed primarily in the male testes and targeting of this gene by RNA interference or gene editing leads to male sterility. [ABSTRACT FROM AUTHOR]