Doublesex is essential for masculinization but not feminization in Lygus hesperus

Authors: Hull, Joe; HEU, CHAN - Oak Ridge Institute For Science And Education (ORISE); Gross, Roni; Leroy, Dannialle; SHUTZE, INANA - Oak Ridge Institute For Science And Education (ORISE); Langhorst, Daniel; Fabrick, Jeffrey; Brent, Colin

Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/24/2024
Publication Date: 2/1/2024
Citation: Hull, J.J., Heu, C.C., Gross, R.J., Leroy, D.M., Shutze, I.X., Langhorst, D.R., Fabrick, J.A., Brent, C.S. 2024. Doublesex is essential for masculinization but not feminization in Lygus hesperus. Insect Biochemistry and Molecular Biology. 166. Article 104085. https://doi.org/10.1016/j.ibmb.2024.104085.
DOI: https://doi.org/10.1016/j.ibmb.2024.104085

Interpretive Summary: As an alternative to conventional control practices for pest insects, a functional genomics approach is being used to identify genes that can be targeted to disrupt key developmental processes. One such essential process is sex determination, which involves a cascade of genes. The gene doublesex (DSX) is typically the most downstream sexual differentiation factor and has a demonstrated role in feminization in most of the model insects studied. However, our knowledge of this gene’s function in many pest insects outside these model species is limited. For Lygus hesperus (western tarnished plant bug), a pest of diverse crops, little is known about sex determination and even less about the role of DSX. To fill this gap, ARS scientists isolated and sequenced the dsx gene in L. hesperus and used expression profiling to determine that the gene is expressed throughout the body and all life stages, but that there are sex-biased differences. Silencing the dsx gene through RNA interference (RNAi) or by gene editing using CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) resulted in changes to external and internal male traits so that more females than males were produced, and the few males exhibited feminine characteristics. These results indicate that for L. hesperus, unlike most model species, DSX functionality is limited to masculinization, and has no impact on female-specific traits. Additionally, DSX was found to have different effects at different stage of developmental, impacting primary sex traits early, but later influencing only secondary sex characteristics (i.e., cuticle color). Because of the central role played by DSX in L. hesperus development, it is a suitable candidate for use in targeted control approaches.

Technical Abstract: 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.