Lygus hesperus Knight, the western tarnished plant bug, is an important pest of numerous agricultural crops throughout the Western U.S., Mexico, and Canada (Scott, 1977, Wheeler, 2001, Strand, 2008, Ritter et al., 2010, Naranjo et al., 2011, Joseph, 2019). In 2022, Lygus species were responsible for approximately $324 million in economic losses in cotton in the U.S. (Cook et al., 2022). In Arizona, an integrated pest management (IPM) program has effectively controlled several Lygus species (including L. hesperus) (Ellsworth, 2000). However, control remains dependent on a few insecticides with limited modes of action, such that evolved resistance to these chemicals threatens the management of this pest species complex (Zhu and Brindley, 1992, Xu and Brindley, 1993, Snodgrass, 1996, Dorman et al., 2020). One new management tactic with great potential is precision genetically-driven sterile insect technique (SIT). Classical SIT, which has been successfully applied to manage and/or eradicate several key agricultural pests (Klassen, 2005, Tabashnik et al., 2021; Pla et al., 2021) relies on bombarding insects with gamma radiation (Bakri et al., 2005, Kandul et al., 2019, Shelton et al., 2020, Chen et al., 2021) to induce sterility but can also have other negative impacts on fitness (Calkins and Parker, 2005, Helinski et al., 2009). In contrast, genetic SIT uses highly targeted, genetically-induced sterility by modifying gene expression and/or protein function. While safe, effective, and efficient, the targeted manipulation of genes involved in spermatogenesis requires functional knowledge, an area of research that is frequently unknown in many insects. However, with the elucidation of such genes, genetic manipulation affecting male sterility and implementation of genetic SIT is now feasible.

Insect spermatogenesis occurs in the follicle cells and involves a germline stem cell undergoing mitosis within the testicular follicle to generate two daughter cells (Fabian and Brill, 2012, Golubkova et al., 2020). One daughter cell continues to propagate the stem line, while the other differentiates into a gonialblast that ultimately forms the spermatogonia. These cells divide meiotically to form spermatids, which then mature through the process of spermiogenesis to produce mature spermatozoa (Fabian and Brill, 2012, Golubkova et al., 2020). Multiple steps within this process, including meiosis and spermiogenesis, require the function of microtubules (Kemphues et al., 1982), polymers of α- and β-tubulin heterodimers that form the basic structural components of the cellular cytoskeleton, function in cell polarity, cell division, and intracellular trafficking (Kemphues et al., 1982, Fackenthal et al., 1995, Popodi et al., 2008, Janke and Magiera, 2020, Nsamba and Gupta, 2022). Because of the crucial roles played by β-tubulin (βtub) in spermatogenesis, they represent logical targets for disruption of male fertility.

Seven phylogenetically distinct β-tubulin clades are known in insects but two appear to be restricted to Hymenoptera, although gene numbers can vary across species depending on gene duplication or loss (Nielsen et al., 2010, Findeisen et al., 2014). All insect β-tubulin isotypes share a conserved amino terminus, which is essential for polymerization during the formation of microtubules. Each isotype also has a unique carboxy terminus that binds to different regulatory proteins, allowing for variation in function (Janke and Magiera, 2020, Nsamba and Gupta, 2022, Popodi et al., 2008). Drosophila melanogaster has five clades or subgroups, each consisting of a single β-tubulin (Findeisen et al., 2014, Krishnan et al., 2022). The first, βtub1 (tub56D), is highly expressed in all developmental stages and in all adult tissues (Kemphues et al., 1982, Bialojan et al., 1984). During embryogenesis, βtub1 functions in neurogenesis and the development and attachment of muscle (Buttgereit et al., 1996). βtub2 (Tub85D) is predominantly expressed in testes (Brown et al., 2014, Leader et al., 2018, Vedelek et al., 2018) and is essential for several stages of spermatogenesis (i.e., meiosis, nucleus formation, sperm tail axoneme formation to generate mature spermatozoa) (Hoyle et al., 1995, Kemphues et al., 1982; Raff, 1994). Mutations to βtub2 result in non-motile sperm in D. melanogaster (Kemphues et al., 1982), but its role in spermatogenesis outside of dipterans is unknown. βtub3 (Tub60D) is expressed in the embryo during mesodermal differentiation, and functions with βtub1 in the development of muscle, sensory organs (e.g., mechanosensory organs, Bolwig’s organ, and nerves), and bristle development (Buttgereit et al., 1996, Dettman et al., 2001, Krishnan et al., 2022). βtub4 (Tub97EF) is found in the gut, hemocyte, and embryo, and is known to stabilize microtubules at low temperature (Myachina et al., 2017). Although βtub5 (tub65B) is predominantly expressed in basal regions of testes and to a lesser extent in brain and imaginal disk (Brown et al., 2014, Leader et al., 2018, Vedelek et al., 2018), functional analyses have largely focused on roles in neuronal and embryonic development (Bhattacharjee et al., 2022, Myachina et al., 2017, Neely et al., 2010).

To examine the potential of targeting L. hesperus spermatogenesis-associated β-tubulins for future genetic pest management, we searched transcriptomic datasets for β-tubulin homologs, profiled their expression, and assessed the in vivo function of testes-dominant homologs via RNA interference (RNAi) and CRISPR/Cas9. Although only four β-tubulin subgroups were identified in Rhodnius prolixus and Acyrthosiphon pisum, (Findeisen et al., 2014), we identified seven unique L. hesperus β-tubulins, two of which (Lhβtub2 and Lhβtub5) are predominantly expressed in testes. Severe impacts on L. hesperus reproduction, including significantly reduced sperm prevalence, sperm length, and egg fertilization were observed following RNAi disruption of Lhβtub2. CRISPR/Cas9 knockout of Lhβtub2 further confirmed its functional role in male L. hesperus reproduction. Our data show that Lhβtub2 is essential for L. hesperus spermatogenesis and could serve as a potential male-specific target of genetic SIT in this agriculturally important pest.