Fluorescent-Based Sex-Separation Technique in Major Invasive Crop Pest, Drosophila suzukii

Abstract

Insect population biocontrol methods, such as the sterile insect technique (SIT), offer promising alternatives to traditional pesticide-based approaches for managing pest populations. Effective use of these strategies requires scal-able sex separation techniques, which are currently nonexistent for Drosophila suzukii, a prominent crop pest species. SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter) is a fluorescence-based sex-sorting technology previously developed for other pest species that enables the identification of females and males based on sex-specific fluorescence. In this study, we investigate the applicability of SEPARATOR to D. suzukii by engineering several strains encoding SEPARATOR constructs that facilitate efficient sex selection in early larval stages. Our findings demonstrate the feasibility of using SEPARATOR in D. suzukii by incorporating transformer introns from three different species: D. suzukii, D. melanogaster, and Ceratitis capitata. This study provides an initial basis for further optimization of SEPARATOR in D. suzukii and indicates that it could be a helpful tool for SIT-based biocontrol strategies. T he spotted wing drosophila, Drosophila suzukii, has recently emerged as a major agricultural pest, particularly affecting cherries, blueberries, and raspberries. 1-3 Since the late 2000s, it has rapidly spread from East Asia to North America and Europe. 4,5 Unlike most of its close relative species, D. suzukii oviposits eggs inside ripening fruit rather than decaying fruit. 6-8 The females use a serrated ovipositor to pierce fruit and deposit eggs, leading to larvae feeding on the fruit. 6,9 Furthermore, the physical damage from oviposition creates entry points for secondary infections by bacteria, fungi, and yeast. 5,10 This results in substantial yield losses and increased labor demands for postharvest fruit sorting, ultimately driving up costs. 11 Moreover, D. suzukii's short generation time accelerates its spread, leading to significant economic losses and increased pesticide usage, including broad-spectrum organo-phosphates, pyrethroids, and spinosyns. 12-15 Most control programs for D. suzukii heavily rely on harmful insecticides. 16,17 Alternative biocontrol methods, such as the sterile insect technique (SIT), offer more sustainable options to reduce chemical use. These methods involve the mass-rearing of insects, which are sterilized and released to suppress wild populations. 18 In traditional SIT programs, such as those targeting the New World screwworm, both sterile males and females are released together due to the lack of sex-sorting technologies. 19,20 Releasing only sterile males, however, is significantly more cost-effective and efficient. 21 The release of sterile females poses several challenges, including additional crop damage from oviposition and reduced suppression efficiency due to sterile females mating. 22-24 For this reason, removing females before release is considered critical in SIT programs. 25-27 Despite its importance, the selective removal of females remains one of the most significant challenges in implementing SIT programs. Manual sex sorting is currently the primary and, in some species , the only available method, making the process labor-intensive, time-consuming, and prone to human error. 19,28 While machine learning has been applied to automate sex sorting in mosquito species such as Aedes albopictus and Aedes aegypti, these methods rely on naturally occurring sexual dimorphisms, limiting their applicability to other pest or vector species. 19,28,29 Large-scale SIT implementation requires a more accurate, efficient, and adaptable alternative. Recently, pgSIT was developed in D. suzukii, eliminating the need for irradiation to produce sterile males and remove females. 30 However , this approach requires manually setting up crosses to generate F1 sterile males for release. Combining pgSIT with Sex-ing Elements Produced by Alternative RNA-splicing of A Trans-genic Observable Reporter (SEPARATOR) could eliminate the