The sterile insect technique (SIT) plays an important role in environmentally sustainable pest management. Its effectiveness hinges on specialized genetic tools called genetic sexing strains (GSSs), which enable the production and release of sterile male insects while excluding females. A collaborative initiative by the International Atomic Energy Agency (IAEA), designated as D44003 “A generic approach for the development of genetic sexing strains for Sterile Insect Technique applications,” aimed to establish a universal framework for developing GSSs applicable to various pest species. This overview summarizes findings from 17 research studies, including laboratory models and field pests. The studies focused on enhancing genetic markers, genome editing, understanding sex determination, creating temperature-sensitive lethal systems, refining radiation protocols, and improving strain components. Collectively, these efforts demonstrate that a versatile, species-independent approach is both feasible and practical in real-world pest control efforts. This progress paves the way for the broader adoption of the SIT, promising a significant advance in integrated pest management.

The Bactrocera genus includes highly invasive fruit and vegetable pest species such as Bactrocera dorsalis, Bactrocera correcta, and Bactrocera oleae. The sterile insect technique (SIT) is a biological control method used to suppress populations of the invasive Bactrocera fruit flies by releasing sterilized male insects that compete with wild males for mates, reducing reproduction and eventually pest numbers. The effectiveness of the SIT against insect pests can be enhanced through male-only releases, achieved via genetic sexing strains (GSS) that enable early-stage sex separation. To overcome limitations faced when developing a new GSS through the classical genetic approach, a novel “neoclassical approach” has been proposed, focusing on the identification of genetic markers, the induction of desired phenotypes through genome editing, and the linkage of selectable markers to male sex. In this study, we evaluated the white pupae gene as a selectable marker for GSS development in 3 Bactrocera species. The white pupae orthologous genes have been identified, and, through CRISPR/Cas9 mutagenesis, the 3rd exon of the white pupae gene was knocked out resulting in white pupae lines in Bactrocera dorsalis, Bactrocera correcta, and Bactrocera oleae species. These results demonstrate the applicability of CRISPR/Cas9-mediated disruption of the conserved white pupae gene as a selectable marker in multiple Bactrocera species, supporting the development of genetic sexing systems for SIT-based pest management.

Bactrocera zonata is a highly invasive agricultural pest that causes extensive damage to fruit crops. The Sterile Insect Technique (SIT), a species-specific and environmentally friendly pest control method, significantly benefits from the availability of Genetic Sexing Strains (GSSs) that enable efficient mass production of males for sterile release. However, no GSS currently exists for B. zonata limiting SIT applications targeting this important invasive pest. Here, we report two key advancements toward GSS development in this species. First, we present a high-quality, chromosome-level genome assembly from male B. zonata, identifying two scaffolds derived from the Y chromosome, which represent potential targets for future male-specific genetic engineering. Second, we demonstrate the feasibility of CRISPR/Cas9 genome editing in B. zonata by generating stable, homozygous white-eye mutants through targeted disruption of the conserved white-eye gene. This visible, recessive phenotype serves as a proof-of-concept for developing selectable markers in this species. Together, these results provide foundational genomic and genetic tools to support the development of GSSs in B. zonata, advancing the potential for sustainable, genetics-based pest control strategies.

Genetic sexing strains (GSS) are important tools for the sterile insect technique (SIT), an environmentally friendly and species-specific insect pest control method. GSS feature sex-specific phenotypes, enabling sex sorting in mass-rearing facilities and male-only releases, which significantly improve the cost-effectiveness and efficiency of SIT programs. In classical GSS, sex linkage of marker gene(s), such as white pupae (wp), is achieved through an irradiation-induced translocation between the marker-carrying autosome and the Y chromosome. However, this approach may render GSS males semisterile. The recently proposed neo-classical GSS concept suggests using genome editing to achieve sex linkage by directly inserting the wild-type marker allele onto the Y chromosome, potentially yielding GSS males with higher fertility. In this study, we examined the Ceratitis capitata wp gene as a genetic marker for the neo-classical GSS concept and developed a minimal, intronless version of this gene, termed mini-wp. We demonstrate that a single copy of mini-wp is sufficient to restore the wild-type brown puparium phenotype and is functional when integrated at various positions within the C. capitata genome, including the X chromosome. Due to its smaller size (4689 bp, including 2000 bp of putative promoter region) relative to the full wild-type wp allele (20868 bp), mini-wp may facilitate its precise insertion into the Y chromosome, representing an important step toward realizing neo-classical GSS. Furthermore, the methodology developed for designing and testing mini-wp in medfly may be adapted to other Tephritid species with an identified wp gene.

The sterile insect technique (SIT) is a highly effective biologically-based method for the suppression of many insect pest populations. SIT efficacy could be improved by methods of male sterilization that avoid the use of irradiation that can result in diminished fitness and mating competitiveness. Alternative sterilization methods include conditional disruption of genes for male fertility, or using their sperm-specific promoters to drive the expression of genes for lethal effectors. Testing has begun for the testis-specific β2-tubulin gene, though additional male fertility genes are required for redundancy or replacement, and for species where the β2-tubulin isoform does not exist or is not testis-specific. Here we had the goal of identifying and characterizing the sequence and transcriptional expression of two genes in the caribfly, Anastrepha suspensa, that are cognates of D. melanogaster spermatocyte-specific male fertility genes. In Drosophila, wampa encodes a coiled-coil dynein subunit required for axonemal assembly essential to microtubule-based sperm motility, while Prosα6T is a proteasome subunit gene required for spermatid individualization and nuclear maturation. In A. suspensa a cognate to wampa exhibited testis-specific transcript expression, which was minimal in both male and female body tissue. A Prosα6T cognate was not apparent in A. suspensa, but its constitutive isoform, Prosα6, expresses in male testes, but also in male and female body tissue. Thus, for A. suspensa, wampa remains a strong candidate gene for male sterility strategies for SIT including a direct target for gene-editing knockout and use of its promoter for testis-specific toxicity or cell death in conditional expression systems.

The Sterile Insect Technique (SIT) is an environmentally friendly, sustainable pest control approach, which uses large-scale releases of sterile insects to suppress or eradicate target populations through infertile matings. The efficiency of SIT is enhanced by male-only releases requiring genetic sexing strains (GSSs) that are classically based on selectable recessive visible markers or temperature-sensitive lethal (tsl) mutations and a rescue by a wild-type allele translocated to the male-determining chromosome. The transfer of identified or designed temperature-sensitive alleles might allow the generation of neoclassical GSSs in additional SIT target species. By using precise genome-editing tools, such as CRISPR/Cas, the creation of specific mutations in target genes and the integration of a wild-type copy is feasible without the introduction of foreign DNA. This might ease regulation of neoclassical GSSs, since they are not considered transgenic. However, integration and expression of genes at male-determining loci or chromosomes is not reliably established. Therefore, additional strategies to link temperature-sensitive phenotypes to female development are required, which could be achieved by targeting genes involved in dosage compensation or sex determination. To create temperature-sensitive alleles, rational protein design using advanced modeling and prediction tools to evaluate and tailor the effect of mutations on protein stability and temperature sensitivity can be used. In addition, emerging synthetic biology strategies such as temperature-inducible N-degrons or temperature-sensitive inteins provide powerful tools to generate temperature sensitivity. Such approaches should enable conditional control over proteins causing female lethality or sex conversion and therefore promise straightforward generic approaches to generate GSSs for male-only production in SIT target species.

Bactrocera tryoni, the Queensland fruit fly, is among the most damaging insect pests to the Australian horticultural industry as larvae infest ripening fruits or vegetables prior to harvest. Genetic biocontrol using Sterile Insect Technique (SIT) programs have been used to successfully suppress populations, via mass release of factory-reared sterile males that mate with wild females. Bi-sex flies are currently used for releases, although the efficiency of these control programs could be improved through using genetic sexing strains that eliminate females early during development, as they are not required for SIT. Here we used CRISPR/Cas9 mutagenesis to modify two nucleotides in the B. tryoni gene shibire, which created a proline to serine amino acid substitution and produced a temperature sensitive phenotype. Shibire is an essential GTPase required in endocytosis and synaptic vesicle recycling, and classical mutagenic screens in the vinegar fly Drosophila melanogaster previously identified temperature sensitive alleles including shi^ts4 that results in adult paralysis. In B. tryoni, the shi^ts4 mutant strain exhibited similar adult paralytic phenotypes when exposed to high temperatures, as well as temperature dependent lethality at egg, larval and pupal stages when subjected to heat treatment above standard rearing temperatures. These temperature sensitive phenotypes could be adapted to develop a SIT genetic sexing strain for conditional elimination of females prior to sterile releases, to improve efficiency and reduce costs.

The genetic network of sex determination in the model organism Drosophila melanogaster was investigated in great detail. Such knowledge not only advances our understanding of the evolution and regulation of sexual dimorphism in insects, but also serves as a basis for developing genetic control strategies for pest species. In this study, we isolated the sex determination gene transformer (Dstra) from a global fruit pest, the spotted-wing Drosophila (Drosophila suzukii), and characterized its gene organization. By comparing the deduced protein sequence of Dstra with its orthologs from 22 species, we found that tra genes from Dipteran species are divergent. Our research demonstrated that Dstra undergoes sex-specific splicing, and we validated its developmental expression profile. We engineered a piggyBac-based transformation vector expressing the complete Dstra coding sequence under the control of the Tetracycline-Off system. Through germ-line transformation, we generated 4 independent transgenic lines, producing pseudo-females from chromosomal males in the absence of tetracycline. This observation indicated ectopic expression of Dstra, confirmed by the detection of female Dstra transcripts in transgenic males. The pseudo-females exhibited altered wing patterns, feminized abdomen, abnormal reproductive organs, and disrupted sexual behavior. Ectopic expression of Dstra affected the sex-specific splicing pattern of the downstream gene fruitless, but not doublesex. In conclusion, our study provides comprehensive genetic, morphological, and behavioral evidence that Dstra controls sexual development in D. suzukii. We discuss the potential applications of this research for genetic control strategies targeting Dstra or using its gene elements.

Globalization and climate change may be driving the spread of the quarantine fruit fly, Bactrocera carambolae, highlighting the need for ecofriendly control methods like the Sterile Insect Technique (SIT), which relies on releasing sterile males to reduce wild populations. However, developing effective strains through genetic manipulation of sex-determination genes remains challenging due to limited knowledge of this pathway in B. carambolae. In this study, the structures of three key orthologous genes—transformer (tra), transformer-2 (tra-2), and doublesex (dsx)—were characterized and compared with those of other Bactrocera species. These genes were found to share conserved structures, following a bottom-up pattern from Bcardsx to Bcartra-2 and Bcartra, with intron retention observed in Bcartra as a structural variation. Developmental expression analysis revealed that the splicing patterns of Bcartra first appeared at 6 hours post oviposition (hpo), with sex-specific patterns established by 10 hpo. The expression profiles in B. carambolae were similar to those in the closely related species B. dorsalis, although gene expression occurred later in B. carambolae. RNAi knockdown of Bcartra resulted in nearly all phenotypic males, supporting the role of BcarTRA in controlling the sex-specific splicing of Bcartra itself and the downstream genes doublesex and fruitless. Additionally, in silico predictions of protein interactions (TRA^F, TRA-2, and RBP1) and putative cis-regulatory elements on Bcartra pre-mRNA suggested specific binding events occur at these conserved sites. These findings contribute to the development of potential genetic tools for pest control and provide insights into the evolutionary relationships among these orthologous genes.

Ceratitis capitata (medfly), a major agricultural pest, is predominantly controlled using chemical insecticides, which pose environmental risks. Ecosustainable alternatives, such as the Sterile Insect Technique (SIT), rely on the mass release of sterile male-only progeny. However, sexing of male offspring requires the elimination of females during development. To overcome the loss of 50% of the progeny, the sex reversal of females into XX fertile males at embryonic stages by dsRNA injections was effective but not scalable. This study demonstrates the efficacy of Dicer-substrate small interfering RNAs (DsiRNAs) as an alternative to long double-stranded RNAs (dsRNAs) for targeting the sex determination genes Cctransformer and Cctransformer-2 in the medfly, which causes full masculinization of XX individuals. Injection of DsiRNAs into XX embryos induced the expression of male-specific Cctra isoforms within a few hours, resulting in the development of adult masculinized XX flies. Additionally, thoracic injection of DsiRNAs in adult females achieved systemic gene silencing, reducing Cctra/Cctra-2 transcript levels in the ovaries by 75%–80% within 48 h. The reduced size of DsiRNAs compared to dsRNAs enhances their potential for alternative delivery methods, including embryo permeabilization, electroporation, and feeding in larvae or adult females. These findings provide a potential foundation for future scalable conditional masculinization of XX individuals rather than relying on female lethality, doubling male-only productivity. Developing novel sexing methods as an alternative to transgenic approaches will expand the applicability of SIT.

Moths and butterflies (Lepidoptera) are the largest group of organisms with female heterogamety and the sex chromosome system WZ/ZZ (female/male) or exceptionally Z0/ZZ. However, the genetic basis of sex determination in Lepidoptera remained unknown for a long time until the sex-determining pathway was discovered in 2014 in the silkworm Bombyx mori. In this species, the dominant W chromosome carries a Feminizer (Fem) gene encoding a precursor of a Fem piRNA that promotes femaleness by downregulating the expression of a Z-linked gene, Masculinizer (Masc). In the W chromosome absence, Masc promotes male development and controls dosage compensation. In the 10 years since this discovery, significant progress has been made in understanding the molecular mechanisms of sex determination in Lepidoptera. Data from recent studies discussed in this review suggest a conserved role for Masc in male sex determination and dosage compensation in the clade Ditrysia, which comprises the majority of Lepidoptera. Although the primary sex-determining signals are not conserved, the presence of feminizing piRNAs of different origins in distantly related species suggests convergent evolution of a similar mechanism of female sex determination. A unique exception is zygosity-based sex determination in the butterfly Bicyclus anynana, where the primary signal is the state of the hypervariable Masc gene. In other species with a dispensable W chromosome, such as the silkmoth Samia cynthia, sex is determined by the Z:A ratio, but a molecular mechanism is not yet known. Overall, the available data suggest considerable diversity in the upstream molecular mechanisms of sex determination in Lepidoptera.

The sterile insect technique, which consists of the mass production and release of sterile insects to control populations of pests and disease vectors, has been effectively used for decades. An important component of sterile insect technique field applications is the availability of sex separation systems that reliably and economically eliminate females from mass-reared sterile insect populations destined for field release. Genetic sexing strains are important for the effectiveness and cost-efficiency of insect population control programs, including sterile insect technique. Classical approaches to generate genetic sexing strains, such as irradiation-induced chromosomal translocations, have yielded stable strains for species like the Mediterranean fruit fly, Ceratitis capitata. However, significant efforts are needed to establish genetic sexing strains using classical genetic methods, as large-scale random mutagenesis and screening are needed. We introduce here a neoclassical genetic approach, leveraging CRISPR-based gene-editing to target known genes to develop selectable genetic markers, followed by genetic rescue in a male-specific manner to speed up the development of genetic sexing strains and enhance their precision, stability, and adaptability. The integration of molecular tools, genetic markers like the white pupae and temperature-sensitive lethal, and strategies for maintaining genetic stability are discussed. We also review the challenges and opportunities in applying classical, transgenic, and neoclassical genetic approaches to improve genetic sexing strains for pest management.

Anastrepha fraterculus is a cryptic species complex with at least eight morphotypes distributed across the Americas. Among them, A. fraterculus sp.1, present in Argentina, is a major pest impacting fresh fruit production. Integrated pest management strategies, including chemical control and trapping, are currently employed to mitigate its effects. Genetic sexing strains of A. fraterculus sp.1 are being evaluated for use in sterile insect technique programs. To support traditional and emerging control methods, this study aimed to enhance the genomic understanding of this morphotype. Individual female and male samples were sequenced using long- and short-read technologies. The female genome (760 Mb) was de novo assembled into 58 scaffolds and the male genome (750 Mb) into 68 scaffolds, with BUSCO completeness scores of 98.8% and 98.7%, respectively. Synteny analysis revealed complete scaffolds of the five autosomes and enabled near-complete reconstruction of the X and Y chromosomes. Gene prediction identified 17 751 and 16 535 protein-coding genes (for female and male genomes, respectively), with repetitive regions representing 46% of both genomes. Additionally, the mitochondrial genome was fully assembled and annotated. This comprehensive genomic resource reveals candidate genes for functional studies, including gene editing and RNA interference, as successfully applied in related tephritid species. These findings lay the foundation for innovative, complementary biocontrol tools against A. fraterculus.

The detection and characterization of sex chromosome sequences is particularly important for major pest families, like the Tephritidae, whereas alternative pest management approaches, mainly involving male-only release programs, rely on the ability to target and manipulate sex-specific genomic regions, particularly those of the Y chromosome. However, resolving and detecting X and Y chromosome sequences at the chromosome level requires careful consideration of algorithmic outputs, especially in species where extensive sex chromosome markers are not available. Here, we present R-CQ and KAMY, two computational methods developed for the detection of sex chromosome-linked sequences through sex-specific short-read DNA sequencing datasets. We evaluate their performance on newly generated chromosome-level assemblies of four important Tephritid pest species: Ceratitis capitata, Bactrocera dorsalis, Bactrocera zonata, and Anastrepha ludens. By combining algorithmic predictions with a manual curation process, we assess the strengths and limitations of each method and provide a robust dataset of curated X- and Y-linked sequences. Overall, our results establish a framework for studying poorly characterized sex chromosome lineages and identifying sex-specific genomic regions, supporting the broader development of sex chromosome-based pest managements systems.

Anastrepha obliqua, a neotropical pest widely distributed in the Americas, attacks mango and other tropical fruits. In Mexico, it is controlled through integrated pest management, using the Sterile Insect Technique (SIT) as a main component. The applicability of SIT is significantly improved with the use of genetic sexing strains (GSS) that allow the possibility to release exclusively sterile males, the primary component of the technique. This study reports the isolation and characterization of two pupal mutations: black pupae (bp) and sphere pupae (sp), allowing for the first time the development of a genetic sexing system based on pupal color in this species. Inheritance analyses from reciprocal crosses between wild-type and mutant individuals showed F₂ phenotypic segregation consistent with a recessive Mendelian inheritance pattern, and linkage analysis indicated that the bp and sp loci are in separate chromosomes. Using the bp mutation, two GSS were developed through gamma irradiation [T(Y;bp⁺)/bp-22](GSS-22) and ethyl methanesulfonate treatment [T(Y;bp⁺)/bp-354](GSS-354). Both GSS exhibited sex-specific pupal differentiation but displayed a high frequency of recombinants. Despite an initial reduction in biological fitness, GSS-22 demonstrated greater genetic stability and a lower frequency of recombinants than GSS-354. Discrepancies between cytogenetic and genomic data, particularly regarding the localization of the gene responsible for the black pupae phenotype, underscore the need to integrate polytene chromosome and genomic analyses to characterize these translocations and improve GSS stability precisely. These results represent a breakthrough in the creation of genetic tools for the management of A. obliqua control.

The Sterile Insect Technique (SIT) is an effective strategy for controlling insect pests, such as the Mediterranean fruit fly (Ceratitis capitata, Wiedemann). The effectiveness of the SIT depends on the ability of the sterile males to mate and their capacity to induce sterility in wild females. This study evaluated how the irradiation age affects their sexual performance, measured by the outcome of female remating events. Males of the GSS VIENNA 8^D53− were irradiated at eleven different ages, from 72 h before emergence (pupal stage) to 72 h after emergence (adult stage) and mated with wild females. These females were subsequently allowed to mate with fertile males from the fluorescent TSS VIENNA 8 1260. The presence of fluorescent offspring was used as indicator of second-male paternity. Results showed that males irradiated at post-emergence ages produced the lowest egg-to-pupae conversion rate, indicating a greater ability to prevent females from producing offspring after remating with a fertile male. In contrast, males irradiated at pre-emergence ages were associated with higher numbers of fluorescent offspring. Although no significant differences were found in mating competitiveness (RSI), the outcome of the remating showed differences in the effectiveness of initial matings. These findings highlight the importance of considering the age of flies at time of irradiation in mass-rearing protocols to enhance the efficacy of SIT programs targeting C. capitata and suggest that irradiating males later in their life cycle, such as adult stages or in pupae close to adult emergence, limit offspring if females remate with fertile males in the field.

Sterile insect technique (SIT) has become a key component of efficient pest control. Fruit fly pests from the Drosophilidae and Tephritidae families pose a substantial and overwhelmingly increasing threat to the agricultural industry, aggravated by climate change and globalization among other contributors. In this review, we discuss the advances in genetic engineering aimed to improve the SIT-mediated fruit fly pest control. This includes SIT enhancement strategies such as novel genetic sexing strain and female lethality approaches. Self-pervasive X-shredding and X-poisoning sex distorters, alongside gene drive varieties are also reviewed. The self-limiting precision-guided SIT, which aims to tackle female removal and male fertility via CRISPR/Cas9, is additionally introduced. By using examples of existing genetic tools in the fruit fly pests of interest, as well as model species, we illustrate that the population control intensity may be modulated depending on strategy selection.

Genetic biocontrol methods are species-specific ways to suppress or modify pest insect populations to mitigate their economic or health impact. Successful genetic biocontrol often requires mass releases of only males of the target species. Reliable and cost-effective sex separation is a major bottleneck to the implementation of genetic biocontrol of many important species of agricultural and medical importance. Conditional selection is critical to resolving this major challenge. A diverse array of tools, such as the temperature-sensitive systems, the Gal4/UAS, QF/QUAS, and Tet-on/off bipartite systems, and the photoactivatable systems, have been established in various insect species. In this review, we focus on how various means of conditional expression have been used to achieve sex separation. We also describe other means of selection and counterselection to achieve sex separation without conditional gene expression. By providing examples across many species and discussing the strengths and weaknesses of each method, we hope to facilitate the design and application of conditional systems to improve genetic biocontrol of insect pests.