Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp ⁻ mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of wp ⁻ mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.

Anastrepha ludens (Loew) is one of the most destructive insect pests damaging several fruits of economic importance. The sterile insect technique (SIT) is used under an area-wide integrated pest management approach, to suppress these pest populations. Mass rearing facilities were initially established to produce sterile males of bi-sexual strains in support of SIT. The first genetic sexing strain (GSS) for A. ludens, Tapachula-7, based on pupal color dimorphism, was a key development since the release of males-only significantly increases the SIT efficiency. In this study, we document the development of a novel pupal color-based GSS. Twelve radiation-induced translocation lines were assessed as potential GSS in terms of recombination rates and rearing efficiency at a small scale. The best one, GUA10, was cytogenetically characterized: it was shown to carry a single translocation between the Y chromosome and chromosome 2, which is known to carry the black pupae marker. This GSS was further evaluated at medium and large scales regarding its genetic stability, productivity and quality versus Tapachula-7. GUA10 presented better genetic stability, fecundity, fertility, production efficiency, flying ability, and male mating, clear indicators that GUA10 GSS can significantly improve the efficacy and cost-effectiveness of SIT applications against this pest species.

Area-wide integrated pest management (AW-IPM) programmes with a sterile insect technique component (SIT) are used to control populations of insect pests worldwide, including the Mediterranean fruit fly, Ceratitis capitata. SIT consists of the mass rearing, radiation-induced sterilization, handling, and release of sterile insects over the target area. Although SIT can be performed by using both sterile males and females, male-only releases significantly increase the efficiency and cost-effectiveness of SIT applications. Male-only releases can be achieved by using genetic sexing strains (GSS). The medfly VIENNA 8 GSS is based on two selectable markers, the white pupae (wp) gene, and the temperature-sensitive lethal (tsl) genes. The latter allows the elimination of females by exposing embryos to elevated temperatures. This study assessed the temperature sensitivity of twenty-seven medfly strains through a TSLT. Our results indicated significant differences among the strains regarding egg hatching as well as pupal and adult recovery rates due to the presence or absence of the tsl mutation and/or the genetic background of the strains. Our findings are discussed in the context of SIT applications, the importance of the tsl gene for developing genetic sexing strains, and climate change.

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. 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.

TheCeratitisFARQ species complex consists of four highly destructive agricultural pests of Africa, namelyC. fasciventris,C. anonae,C. rosa, andC. quilicii. The members of the complex are considered very closely related and the species limits among them are rather obscure. Their economic significance and the need for developing biological methods for their control makes species identification within the complex an important issue, which has become clear that can only be addressed by multidisciplinary approaches. Chromosomes, both mitotic and polytene, can provide a useful tool for species characterization and phylogenetic inference among closely related dipteran species. In the current study, we present the mitotic karyotype and the polytene chromosomes ofC. rosaandC. quiliciitogether within situhybridization data. We performed a comparative cytogenetic analysis among the above two species andC. fasciventris, the only other cytogenetically studied member of the FARQ complex, by comparing the mitotic complement and the banding pattern of the polytene chromosomes of each species to the others, as well as by studying the polytene chromosomes of hybrids between them. Our analysis revealed no detectable chromosomal rearrangements discriminating the three FARQ members studied, confirming their close phylogenetic relationships.