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.

No abstract

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.

Availability of polytene chromosomes and development of polytene chromosome maps have greatly facilitated genetic analysis in Diptera and understanding of chromosomal organization. In tephritids, following the first polytene chromosome maps constructed for the Mediterranean fruit fly, Ceratitis capitata, additional maps have been developed for only few species belonging to the main genera of agricultural importance that are Anastrepha, Bactrocera, Ceratitis, Dacus, Rhagoletis, and Zeugodacus. Comparison of the polytene chromosomes of these species has pointed to the presence of chromosomal rearrangements that can, at least partially, shed light to the chromosomal evolution in this family. Up to now, polytene chromosome maps are available only for one Zeugodacus species, that is Zeugodacus cucurbitae. Here we report the cytogenetic analysis of the mitotic and polytene chromosomes of the pumpkin fly, Zeugodacus tau, along with a comparative analysis with polytene chromosomes of Zeugodacus cucurbitae as well as other tephritids. In situ hybridization experiments resulting to chromosomal localization of selected genes in both species are also presented. The genes used as markers are hsp70, hsp83, scarlet and white pupae. The established homologies presented in this study verify that the two Zeugodacus species are genetically close and support the current taxonomic placement of the Zeugodacus genus. The differences in polytene chromosome level, in combination with results of in situ hybridization experiments, reveal the presence of chromosomal rearrangements, mainly inversions, to both closely and distantly related species, which could potentially be a useful diagnostic tool.