In insects, rapidly evolving primary sex-determining signals are transduced by a conserved regulatory module controlling sexual differentiation. In the agricultural pest Ceratitis capitata (Mediterranean fruit fly, or Medfly), we identified a Y-linked gene, Maleness-on-the-Y (MoY), encoding a small protein that is necessary and sufficient for male development. Silencing or disruption of MoY in XY embryos causes feminization, whereas overexpression of MoY in XX embryos induces masculinization. Crosses between transformed XY females and XX males give rise to males and females, indicating that a Y chromosome can be transmitted by XY females. MoY is Y-linked and functionally conserved in other species of the Tephritidae family, highlighting its potential to serve as a tool for developing more effective control strategies against these major agricultural insect pests.

The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economic impact and has become an emerging model for developing new genetic control strategies as an alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific single guide RNAs (sgRNA) into early embryos. When targeting the eye pigmentation gene white eye (we), a high rate of somatic mosaicism in surviving G0 adults was observed. Germline transmission rate of mutated we alleles by G0 animals was on average above 52%, with individual cases achieving nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end-joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in C. capitata will significantly advance the design and development of new effective strategies for pest control management.

In insects, rapidly evolving primary sex-determining signals are transduced by a conserved regulatory module producing sex-specific proteins that direct sex determination and sexual differentiation 1-4 . In the agricultural pest Ceratitis capitata (medfly), a Y-linked maleness factor (M) is thought to repress the autoregulatory splicing of transformer (Cctra), which is required in XX individuals to establish and maintain female sex determination 5,6 . Despite previous attempts of isolating Y-linked genes using the medfly whole genome, the M factor has remained elusive 7 . Here, we report the identification of a Y-linked gene, Maleness-on the-Y (MoY), and show that it encodes a small novel protein which is both necessary and sufficient for medfly male sex determination. Transient silencing of MoY in XY individuals leads to the development of fertile females while transient expression of MoY in XX individuals results in fertile males. Notably, a cross between these sex reverted individuals gives rise to both fertile males and females indicating that a functional MoY can be maternally transmitted. In contrast to the diversity of M factors found in dipteran species 8-11 , we discovered MoY orthologues in seven other Tephritid species spanning ~111 millions of years of evolution (Mya). We confirmed their male determining function in the olive fly (Bactrocera oleae) and the oriental fruit fly (Bactrocera dorsalis). This unexpected conservation of the primary MoY signal in a large number of important agricultural pests 12will facilitate the development of transferable genetic control strategies in these species, for example sterile male releases or sex-ratio-distorting gene drives.

The olive fruit fly, Bactrocera oleae (Diptera: Tephritidae), is the most destructive insect pest of olive cultivation, causing significant economic and production losses. Here, we present the establishment of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 methodology for gene disruption in this species. We performed targeted mutagenesis of the autosomal gene white (Bo‐we), by injecting into early embryos in vitro preassembled and solubilized Cas9 ribonucleoprotein complexes loaded with two gene‐specific single‐guide RNAs. Gene disruption of Bo‐we led to somatic mosaicism of the adult eye color. Large eye patches or even an entire eye lost the iridescent reddish color, indicating the successful biallelic mutagenesis in somatic cells. Cas9 induced either indels in each of the two simultaneously targeted Bo‐we sites or a large deletion of the intervening region. This study demonstrates the first efficient implementation of the CRISPR/Cas9 technology in the olive fly, providing new opportunities towards the development of novel genetic tools for its control.

In the last decades, the colonization of Mediterranean Europe and of other temperate regions by Aedes albopictus created an unprecedented nuisance problem in highly infested areas and new public health threats due to the vector competence of the species. The Sterile Insect Technique (SIT) and the Incompatible Insect Technique (IIT) are insecticide-free mosquito-control methods, relying on mass release of irradiated/manipulated males, able to complement existing and only partially effective control tools. The validation of these approaches in the field requires appropriate experimental settings, possibly isolated to avoid mosquito immigration from other infested areas, and preliminary ecological and entomological data. We carried out a 4-year study in the island of Procida (Gulf of Naples, Italy) in strict collaboration with local administrators and citizens to estimate the temporal dynamics, spatial distribution, and population size of Ae. albopictus and the dispersal and survival of irradiated males. We applied ovitrap monitoring, geo-spatial analyses, mark-release-recapture technique, and a citizen-science approach. Results allow to predict the seasonal (from April to October, with peaks of 928–9,757 males/ha) and spatial distribution of the species, highlighting the capacity of Ae. albopictus population of Procida to colonize and maintain high frequencies in urban as well as in sylvatic inhabited environments. Irradiated males shown limited ability to disperse (mean daily distance travelled <60m) and daily survival estimates ranging between 0.80 and 0.95. Overall, the ecological characteristics of the island, the acquired knowledge on Ae. albopictus spatial and temporal distribution, the high human and Ae. albopictus densities and the positive attitude of the resident population in being active parts in innovative mosquito control projects provide the ground for evidence-based planning of the interventions and for the assessment of their effectiveness. In addition, the results highlight the value of creating synergies between research groups, local administrators, and citizens for affordable monitoring (and, in the future, control) of mosquito populations.

Background Females of the Mediterranean fruit fly Ceratitis capitata (Medfly) are major agricultural pests, as they lay eggs into the fruit crops of hundreds of plant species. In Medfly, female sex determination is based on the activation of Cctransformer (Cctra). A maternal contribution of Cctra is required to activate Cctra itself in the XX embryos and to start and epigenetically maintain a Cctra positive feedback loop, by female-specific alternative splicing, leading to female development. In XY embryos, the male determining Maleness-on-the-Y gene (MoY) blocks this activation and Cctra produces male-specific transcripts encoding truncated CcTRA isoforms and male differentiation occurs. Results With the aim of inducing frameshift mutations in the first coding exon to disrupt both female-specific and shorter male-specific CcTRA open reading frames (ORF), we injected Cas9 ribonucleoproteins (Cas9 and single guide RNA, sgRNA) in embryos. As this approach leads to mostly monoallelic mutations, masculinization was expected only in G1 XX individuals carrying biallelic mutations, following crosses of G0 injected individuals. Surprisingly, these injections into XX-only embryos led to G0 adults that included not only XX females but also 50% of reverted fertile XX males. The G0 XX males expressed male-specific Cctra transcripts, suggesting full masculinization. Interestingly, out of six G0 XX males, four displayed the Cctra wild type sequence. This finding suggests that masculinization by Cas9-sgRNA injections was independent from its mutagenic activity. In line with this observation, embryonic targeting of Cctra in XX embryos by a dead Cas9 (enzymatically inactive, dCas9) also favoured a male-specific splicing of Cctra, in both embryos and adults. Conclusions Our data suggest that the establishment of Cctra female-specific autoregulation during the early embryogenesis has been repressed in XX embryos by the transient binding of the Cas9-sgRNA on the first exon of the Cctra gene. This hypothesis is supported by the observation that the shift of Cctra splicing from female to male mode is induced also by dCas9. Collectively, the present findings corroborate the idea that a transient embryonic inactivation of Cctra is sufficient for male sex determination.

The colonization of Mediterranean Europe and of other temperate regions by Aedes albopictus created in the last decades an unprecedented nuisance problem in highly infested areas, as well as a new public health threat due to the species competence to transmit exotic arboviruses, such as dengue, chikungunya and zika. The Sterile Insect Technique (SIT) and the Incompatible Insect Technique (IIT) are insecticide-free mosquito-control methods relying on mass release of irradiated/manipulated males which are believed to have a potential in complementing existing and only partially effective control tools. Testing and implementing these approaches are challenging and selection of study sites is an instrumental and crucial step. We carried out a 4-year study in Procida Island (Gulf of Naples, Italy) in strict collaboration with local administrators and citizens to estimate: i) the temporal dynamics, spatial distribution, and population size of Ae. albopictus; and ii) the dispersal and survival of irradiated males. Overall, results provide insights on the bionomics of the mosquito in Southern Europe and draw attention to Procida Island as an ideal site to test innovative control programs against Ae. albopictus which may be used in other Mediterranean and temperate areas.