DsiRNA-mediated silencing of Ceratitis capitata transformer or transformer-2 leads to masculinization of XX embryos and systemic gene silencing in ovaries

Abstract

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.

Keywords

DsiRNAs / Mediterranean fruit fly / sex determination / sexing / Sterile Insect Technique / systemic RNAi

References

[1] Ali, M.W., Zheng, W., Sohail, S., Li, Q., Zheng, W. and Zhang, H. (2017) A genetically enhanced sterile insect technique against the fruit fly, Bactrocera dorsalis (Hendel) by feeding adult double-stranded RNAs. Scientific Reports, 7, 4063.

[2] Amarzguioui, M., Lundberg, P., Cantin, E., Hagstrom, J., Behlke, M.A. and Rossi, J.J. (2006) Rational design and in vitro and in vivo delivery of Dicer substrate siRNA. Nature Protocols, 1, 508–517.

[3] Aumann, R.A., Gouvi, G., Gregoriou, M.E., Rehling, T., Sollazzo, G., Bourtzis, K. et al. (2025) Decoding and engineering temperature-sensitive lethality in Ceratitis capitata for pest control. Proceedings of the National Academy of Sciences USA, 122, e2503604122.

[4] Baum, J.A., Bogaert, T., Clinton, W., Heck, G.R., Feldmann, P., Ilagan, O. et al. (2007) Control of coleopteran insect pests through RNA interference. Nature Biotechnology, 25, 1322–1326.

[5] Bucher, G., Scholten, J. and Klingler, M. (2002) Parental rnai in Tribolium (Coleoptera). Current Biology, 12, R85–R86.

[6] Burtis, K.C. and Baker, B.S. (1989) Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sex-specific polypeptides. Cell, 56, 997–1010.

[7] Chaverra-Rodriguez, D., Macias, V.M., Hughes, G.L., Pujhari, S., Suzuki, Y., Peterson, D.R. et al. (2018) Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing. Nature Communications, 9, 3008.

[8] Clemons, A., Haugen, M., Le, C., Mori, A., Tomchaney, M., Severson, D.W. et al. (2011) siRNA-mediated gene targeting in Aedes aegypti embryos reveals that frazzled regulates vector mosquito CNS development. PLoS ONE, 6, e16730.

[9] Darrington, M., Dalmay, T., Morrison, N.I. and Chapman, T. (2017) Implementing the sterile insect technique with RNA interference—a review. Entomologia Experimentalis et Applicata, 164, 155–175.

[10] Davydova, S., Liu, J., Kandul, N.P., Braswell, W.E., Akbari, O.S. and Meccariello, A. (2023) Next-generation genetic sexing strain establishment in the agricultural pest Ceratitis capitata. Scientific Reports, 13, 19866.

[11] Delli Bovi, P., Pane, A., Polito, C. and Saccone, G. (2004) Cctra gene as a tool to produce male-only progeny in the Mediterranean fruitfly Ceratitis capitata. US20040082032A1. https://patents.google.com/patent/US20040082032A1/en.

[12] DeVault, J.D., Hughes, K.J., Leopold, R.A., Johnson, O.A. and Narang, S.K. (1996) Gene transfer into corn earworm (Helicoverpa zea) embryos. Genome Research, 6, 571–579.

[13] Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494–498.

[14] Fernando, D.D., Korhonen, P.K., Gasser, R.B. and Fischer, K. (2022) An RNA interference tool to silence genes in Sarcoptes scabiei eggs. International Journal of Molecular Sciences, 23, 873.

[15] Flynt, A.S. (2021) Insecticidal RNA interference, thinking beyond long dsRNA. Pest Management Science, 77, 2179–2187.

[16] Franz, G., Bourtzis, K. and Cáceres, C. (2021) Practical and operational genetic sexing systems based on classical genetic approaches in fruit flies, an example for other species amenable to large-scale rearing for the sterile insect technique. In Sterile Insect Technique (eds. V.A. Dyck, J. Hendrichs & A.S. Robinson), pp. 575–604. CRC Press.

[17] Fu, G., Condon, K.C., Epton, M.J., Gong, P., Jin, L., Condon, G.C. et al. (2007) Female-specific insect lethality engineered using alternative splicing. Nature Biotechnology, 25, 353–357.

[18] Gong, L., Wang, Z., Wang, H., Qi, J., Hu, M. and Hu, Q. (2015) Core RNAi machinery and three Sid-1 related genes in Spodoptera litura (Fabricius). International Journal of Agriculture and Biology, 17, 937–944.

[19] Hedley, M.L. and Maniatis, T. (1991) Sex-specific splicing and polyadenylation of dsx pre-mRNA requires a sequence that binds specifically to tra-2 protein in vitro. Cell, 65, 579–586.

[20] Kerremans, P. and Franz, G. (1994) Cytogenetic analysis of chromosome 5 from the Mediterranean fruit fly, Ceratitis capitata. Chromosoma, 103, 142–146.

[21] Kim, D.H., Behlke, M.A., Rose, S.D., Chang, M.S., Choi, S. and Rossi, J.J. (2005) Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nature Biotechnology, 23, 222–226.

[22] Koo, J., Gurusamy, D. and Palli, S.R. (2023) Inefficient uptake of small interfering RNAs is responsible for their inability to trigger RNA interference in Colorado potato beetle cells. Archives of Insect Biochemistry and Physiology, 114, 1–12.

[23] Kumar, M., Gupta, G.P. and Rajam, M.V. (2009) Silencing of acetylcholinesterase gene of Helicoverpa armigera by siRNA affects larval growth and its life cycle. Journal of Insect Physiology, 55, 273–278.

[24] Li, X., Zhang, M. and Zhang, H. (2011) RNA interference of four genes in adult Bactrocera dorsalis by feeding their dsRNAs. PLoS ONE, 6, e17788.

[25] Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method. Methods (San Diego, Calif.), 25, 402–408.

[26] Mao, J., Zhang, P., Liu, C. and Zeng, F. (2015) Co-silence of the coatomer β and v-ATPase A genes by siRNA feeding reduces larval survival rate and weight gain of cotton bollworm, Helicoverpa armigera. Pesticide Biochemistry and Physiology, 118, 71–76.

[27] Meccariello, A., Monti, S.M., Romanelli, A., Colonna, R., Primo, P., Inghilterra, M.G. et al. (2017) Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes. Scientific Reports, 7, 10061.

[28] Meccariello, A., Salvemini, M., Primo, P., Hall, B., Koskinioti, P., Dalíková, M. et al. (2019) Maleness-on-the-Y (MoY) orchestrates male sex determination in major agricultural fruit fly pests. Science, 365, 1457–1460.

[29] Meccariello, A., Hou, S., Davydova, S., Fawcett, J.D., Siddall, A., Leftwich, P.T. et al. (2024) Gene drive and genetic sex conversion in the global agricultural pest Ceratitis capitata. Nature Communications, 15, 372.

[30] Mehlhorn, S., Hunnekuhl, V.S., Geibel, S., Nauen, R. and Bucher, G. (2021) Establishing RNAi for basic research and pest control and identification of the most efficient target genes for pest control: a brief guide. Frontiers in Zoology, 18, 60.

[31] Mysore, K., Sun, L., Hapairai, L.K., Wang, C.W., Roethele, J.B., Igiede, J. et al. (2021) A broad-based mosquito yeast interfering RNA pesticide targeting Rbfox1 represses notch signaling and kills both larvae and adult mosquitoes. Pathogens, 10, 1251.

[32] Olademehin, O.P., Liu, C., Rimal, B., Adegboyega, N.F., Chen, F., Sim, C. et al. (2020) Dsi-RNA knockdown of genes regulated by Foxo reduces glycogen and lipid accumulations in diapausing Culex pipiens. Scientific Reports, 10, 17201.

[33] Ortolá, B., Urbaneja, A., Eiras, M., Pérez-Hedo, M. and Daròs, J.A. (2024) RNAi-mediated silencing of Mediterranean fruit fly (Ceratitis capitata) endogenous genes using orally-supplied double-stranded RNAs produced in Escherichia coli. Pest Management Science, 80, 1087–1098.

[34] Pane, A., Salvemini, M., Bovi, P.D., Polito, C. and Saccone, G. (2002) The transformer gene in Ceratitis capitata provides a genetic basis for selecting and remembering the sexual fate. Development (Cambridge, England), 129, 3715–3725.

[35] Perrotta, M.M., Lucibelli, F., Mazzucchiello, S.M., Fucci, N., Hay Mele, B., Giordano, E. et al. (2023) Female sex determination factors in Ceratitis capitata: molecular and structural basis of TRA and TRA2 recognition. Insects, 14, 605.

[36] Pfaffl, M.W. (2001) A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Research, 29, e45.

[37] Primo, P., Meccariello, A., Inghilterra, M.G., Gravina, A., Del Corsano, G., Volpe, G. et al. (2020) Targeting the autosomal Ceratitis capitata transformer gene using Cas9 or dCas9 to masculinize XX individuals without inducing mutations. BMC Genetics, 21, 150.

[38] Ruiz, N., Abreu, L.A.D., Parizi, L.F., Kim, T.K., Mulenga, A., Braz, G.R.C. et al. (2015) Non-invasive delivery of dsRNA into de-waxed tick eggs by electroporation. PLoS ONE, 10, e0130008.

[39] Saccone, G., Salvemini, M., Pane, A. and Polito, L.C. (2008) Masculinization of XX Drosophila transgenic flies expressing the Ceratitis capitata Doublesex M isoform. International Journal of Developmental Biology, 52, 1051–1057.

[40] Saccone, G. (2022) A history of the genetic and molecular identification of genes and their functions controlling insect sex determination. Insect Biochemistry and Molecular Biology, 151, 103873.

[41] Sagri, E., Koskinioti, P., Gregoriou, M.E., Tsoumani, K.T., Bassiakos, Y.C. and Mathiopoulos, K.D. (2017) Housekeeping in Tephritid insects: the best gene choice for expression analyses in the medfly and the olive fly. Scientific Reports, 7, 45634.

[42] Salvemini, M., Robertson, M., Aronson, B., Atkinson, P., Polito, L.C. and Saccone, G. (2009) Ceratitis capitata transformer-2 gene is required to establish and maintain the autoregulation of Cctra, the master gene for female sex determination. International Journal of Developmental Biology, 53, 109.

[43] Scott, J.G., Michel, K., Bartholomay, L.C., Siegfried, B.D., Hunter, W.B., Smagghe, G. et al. (2013) Towards the elements of successful insect RNAi. Journal of Insect Physiology, 59, 1212–1221.

[44] Siolas, D., Lerner, C., Burchard, J., Ge, W., Linsley, P.S., Paddison, P.J. et al. (2005) Synthetic shRNAs as potent RNAi triggers. Nature Biotechnology, 23, 227–231.

[45] Snead, N.M., Wu, X., Li, A., Cui, Q., Sakurai, K., Burnett, J.C. et al. (2013) Molecular basis for improved gene silencing by Dicer substrate interfering RNA compared with other siRNA variants. Nucleic Acids Research, 41, 6209–6221.

[46] Sollazzo, G., Gouvi, G., Nikolouli, K., Martinez, E.I.C., Schetelig, M.F. and Bourtzis, K. (2022) Temperature sensitivity of wild-type, mutant and genetic sexing strains of Ceratitis capitata. Insects, 13, 943.

[47] Sollazzo, G., Nikolouli, K., Gouvi, G., Aumann, R.A., Schetelig, M.F. and Bourtzis, K. (2024) Deep orange gene editing triggers temperature-sensitive lethal phenotypes in Ceratitis capitata. BMC Biotechnology, 24, 7.

[48] Tan, W., Dai, M., Ye, S., Tang, X., Jiang, D., Chen, D. et al. (2025) ENsiRNA: a multimodality method for siRNA-mRNA and modified siRNA efficacy prediction based on geometric graph neural network. Journal of Molecular Biology, 437, 169131.

[49] Thomas, J.L. (2003) Electroporation, an alternative to biolistics for transfection of Bombyx mori embryos and larval tissues. Journal of Insect Science, 3, 17.

[50] Tian, M. and Maniatis, T. (1992) Positive control of pre-mRNA splicing in vitro. Science, 256, 237–240.

[51] Tian, M. and Maniatis, T. (1993) A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA. Cell, 74, 105–114.

[52] Vogel, E., Santos, D., Huygens, C., Peeters, P., Van den Brande, S., Wynant, N. et al. (2023) The study of cell-penetrating peptides to deliver dsRNA and siRNA by feeding in the desert locust, Schistocerca gregaria. Insects, 14, 597.

[53] Vogel, E., Santos, D., Mingels, L., Verdonckt, T.W. and Broeck, J.V. (2019) RNA interference in insects: protecting beneficials and controlling pests. Frontiers in Physiology, 9, 1912.

[54] Volpe, G., Mazzucchiello, S.M., Rosati, N., Lucibelli, F., Varone, M., Baccaro, D. et al. (2024) Simultaneous silencing of gut nucleases and a vital target gene by adult dsRNA feeding enhances RNAi efficiency and mortality in Ceratitis capitata. Insects, 15, 717.

[55] Ward, C.M., Aumann, R.A., Whitehead, M.A., Nikolouli, K., Leveque, G., Gouvi, G. et al. (2021) White pupae phenotype of tephritids is caused by parallel mutations of a MFS transporter. Nature Communications, 12, 491.

[56] Whyard, S., Erdelyan, C.N., Partridge, A.L., Singh, A.D., Beebe, N.W. and Capina, R. (2015) Silencing the buzz: a new approach to population suppression of mosquitoes by feeding larvae double-stranded RNAs. Parasites & Vectors, 8, 96.

[57] Wiltshire, R.M. and Duman-Scheel, M. (2020) Advances in oral RNAi for disease vector mosquito research and control. Current Opinion in Insect Science, 40, 18–23.

[58] Yamaguchi, J., Mizoguchi, T. and Fujiwara, H. (2011) siRNAs induce efficient RNAi response in Bombyx mori embryos. PLoS ONE, 6, e25469.

[59] Yu, J.Y., Taylor, J., DeRuiter, S.L., Vojtek, A.B. and Turner, D.L. (2003) Simultaneous inhibition of GSK3α and GSK3β using hairpin siRNA expression vectors. Molecular Therapy, 7, 228–236.

[60] Zhu, K.Y. and Palli, S.R. (2020) Mechanisms, applications, and challenges of insect RNA interference. Annual Review of Entomology, 65, 293–311.

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2025 The Author(s). Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.