Molecular characterization of transformer, transformer-2, and doublesex genes in the carambola fruit fly, Bactrocera carambolae, revealing potential for genetic pest management

Abstract

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.

Keywords

gene knockdown / generic approach / genetic sexing strain / sex determination / sustainable agriculture / Tephritidae

References

[1] Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A. et al. (2024) Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 630, 493–500.

[2] Aketarawong, N., Isasawin, S., Sojikul, P. and Thanaphum, S. (2015) Gene flow and genetic structure of Bactrocera carambolae (Diptera, Tephritidae) among geographical differences and sister species, B. dorsalis, inferred from microsatellite DNA data. Zookeys, 239–272.

[3] Aketarawong, N., Isasawin, S., Laohakieat, K. and Thanaphum, S. (2020) Genetic stability, genetic variation, and fitness performance of the genetic sexing Salaya1 strain for Bactrocera dorsalis, under long-term mass rearing conditions. BMC Genetics, 21, 131.

[4] An, W., Cho, S., Ishii, H. and Wensink, P.C. (1996) Sex-specific and non-sex-specific oligomerization domains in both of the doublesex transcription factors from Drosophila melanogaster. Molecular and Cellular Biology, 16, 3106–3111.

[5] Ant, T., Koukidou, M., Rempoulakis, P., Gong, H.-F., Economopoulos, A., Vontas, J., et al. (2012) Control of the olive fruit fly using genetics-enhanced sterile insect technique. BMC Biology, 10, 51.

[6] Aumann, R.A., Häcker, I. and Schetelig, M.F. (2020) Female-to-male sex conversion in Ceratitis capitata by CRISPR/Cas9 HDR-induced point mutations in the sex determination gene transformer-2. Scientific Reports, 10, 18611.

[7] Baruffi, L., Damiani, G., Guglielmino, C.R., Bandi, C., Malacrida, A.R. and Gasperi, G. (1995) Polymorphism within and between populations of Ceratitis capitata: comparison between RAPD and multilocus enzyme electrophoresis data. Heredity (Edinb), 74 (Pt 4), 425–437.

[8] Beukeboom, L.W. and Perrin, N. (2014) The Evolution of Sex Determination. Oxford University Press.

[9] Black, D.L. (2003) Mechanisms of alternative pre-messenger RNA splicing. Annual Review of Biochemistry, 72, 291–336.

[10] Bopp, D., Saccone, G. and Beye, M. (2014) Sex determination in insects: variations on a common theme. Sexual Development, 8, 20–28.

[11] Bourgeois, C.F., Lejeune, F. and Stévenin, J. (2004) Broad specificity of SR (serine/arginine) proteins in the regulation of alternative splicing of pre-messenger RNA. Progress in Nucleic Acid Research and Molecular Biology, 78, 37–88.

[12] Boykin, L.M., Schutze, M.K., Krosch, M.N., Chomič, A., Chapman, T.A., Englezou, A. et al. (2014) Multi-gene phylogenetic analysis of south-east Asian pest members of the Bactrocera dorsalis species complex (Diptera: Tephritidae) does not support current taxonomy. Journal of Applied Entomology, 138, 235–253.

[13] 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.

[14] Chen, S.L., Dai, S.M., Lu, K.H. and Chang, C. (2008) Female-specific doublesex dsRNA interrupts yolk protein gene expression and reproductive ability in oriental fruit fly, Bactrocera dorsalis (Hendel). Insect Biochemistry and Molecular Biology, 38, 155–165.

[15] 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.

[16] de Deus, E., Passos, J., van Sauers-Muller, A., Jesus, C., Silva, J.G. and Adaime, R. (2024) Phylogeography of the invasive fruit fly species Bactrocera carambolae Drew & Hancock (Diptera: Tephritidae) in South America. Insects, 15, 949.

[17] Drosopoulou, E., Syllas, A., Goutakoli, P., Zisiadis, G.A., Konstantinou, T., Pangea, D. et al. (2019) The complete mitochondrial genome of Bactrocera carambolae (Diptera: Tephritidae): genome description and phylogenetic implications. Insects, 10, 429.

[18] Fezza, T.J., Geib, S.M. and Shelly, T.E. (2018) Comparative rearing parameters for bisexual and genetic sexing strains of Zeugodacus cucurbitae and Bactrocera dorsalis (Diptera: Tephritidae) on an artificial diet. Journal of Asia-Pacific Entomology, 21, 283–287.

[19] Franz, G., Bourtzis, K. and Caceres, 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: Principles and Practice in Area-Wide Integrated Pest Management (eds. V.A. Dyck, J. Hendrichs, A.S. Robinson), pp. 575–604. CRC Press.

[20] 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.

[21] Gabrieli, P., Falaguerra, A., Siciliano, P., Gomulski, L.M., Scolari, F., Zacharopoulou, A. et al. (2010) Sex and the single embryo: early development in the Mediterranean fruit fly, Ceratitis capitata. BMC Developmental Biology, 10, 12.

[22] Geuverink, E. and Beukeboom, L.W. (2014) Phylogenetic distribution and evolutionary dynamics of the sex determination genes doublesex and transformer in insects. Sexual Development, 8, 38–49.

[23] Gilles, J.R.L., Schetelig, M.F., Scolari, F., Marec, F., Capurro, M.L., Franz, G., et al. (2014) Towards mosquito sterile insect technique programmes: Exploring genetic, molecular, mechanical and behavioural methods of sex separation in mosquitoes. Acta Tropica, 132, S178–S187.

[24] Häcker, I., Bourtzis, K. and Schetelig, M. (2020) Applying modern molecular technologies in support of the sterile insect technique. In Sterile Insect Technique: Principles and Practice in Area-Wide Integrated Pest Management (eds. V.A. Dyck, J. Hendrichs, A.S Robinson), pp. 657–702. CRC Press.

[25] Hediger, M., Henggeler, C., Meier, N., Perez, R., Saccone, G. and Bopp, D. (2010) Molecular characterization of the key switch F provides a basis for understanding the rapid divergence of the sex-determining pathway in the housefly. Genetics, 184, 155–170.

[26] Heinrichs, V. and Baker, B.S. (1995) The Drosophila SR protein RBP1 contributes to the regulation of doublesex alternative splicing by recognizing RBP1 RNA target sequences. EMBO Journal, 14, 3987–4000.

[27] Hendrichs, J., Franz, G. and Rendon, P. (1995) Increased effectiveness and applicability of the sterile insect technique through male-only releases for control of Mediterranean fruit flies during fruiting seasons. Journal of Applied Entomology, 119, 371–377.

[28] Isasawin, S., Aketarawong, N. and Thanaphum, S. (2012) Characterization and evaluation of microsatellite markers in a strain of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae), with a genetic sexing character used in sterile insect population control. European Journal of Entomology, 109, 331–338.

[29] Isasawin, S., Aketarawong, N., Lertsiri, S. and Thanaphum, S. (2014) Development of a genetic sexing strain in Bactrocera carambolae (Diptera: Tephritidae) by introgression of sex sorting components from B. dorsalis, Salaya1 strain. BMC Genomic Data, 15, S2.

[30] Kandul, N.P., Liu, J., Sanchez, C.H.M., Wu, S.L., Marshall, J.M. and Akbari, O.S. (2019) Transforming insect population control with precision guided sterile males with demonstration in flies. Nature Communications, 10, 84.

[31] Krissinel, E. and Henrick, K. (2007) Inference of macromolecular assemblies from crystalline state. Journal of Molecular Biology, 372, 774–797.

[32] Lagos, D., Ruiz, M.F., Sánchez, L. and Komitopoulou, K. (2005) Isolation and characterization of the Bactrocera oleae genes orthologous to the sex determining Sex-lethal and doublesex genes of Drosophila melanogaster. Gene, 348, 111–121.

[33] Lagos, D., Koukidou, M., Savakis, C. and Komitopoulou, K. (2007) The transformer gene in Bactrocera oleae: the genetic switch that determines its sex fate. Insect Molecular Biology, 16, 221–230.

[34] Laohakieat, K., Aketarawong, N., Isasawin, S., Thitamadee, S. and Thanaphum, S. (2016) The study of the transformer gene from Bactrocera dorsalis and B. correcta with putative core promoter regions. BMC Genetics, 17, 34–34.

[35] Laohakieat, K., Isasawin, S. and Thanaphum, S. (2020) The transformer-2 and fruitless characterisation with developmental expression profiles of sex-determining genes in Bactrocera dorsalis and B. correcta. Scientific Reports, 10, 17938.

[36] Leftwich, P.T., Koukidou, M., Rempoulakis, P., Gong, H.-F., Zacharopoulou, A., Fu, G. et al. (2014) Genetic elimination of field-cage populations of Mediterranean fruit flies. Proceedings of the Royal Society B: Biological Sciences, 281, 20141372.

[37] Li, J. and Handler, A.M. (2017) Temperature-dependent sex-reversal by a transformer-2 gene-edited mutation in the spotted wind drosophila. Drosophila Suzukii. Scientific Reports, 7, 12363.

[38] Li, J. and Handler, A.M. (2019) CRISPR/Cas9-mediated gene editing in an exogenous transgene and an endogenous sex determination gene in the Caribbean fruit fly, Anastrepha suspensa. Gene, 691, 160–166.

[39] Liu, G., Wu, Q., Li, J., Zhang, G. and Wan, F. (2015) RNAi-mediated knock-down of transformer and transformer 2 to generate male-only progeny in the oriental fruit fly, Bactrocera dorsalis (Hendel). PLoS ONE, 10, e0128892.

[40] Long, J.C. and Caceres, J.F. (2009) The SR protein family of splicing factors: master regulators of gene expression. The Biochemical Journal, 417, 15–27.

[41] Luo, Y., Zhao, S., Li, J., Li, P. and Yan, R. (2017) Isolation and molecular characterization of the transformer gene from Bactrocera cucurbitae (Diptera: Tephritidae). Journal of Insect Science, 17.

[42] Lynch, K.W. and Maniatis, T. (1996) Assembly of specific SR protein complexes on distinct regulatory elements of the Drosophila doublesex splicing enhancer. Genes & Development, 10, 2089–2101.

[43] Madeira, F., Madhusoodanan, N., Lee, J., Eusebi, A., Niewielska, A., Tivey, A.R.N. et al. (2024) The EMBL-EBI Job Dispatcher sequence analysis tools framework in 2024. Nucleic Acids Research, 52, W521–W525.

[44] Manley, J.L. and Tacke, R. (1996) SR proteins and splicing control. Genes & Development, 10, 1569–1579.

[45] Marchioro, C.A. (2016) Global potential distribution of Bactrocera carambolae and the risks for fruit production in Brazil. PLoS ONE, 11, e0166142.

[46] Mayeda, A., Screaton, G.R., Chandler, S.D., Fu, X.D. and Krainer, A.R. (1999) Substrate specificities of SR proteins in constitutive splicing are determined by their RNA recognition motifs and composite pre-mRNA exonic elements. Molecular and Cellular Biology, 19, 1853–1863.

[47] McCombs, S.D. and Saul, S.H. (1995) Translocation-based genetic sexing system for the oriental fruit fly (Diptera: Tephritidae) based on pupal color dimorphism. Annals of the Entomological Society of America, 88, 695–698.

[48] 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.

[49] 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.

[50] Morrow, J.L., Riegler, M., Frommer, M. and Shearman, D.C.A. (2014) Expression patterns of sex-determination genes in single male and female embryos of two Bactrocera fruit fly species during early development. Insect Molecular Biology, 23, 754–767.

[51] Ogaugwu, C.E., Schetelig, M.F. and Wimmer, E.A. (2013) Transgenic sexing system for Ceratitis capitata (Diptera: Tephritidae) based on female-specific embryonic lethality. Insect Biochemistry and Molecular Biology, 43, 1–8.

[52] Okonechnikov, K., Golosova, O., Fursov, M. and UGENE Team (2012) Unipro UGENE: a Unified Bioinformatics Toolkit, Bioinformatics, 28, 1166–1167.

[53] Pane, A., Salvemini, M., Delli Bovi, P., 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.

[54] Peng, W., Zheng, W., Handler, A.M. and Zhang, H. (2015) The role of the transformer gene in sex determination and reproduction in the tephritid fruit fly, Bactrocera dorsalis (Hendel). Genetica, 143, 717–727.

[55] Permpoon, R. and Thanaphum, S. (2010) Isolation and characterization of oligomerization domain I and II coding regions of doublesex genes in agricultural fruit flies (Diptera: Tephritidae). European Journal of Entomology, 107, 121–126.

[56] Permpoon, R., Aketarawong, N. and Thanaphum, S. (2011) Isolation and characterization of doublesex homologues in the Bactrocera species: B. dorsalis (Hendel) and B. correcta (Bezzi) and their putative promoter regulatory regions. Genetica, 139, 113–127.

[57] 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.

[58] 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.

[59] Qi, J., Su, S. and Mattox, W. (2007) The doublesex splicing enhancer components Tra2 and Rbp1 also repress splicing through an intronic silencer. Molecular and Cellular Biology, 27, 699–708.

[60] Rendón, P., McInnis, D., Lance, D. and Stewart, J. (2004) Medfly (Diptera: Tephritidae) genetic sexing: large-scale field comparison of males-only and bisexual sterile fly releases in Guatemala. Journal of Economic Entomology, 97, 1547–1553.

[61] Ruiz, M.F., Stefani, R.N., Mascarenhas, R.O., Perondini, A.L.P., Selivon, D. and Sánchez, L. (2005) The gene doublesex of the fruit fly Anastrepha obliqua (Diptera, Tephritidae). Genetics, 171, 849–854.

[62] Ruiz, M.F., Milano, A., Salvemini, M., Eirín-López, J.M., Perondini, A.L.P., Selivon, D. et al. (2007) The gene transformer of Anastrepha fruit flies (Diptera, Tephritidae) and its evolution in insects. PLoS ONE, 2, e1239.

[63] Saccone, G., Peluso, I., Testa, G., DiPaola, F., Pane, A. and Polito, C. (1996) Drosophila sex-lethal and doublesex homologous genes in Ceratitis capitata: searching for sex-specific genes to develop a medfly transgenic sexing strain. In First Research Co-ordination Meeting on Enhancement of the Sterile Insect Technique through Genetic Transformation using Nuclear Techniques Report, Joint IAEA/FAO, pp. 16–32.

[64] 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. The International Journal of Developmental Biology, 53, 109–120.

[65] Sarno, F., Ruiz, M.F., Eirín-López, J.M., Perondini, A.L.P., Selivon, D. and Sánchez, L. (2010) The gene transformer-2 of Anastrepha fruit flies (Diptera, Tephritidae) and its evolution in insects. BMC Evolutionary Biology, 10, 140.

[66] Schetelig, M.F. and Handler, A.M. (2012) A transgenic embryonic sexing system for Anastrepha suspensa (Diptera: Tephritidae). Insect Biochemistry and Molecular Biology, 42, 790–795.

[67] Schetelig, M.F., Milano, A., Saccone, G. and Handler, A.M. (2012) Male only progeny in Anastrepha suspensa by RNAi-induced sex reversion of chromosomal females. Insect Biochemistry and Molecular Biology, 42, 51–57.

[68] Schetelig, M.F., Schwirz, J. and Yan, Y. (2021) A transgenic female killing system for the genetic control of Drosophila suzukii. Scientific Reports, 11, 12938.

[69] Shearman, D.C. and Frommer, M. (1998) The Bactrocera tryoni homologue of the Drosophila melanogaster sex-determination gene doublesex. Insect Molecular Biology, 7, 355–366.

[70] Shukla, J.N. and Nagaraju, J. (2010) Doublesex: a conserved downstream gene controlled by diverse upstream regulators. Journal of Genetics, 89, 341–356.

[71] Skendžić, S., Zovko, M., Živković, I.P., Lešić, V. and Lemić, D. (2021) The impact of climate change on agricultural insect pests. Insects, 12, 440.

[72] Vreysen, M.J. (2001) Principles of area-wide integrated tsetse fly control using the sterile insect technique. Medecine Tropicale: Revue Du Corps De Sante Colonial (Mars), 61, 397–411.

[73] Wee, S.L. and Tan, K.H. (2005) Evidence of natural hybridization between two sympatric sibling species of Bactrocera dorsalis complex based on pheromone analysis. Journal of Chemical Ecology, 31, 845–858.

[74] White, I.M. and Elson-Harris, M.M. (1992) Fruit Flies of Economic Significance: Their Identification and Bionomics. CAB International.

[75] Wishard, R., Karuppannasamy, A., Asokan, R., Chikmagalur Nagaraja, B., Chalapathi, P., Dhawane, Y. et al. (2023) CRISPR/Cas9 editing of transformer2 gene of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) leads to intersex phenotype. Journal of Asia-Pacific Entomology, 26, 102105.

[76] Xu, J. and Zhang, Y. (2010) How significant is a protein structure similarity with TM-score = 0.5? Bioinformatics, 26, 889–895.

[77] Yan, Y., Aumann, R.A., Häcker, I. and Schetelig, M.F. (2023) CRISPR-based genetic control strategies for insect pests. Journal of Integrative Agriculture, 22, 651–668.

[78] Yan, Y., Ahmed, H.M.M., Wimmer, E.A. and Schetelig, M.F. (2024) Biotechnology-enhanced genetic controls of the global pest Drosophila suzukii. Trends in Biotechnology, 43, 826–837.

[79] Yan, Y., Zhao, J., Schwirz, J., Borghesi, C., Liu, C., Liu, B. et al. (2025) The transformer gene controls sexual development in Drosophila suzukii. Insect Science, 0, 1–19. https://doi.org/10.1111/1744-7917.70031.

[80] Zhao, S., Xing, Z., Liu, Z., Liu, Y., Liu, X., Chen, Z. et al. (2019) Efficient somatic and germline genome engineering of Bactrocera dorsalis by the CRISPR/Cas9 system. Pest Management Science, 75, 1921–1932.

[81] Zimowska, G.J., Xavier, N., Qadri, M. and Handler, A.M. (2024) A transposon-based genetic marker for conspecific identity within the Bactrocera dorsalis species complex. Scientific Reports, 14, 1924.

[82] Zhang, Y. and Skolnick, J. (2004) Scoring function for automated assessment of protein structure template quality. Proteins, 57, 702–710.

[83] Zhu, L., Wilken, J., Phillips, N.B., Narendra, U., Chan, G., Stratton, S.M. et al. (2000) Sexual dimorphism in diverse metazoans is regulated by a novel class of intertwined zinc fingers. Genes & Development, 14, 1750–1764.

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