The shibire^ts4 mutation causes temperature sensitive paralytic and lethal phenotypes in the Queensland fruit fly, Bactrocera tryoni

Abstract

Bactrocera tryoni, the Queensland fruit fly, is among the most damaging insect pests to the Australian horticultural industry as larvae infest ripening fruits or vegetables prior to harvest. Genetic biocontrol using Sterile Insect Technique (SIT) programs have been used to successfully suppress populations, via mass release of factory-reared sterile males that mate with wild females. Bi-sex flies are currently used for releases, although the efficiency of these control programs could be improved through using genetic sexing strains that eliminate females early during development, as they are not required for SIT. Here we used CRISPR/Cas9 mutagenesis to modify two nucleotides in the B. tryoni gene shibire, which created a proline to serine amino acid substitution and produced a temperature sensitive phenotype. Shibire is an essential GTPase required in endocytosis and synaptic vesicle recycling, and classical mutagenic screens in the vinegar fly Drosophila melanogaster previously identified temperature sensitive alleles including shi^ts4 that results in adult paralysis. In B. tryoni, the shi^ts4 mutant strain exhibited similar adult paralytic phenotypes when exposed to high temperatures, as well as temperature dependent lethality at egg, larval and pupal stages when subjected to heat treatment above standard rearing temperatures. These temperature sensitive phenotypes could be adapted to develop a SIT genetic sexing strain for conditional elimination of females prior to sterile releases, to improve efficiency and reduce costs.

Keywords

CRISPR/Cas9 / genetic sexing strain / sterile insect technique

References

[1] Anderson, E.M., Haupt, A., Schiel, J.A., Chou, E., Machado, H.B., Strezoska, Ž. et al. (2015) Systematic analysis of CRISPR-Cas9 mismatch tolerance reveals low levels of off-target activity. Journal of Biotechnology, 211, 56–65.

[2] 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.

[3] Belote, J.M. and Lucchesi, J.C. (1980) Control of X chromosome transcription by the maleless gene in Drosophila. Nature, 285, 573–575.

[4] Bourtzis, K. and Vreysen, M.J.B. (2021) Sterile Insect Technique (SIT) and its applications. Insects, 12, 638.

[5] Caceres, C. (2002) Mass rearing of temperature sensitive genetic sexing strains in the Mediterranean fruit fly (Ceratitis capitata). Genetica, 116, 107–116.

[6] Chen, M.S., Obar, R.A., Schroeder, C.C., Austin, T.W., Poodry, C.A., Wadsworth, S.C. et al. (1991) Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis. Nature, 351, 583–586.

[7] Choo, A., Fung, E., Chen, I.Y., Saint, R., Crisp, P. and Baxter, S.W. (2020) Precise single base substitution in the shibire gene by CRISPR/Cas9-mediated homology directed repair in Bactrocera tryoni. BMC Genetics, 21, 127.

[8] Choo, A., Fung, E., Nguyen, T. and Okada, A. (2022) CRISPR/Cas9 mutagenesis to generate novel traits in Bactrocera tryoni for Sterile Insect Technique. In Applications of Genome Modulation and Editing. Methods in Molecular Biology (eds. P.J. Verma, H. Sumer & J. Liu), pp. 151–171. Humana, New York, NY.

[9] Clarke, A.R., Merkel, K., Hulthen, A.D. and Schwarzmueller, F. (2019) Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) overwintering: an overview. Austral Entomology, 58, 3–8.

[10] Collins, S.R., Weldon, C.W., Banos, C. and Taylor, P.W. (2009) Optimizing irradiation dose for sterility induction and quality of Bactrocera tryoni. Journal of Economic Entomology, 102, 1791–1800.

[11] Concordet, J.P. and Haeussler, M. (2018) CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Research, 46, W242–W245.

[12] Delprat, M.A., Stolar, C.E., Manso, F.C. and Cladera, J.L. (2002) Genetic stability of sexing strains based on the locus sw of Ceratitis capitata. Genetica, 116, 85–95.

[13] Dominiak, B.C., Campbell, A.J., Worsley, P. and Nicol, H.I. (2011) Evaluation of three ground release methods for sterile Queensland fruit fly Bactrocera tryoni (Froggatt) (Diptera: Tephritidae). Crop Protection, 30, 1541–1545.

[14] Dominiak, B.C., Taylor, P.W. and Rempoulakis, P. (2023) Marking and identification methodologies for mass releases of sterile Queensland fruit fly Bactrocera tryoni (Diptera: Tephritidae): an overview. Crop Protection, 166, 106173.

[15] Dyck, V.A., Hendrichs, J. and Robinson, A.S. (2021) Sterile Insect Technique: Principles and Practice in Area-Wide Integrated Pest Management. 2nd edn. CRC Press, Boca Raton, Florida, USA.

[16] 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. 2nd edn. (eds. V.A. Dyck, J. Hendrichs & A.S. Robinson), pp. 1–20. CRC Press, Boca Raton, Florida, USA.

[17] Grant, D., Unadkat, S., Katzen, A., Krishnan, K.S. and Ramaswami, M. (1998) Probable mechanisms underlying interallelic complementation and temperature-sensitivity of mutations at the shibire locus of Drosophila melanogaster. Genetics, 149, 1019–1030.

[18] Grigliatti, T.A., Hall, L., Rosenbluth, R. and Suzuki, D.T. (1973) Temperature-sensitive mutations in Drosophila melanogaster. Molecular and General Genetics, 120, 107–114.

[19] Hancock, D.L., Hamacek, E.L., Lloyd, A.C. and Elson-Harris, M.M. (2000) The distribution and host plants of fruit flies (Diptera: Tephritidae) in Australia. Queensland Department of Primary Industries. http://era.daf.qld.gov.au/id/eprint/3593/

[20] 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.

[21] Hendrichs, J., Robinson, A.S., Cayol, J.P. and Enkerlin, W. (2002) Medfly area wide sterile insect technique programmes for prevention, suppression or eradication: The importance of mating behavior studies. Florida Entomologist, 85, 1–13.

[22] Hoskins, J.L., Rempoulakis, P., Stevens, M.M. and Dominiak, B.C. (2023) Biosecurity and management strategies for economically important exotic tephritid fruit fly species in Australia. Insects, 14, 801.

[23] Kelley, L.A., Mezulis, S., Yates, C.M., Wass, M.N. and Sternberg, M.J. (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols, 10, 845–858.

[24] Khan, M.A.M., Shuttleworth, L.A., Osborne, T., Collins, D., Gurr, G.M. and Reynolds, O.L. (2019) Raspberry ketone accelerates sexual maturation and improves mating performance of sterile male Queensland fruit fly, Bactrocera tryoni (Froggatt). Pest Management Science, 75, 1942–1950.

[25] Kim, Y.-T. and Wu, C.-F. (1990) Allelic interactions at the Shibire locus of Drosophila: effects on behavior. Journal of Neurogenetics, 7, 1–14.

[26] Knipling, E.F. (1955) Possibilities of insect control or eradication through the use of sexually sterile males. Journal of Economic Entomology, 48, 459–462.

[27] Knipling, E.F. (1959) Sterile-male method of population control. Science, 130, 902–904.

[28] Koskinioti, P., Augustinos, A.A., Carvalho, D.O., Misbah-ul-Haq, M., Pillwax, G., de la Fuente, L.D. et al. (2021) Genetic sexing strains for the population suppression of the mosquito vector Aedes aegypti. Philosophical Transactions of the Royal Society B-Biological Sciences, 376, 20190808.

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

[30] Lin, Y., Cradick, T.J., Brown, M.T., Deshmukh, H., Ranjan, P., Sarode, N. et al. (2014) CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Research, 42, 7473–7485.

[31] Lloyd, A.C., Hamacek, E.L., Kopittke, R.A., Peek, T., Wyatt, P.M., Neale, C.J. et al. (2010) Area-wide management of fruit flies (Diptera: Tephritidae) in the Central Burnett district of Queensland, Australia. Crop Protection, 29, 462–469.

[32] McGuire, A.V., Edwards, W. and Northfield, D.T. (2023) The infection efficacy of Metarhizium strains (Hypocreales: Clavicipitaceae) against the Queensland fruit fly Bactrocera tryoni (Diptera: Tephritidae). Journal of Economic Entomology, 116, 627–631.

[33] McInnis, D.O., Tam, S., Grace, C. and Miyashita, D. (1994) Population suppression and sterility rates induced by variable sex ratio, sterile insect releases of Ceratitis capitata (Diptera: Tephritidae) in Hawaii. Annals of the Entomological Society of America, 87, 231–240.

[34] McInnis, D.O., Tam, S., Lim, R., Komatsu, J., Kurashima, R. and Albrecht, C. (2004) Development of a pupal color-based genetic sexing strain of the melon fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae). Annals of the Entomological Society of America, 97, 1026–1033.

[35] Meats, A., Maheswaran, P., Frommer, M. and Sved, J. (2002) Towards a male-only release system for SIT with the Queensland fruit fly, Bactrocera tryoni, using a genetic sexing strain with a temperature-sensitive lethal mutation. Genetica, 116, 97–106.

[36] Meza, J.S., Bourtzis, K., Zacharopoulou, A., Gariou-Papalexiou, A. and Cáceres, C. (2020) Development and characterization of a pupal-colour based genetic sexing strain of Anastrepha fraterculus sp. 1 (Diptera: Tephritidae). BMC Genetics, 21, 134.

[37] Meza, J.S., Cáceres, C. and Bourtzis, K. (2019) Slow larvae mutant and its potential to improve the pupal color-based genetic sexing system in Mexican fruit fly, (Diptera: Tephritidae). Journal of Economic Entomology, 112, 1604–1610.

[38] Meza, J.S., ul Hag, I., Vreysen, M.J.B., Bourtzis, K., Kyritsis, G.A. and Cáceres, C. (2018) Comparison of classical and transgenic genetic sexing strains of Mediterranean fruit fly (Diptera: Tephritidae) for application of the sterile insect technique. PLoS ONE, 13, e0208880.

[39] Moadeli, T., Taylor, P.W. and Ponton, F. (2017) High productivity gel diets for rearing of Queensland fruit fly, Bactrocera tryoni. Journal of Pest Science, 90, 507–520.

[40] Nguyen, T.N.M., Choo, A. and Baxter, S.W. (2021) Lessons from Drosophila: engineering genetic sexing strains with temperature-sensitive lethality for Sterile Insect Technique applications. Insects, 12, 243.

[41] Nguyen, T.N.M., Mendez, V., Ward, C., Crisp, P., Papanicolaou, A., Choo, A. et al. (2020) Disruption of duplicated yellow genes in Bactrocera tryoni modifies pigmentation colouration and impacts behaviour. Journal of Pest Science, 94, 917–932.

[42] Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M. et al. (2004) UCSF Chimera—a visualization system for exploratory research and analysis. Journal of Computational Chemistry, 25, 1605–1612.

[43] Porras, M.F., Meza, J.S., Rajotte, E.G., Bourtzis, K. and Cáceres, C. (2020) Improving the phenotypic properties of the Ceratitis capitata (Diptera: Tephritidae) temperature-sensitive lethal genetic sexing strain in support of Sterile Insect Technique applications. Journal of Economic Entomology, 113, 2688–2694.

[44] Ramírez-Santos, E., Rendon, P., Gouvi, G., Zacharopoulou, A., Bourtzis, K., Cáceres, C. et al. (2021) A novel genetic sexing strain of Anastrepha ludens for cost-effective Sterile Insect Technique applications: improved genetic stability and rearing efficiency. Insects, 12, 499.

[45] 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.

[46] Robinson, A.S. (2002) Genetic sexing strains in medfly, Ceratitis capitata, sterile insect technique programmes. Genetica, 116, 5–13.

[47] Robinson, A.S. (2005) Genetic basis 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. 95–114. Springer, Dordrecht.

[48] Söding, J. (2005) Protein homology detection by HMM-HMM comparison. Bioinformatics, 21, 951–960.

[49] Sollazzo, G., Gouvi, G., Nikolouli, K., Aumann, R.A., Djambazian, H., Whitehead, M.A. et al. (2023) Genomic and cytogenetic analysis of the Ceratitis capitata temperature-sensitive lethal region. G3-Genes Genomes Genetics, 13, jkad074.

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

[51] 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.

[52] Staples, R.R. and Ramaswami, M. (1999) Functional analysis of dynamin isoforms in Drosophila melanogaster. Journal of Neurogenetics, 13, 119–143.

[53] Sultana, S., Baumgartner, J.B., Dominiak, B.C., Royer, J.E. and Beaumont, L.J. (2017) Potential impacts of climate change on habitat suitability for the Queensland fruit fly. Scientific Reports, 7, 13025.

[54] Sultana, S., Baumgartner, J.B., Dominiak, B.C., Royer, J.E. and Beaumont, L.J. (2020) Impacts of climate change on high priority fruit fly species in Australia. PLoS ONE, 15, e0213820.

[55] Sutherst, R.W., Collyer, B.S. and Yonow, T. (2000) The vulnerability of Australian horticulture to the Queensland fruit fly, Bactrocera (Dacus) tryoni, under climate change. Australian Journal of Agricultural Research, 51, 467–480.

[56] Suzuki, D.T., Grigliatti, T. and Williamson, R. (1971) Temperature-sensitive mutations in Drosophila melanogaster, VII. A mutation (parats) causing reversible adult paralysis. Proceedings of the National Academy of Sciences USA, 68, 890.

[57] van der Bliek, A.M. and Meyerowitz, E.M. (1991) Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic. Nature, 351, 411–414.

[58] 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.

RIGHTS & PERMISSIONS

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