Genetic engineering of sex chromosomes for batch cultivation of non-transgenic, sex-sorted males

Autoři: Siba R. Das aff001;  Maciej Maselko aff001;  Ambuj Upadhyay aff001;  Michael J. Smanski aff001
Působiště autorů: Department of Biochemistry, Molecular Biology, and Biophysic, Saint Paul, MN, United States of America aff001;  Biotechnology Institute University of Minnesota, Saint Paul, MN, United States of America aff002
Vyšlo v časopise: Genetic engineering of sex chromosomes for batch cultivation of non-transgenic, sex-sorted males. PLoS Genet 16(11): e32767. doi:10.1371/journal.pgen.1009180
Kategorie: Research Article
doi: 10.1371/journal.pgen.1009180


The field performance of Sterile Insect Technique (SIT) is improved by sex-sorting and releasing only sterile males. This can be accomplished by resource-intensive separation of males from females by morphology. Alternatively, sex-ratio biasing genetic constructs can be used to selectively remove one sex without the need for manual or automated sorting, but the resulting genetically engineered (GE) control agents would be subject to additional governmental regulation. Here we describe and demonstrate a genetic method for the batch production of non-GE males. This method could be applied to generate the heterogametic sex (XY, or WZ) in any organism with chromosomal sex determination. We observed up to 100% sex-selection with batch cultures of more than 103 individuals. Using a stringent transgene detection assay, we demonstrate the potential of mass production of transgene free males.

Klíčová slova:

Drosophila melanogaster – Genetic engineering – Genetics – Insect pests – Insects – Tetracyclines – X chromosomes – Y chromosomes


1. Dyck VA, Hendrichs J, Robinson AS. Sterile insect technique: principles and practice in area-wide integrated pest management. Springer; 2005.

2. Wyss JH. Screwworm eradication in the Americas. Ann N Y Acad Sci. 2000;916:186–93. doi: 10.1111/j.1749-6632.2000.tb05289.x 11193620

3. Vargas-Terán M, Hofmann HC, Tweddle NE. Impact of Screwworm Eradication Programmes Using the Sterile Insect Technique. Sterile Insect Technique. Berlin/Heidelberg: Springer-Verlag; 2005. pp. 629–650. doi: 10.1007/1-4020-4051-2_24

4. Hendrichs J, Robinson AS, Cayol JP, Enkerlin W. Medfly areawide sterile insect technique programmes for suppression or eradication: The importance of mating behavior studies. Florida Entomol. Florida Entomological Society; 2002;85:1–13. doi: 10.1653/0015-4040(2002)085[0001:MASITP]2.0.CO;2

5. Dominiak BC, Sundaralingam S, Jiang L, Fanson BG, Collins SR, Banos C, et al. Evaluating irradiation dose for sterility induction and quality control of mass-produced fruit fly Bactrocera tryoni (Diptera: Tephritidae). J Econ Entomol. 2014;107:1172–8. doi: 10.1603/ec13421 25026679

6. Rendón P, McInnis D, Lance D, Stewart J. Medfly (Diptera:Tephritidae) Genetic Sexing: Large-Scale Field Comparison of Males-Only and Bisexual Sterile Fly Releases in Guatemala. J Econ Entomol. 2004;97:1547–1553. doi: 10.1603/0022-0493-97.5.1547 15568342

7. Lutrat C, Giesbrecht D, Marois E, Whyard S, Baldet T, Bouyer J. Sex Sorting for Pest Control: It’s Raining Men! Trends Parasitol. 2019;35:649–662. doi: 10.1016/ 31255488

8. Carvalho DO, Nimmo D, Naish N, McKemey AR, Gray P, Wilke ABB, et al. Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil. J Vis Exp. 2014;e3579. doi: 10.3791/3579 24430003

9. Marois E, Scali C, Soichot J, Kappler C, Levashina EA, Catteruccia F. High-throughput sorting of mosquito larvae for laboratory studies and for future vector control interventions. Malar J. BioMed Central; 2012;11:302. doi: 10.1186/1475-2875-11-302 22929810

10. Caceres C. Mass rearing of temperature sensitive genetic sexing strains in the Mediterranean fruit fly (Ceratitis capitata). Genetica. 2002;116:107–16. doi: 10.1023/a:1020967810703 12484530

11. Fu G, Lees RS, Nimmo D, Aw D, Jin L, Gray P, et al. Female-specific flightless phenotype for mosquito control. Proc Natl Acad Sci. 2010;107:4550–4554. doi: 10.1073/pnas.1000251107 20176967

12. Concha C, Palavesam A, Guerrero FD, Sagel A, Li F, Osborne JA, et al. A transgenic male-only strain of the New World screwworm for an improved control program using the sterile insect technique. BMC Biol. 2016;14. doi: 10.1186/s12915-016-0236-7 26934976

13. Yan Y, Linger RJ, Scott MJ. Building early-larval sexing systems for genetic control of the Australian sheep blow fly Lucilia cuprina using two constitutive promoters. Sci Rep. Nature Publishing Group; 2017;7:2538. doi: 10.1038/s41598-017-02763-4 28566730

14. Jin L, Walker AS, Fu G, Harvey-Samuel T, Dafa’Alla T, Miles A, et al. Engineered female-specific lethality for control of pest lepidoptera. ACS Synth Biol. 2013;2:160–166. doi: 10.1021/sb300123m 23802263

15. Tan A, Fu G, Jin L, Guo Q, Li Z, Niu B, et al. Transgene-based, female-specific lethality system for genetic sexing of the silkworm, Bombyx mori. Proc Natl Acad Sci U S A. National Academy of Sciences; 2013;110:6766–70. doi: 10.1073/pnas.1221700110 23569267

16. Smanski MJ, Zarkower D. Genetic manipulation of sex ratio in mammals: the Reaper comes for Mickey. EMBO Rep. John Wiley & Sons, Ltd; 2019;20. doi: 10.15252/embr.201948577 31267656

17. Li F, Wantuch HA, Linger RJ, Belikoff EJ, Scott MJ. Transgenic sexing system for genetic control of the Australian sheep blow fly Lucilia cuprina. Insect Biochem Mol Biol. Elsevier Ltd; 2014;51:80–88. doi: 10.1016/j.ibmb.2014.06.001 24928635

18. Heinrich JC, Scott MJ. A repressible female-specific lethal genetic system for making transgenic insect strains suitable for a sterile-release program. Proc Natl Acad Sci U S A. National Academy of Sciences; 2000;97:8229–32. doi: 10.1073/pnas.140142697 10890889

19. Fu G, Condon KC, Epton MJ, Gong P, Li J, Condon GC, et al. Female-specific insect lethality engineered using alternative splicing. Nat Biotechnol. 2007;25. doi: 10.1038/nbt0107-25 17211383

20. Pfeiffer BD, Truman JW, Rubin GM. Using translational enhancers to increase transgene expression in Drosophila. Proc Natl Acad Sci U S A. National Academy of Sciences; 2012;109:6626–31. doi: 10.1073/pnas.1204520109 22493255

21. Buchman A, Akbari OS. Site-specific transgenesis of the Drosophila melanogaster Y-chromosome using CRISPR/Cas9. Insect Mol Biol. John Wiley & Sons, Ltd (10.1111); 2019;28:65–73. doi: 10.1111/imb.12528 30079589

22. Bain C, Selfa T. Non-GMO vs organic labels: purity or process guarantees in a GMO contaminated landscape. Agric Human Values. Springer Netherlands; 2017;34:805–818. doi: 10.1007/s10460-017-9776-x

23. Fisher K. Genetic Sexing Strains of Mediterranean Fruit Fly (Diptera: Tephritidae): Quality in Mass-Reared Temperature-Sensitive Lethal Strains Treated at High Temperatures. J. Econ. Entomol. 2000. doi: 10.1603/0022-0493-93.2.394 10826191

24. Caceres C, Cayol JP, Enkerlin W, Franz G, Cáceres-Barrios C. Hendrichs J, Robinson AS. Comparison of Mediterranean fruit fly (Ceratitis capitata) (Tephritidae) bisexual and genetic sexing strains: development, evaluation and economics. Proc 6th Int Fruit Fly Symp. 2002;367–381.

25. Eckermann KN, Dippel S, Ranjbar MKN, Ahmed HM, Curril IM, Wimmer EA. Perspective on the combined use of an independent transgenic sexing and a multifactorial reproductive sterility system to avoid resistance development against transgenic Sterile Insect Technique approaches. BMC Genet. 2014;15. doi: 10.1186/1471-2156-15-15 24491120

26. Handler AM. Enhancing the stability and ecological safety of mass-reared transgenic strains for field release by redundant conditional lethality systems. Insect Sci. 2016;23:225–234. doi: 10.1111/1744-7917.12245 26097098

27. Leftwich PT, Clarke NVE, Matthew I, Leftwich PT, Clarke NVE, Hutchings MI, et al. Gut microbiomes and reproductive isolation in Drosophila. Proc Natl Acad Sci. 2018;115:E2487. doi: 10.1073/pnas.1801321115 29463715

28. Schetelig MF, Scolari F, Handler AM, Kittelmann S, Gasperi G, Wimmer EA. Site-specific recombination for the modification of transgenic strains of the Mediterranean fruit fly Ceratitis capitata. Proc Natl Acad Sci U S A. National Academy of Sciences; 2009;106:18171–6. doi: 10.1073/pnas.0907264106 19828439

29. Pfeiffer BD, Ngo T-TB, Hibbard KL, Murphy C, Jenett A, Truman JW, et al. Refinement of Tools for Targeted Gene Expression in Drosophila. Genetics. 2010;186:735–755. doi: 10.1534/genetics.110.119917 20697123

30. Szabad J, Bellen HJ, Venken KJT. An assay to detect in vivo Y chromosome loss in Drosophila wing disc cells. G3 Genes, Genomes, Genet. 2012;2:1095–1102. doi: 10.1534/g3.112.002899 22973547

31. Venken KJT, He Y, Hoskins RA, Bellen HJ. P[acman]: A BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science. 2006;314:1747–1751. doi: 10.1126/science.1134426 17138868

Článek vyšel v časopise

PLOS Genetics

2020 Číslo 11
Nejčtenější tento týden
Nejčtenější v tomto čísle
Zapomenuté heslo

Nemáte účet?  Registrujte se

Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se