Abstract
CRISPR/Cas9 mediated mutagenesis has revolutionized the testing of gene function in both model and non-model organisms. The red imported fire ant, Solenopsis invicta, is the best-studied ant species because of their painful sting, aggressive nature, and their detrimental effects on invaded ecosystems. We have developed a microinjection protocol for CRISPR/Cas9 mutagenesis of fire ant embryos. As a proof-of-principle we generated individuals mosaic for the knockout of Sinv-spitz or Gp-9. We verified that many injected individuals carry mutations, often to high frequency of the cells within the individual. Observations at the first instar larval stage indicate that mutating Sinv-spitz does not produce a loss of oenocyte phenotype, as would have been expected based on the homologous function of the fruit fly, Drosophila melanogaster, spitz. Our success indicates that CRISPR/Cas9 mutagenesis should be a useful technique for studying gene function in the fire ant at the individual and possibly social levels.
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17 July 2021
A Correction to this paper has been published: https://doi.org/10.1007/s00040-021-00825-5
References
Adli M (2018) The CRISPR tool kit for genome editing and beyond. Nat Commun 9:1911
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Ascunce MS et al (2011) Global invasion history of the fire ant Solenopsis invicta. Science 331:1066–1068
Callcott AMA, Collins HL (1996) Invasion and range expansion of imported fire ants (Hymenoptera: Formicidae) in North America from 1918–1995. Fla Entomol 79:240–251
Chen JSC, Shen CH, Lee HJ (2006) Monogynous and polygynous red imported fire ants, Solenopsis invicta buren (Hymenoptera: Formicidae), in Taiwan. Environ Entomol 35:167–172
Choi MY, Meer RKV, Coy M, Scharf ME (2012) Phenotypic impacts of PBAN RNA interference in an ant, Solenopsis invicta, and a moth, Helicoverpa zea. J Insect Physiol 58:1159–1165
Cong L et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
Cridge AG, Lovegrove MR, Skelly JG, Taylor SE, Petersen GEL, Cameron RC, Dearden PK (2017) The honeybee as a model insect for developmental genetics. Genesis 55:1–12
Elstob PR, Brodu V, Gould AP (2001) spalt-dependent switching between two cell fates that are induced by the Drosophila EGF receptor. Development 128:723–732
Fish MP, Groth AC, Calos MP, Nusse R (2007) Creating transgenic Drosophila by microinjecting the site-specific phiC31 integrase mRNA and a transgene-containing donor plasmid. Nat Protoc 2:2325–2331
Friedland AE, Tzur YB, Esvelt KM, Colaiacovo MP, Church GM, Calarco JA (2013) Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods 10:741–743
Gotzek D, Ross KG (2009) Current status of a model system: The gene Gp-9 and its association with social organization in fire ants. PLoS ONE 4:e7713
Hartenstein AY, Rugendorff A, Tepass U, Hartenstein V (1992) The function of the neurogenic genes during epithelial development in the Drosophila embryo. Development 116:1203–1220
Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278
Huang YC, Wang J (2014) Did the fire ant supergene evolve selfishly or socially? BioEssays 36:200–208
Huang YC, Lee CC, Kao CY, Chang NC, Lin CC, Shoemaker D, Wang J (2016) Evolution of long centromeres in fire ants. BMC Evol Biol 16:189
Huang YC, Dang VD, Chang NC, Wang J (2018) Multiple large inversions and breakpoint rewiring of gene expression in the evolution of the fire ant social supergene. Proc Biol Sci 285:1–8
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821
Jouvenaz DP, Allen GE, Banks WA, Wojcik DP (1977) Survey for pathogens of fire ants, Solenopsis-spp-(Hymenoptera-Formicidae) in the Southeastern United-States. Fla Entomol 60:275–279
Keller L, Ross KG (1998) Selfish genes: a green beard in the red fire ant. Nature 394:573–575
Kohno H, Suenami S, Takeuchi H, Sasaki T, Kubo T (2016) Production of knockout mutants by CRISPR/Cas9 in the European honeybee, Apis mellifera L. Zoolog Sci 33:505–512
Krieger MJB, Ross KG (2002) Identification of a major gene regulating complex social behavior. Science 295:328–332
Kriventseva EV et al (2015) OrthoDB v8: update of the hierarchical catalog of orthologs and the underlying free software. Nucleic Acids Res 43:D250–256
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549
Leal WS, Ishida Y (2008) GP-9s are ubiquitous proteins unlikely involved in olfactory mediation of social organization in the red imported fire ant, Solenopsis invicta. PLoS One 3:e3762
Li-Byarlay H et al (2013) RNA interference knockdown of DNA methyltransferase 3 affects gene alternative splicing in the honey bee. Proc Natl Acad Sci USA 110:12750–12755
Lu HL, Vinson SB, Pietrantonio PV (2009) Oocyte membrane localization of vitellogenin receptor coincides with queen flying age, and receptor silencing by RNAi disrupts egg formation in fire ant virgin queens. Febs J 276:3110–3123
Makki R, Cinnamon E, Gould AP (2014) The development and functions of oenocytes. Annu Rev Entomol 59:405–425
McDonald JA, Pinheiro EM, Kadlec L, Schupbach T, Montell DJ (2006) Multiple EGFR ligands participate in guiding migrating border cells. Dev Biol 296:94–103
Naito Y, Hino K, Bono H, Ui-Tei K (2015) CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics 31:1120–1123
Reich A, Shilo BZ (2002) Keren, a new ligand of the Drosophila epidermal growth factor receptor, undergoes two modes of cleavage. EMBO J 21:4287–4296
Rittschof CC, Robinson GE (2016) Behavioral genetic toolkits: toward the evolutionary origins of complex phenotypes. Curr Top Dev Biol 119:157–204
Ross KG, Keller L (1998) Genetic control of social organization in an ant. Proc Natl Acad Sci USA 95:14232–14237
Roth A et al (2019) A genetic switch for worker nutrition-mediated traits in honeybees. PLoS Biol 17:e3000171
Rutledge BJ, Zhang K, Bier E, Jan YN, Perrimon N (1992) The Drosophila spitz gene encodes a putative EGF-like growth factor involved in dorsal-ventral axis formation and neurogenesis. Genes Dev 6:1503–1517
Schulte C, Theilenberg E, Muller-Borg M, Gempe T, Beye M (2014) Highly efficient integration and expression of piggyBac-derived cassettes in the honeybee (Apis mellifera). Proc Natl Acad Sci USA 111:9003–9008
Shilo BZ (2014) The regulation and functions of MAPK pathways in Drosophila. Methods 68:151–159
Smith CR, Dolezal A, Eliyahu D, Holbrook CT, Gadau J (2009) Ants (Formicidae): models for social complexity. Cold Spring Harb Protoc 2009:pdb emo125
Stolle E et al (2019) Degenerative expansion of a young supergene. Mol Biol Evol 36:553–561
Team (2013) A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. https://www.R-project.org/
Thurmond J et al (2019) FlyBase 2.0: the next generation. Nucleic Acids Res 47:D759–D765
Trible W et al (2017) orco mutagenesis causes loss of antennal lobe glomeruli and impaired social behavior in ants. Cell 170:727–735
Tschinkel WR (2006) The fire ants. The Belknap Press of Harvard University Press, Cambridge
Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3—new capabilities and interfaces. Nucleic Acids Res 40:e115
Urban S, Lee JR, Freeman M (2002) A family of Rhomboid intramembrane proteases activates all Drosophila membrane-tethered EGF ligands. EMBO J 21:4277–4286
Wang J, Ross KG, Keller L (2008) Genome-wide expression patterns and the genetic architecture of a fundamental social trait. PLoS Genet 4:e1000127
Wang J, Wurm Y, Nipitwattanaphon M, Riba-Grognuz O, Huang YC, Shoemaker D, Keller L (2013) A Y-like social chromosome causes alternative colony organization in fire ants. Nature 493:664–668
Wiedenheft B, Sternberg SH, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482:331–338
Wurm Y et al (2011) The genome of the fire ant Solenopsis invicta. Proc Natl Acad Sci USA 108:5679–5684
Yan H et al (2017) An engineered orco mutation produces aberrant social behavior and defective neural development in ants. Cell 170:736–747
Acknowledgements
We thank W. Trible and the members of the laboratory for helpful discussions and support in the experiments. We also thank two anonymous reviewers and the editor for comments that improved the manuscript. This work was supported by the Biodiversity Research Center, Academia Sinica; the Taiwan Ministry of Science and Technology (MOST) (104-2314-B-001-009-MY5 and 103-2311-B-001-018-MY3); and an Academia Sinica Career Development Grant to J.W.
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Chiu, YK., Hsu, JC., Chang, T. et al. Mutagenesis mediated by CRISPR/Cas9 in the red imported fire ant, Solenopsis invicta. Insect. Soc. 67, 317–326 (2020). https://doi.org/10.1007/s00040-020-00755-8
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DOI: https://doi.org/10.1007/s00040-020-00755-8