Skip to main content
Log in

Mutagenesis mediated by CRISPR/Cas9 in the red imported fire ant, Solenopsis invicta

  • Technical Article
  • Published:
Insectes Sociaux Aims and scope Submit manuscript

A Correction to this article was published on 17 July 2021

This article has been updated

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Change history

References

  • Adli M (2018) The CRISPR tool kit for genome editing and beyond. Nat Commun 9:1911

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  PubMed  Google Scholar 

  • Ascunce MS et al (2011) Global invasion history of the fire ant Solenopsis invicta. Science 331:1066–1068

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Cong L et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huang YC, Wang J (2014) Did the fire ant supergene evolve selfishly or socially? BioEssays 36:200–208

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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

    Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  Google Scholar 

  • Keller L, Ross KG (1998) Selfish genes: a green beard in the red fire ant. Nature 394:573–575

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Krieger MJB, Ross KG (2002) Identification of a major gene regulating complex social behavior. Science 295:328–332

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Makki R, Cinnamon E, Gould AP (2014) The development and functions of oenocytes. Annu Rev Entomol 59:405–425

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rittschof CC, Robinson GE (2016) Behavioral genetic toolkits: toward the evolutionary origins of complex phenotypes. Curr Top Dev Biol 119:157–204

    Article  CAS  PubMed  Google Scholar 

  • Ross KG, Keller L (1998) Genetic control of social organization in an ant. Proc Natl Acad Sci USA 95:14232–14237

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Roth A et al (2019) A genetic switch for worker nutrition-mediated traits in honeybees. PLoS Biol 17:e3000171

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shilo BZ (2014) The regulation and functions of MAPK pathways in Drosophila. Methods 68:151–159

    Article  CAS  PubMed  Google Scholar 

  • Smith CR, Dolezal A, Eliyahu D, Holbrook CT, Gadau J (2009) Ants (Formicidae): models for social complexity. Cold Spring Harb Protoc 2009:pdb emo125

    PubMed  Google Scholar 

  • Stolle E et al (2019) Degenerative expansion of a young supergene. Mol Biol Evol 36:553–561

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Trible W et al (2017) orco mutagenesis causes loss of antennal lobe glomeruli and impaired social behavior in ants. Cell 170:727–735

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tschinkel WR (2006) The fire ants. The Belknap Press of Harvard University Press, Cambridge

    Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang J, Ross KG, Keller L (2008) Genome-wide expression patterns and the genetic architecture of a fundamental social trait. PLoS Genet 4:e1000127

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Wiedenheft B, Sternberg SH, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482:331–338

    Article  CAS  PubMed  Google Scholar 

  • Wurm Y et al (2011) The genome of the fire ant Solenopsis invicta. Proc Natl Acad Sci USA 108:5679–5684

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yan H et al (2017) An engineered orco mutation produces aberrant social behavior and defective neural development in ants. Cell 170:736–747

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Y.-C. Huang or J. Wang.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 266 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00040-020-00755-8

Keywords

Navigation