Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Honey bee functional genomics using symbiont-mediated RNAi

Nature Protocols volume 18, pages 902–928 (2023)Cite this article

Subjects

Abstract

Honey bees are indispensable pollinators and model organisms for studying social behavior, development and cognition. However, their eusociality makes it difficult to use standard forward genetic approaches to study gene function. Most functional genomics studies in bees currently utilize double-stranded RNA (dsRNA) injection or feeding to induce RNAi-mediated knockdown of a gene of interest. However, dsRNA injection is laborious and harmful, and dsRNA feeding is difficult to scale cheaply. Further, both methods require repeated dsRNA administration to ensure a continued RNAi response. To fill this gap, we engineered the bee gut bacterium Snodgrassella alvi to induce a sustained host RNA interference response that reduces expression of a targeted gene. To employ this functional genomics using engineered symbionts (FUGUES) procedure, a dsRNA expression plasmid is cloned in Escherichia coli using Golden Gate assembly and then transferred to S. alvi. Adult worker bees are then colonized with engineered S. alvi. Finally, gene knockdown is verified through qRT–PCR, and bee phenotypes of interest can be further assessed. Expression of targeted genes is reduced by as much as 50–75% throughout the entire bee body by 5 d after colonization. This protocol can be accomplished in 4 weeks by bee researchers with microbiology and molecular cloning skills. FUGUES currently offers a streamlined and scalable approach for studying the biology of honey bees. Engineering other microbial symbionts to influence their hosts in ways that are similar to those described in this protocol may prove useful for studying additional insect and animal species in the future.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Workflow for using symbiont-mediated RNAi to study gene function in bees.
Fig. 2: dsRNA expression vector cloning.
Fig. 3: Plasmid conjugation into S. alvi.
Fig. 4: Honey bee inoculation with S. alvi.
Fig. 5: Validation of gene knockdown.

Similar content being viewed by others

Data availability

Plasmid sequences are available on the Addgene website under the accession numbers provided in the Reagents section. Other data supporting the results of this study are available within the article.

References

  1. Patterson Rosa, L., Eimanifar, A., Kimes, A. G., Brooks, S. A. & Ellis, J. D. Attack of the dark clones the genetics of reproductive and color traits of South African honey bees (Apis mellifera spp.). PLoS ONE 16, e0260833 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Zayed, A. & Robinson, G. E. Understanding the relationship between brain gene expression and social behavior: lessons from the honey bee. Annu. Rev. Genet. 46, 591–615 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Ament, S. A., Corona, M., Pollock, H. S. & Robinson, G. E. Insulin signaling is involved in the regulation of worker division of labor in honey bee colonies. Proc. Natl Acad. Sci. USA 105, 4226–4231 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Chen, D. et al. Uncovering the immune responses of Apis mellifera ligustica larval gut to Ascosphaera apis infection utilizing transcriptome sequencing. Gene 621, 40–50 (2017).

    Article  CAS  PubMed  Google Scholar 

  5. Vogel, E., Santos, D., Mingels, L., Verdonckt, T.-W. & Broeck, J. V. RNA interference in insects: protecting beneficials and controlling pests. Front. Physiol. 9, (2019).

  6. Darryl Conte, J. RNA interference in Caenorhabditis elegans. Curr. Protoc. Mol. Biol. 109, 26.3.1–26.330 (2015).

    PubMed Central  Google Scholar 

  7. Das, P. R. & Sherif, S. M. Application of exogenous dsRNAs-induced RNAi in agriculture: challenges and triumphs. Front. Plant Sci. 11, 946 (2020).

  8. Perrimon, N., Ni, J.-Q. & Perkins, L. In vivo RNAi: today and tomorrow. Cold Spring Harb. Perspect. Biol. 2, a003640 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Whitten, M. M. A. et al. Symbiont-mediated RNA interference in insects. Proc. Biol. Sci. 283, 20160042 (2016).

    PubMed  PubMed Central  Google Scholar 

  10. Leonard, S. P. et al. Engineered symbionts activate honey bee immunity and limit pathogens. Science 367, 573–576 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kwong, W. K. & Moran, N. A. Gut microbial communities of social bees. Nat. Rev. Microbiol. 14, 374–384 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Martinson, V. G. et al. A simple and distinctive microbiota associated with honey bees and bumble bees. Mol. Ecol. 20, 619–628 (2011).

    Article  PubMed  Google Scholar 

  13. Martinson, V. G., Moy, J. & Moran, N. A. Establishment of characteristic gut bacteria during development of the honeybee worker. Appl. Environ. Microbiol. 78, 2830–2840 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Horak, R. D., Leonard, S. P. & Moran, N. A. Symbionts shape host innate immunity in honeybees. Proc. Biol. Sci. 287, 20201184 (2020).

    PubMed  PubMed Central  Google Scholar 

  15. Kwong, W. K. & Moran, N. A. Cultivation and characterization of the gut symbionts of honey bees and bumble bees: description of Snodgrassella alvi gen. nov., sp. nov., a member of the family Neisseriaceae of the Betaproteobacteria, and Gilliamella apicola gen. nov., sp. nov., a member of Orbaceae fam. nov., Orbales ord. nov., a sister taxon to the order ‘Enterobacteriales’ of the Gammaproteobacteria. Int. J. Syst. Evol. Microbiol. 63, 2008–2018 (2013).

    Article  CAS  PubMed  Google Scholar 

  16. Kwong, W. K., Engel, P., Koch, H. & Moran, N. A. Genomics and host specialization of honey bee and bumble bee gut symbionts. Proc. Natl Acad. Sci. USA 111, 11509–11514 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Leonard, S. P. et al. Genetic engineering of bee gut microbiome bacteria with a toolkit for modular assembly of broad-host-range plasmids. ACS Synth. Biol. 7, 1279–1290 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Elston, K. M., Perreau, J., Maeda, G. P., Moran, N. A. & Barrick, J. E. Engineering a culturable Serratia symbiotica strain for aphid paratransgenesis. Appl. Environ. Microbiol. https://doi.org/10.1128/AEM.02245-20 (2021).

  19. Koch, H., Abrol, D. P., Li, J. & Schmid-Hempel, P. Diversity and evolutionary patterns of bacterial gut associates of corbiculate bees. Mol. Ecol. 22, 2028–2044 (2013).

    Article  CAS  PubMed  Google Scholar 

  20. Kwong, W. K. et al. Dynamic microbiome evolution in social bees. Sci. Adv. 3, e1600513 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Li, Y., Leonard, S. P., Powell, J. E. & Moran, N. A. Species divergence in gut bacteria of social bees. Proc. Natl Acad. Sci. USA (2022).

  22. Hammer, T. J., Le, E., Martin, A. N. & Moran, N. A. The gut microbiota of bumblebees. Insectes Soc. 68, 287–301 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Tang, Q.-H. et al. The composition of bacteria in gut and beebread of stingless bees (Apidae: Meliponini) from tropics Yunnan, China. Antonie Van. Leeuwenhoek 114, 1293–1305 (2021).

    Article  CAS  PubMed  Google Scholar 

  24. Klein, A.-M., Boreux, V., Fornoff, F., Mupepele, A.-C. & Pufal, G. Relevance of wild and managed bees for human well-being. Curr. Opin. Insect Sci. 26, 82–88 (2018).

    Article  PubMed  Google Scholar 

  25. Taracena, M. L. et al. Genetically modifying the insect gut microbiota to control Chagas disease vectors through systemic RNAi. PLoS Negl. Trop. Dis. 9, e0003358 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Jiang, Y.-X. et al. The combination of Bacillus thuringiensis and its engineered strain expressing dsRNA increases the toxicity against Plutella xylostella. Int. J. Mol. Sci. 23, 444 (2022).

    Article  CAS  Google Scholar 

  27. Elston, K. M. et al. Engineering insects from the endosymbiont out. Trends Microbiol. 30, 79–96 (2022).

    Article  CAS  PubMed  Google Scholar 

  28. Jain, A. & Srivastava, P. Broad host range plasmids. FEMS Microbiol. Lett. 348, 87–96 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. Kohno, H. & Kubo, T. Genetics in the honey bee: achievements and prospects toward the functional analysis of molecular and neural mechanisms underlying social behaviors. Insects 10, 348 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Otte, M. et al. Improving genetic transformation rates in honeybees. Sci. Rep. 8, 16534 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Schulte, C., Theilenberg, E., Müller-Borg, M., Gempe, T. & Beye, M. Highly efficient integration and expression of piggyBac-derived cassettes in the honeybee (Apis mellifera). Proc. Natl Acad. Sci. USA 111, 9003–9008 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Chen, Z. et al. Neurodevelopmental and transcriptomic effects of CRISPR/Cas9-induced somatic orco mutation in honey bees. J. Neurogenet. 35, 320–332 (2021).

    Article  CAS  PubMed  Google Scholar 

  33. Değirmenci, L. et al. CRISPR/Cas 9-mediated mutations as a new tool for studying taste in honeybees. Chem. Senses 45, 655–666 (2020).

    Article  PubMed  Google Scholar 

  34. Hu, X. F., Zhang, B., Liao, C. H. & Zeng, Z. J. High-efficiency CRISPR/Cas9-mediated gene editing in honeybee (Apis mellifera) embryos. G3 9, 1759–1766 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kohno, H., Suenami, S., Takeuchi, H., Sasaki, T. & Kubo, T. Production of knockout mutants by CRISPR/Cas9 in the European honeybee, Apis mellifera L. Zool. Sci. 33, 505–512 (2016).

    Article  CAS  Google Scholar 

  36. Kohno, H. & Kubo, T. mKast is dispensable for normal development and sexual maturation of the male European honeybee. Sci. Rep. 8, 11877 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Liang, L. et al. Expansion of CRISPR targeting sites using an integrated gene-editing system in Apis mellifera. Insects 12, 954 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Nie, H.-Y. et al. CRISPR/Cas9 mediated knockout of Amyellow-y gene results in melanization defect of the cuticle in adult Apis mellifera. J. Insect Physiol. 132, 104264 (2021).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Müßig, L. et al. Acute disruption of the NMDA receptor subunit nr1 in the honeybee brain selectively impairs memory formation. J. Neurosci. 30, 7817–7825 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Rodríguez-García, C. et al. Transferrin-mediated iron sequestration suggests a novel therapeutic strategy for controlling Nosema disease in the honey bee, Apis mellifera. PLoS Pathog. 17, e1009270 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Niu, J., Meeus, I., Cappelle, K., Piot, N. & Smagghe, G. The immune response of the small interfering RNA pathway in the defense against bee viruses. Curr. Opin. Insect Sci. 6, 22–27 (2014).

    Article  PubMed  Google Scholar 

  43. Brutscher, L. M. & Flenniken, M. L. RNAi and antiviral defense in the honey bee. J. Immunol. Res. 2015, e941897 (2015).

    Article  Google Scholar 

  44. Flenniken, M. L. & Andino, R. Non-specific dsRNA-mediated antiviral response in the honey bee. PLoS ONE 8, e77263 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. McMenamin, A. J., Daughenbaugh, K. F. & Flenniken, M. L. The heat shock response in the Western honey bee (Apis mellifera) is antiviral. Viruses 12, 245 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Gatehouse, H. S. et al. Amylase activity in honey bee hypopharyngeal glands reduced by RNA interference. J. Apic. Res. 43, 9–13 (2004).

    Article  CAS  Google Scholar 

  47. Amdam, G. V., Simões, Z. L. P., Guidugli, K. R., Norberg, K. & Omholt, S. W. Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA. BMC Biotechnol. 3, 1 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Amdam, G. V., Norberg, K., Page, R. E., Erber, J. & Scheiner, R. Downregulation of vitellogenin gene activity increases the gustatory responsiveness of honey bee workers (Apis mellifera). Behav. Brain Res. 169, 201–205 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ihle, K. E., Page, R. E., Frederick, K., Fondrk, M. K. & Amdam, G. V. Genotype effect on regulation of behaviour by vitellogenin supports reproductive origin of honeybee foraging bias. Anim. Behav. 79, 1001–1006 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Guo, X., Wang, Y., Sinakevitch, I., Lei, H. & Smith, B. H. Comparison of RNAi knockdown effect of tyramine receptor 1 induced by dsRNA and siRNA in brains of the honey bee, Apis mellifera. J. Insect Physiol. 111, 47–52 (2018).

    Article  CAS  PubMed  Google Scholar 

  51. Nelson, C. M., Ihle, K. E., Fondrk, M. K., Page, R. E. & Amdam, G. V. The gene vitellogenin has multiple coordinating effects on social organization. PLoS Biol. 5, e62 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Farooqui, T., Vaessin, H. & Smith, B. H. Octopamine receptors in the honeybee (Apis mellifera) brain and their disruption by RNA-mediated interference. J. Insect Physiol. 50, 701–713 (2004).

    Article  CAS  PubMed  Google Scholar 

  53. Guidugli, K. R. et al. Vitellogenin regulates hormonal dynamics in the worker caste of a eusocial insect. FEBS Lett. 579, 4961–4965 (2005).

    Article  CAS  PubMed  Google Scholar 

  54. Mackert, A., do Nascimento, A. M., Bitondi, M. M. G., Hartfelder, K. & Simões, Z. L. P. Identification of a juvenile hormone esterase-like gene in the honey bee, Apis mellifera L.—expression analysis and functional assays. Comp. Biochem. Physiol. B 150, 33–44 (2008).

    Article  PubMed  Google Scholar 

  55. Li-Byarlay, H. et al. RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee. Proc. Natl Acad. Sci. USA 110, 12750–12755 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Leboulle, G. et al. Characterisation of the RNA interference response against the long-wavelength receptor of the honeybee. Insect Biochem. Mol. Biol. 43, 959–969 (2013).

    Article  CAS  PubMed  Google Scholar 

  57. Wang, Y., Brent, C. S., Fennern, E. & Amdam, G. V. Gustatory perception and fat body energy metabolism are jointly affected by vitellogenin and juvenile hormone in honey bees. PLoS Genet. 8, e1002779 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Wang, Y. et al. Down-regulation of honey bee IRS gene biases behavior toward food rich in protein. PLoS Genet. 6, e1000896 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  59. Wang, Y., Baker, N. & Amdam, G. V. RNAi-mediated double gene knockdown and gustatory perception measurement in honey bees (Apis mellifera). J. Vis. Exp. https://doi.org/10.3791/50446 (2013).

  60. Nilsen, K.-A. et al. Insulin-like peptide genes in honey bee fat body respond differently to manipulation of social behavioral physiology. J. Exp. Biol. 214, 1488–1497 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Jarosch, A. & Moritz, R. F. A. Systemic RNA-interference in the honeybee Apis mellifera: tissue dependent uptake of fluorescent siRNA after intra-abdominal application observed by laser-scanning microscopy. J. Insect Physiol. 57, 851–857 (2011).

    Article  CAS  PubMed  Google Scholar 

  62. Jarosch, A., Stolle, E., Crewe, R. M. & Moritz, R. F. A. Alternative splicing of a single transcription factor drives selfish reproductive behavior in honeybee workers (Apis mellifera). Proc. Natl Acad. Sci. USA 108, 15282–15287 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Mustard, J. A., Pham, P. M. & Smith, B. H. Modulation of motor behavior by dopamine and the D1-like dopamine receptor AmDOP2 in the honey bee. J. Insect Physiol. 56, 422–430 (2010).

    Article  CAS  PubMed  Google Scholar 

  64. Marco Antonio, D. S., Guidugli-Lazzarini, K. R., do Nascimento, A. M., Simões, Z. L. P. & Hartfelder, K. RNAi-mediated silencing of vitellogenin gene function turns honeybee (Apis mellifera) workers into extremely precocious foragers. Sci. Nat. 95, 953–961 (2008).

    Article  CAS  Google Scholar 

  65. Seehuus, S.-C., Norberg, K., Gimsa, U., Krekling, T. & Amdam, G. V. Reproductive protein protects functionally sterile honey bee workers from oxidative stress. Proc. Natl Acad. Sci. USA 103, 962–967 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Li, W. et al. Silencing the honey bee (Apis mellifera) naked cuticle gene (nkd) improves host immune function and reduces Nosema ceranae infections. Appl. Environ. Microbiol. 82, 6779–6787 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Costa, C. P. et al. RNAi-mediated functional analysis of bursicon genes related to adult cuticle formation and tanning in the honeybee, Apis mellifera. PLoS ONE 11, e0167421 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  68. Lourenço, A. P., Florecki, M. M., Simões, Z. L. P. & Evans, J. D. Silencing of Apis mellifera dorsal genes reveals their role in expression of the antimicrobial peptide defensin-1. Insect Mol. Biol. 27, 577–589 (2018).

    Article  PubMed  Google Scholar 

  69. Mello, T. R. P. et al. Hormonal control and target genes of ftz-f1 expression in the honeybee Apis mellifera: a positive loop linking juvenile hormone, ftz-f1, and vitellogenin. Insect Mol. Biol. 28, 145–159 (2019).

    Article  CAS  PubMed  Google Scholar 

  70. Schlüns, H. & Crozier, R. H. Relish regulates expression of antimicrobial peptide genes in the honeybee, Apis mellifera, shown by RNA interference. Insect Mol. Biol. 16, 753–759 (2007).

    Article  PubMed  Google Scholar 

  71. Mello, T. R. P. et al. Developmental regulation of ecdysone receptor (EcR) and EcR-controlled gene expression during pharate-adult development of honeybees (Apis mellifera). Front. Genet. 5, 445 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  72. Paldi, N. et al. Effective gene silencing in a microsporidian parasite associated with honeybee (Apis mellifera) colony declines. Appl. Environ. Microbiol. 76, 5960–5964 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. He, N. et al. RNA Interference-mediated knockdown of genes encoding spore wall proteins confers protection against Nosema ceranae infection in the European honey bee, Apis mellifera. Microorganisms 9, 505 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Wolschin, F., Mutti, N. S. & Amdam, G. V. Insulin receptor substrate influences female caste development in honeybees. Biol. Lett. 7, 112–115 (2011).

    Article  PubMed  Google Scholar 

  75. Patel, A. et al. The making of a queen: TOR pathway is a key player in diphenic caste development. PLoS ONE 2, e509 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  76. Garbian, Y., Maori, E., Kalev, H., Shafir, S. & Sela, I. Bidirectional transfer of RNAi between honey bee and Varroa destructor: Varroa gene silencing reduces Varroa population. PLoS Pathog. 8, e1003035 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Barchuk, A. R., Figueiredo, V. L. C. & Simões, Z. L. P. Downregulation of ultraspiracle gene expression delays pupal development in honeybees. J. Insect Physiol. 54, 1035–1040 (2008).

    Article  CAS  PubMed  Google Scholar 

  78. Nunes, F. M. F. & Simões, Z. L. P. A non-invasive method for silencing gene transcription in honeybees maintained under natural conditions. Insect Biochem. Mol. Biol. 39, 157–160 (2009).

    Article  CAS  PubMed  Google Scholar 

  79. Mutti, N. S., Wang, Y., Kaftanoglu, O. & Amdam, G. V. Honey bee PTEN – description, developmental knockdown, and tissue-specific expression of splice-variants correlated with alternative social phenotypes. PLoS ONE 6, e22195 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Nunes, F. M. F., Ihle, K. E., Mutti, N. S., Simões, Z. L. P. & Amdam, G. V. The gene vitellogenin affects microRNA regulation in honey bee (Apis mellifera) fat body and brain. J. Exp. Biol. 216, 3724–3732 (2013).

    CAS  PubMed  Google Scholar 

  81. Scott, J. G. et al. Towards the elements of successful insect RNAi. J. Insect Physiol. 59, 1212–1221 (2013).

    Article  CAS  PubMed  Google Scholar 

  82. Aronstein, K. & Saldivar, E. Characterization of a honey bee Toll related receptor gene Am18w and its potential involvement in antimicrobial immune defense. Apidologie 36, 3–14 (2005).

    Article  CAS  Google Scholar 

  83. Whitten, M. & Dyson, P. Gene silencing in non-model insects: overcoming hurdles using symbiotic bacteria for trauma-free sustainable delivery of RNA interference. BioEssays 39, 1600247 (2017).

    Article  Google Scholar 

  84. Hunter, W. et al. Large-scale field application of RNAi technology reducing Israeli acute paralysis virus disease in honey bees (Apis mellifera, Hymenoptera: Apidae). PLoS Pathog. 6, e1001160 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  85. Maori, E. et al. IAPV, a bee-affecting virus associated with colony collapse disorder can be silenced by dsRNA ingestion. Insect Mol. Biol. 18, 55–60 (2009).

    Article  CAS  PubMed  Google Scholar 

  86. Thoma, S. & Schobert, M. An improved Escherichia coli donor strain for diparental mating. FEMS Microbiol. Lett. 294, 127–132 (2009).

    Article  CAS  PubMed  Google Scholar 

  87. Deng, Y. et al. Screening and validation of reference genes for RT-qPCR under different honey bee viral infections and dsRNA treatment. Front. Microbiol. 11, (2020).

  88. Jeon, J. H., Moon, K., Kim, Y. & Kim, Y. H. Reference gene selection for qRT-PCR analysis of season- and tissue-specific gene expression profiles in the honey bee Apis mellifera. Sci. Rep. 10, 13935 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Ahmed, M. & Kim, D. R. pcr: an R package for quality assessment, analysis and testing of qPCR data. PeerJ 6, e4473 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  90. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408 (2001).

    Article  CAS  PubMed  Google Scholar 

  91. Evans, J. D., Chen, Y. P., Prisco, G., di, Pettis, J. & Williams, V. Bee cups: single-use cages for honey bee experiments. J. Apic. Res. 48, 300–302 (2009).

    Article  Google Scholar 

  92. Green, M. R., Sambrook, J. & Sambrook, J. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2012).

  93. Svec, D., Tichopad, A., Novosadova, V., Pfaffl, M. W. & Kubista, M. How good is a PCR efficiency estimate: recommendations for precise and robust qPCR efficiency assessments. Biomol. Detect. Quantif. 3, 9–16 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Kralik, P. & Ricchi, M. A basic guide to real time PCR in microbial diagnostics: definitions, parameters, and everything. Front. Microbiol. 8, (2017).

  95. Pfaffl, M. W. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res. 29, e45 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Ritz, C. & Spiess, A.-N. qpcR: an R package for sigmoidal model selection in quantitative real-time polymerase chain reaction analysis. Bioinformatics 24, 1549–1551 (2008).

    Article  CAS  PubMed  Google Scholar 

  97. Deshwal, S. & Mallon, E. B. Antimicrobial peptides play a functional role in bumblebee anti-trypanosome defense. Dev. Comp. Immunol. 42, 240–243 (2014).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank N. Moran for providing invaluable guidance and resources during the development of this protocol. We also thank B. Jack for his help in graphic creation. Development of this protocol was supported by funding from the National Science Foundation (IOS-2103208) and the Defense Advanced Research Projects Agency (HR0011-15-C0095).

Author information

Author notes

  1. These authors contributed equally: Patrick J. Lariviere, Sean P. Leonard.

Authors and Affiliations

  1. Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA

    Patrick J. Lariviere, Sean P. Leonard & Jeffrey E. Barrick

  2. Department of Integrative Biology, The University of Texas at Austin, Austin, TX, USA

    Patrick J. Lariviere, Sean P. Leonard, Richard D. Horak & J. Elijah Powell

Authors
  1. Patrick J. Lariviere
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sean P. Leonard
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Richard D. Horak
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. Elijah Powell
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jeffrey E. Barrick
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization: S.P.L. and J.E.B.; methodology: S.P.L., J.E.P. and J.E.B.; investigation: P.J.L., S.P.L., J.E.P. and R.D.H.; writing: P.J.L., S.P.L. and J.E.B.; editing: P.J.L., S.P.L., J.E.P., R.D.H. and J.E.B.

Corresponding author

Correspondence to Jeffrey E. Barrick.

Ethics declarations

Competing interests

S.P.L. and J.E.B. are co-inventors on a patent (US 11,382,989) covering the use of engineered symbionts to improve bee health. J.E.B. is the owner of Evolvomics LLC.

Peer review

Peer review information

Nature Protocols thanks Hiroki Kohno, Zila L.P. Simoes and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Leonard, S. P. et al. Science 367, 573–576 (2020): https://doi.org/10.1126/science.aax9039

Leonard, S. P. et al. ACS Synth. Biol. 7, 1279–1290 (2018): https://doi.org/10.1021/acssynbio.7b00399

Supplementary information

Reporting Summary

Supplementary Video 1

Technique for scraping S. alvi colonies from a Columbia Blood agar plate

Source data

Source Data Fig. 1

Unprocessed gel for Fig. 2c.

Source Data Fig. 5

Statistical source data for Fig. 5b,c.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lariviere, P.J., Leonard, S.P., Horak, R.D. et al. Honey bee functional genomics using symbiont-mediated RNAi. Nat Protoc 18, 902–928 (2023). https://doi.org/10.1038/s41596-022-00778-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-022-00778-4

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology