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Insectes Sociaux

, Volume 60, Issue 4, pp 475–484 | Cite as

Systemic gene knockdown in Camponotus floridanus workers by feeding of dsRNA

  • C. Ratzka
  • R. Gross
  • H. Feldhaar
Research Article

Abstract

RNA interference (RNAi) technology enables to study specific gene functions also in social insects, which are otherwise difficult to access for genetic manipulations. The recent sequencing of the genomes from seven ant species made these members of the Formicidae available for knockdown studies. However, for this purpose the RNAi technology first needs to be adapted for application in ants. Studies on other insects show that the effectiveness of RNAi is quite species-specific and can depend on several experimental parameters such as the investigated stage of the insect, the target gene and/or the dsRNA delivery method. RNAi in ants through feeding of dsRNA is a preferable approach, since knockdown can be achieved in individuals without interfering with the animal’s physiology in contrast to injection of dsRNA. Here, we present a protocol for gene knockdown in Formicidae by feeding of dsRNA to worker animals. The expression of a peptidoglycan recognition protein gene, PGRP-LB, was efficiently knocked down in the body of Camponotus floridanus worker ants. Moreover, we describe a relatively cheap method to extract dsRNA from bacteria in order to obtain large quantities needed for feeding experiments.

Keywords

RNAi Camponotus floridanus dsRNA feeding Immunity Peptidoglycan recognition protein 

Notes

Acknowledgments

This work was funded by the priority program SFB567/C2 and the grant GR1243/8-1 of the Deutsche Forschungsgemeinschaft (DFG), and by the EU COST action FA0701 “Arthropod symbioses: from fundamental studies to pest management”. C. Ratzka was kindly supported by the program “Chancengleichheit für Frauen in Forschung und Lehre”. We thank Andreas Vilcinskas as well as Eileen Knorr for the opportunity to learn the application of RNAi at the University of Gießen. Moreover, Carolin Ratzka thanks Gro Amdam and Christina Grozinger for the chance to participate at the International Short Course on RNAi-Mediated Functional Genetics in Honey Bees.

Supplementary material

40_2013_314_MOESM1_ESM.doc (742 kb)
Supplementary material 1 DOC (742 KB)

References

  1. Amdam G.V., Simoes Z.L., Guidugli K.R., Norberg K. and Omholt S.W. 2003. Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA. BMC Biotechnol. 3: 1Google Scholar
  2. Bischoff V., Vignal C., Duvic B., Boneca I.G., Hoffmann J.A. and Royet J. 2006. Downregulation of the Drosophila immune response by peptidoglycan-recognition proteins SC1 and SC2. PLoS Pathog. 2: e14Google Scholar
  3. Bonasio R., Zhang G., Ye C. et al. 2010. Genomic comparison of the ants Camponotus floridanus and Harpegnathos saltator. Science 329: 1068-1071Google Scholar
  4. Carlin N.F. and Hölldobler B. 1986. The kin recognition system of carpenter ants (Camponotus spp.).1. Hierarchical cues in small colonies. Behav. Ecol. Sociobiol. 19: 123-134Google Scholar
  5. Choi M.Y. and Vander Meer R.K. 2012. Ant trail pheromone biosynthesis is triggered by a neuropeptide hormone. PLoS ONE 7: e50400Google Scholar
  6. Choi M.Y., Vander Meer R.K., Coy M. and Scharf M.E. 2012. Phenotypic impacts of PBAN RNA interference in an ant, Solenopsis invicta, and a moth, Helicoverpa zea. J. Insect Physiol. 58: 1159-1165Google Scholar
  7. Erthal M., Jr., Silva P.C. and Samuels I.R. 2007. Digestive enzymes in larvae of the leaf cutting ant, Acromyrmex subterraneus (Hymenoptera: Formicidae: Attini). J. Insect Physiol. 53: 1101-1111Google Scholar
  8. Feinberg E.H. and Hunter C.P. 2003. Transport of dsRNA into cells by the transmembrane protein SID-1. Science 301: 1545-1547Google Scholar
  9. Feldhaar H., Straka J., Krischke M., Berthold K., Stoll S., Mueller M.J. and Gross R. 2007. Nutritional upgrading for omnivorous carpenter ants by the endosymbiont Blochmannia. BMC Biol. 5: 48Google Scholar
  10. Fire A., Xu S., Montgomery M.K., Kostas S.A., Driver S.E. and Mello C.C. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806-811Google Scholar
  11. Hamilton C., Lejeune B.T. and Rosengaus R.B. 2011. Trophallaxis and prophylaxis: social immunity in the carpenter ant Camponotus pennsylvanicus. Biol. Lett. 7: 89-92Google Scholar
  12. Hölldobler B. and Wilson E.O. 1990. The Ants. Belknap Press of Harvard University Press, Cambridge, MassGoogle Scholar
  13. Huvenne H. and Smagghe G. 2010. Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a review. J. Insect Physiol. 56: 227-235Google Scholar
  14. Ihle K.E., Page R.E., Frederick K., Fondrk M.K. and Amdam G.V. 2010. Genotype effect on regulation of behaviour by vitellogenin supports reproductive origin of honeybee foraging bias. Anim. Behav. 79: 1001-1006Google Scholar
  15. Jarosch A. and Moritz R.F. 2011. 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: 851-857Google Scholar
  16. Kamath R.S. and Ahringer J. 2003. Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30: 313-321Google Scholar
  17. Kamath R.S., Martinez-Campos M., Zipperlen P., Fraser A.G. and Ahringer J. 2001. Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol. 2: RESEARCH0002Google Scholar
  18. Kulkarni M.M., Booker M., Silver S.J., Friedman A., Hong P., Perrimon N. and Mathey-Prevot B. 2006. Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays. Nat. Methods 3: 833-838Google Scholar
  19. Livak K.J. and Schmittgen T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402-408Google Scholar
  20. Lu H.L., Vinson S.B. and Pietrantonio P.V. 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: 3110-3123Google Scholar
  21. Martinez J., Patkaniowska A., Urlaub H., Luhrmann R. and Tuschl T. 2002. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110: 563-574Google Scholar
  22. Mello C.C. and Conte D., Jr. 2004. Revealing the world of RNA interference. Nature 431: 338-342Google Scholar
  23. Mellroth P., Karlsson J. and Steiner H. 2003. A scavenger function for a Drosophila peptidoglycan recognition protein. J. Biol. Chem. 278: 7059-7064Google Scholar
  24. Mellroth P. and Steiner H. 2006. PGRP-SB1: an N-acetylmuramoyl l-alanine amidase with antibacterial activity. Biochem. Biophys. Res. Commun. 350: 994-999Google Scholar
  25. Mito T., Nakamura T., Bando T., Ohuchi H. and Noji S. 2011. The advent of RNA interference in Entomology. Entomol. Sci. 14: 1-8Google Scholar
  26. Nelson C.M., Ihle K.E., Fondrk M.K., Page R.E. and Amdam G.V. 2007. The gene vitellogenin has multiple coordinating effects on social organization. PLoS Biol. 5: e62Google Scholar
  27. Nunes F.M. and Simoes Z.L. 2009. A non-invasive method for silencing gene transcription in honeybees maintained under natural conditions. Insect Biochem. Mol. Biol. 39: 157-160Google Scholar
  28. Nygaard S., Zhang G., Schiott M. et al. 2011. The genome of the leaf-cutting ant Acromyrmex echinatior suggests key adaptations to advanced social life and fungus farming. Genome Res. 21: 1339-1348Google Scholar
  29. Paredes J.C., Welchman D.P., Poidevin M. and Lemaitre B. 2011. Negative regulation by amidase PGRPs shapes the Drosophila antibacterial response and protects the fly from innocuous infection. Immunity 35: 770-779Google Scholar
  30. Patel A., Fondrk M.K., Kaftanoglu O., Emore C., Hunt G., Frederick K. and Amdam G.V. 2007. The making of a queen: TOR pathway is a key player in diphenic caste development. PLoS ONE 2: e509Google Scholar
  31. Rajagopal R., Sivakumar S., Agrawal N., Malhotra P. and Bhatnagar R.K. 2002. Silencing of midgut aminopeptidase N of Spodoptera litura by double-stranded RNA establishes its role as Bacillus thuringiensis toxin receptor. J. Biol. Chem. 277: 46849-46851Google Scholar
  32. Ratzka C., Förster F., Liang C., Kupper M., Dandekar T., Feldhaar H. and Gross R. 2012. Molecular characterization of antimicrobial peptide genes of the carpenter ant Camponotus floridanus. PLoS ONE 7 (8): e43036Google Scholar
  33. Ratzka C., Gross R. and Feldhaar H. 2013. Gene expression analysis of the endosymbiont-bearing midgut tissue during ontogeny of the carpenter ant Camponotus floridanus. J. Insect Physiol. 59: 611-623Google Scholar
  34. Ratzka C., Liang C., Dandekar T., Gross R. and Feldhaar H. 2011. Immune response of the ant Camponotus floridanus against pathogens and its obligate mutualistic endosymbiont. Insect Biochem. Mol. Biol. 41: 529-536Google Scholar
  35. Rodriguez-Cabrera L., Trujillo-Bacallao D., Borras-Hidalgo O., Wright D.J. and Ayra-Pardo C. 2010. RNAi-mediated knockdown of a Spodoptera frugiperda trypsin-like serine-protease gene reduces susceptibility to a Bacillus thuringiensis Cry1Ca1 protoxin. Environ. Microbiol. 12: 2894-2903Google Scholar
  36. Rozen S. and Skaletsky H. 2000. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132: 365-386Google Scholar
  37. Sauer C., Dudaczek D., Hölldobler B. and Gross R. 2002. Tissue localization of the endosymbiotic bacterium “Candidatus Blochmannia floridanus” in adults and larvae of the carpenter ant Camponotus floridanus. Appl. Environ. Microbiol. 68: 4187-4193Google Scholar
  38. Schlüns H. and Crozier R.H. 2007. Relish regulates expression of antimicrobial peptide genes in the honeybee, Apis mellifera, shown by RNA interference. Insect Mol. Biol. 16: 753-759Google Scholar
  39. Smith C.D., Zimin A., Holt C. et al. 2011a. Draft genome of the globally widespread and invasive Argentine ant (Linepithema humile). Proc. Natl Acad. Sci. USA 108: 5673-5678Google Scholar
  40. Smith C.R., Smith C.D., Robertson H.M. et al. 2011b. Draft genome of the red harvester ant Pogonomyrmex barbatus. Proc. Natl Acad. Sci. USA 108: 5667-5672Google Scholar
  41. Stoll S., Feldhaar H. and Gross R. 2008. Transcriptional profiling of the endosymbiont Blochmannia floridanus during different developmental stages of its holometabolous ant host. Environ. Microbiol. 11: 877-888Google Scholar
  42. Suen G., Teiling C., Li L. et al. 2011. The genome sequence of the leaf-cutter ant Atta cephalotes reveals insights into its obligate symbiotic lifestyle. PLoS Genet. 7: e1002007Google Scholar
  43. Terenius O., Papanicolaou A., Garbutt J.S. et al. 2011. RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design. J. Insect Physiol. 57: 231-245Google Scholar
  44. Terra W.R., Espinozafuentes F.P. and Ferreira C. 1988. Midgut amylase, lysozyme, aminopeptidase, and trehalase from larvae and adults of Musca domestica. Arch. Insect Biochem. Physiol. 9: 283-297Google Scholar
  45. Tian H., Peng H., Yao Q., Chen H., Xie Q., Tang B. and Zhang W. 2009. Developmental control of a lepidopteran pest Spodoptera exigua by ingestion of bacteria expressing dsRNA of a non-midgut gene. PLoS ONE 4: e6225Google Scholar
  46. Timmons L., Court D.L. and Fire A. 2001. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263: 103-112Google Scholar
  47. Timmons L. and Fire A. 1998. Specific interference by ingested dsRNA. Nature 395: 854Google Scholar
  48. Walshe D.P., Lehane S.M., Lehane M.J. and Haines L.R. 2009. Prolonged gene knockdown in the tsetse fly Glossina by feeding double stranded RNA. Insect Mol. Biol. 18: 11-19Google Scholar
  49. Wang Y., Mutti N.S., Ihle K.E., Siegel A., Dolezal A.G., Kaftanoglu O. and Amdam G.V. 2010. Down-regulation of honey bee IRS gene biases behavior toward food rich in protein. PLoS Genet. 6: e1000896Google Scholar
  50. Winston W.M., Molodowitch C. and Hunter C.P. 2002. Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. Science 295: 2456-2459Google Scholar
  51. Wolschin F., Mutti N.S. and Amdam G.V. 2011. Insulin receptor substrate influences female caste development in honeybees. Biol. Lett. 7: 112-115Google Scholar
  52. Wurm Y., Wang J., Riba-Grognuz O. et al. 2011. The genome of the fire ant Solenopsis invicta. Proc. Natl Acad. Sci. USA 108: 5679-5684Google Scholar
  53. Zaidman-Remy A., Herve M., Poidevin M., Pili-Floury S., Kim M.S., Blanot D., Oh B.H., Ueda R., Mengin-Lecreulx D. and Lemaitre B. 2006. The Drosophila amidase PGRP-LB modulates the immune response to bacterial infection. Immunity 24: 463-473Google Scholar
  54. Zaidman-Remy A., Poidevin M., Herve M., Welchman D.P., Paredes J.C., Fahlander C., Steiner H., Mengin-Lecreulx D. and Lemaitre B. 2011. Drosophila Immunity: Analysis of PGRP-SB1 Expression, Enzymatic Activity and Function. PLoS ONE 6: e17231Google Scholar
  55. Zhu F., Xu J., Palli R., Ferguson J. and Palli S.R. 2011. Ingested RNA interference for managing the populations of the Colorado potato beetle, Leptinotarsa decemlineata. Pest Manag. Sci. 67: 175-182Google Scholar

Copyright information

© International Union for the Study of Social Insects (IUSSI) 2013

Authors and Affiliations

  1. 1.Lehrstuhl für MikrobiologieBiozentrum, Universität WürzburgWürzburgGermany
  2. 2.Lehrstuhl für Tierökologie IUniversität BayreuthBayreuthGermany

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