Eur. J. Entomol. 114: 170-178, 2017 | DOI: 10.14411/eje.2017.022

Double strand RNA-mediated RNA interference through feeding in larval gypsy moth, Lymantria dispar (Lepidoptera: Erebidae)

Saikat Kumar B. GHOSH, Dawn E. GUNDERSEN-RINDAL*
United States Department of Agriculture - ARS Invasive Insect Biocontrol and Behavior Laboratory, Beltsville, Maryland, USA; e-mails: saikat.ghosh@ars.usda.gov, dawn.gundersen-rindal@ars.usda.gov

RNA interference (RNAi) technology uses dsRNAs to silence specific targeted genes by downregulating their expression. It has become a potent tool for functional and regulatory studies of insect genes and has potential to be applied for insect control. Though it has been challenging to generate effective RNAi in lepidopteran insects, in the current study this technology was applied to develop specific RNAi-based molecular tools that could be used to negatively impact the invasive lepidopteran forest pest, gypsy moth (GM). GM midgut-specific genes were selected for dsRNA design from larval transcriptome profiles. Two methods were used to produce specific dsRNAs, bacterial expression and in vitro synthesis, which were then fed per os to GM larvae. Depletion of uncharacterized gene targets known as locus 365 and locus 28365, or their stacked combination, depleted target transcripts in a sequence specific manner and resulted in 60% reduction in body mass. Treated GM females that were able to moult to the adult stage displayed an approximately two-fold reduction in egg masses. These have potential to be developed as molecular biopesticides for GM.

Keywords: Lepidoptera, Erebidae, Lymantria dispar, gypsy moth larvae, RNAi, dsRNA, ingestion

Received: December 20, 2016; Accepted: March 10, 2017; Published online: April 5, 2017Show citation

ACS AIP APA ASA Harvard Chicago IEEE ISO690 MLA NLM Turabian Vancouver
GHOSH, S.K.B., & GUNDERSEN-RINDAL, D.E. (2017). Double strand RNA-mediated RNA interference through feeding in larval gypsy moth, Lymantria dispar (Lepidoptera: Erebidae). Eur. J. Entomol.114(1), 2017.000. doi: 10.14411/eje.2017.022.
Download citation

References

  1. Baigude H. & Rana T.M. 2009: Delivery of therapeutic RNAi by nanovehicles. - ChemBioChem 10: 2449-2454. Go to original source...
  2. Bartel D.P. 2004: MicroRNAs: genomics, biogenesis, mechanism, and function. - Cell 116: 281-297. Go to original source...
  3. Bernstein E., Caudy A.A., Hammond S.M. & Hannon G.J. 2001: Role for a bidentate ribonuclease in the initiation step of RNA interference. - Nature 409: 363-366. Go to original source...
  4. Cheng T., Lin P., Huang L., Wu Y., Jin S., Liu C. & Xia Q. 2016: Genome-wide analysis of host responses to four different types of microorganisms in Bombyx mori (Lepidoptera: Bombycidae). - J. Insect Sci. 16: 1-11. Go to original source...
  5. Clemens J.C., Worby C.A., Simonson-Leff N., Muda M., Maehama T., Hemmings B.A. & Dixon J.E. 2000: Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. - Proc. Natn. Acad. Sci. U.S.A. 97: 6499-6503. Go to original source...
  6. Doane C.C. & McManus M.L. 1981: The Gypsy Moth: Research toward Integrated Pest Management. USDA For. Serv. Tech. Bull. No. 1585, Washington, 777 pp.
  7. Draz M.S., Fang B.A., Zhang P., Hu Z., Gu S., Weng K.C., Gray J.W. & Chen F.F. 2014: Nanoparticle-mediated systemic delivery of siRNA for treatment of cancers and viral infections. - Theranostics 4: 872-892. Go to original source...
  8. Fire A., Xu S., Montgomery M.K., Kostas S.A., Driver S.E. & Mello C.C. 1998: Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. - Nature 391: 806-811. Go to original source...
  9. Garbutt J.S. & Reynolds S.E. 2012: Induction of RNA interference genes by double-stranded RNA; implications for susceptibility to RNA interference. - Insect Biochem. Mol. Biol. 42: 621-628. Go to original source...
  10. Garner K.J., Hiremath S., Lehtoma K. & Valaitis A.P. 1999: Cloning and complete sequence characterization of two gypsy moth aminopeptidase-N cDNAs, including the receptor for Bacillus thuringiensis Cry1Ac toxin. - Insect Biochem. Mol. Biol. 29: 527-535. Go to original source...
  11. Grishok A., Sinskey J.L. & Sharp P.A. 2005: Transcriptional silencing of a transgene by RNAi in the soma of C. elegans. - Genes Devel. 19: 683-696. Go to original source...
  12. Hagedorn H.H. & Kunkel J.G. 1979: Vitellogenin and vitellin in insects. - Annu. Rev. Entomol. 24: 475-505. Go to original source...
  13. Höfte H. & Whiteley H.R. 1989: Insecticidal crystal proteins of Bacillus thuringiensis. - Microbiol. Rev. 53: 242-255.
  14. Honeybee Genome Sequencing Consortium 2006: Insights into social insects from the genome of the honeybee Apis mellifera. - Nature 443: 931-949. Go to original source...
  15. Huvenne H. & Smagghe G. 2010: Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a review. - J. Insect Physiol. 56: 227-235. Go to original source...
  16. Ivashuta S., Zhang Y., Wiggins B.E., Ramaseshadri P., Segers G.C., Johnson S., Meyer S.E., Kerstetter R.A., McNulty B.C., Bolognesi R. & Heck G.R. 2015: Environmental RNAi in herbivorous insects. - RNA (New York) 21: 840-850. Go to original source...
  17. Kamath R.S., Martinez-Campos M., Zipperlen P., Fraser A.G. & Ahringer J. 2001: Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. - Genome Biol. 2(1): Research0002, 10 pp.
  18. Kennerdell J.R. & Carthew R.W. 1998: Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. - Cell 95: 1017-1026. Go to original source...
  19. Ketting R.F., Fischer S.E., Bernstein E., Sijen T., Hannon G.J. & Plasterk R.H. 2001: Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. - Genes Devel. 15: 2654-2659. Go to original source...
  20. Kolliopoulou A. & Swevers L. 2014: Recent progress in RNAi research in Lepidoptera: Intracellular machinery, antiviral immune response and prospects for insect pest control. - Curr. Opin. Insect Sci. 6: 28-34. Go to original source...
  21. Li X., Zhang M. & Zhang H. 2011: RNA interference of four genes in adult Bactrocera dorsalis by feeding their dsRNAs. - PLoS one 6(3): e17788, 11 pp. Go to original source...
  22. Liu R.-Y., Fioravante D., Shah S. & Byrne J.H. 2008: cAMP response element-binding protein 1 feedback loop is necessary for consolidation of long-term synaptic facilitation in Aplysia. - J. Neurosci. 28: 1970-1976. Go to original source...
  23. Livshits M.A., Amosova O.A. & Lyubchenko Yu.L. 1990: Flexibility difference between double-stranded RNA and DNA as revealed by gel electrophoresis. - J. Biomol. Struct. Dynam. 7: 1237-1249. Go to original source...
  24. Mao Y.-B., Cai W.-J., Wang J.-W., Hong G.-J., Tao X.-Y., Wang L.-J., Huang Y.-P. & Chen X.-Y. 2007: Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. - Nature Biotechnol. 25: 1307-1313. Go to original source...
  25. Martin P.A. 2004: A freeze-dried diet to test pathogens of Colorado potato beetle. - Biol. Contr. 29: 109-114. Go to original source...
  26. Martinez J., Patkaniowska A., Urlaub H., Lührmann R. & Tuschl T. 2002: Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. - Cell 110: 563-574. Go to original source...
  27. Rajagopal R., Sivakumar S., Agrawal N., Malhotra P. & 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: 46849-46851. Go to original source...
  28. Shah N., Dorer D.R., Moriyama E.N. & Christensen A.C. 2012: Evolution of a large, conserved, and syntenic gene family in insects. - G3 (Bethesda) 2: 313-319.
  29. Shu Y.H., Wang J.W., Lu K., Zhou J.L., Zhou Q. & Zhang G.R. 2011: The first vitellogenin receptor from a Lepidopteran insect: molecular characterization, expression patterns and RNA interference analysis. - Insect Mol. Biol. 20: 61-73. Go to original source...
  30. Sparks M.E. & Gundersen-Rindal D.E. 2011: The Lymantria dispar IPLB-Ld652Y cell line transcriptome comprises diverse virus-associated transcripts. - Viruses 3: 2339-2350. Go to original source...
  31. Sparks M.E., Blackburn M.B., Kuhar D. & Gundersen-Rindal D.E. 2013: Transcriptome of the Lymantria dispar (gypsy moth) larval midgut in response to infection by Bacillus thuringiensis. - PLoS one 8(5): e61190, 9 pp. Go to original source...
  32. Suetsugu Y., Futahashi R., Kanamori H., Kadono-Okuda K., Sasanuma S.-I., Narukawa J., Ajimura M., Jouraku A., Namiki N., Shimomura M. et al. 2013: Large scale full-length cDNA sequencing reveals a unique genomic landscape in a lepidopteran model insect, Bombyx mori. - G3 (Bethesda) 3: 1481-1492.
  33. Terenius O., Papanicolaou A., Garbutt J.S., Eleftherianos I., Huvenne H., Kanginakudru S., Albrechtsen M., An C., Aymeric J.-L., Barthel A. et al. 2011: RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design. - J. Insect Physiol. 57: 231-245. Go to original source...
  34. Tian H., Peng H., Yao Q., Chen H., Xie Q., Tang B. & 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(7): e6225, 13 pp. Go to original source...
  35. Timmons L. & Fire A. 1998: Specific interference by ingested dsRNA. - Nature 395: 854. Go to original source...
  36. Timmons L., Court D.L. & Fire A. 2001: Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. - Gene 263: 103-112. Go to original source...
  37. Turner C.T., Davy M.W., MacDiarmid R.M., Plummer K.M., Birch N.P. & Newcomb R.D. 2006: RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding. - Insect Mol. Biol. 15: 383-391. Go to original source...
  38. Wang K., Peng Y., Pu J., Fu W., Wang J. & Han Z. 2016: Variation in RNAi efficacy among insect species is attributable to dsRNA degradation in vivo. - Insect Biochem. Mol. Biol. 77: 1-9. Go to original source...
  39. Winston W.M., Molodowitch C. & Hunter C.P. 2002: Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. - Science (New York) 295: 2456-2459. Go to original source...
  40. Wynant N., Santos D., Verdonck R., Spit J., Van Wielendaele P. & Vanden Broeck J. 2014: Identification, functional characterization and phylogenetic analysis of double stranded RNA degrading enzymes present in the gut of the desert locust, Schistocerca gregaria. - Insect Biochem. Mol. Biol. 46: 1-8. Go to original source...
  41. Xiao D., Gao X., Xu J., Liang X., Li Q., Yao J. & Zhu K.Y. 2015: Clathrin-dependent endocytosis plays a predominant role in cellular uptake of double-stranded RNA in the red flour beetle. - Insect Biochem. Mol. Biol. 60: 68-77. Go to original source...
  42. Yoon J.-S., Shukla J.N., Gong Z.J., Mogilicherla K. & Palli S.R. 2016: RNA interference in the Colorado potato beetle, Leptinotarsa decemlineata: Identification of key contributors. - Insect Biochem. Mol. Biol. 78: 78-88. Go to original source...
  43. Yu N., Christiaens O., Liu J., Niu J., Cappelle K., Caccia S., Huvenne H. & Smagghe G. 2012: Delivery of dsRNA for RNAi in insects: an overview and future directions. - Insect Sci. 20: 4-14. Go to original source...
  44. Zamore P.D. 2001: RNA interference: listening to the sound of silence. - Nature Struct. Biol. 8: 746-750. Go to original source...
  45. Zhang X., Zhang J. & Zhu K.Y. 2010: Chitosan/double-stranded RNA nanoparticle-mediated RNA interference to silence chitin synthase genes through larval feeding in the African malaria mosquito (Anopheles gambiae). - Insect Mol. Biol. 19: 683-693. Go to original source...