Non-Invasive Delivery of dsRNA into De-Waxed Tick Eggs by Electroporation

PLoS One. 2015 Jun 19;10(6):e0130008. doi: 10.1371/journal.pone.0130008. eCollection 2015.

Abstract

RNA interference-mediated gene silencing was shown to be an efficient tool for validation of targets that may become anti-tick vaccine components. Here, we demonstrate the application of this approach in the validation of components of molecular signaling cascades, such as the Protein Kinase B (AKT)/Glycogen Synthase Kinase (GSK) axis during tick embryogenesis. It was shown that heptane and hypochlorite treatment of tick eggs can remove wax, affecting corium integrity and but not embryo development. Evidence of AKT and GSK dsRNA delivery into de-waxed eggs of via electroporation is provided. Primers designed to amplify part of the dsRNA delivered into the electroporated eggs dsRNA confirmed its entry in eggs. In addition, it was shown that electroporation is able to deliver the fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI). To confirm gene silencing, a second set of primers was designed outside the dsRNA sequence of target gene. In this assay, the suppression of AKT and GSK transcripts (approximately 50% reduction in both genes) was demonstrated in 7-day-old eggs. Interestingly, silencing of GSK in 7-day-old eggs caused 25% reduction in hatching. Additionally, the effect of silencing AKT and GSK on embryo energy metabolism was evaluated. As expected, knockdown of AKT, which down regulates GSK, the suppressor of glycogen synthesis, decreased glycogen content in electroporated eggs. These data demonstrate that electroporation of de-waxed R. microplus eggs could be used for gene silencing in tick embryos, and improve the knowledge about arthropod embryogenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arthropod Proteins / genetics
  • Arthropod Proteins / metabolism
  • Electroporation
  • Female
  • Gene Expression
  • Gene Knockdown Techniques*
  • Glycogen Synthase Kinases / genetics
  • Glycogen Synthase Kinases / metabolism
  • Heptanes / chemistry
  • Ovum / chemistry
  • Ovum / physiology
  • Proto-Oncogene Proteins c-akt / genetics
  • Proto-Oncogene Proteins c-akt / metabolism
  • RNA Interference
  • RNA, Double-Stranded / genetics*
  • Rhipicephalus / genetics*
  • Solvents / chemistry
  • Tissue Culture Techniques
  • Waxes / chemistry

Substances

  • Arthropod Proteins
  • Heptanes
  • RNA, Double-Stranded
  • Solvents
  • Waxes
  • Glycogen Synthase Kinases
  • Proto-Oncogene Proteins c-akt

Grants and funding

This work was supported by grants from Conselho Nacional de Desenvolvimento Científico (CNPq, INCT de Entomologia Molecular), Fundação de Coordenação de Aperfeiçoamento do Pessoal de Nível Superior (CAPES)and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). The funders had no role in study design, data colletion and analysis, decision to publish, or preparation of the manuscript.