Location: Mosquito and Fly ResearchTitle: Reproducible dsRNA microinjection and oviposition bioassay in mosquitoes and house flies
|WAITS, CHRISTY - Navy Entomology Center Of Excellence, Cmave Detachment|
|Geden, Christopher - Chris|
|ESTEP, ALDEN - Navy Entomology Center Of Excellence, Cmave Detachment|
Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/5/2018
Publication Date: 11/8/2018
Citation: Sanscrainte, N.D., Waits, C.M., Geden, C.J., Estep, A.S., Becnel, J.J. 2018. Reproducible dsRNA microinjection and oviposition bioassay in mosquitoes and house flies. Journal of Visualized Experiments. (141):e58650. https://doi.org/10.3791/58650.
Interpretive Summary: Scientists at the Center for Medical, Veterinary and Agricultural Entomology and collaborators have developed a protocol that describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids or other chemicals directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.
Technical Abstract: Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds. Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe & Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts. This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of the most effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.