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Abstract

Biology

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

Published: March 1st, 2022

DOI:

10.3791/63266

1Division of Parasitic Diseases and Malaria, Entomology Branch, Centers for Disease Control and Prevention, 2Department of Entomology, Cornell University, 3Centro de Estudios en Biotecnologia, Universidad del Valle de Guatemala, 4Department of Cell Biology, Johns Hopkins School of Medicine, 5Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, 6Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health and Malaria Research Institute, 7Department of Cell Biology, Johns Hopkins School of Medicine, 8Biomedical Sciences Department, Idaho College of Osteopathic Medicine

Abstract

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

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Keywords RNA Interference

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