RNA interference is widely used to analyze gene functions via phenotypic knockdown of target transcripts in mosquitoes, which transmit numerous mosquito‐borne diseases. Functional analysis of mosquito genes is indispensable to understand and reduce transmission of mosquito‐borne diseases in mosquitoes. Intrathoracic injection of double‐stranded RNA (dsRNA) remains the simplest and most customizable method in mosquitoes for functional analysis of the genes of interest. However, achieving consistent and effective knockdown by dsRNAi is often elusive and may require extensive optimization. We tested the effectiveness of gene silencing by intrathoracic injection of four different quantities of dsRNA targeting two Aedes aegypti genes, cysteine desulfurylase (Nfs1) and short‐chain dehydrogenase (SDH). We found that Nfs1 gene has a lower expression level upon silencing than SDH gene. In the case of the gene that is easier to silence, Nfs1 gene expression was significantly silenced by all four tested quantities of dsRNA up to 21 days post infection (d.p.i.), but silencing of SDH, the gene that is difficult to silence, was less effective, with knockdown lasting up to 9 d.p.i. only when 1,000 ng of dsRNA was used. Based on our observation, intrathoracic injection of 500 ng of dsRNAs per mosquito is recommended to achieve effective knockdown for well‐silenced transcripts such as Nfs1 for up to 3 weeks. This includes most in vivo bioassays involving arboviral infections in Ae. aegypti. The estimated quantities of dsRNA described in this study should be applicable to most Ae. aegypti dsRNAi studies and thus provide a guideline to develop efficient dsRNAi in other experimental investigations.

中文翻译：

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埃及伊蚊双链RNAi胸腔内注射方法的优化

RNA 干扰被广泛用于通过蚊子中目标转录本的表型敲低来分析基因功能，蚊子会传播多种蚊媒疾病。蚊子基因的功能分析对于了解和减少蚊子传播疾病在蚊子中的传播是必不可少的。胸腔内注射双链 RNA (dsRNA) 仍然是蚊子中用于目标基因功能分析的最简单和最可定制的方法。然而，通过 dsRNAi 实现一致和有效的击倒通常是难以捉摸的，可能需要广泛的优化。我们通过胸腔内注射四种不同数量的 dsRNA 来测试基因沉默的有效性，这些 dsRNA 靶向两种埃及伊蚊基因，半胱氨酸脱硫酶 (Nfs1) 和短链脱氢酶 (SDH)。我们发现 Nfs1 基因在沉默时的表达水平低于 SDH 基因。在更容易沉默的基因的情况下，Nfs1 基因表达在感染后 21 天 (dpi) 内被所有四种测试量的 dsRNA 显着沉默，但难以沉默的基因 SDH 的沉默较少有效，仅当使用 1,000 ng dsRNA 时，击倒持续时间长达 9 dpi。根据我们的观察，建议每只蚊子胸腔内注射 500 ng dsRNA，以实现对良好沉默的转录本（如 Nfs1）的有效敲低长达 3 周。这包括大多数涉及 Ae 虫媒病毒感染的体内生物测定。埃及人。本研究中描述的 dsRNA 估计量应适用于大多数 Ae。