RNA interference in the cat flea, Ctenocephalides felis: Approaches for sustained gene knockdown and evidence of involvement of Dicer-2 and Argonaute2.
RNA interference in the cat flea, Ctenocephalides felis: Approaches for sustained gene knockdown and evidence of involvement of Dicer-2 and Argonaute2.
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DOI：
10.1016/j.ijpara.2018.04.006
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发表时间：
2018-11
影响因子：
4
通讯作者：
Bowman AS
中科院分区：
文献类型：
--
作者：
Edwards CH;Baird J;Zinser E;Woods DJ;Shaw S;Campbell EM;Bowman AS
首次在跳蚤，猫蚤（Ctenocephalides felis）中发现基因敲低，通过浸泡在双链（ds） RNA溶液中进行强烈的瞬时基因敲低。通过持续喂食血液中的dsRNA，强而持续的基因敲低。dsRNA处理诱导Dicer-2和Argonaute-2基因表达增加。基因敲除方法对农药和病原体在跳蚤中的传播研究是有用的。有效的RNA干扰（RNAi）方法已经在许多害虫物种中发展起来，使基因功能的探索成为可能。尽管RNAi方法的发展将为控制这种重要害虫开辟潜在的途径，但到目前为止，RNAi还没有在猫蚤中进行过尝试。本研究旨在确定成年猫C. felis暴露于双链RNA （dsRNA）后是否会发生RNAi反应，哪种给药方法可以有效地敲低dsRNA基因，并研究任何RNAi反应的动力学。通过血腔内显微注射dsGSTσ可降低80%的GSTσ，但这种侵入性技术与较高的死亡率相关。将C. felis浸泡在dsGSTσ或dsDicer-2中过夜，GSTσ和Dicer-2分别被下调65%和60%，并且随着浸泡液中dsRNA浓度的增加，下调程度并没有提高。出乎意料的是，通过膜喂养系统在全血中连续给药dsRNA实现了最大程度的下调，导致GSTσ在2 天内下调96%，并持续至少7 天。因此，与许多其他物种不同，肠核酸酶不会损害猫绒猴对摄入的dsRNA的RNAi反应。暴露于外源性dsRNA后3 h检测到Dicer-2和Argonaute2适度但显著上调，暗示短干扰RNA途径。据我们所知，这项研究首次展示了猫跳蚤中实验诱导的RNAi，并深入了解了基因敲低反应的进展。
First known demonstration of gene knockdown in a flea, the cat flea (Ctenocephalides felis) Strong, transient gene knockdown by immersion in double-stranded (ds) RNA solution. Strong, sustained gene knockdown by continuous feeding of dsRNA in blood. dsRNA treatment induces an increase in Dicer-2 and Argonaute-2 gene expression. The knockdown approach is useful for pesticide and pathogen transmission studies in fleas. Effective RNA interference (RNAi) methods have been developed in many pest species, enabling exploration of gene function. Until now RNAi had not been attempted in the cat flea, Ctenocephalides felis, although the development of RNAi approaches would open up potential avenues for control of this important pest. This study aimed to establish if an RNAi response occurs in adult C. felis upon exposure to double-stranded RNA (dsRNA), which administration methods for dsRNA delivery could bring about effective gene knockdown and to investigate dynamics of any RNAi response. Knockdown of 80% of GSTσ was achieved by intrahaemoceolic microinjection of dsGSTσ but this invasive technique was associated with relatively high mortality rates. Immersing C. felis in dsGSTσ or dsDicer-2 overnight resulted in 65% knockdown of GSTσ or 60% of Dicer-2, respectively, and the degree of knockdown was not improved by increasing the dsRNA concentration in the bathing solution. Unexpectedly, the greatest degree of knockdown was achieved with the continuous administration of dsRNA in whole blood via a membrane feeding system, resulting in 96% knockdown of GSTσ within 2 days and sustained up to, at least, 7 days. Thus, unlike in many other species, the gut nucleases do not impair the RNAi response to ingested dsRNA in C. felis. A modest, but significant, upregulation of Dicer-2 and Argonaute2 was detectable 3 h after exposure to exogenous dsRNA, implicating the short-interfering RNA pathway. To our knowledge this study represents the first demonstration of experimentally induced RNAi in the cat flea as well as giving insight into how the gene knockdown response progresses.
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影响因子：
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