有效的RNA干扰(RNAi)方法已经在许多害虫物种中得到发展,探索基因功能。到目前为止,还没有在猫跳蚤中尝试RNAi,Ctenocephalidesfelis,尽管RNAi方法的发展将开辟控制这种重要害虫的潜在途径。这项研究旨在确定在暴露于双链RNA(dsRNA)的成年C.felis中是否发生RNAi反应,用于dsRNA递送的施用方法可以带来有效的基因敲低并研究任何RNAi应答的动力学。通过血头腔内微量注射dsGSTσ可以实现80%的GSTσ击倒,但这种侵入性技术与相对较高的死亡率相关。将C.felis浸入dsGSTσ或dsDicer-2过夜分别导致GSTσ或Dicer-2的65%敲低,并且敲低的程度没有通过增加沐浴液中的dsRNA浓度而得到改善。出乎意料的是,通过膜饲喂系统在全血中连续施用dsRNA,实现了最大程度的敲除,在2天内导致96%的GSTσ击倒,并持续到,至少,7天因此,与许多其他物种不同,肠核酸酶不会损害对C.felis中摄入的dsRNA的RNAi应答。一个谦虚的,但意义重大,暴露于外源dsRNA后3小时可检测到Dicer-2和Argonaute2的上调,涉及短干扰RNA途径。据我们所知,这项研究代表了猫跳蚤中实验诱导的RNAi的首次证明,以及深入了解基因敲除反应的进展。
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.