Abstract: Objective　To investigate the role of angiotensin⁃converting enzyme (ACE) in the reproduction of Culex pipiens pallens, so as to provide insights into selection of targets for controlling mosquito vector populations. Methods　Cx. pipiens pallens was collected from Tangkou County, Shandong Province in 2009. Female and male mosquitoes were selected at 72 hours post⁃eclosion, and quantitative real⁃time reverse transcription PCR (qPCR) assay was used to detect the expression of ACE gene in the whole body and reproductive tissues of male mosquitoes and fertilized female mosquitoes before (0 h) and after blood meals (24, 48, 72 h), respectively. Then, 150 female and 150 male mosquitoes at 0 to 4 hours post⁃eclosion were selected and divided into the wild⁃type group (WT group), small interfering RNA⁃negative control group (siNC group) and small interfering RNA⁃ACE group (siACE group), of 50 mosquitoes in each group. Mosquitoes in the WT group were given no treatment, and mosquitoes in the siNC and siACE groups were given microinjection of siNC and siACE into the hemolymph at a dose of 0.3 μg per mosquito. The knockdown efficiency was checked using qPCR assay, and the reproductive phenotype of mosquitoes was observed. Results　The relative ACE gene expression was higher in the whole body of male mosquitoes (5.467 ± 1.006) relative to females (1.199 ± 0.241) (t = 5.835, P = 0.004) at 72 h post⁃eclosion, and the highest ACE expression was seen in reproductive tissues of male mosquitoes (199.100 ± 24.429), which was 188.3 times higher than in remaining tissues (1.057 ± 0.340) (t = 6.602, P = 0.002). Blood meal induced high ACE expression in all body tissues of fertilized female mosquitoes, with peak expression at 24 h after blood meals (14.957 ± 2.815), which was 14.8 times higher than that before blood meals (1.009 ± 0.139) (P = 0.002). The transcriptional level of ACEs continued to increase in the ovaries of female mosquitoes after blood meals during the vitellogenesis phase, peaking at 48 h after blood meals (5.500 ± 0.734), which was 5.1 times higher than that before blood meals (1.072 ± 0.178) (P = 0.002). Small RNA interference targeting ACE resulted in a 57.2% reduction in ACE expression in female mosquitoes in the siACE group (0.430 ± 0.070) relative to the siNC group (1.002 ± 0.070) (P = 0.001), and a 41.1% reduction in male mosquitoes in the siACE group (0.588 ± 0.067) relative to the siNC group (1.008 ± 0.131) (P = 0.016). Knockdown of ACE expression resulted in a 48.0% decrease in the number of eggs laid by female mosquitoes in the siACE group [(94.000 ± 27.386) eggs] relative to the siNC group [(180.800 ± 27.386)] (P < 0.001), and a 45.0% decrease in the number of eggs laid by wild female mosquitoes mated with males in the siACE group [(104.500 ± 20.965) eggs] relative to the siNC group [(190.050 ± 10.698) eggs] (P < 0.001). Conclusions　 Reduced ACE expression may inhibit the fecundity of male and female mosquitoes, and ACE may be as a potential target for mosquito vector population suppression.

Key words: Culex pipiens pallens, Angiotensin?converting enzyme, RNA interference, Reproduction

摘要： 目的　探讨血管紧张素转换酶（angiotensin⁃converting enzyme，ACE）在淡色库蚊（Culex pipiens pallens）生殖中的作用，为蚊媒种群控制靶标的选择提供科学依据。方法　淡色库蚊于2009年采集于山东省唐口县。选择羽化后72 h雌、雄蚊，采用实时荧光定量PCR（quantitative real⁃time reverse transcription PCR，qPCR）技术检测雄蚊及吸血前（0 h）、吸血后（24、48、72 h）受精雌蚊全身和生殖组织中ACE基因表达水平。随后，选择羽化0～4 h的雌、雄库蚊各150只，分别分为野生（wild type, WT）组、RNA干扰⁃阴性对照 （small interfering RNA⁃negative control group, siNC）组和RNA干扰⁃ACE (small interfering RNA⁃ACE group，siACE)组，每组50只。WT组淡色库蚊不作任何处理；siNC组和siACE组分别通过显微注射仪注射siNC和siACE至蚊血淋巴中，注射剂量为0.3 μg/只。利用qPCR技术验证各组敲低效率，并观察各组生殖表型。结果　羽化后72 h，雄蚊全身组织ACE表达量（5.467 ± 1.006）高于雌蚊（1.199 ± 0.241）（t = 5.835, P = 0.004）；雄蚊生殖组织中ACE表达量最高（199.100 ± 24.429），为剩余组织（1.057 ± 0.340）的188.3倍（t = 6.602，P = 0.002）。吸血触发ACE在受精雌蚊全身组织中高表达，吸血后24 h（14.957 ± 2.815）达到表达量高峰，为未吸血时（1.009 ± 0.139）的14.8倍（P = 0.002）；雌蚊吸血后卵巢中ACE转录水平在卵黄合成期持续升高，在吸血后48 h（5.500 ± 0.734）达高峰，是未吸血雌蚊（1.072 ± 0.178）的5.1倍（P = 0.002）。靶向ACE的小RNA干扰导致siACE组雌蚊中ACE表达量（0.430 ± 0.070）较siNC组（1.002 ± 0.070）降低57.2%（P = 0.001），siACE组雄蚊ACE表达量（0.588 ± 0.067）较siNC组（1.008 ± 0.131）降低41.1%（P = 0.016）。敲低ACE导致siACE组雌蚊产卵量[（94.000 ± 27.386）只]较siNC组[（180.800 ± 27.386）只]降低48.0%（P < 0.001）；与siACE组雄蚊交配的野生雌蚊产卵量[（104.500 ± 20.965）只]较siNC组[（190.050 ± 10.698）只]降低45.0%（P < 0.001）。 结论　ACE表达降低可同时抑制雌、雄蚊虫生殖力，ACE可作为蚊媒种群抑制的潜在靶点。

关键词: 淡色库蚊, 血管紧张素转换酶, RNA干扰, 生殖