Hormonal regulation of microRNA expression dynamics in the gut of the yellow fever mosquito <i>Aedes aegypti</i>

The yellow fever mosquito <i>Aedes aegypti</i> is an obligatory blood feeder and a major arboviral disease vector, evoking severe public health concerns worldwide. In adult female mosquitoes, the gut is critical for blood digestion and pathogen entry. We aimed for a systematic exploration of microRNA expression dynamics in the gut during the gonadotrophic cycle. Small RNA libraries were constructed from female mosquito gut tissues at five time points. Unsupervised hierarchical clustering revealed three expression clusters (early, mid and late) peaking at sequential time points – 24, 48 and 72 h posteclosion. Differentially expressed miRNAs were identified at 24 h post-blood meal (PBM). Depletions of Methoprene-tolerant [Met; the juvenile hormone (JH) receptor] and Ecdysone receptor [EcR; the receptor to 20-hydroxyecdysone (20E)] were performed using dsRNA to these genes to investigate impacts on microRNA expressions. Our results suggest that Met-mediated signalling downregulates miRNA expression from the early cluster and upregulates that from the late cluster. EcR signalling either up- or downregulated miRNA levels at 24 h PBM, indicating a differential effect of this receptor in miRNA gene expression. Furthermore, miR-281, which is the most abundant miRNA in the gut tissue, is induced and repressed by Met- and EcR-mediated signalling, respectively. Systematic depletion using synthetic antagomir and phenotype examinations indicate that miR-281 is obligatory for the normal progression of blood digestion, ovarian development and reproduction. Collectively, this study unveils expression dynamics of microRNAs in the female gut tissue during the gonadotrophic cycle and demonstrates that they are affected by JH and 20E signalling.

黄热病蚊（yellow fever mosquito）埃及伊蚊（Aedes aegypti）是专性吸血昆虫，亦是主要的虫媒病毒病传播媒介，在全球范围内引发严峻的公共卫生关切。对于成年雌性蚊子而言，肠道是血液消化与病原体入侵的关键组织。本研究旨在系统探究生殖周期（gonadotrophic cycle）中雌性伊蚊肠道内微小RNA（microRNA，miRNA）的表达动态。研究人员于五个时间点采集雌性伊蚊肠道组织，构建了小RNA文库。无监督层次聚类分析显示，存在三个表达簇（早期、中期与晚期），其表达峰值分别出现在羽化后24、48及72小时。在吸血后24小时（post-blood meal，简称PBM），研究人员鉴定得到了差异表达的miRNAs。本研究针对这两个基因设计双链RNA（double-stranded RNA，简称dsRNA），分别敲低保幼激素受体（juvenile hormone receptor，JH受体）Methoprene-tolerant（Met）以及蜕皮激素受体（Ecdysone receptor，EcR，即20-羟基蜕皮激素（20-hydroxyecdysone，20E）的受体）的表达，以探究二者对miRNA表达的调控作用。研究结果显示，Met介导的信号通路可下调早期表达簇的miRNA水平，并上调晚期表达簇的miRNA水平。EcR信号通路在吸血后24小时可上调或下调miRNA的表达水平，提示该受体对miRNA基因的表达存在差异化调控效应。此外，肠道组织中丰度最高的miR-281，分别受到Met和EcR介导的信号通路的诱导与抑制。通过合成的miRNA拮抗剂（antagomir）进行系统性敲低，并结合表型检测实验，结果表明miR-281对于血液消化、卵巢发育及繁殖的正常进程是必需的。综上，本研究揭示了雌性伊蚊肠道在生殖周期内miRNA的表达动态，并证实其表达受保幼激素（juvenile hormone，JH）与20-羟基蜕皮激素（20E）信号通路的调控。