Transcriptome Sequencing Analysis and Functional Identification of Sex Differentiation Genes from the Mosquito Parasitic Nematode, Romanomermis wuchangensis

Mosquito-transmitted diseases like malaria and dengue fever are global problem and an estimated 50–100 million of dengue or dengue hemorrhagic fever cases are reported worldwide every year. The mermithid nematode Romanomermis wuchangensis has been successfully used as an ecosystem-friendly biocontrol agent for mosquito prevention in laboratory studies. However, this nematode can not undergo sex differentiation in vitro culture, which has seriously affected their application of biocontrol in the field. In this study, based on transcriptome sequencing analysis of R. wuchangensis, Rwucmab-3, Rwuclaf-1 and Rwuctra-2 were cloned and used to investigate molecular regulatory function of sex differentiation. qRT-PCR results demonstrated that the expression level of Rwucmab-3 between male and female displayed obvious difference on the 3rd day of parasitic stage, which was earlier than Rwuclaf-1 and Rwuctra-2, highlighting sex differentiation process may start on the 3rd day of parasitic stage. Besides, FITC was used as a marker to test dsRNA uptake efficiency of R. wuchangensis, which fluorescence intensity increased with FITC concentration after 16 h incubation, indicating this nematode can successfully ingest soaking solution via its cuticle. RNAi results revealed the sex ratio of R. wuchangensis from RNAi treated groups soaked in dsRNA of Rwucmab-3 was significantly higher than gfp dsRNA treated groups and control groups, highlighting RNAi of Rwumab-3 may hinder the development of male nematodes. These results suggest that Rwucmab-3 mainly involves in the initiation of sex differentiation and the development of male sexual dimorphism. Rwuclaf-1 and Rwuctra-2 may play vital role in nematode reproductive and developmental system. In conclusion, transcript sequences presented in this study could provide more bioinformatics resources for future studies on gene cloning and other molecular regulatory mechanism in R. wuchangensis. Moreover, identification and functional analysis of sex differentiation genes may clarify the sex differentiation mechanism of R. wuchangensis, which are helpful to solve the uncompleted sex differentiation problem in vitro culture and the potential large-scale field application controlling the larvae of C. quinquefasciatus, A. aegypti and A. albopictus.疟疾、登革热等蚊媒传染病是全球性公共卫生问题，据估计全球每年报告的登革热及登革出血热病例达5000万至1亿例。武昌罗索线虫（Romanomermis wuchangensis）作为一种生态友好型生物防治剂，已在实验室研究中成功应用于蚊虫防治。然而，该线虫在体外培养条件下无法完成性别分化，这严重制约了其在田间生物防治中的应用。本研究基于武昌罗索线虫的转录组测序分析，克隆得到Rwucmab-3、Rwuclaf-1及Rwuctra-2三个基因，并对其在性别分化中的分子调控功能展开研究。实时荧光定量PCR（qRT-PCR）结果显示，在寄生期第3天时，Rwucmab-3的雌雄表达量差异最为显著，早于Rwuclaf-1和Rwuctra-2，提示性别分化过程可能始于寄生期第3天。此外，本研究以异硫氰酸荧光素（FITC）为标记物，检测武昌罗索线虫对双链RNA（dsRNA）的摄取效率：孵育16小时后，线虫的荧光强度随FITC浓度升高而增强，表明该线虫可通过体壁成功摄取浸泡液。RNA干扰（RNAi）实验结果显示，经Rwucmab-3的dsRNA浸泡处理组的武昌罗索线虫性别比例显著高于绿色荧光蛋白（GFP）dsRNA处理组及空白对照组，提示靶向Rwucmab-3的RNAi可能会阻碍雄线虫的发育。上述结果表明，Rwucmab-3主要参与性别分化的启动以及雄性二态性的发育过程。Rwuclaf-1与Rwuctra-2则可能在线虫的生殖与发育系统中发挥关键作用。综上，本研究获得的转录组序列可为未来武昌罗索线虫的基因克隆及其他分子调控机制研究提供更多生物信息学资源。此外，对性别分化基因的鉴定与功能分析，可阐明武昌罗索线虫的性别分化机制，有助于解决其体外培养中性别分化不完全的问题，并推动其在田间大规模防治致倦库蚊（Culex quinquefasciatus）、埃及伊蚊（Aedes aegypti）及白纹伊蚊（Aedes albopictus）幼虫的潜在应用。