| Mosquito-transmitted disease like malaria and dengue fever was a global problem and an estimated 50～100 millions of dengue fever cases were reported worldwide every year. The mermithid nematode Romanomermis wuchangensis has been successfully used as an ecological-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. Mermithidae usually get nutrients from the hemolymph of host during the parasitic stage, and the sex differentiation is related to the abundance of nutrients. Thus, study on sex differentiation genes of R. wuchangensis is helpful to elucidate molecular mechanism in the critical developmental period of the nematode, which can provide useful information for exploring efficiency monitoring and integrated pest management strategies of mosquito in the field.Based on transcriptome sequencing analysis of R. wuchangensis, Rwucmab-3, Rwuclaf-1 and Rwuctra-2 were cloned and used to investigate molecular regulatory function in sex differentiation. qRT-PCR results demonstrated (1) The expression level of Rwucmab-3 between male and female displayed obvious difference on the 3rd day of parasitic stage (p<0.05), which was earlier than Rwuclaf-1 and Rwuctra-2, highlighting sex differentiation process already started on the 3rd day of parasitic stage; (2) The expression level of Rwuclaf-1 between male and female nematode presented difference on the 5th day of parasitic stage for the first time (p<0.05), and the expression level of Rwuclaf-1 in both male and female nematodes were significantly higher than other developmental stages on the 1st day of late parasitic stage; (3) On the 1st day of the late parasitic stage, the expression level of Rwuctra-2 first presented differences between male and female nematodes (p< 0.05), and the expression level in female nematodes were significantly higher than other developmental stages.Besides, FITC was used as a marker to test the uptake efficiency of R. wuchangensis’s cuticle. The fluorescence intensity increased with FITC concentration increasing after 16 h incubation, indicating this nematode can successfully ingest soaking solution via the cuticle. RNAi results revealed the sex ratio of R. wuchangensis from RNAi treated groups soaked in dsRNA of Rwucmab-3 was significantly higher than gfpdsRNA treated groups and control groups (p<0.01), highlighting RNAi of Rwucmab-3 may hinder the nematode develop into male. RNAi results of Rwuclaf-1 and Rwuctra-2 from R. wuchangensis demonstrated a slight, but statistically insignificant increase or decrease in sex ratio with both of control groups (P>0.05). One possible explanation is that the complex interactions between sex differentiation genes may influence the RNAi effects. Another possible explanation is the length and position of Rwuclaf-1 and Rwuctra-2 dsRNA in target gene are improper, which can also affect the RNAi efficiency of the nematodes.To further explore the tissue localization of Rwucmab-3, in situ hybridization was carried out on post-parasitic stage R.wuchangensis. The results of in situ hybridization showed that Rwucmab-3 mRNA was present in the oocytes of females, indicating Rwucmab-3 also involved in oogenesis in post-parasitic stage R. wuchangensis, which was consistent with research in Mus musculus and Drosophila melanogaster.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 three key sex differentiation genes are helpful for large-scale cultivation of R. wuchangensis in vitro and field application to control the larvae of Culex quinquefasciatus and Aedes aegypti in water environment. |
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