Sex-Biased PiRNA And Its Roles In Ovarian Development Of Aedes Albopictus

Background:Aedes albopictus is currently considered as one of the most invasive and aggressive species in the world,and is the vector of Zika virus(ZIKV),Dengue virus(DENV),and yellow fever virus(YFV)and other arboviruses.With typical sexual dimorphism characteristics,females are the main protagonists of blood-sucking and disease transmission.Only female mosquitoes can develop and lay eggs after blood meals,and the population can reproduce.Therefore,understanding the regulatory mechanism of ovarian development after mosquito blood meal is not only beneficial to our in-depth understanding of mosquito physiology and embryonic development,but also can provide new targets for the application of female mosquito infertility technology in mosquito control.Piwi-interacting RNAs(piRNAs)are a class of newly discovered small non-coding RNAs(sncRNAs)with a length of 26-32 nt in 2006,most of which are concentrated in 29-30 nt.The 5’ terminal base has a strong bias to uridine(U),the 3’terminal has 2’-O-methyl modification,and the tenth position has a strong bias to adenosine(A),which can bind to PIWI protein to form piRNA complex(piRC)to perform its biological function.Currently,in eukaryotes,more and more studies have demonstrated that non-coding RNAs play a key role in sexually dimorphic development,especially piRNAs are extensively involved in pre-transcriptional and post-transcriptional gene regulation and have been confirmed to be closely related to reproduction.Ae.albopictus is an important vector of arbovirus.Our previous studies have shown that Ae.albopictus has a huge genome with a large number of piRNAs.However,studies on the functions of piRNAs related to the regulation of sexual dimorphism such as female ovarian development in mosquitoes is relatively delayed at present.Exploring the molecular regulatory mechanism of piRNA in Ae.albopictus will not only helps us to further understand the molecular regulatory network of mosquito ovarian development,but also provide new targets and methods for the application of mosquito infertility technology in mosquito vector control.Objective:To explore the sex-biased expression pattern of piRNAs in Ae.albopictus,focusing on the function of female piRNAs highly expressed in the molecular regulatory network of mosquito ovarian development and embryonic development.By screening female with high expression,in-depth study of their functions,understanding the effects of piRNAs on ovarian and embryonic development,and further study of their internal molecular mechanisms can provide new targets for the prevention and treatment of mosquito infertility,and provide new ideas for the development of new mosquito control strategies,which has important social and economic significance.Methods:1.The virgin and bloodless adult female and male Ae.albopictus after emergence were collected.The samples were sequenced by sRNA high-throughput sequencing method.The piRNAs were predicted by bioinformatics method,and the piRNAs with male and female differences were screened and verified by stem-loop RT-PCR and qRT-PCR to identify candidate piRNAs.2.The tissues of Ae.albopictus at different developmental stages,female mosquito tissues and ovarian tissues at different time points after blood meal were dissected and collected.The expression of piRNA in the ovaries of each developmental stage and tissue,and the distribution of piRNA in the cytoplasm and nucleus of ovarian cells was detected by qPCR.The correlation between piRNA and PIWI protein was detected by RNAi and RIP-qPCR.3.Through overexpression or inhibition,it was verified that overexpression or interference significantly affected the phenotype of piRNA in the ovarian development of female mosquitoes after blood meal.The female mosquito samples of Ae.albopictus mosquitoes after overexpression or inhibition of piRNA were collected for transcriptome sequencing analysis of relevant target genes.The target gene prediction database RNAhybrid was used to predict the binding sites of the candidate piRNA target genes.The relationship with the target genes was verified by RNAi and dual luciferase reporter gene activity experiments.Results:1.The analysis of RNA sequencing results showed a sex-biased expression pattern of piRNAs in Ae.albopictus.The spatio-temporal expression profile showed that Aalpi-18529 was enriched in the ovary of female mosquitoes,and the expression level began to decline continuously after blood meal,indicating that it had a potential correlation with the ovarian development after blood meal in female mosquitoes.RNAi and RIP-qPCR experiments of AGO3,PIWI5 and VASA proteins in the Ven body protein complex associated with piRNAs generation confirmed that the generation of Aalpi-18529 depends on PIWI5,AGO3 and VASA,and directly binds to PIWI5,which is a classic piRNA amplification via the ping-pong cycle.2.Compared with the negative control group(NC group)and the blank control group(WT group),overexpression of Aalpi-18529 inhibited the ovarian development of female mosquitoes,with a reduction of about 15 follicles per female and a significant reduction of about 75 μm in average follicle size(length of the long axis).At the same time,overexpression of Aalpi-18529 reduced daily mean number of eggs production of per female by about 20%,the mean cumulative egg number laid by per Ae.albopictus adults was significantly lower than that of the WT group and the NC group,and the hatching rate of eggs was also reduced by about 30%.3.Overexpression of Aalpi-18529 was able to reduce the level of growth arrest and DNA damage-induced 45 alpha(GADD45α)gene in ovary,and dual luciferase assay showed that Aalpi-18529 could directly act on GADD45α and inhibit its expression.When the GADD45α was interfered in Ae.albopictus female mosquito,it was basically consistent with the ovarian phenotype after overexpression of Aalpi-18529:the ovarian development of some female mosquitoes was inhibited,the number of developing follicles was reduced,the length and diameter of follicles were significantly reduced,and the egg production and hatching rate were significantly reduced.Therefore,GADD45α protein is a direct target gene of Aalpi-18529.Conclusions:Female highly expressed Aalpi-18529 could specifically bind to GADD45α gene and regulate its expression at the post-transcriptional level,which in turn affected the postprandial ovarian development of Ae.albopictus,laying a foundation for further research on the function and molecular mechanism of mosquito piRNA,providing new ideas for finding potential targets for mosquito control,and having great significance for the development of new mosquito vector control strategies.