| Objective: Through different climate zones of Aedes albopictus suffering the oral infection of Zika virus,the vertical transmission and transmission efficiency to ZIKV of Aedes albopictus are clarified and the preservation and the ability to transmit ZIKV for Aedes albopictus diapause eggs is determined,to explore the role of vector mosquito in a long-distance spread of virus and the preservation mechanism of maintaining Zika virus survival during non-epidemic periods in nature;After the analysis of transcriptome mRNA expression profiles and microRNA expression profiles of diapause eggs and non-diapause eggs after ZIKV infection,the molecular mechanism of viral infection and diapause in Aedes albopictus was explored,providing new ideas for finding noval vector control targets and strategies.Methods:1.The transmission efficiency of different geographic strains of Aedes albopictus against ZIKV was tested: three geographic strains of Aedes albopictus collected from the temperate zone,northern subtropical and southern subtropical zones were infected with oral ZIKV and dissected on 4d,7d,10 d,14d post-infection(dpi).Midguts,salivary glands and ovaries,and saliva are collected for extracted RNA,and the infection rate and viral load of tissues are detected by fluorescence real-time quantitative PCR(qRT-PCR);saliva is collected and inoculated into C6/36 cells to observe cytopathic effect.2.Study on the transmission of ZIKV from Aedes albopictus of different geographic strains via diapause eggs: detection of the diapause incidence and hatching rate of eggs from the first and second gonotrophic cycle under short-day photoperiod and long-day photoperiod;The ZIKV infection of offspring(eggs,larvae,adults)and the transmission experiment of F1 adult mosquito bitten baby mice after the diapause was terminated by stimulating diapause eggs with hypoxic water.3.The transcriptome sequencing and Small RNA sequencing analysis of the diapause eggs of Aedes albopictus of different geographic strains in the early diapause,middle diapause and late diapause stages after ZIKV infection: Aedes albopictus simultaneously fed with the blood meal containing ZIKV was used as the ZIKV group and fed with the only blood meal as the control group.Diapause eggs in the early diapause,middle diapause and late diapause stages were collected under short-day photoperiod and long-day photoperiod,and total RNA was extracted to construct Small RNA library and mRNA library.Use RNA-seq sequencing technology and bioinformatics methods to analyze the expression profiles of miRNA and mRNA in both ZIKV group and control group,and conduct joint analysis of miRNA and mRNA expression profiles to construct a miRNA-mRNA regulatory network and choose key miRNAs and target genes associated with ZIKV infection and diapause.4.Verification by qRT-PCR: Data of Small RNA and RNA-Seq sequencing was verified by qRT-PCR.Result:1.Detection of the vector competence of Aedes albopictus of different geographic strains after oral ZIKV infection: on 4d,7d,10 d,14d post-infection,the infection rate(IR)and dissemination rate(DR)and transmission rate(TR)were increasing over time.The TR of Aedes albopictus of different geographic strains failed to detect ZIKV on 4 dpi.The TR of Aedes albopictus from Beijing strain was lower than the TR of the Hangzhou strain and the Guangzhou strain of Aedes albopictus,and there was a significant difference on 7 dpi(Fisher's exact tests,P < 0.05);the IR trends of the three Aedes albopictus strains were the same,and there was no significant difference at any timepoint(P > 0.05);After 10 dpi,the infection rate of salivary glands from the Beijing strain Aedes albopictus was significantly higher than the infection rates of which from the Hangzhou strain and Guangzhou strain(P < 0.05).The saliva,midgut,salivary glands and ovaries of Aedes albopictus gradual of three geographic strains showed an increase in viral loads.ZIKV was not detected in the salivary glands on 4 dpi,and on 10 dpi,the ZIKV copy number of in the saliva of Aedes albopictus from the Hangzhou and Guangzhou strains was significantly higher than that of Aedes albopictus from the Beijing strain(P < 0.05);The ZIKV copy number in the midguts of the three geographic strains of Aedes albopictus had no significant difference at any timepoint(P > 0.05);The ZIKV copy numbers in salivary glands of three geographic strains of Aedes albopictus had significant difference on 4 dpi and 14 dpi after infection(P < 0.05),indicating that ZIKV was effectively replicated in Aedes albopictus,but The ZIKV copy numbers of the salivary glands and ovaries of Aedes albopictus of the three geographic strains were not significantly different at the same time point(P > 0.05).2.Short-day photoperiod could induce Aedes albopictus to lay diapause eggs:Compared with the long-day photoperiod control group,the diapause incidence of Aedes albopictus eggs in the three geographical strains of Aedes albopictus under short-day photoperiod was significantly higher.There was a significant difference(P <0.05);The diapause incidence of the three geographical strains of Aedes albopictus in the short-day photoperiod gradually increased from 7 dpov to 21 dpov,and tended to flat and reached the peak on 40 dpov.The diapause incidences of the diapause eggs of the same geographic strain of Aedes albopictus were significant between the early diapause and late diapause stages(P <0.05).The diapause incidence of Aedes albopictus from the Beijing strain was higher than that of Aedes albopictus from the Guangzhou strain at any stage and the difference was significant(P < 0.05).The diapause incidence of Aedes albopictus from the Hangzhou strain and the Guangzhou strain had significant difference between 21 dpov in the middle diapause and 40 dpov in the late diapause(P < 0.05),but there was no significant difference in 11 dpov in early diapause(P > 0.05).3.The effect of light photoperiod on the hatch of diapause eggs of Aedes albopictus: Under short-day photoperiod,Aedes albopictus eggs from three geographic strains suffered the combined effect of low temperature and photoperiod,from 7 dpov to40 dpov,the hatching rate gradually decreased,and there was a significant difference between the early and late diapause(P < 0.05);The hatching rate of Aedes albopictus from the three geographic strains under the short-day photoperiod was significantly higher than that of the long-day photoperiod control group(P < 0.05).When comparing the hatching rate of Aedes albopictus from different geographic strains,the hatching rate(21.45%)of Aedes albopictus from the Beijing strain in the temperate zone was significantly higher than that(8.15%)of Aedes albopictus from the Guangzhou strain in the subtropical zone(P < 0.05)in the late diapause under short-day photoperiod.4.The diapause incidence of Aedes albopictus from different geographic strains after ZIKV infection: The diapause incidence of Aedes albopictus from the three geographic strains under short-day photoperiod was higher than that of Aedes albopictus under the long-day photoperiod control group.The diapause incidence of the diapause eggs of Aedes albopictus from the three geographic strains in late diapause was higher than that of the early diapause(P < 0.05);The diapause incidence of Aedes albopictus from the Beijing strain was significantly higher than that of Aedes albopictus from the Guangzhou strain(P < 0.05);The diapause incidences of Aedes albopictus from Hangzhou strain and Guangzhou strain were significantly different in the middle diapause and late diapause(P < 0.05).5.Hatching rate of Aedes albopictus from the three geographic strains after ZIKV infection: The average hatching rate of Aedes albopictus from the three geographic strains in the long-day photoperiod control group was more than 40% on 40 dpov,and the hatching rate of Aedes albopictus from three geographic strains was not significant(P > 0.05);The hatching rates of eggs were lower in the late diapause of short-day photoperiod condition because of the combination of low temperature and photoperiod.than hatching rate in the long-day photoperiod control group(P < 0.05);And the hatching rates of Aedes albopictus from Hangzhou strain and Guangzhou strain in the subtropical zone were significantly lower than that of Beijing strain(P < 0.05)under short-day photoperiod.Compared with the hatching rate in the uninfected group,the hatching rate of infected eggs was significantly reduced(P < 0.05).6.Infection rate of diapause eggs: Under short-day photoperiod,the trend of the MIRs of the second gonotrophic cycle of Aedes albopictus from the three geographic strains was similar,and there was no significant difference in MIR changes in early diapause 11 dpov and the late 40 dpov(P > 0.05).However,on 40 dpov in late diapause,the MIR of the eggs of Aedes albopictus from the Guangzhou strain was significantly higher than the MIR of Aedes albopictus from the Beijing strain the eggs of the strain(P< 0.05).The RNA copy number of eggs of Aedes albopictus from Hangzhou strain and Guangzhou strain was higher than that of Aedes albopictus from Beijing strain on 11 dpov in the long-day photoperiod control group(F = 15.60,P < 0.01);There was no significant difference in RNA copy number in the second gonotrophic cycle of Aedes albopictus from the three geographical strains in both the middle diapause and late diapause(P > 0.05).7.Detection of the vertical transmission to ZIKV of Aedes albopictus from different geographical strains: The Filial infection rates(FIRs)of the offspring Aedes albopictus of three Aedes albopictus strains were detected by qRT-PCR,and the results showed that the offspring of the second gonotrophic cycle of the Beijing Aedes albopictus under short-day photoperiod: The MIRs were 8/120 for the larvae and 7/120 for the adult;the MIRs of the FIR of the Hangzhou Aedes albopictus were 7/120 for the larva and 4/120 for the adult;the MIRs of the FIR of the offspring of the Guangzhou Aedes albopictus were 13 /120 for the larva,8/120 for the adult;FIR of progeny larvae of Guangzhou Aedes albopictus strain was significantly higher than that of Beijing Aedes albopictus strain and Hangzhou Aedes albopictus strain(P < 0.05),but there were no significant differences in MIRs for adult Aedes albopictus(P > 0.05).After inoculating to C6/36 cells with samples of adult mosquitoes and larvae of the second gonotrophic cycle of three Aedes albopictus strains caused C6/36 cells cytopathic effect;The brain tissues of suckling mice bitten after adult mosquitoes of the second gonotrophic cycle from the three Aedes albopictus strains were tested positive for ZIKV.8.mRNA sequencing analysis of diapause eggs of Aedes albopictus after ZIKV infection: A total of 20 differentially expressed genes were selected for qRT-PCR verification,and the results showed that the results of RNA-seq were consistent with qRT-PCR.The GO and KEGG enrichment analysis of differentially expressed genes revealed that some pathways related to diapause such as circadian rhythm and immune response and inflammatory response such as MAPK signaling pathway,and metabolism pathways such as Amino acid synthesis pathways and metabolism pathways,energy synthesis pathways and fat metabolism pathways,redox reaction pathways.Using Small RNA sequencing technology to select differentially expressed miRNAs,the results showed that through screening of GO functional annotations related to viral infection,multiple anti-viral related pathways were found,such as immune system process,transcription regulation activity,virus response;The signal pathway is in Toll signal pathway,MAPK signal pathway,cell cycle and so on.According to the enrichment of KEGG pathway and MAPK signaling pathway and Fc?R-mediated phagocytosis were enriched in target genes of diapause eggs after ZIKV infection,which were related to immune response and inflammatory response.9.miRNAs-mRNAs association analysis: A total of 49 miRNAs-mRNAs pairs were obtained from the differentially expressed miRNAs and mRNAs of infected Aedes albopictus diapause eggs with ZIKV,of which 24 pairs were up-regulated and 25 pairs were down-regulated.There were 10 pairs of novel discovered miRNAs-mRNAs pairs,and 5 pairs were up and down.Conclusion:1.The Beijing Aedes albopictus strain in the temperate zone,the Hangzhou Aedes albopictus strain in the northern subtropical zone and the Guangzhou Aedes albopictus strain in the southern subtropical zone can infect and transmit Zika virus,and the vector competence of the Guangzhou Aedes albopictus strain in the southern subtropical zone is higher than that of Aedes albopictus in Beijing in the temperate zone.2.Zika virus can survive in the diapause eggs of Aedes albopictus from Beijing in the temperate zone,Aedes albopictus from Hangzhou and Guangzhou in the subtropical zone.Zika virus can be vertically transmitted to the offspring adults through the diapause eggs of Aedes albopictus.Zika virus can also be detected in the brain of suckling mice after biting.3.Short-day photoperiod can induce Aedes albopictus to lay diapause eggs,and the diapause incidence of Aedes albopictus induced by short-day photoperiod increased gradually from pre-diapause to late-diapause.The diapause incidence and hatching rate of Aedes albopictus from Beijing in the temperate zone are relatively higher,which indicates that Aedes albopictus in the temperate zone can survive in lower temperature in winter.After oral infection with Zika virus,the hatching rate of Aedes albopictus from Beijing in the temperate zone is higher.Compared with the non-infected group,the diapause incidence of Aedes albopictus eggs in the infected group is not significantly different,but the hatching rate is significantly lower.4.Through transcriptome analysis of diapause eggs infected with Zika virus,it was found that some pathways related to diapause and infection immunity,such as circadian rhythm,amino acid anabolism,energy synthesis and fat metabolism.After association analysis,10 novel miRNAs-mRNAs were found. |
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