| Objective:In this study,we investigated the effects of fluctuating and constant temperatures on the vector competence of Zika virus(ZIKV)and Dengue virus(DENV)infection in Ae.albopictus in the laboratory and conducted transcriptome analysis to explore the target genes related to the infection process of mosquito-borne viruses under different temperature patterns,and to analyze the molecular mechanism of virus infection in Ae.albopictus under fluctuating and constant temperatures,to provide a theoretical basis for targeting and regulating the infection and transmission of mosquito-borne viruses.Methods:1.According to the average minimum temperature,average maximum temperature,and average temperature of the high mosquito-borne disease epidemic season of 2015-2019 in Guangdong Province,four temperature conditions were set:23℃,27℃,31℃ and diurnal fluctuation temperature(DTR).The DTR fluctuated between the maximum and minimum temperature daily according to the daily temperature fluctuation rule.reaching the minimum temperature at 1:00 and the maximum temperature at 13:00.2.After oral infection with ZIKV and DENV-2 respectively,Ae.albopictus strains in Guangzhou were placed at four temperature groups of 23℃,27℃,31℃ and DTR,and the midgut and salivary gland tissues of female mosquitoes were taken on 4,7,10 and 14 days post-infection(dpi),and virus detection was performed by RT-q PCR to calculate the infection rate and virus copy number of mosquito midgut and salivary gland tissues;the number of deaths of Ae.albopictus at the four temperature groups was observed and recorded at the same time point every day from day 1 to 14 dpi,and the survival curves of Ae.albopictus at the four temperature groups were drawn.3.At four sets of temperatures,23℃,27℃,31℃,and DTR,Ae.albopictus mosquitoes were fed with viral and non-viral blood meals,The midgut tissues were used for transcriptome sequencing on the second day after feeding.Differentially expressed genes(DEGs)were screened using bioinformatics analysis,and RT-q PCR was applied to validate selected differentially expressed genes and further analyze the biological processes involved in differential genes.Results:1.At fluctuating temperatures,the ZIKV infection rate of Ae.albopictus showed a gradual increase with time,and the transmission rate showed a trend of increasing,then decreasing and then increase with time.The infection rate and transmission rate of DTR reached the highest on the 14 dpi,and the infection rate was 96.7%,which was significantly greater than 23℃(P<0.01)and 27℃(P<0.001);the transmission rate was78.57%,which was significantly greater than 31℃(P<0.05).And the virus copy number of midgut and salivary gland tissues at fluctuating temperatures was significantly greater than the constant temperature at 7,10 dpi(P<0.05).2.At fluctuating temperatures,the DENV-2 infection rate of Aedes albopictus showed a decreasing and then increasing trend over time,and the transmission rate increased gently throughout the infection cycle.On 7-14 dpi,the DTR infection rate was less than 23℃ and 27℃;on 7 dpi,the DTR transmission rate was less than 23℃ and 27℃.And on 4 and 7 dpi,the midgut and salivary adenovirus copy numbers under DTR were significantly smaller than the constant temperature(P<0.05).3.After Ae.albopictus infection with ZIKV,DTR and 23℃ survival were greater than 27℃ and 31℃ and statistically significant(P< 0.0001);after infection with DENV-2,DTR survival was the lowest and significantly lower than constant temperature(P< 0.01).4.Analysis of the transcriptome data of Ae.albopictus infected with ZIKV and uninfected with ZIKV revealed that there were 1252 differential genes at 23℃,mainly enriched in adipocyte lipolysis regulation,luteinizing hormone-mediated oocyte maturation,neurotrophic factor signaling and other pathways;662 differential genes at31℃,mainly enriched in longevity regulation pathway,olfactory transduction,protein digestion and absorption,amino acid biosynthesis and other pathways;1104 differential genes at DTR,mainly enriched in apoptosis,AMPK signaling and so on.Analysis of transcriptomic data from ZIKV-infected 23℃,31℃ and DTR revealed that,after comparing DTR with 23℃,the 23℃-specific differential genes were mainly enriched in thermogenesis,pyrimidine metabolism,carbon metabolism,butyrate metabolism porphyrin metabolism and other pathways;after comparing DTR with 31℃,the 31℃-specific differential genes were mainly enriched in protein digestion and absorption,insect hormone biosynthesis,and DTR is mainly enriched in PI3K-Akt signaling pathway,AMPK signaling pathway,T cell receptor signaling pathway,apoptosis,IL-17 signaling and so on.5.Analysis of the transcriptome data of DENV-2 and non-DENV-2 infected Ae.albopictus showed that there were 786 differential genes at 23℃,mainly in purine metabolism,pyrimidine metabolism and cell cycle pathways;333 differential genes at31℃,mainly in estrogen signaling pathway,longevity regulation pathway and apoptosis pathway;248 differential genes at DTR,mainly in antigen processing presentation,NF-kappa signaling and other pathways.Analysis of transcriptomic data from 23℃,31℃ and DTR infected with DENV-2 revealed that,after comparing DTR with 23℃,the differential genes specific to 23℃were mainly enriched in Fox O signaling pathway,autophagy,longevity regulation pathway,p53 signaling pathway and other pathways;after comparing DTR with 31℃,the differential genes specific to 31℃ were mainly enriched in c GMP-PKG signaling pathway,After comparing DTR with 31℃,the difference genes specific to 31℃ were mainly enriched in c GMP-PKG signaling pathway,estrogen signaling pathway,Erb B signaling pathway,Fox O signaling pathway,cell cycle and other pathways;the difference genes specific to DTR were mainly enriched in melanogenesis,TNF signaling pathway,insulin signaling pathway,fatty acid metabolism,protein digestion and absorption,starch and sucrose metabolism.6.RT-qPCR validation of differentially expressed genes showed that the RTq PCR results of differential genes were consistent with the transcriptome expression trends,which indicated that the transcriptome data were reliable.Conclusions:1.The fluctuating temperature pattern enhanced the vector competence of Ae.albopictus for ZIKV and reduced the vector competence for DENV compared to constant temperature.Moreover,the survival rate of Ae.albopictus after ZIKV infection was significantly greater at fluctuating temperatures than at constant temperatures,which prolonged mosquito transmission of the virus and increased the risk of ZIKV epidemics.2.Genes associated with antiviral pathways,such as the Toll and Imd signaling pathway,were significantly upregulated in Ae.albopictus mosquitoes after infection with ZIKV and DENV compared to uninfected Ae.albopictus mosquitoes.3.Mosquito antiviral pathways are activated at fluctuating temperatures,such as the melanogenic pathway,a pathway associated with mosquito defense against pathogens such as parasites and viruses.Genes associated with this pathway were significantly up-regulated at fluctuating temperatures,especially after infection with DENV-2(P<0.0001). |
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