| Background:Circadian rhythm is a long-term adaptation of organisms to the periodic changes of environmental factors such as light and temperature caused by the rotation and revolution of the earth,resulting in an inherent spontaneous timing mechanism-The biological clock system consists of an input pathway,a central pacemaker or oscillator,and an output pathway.The input pathway is used to receive environmental signals such as external light and temperature and transmit them to the core oscillator to synchronize the biological clock with the environment.The central pacemaker generates molecular oscillations through transcription,translation and modification,while the output pathway regulates downstream biological behaviors and physiological rhythm changes through molecular oscillations.Circadian rhythm generally exist in various species from humans to cyanobacteria in the biological world,from organisms to various biological levels of tissues and cells.Although cells and tissues cultured in vitro are separated from the regulation of the central nervous system of the organism,they also have three original biological clocks.The molecular regulation mechanism of the biological clock has always been the subject of intense investigation in Drosophila,but little research has been done on the clock mechanism of other non-drosophilid insects.Mosquitoes are a class of important medical vector insects that can transmit malaria,dengue fever,Zika virus disease,filariasis,yellow fever,Japanese encephalitis and other diseases,cause great harm to human life and health.Different mosquito species have evolved distinct circadian behaviors based on their ecological niches,and many mosquito behaviors change with time of day,including flight activity,mating,oviposition,and biting.As mating activity is an important physiological behavior of mosquito reproduction after emergence,It is of great significance for mosquito control to clarify the regulation mechanism of mosquito biological clock on mating rhythm.Objective:1.To explore whether there is a circadian rhythm in Ae.albopictus C6/36 cells,and to analyze the characteristics of non-clock genes and core clock genes in the cells,and to clarify the circadian clock regulation mechanism at the cellular level of Ae.albopictus.2.To observe the mating behavior and regularity of daytime-active Ae.albopictus and nocturnal-active Culex quinquefasciatus under day and night conditions,and explore the regulatory effect and mechanism of core clock genes on the mating behavior of the two mosquitoes.It provides a theoretical basis for accurate monitoring and control of mosquitoes.Methods:1.The circadian rhythm model of Ae.albopictus C6/36 cells was established,After synchronizing for one week under the conditions of day and night(12h Light:12h Dark,LD).Collected at the beginning of light and at intervals of 6 hours,a total of 5 time points within 24 hours were collected for transcriptome sequencing,and the transcriptome sequencing data were analyzed and verified,and the characteristics of oscillating genes were analyzed.2.Per gene was knocked down in Ae.albopictus C6/36 cells by RNAi technology,Cell samples at different time points were collected on the second day with the highest knockdown efficiency,and the effect of clock gene per on the rhythmic expression of cell metabolic genes was analyzed by RT-qPCR.3.Ae.albopictus and Cx.quinquefasciatus were exposed to different light conditions,including LD(light on at 8:00,12h Light:12h Dark),LDDD(12h Dark:12h Dark),DL(light on at 20:00,12h Dark:12h Light)and DLDD.The spermatheca of female mosquitoes at each time period were dissected to calculate the insemination rate of female mosquitoes,and the mating behavior rhythm of the two strains of mosquitoes within 24 hours was compared.4.RT-qPCR was used to detect the expression profile of desat1 gene in the head and body of Ae.albopictus and Cx.quinquefasciatus male and female under LD conditions within 24 hours.Knockdown of the desat1 by dsRNA was used to compare the effects on the mating behavior of two mosquito species,and the effect of the desat1 gene on cuticular hydrocarbon synthesis was compared by gas chromatography mass spectrometry.5.The per and clk core clock gene knockout strains of Ae.albopictus and Cx.quinquefasciatus constructed in our laboratory were used to compare the mating rhythm,and explore the regulation and mechanism of the core clock gene on the mating rhythm.Results:1.Under the circadian model of Ae.albopictus C6/36 cells,the samples collected at different time points were sequenced and screened for rhythmically oscillating genes.It was found that except for the core clock gene per,which exhibited circadian oscillatory expression,none of the other core clock genes exhibited oscillations,and many non-core clock genes were mainly involved in metabolism were screened to exhibit rhythmic oscillation.2.After knocking down per by siRNA silencing technology,the expression of per gene in one day tended to be flat,the rhythm disappeared.In addition,three key rate-limiting enzymes involved in the glycolysis pathway,eno,imp3L and Gapdh2,were found to have different degrees of disturbance in oscillation rhythm..3.The mating behaviors of Ae.albopictus and Cx.quinquefasciatus under LD,LDDD,DL and DLDD conditions showed a circadian rhythm.Ae.albopictus can mate throughout a day,but the mating activity was more active 3 hours after turning on the lights(early morning)and 3 hours before turning off the lights(evening);and the mating rate was up to 90%.Cx.quinquefasciatus reached the mating peak only 3 hours after the lights were turned off within 24 hours.The highest mating rate was 40%within 3 hours,and there was almost no mating under the light.4.The expression profile of desatl gene showed rhythmic expression in both species.The expression peak of desatl in Ae.albopictus was observed in both day and night,while that in Cx.quinquefasciatus was observed in the transition period from light to darkness.In addition,knocking down desat1 with dsRNA can significantly reduce the mating rate of the two mosquito species.Compared with the 90%mating rates of Ae.albopictus and Cx.quinquefasciatus control group,the mating rates of Ae.albopictus(65.5%)and Cx.quinquefasciatus(62.9%)were significantly decreased.Knockdown of desat1 expression lead to changes in related pheromones.Hydrocarbon levels of Ae.albopictus C19,C23,C29 and C31 were decreased,while C27 was increased.In Cx.quinquefasciatus,C21 and C23 were significantly decreased,while C27 and C29 were significantly increased.5.The mating rhythm of the per and clk mutant strains of the Ae.albopictus and clk mutant strains of Cx.quinquefasciatus changed to varying degrees.The mating rate of Ae.albopictus perΔ207 mutant strain decreased significantly at each time point,and the mating rate at each time point ranged from 0 to 40%.The mating rate of Ae.albopictus clkΔ61 mutant strain was lower than that of the WT control group at each time point,but there was no significant change in the mating rhythm of Ae.albopictus,and the peak phase of the mating rhythm of Ae.albopictus clkΔ293 mutant strain was delayed.The mating peak was delayed by 3h.The mating rate of Cx.quinquefasciatus clkΔ98 mutant strain was 5-10%during daytime,and there was no mating at any time point at night,which was contrary to the mating rhythm of WT.In addition,the amplitudes and phases of desat1 gene expression profiles of Ae.albopictus and Cx.quinquefasciatus mutant strains were changed to varying degrees.Conclusion:1.Both core clock genes and non-core clock genes in Ae.albopictus C6/36 cells showed oscillating rhythmic expression,and only per showed oscillating rhythmic expression in the core clock genes,suggesting that Ae.albopictus C6/36 cells may have a clock mechanism different from the typical core clock gene oscillator.Noncore clock genes are mainly involved in metabolic rhythms.These results suggest that c6/36 cells may regulate their metabolic rhythms independently of typical core clock gene oscillators.2.After per gene was knocked down by siRNA interference in C6/36 cells,the rhythm of three key rate-limiting enzymes involved in glycolysis pathway was also found to be disregulated to varying degrees,suggesting that per was involved in regulating metabolic rhythm in C6/36 cells.3.Ae.albopictus and Cx.quinquefasciatus had completely different mating rhythms.Ae.albopictus mainly mated during daytime and had two mating peaks,while Cx.quinquefasciatus mainly mated at 3 h after turn off the lights and had only one mating peak.4.The expression of desat1 gene is closely related to mating activity,and inhibition of desat1 gene expression results in significantly decreased insemination rate of mosquitoes.The change of cuticular hydrocarbons content after desatl knockdown and the effects of hydrocarbons on mating indicate that desat1 can regulate the synthesis of cuticular hydrocarbons in two mosquito species and affect mating activities.The difference in the temporal expression of desat1 gene between Ae.albopictus and Cx.quinquefasciatus may be one of the reasons for the different mating rhythms of the two mosquitoes.5.The changes in mating rhythm of Ae.albopictus per and clk mutant strains and Cx.quinquefasciatus clk mutant strains indicated that core clock genes were involved in regulating the mating rhythm of Ae.albopictus and Cx.quinquefasciatus.The expression profile of desatl in mutant strains indicated that the core clock genes was involved in regulating the rhythmic oscillation of desat1 gene. |
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