| Background:Aedes albopictus is a diurnal mosquito that transmits viruses,including dengue virus,and is the major vector of the outbreak of dengue fever in China.Culex quinquefasciatus is a nocturnal mosquito that can transmit diseases such as Japanese encephalitis.To better adapt to the circadian rhythms of natural environments,organisms have developed circadian rhythm.It refers to the oscillation of physiological or behavioral activity for a period of about 24 hours.If the circadian rhythm is out of whack,it can cause a variety of pathological changes.In addition to the most deeply studied locomotor activity rhythm,phenotypic rhythm such as egg-laying rhythm also belong to circadian rhythm.Circadian rhythm is usually regulated by core clock genes,and some studies have found that microRNAs are also involved in the regulation of circadian rhythm,but there are few studies on the egg-laying rhythm and its regulation mechanism of mosquitoes.Elucidating the egg-laying rhythm and its regulation mechanism of mosquitoes is of great significance for developing new vector mosquito control methods.Objectives:By constructing oviposition models of mosquitoes,monitoring the egg-laying rhythm of the two vectors,Ae.albopictus and Cx.quinquefasciatus,and studying the regulation effect of the core clock gene and miRNA on the egg-laying rhythm of Ae.albopictus,we can make the foundation for using egg-laying rhythm as a new technology for mosquito control.Methods:1.Ae.albopictus and Cx.quinquefasciatus were placed under LD(Light:Dark=12 h:12 h)for synchrony and mating.After the blood-meal,the egg-laying cups were collected and changed to record the number of eggs laid each day.The peak day was used as the follow-up experiment day.According to the above results,the mosquitoes were placed in different conditions of illumination and forced to retain eggs,ZT0(CT0)began to record the number of eggs laid in different periods within 24 hours(Cx.quinquefasciatus was within 48 hours)every 3 hours.The egg-laying rhythm model of the two mosquitoes was constructed to analyze the characteristics and compare the differences between the two mosquitoes.2.The core clock genes period and clock gene mutated strains of Ae.albopictus constructed in the laboratory were compared egg-laying rhythm with those of the wild-type under different light conditions.3.TA cloning sequencing was used to verify the consistency of miR-2940-1 in Ae.albopictus with RNA-seq data and miR-2940-3p sequence in Ae.aegypti.The expression profile of miR-2940-1 in tissues,head and body at time points was oscillated by RT-qPCR.4.RT-qPCR was used to compare the temporal expression profile of miR-2940-1 of female Ae.albopictus with the egg-laying rhythm under different condition.In addition,female mosquitos were injected with antagomir-2940-1 and Negative control at the optimal concentration respectively by thorax injection.The effects of antagomir-2940-1 on ovary development were verified by anatomical observation,and the egg-laying rhythm was studied within the duration of sustained antagonistic effect of the antagomir.5.RT-qPCR was used to compare the temporal expression profiles of the core clock genes,period and clock in female Ae.albopictus after blood-meal with the temporal expression profiles of miR-2940-1 under LD condition.Results:1.Both Ae.albopictus and Cx.quinquefasciatus showed circadian oscillations in egg-laying activity and quantity under different light conditions.Under LD conditions,the oviposition activity of Ae.albopictus was unimodal,with the oviposition peak in ZT9-ZT12.The oviposition activity of Cx.quinquefasciatus was bimodal with less day and more night,and the oviposition main peak was ZT12-ZT15.2.The egg-laying rhythm of period gene mutated strains of Ae.albopictus under LD condition,and the oviposition activity and number of ZT0-ZT3 were significantly lower than those of the wild-type.The phase of clock gene mutation was significantly advanced compared with that of wild-type.Under the condition of DD,the number of day and night spawning of period gene mutation was lower and higher than LD,and the phase of the spawning peak was later than that of wild-type.When clock gene was mutated,the number of eggs laid day and night was the same,and the spawning peak shifted backward and presented a bimodal pattern.3.The results of miR-2940-1 in Ae.albopictus were consistent with those of RNA-seq and the sequence of miR-2940-3p in Aedes aegypti.The expression level of miR-2940-1 in the body of female Ae.albopictus was higher than that in the head,and both showed oscillatory expression.The peak value of the former was ZT15 and the trough value was ZT3,while the peak value of the latter was ZT12 and the trough value was ZT6.The peak value of the body was earlier than that in the head,while the trough value of the head was earlier than that in the body.4.The expression of miR-2940-1 in wild-type female Ae.albopictus after blood-meal was rhythmical under LD condition,corresponding to the egg-laying rhythm.Knocked down miR-2940-1 by antagomir affected the ovary development of female Ae.albopictus.Compared with the No-injection and Negative control,the number and length of follicles were statistical decreased.In terms of egg-laying rhythm,the phase of peak moved forward and the number of oviposition decreased under LD and DD condition,as well as the oviposition activity and number of ZT0-ZT3 decreased significantly.5.The clock gene of female Ae.albopictus was no rhythmically expressed after blood-meal under LD condition,and had no corresponding relationship with miR-2940-1.However,period gene was expressed in circadian rhythm,which may have a corresponding trend with miR-2940-1.Conclusions:1.Both Ae.albopictus and Cx.quinquefasciatus have egg-laying rhythms,but their models and habits of activity are opposite,the former is unimodal and have diurnal habit,while the latter is bimodal and have nocturnal habit.2.The core clock genes period and clock play an important role in maintaining the regulation of egg-laying rhythm of Ae.albopictus.3.miR-2940-1 is closely related to oviposition activities of Ae.albopictus,and knocked down miR-2940-1 made the effects of ovary development and egg-laying rhythm,suggesting that it is involved in the regulation of ovary development and egg-laying rhythm of female Ae.albopictus.4.miR-2940-1 may be involved in the expression of the core clock genes period rhythmic oscillation to regulate the egg-laying rhythm. |
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