| Honeybees is a classical eusocial insect species,generally containing a single mated queen,hundreds of males and thousands of sterile female workers.It is characterized by a rich variety of social behavior and a sophisticated division of labor.A delicate language communication system in honeybee colonies is the prerequisite of social management.Pheromone,as a chemical language,is one of the most important mediators for coordination of colony activities.Many social activities are all realized through pheromone communication,such as queen mating flight,regulation of worker bee behavior,larval rearing,food collection,social organization and defense.Currently,people have a preliminary understanding on pheromone components,cells and physiological pathways regulated by pheromone response.However,the understanding on molecular mechanisms of honeybee drone mating behavior and pheromone communication is not clear.Therefore,this study carried out the research on the behavioral response and molecular mechanism of queen mandibular pheromone(QMP)on drone.Firstly,this study detected the behavioral effect of QMP and(E)-9-oxodec-2-enoic acid(9-ODA,one of the predominant compounds of QMP)on Apis mellifera ligustica and Apis cerana cerana mature drones at different physiological statuses(flying and crawling)by an indoor olfactometer.Behavioural results showed that QMP at 0.04?0.2?1.0?7.0?g·?L-1concentrations all significantly reduced the selection rate of flying and crawling A.mellifera drones and flying A.cerana drones,but no difference on crawling A.cerana ones.However,there was no difference among three doses of 9-ODA(3.5?7.0?14.0?g·?L-1)on behavioral selection of both A.mellifera and A.cerana drones.The further results showed that 7.0?g·?L-1 QMP significantly reduced the selection of flying and crawling A.mellifera drones but no effect on flying or crawling A.cerana drones,when two species were placed in the indoor olfactometer together.The molecular mechanism of drones recognzing QMP at different environment is still unknown.The odorant receptor 11 gene(Ac Or11)from A.cerana was cloned in this study.Its c DNA(Gen Bank accession:MG793195)contains an open reading frame(ORF)of 1185bp encoding a polypeptide of 394 amino acids.Moreover,the Ac Or11 protein belongs to the 7-transmembrane?6 receptor(7TM?6)super-family.Then this study also compared and analyzed the expression characteristics of four candidate pheromone receptor genes Ors(Or10,Or11,Or18,Or170)from A.mellifera and A.cerana dornes during two developmental stages(immature and sexually mature)and different physiological statuses(flying and crawling)by used q RT-PCR.The results showed that expression levels of these four Ors in drone antennae were higher than those of brains in both immature and mature stages.At immature stage,expression levels of four Ac Ors in antennae of flying drones were significantly higher than those of crawling drones.However,the expression of these genes in drone antennae were not different between two physiological statuses.Interestingly,four Am Ors expression in brains of mature flying drones was dramatically higher than those of mature crawling drones.Only Ac Or11 expression was obviously higher in brains of mature flying drones.Is there some kind of Ors playing a key role in the mating flight of drones?A q RT-PCR system was employed to detect and analyze the expression characteristics of QMP and 9-ODA on the above four Ors A.mellifera and A.cerana drones at flying and crawling statuses.The results showed that QMP and 9-ODA both inhibited the transcriptional expression of Or11 in antennae and brain of A.mellifera and A.cerana drones.The expression levels of Ac Or18 and Ac Or170 in drone antennae were significantly down-regulated by QMP and 9-ODA,but the Am Or18 and Am Or170 in drone antennae were not affected by QMP or 9-ODA.Moreover,QMP and 9-ODA effectively decreased the expression levels of Or170 in brain of A.mellifera and A.cerana drones,but had no effect on Or10 in drone brain of A.mellifera and A.cerana.It is not clear that the downstream signaling genes of Or11 recognize 9-ODA.The effect of 9-ODA on Or11 and Kr-h1(Krüppel-homolog 1,a candidate transcription factor for Or11)in 4 d and 14 d A.mellifera drones were measured by q RT-PCR.The results showed that 9-ODA significantly reduced the transcription levels of Or11 and Kr-h1 genes.Subsequently,si RNA-Or11 was injected into antennae and brains of the drone pupae respectively,and brooded for 72 h in an incubator until eclosion.The expression characteristics of Or11,Kr-h1 and Br-c(Broad-Complex,the downstream gene of Kr-h1)in antennae and brain of these drone were detected by q RT-PCR.The results showed that si RNA-Or11 significantly decreased the expression of three genes in both antennae and brains of these injected drones,suggesting that the downstream gene of Or11 is the transcription factor Kr-h1 and there may be a molecular pathway in drones responding to queen sex pheromones,i.e.Or11-Kr-h1-Br-c.This study provides a theoretical basis for further studies on mechanisms of olfactory recognition in honeybee drone mating flight. |
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