Composition Analysis And Molecular Mechanism Of Capping Pheromone In Honeybee Larvae

In honeybees,methyl palmitate(MP),methyl oleate(MO),methyl linoleate(ML)and methyl linolenate(MLN)are important pheromone components of the capping pheromones triggering the capping behavior of worker.Using GC/MS compared the amounts of these four pheromone components in the larvae of workers and drones of A pis mellifera ligu stica and A pis ce rnan a c e rn a n a,prior to be capped(4th instar),in the process of being capped(capping)and had been capped(capped).In A pis mellifera ligust ica,the amounts of MP,MO and MLN peaked at the capping larval stage and ML was highest at capped larvae in worker larvae,whereas in drone larvae the amounts of the four pheromone components were higher overall and increased with aging.In A pis cernana cerna na,the amounts of the four capping pheromone components were significantly higher in the larvae at the capping stage and capped stage than at the prior to be capped.Among them,the amounts of MP and MO increased with the age of the larvae,while the amounts of ML and MLN were not significantly different in the larvae at the capping stage and after capping.The amounts of four pheromone components in the drones all increased with age.The release of MP,MO,ML and MLN was increased during the critical stage of capping in worker and drone larvae of A pis mellifera lig usti ca and A p is ce rnana ce rnana,which further verified that MP,MO,ML and MLN were pheromones related to capping behavior of honeybees.Using A pis mellifera ligu stica as the experimental material,the larvae of workers and drones of prior to be capped(4th instar),in the process of being capped(capping)and had been capped(capped)were collected for transcriptome sequencing analysis using Illumina high-throughput sequencing platform.The results showed that there were 4413DEGs and 1358 DEGs among the larvae groups of three stages of worker and drone,respectively.In the comparison groups of 4th instar vs capping,capping vs capped and 4th instar vs capped,there were 1504,2952 and 4016 DEGs in the larvae of worker,respectively,among which 776,1600 and 2073 DEGs were up-regulated and 728,1352and 1943 DEGs were down-regulated.There were 500,21 and 1397 DEGs in drone larvae,respectively,among which 293,21 and 787 DEGs were up-regulated and 207,0 and 610DEGs were down-regulated.Furthermore,we proposed de novo biosynthetic pathways for MP,MO,ML and ML,from acetyl-Co A,and 12 related candidate genes.Besides,stable isotope tracer ¹³C and deuteriumwere used to confirm that these capping pheromone components were de novo synthesized by larvae themselves rather than from their diets.The biosynthetic pathway of the capping pheromone of honeybee larvae was proposed for the first time in this study,providing a new idea and theoretical basis for the future research on the molecular mechanism of honeybee pheromone.Using A pis c erna na c erna na as the experimental material,the larvae of workers and drones of prior to be capped(4th instar),in the process of being capped(capping)and had been capped(capped)were collected for gene expression analysis using RNA-seq.The results showed that there were 4299 DEGs and 3926 DEGs among the larvae groups of three stages of worker and drone,respectively.In the comparison groups of 4th instar vs capping,capping vs capped and 4th instar vs capped,there were 62,3288 and 3701 DEGs in the larvae of worker,respectively,among which 20,1812 and 2022 DEGs were up-regulated and 42,1476 and 1679 DEGs were down-regulated.There were 355,2343and 3489 DEGs in drone larvae,respectively,among which 204,1517 and 1936 DEGs up-regulated and 151,826 and 1553 DEGs were down-regulated.Furthermore,we proposed de novo biosynthetic pathways for MP,MO,ML and ML,from acetyl-Co A,and11 related candidate genes.These pathways and related candidate genes were the same as those of A pis mellifera ligu stica,which further demonstrated the reliability of the experimental results.This experiment showed that it is very possible for A pis mellifer a l i g u st i c a and A p i s c e rn a n a c e rn a n a to use the same pathway for pheromone biosynthesis,but it still needs to be further verified by interference test combined with capping behavior of worker.Using A pis mellifera ligu stica as the experimental material,the larvae of workers and drones of prior to be capped,in the process of being cappedand had been capped were collected for Genome-wide DNA methylation analysis using WGBS technology.The results showed that there were differences in the DNA methylation levels among the larvae of different capping period of worker and drone.96-120 differentially methylated regions(DMRs)were found in the groups of worker and drone larvae at different capping process,and 41-54 associated genes were found in these DMRs.However,no pathway related to pheromone biosynthesis was found in KEGG enrichment analysis.The regulation mechanism of DNA methylation on pheromone biosynthesis of honeybee larvae remains to be further studied.