| Honey bees are important eusocial insects.They share resources,have a clear division of labor,and exchange information accurately.Honey bees are not only important pollinating insects,who play an important role in improving agricultural yields and maintaining global ecological balance,they are also typical model insect,and are of great significance for studying social behavioral characteristics.Experiment 1: We transferred eggs(E),1-day-old larvae(L1),2-day-old larvae(L2)and 3-day-old larvae(L3)to queen cells to rear queens.We measured 5 indicators(queen cell length,remaining royal jelly,duration of queens development,weight,number of ovarioles)related to the development of queen bees.We performed single-gene methylation detection on 14 DNA fragments of 5 queen development-related genes(Cyt P450,Defensin-1,Dynactin P62,JH and Vg)by using Agena Mass ARRAY? Methylation analysis.The results showed that as the age of the brood increased,the quality of the queen bees declined.However,DNA methylation levels of 14 fragments of 5 genes in the 4experimental groups(E,L1,L2,L3)were not significantly different.Experiment 2: Sampling method is the same as experiment 1.We sequenced the transcriptome of queen bee samples through an Illumina high-throughput sequencing platform.The results showed that as the age of the brood increased,the number of differentially expressed genes(DEGs)between queen groups that were reared from larvae,and queen groups that reared from eggs,gradually increased.Among them,a large number of genes related to immunity,metabolism,individual development,reproduction,and life span were down-regulated.In addition,all of the four queen groups in this experiment had significantly different numbers of five alternative splicing,which may regulate gene expression.Experiment 3: We transferred eggs,1-day-old larvae,2-day-old larvae and 3-day-old larvae to queen cells to rear queens.We measured 4 indicators(queen cell length,remaining royal jelly,weight,number of ovarioles)of queen bee quality.We tested the whole genome of the for the 4 queen groups for methylation by using whole genome Bisulfite sequencing.The results showed that the number of ovarian tubes in the queen bees of group E were significantly higher than those in the groups L1,L2,and L3,and the number of ovarian tubes decreased significantly with the increase of the age of the brood.As the age of the brood increased,the number of differentially methylated genes(DMGs)between larvae groups and egg groups gradually increased.Among them,there were a lot of functional genes involved in caste differentiation,longevity,immunity,physical development or metabolism.Experiment 4: We transferred eggs(E),1-day-old larvae(L1)and 2-day-old larvae(L2)to queen cells to rear queen samples for 4 generations(G1-G4).We measured 4morphological indicators(same as above)of queen bees.We tested the whole genome for methylation,for all queen groups in 4 generations,by using whole genome Bisulfite sequencing.Considering both weight and ovarioles,our results show that the quality of the queens of group E was the best and that the quality of the queens from group L2 was the worst.There was no significant difference in whole genome DNA methylation levels of the larvae groups(L1,L2)with egg group(E)within each generation(G1-G4).However,compared with G0 group,the result indicates that the DNA methylation of the group L2 accumulated faster than in the groups E and L1.In addition,we also found that the number of queen DMGs in L1 vs E and L2 vs E within each generation(G1-G4)had a cumulative effect trend with successive generations.When comparing adjacent generations of the same breeding method,the number of DMG in group E is very stable as the number of generations increases,while the number of DMG in the groups L1 and L2 also increases as the number of generations increases.Over four generations the methylomes of lines of queens reared from eggs and worker larvae diverged,accumulating increasing differences in exons of genes related to caste differentiation,growth and immunity,which indicates that honey bee DNA methylation could have a accumulative effect. |
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