Multi-omics Joint Analysis Of The Epigenetic Mechanism Of Honeybee Caste Differentiation

Honey bees are eusocial insects and an important model organism for studies of caste development and caste differentiation in social insects.Their division of labor is mainly based on the differentiation of castes（queen and worker）.After divergence,queens and workers have different morphological,physiological,longevity,and behavioral traits,despite sharing the same genome.The mechanism underlying caste differentiation is not fully understood.However,there is evidence that differences in nutritional status between queens and workers modulate caste differentiation by altering DNA methylation patterns.In addition,various signaling pathways,such as the wingless-type protein（Wnt）signaling pathway,target of rapamycin（TOR）nutrient sensing pathway,and mitogen-activated protein kinase（MAPK）signaling pathway are related to honey bee caste differentiation.In this study,based on chromosome conformation capture technology（Hi-C technology）,assay for transposase accessible chromatin with high-throughput sequencing（ATAC-seq）,Chromatin Immunoprecipitation with high-throughput sequencing（ChIP-seq）,and transcriptome sequencing（RNA-seq）,we used western honey bees（Apis mellifera）as the study material,and selected 2^nd instar larvae of queen and worker bees at the early stage of development and 4^th instar larvae at the critical stage of hierarchical differentiation.Hi-C,ATAC-seq,ChIP-seq and RNA-seq were performed on the 2nd instar larvae and 4th instar larvae of queen and worker bees,respectively,and the results of the joint analysis and validation were as follows.Hi-C analysis revealed no significant differences in Hi-C（intrachromosomal Cis and interchromosomal Trans）between 2^nd instar queen larvae and worker larvae,while 4^thinstar larvae showed significant differences,with stronger intrachromosomal interactions in queen larvae and interchromosomal interactions in worker larvae.Especially interesting was the fact that 4^th instar queen larvae had stronger interactions at the terminal junctions of the two chromosomes（telomeres）.Changes in A/B compartments,TAD boundaries and loops play a role in the regulation of gene expression.The A/B compartment,TAD boundary and loop play a key role in the regulation of gene expression.Many DEGs associated with A/B transition regions,TAD boundaries and loops are enriched in key pathways such as TOR,IRS,Notch,and Hippo,suggesting that differences in chromosome structure are associated with honeybee caste differentiation.The results of ATAC analysis revealed that both unique and shared peaks were more frequent in 4^th instar larvae than in 2^nd insatr larvae.The proportions of mononucleosomes,binucleosomes and trinucleosomes increased with time in queens,while those in workers decreased with time,indicating that different transcriptional activities existed between queen and worker during larval development.Among the unique peak and differential peak-associated genes,4^th instar larvae had more genes associated with caste differentiation than 2^nd instar larvae,suggesting that differences in chromatin accessibility are associated with honeybee caste differentiation.Analysis of the ChIP results found that:Compared with the 2^nd instar,the numbers of DEGs and H3K4me1 markers were significantly increased at the 4^th instar,and gene expression was negatively correlated with H3K4me1.Further analysis revealed that the chromatin patterns of queens and workers differed significantly at the 4^th instar.H3K4me1modification promotes larval development towards worker bees.Compared with the 2^ndinstar,the numbers of DEGs and H3K27ac markers were significantly increased at the 4^thinstar,and gene expression was postively correlated with H3K27ac.Further analysis revealed that the chromatin patterns of queens and workers differed significantly at the 4^thinstar.H3K27ac modification promotes larval development towards queen bees.These findings illustrate the important role of H3K27ac in honey bee larval development and caste differentiation.RNA-seq analysis revealed that more genes were down-regulated in queens at 2^ndinstar and up-regulated in queens at 4^th instar compared to workers.The number of DEGs was significantly higher in 4^th instars compared to 2^nd instars.The combined Hi-C,ATAC,ChIP and RNA-seq analyses revealed that Hi-C,ATAC-seq and ChIP-seq results were all positively correlated with RNA-seq results,and that 4^thinstar queen larvae had more DEGs than 2^nd instar larvae,and these genes were mainly associated with caste differentiation.The common differential gene in the four omics:4-coumarate-Co A ligase（4CL）was disturbed,and the queen birth weight and ovarian tube number were significantly lower compared to the control group,suggesting that this gene may be related to honey bee caste differentiation.The joint analysis of four omics showed that honey bee caste differentiation is regulated by complex multi-omics interactions,and in the early larval development,epigenetic modifications are less and less involved in regulating gene expression,so 2^ndinstar larvae development is plastic;however,at the critical larval development stage,epigenetic modifications change intensify and multiple epigenetic modifications are regulated by synergistic interactions,especially with key genes of caste differentiation by epigenetic However,the changes in epigenetic modifications intensify at the critical stage of larval development,and multiple epigenetic modifications interact synergistically,especially with key genes of caste differentiation,which are regulated by multiple epigenetic modifications,so that the development of 4th instar larvae is irreversible.For the first time,it is proposed that epigenetic modifications can automatically initiate their own epigenetic regulatory programs according to the developmental state of the organism,and thus regulate the development of the organism.The results explain the molecular mechanism of epigenetic modifications regulating phenotypic plasticity in honeybees.