Integrating Of Solexa High-abundance Mrnas And Mirnas In Apis Mellifera:Comparison Between Nurses And Foragers To Identify Regulatory Network

Honeybee (Apis mellifera), an ensocial insect, is universally acknowledged to be a good model for studying the evolution and mechanism of complex social behavior. Generally, adult honeybees take care of larvae and queen during2-3weeks after emergence, while they start to forage for honey or pollen during the following1-3weeks. In this study, the next generation sequencing technology was applied to detect the mRNAs and sRNAs of honeybee’s head. Many differentially expressed genes were obtained, which could be associated with the behaviorral transition of honeybee. The regulatory network of miRNA and mRNA were also analyzed. The main results were summarized as follows:1. High-abundance mRNAs in Apis mellifera:comparison between nurses and foragersA large volume of honey bee (Aips mellifera) tag-seq was obtained to identify the differential gene expression via Solexa/Illumina Digital Gene Expression tag profiling (DGE) based on next generation sequencing. In total,3422327(nurses) and4286250(foragers) clean tags were sequenced,173532(nurses) and426047(foragers) total clean tags could not be match to the reference database, and7508and6875mapped genes were detected in foragers and nurses, showing72.47%and79.14%of total reference gene number respectively. The top ten high expression genes includes several major royal jelly proteins, glucose oxidase and defensin.1434genes were considered having significantly different expression, that is, showing an expression ratio (foragers/nurses) of more than2and FDR of less than0.001,1337genes were up-regulated in foragers, and97genes were down-regulated in foragers. Furthermore, we performed the Gene Ontology (GO) category and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis,415genes with annotation terms were linked to the GO cellular component category.200KEGG pathways were obtained, including21signaling pathways. The PPAR signaling pathway was the most highly enriched with the lowest Q-value. 2. High-abundance miRNAs in Apis mellifera:comparison between nurses and foragersMicroRNAs (miRNAs) are endogenous small (-22nt) non-coding RNAs regulating gene expression in animals and plants by targeting mRNAs for cleavage or translational repression. To find some differentially expressed miRNAs that may be associated with age-dependent behavioral changes in honeybees, we applied the next generation sequencing technology (Solexa) to detect small RNAs in nurses and foragers. Our results showed that both of the nurses and foragers had a complicated small RNA population, and that the length of small RNAs varied,22-nt being the predominant length. Combining deep sequencing and bioinformatics analysis, we discovered9known miRNAs were significantly different between nurses and foragers with a p-value less than0.01and the absolute value of fold-change more than1. Some of their target genes were related to neural function. Moreover,67novel miRNAs were identified in nurses and foragers, of which,16ones were confirmed by step-loop RT-PCR. Ame-miR-31a and ame-miR-13b were further validated using quantitative reverse transcription polymerase chain reaction assays. This study provides new information on the miRNA abundance of honeybees, helps us to understand the miRNA function in the regulation of honeybee development.3. Regulatory network analyses of miRNA and mRNA between nurses and foragersThe targets of9differentially expressed miRNAs between nurses and foragers were predicted by using Miranda v3.3a, all of the miRNAs target a lot of mRNA. Then integrated analysis of miRNA and mRNA was done by using Cytoscape2.8.2software combining differentially expressed mRNAs, four up-regulated miRNAs in nurses target37down-regulated genes and two up-regulated genes, while five down-regulated miRNAs target55genes, only2of these genes were up-regulated, others were down-regulated genes. Their potential ontology and pathway were analyzed by using the DAVID Bioinformatics Resources. Go analyses of target mRNAs revealed that only7mRNA belonging to cellular component which include Cytoplasm and Cytoplasmic part. Pathway analyses showed that28out of90down-regulated target mRNAs belonging to45Pathway categories, while2up-regulated target mRNAs belonging to2Pathway categories.