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Differential gene expression underlying ovarian phenotype determination in honey bee, Apis mellifera, caste development

ABSTRACT: Adult honey bee queens and workers drastically differ in ovary size. This adult ovary phenotype difference becomes established during the last two larval instars, when massive programmed cell death in the ovaries of worker larvae leads to the degeneration and removal of 95-99% of the ovariole anlagen. The higher juvenile hormone (JH) levels in queen larvae protect their ovaries against such degeneration. To gain insights into the molecular architecture underlying this divergence critical for adult caste fate we performed a microarray analysis contrasting RNA extracts from fourth and early fifth instar queen and worker ovaries. While for the fourth instar we found differential expression (log2FC > 1.0) for only nine genes, the number of differentially represented transcripts (DRTs) increased to 56 in early fifth instar ovaries. From these, 18 had their expression levels further analyzed by real-time PCR (RT-qPCR). For 13 of these the expression differed significantly between queen and worker ovaries at least one time point in development, and interestingly, genes with enzyme functions were overexpressed in workers, while genes related to transcription and signaling were so in queens. For the RT-qPCR confirmed genes we further analyzed their response to JH, revealing a significant up-regulation for two genes, one encoding a short chain dehydrogenase (SDR) and the other heat shock protein 90 (Hsp90). Five other genes, including Hsp60 and hexamerin 70b, were significantly down-regulated by JH. As SDR genes have previously come up as differentially expressed in different transcriptome assays in honey bee larvae, and heat shock proteins are involved in hormone responses, these are interesting candidates for further functional assays.

ORGANISM(S): Apis mellifera

PROVIDER: GSE81118 | GEO | 2016/10/06

SECONDARY ACCESSION(S): PRJNA320633

REPOSITORIES: GEO

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Project description:Honeybees are very important eusocial insects and are involved in the pollination of many plants. Queen bees and worker bees develop from the same fertilized eggs, and are thus genetically identical despite their substantial behavioural and physiological differences. The mechanism governing developmental differences between worker and queen bees has always attracted much interest. While there are several reports on mRNA expression related to caste differentiation, no systematic investigation of small RNAs has thus far been carried out. Results: Using deep sequencing we systematically profiled small RNA expression in 4th-6th day worker larvae and queen larvae (the critical stages at which the fates of workers and queens are determined), and found that 38 miRNAs were differentially expressed between worker and queen larvae. In addition, 639 mature miRNA candidates were identified in our work for the first time, of which, 526 were expressed only in workers (318) or queens (208). Conclusion: We present the first profile of honeybee small RNAs and explore the mechanism of caste differentiation between worker and queen bees. Caste-specific expression patterns and large discrepancies in small RNA profiles between worker and queen bees indicate that small RNAs may be related to the differential development of worker and queen bee larvae. Results presented here will make a valuable contribution to understanding the caste switch between worker and queen bees. Three healthy 10-frame colonies of ‘Zhenongda No.1’- a high-yielding royal jelly breed of Apis mellifera ligustica , were maintained at the Huajiachi campus of Zhejiang University. In each colony, the queen laid eggs over a period of 24 hours in one empty frame which was subsequently moved to an egg-free super-chamber. After 66 hours (less than 18 hours after hatching), we transferred 150 larvae into queen cups to rear queens in each colony and put the queen cup frames into their corresponding colonies. 40-60 worker larvae and queen larvae were collected from each colony after 4 days (73~90 h after hatching), 5 days (97~114 h after hatching) and 6 days (121~138 h after hatching). The larval samples were collected into 50 ml tubes, immediately frozen in liquid nitrogen and stored at -80C until being used for RNA extraction. After total RNA was extracted and quantified , relative equal amounts of total RNAs from each of the three sampling days were pooled into respectively worker and queen samples, and the fraction of small RNAs less than 30nt long was retained and sequenced on the Illumina/Solexa high-throughput platform (HTP).

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Differential gene expression underlying ovarian phenotype determination in honey bee, Apis mellifera, caste development

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