It takes 72 hours of embryogenesis for the most prevalent and active hive member, the honeybee worker (Apis mellifera linguistica), to hatch from incubation and start its journey towards honey production. During these 72 hours from egg to hatchling larva, the bee’s body organs develop, laying the framework for the fully functional adult that emerges almost three weeks later. Fang et al. (2014) have documented changes in the proteome with a view to understanding more about the molecular processes taking place during embryogenesis, along with the potential for future genetic manipulation.1
The researchers examined 1,000 eggs incubating in experimental hive setups (n=5 colonies) at 24, 48 and 72 hours post laying. They repeated the experiment three times, ending up with a total of 5.000 eggs for each time point. Fang et al. pooled the eggs for each experiment per time point and then extracted the proteins in preparation for proteome characterization. Liquid chromatography–tandem mass spectrometric (LC-MS/MS) analysis was carried out using an Orbitrap Elite hybrid ion trap-Orbitrap mass spectrometer coupled with an EASY-nLC 1000 liquid chromatograph (both Thermo Scientific). The team examined MS/MS raw data files, searching them against the A. mellifera protein database for identification.
Fang and colleagues identified a total of 1,460 proteins across the three time points. Protein numbers increased with embryogenesis, with 846 identified at 24 hours, 1,080 at 48 hours and 1,118 at 72 hours. Of these, 585 (40%) were consistently expressed across all time points; the researchers considered these to be core housekeeping proteins. They also observed that certain proteins were unique to each time point: 156 at 24 hours, 135 at 48 hours, and 170 at 72 hours.
Using a label-free proteomics approach, the researchers quantified protein expression across the time points, showing proteome changes with embryogenesis. They verified these results using quantitative real-time polymerase chain reactions and Western blotting with specific antibodies.
The team also investigated protein–protein interactions and functionality through the gene ontology (GO) tool via BLAST2GO, functional pathway analysis by KEGG Orthology-Based Annotation System (KOBAS) tools, and interaction prediction by Interologous Interaction Database (I2D) annotation. Their results show that at 24 hours, the majority of proteins are assigned to nutritional storage, nucleic acid metabolism and cell proliferation; at 48 hours, the predominant function is organogenesis; and at 72 hours, most proteins are involved in fatty acid metabolism and RNA transport.
With the results and discussion on the proteins identified and their roles in cellular activity, Fang and co-authors are confident that they have enhanced proteomic coverage for A. mellifera embryogenesis, thereby increasing the potential for future genetic manipulation.
1. Fang, Y., et al. (2014, June) “In-depth Proteomics Characterization of Embryogenesis of the Honey Bee Worker (Apis mellifera L.),” Molecular and Cellular Proteomics, pii: mcp.M114.037846 [e-pub ahead of print].
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