Two-dimensional gel proteome analysis of honeybee, Apis mellifera, worker red-eye pupa hemolymph

Abstract

Apis mellifera Linnaeus is a holometabolous insect that undergoes complete metamorphosis in its nonfeeding pupal stage before transitioning to the adult stage. Its pupal stages are classifiable by the unique color pigmentation of its compound eyes and thorax; notably, there is a red-eye stage involving an unpigmented body that has a relatively short duration and is easy to recognize. The aim of the current study was to create a proteomic reference map of the worker red-eye pupa hemolymph. Hemolymph was collected from dorsal vessels using glass capillary tubes and was examined using pI 3–10 two-dimensional gel electrophoresis (2DE; 10 and 14 %) and matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF protein identification. This experimental approach allowed us to identify 129 different proteins organized into orthologous groups. Overall, the predominant category was post-translational modifications, protein turnover and chaperones (23.3 % of the identified proteins). In addition, we identified proteins in the non-orthologous groups of olfaction (2.3 % of the identified proteins) and storage hexamerins (3.1 % of the identified proteins). Quantitatively, the major protein isoforms that were accurately identified via 10 % 2DE were four forms of storage hexamerin: the 110, 70a, 70b, and 70c forms. The most abundant enzymes identified were short-chain dehydrogenases/reductases with pivotal developmental roles in ecdysteroidogenesis and a sigma class glutathione-S-transferase that most likely serves as a major protectant against the by-products of oxidative stress. Many of the identified proteins are known to be involved in the mechanisms of metamorphosis. All of the identified proteins are useful as markers for future comparative physiological and developmental studies.