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 posttranslational 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.

Detailed proteome mapping of newly emerged honeybee worker hemolymph and comparison with the red-eye pupal stage

“…In addition to the proteomic 2D-E-MS/MS mapping of emerged honeybees, we aimed to compare these results with the hemolymph protein composition of the red-eye pupal stage described in our previous study (Erban et al 2014). The results indicated that at the end of metamorphosis/time of emergence, the honeybee hemolymph became depleted of proteins and only a few proteins remained at a higher concentration.…”

“…We did not process the hemolymph before analysis, analogous to Erban et al (2014), as the samples mixed with DeStreak containing 0.5 % IPG buffer (pH 3-10 or 4-7) were directly used for isoelectrofocusing (IEF). IEF was performed on an Ettan IPGphor 3 (GE).…”

“…The sample preparation procedures and subsequent protein identification of trypsin digests followed the methodology described by Erban et al (2014). The spectra were searched against the non-redundant (nr) NCBI database using MASCOT 2.2 (Boston, MA, USA).…”

“…During this period, the body is reconstructed and, finally, the capping is eaten away and the adult emerges (Winston 1987). The just/newly emerged honeybee, like the redeye pupa stage (Erban et al 2014), is a well-defined, easily recognizable developmental stage in the honeybee life cycle and, therefore, very useful for studying physiological and developmental factors that can be caused by aging, entomopathogens (such as viruses), bacteria, Nosema , the hemolymph-sucking mite Varroa , or the influence of pesticides.…”

“…of the red-eye pupal stage at the proteome level (Erban et al 2014). In addition, honeybee hemolymph proteins of different developmental stages were detected in the ectoparasite Varroa destructor (Erban et al 2015).…”