SDS-PAGE-Based Quantitative Assay of Hemolymph Proteins in Honeybees: Progress and Prospects for Field Application

Abstract

In human and veterinary medicine, serum proteins are considered to be useful biomarkers for assessing the health and nutritional status of the organism. Honeybee hemolymph has a unique proteome that could represent a source of valuable biomarkers. Therefore, the aims of this study were to separate and identify the most abundant proteins in the hemolymph of worker honeybees to suggest a panel of these proteins that could represent useful biomarkers for assessing the nutritional and health status of the colonies and, finally, to analyze them in different periods of the year. Four apiaries were selected in the province of Bologna, and the bees were analyzed in April, May, July, and November. Thirty specimens from three hives of each apiary were sampled and their hemolymph was collected. The most represented bands obtained after 1D sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were cut from the gel, and the proteins were identified using an LC-ESI-Q-MS/MS System. A total of twelve proteins were unmistakably identified; the two most abundant proteins were apolipophorin and vitellogenin, which are known biomarkers of bee trophic and health status. The two other proteins identified were transferrin and hexamerin 70a, the first being involved in iron homeostasis and the second being a storage protein. Most of these proteins showed an increase from April to November, mirroring the physiological changes of honeybees during the productive season. The current study suggests a panel of biomarkers from honeybee hemolymph worth testing under different physiological and pathological field conditions.

Keywords: apolipophorin; hexamerin 70a; nutritional biomarkers; proteomics; transferrin; vitellogenin.

Figure 1

Seasonal variations of total protein concentrations in hemolymph of honeybees from four apiaries (A, B, C, and D) of the province of Bologna. The data are expressed in mg/mL and reported as the mean ± SD (n = 3). Different lower-case letters indicate significant differences (p < 0.05) among the time points (months) within the same apiary. Different symbols (*,†) indicate significant differences (p < 0.05) among the apiaries at the same time point (May).

Figure 2

Representative gel obtained after SDS-PAGE (4–12%, Coomassie staining) of hemolymph proteins from healthy worker bees (apiary D, April sampling) at different dilutions (lanes 1–2, 15 µg; lanes 3–5, 3 µg). The first lane shows the molecular mass marker (kDa). The rectangles indicate the bands cut and analyzed using mass spectrometry that led to the protein identifications (Table 1). The numbers next to the rectangles correspond to those reported in Table 1.

Figure 3

(A) Representative gels obtained after SDS-PAGE (4–12%, Coomassie staining) of hemolymph from worker bees sampled in April and November from the same 3 hives located in the province of Bologna (apiary A); the dashed black box indicates the internal standard of quantity (1 µg). (B) The pherograms of lanes 2 and 5 are reported as an example. The arrows indicate the bands identified as apolipophorin-I (ApoLp-I), vitellogenin (Vg), apolipophorin-II (ApoLp-II), transferrin (Tf), and hexamerin 70a (Hex 70a).

Figure 4

Seasonal variations of vitellogenin, apolipophorin-I, transferrin, and hexamerin 70a in the hemolymph of honeybees from four apiaries (A–D) of the province of Bologna. The data are expressed as mg/mL and reported as the mean ± SD (n = 3). For each analyte, different lower-case letters indicate significant differences (p < 0.05) among time points (months) within the same apiary.