Shotgun proteomics deciphered age/division of labor-related functional specification of three honeybee (Apis mellifera L.) exocrine glands

Abstract

The honeybee (Apis mellifera L.) uses various chemical signals produced by the worker exocrine glands to maintain the functioning of its colony. The roles of worker postcerebral glands (PcGs), thoracic glands (TGs), and mandibular glands (MGs) and the functional changes they undergo according to the division of labor from nursing to foraging are not as well studied. To comprehensively characterize the molecular roles of these glands in workers and their changes according to the division of labor of workers, we analyzed the proteomes of PcGs, TGs, and MGs from nurse bees and foragers using shotgun proteomics technology. We identified approximately 2000 proteins from each of the nurse bee or forager glands and highlighted the features of these glands at the molecular level by semiquantitative enrichment analyses of frequently detected, gland-selective, and labor-selective proteins. First, we found the high potential to produce lipids in PcGs and MGs, suggesting their relation to pheromone production. Second, we also found the proton pumps abundant in TGs and propose some transporters possibly related to the saliva production. Finally, our data unveiled candidate enzymes involved in labor-dependent acid production in MGs.

Fig 1. Experimental overview of the shotgun proteomics strategy.

(A) The protein extracts from the three exocrine glands separated according to division of labor; nPcGs, fPcGs, nTGs, fTGs, nMGs, and fMGs, were in-solution digested and subjected to shotgun LC-MS/MS [67]. (B) The number of proteins identified according to the criteria described in the Methods section and the total number of spectral counts detected in each gland (nPcG, fPcG, nTG, fTG, nMG, and fMG) are shown. SC, spectral count. (C) Distribution of spectral counts for the six glands: nPcG, fPcG, nTG, fTG, nMG, and fMG. (D) Distribution of spectral counts for each gland based on the data merged from nurse bees and foragers in panel (C).

Fig 2. Functional analysis of the three exocrine glands based on the frequently detected proteins identified from the PcGs, TGs, and MGs.

(A) Semiquantitative comparison of the KEGG pathway categories of the frequently detected proteins identified from the PcGs, TGs, and MGs. (B) Semiquantitative comparison of the KEGG pathway subcategories of the frequently detected proteins categorized as “Metabolism” in panel (A).

Fig 3. Functional analysis of the three glands based on the gland-selective proteins identified from the PcGs, TGs, and MGs.

(A) The venn diagram shows the numbers of the proteins specifically detected from the glands and the small circles expanded on the boundaries indicate an abundance at least 5-fold higher than the same-color circles. The number of the corresponding proteins is indicated in each partition. (B) Semiquantitative comparison of the KEGG pathway categories of the gland-selective proteins identified from the PcGs, TGs, and MGs. (C) Semiquantitative comparison of the KEGG pathway subcategories of the gland-selective proteins categorized as “Metabolism” in panel (B).

Fig 4. Schematic cartoon and candidate transporters of TG secretory epithelia.

(A) The assumed ion transport mechanisms in the insect salivary gland. V-ATPase on the apical membrane is coupled with a cation/proton exchanger on the same side and related to an anion exchanger, aquaporin, and chloride channel. (B) The spectral counts of the related proteins identified from each gland. These transporters satisfy our TG-selective protein criteria. p., predicted.

Fig 5. Labor-related functional analysis of the three exocrine glands based on the labor-selective proteins identified from the nPcGs, fPcGs, nTGs, fTGs, nMGs, and fMGs.

(A-C) The venn diagrams show the numbers of the proteins specifically detected from the glands of either nurse bees or foragers, and the small circles expanded on the boundaries indicate an abundance at least 5-fold greater than the same-color circles. The number of the corresponding proteins is indicated in each partition. (D-F) Semiquantitative comparison of the KEGG pathway categories of the labor-selective proteins identified from the PcGs (D), TGs (E), and MGs (F). (G-I) Semiquantitative comparison of the KEGG pathway subcategories of the labor-selective proteins categorized as “Metabolism” in panels (D-F).

Fig 6. Glycolysis and ether lipid synthesis pathway.

(A) The steps of glycolysis and ether lipid synthesis. The numbers indicate enzymes linked to panel (B). The question mark indicates that no homolog is identified in the previous study [49]. (B) The spectral counts of the related proteins identified from each gland. The pathway numbers are linked to panel (A). p., predicted.

Fig 7. Validation of variable protein expression levels based on immunoblotting analyses.

Immunoblotting analysis for MRJP2, aldolase, acetyl-CoA acyltransferase 2, and IDGF4 were performed with two independent biologic samples (red and green bars), and the band intensity for each protein in each gland was normalized to that of beta-actin. The normalized protein expression levels in each gland were compared with the spectral counts (SC) of each protein in each gland normalized to that of beta-actin (blue bars), taking the protein expression levels and spectral counts of nPcG as 1.