Proteomic analysis in the Dufour’s gland of Africanized Apis mellifera workers (Hymenoptera: Apidae)

Abstract

The colony of eusocial bee Apis mellifera has a reproductive queen and sterile workers performing tasks such as brood care and foraging. Chemical communication plays a crucial role in the maintenance of sociability in bees with many compounds released by the exocrine glands. The Dufour’s gland is a non-paired gland associated with the sting apparatus with important functions in the communication between members of the colony, releasing volatile chemicals that influence workers roles and tasks. However, the protein content in this gland is not well studied. This study identified differentially expressed proteins in the Dufour’s glands of nurse and forager workers of A. mellifera through 2D-gel electrophoresis and mass spectrometry. A total of 131 spots showed different expression between nurse and forager bees, and 28 proteins were identified. The identified proteins were categorized into different functions groups including protein, carbohydrate, energy and lipid metabolisms, cytoskeleton-associated proteins, detoxification, homeostasis, cell communication, constitutive and allergen. This study provides new insights of the protein content in the Dufour’s gland contributing to a more complete understanding of the biological functions of this gland in honeybees.

Full text

RESEARCH ARTICLE
Proteomic analysis in the Dufour’s gland of
Africanized Apis mellifera workers
(Hymenoptera: Apidae)
Aparecida das Dores Teixeira
1☯
, Patricia D. Games
1☯
, Benjamin B. Katz
2☯
, John
M. Tomich
2☯
, Jose
´C. Zanuncio
3☯
, Jose
´Eduardo Serrão
1☯
*
1Department of General Biology, Universidade Federal de Vic¸osa, Vic¸osa, Minas Gerais, Brazil,
2Biotechnology Core Facility and Department of Biochemistry and Molecular Biophysics, Kansas State
University, Manhattan, Kansas, United States of America, 3Department of Entomology, Universidade
Federal de Vic¸osa, Vic¸osa, Minas Gerais, Brazil
☯These authors contributed equally to this work.
*jeserrao@ufv.br
Abstract
The colony of eusocial bee Apis mellifera has a reproductive queen and sterile workers per-
forming tasks such as brood care and foraging. Chemical communication plays a crucial
role in the maintenance of sociability in bees with many compounds released by the exo-
crine glands. The Dufour’s gland is a non-paired gland associated with the sting apparatus
with important functions in the communication between members of the colony, releasing
volatile chemicals that influence workers roles and tasks. However, the protein content in
this gland is not well studied. This study identified differentially expressed proteins in the
Dufour’s glands of nurse and forager workers of A.mellifera through 2D-gel electrophoresis
and mass spectrometry. A total of 131 spots showed different expression between nurse
and forager bees, and 28 proteins were identified. The identified proteins were categorized
into different functions groups including protein, carbohydrate, energy and lipid metabo-
lisms, cytoskeleton-associated proteins, detoxification, homeostasis, cell communication,
constitutive and allergen. This study provides new insights of the protein content in the
Dufour’s gland contributing to a more complete understanding of the biological functions of
this gland in honeybees.
Introduction
Bees play a key role in agriculture and global ecosystems, pollinating plants in native areas as
well as high value agricultural crops [1,2]. They have different levels of social behavior, from
solitary to eusocial, whose colony has a reproductive queen and thousands of sterile or semi-
sterile workers, performing all other tasks for colony maintenance [3–5].
The honeybee Apis mellifera (Hymenoptera: Apidae) is a eusocial insect with a remarkable
labor division [6]. Until two-three weeks of age, adult workers perform tasks inside the nest,
including brood care and are named nurses [5]. After this period, the workers perform tasks
PLOS ONE | https://doi.org/10.1371/journal.pone.0177415 May 24, 2017 1 / 16
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OPEN ACCESS
Citation: Teixeira AdD, Games PD, Katz BB,
Tomich JM, Zanuncio JC, Serrão JE (2017)
Proteomic analysis in the Dufour’s gland of
Africanized Apis mellifera workers (Hymenoptera:
Apidae). PLoS ONE 12(5): e0177415. https://doi.
org/10.1371/journal.pone.0177415
Editor: Stephen J. Martin, University of Salford,
UNITED KINGDOM
Received: February 6, 2017
Accepted: April 26, 2017
Published: May 24, 2017
Copyright: ©2017 Teixeira et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
whithin the paper and its Supporting Information
file.
Funding: This study was supported by Brazilian
research agencies CNPq and FAPEMIG. These
funder supplied grants for usage in reagents. All
the funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared
that no competing interest exist.

out of colony including water, nectar, pollen and/or resin collection and are named foragers
[5].
The social organization of the bees is maintained by a complex communication system that
involves the pheromones release by various exocrine glands [6]. Among the exocrine gland of
the bees, are those attached to the sting apparatus, including venom and Dufour’s glands [7].
The venom gland originates from glands associated with the ovipositor of ancestors Hyme-
noptera, whereas the Dufour’s gland is homologous to the lateral glands of other insects [8,9].
A set of functions has been attributed to the Dufour’s gland secretion in different Hyme-
noptera, such as lining brood cells, nest building, larval feeding, in addition to its role in com-
munication including hosts marker, nest and nest-mate recognition, fertility sign, trail [10],
alarm [11] and sexual pheromones [12,13]. Although the main compounds released by
Dufour’s gland have been characterized as volatiles [10,11], the proteins present in the gland
remains poorly studied.
The objective of this study was to characterize those proteins expressed differentially in the
Dufour’s gland of nurse and forager workers of A.mellifera, contributing with new data for the
comprehension of the biological functions of this gland in the labor division.
Materials and methods
Biological samples
Africanized Apis mellifera were collected from Central Apiary of the Universidade Federal de
Vic¸osa, Vic¸osa, Minas Gerais, Brazil (20˚ 45’ S 42˚ 52’ W). For this study, we analyzed 400
workers randomly collected from six colonies and in different dates. Two hundred nurse
workers were collected in the brood areas inside the nest and 200 forager ones were collected
in the nest entrance with pollen grains loaded in their hind legs. Nurse and forager workers
were cryo-anesthetized, dissected and their Dufour’s glands were removed and stored in 1%
(v/v) aqueous solution of protease inhibitors (Sigma P2714) at -20˚C until use.
According Brazilian laws there is no need for ethical requirements for studies on
invertebrates.
Protein extraction
The samples of Dufour’s glands were dried using a Speed Vac concentrator (Concentrator
Plus 5305, Eppendorf, Hamburg, Germany) and resuspended in 100 μL of lysis buffer (7 M
urea, 2 M thiourea, 4% (w/v) 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate
(CHAPS), and 40 mM dithiothreitol (DTT). Then, the samples immersed in an ice bath were
submitted to successive ultrasound pulses (2 min) centrifuged at 15,000 x g for 30 minutes at
4˚C and the supernatant collected. Total protein concentration was determined according to
Bradford assay [14] using BSA as a standard.
Two-dimensional electrophoresis and image acquisition
The samples were loaded onto immobilized pH gradient (IPG) strips (linear pH 3–10, 7 cm,
GE Healthcare, USA). The strips were rehydrated for 12 h in 125 μL of rehydration solution
(DeStreak—GE Healthcare, USA) containing 150 μg protein, 2% (v/v) ampholyte pH 3–10
(GE Healthcare) and 40 mM DTT. Isoelectric focusing (IEF) was performed in Ettan IPGphor
III apparatus (GE Healthcare) at 20˚C, with a maximum current of 50 mA/strip for approxi-
mately 14 h using the following program: 12 h at 300 V, 1000 V for 30 min, 5000 V for 80 min
and 5000 V for 25 min. The IPG strips were first equilibrated in 5 mL equilibration buffer I [75
mM Tris-HCl, pH 8.8, 6 M urea, 25.5% (v/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS),
Proteomic analysis in the Dufour’s gland of the honeybee worker
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0.002% (w/v) bromophenol blue and 1% (w/v) DTT] for 15 min and later in 5 mL equilibra-
tion buffer II [75 mM Tris-HCl, pH 8.8, 6 M urea, 25.5% (v/v) glycerol, 2% (w/v) sodium
dodecyl sulfate (SDS), 0.002% (w/v) bromophenol blue and 2.5% (w/v) iodoacetamide] for 15
min. After equilibration, the strips were transferred to an SDS-polyacrylamide, 14% separating
gel in Mini-PROTEAN Tetra cell (Bio-Rad, USA). The electrophoresis was performed at 100
V for 40 min and 60 V until the dye front reached the bottom of the gel. Gels were fixed in
40% (v/v) methanol with 10% (v/v) acetic acid for 30 min, washed in water, and stained with
staining solution [0.8% (v/v) phosphoric acid, 20% (v/v) methanol, 8% (w/v) ammonium sul-
fate and 0.08% (w/v) Coomassie Brilliant Blue G-250 for 36 h. After this time, the gels were
stored in solution containing 5% (v/v) acetic acid.
For each nurse or forager workers were carried out three gels, representing a triplicate.
The gels were digitized with Image Scanner III (GE Healthcare) at 16 bit and 600 dpi resolu-
tion. The images were analyzed using Image Master 2D Platinum 7.0 software (GE Healthcare,
USA). ANOVA was carried out a threshold of p 0.05, and those spots with 1.2-fold change
in average spot volume between nurse and forager workers were used for the mass spectrome-
try analyses.
In-gel extraction and trypsin digestion of proteins for MALDI analysis
The differentially expressed spots in the Dufour’s gland of A.mellifera nurse and forager work-
ers were manually excised from each gel and transferred to 0.6 mL microcentrifuge tubes con-
taining solution of 50% (v/v) acetonitrile (ACN) and 25 mM ammonium bicarbonate
(NH
4
HCO
3
). The samples were washed 1–4 times in this last solution until the gel pieces
become translucent white. The gel slices were dehydrated for 5 min with 100% (v/v) ACN
twice and dried at Speed Vac. Dried gel pieces were reduced with 65 mM DTT in 100 mM
NH
4
HCO
3
for 30 min at 56˚C and alkylated with 200 mM iodoacetamide for 30 min in the
dark at room temperature. The gel pieces were incubated with 100 mM NH
4
HCO
3
and rehy-
drated with 100% (v/v) ACN for 10 min and dried using Speed Vac. Then the gel slices were
digested in solution containing 40 mM NH
4
HCO
3
, 10% (v/v) ACN and 25 ng μL
-1
trypsin
(Trypsin Gold, Mass Spectrometry Grade, Promega, USA) overnight at 37˚C. Peptides were
extracted from the gel slices by incubating them with 5% (v/v) formic acid, 50% (v/v) ACN for
15 min. After incubation, gel slices were sonicated, and then the peptide solutions were trans-
ferred to a clean tube and dried in the Speed Vac to a final volume of 10 μL. The samples were
desalted using a Zip-Tip C18 cartridge (Millipore, USA), dried in the Speed Vac and resus-
pended in 0.1% (v/v) aqueous trifluoroacetic acid (TFA) for MALDI mass analysis.
Mass spectrometry and bioinformatics analyses
Desalted peptides samples from Dufour’s glands of both nurse and forager workers were
mixed with α-cyano-4-hydroxycinnamic acid matrix [10 mg mL
-1
in 50% (v/v) ACN, 0.1%
(v/v) TFA] prepared in 1:1 ratio and the resulting 2 μL was spotted onto the MALDI plate
MTP Anchor Chip TM 600/384 TF (Bruker Daltonics, Alemanha). The mass spectrometry
(MS) analyses were performed on Ultraflex III MALDI TOF/TOF mass spectrometer (Bruker
Daltonics, Germany). Spectra were acquired in the positive-ion reflectron mode with a mass
range at 66.7 Hz with a 1000 laser shots added per spectrum. The MS/MS spectra were
acquired in LIFT mode and 3000 laser shots. MS and MS/MS spectra were analyzed using
FlexAnalysis 3.3 software (Bruker Daltonics, Germany) and calibrated internally with auto-
proteolysis peptides of trypsin. All known contaminants (trypsin autoproteolysis and known
keratin peaks) were excluded during the process. MS/MS spectra were processing using Sort
Neaten Assign and Place (SNAP). Spectra were searched against the A.mellifera database
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(downloaded from uniprot.org, last update April 13, 2015) using the computer program Mas-
cot (Mascot Daemon). Search parameters were as follows: taxonomy, other Metazoa; trypsin
cleavage; allow up to one missed cleavage; peptide mass tolerance for MS, 0.5 Da; peptide mass
tolerance for MS/MS, 0.2; fixed modification, carbamidomethyl (C); variable modification,
oxidation (M). After identification by Mascot, the proteins were validated using Scaffold v.4.0
software. Peptide and Protein Prophet algorithms were used to establish the peptide and pro-
tein threshold at 90% respectively, with at least one unique peptide matches required in each
sample. Proteins identified and validated were then analyzed using Uniprot database (http://
www.uniprot.org/) to suggest probable function. Further, all the significant proteins were ana-
lyzed using various bioinformatics programme includes CELLO v.2.5 subcellular localization
predictor (http://cello.life.nctu.edu.tw/), SIGNAL P v.4.1 signal peptide predictor (http://www.
cbs.dtu.dk/services/SignalP/) and STRING v.10.0 database protein-protein interaction predic-
tor (http://www.string-db.org/).
Results
Comparative 2D gel electrophoresis
The 2D-electrophoresis profiles obtained from the Dufour’s gland of nurse and forager work-
ers of A.mellifera revealed 279 spots (Fig 1). The set of ANOVA (p <0.05) analyses detected
131 spots showing differential expression between nurse and forager workers (see S1 Table).
Mass spectrometry
A total of 80 protein spots from Dufour’s glands of A.mellifera workers were excised for diges-
tion and mass spectrometry analyses. The MS/MS analyses resulted in the identification of 28
spots with 24 different proteins (Fig 1A and 1B,Table 1).
Proteins in the up-regulated group in nurse workers (a total of 20 proteins identified)
included proteins that were more expressed higher in nurse workers compared to forager bees
(c.a. 15 proteins) as well as those proteins that were present only in nurse workers (c.a. 5 pro-
teins) (Fig 2,Table 2). These proteins belong to different functional categories playing roles in
the metabolisms of protein (spot 1, 23, 26, 36, 37, 61 and 62), carbohydrates (spot 18), energy
Fig 1. Representative 2-D electrophoresis proteins profile from Dufour’s gland of Apis mellifera nurse (A) and forager (B)
workers. All identified proteins by MALDI-TOF/TOF are numbered and spot numbers correspond to Table 1.
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(spots 10, 13 and 56), and lipid (spots 16, 68 and 71), detoxification (spots 32, 51 and 70),
homeostasis (spot 5), constituent protein (spot 27) and cell communication (spot 28) (Fig 2A,
Table 2).
Proteins down-regulated group in nurse workers (a total of eight proteins identified),
included those proteins with lower expression levels in the nurse workers in relation to forager
ones (c.a. one protein) as well as those different proteins presents only in forager workers (c.a.
five proteins) (Table 2). These proteins have been classified into carbohydrate (spot 2 and 14)
and lipid (78) metabolisms, cytoskeleton-associated proteins (spots 20, 33, 38 and 40) and
allergen (spot 45) (Fig 2B,Table 2).
Subcellular localization and signal peptide
Data on subcellular localization and the presence of signal peptide in the identified proteins
were evaluated taking into account secretory function of Dufour’s gland (Fig 3,Table 2). Most
Table 1. List of identified proteins spots in Dufour’s glands of Apis mellifera nurse and forager workers.
Spot number
a
Accession number Protein name MM (Da)/pI
Experimental
MM (Da)/pI
Theorical
ANOVA
b
Score
c
01 V9II55_APICE Heat Shock protein (HSP60) 57139/5.61 60546/5.64 0.000059 82
02 A0A088A4K0_APIME Phosphoglycerate mutase 31688/7.77 35396/9.36 0.000068 156
05 J7F0F7_APICC Ferritin 23473/6.42 25718/5.9 0.00037 79
10 V9IJ68_APICE Transitional endoplasmic reticulum ATPase 86885/5.57 89467/5.18 0.0012524 74
13 A0A088ARZ8_APIME Arginine kinase 42360/6.16 43726/6.11 0.0013432 414
14 V9II98_APICE Chitinase-like protein Idgf4 56395/8.13 48921/8.06 0.0014878 162
16 Q76LA4_APIME Fatty acid binding protein 10636/7.66 15140/6.37 0.0021074 61
18 A0A088AST9_APIME Enolase 49088/7.10 39821/6.54 0.0024789 336
20 V9IE14_APICE Actin-42A 45085/5.27 42121/5.3 0.004775 169
23 V9IEZ2_APICE Heat shock protein (HSP70) 65541/5.80 71383/5.43 0.0052428 504
26 A0A088ATC7_APIME Proteasome subunit alpha type 36131/6.31 31250/5.78 0.0063259 56
27 A0A088AJH1_APIME Annexin 35612/4.53 36088/4.6 0.0072667 108
28 Q1W641_APIME Odorant Binding Protein 13 (OBP13) 7873/5.95 15494/6.37 0.0090299 123
32 G0WRL1_APICC thioredoxin peroxidase 1(Tpx-1) 20221/5.82 22065/6.31 0.0111726 106
33 L0HT99_APICE Actin (Fragment) 30141/7.54 31458/4.94 0.01178530 41
36 V9IK10_APICE Stress-70 protein (HSP70) 65221/6.30 75642/6.38 0.0132579 114
37 V9IGA1_APICE Glucose-regulated protein (HSP70) 68495/5.44 72878/5.29 0.0136561 298
38 V9IE14_APICE Actin-42A 44934/5.51 42121/5.3 0.0138284 94
40 V9IE14_APICE Actin-42A 43919/5.57 42121/5.3 0.0230893 253
45 ACPH1_APIME Venom acid phosphatase Acph-1 46005/5.99 45588/5.63 0.025984 135
51 V9IJF3_APICE Glutathione S-transferase 22562/5.81 22997/5.49 0.0312186 245
56 A0A088AMB8_APIME ATP synthase subunit beta 48372/5.37 55096/5.25 0.0384374 471
61 A0A088A7Y2_APIME Protein disulfide-isomerase 55213/6.05 6220/5.57 0.0423643 157
62 V9IG54_APICE Protein lethal(2)essential for life (HSP20) 21819/6.93 21882/6.32 0.0447684 86
68 I1VC86_APIME Phospholipase A2 18400/7.66 19585/7.05 0.0200282 50
70 V9IJF3_APICE Glutathione S-transferase 23544/5.80 22997/5.49 0.0152591 141
71 A0A088A9C4_APIME Carboxylic ester hydrolase 59133/5.97 59330/5.55 0.0173495 139
78 I1VC86_APIME Phospholipase A2 18302/9.30 19585/7.05 0.0219103 107
a
Spot numbers as in 2D reference gel (see Fig 1).
b
ANOVA values from Image Master software.
c
Mascot scores; Individual ions scores >27 indicate identity or extensive homology (p<0.05).
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of the identified proteins (c.a. 57%) showed cytoplasmic subcellular localization (Fig 3A) and a
small group (c.a. 25%) of the proteins contained signal peptides (Fig 3B).
Interaction of expressed proteins
Protein-protein predicted functional interactions were assessed to obtain additional informa-
tion about the functions of the protein of the Dufour’s gland in A.mellifera workers (Fig 4).
Most of the up-regulated proteins of Dufour’s gland of nurse workers showed functional
interactions among them: heat shock protein (LOC409384), transitional endoplasmic
Fig 2. Identified proteins from Dufour’s gland of Apis mellifera workers. Up-regulated (A) and down-regulated (B)
proteins in nurse workers. Classification into functional categories was obtained from the Uniprot database.
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reticulum ATPase (TER94), enolase (LOC552678), heat shock protein (Hsc70-4), proteasome
subunit alpha type (LOC552654), thioredoxin peroxidase 1 (Tpx-1), stress-70 protein (Hsc70-
5), glucose-regulated protein (Hsc70-3), ATP synthase subunit beta (Atp5b), protein lethal (2)
essential for life (LOC410087), protein disulfide-isomerase (ERp60) and ferritin (Fer1HCH)
(Fig 4A).
The heat shock protein (LOC409384) exhibited functional interactions with most of the
protein interaction network, but there was a predominance of stronger interactions with the
glucose-regulated protein (Hsc70-3), heat shock protein (Hsc 70–4) and stress protein-70
Table 2. List of identified proteins spots in Dufour’s glands of Apis mellifera workers with their fold expression values, functional categories, sub-
cellular localization and signal peptide analyses.
Spots
number
a
Protein name Expression Fold
change
b
Functional categories Subcellular
localization
Signal
peptide
01 Heat Shock protein (HSP60) Up 16.49 Protein metabolism Cytoplasmic No
23 Heat shock protein (HSP70) Up 7.41 Protein metabolism Cytoplasmic No
26 Proteasome subunit alpha type Up 5.21 Protein metabolism Cytoplasmic/nucleus No
36 Stress-70 protein (HSP70) Up 9.92 Protein metabolism Mitochondria No
37 Glucose-regulated protein (HSP70) Up 2.93 Protein metabolism Endoplasmic reticulum No
61 Protein disulfide-isomerase Up 1Protein metabolism Endoplasmic reticulum Yes
62 Protein lethal(2)essential for life
(HSP20)
Up 2.89 Protein metabolism Mitochondria /
Cytoplasmic
No
02 Phosphoglycerate mutase Down 1Carbohydrate
metabolism
Cytoplasmic No
14 Chitinase-like protein Idgf4 Down 1Carbohydrate
metabolism
Extracellular Yes
18 Enolase Up 4.70 Carbohydrate
metabolism
Cytoplasmic No
10 Transitional endoplasmic reticulum
ATPase
Up 2.46 Energy metabolism Endoplasmic reticulum No
13 Arginine kinase Up 1.84 Energy metabolism Cytoplasmic No
56 ATP synthase subunit beta Up 1.54 Energy metabolism Mitochondria No
16 Fatty acid binding protein Up 1Lipid metabolism Cytoplasmic No
68 Phospholipase A2 Up 2.62 Lipid metabolism Extracellular Yes
71 Carboxylic ester hydrolase Up 1Lipid metabolism Cytoplasmic No
78 Phospholipase A2 Down 1Lipid metabolism Extracellular Yes
20 Actin-42A Down 1Cytoskeletal Cytoplasmic No
33 Actin (Fragment) Down 1Cytoskeletal Cytoplasmic No
38 Actin-42A Down 1Cytoskeletal Cytoplasmic No
40 Actin-42A Down 3.49 Cytoskeletal Cytoplasmic No
32 Thioredoxin peroxidase 1 (TPX
protein)
Up 3.23 Detoxification Cytoplasmic No
51 Glutathione S-transferase Up 1.67 Detoxification Cytoplasmic No
70 Glutathione S-transferase Up 4.79 Detoxification Cytoplasmic No
05 Ferritin Up 1Homeostasis Extracellular Yes
27 Annexin Up 3.16 Constitutive protein Cytoplasmic No
28 Odorant Binding Protein 13 (OBP13) Up 1Cell communication Extracellular Yes
45 Venom acid phosphatase Acph-1 Down 1Allergen Extracellular Yes
a
Spot numbers as in 2D reference gel (see Fig 1).
b
Fold change is the ratio between average normalized volume of each protein in nurse workers that of the corresponding spot in forager ones gel. 1=
means that spots of the all replicates are presents only in nurse or forager workers.
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(Hsc70-5), all them a family of heat shock proteins (HSPs), besides of interaction with ATP
synthase subunit beta (Atp5b) (Fig 4A).
The thioredoxin peroxidase 1 (Tpx-1) exhibited strong functional interactions with the
protein disulfide isomerase (ERp60), which in turn showed interactions with heat shock pro-
teins (Hsc70-3 and Hsc70-4) (Fig 4A). In addition, Tpx-1 interacted weakly with ferritin
(Fer1HCH).
All down-regulated proteins in Dufour’s gland of nurse workers including chitinase-like
protein Idgf4-like (LOC413324), phosphoglycerate mutase (LOC552736), actin fragment
Fig 3. Identified proteins from Dufour’s gland of Apis mellifera workers. (A) Classification into subcellular
localizations obtained from the CELLO v.2.5 subcellular localization predictor. (B) Peptide signal presence obtained from
the SIGNAL P v.4.1.
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(GB18527-PA), actin 42A (Arp 1), phospholipase A2 (Pla2) and venom acid phosphatase
Acph-1 (Acph-1) showed no functional interactions among themselves (Fig 4B).
Discussion
All of the proteins identified that exhibited activity in protein metabolism showed different
levels in the Dufour’s gland of nurse workers of A.mellifera such as heat shock proteins
(HSPs) superfamily: heat shock protein (HSP60), heat shock protein (HSP70), stress-70 pro-
tein (HSP70), glucose-regulated protein (HSP70) and protein lethal (2) essential for life
(HSP20). On the other hand, protein disulfide isomerase was expressed solely in the Dufour’s
gland of nurse workers. Molecular chaperones and proteases are required to the normal func-
tionality of proteins in a living cell showing synthesis/degradation activities [15], controlling
the balance between native folded functional proteins and aggregation-prone misfolded or
invalidated proteins [16]. To this effect, proteins involved in configuration of nascent peptide
chains are expressed as heat shock proteins (HSPs) and protein disulfide-isomerase. In addi-
tion, proteins with degradative function [17] such as the proteasome subunit alpha type are
also required to keep this balance.
In honeybees, HSPs are induced at high temperatures [18], under pathogenic infections
conditions [19,20] and processes of embryogenesis [21].
The protein lethal (2) essential for life in A.mellifera is up-regulated in the brain of forager
workers [22,23] and seems play some role during bee aging. However, the function of this pro-
tein in the Dufour’s gland of A.mellifera workers needs further studies.
Fig 4. Protein-protein interactions of the identified proteins from Dufour’s gland of Apis mellifera workers. Up-regulated (A) and
down-regulated (B) proteins in nurse workers in comparison with forager ones. Proteins were analyzed using the STRING database. The
numbers correspond to the protein accession numbers in the Uniprot database and are described in the text. The interactions networks
are shown in the confidence view where thicker and thinner lines represent the stronger and weaker interactions, respectively.
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All proteins discussed above are functionally related among themselves (see Fig 4A) and
seem play some role in protein metabolism of Dufour’s gland. Although most of the secreted
products released by Dufour’s gland of the bees are lipids [24,25], these proteins may be
involved in the metabolism of other proteins, with their metabolic products used in the pro-
duction of hydrocarbons, alcohols and esters, which have been reported in isolated secretions
of this gland from nurse workers [26–29].
Within proteins of the carbohydrate metabolism family, phosphoglycerate mutase and chit-
inase-like protein Idgf4 were found only in the Dufour’s gland of forager workers, whereas
enolase was more expressed in nurse ones. Both phosphoglycerate mutase and enolase are
involved in the glucose oxidation [30,31] which is the major energy substrate for various tis-
sues of bees [32,33]. The chitinase-like protein Idgf4 is an imaginal disc growth factors, related
to chitinase, but unlike these, chitinase-like protein Idgf4 has not hydrolase activity [34]. In A.
mellifera, Idgf4 has been reported to act in the larval development [35,36] and it seems to be
released and transported to target tissues via insect hemolymph [37].
The abundance of these energy conversion proteins from carbohydrate in the Dufour’s
glands of forager (phosphoglycerate mutase) and nurse (enolase) workers, suggests that this
gland in both workers have an important function in cellular processes with high energy con-
sumption, showing that Dufour’s gland remains active throughout the bee lifespan [38]. The
function of the chitinase-like protein Idgf4 found only in the Dufour’s gland of forager work-
ers remains unknown.
Proteins of energy metabolism as arginine kinase, ATP synthase subunit beta and transi-
tional endoplasmic reticulum ATPase were more highly expressed in Dufour’s gland of nurse
workers. Arginine kinase has been reported to be both a cytoplasmic and mitochondrial pro-
tein in arthropods [39,40] and is up-regulated in Apis cerana cerana workers under biotic and
abiotic stresses [41] while ATP synthase subunit beta, in addition to its role energy production,
has been reported to act as a lipophorin binding protein, the main lipoprotein in the circula-
tion of the insects [42], which cycles among peripheral tissues to exchange its lipid cargo at the
plasma membrane of target cells [43]. The transitional endoplasmic reticulum ATPase, in
turn, has been characterized as key mediator involved in ER-associated degradation (ERAD), a
major quality control process in the protein secretary pathway [44,45].
These enzymes may function in high energy reactions required for the transport of sub-
stances from the hemolymph, which mediates chemical exchanges among tissues and organs,
besides to serving in water storage [46,47] or even modifying compounds in the gland cells
prior their release into the gland lumen. The morphology of cells in the Dufour’s gland of
queens and workers honeybees, suggest that they are able to uptake substances from hemo-
lymph, as circulating fatty acids [25,48–51], which may be used as substrates in the production
of hydrocarbons and esters found in the Dufour’s gland secretions [26–29,52,53].
The presence of proteins involved in lipid metabolism include fatty acid binding protein
and hydrolase carboxylic ester, present only in nurse workers, and phospholipase A2, present
in both nurse as forager in Dufour’s gland of A.mellifera is logical because the principal prod-
ucts released by the gland are lipid-based compounds [24,25,52,53].
Fatty acid binding proteins transfer fatty acids for cell membranes and mediate the effect of
fatty acids on gene expression in different tissues [54] while carboxylic ester hydrolases cata-
lyze the hydrolysis of an ester bond resulting in the formation of an alcohol and a carboxylic
acid [55]. In addition, phospholipases A2 (Pla2s) catalyze the hydrolysis of the sn-2 acyl glycer-
ophospholipd releasing free fatty acids and lysophospholipids [56]. All proteins described
above seem be involved in the production of lipids compound and its precursors found in the
Dufour’s gland secretion in honey bees [26–29,52,53].
Proteomic analysis in the Dufour’s gland of the honeybee worker
PLOS ONE | https://doi.org/10.1371/journal.pone.0177415 May 24, 2017 10 / 16

A hypothetical model has been suggested for esters production in Dufour’s gland of queens
and workers of A.mellifera, in which esters are synthesized by the elongation of fatty acids into
long chain acyl groups, following by their conversion to alcohols and subsequent esterification
by acyl-CoAs [57–59]. Eicosanol, an alarm pheromone [28,60] from workers may be an inter-
mediate in the secretory pathway of esters in the Dufour’s gland [59]. In the presence of the
queen, this secretory pathway is stopped, and the workers produce only precursor esters, but
in queenless colonies, this route proceeds to esters [59]. This may explain the increase in the
expression of lipid metabolism proteins in Dufour’s gland of nurse workers that potentially
may lay eggs in the absence of the queen [61,62] since the secretion of this gland has been
reported to be related to the reproductive status of eusocial bees [59].
The detoxification and oxidative stress proteins, thioredoxin peroxidase-1 (Tpx-1) and glu-
tathione S-transferase (GST) were up-regulated in Dufour’s gland of nurse workers. These pro-
teins are functionally related (see Fig 4A), degrading potentially harmful substances such as
reactive oxygen species and acting in the detoxification of endogenous and xenobiotic com-
pounds [63,64]. They are also important in intercellular transport, horm one biosynthesis and
insect resistance to insecticides [65,66]. In adult worker bees, GSTs have been reported in the
muscle, brain, fat body, epidermis, midgut, and fat body [67] and venom [68].
The up-regulation of these detoxification proteins in nurse workers, may be due to the
higher metabolic activity of Dufour’s gland than in forager ones, because the cell metabolism
results in the production of reactive oxygen species that are toxic for cells [69].
Ferritin was expressed only in the Dufour’s gland of nurse worker. This protein transport
iron ions contributing to cell homeostasis [70]. Furthermore, it seems to play role in detoxifi-
cation process in some insects [71], sharing biological functions with Tpx-1 (see Fig 4A). Stud-
ies on insect ferritins revealed that most insects have abundant ferritin in the hemolymph and
in the secretory pathways of the cells [72]. Its high expression in Dufour’s gland of A.mellifera
nurse workers is not clear.
Odorant binding protein OBP-13 plays important role in chemical communication in
insects [73]. The genome of A.mellifera predict 21 OBPs (OBP 1–21) [74,75], which are small
water soluble molecules, binding reversibly to messenger molecules such as pheromones that
are generally hydrophobic molecules with low solubility [76,77]. Although OBPs are com-
monly expressed in sensory organ of insects such as gustatory and olfactory sensilla [77], they
have been also reported in ovary, brain, tegument [74,78], mandibular gland [79], hemolymph
[37] and head salivary gland [80] of social bees. In the present work, OBP-13 was up-regulated
in the Dufour’s gland of younger nurse workers similarly to that previously reported to A.mel-
lifera which OBP-13 expression is caste and age regulated, with high expression in younger
workers [81]. Its high expression in nurse workers may be associated with increased produc-
tion of pheromones by the Dufour’s gland in these bees.
An intriguing finding is the presence of venom acid phosphatase Acph-1 in the Dufour’s
gland of A.mellifera forager workers. This is an enzyme of bee venom [68,82] that hydrolyzes
phosphomonoesters at acidic pHs. The Acph-1 of bee venom is a potent allergen, triggering
strong reactions in humans [83]. The presence of this protein in the Dufour’s gland of forager
workers might be due a possible contamination from the venom gland, but during the removal
of the Dufour’s gland there were no apparent disruptions of the venom reservoirs. Thus, this
protein may have another function in these worker bees.
The chemical composition of Dufour’s gland secretion in insects has been identified mainly
by gas chromatography and mass spectrometry, as being composed for saturated or unsatu-
rated long chain hydrocarbons, although other compounds may also occur in this gland [10,
24,52,53,84–86]. The principal enzymes for the hydrocarbons biosynthesis are fatty acid
synthases, elongases, desaturases, reductases and P450 decarboxylase [12,87]. In the honey bee
Proteomic analysis in the Dufour’s gland of the honeybee worker
PLOS ONE | https://doi.org/10.1371/journal.pone.0177415 May 24, 2017 11 / 16

A.mellifera, six desaturase and 10 elongases genes have been predict with some them expressed
differentially in nurse and forager workers [88]. However, these hydrocarbon metabolism
enzymes were not identified in this study, perhaps because as small they were not detected
using a 2D gel/MALDI-TOF/TOF protocol or because we only analyzed those spots with sig-
nificant difference in the two-dimensional electrophoresis suggesting that these enzymes are
expressed equally in both the Dufour’s gland of nurse and forager workers. This result suggests
that further studies are necessary to identify the complete profile of proteins of the Dufour’s
gland of A.mellifera.
This is the first descriptive and comparative proteomic study to characterize proteins in the
Dufour’s gland of A.mellifera. Our results showed differences in the quantity and types of pro-
teins in the Dufour’s gland when nurse workers change to forager ones, suggesting that this
gland has different roles according to bee age and social behavior. Despite its simple structural
complexity, the Dufour’s gland of bees seem to have a great functional complexity, since until
now, its major role has been reported as the production of lipid other than proteins. This study
can be useful for the comprehension of some molecular mechanisms triggered the labor divi-
sion in bees.
Supporting information
S1 Table. List of identified proteins spots in Dufour’s gland of Apis mellifera workers with
Image Master. Up-regulated and down-regulated refer to proteins with higher and lower
expression, respectively, in nurse worker in comparison with forager ones.
(PDF)
Acknowledgments
The authors thank the Nu
´cleo de Ana
´lises de Biomole
´culas of Universidade Federal de Vic¸osa,
Brazil, and Biotechnology Core Facility of Kansas State University, USA for technical
assistance.
Author Contributions
Conceptualization: ADT JES.
Data curation: ADT PDG BBK.
Formal analysis: ADT PDG JMT JES.
Funding acquisition: JES JCZ.
Investigation: ADT PDG BBK.
Project administration: JES.
Resources: JES JCZ JMT.
Supervision: JES.
Visualization: ADT PDG JMT JCZ JES.
Writing – original draft: JCZ JMT JES.
Writing – review & editing: ADT JCZ JMT JES.
Proteomic analysis in the Dufour’s gland of the honeybee worker
PLOS ONE | https://doi.org/10.1371/journal.pone.0177415 May 24, 2017 12 / 16

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Proteomic analysis in the Dufour’s gland of the honeybee worker
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