Article
pubs.acs.org/jpr
Proteomic Analysis Reveals the Molecular Underpinnings of
Mandibular Gland Development and Lipid Metabolism in Two Lines
of Honeybees (Apis mellifera ligustica)
Xinmei Huo,†,# Bin Wu,†,# Mao Feng,† Bin Han,† Yu Fang,† Yue Hao,† Lifeng Meng,†
Abebe Jenberie Wubie,‡ Pei Fan,† Han Hu,† Yuping Qi,† and Jianke Li*,†
†
Institute of Apicultural Research/Key Laboratory of Pollinating Insect Biology, Ministry of Agriculture, Chinese Academy of
Agricultural Science, Beijing 100093, China
‡
Department of Animal production and Technology, College of Agriculture and Environmental Sciences, Bahir Dar University, Bahir
Dar, Ethiopia
S Supporting Information
*
ABSTRACT: The mandibular glands (MGs) of honeybee workers are vital for the
secretion of lipids, for both larval nutrition and pheromones. However, knowledge of
how the proteome controls MG development and functionality at the different
physiological stages of worker bees is still lacking. We characterized and compared the
proteome across different ages of MGs in Italian bees (ITBs) and Royal Jelly (RJ) bees
(RJBs), the latter being a line bred for increasing RJ yield, originating from the ITB.
All 2000 proteins that were shared by differently aged MGs in both bee lines (>4000
proteins identified in all) were strongly enriched in metabolizing protein, nucleic acid,
small molecule, and lipid functional groups. The fact that these shared proteins are
enriched in similar groups in both lines suggests that they are essential for basic
cellular maintenance and MG functions. However, great differences were found when
comparing the proteome across different MG phases in each line. In newly emerged
bees (NEBs), the unique and highly abundant proteins were enriched in protein
synthesis, cytoskeleton, and development related functional groups, suggesting their
importance to initialize young MG development. In nurse bees (NBs), specific and
highly abundant proteins were mainly enriched in substance transport and lipid
synthesis, indicating their priority may be in priming high secretory activity in lipid
synthesis as larval nutrition. The unique and highly abundant proteins in forager bees (FBs) were enriched in lipid metabolism,
small molecule, and carbohydrate metabolism. This indicates their emphasis on 2-heptanone synthesis as an alarm pheromone to
enhance colony defense or scent marker for foraging efficiency. Furthermore, a wide range of different biological processes was
observed between ITBs and RJBs at different MG ages. Both bee stocks may adapt different proteome programs to drive gland
development and functionality. The RJB nurse bee has reshaped its proteome by enhancing the rate of lipid synthesis and
minimizing degradation to increase 10-hydroxy-2-decenoic acid synthesis, a major component of RJ, to maintain the desired
proportion of lipids in increased RJ production. This study contributes a novel understanding of MG development and lipid
metabolism, and a potential starting point for lipid or pheromone biochemists as well as developmental geneticists.
KEYWORDS: proteome, mandibular glands, honeybee workers, 10-hydroxy-2-decenoic acid
1. INTRODUCTION
The pheromonal communication system of the honeybees is
one of the most complex systems in nature, utilizing a dozen
known glands and a vast array of different pheromones, which
are released by individual bees and result in changes to the
physiology and behavior of other bees.5,6 Among these
pheromonal glands, the twin mandibular glands (MGs) are
one of the primary pheromone-producing glands of the
honeybee. The MGs are located inside the head, above the
base of the mandible, and consist of a pair of sack-like glands
The honeybee (Apis mellifera L.) is a highly organized eusocial
insect.1 The members of a honeybee colony, including one
queen, a few drones, and thousands of workers (infertile
females), carry out their respective functions for the good of the
species. These functions are selected based on caste, sex, age,
and environmental conditions.2,3 As the most dominant
population and labor force in a colony, worker bees generally
perform a wide variety of age-dependent tasks.4 Their highly
organized social order is achieved by a range of communication
mechanisms including pheromones, the dance language, visual
and mechanical senses, and others.5,6
© 2016 American Chemical Society
Received: June 7, 2016
Published: August 12, 2016
3342
DOI: 10.1021/acs.jproteome.6b00526
J. Proteome Res. 2016, 15, 3342−3357
Article
Journal of Proteome Research
proteome-wide understanding of honeybee biology, including
embryogenesis,37,38 brain functions,34,39 and those systems
responsible for HG secretions and functions.40 To date,
however, only a few studies have been published regarding
the caste-specific genes and proteins expressed in the MGs of
queens, drones, and worker bees. For instance, caste-selective
gene expression is found in the MGs of queens and worker
bees,41−43 and odorant-binding proteins and chemosensory
proteins are also selectively expressed in the MGs of queen,
worker, and drone bees.44 Despite the organ’s functional
importance in producing fatty acid larval food and pheromones,
knowledge of how MG proteome changes in worker bees drive
gland development and lipid synthesis was still very limited.
Thus, we carried out an in-depth characterization and
comparison of the MG proteomes of both ITB and RJB to
determine the regulatory mechanisms of MG development, and
lipid or pheromone metabolism that affect the adult lives of
both bee varieties.
covered entirely with secretory cells, which contribute to castedependent social communication.7,8
The pheromones produced in the MGs of a queen bee have
numerous functions, such as inhibiting excess queen
production, swarming,9 and worker ovary development.10 On
the other hand, the MG secretions of the worker bees contain
mainly volatile substances used as trail pheromones for intraand interspecific communication.7,11,12 For example, 2heptanone, which is produced in the MGs of guard and
forager bees as an alarm substance, when combined with
isopentyl acetate from the Koschevnikov gland and 15 alarm
components of the sting gland,13,14 induces the stinging
behavior of the bees to defend the colony.14 2-Heptanone
also compels other worker bees to strongly reject all flowers
that have been recently visited to improve their foraging
efficiency;7,12 its amount increases progressively with age and is
typically higher in foraging bees than in either newly emerged
(NEBs) or nurse bees (NBs).7 In addition to the above roles in
regulating social activity, a major function of the worker MGs is
the secretion of fatty acids, which are used as larval nutrition.15
At least 6 kinds of fatty acids have been found in the MGs of
worker bees; 10-hydroxy-2-decenoic acid (10-HDA); its
precursor 10-hydroxydecanoic acid (10-HDAA); 2(E)-decenedioic acids; decanedioic acids; 9-keto-2(E)-decenoic acid (9ODA); and its precursor 9-hydroxy-2(E)-decenoic acid (9HDA).16,17 Among these fatty acids, 10-HDA and its precursor
10-HDAA are the most abundant. 10-HDA mainly functions as
a means of larval nutrition and an antiseptic for preserving
larval food,18,19 but also acts as a pheromone to regulate other
honeybee behavior.20,21
Not only do the composition and amounts of MG secretions
vary depending on the caste of bees, as described above, but
also depending on the age of the worker bees. The NBs tend to
have greater quantities of 10-HDA and 10-HDAA than forager
bees (FBs)22 and the ω-hydroxy acids 10-HDA, 10-HDAA and
their corresponding diacids are found in the RJ secreted by
NBs.23,24 These acids are major lipid components in RJ. In
addition to its various functions as a pheromone and nutrient
for honeybees, 10-HDA, often referred to as “RJ acid,” is a fatty
acid unique to RJ and widely regarded as the most important
quality criterion in RJ assessment.25 10-HDA has also been
reported to have a wide variety of pharmacological activities,
which promote neurogenesis,26 increased lifespan,27 inhibition
of angiogenesis,28 modulating the immune system,29 and
antitumor functions in mammals.30,31
RJ is secreted by the hypopharyngeal glands (HGs) in the
NBs, and is important for the promotion of human health,
particularly in Asia.32 Hence, breeding honeybees to increase RJ
yield is a major goal in Asia, although globally, increasing
pollination would be a greater economic driver. Since the
1980s, higher RJ yield has derived from breeding a strain of
high-RJ producing honeybee (RJB) from the Italian bee (ITB,
Apis mellifera L.) in China, which can produce 10 times more
RJ than the ITB.32−34 The RJB is now a major producer of RJ
in China, with a yearly production of ∼3500 tons of RJ,
accounting for >90% of the world total.34,35 The increased RJ
yield by RJBs was identified as an inheritable trait in 2003 by
our group.36 Our recent studies have revealed that the RJB has
reshaped the proteome settings of the HGs and nervous system
to support its biological performance, thus increasing the
secretion of RJ.32,34
Recent advances in mass-spectrometry (MS)-based proteomics have allowed the investigation of mechanisms underlying a
2. MATERIALS AND METHODS
2.1. Chemicals
Modified sequencing-grade trypsin was bought from Promega
(Madison, WI, USA) and all the other chemical reagents were
bought from Sigma-Aldrich (St. Louis, MO, USA) unless
otherwise specified. All the reagents were analytical grade or
better.
2.2. Protein Preparation and Digestion
Artificially inseminated queens (Apis mellifera ligustica) of
Italian bees (ITB) and high royal jelly producing bees (RJB) of
similar ages by the semen of each bee stock itself were obtained
from Bologna, Italy, and Zhejiang Province, China, respectively.
Five colonies of each stock with similar colony strength, headed
by those queens, were used for egg-laying and colony build-up.
All the bees were maintained at the experimental apiary of the
Institute of Apicultural Research, Chinese Academy of
Agricultural Sciences, Beijing. Some NEBs (emerged from
comb cells >10 h in an incubator) were marked on the thorax
and then placed back into colonies for subsequent development, and others were directly sampled. The NEBs that were
marked were later collected between day 8 to day 10 as samples
of NBs, once head extension into young larval cells was
observed. The FBs, carrying pollen loads, were collected at the
entrances of hives (∼20 days old). Once the bee samples were
obtained, the MGs were dissected from the heads using a
binocular microscope. In total, more than 300 MGs from five
colonies of each stock were sampled at each life stage of NEBs,
NBs and FBs.
Protein extraction was carried out according to the method
previously described.45 In brief, the MGs were homogenized
with lysis buffer (8 M urea, 2 M thiourea, 4% CHAPS, 20 mM
Tris-base, 30 mM dithiothreitol (DTT), 1 mg/10 μL) and
protease inhibitors (Roche, Basel, Switzerland) on ice for 30
min. The homogenate was centrifuged at 15 000g, 4 °C for 20
min. The collected supernatant was precipitated with three
volumes of ice-cold acetone for 30 min. Then the mixture was
centrifuged at 15 000g, 4 °C for 20 min. The precipitated
pellets were resuspended in 100 μL of 5 M urea, followed by
dissolving in four volumes of 40 mM NH4HCO3. The final
protein concentration was quantified using a Bradford assay.
3343
DOI: 10.1021/acs.jproteome.6b00526
J. Proteome Res. 2016, 15, 3342−3357
Article
Journal of Proteome Research
2.3. LC−MS Analysis
the expectation-maximization algorithm, and feature alignment
of the same peptide from three replicates of each sample were
done by an algorithm for high-performance retention time.47
Then, the protein abundance levels of MGs in all samples of
both bee lines was quantified by the sum of the three most
abundant ion peak intensities of the tryptic peptides. The
protein abundance levels were compared over three stages in
each bee stock and at three stages between both bee lines.
Statistically, proteins and peptide features were considered to
be significantly changed between different samples only when
they had both p-value < 0.05 and the fold change ≥2. The
protein expression profile was done by hierarchical clustering to
create an expressional profile of differentially expressed protein
groups during MG development, and visualized by PEAKS Q
module (version 7.0, Bioinformatics Solutions Inc.).
The protein sample was mixed with a solution of 10 mM DTT
for 1 h to break the proteins’ disulfide bonds. Then, it was
incubated in 50 mM iodoacetamide for alkylation at room
temperature for 1 h in the dark. Proteins were digested using
trypsin (sequencing grade) in a volume ratio of 1:50 (enzyme/
protein) at 37 °C for 14 h. The enzymatic digestion was
stopped by adding 1 μL of formic acid to the solution. Finally,
peptides were extracted using a SpeedVac system (RVC 2−18,
Marin Christ, Osterod, Germany) for the following LC−MS
analysis.
The peptide sample was loaded onto an LC−MS system with
three replicates. The nano scale liquid chromatography system
EASY-nLC 1000 (Thermo Fisher Scientific) was coupled with a
mass spectrometer Q-Exactive (Thermo Fisher Scientific) via
the nano electrospray source. Reverse-phase chromatography
and a trap column packed with 2 cm long, 100 μm inner
diameter fused silica trap column containing 5.0 μm Aqua C18
beads (Thermo Fisher Scientific) were used for peptide
enrichment. The peptides were loaded onto the trap column
at a flow rate of 5 μL/min, and then eluted from the analytical
column (15 cm long, 75 μm inner diameter fused silica column
filing with 3.0 μm Aqua C18 beads, Thermo Fisher Scientific).
Peptides were gradient eluted at a flow rate of 350 nL/min with
the following conditions: 100% buffer A (0.1% formic acid) to
8% buffer B (0.1% formic acid, 80% acetonitrile) for 10 min, 8%
to 20% buffer B for 80 min, 20 to 30% buffer B for 20 min, 30%
to 90% buffer B for 5 min, then 90% buffer B for 10 min. The
eluted peptides from the analytical column were directly
injected into the mass spectrometer via nano-ESI source. Ion
signals were collected in a data-dependent mode with the
following settings: full scan resolution at 70 000, scan range: m/
z 300−1800; precursor ions were fragmented by high energy
collision-induced dissociation mode with MS/MS scan
resolution of 17 500, isolation window 2 m/z, normalized
collision energy of 27, loop count 20. Dynamic exclusion was
also used (charge exclusion: unassigned 1 > 8; peptide match:
preferred; exclude isotopes: on; dynamic exclusion: 10 s). The
MS/MS data was collected and saved in raw files through the
Xcalibur software (version 2.2, Thermo Fisher Scientific).
2.6. Bioinformatics Analysis
To enrich the biological groups and KEGG pathway, the
identified proteins were submitted to ClueGOv2.1.6, a
Cytoscape plug-in (http://www.ici.upmc.fr/cluego/) software.48 The significantly enriched gene ontology (GO)
categories were reported using a right-sided hypergeometric
test, which compares the background set of GO annotations in
the whole genome of Apis mellifera L. The false discovery rate
(FDR) was controlled by the Bonferroni step-down test to
correct the p-value.
To better understand the protein−protein interactions
among the highly expressed proteins of each time point, we
constructed protein−protein interaction (PPI) networks
through GeneMANIA, a Cytoscape plug-in.49 The available
integrated and predicted PPI data sets of Drosophila
melanogaster were embedded into GeneMANIA with the
following settings: all networks enabled, equal weighting by
GO biological process, and the top 20 related genes displayed.
The GO category enrichment of the input data set was
employed using a right-sided hypergeometric test in
GeneMANIA and the FDR was done by q-values. Proteins
were then grouped based on their GO annotations and
networks were visualized in Cytoscape.
2.7. RJ Collection and Determination of 10-HDA Content in
RJ
2.4. Protein Identification
To produce RJ for quantification of 10-HDA content, the same
five colonies of RJB and ITB mentioned above, were managed
for RJ production with almost identical populations, food, and
brood levels at the experimental apiary of the Institute of
Apicultural Research, Chinese Academy of Agricultural
Sciences, Beijing, as previously described.50 Each batch of RJ
was collected from 60 artificial plastic queen cell cups around
70 h after larvae (∼24 h old) were grafted and RJ weight was
measured with a digital scale (Mettler Toledo). A total of 10
batches of RJ samples were produced.
Before determining the 10-HDA content, an aliquot of 0.5 g
homogenated RJ from each RJB and ITB was weighed in a
polypropylene centrifuge tube and dissolved by adding 3 mL of
0.03 M HCl in a 50 mL volumetric flask. Then, 30 mL of
ethanol and 5 mL of methyl4-hydroxybenzoate solution were
added (with a final concentration of 128 μg/mL), and the
volume was compensated with ethanol. The analyte was
extracted by ultra sonification with occasional shaking. The
solution was filtered through a 0.22 μm membrane, and 5 μL of
sample solution was injected in the Agilent Technologies 1200
series liquid chromatographic system. The quality control of
10-HDA content detection was performed by the methods
The raw MS/MS data was searched against a database using
PEAKS software (version 7.0, Bioinformatics Solutions Inc.
Waterloo, Canada). The sequence database was generated by
downloading protein sequences of Apis mellifera L. from NCBI
(downloaded Feb, 8, 2015) and the common contaminants,
with a total of 27,759 entries. Search parameters were trypsin
specificity; carbamidomethyl as a fixed modification; and
oxidation as a variable modification, with two allowed missed
cleavages per peptide; three maximum allowed variable PTM
per peptide. Precursor mass tolerance was set at 30.0 ppm, and
fragment ion tolerance at 0.05 Da. The false discovery rate
(FDR) was controlled at the protein and peptide levels using a
fusion-decoy database search strategy with a threshold ≤1.0%,
an enhanced target-decoy approach that makes more
conservative FDR estimations.46 Protein identifications were
only considered confident if at least one unique peptide with at
least two spectra were identified.
2.5. Label-free Quantitation of Protein Abundance
Raw MS data was processed in the PEAKS Q module (version
7.0, Bioinformatics Solutions Inc.) to quantify the abundance of
proteins. Feature detection was conducted on each sample by
3344
DOI: 10.1021/acs.jproteome.6b00526
J. Proteome Res. 2016, 15, 3342−3357
Article
Journal of Proteome Research
Figure 1. Global proteomic view of mandibular gland (MG) development across the three stages in the Italian bees. (A) The number of shared and
unique proteins identified at three time points of MG development. (B) Enriched functional groups of the shared proteins. (C, D and E) Enriched
functional groups and pathways of the unique proteins identified in the newly emerged, nurse and foraging bees, respectively. % genes/Term stands
for the proportion of genes enriched in corresponding functional groups. The bars with the same color represent they belong to the same functional
group. The numbers stand for the genes enriched to the corresponding functional group. *, P < 0.05; **, P < 0.01.
3345
DOI: 10.1021/acs.jproteome.6b00526
J. Proteome Res. 2016, 15, 3342−3357
Article
Journal of Proteome Research
Figure 2. Quantitative proteome comparison during the mandibular gland (MG) development in Italian bees (ITBs) (fold change ≥2 and p < 0.05).
(A) Hierarchical clustering of the differentially expressed proteins in the three development stages of MGs in ITBs. The highly- and low-abundant
proteins are distinguished by red and green color, respectively. The color intensity changes with the protein expressional level as indicated on the bar.
(B, C and D) Enriched functional groups and pathways of highly abundant proteins in the newly emerged, nurse and foraging bees of ITB,
respectively. (E) Representative protein expressional level between the three MG development stages in ITBs, and a is significantly higher than b and
c; and b is significantly higher than c. % genes/Term stands for the proportion of genes enriched in corresponding functional groups. The bars with
the same color represent they belong to the same functional group. The numbers stand for the genes enriched to the corresponding functional
group. *, P < 0.05; **, P < 0.01.
described.51 A standard calibration curve was constructed with
four points of concentrations (52.8 μg/mL, 105.6 μg/mL,
211.2 μg/mL, 316.8 μg/mL of 10-HDA, and 128.0 μg/mL of
methyl 4-hydroxybenzoate as internal standard) by plotting the
peak area ratios of 10-HDA and methyl 4-hydroxybenzoate (Yaxis) against the nominal concentration of 10-HDA (X-axis).
The 10-HDA concentration of RJ was determined by Agilent
Technologies 1200 series high-performance liquid chromatography equipped with an Agilent G1312A pump, Agilent diode
array detector G1315D, and a reversed phase C18 column
(EclipseXDB C18, 5 μm, with dimensions of 4.0 × 150 mm,
Agilent Technologies). Mobile phase was composed of
methanol/0.05% H3PO4 (v/v = 55:45) at a flow rate of 1
mL/min and the pH was adjusted to 2.5 with H3PO4. The
column temperature was adjusted to 35 °C during the
experiment and the detector was adjusted to 210 nm. Finally,
data were collected through the Agilent software, ChemStation.
2.8. Quantitative Real-Time PCR
To compare the differentially expressed proteins implicated in
fatty acid metabolism at the gene level in the MGs of ITB and
RJB NBs, the MG tissue (around 20 mg for each sample) was
homogenated on ice and total RNA was extracted by an
3346
DOI: 10.1021/acs.jproteome.6b00526
J. Proteome Res. 2016, 15, 3342−3357
Article
Journal of Proteome Research
Figure 3. Global proteomic view of mandibular gland (MG) development across the three stages in high royal jelly producing bees. (A) The number
of shared and unique proteins identified across the three stages of MG development. (B) Functional groups enriched in the core proteome (shared
by three ages). (C, D and E) Functional groups enriched by the unique proteins identified in the newly emerged, nurse and foraging bees,
respectively. % genes/Term stands for the proportion of genes enriched in corresponding functional groups. The bars with the same color represent
they belong to the same functional group. The numbers stand for the genes enriched to the corresponding functional group. *, P < 0.05; **, P <
0.01.
3347
DOI: 10.1021/acs.jproteome.6b00526
J. Proteome Res. 2016, 15, 3342−3357
Article
Journal of Proteome Research
Figure 4. Quantitative proteome comparison during the mandibular gland (MG) development in high royal jelly producing bees (RJBs) (fold
change ≥2 and p < 0. 05). (A) Hierarchical clustering of the differentially expressed proteins in the three development stages of MGs in RJB. The
highly- and low-abundant proteins are distinguished by red and green color, respectively. The color intensity changes with the protein expressional
level as indicated on the bar. (B, C and D) Enriched functional groups and pathways of highly abundant proteins in the newly emerged, nurse and
foraging bees of RJB, respectively. (E) Representative protein expressional level between the three time points of MG development in RJB, and a is
significantly higher than b and c; and b is significantly higher than c. % genes/Term stands for the proportion of genes enriched in corresponding
functional groups. The bars with the same color represent they belong to the same functional group. The numbers stand for the genes enriched to
the corresponding functional group. *, P < 0.05; **, P < 0.01.
real-time PCR was conducted on iQ5Multicolor Real-Time
PCR Detection System (Bio-Rad, Hercules, USA).
RNeasy Mini kit (QIAGEN, Germany). The total RNA was
quantified by NanoDrop ND91000 spectrophotometer (Thermo Fisher Scientific) and the quality of total RNA was assessed
by 1.0% denaturing agarose gel electrophoresis by visualizing
the bands of 28S and 18S rRNA. Reverse transcription was
performed to generate cDNA by the PrimeScript RT reagent
kit (Takara Bio, Kyoto, Japan). Each sample was analyzed
individually and processed in triplicate. Four differentially
expressed proteins involved in fatty acid biosynthesis and betaoxidation in both IT and RJ NBs were selected to detect the
corresponding mRNA levels by quantitative real-time PCR.
The gene of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was used as an internal control. The differences in
gene expression were calculated and normalized with the
reference gene (GAPDH) using 2−ΔΔCt method.52 The
statistical analysis of gene expression was performed by oneway ANOVA. An error probability p < 0.05 was considered
statistically significant. The gene name, accession number, and
primer sequence are provided in Table S1. The quantitative
3. RESULTS
3.1. Comprehensive Proteome Profiling During MG
Development in the ITB
To improve our understanding of MG development and
functionality in the ITB, the MG proteome was comprehensively profiled across three stages of adult bee life. Of the 4070
proteins (FDR < 1% at peptide and protein level) identified
over the three stages, 3135, 3463, and 2595 proteins were
found in NEBs, NBs, and FBs, respectively (Figure 1A and
Table S2−4). 1840 proteins (45.2% of total) were shared by all
three stages (Figure 1A). Functional groups implicated in
substance transportation and the metabolism of proteins,
nucleic acids, small molecules, organic substances and complex
were significantly enriched by the common proteins. Pathways
related to the metabolism of proteins, carbohydrates, energy,
3348
DOI: 10.1021/acs.jproteome.6b00526
J. Proteome Res. 2016, 15, 3342−3357
Article
Journal of Proteome Research
Figure 5. Protein−protein interaction (PPI) networks of highly abundant proteins at three time points of mandibular gland development of Italian
bees (ITBs) and high royal jelly producing bees (RJBs). (A, B and C) PPI networks of highly abundant proteins in the newly emerged, nurse and
foraging bees of ITB, respectively, and (D, E and F) PPI networks of highly abundant proteins in the newly emerged, nurse and foraging bees of RJB,
respectively.
informative fact was that the proteins higher in abundance in
NEBs than in NBs and FBs were related to the translational
machinery, including 44 ribosomal protein subunits, 7 translation initiation/elongation factors and tRNA ligase, ATPdependent RNA helicase, and splicing factors (Table S6).
Moreover, 12 proteins involved in cytoskeleton formation, such
as actin-related protein 3-like, actin-interacting protein, dynein
light chain 2, paramyosin long form-like were also enhanced in
NEBs (Table S6). 53 of the highly abundant proteins in the
NBs were functionally enriched in glycolysis/gluconeogenesis
and pyruvate metabolism (Figure 2C, Table S7). Furthermore,
the highly abundant proteins in the FBs were significantly
enriched in functional groups implicated in energy, small
molecule, nucleic acid, carbohydrate and protein metabolism
functional classes and pathways (Figure 2D, Table S7). The
protein−protein interaction (PPI) network analysis further
revealed that the highly abundant proteins in the NEBs were
mainly linked to the ribosome and lipid particle functional
groups, whereas the proteins in higher abundance in the NBs
were significantly enriched in ribosome, protein transport, tube
development, and the oxidoreduction coenzyme metabolic
process. For the FBs, the highly abundant proteins were
enriched in functional groups of carbohydrate catabolic
processes, lipid particle, antioxidant activity, translational
initiations, and the extracellular matrix part in the network
(see Figure 5A, B and C).
fatty acids, and small molecules were also significantly enriched
by overlapped proteins (Figure 1B, Table S5).
Additionally, among the three stages of NEBs, NBs, and FBs,
there were 535, 724, and 248 exclusively expressed proteins,
respectively. The functional groups associated with protein
biosynthesis were significantly enriched by uniquely expressed
proteins in NEBs; including aminoacyl-tRNA biosynthesis;
translation; cellular amino acid metabolic processes; amino acid
activation; tRNA aminoacylation; and tRNA aminoacylation for
protein translation (Figure 1C, Table S5). The exclusively
expressed proteins in the NBs were significantly enriched in
substance transport, nucleic acid transport, and peptide
biosynthesis (Figure 1D, Table S5). Moreover, the exclusively
expressed proteins in FBs were significantly implicated in lipid
and isoprenoid metabolism, and aminoglycan metabolic
processes (Figure 1E, Table S5).
3.2. Quantitative Proteome Comparison During the MG
Development in the ITB
To generate a holistic view of profile changes in protein
expression during MG development, a label-free quantitative
approach was employed. Among the 4070 identified proteins,
339 proteins were differentially expressed during the MG
development, and 180, 53, and 106 proteins were expressed in
high abundance in the NEBs, NBs, and FBs, respectively
(Figure 2A, Table S6). The 180 highly abundant proteins in the
NEBs were significantly enriched in macromolecule biosynthesis, organic substances, cellular biosynthetic processes, and
protein metabolism (Figure 2B, Table S7). The most
3349
DOI: 10.1021/acs.jproteome.6b00526
J. Proteome Res. 2016, 15, 3342−3357
Article
Journal of Proteome Research
Figure 6. Proteome comparison of mandibular glands (MG) between the nurse bees of Italian bees (ITBs) and high royal jelly producing bees
(RJBs). (A) Hierarchical clustering of the differentially expressed proteins in the MGs of nurse ITBs and nurse RJBs. (B) The number of shared and
unique proteins identified in the MGs of nurse ITBs and nurse RJBs. (C) Functional groups and pathways enriched by the unique proteins identified
in nurse ITBs and nurse RJBs, respectively. (D, E) Enriched functional groups and pathways of highly abundant proteins in nurse ITBs and RJBs,
respectively. % genes/Term stands for the proportion of genes enriched in corresponding functional groups. The bars with the same color represent
they belong to the same functional group. The numbers stand for the genes enriched to the corresponding functional group. *, P < 0.05; **, P <
0.01.
3.3. Comprehensive Proteome Profiling During MG
Development in the RJB
3.4. Quantitative Proteome Comparison during the MG
Development in the RJB
Similarly, the MG proteomes of the RJB across the three ages
were also profiled. A total of 4346 proteins were identified, and
3277, 3198, and 3095 proteins were identified in NEBs, NBs
and FBs, respectively (Figure 3A and Table S8−10). There
were 2102 shared proteins (Figure 3A) overall, which were
enriched in similar functional groups as in the ITBs (Figure 3B
and Table S11).
There were 617, 278, and 329 exclusive proteins in NEBs,
NBs, and FBs, respectively. Regarding the specific proteins in
the NEBs, only the spliceosome was significantly enriched
(Figure 3C, Table S11). However, the unique proteins in the
NBs were significantly enriched in the functional groups and
pathways associated with substance transport, including protein
export, hydrogen transport, proton transport, vesicle-mediated
transport, and hydrogen ion transmembrane transport (Figure
3D, Table S11). The unique proteins in the FBs were
significantly enriched in the functional groups implicated in
lipid, small molecule, amino acid and nucleic acid metabolism
(Figure 3E, Table S11).
The proteome changes during MG development in RJBs were
also analyzed and the protein expression profiles over three
ages were represented by a heat map (Figure 4A). Among those
100 proteins that altered their expressions across the three
phases, 40, 29, and 31 proteins were highly abundant in the
NEBs, NBs, and FBs, respectively (Table S12). The 40 highly
abundant proteins in the NEBs were significantly enriched in
the translation functional group and pathways of ribosome,
valine, leucine and isoleucine degradation (Figure 4B, Table
S13). While the 29 proteins with high abundance levels in the
NBs were significantly enriched in the functional group of
protein folding (Figure 4C, Table S13), the 31 highly abundant
proteins in the FBs were significantly enriched in the fatty acid
biosynthesis pathway and functional groups, including small
molecule, carbohydrate, fatty acid, and nucleic acid metabolisms
(Figure 4D, Table S13).
Further analysis of the highly abundant proteins in the PPI
network showed that only one functional group was
significantly enriched in the NEBs (ribosome) and FBs
(carbohydrate metabolic processes). In contrast, four other
functional groups including secretion, skeletal muscle organ
3350
DOI: 10.1021/acs.jproteome.6b00526
J. Proteome Res. 2016, 15, 3342−3357
Article
Journal of Proteome Research
Figure 7. Measurement of royal jelly (RJ) yield and 10-HDA concentration. (A) A royal jelly frame contains plastic queen cells with the adhering
honeybees. (B) A royal jelly frame removed from the honeybee but wax caps on the queen cell cups. (C) Wax caps removed and RJ left in the queen
cell cups. (D) The weight of 10 batches of RJ collected from the Italian bees (ITBs) and high royal jelly producing bees (RJBs). Every batch of RJ
was collected from 60 queen cell cups of five colonies and represents as mean ± SE (n = 10). Asterisks indicate the statistically significant differences
between RJ yield of the two honeybee lines at different batches (p < 0.01). (E) 10-HDA concentration of RJ derived from ITB and RJB is shown as
mean ± SE (n = 3).
differently expressed between two bees, 60 and 105 proteins
were highly abundant in the forager ITB and RJB, respectively
(Figure S2A, Table S20). The 60 highly abundant proteins in
ITBs were enriched in the translation and ribosome pathway
(Figure S2E, Table S21). The 105 highly abundant proteins in
RJBs were enriched in protein biosynthesis, cytoskeleton
metabolism, and lipid transport (Figure S2F, Table S21).
development, CoA ligase, and oxidoreductase activities were
significantly enriched in NBs (Figure 5D, E and F).
3.5. Proteome Comparison of MG Development at Three
Stages between ITB and RJB
To better understand functional differences between both bee
lines, the proteomes of MGs at each developmental stage of
both ITBs and RJBs was compared. In the NEBs, 3135 and
3277 were identified in ITBs and RJBs, respectively (Figure
S1B, Table S2 and S8). Among the differentially expressed 148
proteins, 120 and 28 proteins were highly abundant in ITBs
and RJBs, respectively (Figure S1A, Table S14). The 120 highly
abundant proteins in ITBs were strongly enriched in protein
biosynthesis, nucleic acid biosynthesis, and energy metabolism
(Figure S1C, Table S15). The 28 highly abundant proteins in
RJBs were not enriched in any functional group and pathway.
There were 3463 and 3198 proteins found in the nurse ITB
and RJB, respectively (Figure 6B, Table S3 and S9). The 755
proteins uniquely expressed in ITBs were significantly enriched
in nucleic acid and organic substance transport (Figure 6C,
Table S16), while the 490 proteins uniquely expressed in RJBs
were enriched in selenocompound metabolism (Figure 6C,
Table S16). Among the 192 proteins that differed in abundance
between both bee strains, 139 and 53 proteins were highly
abundant in ITBs and RJBs, respectively (Table S17). The 139
highly abundant proteins in ITBs were enriched in energy,
nucleic acid, phagosome, and small molecule metabolism
(Figure 6D, Table S18). The 53 highly abundant proteins in
the nurse RJB were strongly enriched in nucleic acid
metabolism, protein biosynthesis, and small molecule metabolism (Figure 6E, Table S18).
In FBs, 2595 and 3095 proteins were identified in ITBs and
RJBs, respectively (Figure S2B, Table S4 and S10). The 325
uniquely expressed proteins in ITBs were mainly enriched in
organonitrogen compound biosynthetic process and endocytosis (Figure S2C, Table S19). However, the 825 uniquely
expressed proteins in RJB were strongly enriched in the small
molecule metabolic process, lipid metabolism, and substance
transport (Figure S2D, Table S19). Among the 165 proteins
3.6. Measurement of RJ Yield and 10-HDA Concentration
To measure the relationship between RJ output and 10-HDA
concentration, the RJ yield and 10-HDA were quantified in
both ITB and RJB. RJ production from the queen cell cups in
the RJB was measured at 0.824 ± 0.038 g and 0.084 ± 0.004 g
in the ITB, a 10-fold difference (Figure 7D). However, the 10HDA concentration in RJ pooled from 10 batches of both bee
strains was not significantly different between the two samples,
i.e., 2.16 ± 0.14% and 2.18 ± 0.12% for RJB and ITB,
respectively (Figure 7E).
3.7. Verification of Differentially-Expressed Proteins in
Lipid Metabolism between Nurse ITBs and RJBs at mRNA
Level
When comparing the proteins involved in lipid metabolism
produced by both nurse ITBs and RJBs, the expression level of
the fatty acid synthase, involved in fatty acid synthesis, was 4.47
fold greater in the RJBs in than in the ITBs in our proteome
data. However, the expression levels of three other proteins
related to lipid degradation (carnitine O-palmitoyltransferase,
trifunctional enzyme, and 3-hydroxyacyl-CoA dehydrogenase)
were 12.5, 7.69, and 2.38 times higher in ITBs than in RJBs
respectively (Table S22).
Given the association of these four differently expressed
proteins with lipid metabolism and 10-HDA synthesis,15
mRNA expression levels in the MGs of ITB and RJB NBs
were analyzed by qPCR. As a result, mRNA expression trends
of these proteins (fatty acid synthase, trifunctional enzyme, and
3-hydroxyacyl-CoA dehydrogenase) were consistent with their
protein expression trends. However, mRNA level of carnitine
O-palmitoyltransferase was not consistent with its protein
expression level in the two honeybee lines (Figure 8).
3351
DOI: 10.1021/acs.jproteome.6b00526
J. Proteome Res. 2016, 15, 3342−3357
Article
Journal of Proteome Research
Figure 8. Test the mRNA expression level of differentially expressed proteins (fold change ≥2 and p < 0. 05) related fatty acid metabolism between
the nurse bees of Italian bees (ITBs) and high royal jelly producing bees (RJBs) by quantitative PCR analysis. Error bar is standard deviation. *, p <
0.05.
4. DISCUSSION
We report here the most depth proteome coverage of MGs
(>4000 proteins), covering ∼30% of the honeybee proteome.
The major findings are the different proteome settings
employed by the MGs of NEBs, NBs, and FBs, confirming
their specific roles in the hive and the difference in lipid
production in RJBs. Moreover, the ITBs and RJBs adapt quite
divergent proteome programs to sustain gland growth and
functionality at different physiological stages.
transport in RJBs and monovalent inorganic cation transport in
ITBs, were enriched. These observations suggest their roles are
key for delivering the biomolecules to the desired cellular
location to support gland functions.
As in other honeybee organs and tissues,32,37,57,58 the MGs
demand a great amount of biological fuel to maintain their key
functions and development. This demand is met by the
enriched energy metabolism related biological processes,
including glycolysis/gluconeogenesis and citrate cycle fatty
acid degradation in both strains; carbohydrate metabolic
processes in RJBs; and pentose and glucuronate interconversions in ITBs.
4.1. Basic Functionality and Development of MGs Require
a Shared Proteome
Although morphology and function vary depending on age,8 a
shared proteome in the MGs of both stocks across the adult
stages may indicate central roles for cellular maintenance and
lipid synthesis in worker bees. The enriched ribosome,
translation, protein folding, and protein processing in the
endoplasmic reticulum by the shared proteins suggest their
centrality in maintaining cell structure and functions by
synthesizing new proteins for the MGs in both bee lines.
Living cells require a dynamic balance between protein
synthesis and degradation, where molecular chaperones and
proteases are involved.53 Hence, the enriched proteasome and
phagosome in both strains indicate their roles are key for
controlling the balance between native folded and unfolded
proteins.54 Moreover, the lipids, small molecule, oxoacid, and
organic acids that are enriched in the shared proteome signify
their roles are critical for volatile chemical substance biosynthesis in the MGs, including 10-HDA, 2-heptanone, and other
pheromone molecules.55,56
The main function of the MGs is the secretion of lipids into
RJ as larval nutrition and into pheromones to signal nest
mates.15 Molecular transporters must deliver these lipid and
pheromone molecules to the desired cellular locations for their
functions. Here, molecule transport groups, such as protein
localization, protein transport in both strains, organic substance
4.2. Proteins Expressed in NEBs are Mainly to Initiate MG
Development
The physiological maturity of MGs is vital to performing agedependent tasks and they are morphologically and physiologically immature in the NEBs.7 To drive development and
functionality, the MGs have to develop as age advances. To this
effect, a multitude of proteins have to be synthesized as tissue
blocks to promote construction and cell growth of the young
glands as in embryos and HGs.37,38,57 Here, protein biosynthesis in the NEBs is enriched by the proteins identified in both
bee lines, suggesting that protein blocks are vital for priming
the gland growth. Moreover, the importance of protein
materials for supporting young gland development is further
underscored by the fact that such proteins are unique to NEBs
of both bee strains (not found in NBs and FBs). For instance,
protein biosynthesis machinery, aminoacyl-tRNA biosynthesis,
translation, and tRNA aminoacylation for translation were
enriched by proteins unique to ITBs and spliceosome was
enriched by proteins unique to RJBs. Additionally, for proteins
in high abundance in NEBs relative to NBs and FBs, the
ribosome and translation process were enriched in both strains,
and translation elongation and protein folding were enriched in
the ITBs. These data indicate that the pathways implicated in
protein biosynthesis may be functionally enhanced to produce a
3352
DOI: 10.1021/acs.jproteome.6b00526
J. Proteome Res. 2016, 15, 3342−3357
Article
Journal of Proteome Research
compared to NEBs; however, the amount is much lower than
in foragers.7
To support the high secretory activity of MGs in NBs, the
enriched protein export, vesicle-mediated transport, and
organic substance transport were enriched by specific proteins
in both strains of NBs. These data suggest the importance of
transporting molecular substances to desired cellular locations,
which provides the secretory activity of the glands. Additionally,
the proteins in high abundance are associated with molecule
delivery and secretory activity in the MGs of NBs, activities
including voltage-dependent anion-selective channel, and
calcium-transporting ATPase.72,73 This activity is also supported by the enriched tube development and protein transport
in ITB NBs, and functional groups related to secretion in RJB
NBs in the PPI network. For instance, tube development can
promote the formation of an excretory canal, through which the
MG secretion could be delivered to the outer surface of the
integument.7,74
sufficient amount of protein molecules as cellular building
blocks for tissue construction and cell growth in young
glands.59 For instance, 44 ribosomal proteins were expressed at
a higher abundance in the ITB NEBs than in NBs and FBs, and
9 ribosomal proteins and eukaryotic translation initiation
factors were highly expressed in the RJB NEBs compared to
NBs and FBs.
As the gland grows with the age of the bees,7 MG
development requires the expression of cytoskeletal proteins
at a high abundance to support the cell shape, cell stability, and
cellular division as in the HGs and embryos.37,57,60 The higher
abundance of cytoskeletal proteins and development related
proteins in NEBs than NBs and FBs further emphasize their
significance. These proteins include protein l (2)37Cc, AFG3like protein 2, and papilin in both strains; actin, myosin, and
fibrillin in the ITB NEBs and fibrillin in the RJB NEBs. All
these data imply the importance of the early elevated activities
of cytoskeletal and development related proteins to promote
MG growth.60−63 In addition, those proteins related to
morphogensis, including endocuticle structural glycoprotein
SgAbd-8, are more highly expressed in NEBs than in NBs and
FBs in both strains, and thus may be vital for building the basic
morphological configuration of MGs.
4.4. Proteins Expressed in MGs of FBs Could Enhance
Foraging and Colony Defense Efficiency
Foraging and colony defense are the main duties of the FBs.75
To improve foraging efficiency, the FBs employ a short-term
repellent pheromone, 2-heptanone, deposited on flowers on
previous visits.7 It also acts as an alarm pheromone, which
stimulates sting behavior.13 The fact that lipid metabolism was
enriched by unique proteins of FBs relative to NEBs and NBs
in both strains suggests it is vital for metabolizing small
molecular pheromones 2-heptanone which could be derived
from keto acid produced by lipase via β-oxidation of long-chain
fatty acids. Moreover, proteins in high abundance enriched in
small molecule, oxoacid, and organic acid metabolism in ITB
FBs, and small molecule and lipid metabolism in RJB FBs,
suggest their roles may be in enhancing the production of small
molecular pheromones including 2-heptanone. This is consistent with the higher 2-heptanone concentration in FBs than
in NBs.7 Moreover, oxoacid metabolism is enriched in ITB FBs,
suggesting its importance in catalyzing octanoic acids to 2heptanone.71 The highly expressed proteins in FBs involved in
lipid biosynthesis and β-oxidation suggest their elevated roles in
providing materials and metabolic energy for 2-heptanone
synthesis, including fatty acid synthase in both strains, esterase
in ITBs, and long-chain-fatty-acid-CoA ligase in RJBs.66,76,77
4.3. Proteins Expressed in the NBs May Implicate in
Promoting Secretory Activity in MG
The major time frame for the secretion of lipids and
pheromones in the MGs of adult workers is in NBs.7,64
Generally, a great amount of lipids are synthesized and secreted
into RJ as a major component of larval and queen food.23 The
stronger expressions of fatty acid synthase, acetyl-CoA
carboxylase in the NBs of ITBs, and fatty acid synthase, verylong-chain enoyl-CoA reductase in the NBs of RJBs, are
supposed to promote voluminous lipid synthesis.65−67 This
supposition is supported by the up-regulated genes for fatty
acid synthase in the MGs of worker bees.42,43 10-HDA is the
most important lipid produced by hydroxylation of octadecanoic acids at the ω-position,15 which is catalyzed by P450
enzymes.56 For the conversion of 10-HDA, the highly abundant
level of cytochrome P450 family members in the NBs of both
strains imply its pivotal enzymatic role in hydroxy group
introduction at the ω position to produce 10-HDA.56 This is in
accordance with the fact that several genes encoding P450
enzymes are up-regulated in the NBs.41,43 The high levels of
peroxisomal multifunctional enzyme type 2 and carnitine Opalmitoyltransferase in the nurse ITBs reflect that they are
likely involved in the peroxisomal β-oxidation for the
conversion of 10-HDA. This is consistent with the up-regulated
gene of peroxisomal multifunctional enzyme type 2 in the MGs
of worker bees.43 Moreover, the highly abundant long-chain
fatty acid transport protein in ITB NBs and apolipophorin in
RJB NBs may enhance the activity related to lipid secretion and
transport.68,69 Furthermore, the highly expressed adipocyte
plasma membrane-associated protein in NBs of both strains
may function in the adipocyte differentiation and further in
fatty acid storage.70
In addition to lipid synthesis, the MGs of NBs produces 2heptanone as an alarm pheromone.7 2-heptanone is derived
from keto acid produced by lipase via β-oxidation of long-chain
fatty acids.15,71 The proteins in high abundance associated with
lipid biosynthesis and oxidation in NBs of both lines suggest
that 2-heptanone synthesis drives sting behavior in NBs
4.5. ITBs and RJBs Adapt Different Proteome Program
during MG Development
In the NEBs, the ITB MGs expressed a large number of
proteins in high abundances involved in protein biosynthesis
and energy metabolism, as compared with RJB. In ITBs, the
120 highly abundant proteins in ITBs, translation elongation,
ATP biosynthetic process, and ribosome were enriched.
However, the 28 highly abundant proteins in RJBs were not
enriched in any functional group or pathway. Notably, both the
high abundance of proteins related to transcription and
translation and proteins related to morphogenesis and
development in ITBs relative to RJBs (such as cuticular
protein, apidermin, atlastin) suggest that proteome rearrangement may drive the young MGs in distinct developing
trajectories in both bee stocks.
For NBs, the divergence of MGs between the two strains is
heightened further. The highly abundant proteins in RJBs that
are enriched in nucleic acid metabolism and protein synthesis
suggest a high demand of nucleic acid and protein for gland
development. For instance, the highly abundant cuticle proteins
3353
DOI: 10.1021/acs.jproteome.6b00526
J. Proteome Res. 2016, 15, 3342−3357
Article
Journal of Proteome Research
Notably, the RJ samples were collected under similar
environmental conditions, including colony strength and nectar
flow.85 The same concentration of 10-HDA was detected in the
RJ of both strains, which may indicate that genetic variation
played a major role and that selection has shaped RJBs to adapt
their MGs to produce the quantity of 10-HDA that keeps pace
with increased RJ production.
and cytoskeletal proteins, such as troponin, and laminin in RJB
NBs suggest that their roles are vital to MG development and
cell division.78,79 In particular, the abundance of endocuticle
structural glycoprotein SgAbd-1 in RJBs, a critical component
of endocuticle,80 was 255 times higher in RJBs than in ITBs.
Moreover, the small molecule metabolism group enriched by
the highly abundant proteins in RJB NBs relative to ITBs
indicates its enhanced roles in small molecule synthesis
including 10-HDA. For ITB NBs, however, the function and
secretory activity of MGs may be accomplished via organonitrogen compound biosynthesis, small molecule metabolic
process, and organic substance transport by highly abundant
and unique proteins.
For the FBs, the divergent proteome signatures of MGs
between both strains still remain. The lipid biosynthesis and
transport pathways enriched by highly abundant and unique
proteins in RJBs suggest lipid synthesis and secretory activity in
RJBs remain at a high level as compared to ITBs. The
cytoskeleton metabolism, protein biosynthesis, and protein
transport pathways are enriched by highly abundant and unique
proteins in RJB FBs, relative to ITB FBs, suggesting that
mitosis of MGs may still underway. However, the ITB FBs was
observed to have a distinct proteome profile as compared to
RJB FBs. For instance, protein synthesis enriched by highly
abundant proteins of ITB FBs indicates a dynamic balance of
proteins for normal tissue function.
5. CONCLUSIONS
This work reports an unprecedented depth of proteome
coverage on the MGs of ITBs and RJBs. The MGs of both bee
stocks have evolved unique proteome signatures to fit with
distinct age-dependent physiology. In NEBs, the proteome
plays a key role in initiating young gland development. The
proteome of NBs, however, is mainly focused on priming high
secretory activities by enriched lipid synthesis and transport,
thus providing lipid nutrition for the bee brood. The proteome
of FBs is essential to alarm pheromone synthesis, which allows
for defending the colony and improving foraging performance.
Moreover, selection for high RJ production has altered the
proteome program between ITBs and RJBs during the MG
development in a plethora of biological processes. The tailored
proteome programs at different ages of MGs may also drive
different development trajectories in both bee lines. RJBs have
adapted their proteome settings by enhancing lipid biosynthesis
and minimizing lipid degradation to maintain a reasonable 10HDA concentration in sync with the enhanced RJ production.
Our data unveil a novel understanding of the regulatory
mechanisms governing MG development and functions, and
provide valuable resources and starting points for lipid or
pheromone biochemists as well as developmental geneticists.
4.6. MGs of RJB NBs Enhance Lipid Synthesis and Minimize
Degradation to Maintain Reasonable 10-HDA
Concentration in RJ
Maintaining a reasonable 10-HDA concentration in RJ is vital
for both honeybees and humans. In this study, the measured RJ
production by RJBs was 10-fold that of the ITB, which is
consistent with our previously reports.32,35 Interestingly, the
same 10-HDA concentration was measured in both RJ samples,
suggesting that RJB NBs may have enhanced pathway activity
related to 10-HDA synthesis. For instance, the highly expressed
fatty acid synthase in RJB NBs may strengthen long-chain fatty
acids synthesis to provide materials for 10-HDA synthesis,
which fits with its up-regulated gene expression in NBs.42,43
However, the degradation rate of lipids was significantly
enhanced in ITB NBs relative to RJB NBs. This is supported
by the stronger expression of carnitine O-palmitoyl transferase
(12.5 folds), trifunctional enzyme (7.69 folds), and 3hydroxyacyl-CoA dehydrogenase (2.38 folds) in ITB NBs.
These three proteins participate in lipid β-oxidation to induce
high rate of lipid degradation,81−83 of which the latter two were
validated by their gene up-regulation here. These observations
suggest that lipid biosynthesis is likely enhanced in ITBs, while
their degradation rate is minimized in RJB NBs, thereby
maintaining a proper proportion of lipids in the increased RJ
production. This is because the lipid synthesis and degradation
do not occur through a bidirectional reversible reaction, but
through different pathways.84 The inconsistent mRNA and
protein expression trend of carnitine O-palmitoyl transferase in
both bee lines may due to splicing and/or post translational
modification.
During the selection for higher RJ production in HGs, lipid
synthesis in the MGs of RJBs was not considered. However, the
enhanced lipid synthesis capacity in MGs may have a
coselective effect for increasing the baseline expression of
lipid genes, implying that genes controlling lipid and protein
synthesis are under the control of the same regulatory elements.
■
ASSOCIATED CONTENT
S Supporting Information
*
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jproteome.6b00526.
Supporting figures (PDF)
Supporting tables (XLSX)
■
AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +86 10 6259 1449. E-mail: apislijk@126.com.
Author Contributions
#
XH and BW contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work is supported by the Agricultural Science and
Technology Innovation Program (CAAS-ASTIP-2015-IAR),
and the earmarked fund for Modern Agro-Industry Technology
Research System (CARS-45) in China.
■
REFERENCES
(1) Winston, M. L. The honey bee colony: life history. In The Hive
and the Honey Bee, 5th ed.; Graham, J. M., Ed.; Dadant: Hamilton, IL,
1992; pp 73−101.
(2) Huang, Z. Y.; Robinson, G. E. Regulation of honey bee division
of labor by colony age demography. Behavioral Ecology and Sociobiology
1996, 39 (3), 147−58.
3354
DOI: 10.1021/acs.jproteome.6b00526
J. Proteome Res. 2016, 15, 3342−3357
Article
Journal of Proteome Research
(3) Robinson, G. E. Genomics and integrative analyses of division of
labor in honeybee colonies. Am. Nat. 2002, 160 (Suppl 6), S160−72.
(4) Robinson, G. E.; Huang, Z. Y. Colony integration in honey bees:
genetic, endocrine and social control of division of labor. Apidologie
1998, 29 (1−2), 159−70.
(5) Slessor, K. N.; Winston, M. L.; Le Conte, Y. Pheromone
communication in the honeybee (Apis mellifera L.). J. Chem. Ecol.
2005, 31 (11), 2731−45.
(6) Trhlin, M.; Rajchard, J. Chemical communication in the
honeybee (Apis mellifera L.): a review. Vet. Med. 2011, 56 (6), 265−73.
(7) Vallet, A.; Cassier, P.; Lensky, Y. Ontogeny of the fine structure
of the mandibular glands of the honey bee (Apis mellifera L.) workers
and the pheromonal activity of 2-heptanone. J. Insect Physiol. 1991, 37,
789−804.
(8) Blum, M. S. Mandibular gland pheromones. In The Hive and the
Honey Bee, 5th ed.; Graham, J. M., Ed.; Dadant: Hamilton, IL, 1992;
pp 380−385.
(9) Winston, M. L.; Higo, H. A.; Colley, S. J.; Pankiw, T.; Slessor, K.
N. The role of queen mandibular pheromone and colony congestion
in honey bee (Apis mellifera L.) reproductive swarming (Hymenoptera: Apidae). J. Insect Behav 1991, 4, 649−60.
(10) Hoover, S. E.; Keeling, C. I.; Winston, M. L.; Slessor, K. N. The
effect of queen pheromones on worker honey bee ovary development.
Naturwissenschaften 2003, 90 (10), 477−80.
(11) Stout, J. C.; Goulson, D. The use of conspecific and interspecific
scent marks by foraging bumblebees and honeybees. Anim. Behav.
2001, 62, 183−9.
(12) Giurfa, M. The repellent scent-mark of the honey bee Apis
mellifera ligustica and its role as communication cue during foraging.
Insectes Soc. 1993, 40, 59−67.
(13) Pankiw, T. Cued in: honey bee pheromones as information flow
and collective decision-making. Apidologie 2004, 35 (2), 217−26.
(14) Kerr, W. E.; Blum, M. S.; Pisani, J. F.; Stort, A. C. Correlation
between amounts of 2-heptanone and iso-amyl acetate in honeybees
and their aggressive behaviour. J. Apic Res. 1974, 13, 173−6.
(15) Plettner, E.; Slessor, K. N.; Winston, M. L. Caste-selective
pheromone biosynthesis in honeybees. Science 1996, 272, 1851−3.
(16) Plettner, E.; Slessor, K. N.; Winston, M. L.; Robinson, G. E.;
Page, R. E. Mandibular gland components and ovarian development as
measures of caste differentiation in the honey bee (Apis mellifera L.). J.
Insect Physiol. 1993, 39 (3), 235−40.
(17) Plettner, E.; Otis, G. W.; Wimalaratne, P. D. C.; Winston, M. L.;
Slessor, K. N.; Pankiw, T.; Punchihewa, P. W. K. Species- and castedetermined mandibular gland signals in honeybees (Apis). J. Chem.
Ecol. 1997, 23 (2), 363−77.
(18) Blum, M. S.; Novak, A. F.; Taber, S. 10-Hydroxy-delta 2decenoic acid, an antibiotic found in royal jelly. Science 1959, 130,
452−3.
(19) Kinoshita, G.; Shuel, R. W. Mode of action of royal jelly in
honeybee development. Some aspects of lipid nutrition. Can. J. Zool.
1975, 53 (3), 311−9.
(20) Nagaraja, N.; Brockmann, A. Drones of the dwarf honey bee
Apis florea are attracted to (2E)-9-oxodecenoic acid and (2E)-10hydroxydecenoic acid. J. Chem. Ecol. 2009, 35 (6), 653−5.
(21) Brockmann, A.; Dietz, D.; Spaethe, J.; Tautz, J. Beyond 9-ODA:
sex pheromone communication in the European honey bee. J. Chem.
Ecol. 2006, 32 (3), 657−67.
(22) Keeling, C. I.; Otis, G. W.; Hadisoesilo, S.; Slessor, K. N.
Mandibular gland component analysis in the head extracts of Apis
cerana and Apis nigrocincta. Apidologie 2001, 32, 243−52.
(23) Barker, S. A.; Foster, A. B.; Lamb, D. C.; Hodgson, N.
Identification of 10-hydroxy-delta 2-decenoic acid in royal jelly. Nature
1959, 183 (4666), 996−7.
(24) Weaver, N.; Johnston, N. C.; Benjamin, R.; Law, J. H. Novel
fatty acids from the royal jelly of honeybees (Apis mellifera, L.). Lipids
1968, 3 (6), 535−8.
(25) Sabatini, A. G.; Marcazzan, G. L.; Caboni, M. F.; Bogdanov, S.;
Almeida-Muradian, L. B. d. Quality and standardisation of Royal Jelly.
J. ApiProd. ApiMed. Sci. 2009, 1 (1), 1−6.
(26) Hattori, N.; Nomoto, H.; Fukumitsu, H.; Mishima, S.;
Furukawa, S. Royal jelly and its unique fatty acid, 10-hydroxy-trans2-decenoic acid, promote neurogenesis by neural stem/progenitor
cells in vitro. Biomed. Res. 2007, 28 (5), 261−6.
(27) Honda, Y.; Fujita, Y.; Maruyama, H.; Araki, Y.; Ichihara, K.;
Sato, A.; Kojima, T.; Tanaka, M.; Nozawa, Y.; Ito, M.; Honda, S.
Lifespan-extending effects of royal jelly and its related substances on
the nematode Caenorhabditis elegans. PLoS One 2011, 6 (8), e23527.
(28) Izuta, H.; Chikaraishi, Y.; Shimazawa, M.; Mishima, S.; Hara, H.
10-Hydroxy-2-decenoic acid, a major fatty acid from royal jelly, inhibits
VEGF-induced angiogenesis in human umbilical vein endothelial cells.
Evid Based Complement Alternat Med. 2009, 6 (4), 489−94.
(29) Vucevic, D.; Melliou, E.; Vasilijic, S.; Gasic, S.; Ivanovski, P.;
Chinou, I.; Colic, M. Fatty acids isolated from royal jelly modulate
dendritic cell-mediated immune response in vitro. Int. Immunopharmacol. 2007, 7 (9), 1211−20.
(30) Townsend, G. F.; Morgan, J. F.; Tolnai, S.; Hazlett, B.; Morton,
H. J.; Shuel, R. W. Studies on the in vitro antitumor activity of fatty
acids. I. 10-Hydroxy-2-decenoic acid from royal jelly. Cancer Res. 1960,
20, 503−10.
(31) Townsend, G. F.; Brown, W. H.; Felauer, E. E.; Hazlett, B.
Studies on the in vitro antitumor activity of fatty acids. IV. The esters
of acids closely related to 10-hydroxy-2-decenoic acids from royal jelly
against transplantable mouse leukemia. Biochem. Cell Biol. 1961, 39,
1765−70.
(32) Li, J. K.; Feng, M.; Begna, D.; Fang, Y.; Zheng, A. J. Proteome
comparison of hypopharyngeal gland development between Italian and
royal jelly producing worker honeybees (Apis mellifera L.). J. Proteome
Res. 2010, 9 (12), 6578−94.
(33) Li, J.; Li, H.; Zhang, Z.; Pan, Y. Identification of the proteome
complement of high royal jelly producing bees (Apis mellifera) during
worker larval development. Apidologie 2007, 38 (6), 545−57.
(34) Han, B.; Fang, Y.; Feng, M.; Hu, H.; Qi, Y.; Huo, X.; Meng, L.;
Wu, B.; Li, J. Quantitative neuropeptidome analysis reveals neuropeptides are correlated with social behavior regulation of the honeybee
workers. J. Proteome Res. 2015, 14 (10), 4382−93.
(35) Feng, M.; Fang, Y.; Han, B.; Xu, X.; Fan, P.; Hao, Y.; Qi, Y.; Hu,
H.; Huo, X.; Meng, L.; Wu, B.; Li, J. In-Depth N-Glycosylation
Reveals Species-Specific Modifications and Functions of the Royal
Jelly Protein from Western (Apis mellifera) and Eastern Honeybees
(Apis cerana). J. Proteome Res. 2015, 14 (12), 5327−40.
(36) Li, J. K.; Chen, S. L.; Zhong, B. X.; Su, S. K. Genetic analysis for
developmental behavior of honeybee colony’s royal jelly production
traits in western honeybees. Acta Genet. Sin. 2003, 30 (6), 547−54.
(37) Fang, Y.; Feng, M.; Han, B.; Lu, X.; Ramadan, H.; Li, J. In-depth
proteomics characterization of embryogenesis of the honey bee worker
(Apis mellifera ligustica). Mol. Cell. Proteomics 2014, 13 (9), 2306−20.
(38) Fang, Y.; Feng, M.; Han, B.; Qi, Y.; Hu, H.; Fan, P.; Huo, X.;
Meng, L.; Li, J. Proteome analysis unravels mechanism underling the
embryogenesis of the honeybee drone and its divergence with the
worker (Apis mellifera lingustica). J. Proteome Res. 2015, 14 (9), 4059−
71.
(39) Hernandez, L. G.; Lu, B.; da Cruz, G. C.; Calabria, L. K.;
Martins, N. F.; Togawa, R.; Espindola, F. S.; Yates, J. R.; Cunha, R. B.;
de Sousa, M. V. Worker honeybee brain proteome. J. Proteome Res.
2012, 11 (3), 1485−93.
(40) Qi, Y.; Fan, P.; Hao, Y.; Han, B.; Fang, Y.; Feng, M.; Cui, Z.; Li,
J. Phosphoproteomic analysis of protein phosphorylation networks in
the hypopharyngeal gland of honeybee workers (Apis mellifera
ligustica). J. Proteome Res. 2015, 14 (11), 4647−61.
(41) Malka, O.; Karunker, I.; Yeheskel, A.; Morin, S.; Hefetz, A. The
gene road to royalty - differential expression of hydroxylating genes in
the mandibular glands of the honeybee. FEBS J. 2009, 276 (19),
5481−90.
(42) Hasegawa, M.; Asanuma, S.; Fujiyuki, T.; Kiya, T.; Sasaki, T.;
Endo, D.; Morioka, M.; Kubo, T. Differential gene expression in the
mandibular glands of queen and worker honeybees, Apis mellifera L.:
implications for caste-selective aldehyde and fatty acid metabolism.
Insect Biochem. Mol. Biol. 2009, 39 (10), 661−7.
3355
DOI: 10.1021/acs.jproteome.6b00526
J. Proteome Res. 2016, 15, 3342−3357
Article
Journal of Proteome Research
Drosophila: possible translational regulation. Nucleic Acids Res. 1986,
14 (15), 6169−83.
(62) Kramerova, I. A.; Kawaguchi, N.; Fessler, L. I.; Nelson, R. E.;
Chen, Y.; Kramerov, A. A.; Kusche-Gullberg, M.; Kramer, J. M.;
Ackley, B. D.; Sieron, A. L.; Prockop, D. J.; Fessler, J. H. Papilin in
development; a pericellular protein with a homology to the ADAMTS
metalloproteinases. Development 2000, 127 (24), 5475−85.
(63) Maltecca, F.; Aghaie, A.; Schroeder, D. G.; Cassina, L.; Taylor,
B. A.; Phillips, S. J.; Malaguti, M.; Previtali, S.; Guénet, J. L.; Quattrini,
A.; Cox, G. A.; Casari, G. The mitochondrial protease AFG3L2 is
essential for axonal development. J. Neurosci. 2008, 28 (11), 2827−36.
(64) Gracioli-Vitti, L. F.; Abdalla, F. C. Comparative ultrastructure of
the mandibular gland in Scaptotrigona postica (Hymenoptera, Apidae,
Melliponini) workers and males. Braz J. Morphol. Sci. 2006, 23 (3−4),
415−24.
(65) Hoja, U.; Marthol, S.; Hofmann, J.; Stegner, S.; Schulz, R.;
Meier, S.; Greiner, E.; Schweizer, E. HFA1 encoding an organellespecific acetyl-CoA carboxylase controls mitochondrial fatty acid
synthesis in Saccharomyces cerevisiae. J. Biol. Chem. 2004, 279 (21),
21779−86.
(66) Jayakumar, A.; Tai, M. H.; Huang, W. Y.; al-Feel, W.; Hsu, M.;
Abu-Elheiga, L.; Chirala, S. S.; Wakil, S. J. Human fatty acid synthase:
properties and molecular cloning. Proc. Natl. Acad. Sci. U. S. A. 1995,
92 (19), 8695−9.
(67) Moon, Y. A.; Horton, J. D. Identification of two mammalian
reductases involved in the two-carbon fatty acyl elongation cascade. J.
Biol. Chem. 2003, 278 (9), 7335−43.
(68) Kutty, R. K.; Kutty, G.; Kambadur, R.; Duncan, T.; Koonin, E.
V.; Rodriguez, I. R.; Odenwald, W. F.; Wiggert, B. Molecular
characterization and developmental expression of a retinoid- and
fatty acid-binding glycoprotein from Drosophila. A putative lipophorin.
J. Biol. Chem. 1996, 271 (34), 20641−9.
(69) Hirsch, D.; Stahl, A.; Lodish, H. F. A family of fatty acid
transporters conserved from mycobacterium to man. Proc. Natl. Acad.
Sci. U. S. A. 1998, 95 (15), 8625−9.
(70) Albrektsen, T.; Richter, H. E.; Clausen, J. T.; Fleckner, J.
Identification of a novel integral plasma membrane protein induced
during adipocyte differentiation. Biochem. J. 2001, 359 (Pt2), 393−
402.
(71) Kinsella, J. E.; Dan, H. Biosynthesis of flavors by Penicillium
roqueforti. Biotechnol. Bioeng. 1976, 18 (7), 927−38.
(72) Rudolph, H. K.; Antebi, A.; Fink, G. R.; Buckley, C. M.;
Dorman, T. E.; LeVitre, J.; Davidow, L. S.; Mao, J. I.; Moir, D. T. The
yeast secretory pathway is perturbed by mutations in PMR1, a member
of a Ca2+ ATPase family. Cell 1989, 58 (1), 133−45.
(73) Okada, S. F.; O’Neal, W. K.; Huang, P.; Nicholas, R. A.;
Ostrowski, L. E.; Craigen, W. J.; Lazarowski, E. R.; Boucher, R. C.
Voltage-dependent anion channel-1 (VDAC-1) contributes to ATP
release and cell volume regulation in murine cells. J. Gen. Physiol. 2004,
124 (5), 513−26.
(74) Cruz-Landim, C. D.; Gracioli-Vitti, L. F.; Abdalla, F. C.
Ultrastructure of the intramandibular gland of workers and queens of
the stingless bee Melipona quadrifasciata. J. Insect Sci. 2011, 11 (3),
373−7.
(75) xWhiffler, L. A.; Drusedau, M. U. H.; Crewe, R. M.; Hepburn,
H. R. Defensive behaviour and the division of labour in the African
honeybee (Apis mellifera scutettata). J. Comp. Physiol., A 1988, 163 (3),
401−11.
(76) Levisson, M.; van der Oost, J.; Kengen, S. W. Characterization
and structural modeling of a new type of thermostable esterase from
Thermotoga maritima. FEBS J. 2007, 274 (11), 2832−42.
(77) Min, K. T.; Benzer, S. Preventing neurodegeneration in the
Drosophila mutant bubblegum. Science 1999, 284 (5422), 1985−8.
(78) Henchcliffe, C.; García-Alonso, L.; Tang, J.; Goodman, C. S.
Genetic analysis of laminin A reveals diverse functions during
morphogenesis in Drosophila. Development 1993, 118 (2), 325−37.
(79) Barbas, J. A.; Galceran, J.; Krah-Jentgens, I.; de la Pompa, J. L.;
Canal, I.; Pongs, O.; Ferrús, A. Troponin I is encoded in the
(43) Malka, O.; Nino, E.; Grozinger, C.; Hefetz, A. Genomic analysis
of the interactions between social environment and social
communication systems in honey bees (Apis mellifera). Insect Biochem.
Mol. Biol. 2014, 47, 36−45.
(44) Iovinella, I.; Dani, F. R.; Niccolini, A.; Sagona, S.; Michelucci, E.;
Gazzano, A.; Turillazzi, S.; Felicioli, A.; Pelosi, P. Differential
expression of odorant-binding proteins in the mandibular glands of
the honey bee according to caste and age. J. Proteome Res. 2011, 10
(8), 3439−49.
(45) Han, B.; Fang, Y.; Feng, M.; Lu, X.; Huo, X.; Meng, L.; Wu, B.;
Li, J. In-depth phosphoproteomic analysis of royal jelly derived from
Western and eastern honeybee species. J. Proteome Res. 2014, 13 (12),
5928−43.
(46) Zhang, J.; Xin, L.; Shan, B. Z.; Chen, W. W.; Xie, M. J.; Yuen,
D.; Zhang, W. M.; Zhang, Z. F.; Lajoie, G. A.; Ma, B.; PEAKS, D. B. de
novo sequencing assisted database search for sensitive and accurate
peptide identification. Mol. Cell. Proteomics 2012, 11 (4),
M111010587.
(47) Lin, H.; He, L.; Ma, B. A combinatorial approach to the peptide
feature matching problem for label-free quantification. Bioinformatics
2013, 29 (14), 1768−75.
(48) Bindea, G.; Mlecnik, B.; Hackl, H.; Charoentong, P.; Tosolini,
M.; Kirilovsky, A.; Fridman, W. H.; Pages, F.; Trajanoski, Z.; Galon, J.
ClueGO: a Cytoscape plug-in to decipher functionally grouped gene
ontology and pathway annotation networks. Bioinformatics 2009, 25
(8), 1091−3.
(49) Warde-Farley, D.; Donaldson, S. L.; Comes, O.; Zuberi, K.;
Badrawi, R.; Chao, P.; Franz, M.; Grouios, C.; Kazi, F.; Lopes, C. T.;
Maitland, A.; Mostafavi, S.; Montojo, J.; Shao, Q.; Wright, G.; Bader,
G. D.; Morris, Q. The GeneMANIA prediction server: biological
network integration for gene prioritization and predicting gene
function. Nucleic Acids Res. 2010, 38 (13), W214−20.
(50) Jianke, L. Technology for royal jelly production. Am. Bee J.
2000, 140 (6), 469−72.
(51) Bloodworth, B. C.; Harn, C. S.; Hock, C. T.; Boon, Y. O. Liquid
chromatographic determination of trans-10-hydroxy-2-decenoic acid
content of commercial products containing royal jelly. J. AOAC Int.
1995, 78 (4), 1019−23.
(52) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene
expression data using real-time quantitative PCR and the 2−ΔΔct
method. Methods 2001, 25 (4), 402−8.
(53) Miura, E.; Kato, Y.; Matsushima, R.; Albrecht, V.; Laalami, S.;
Sakamoto, W. The balance between protein synthesis and degradation
in chloroplasts determines leaf variegation in Arabidopsis yellow
variegated mutants. Plant Cell 2007, 19 (4), 1313−28.
(54) Trombetta, E. S.; Parodi, A. J. Quality control and protein
folding in the secretory pathway. Annu. Rev. Cell Dev. Biol. 2003, 19,
649−76.
(55) Hawke, J. C. The formation and metabolism of methyl ketones
and related compounds. J. Dairy Res. 1966, 33, 225−42.
(56) Plettner, E.; Slessor, K. N.; Winston, M. L. Biosynthesis of
mandibular acids in honey bees (Apis mellifera): de novo synthesis,
route of fatty acid hydroxylation and caste selective β-oxidation. Insect
Biochem. Mol. Biol. 1998, 28, 31−42.
(57) Feng, M.; Fang, Y.; Li, J. Proteomic analysis of honeybee worker
(Apis mellifera) hypopharyngeal gland development. BMC Genomics
2009, 10, 645.
(58) Feng, M.; Fang, Y.; Han, B.; Zhang, L.; Lu, X.; Li, J. Novel
aspects of understanding molecular working mechanisms of salivary
glands of worker honeybees (Apis mellifera) investigated by proteomics
and phosphoproteomics. J. Proteomics 2013, 87, 1−15.
(59) Grewal, S. S.; Li, L.; Orian, A.; Eisenman, R. N.; Edgar, B. A.
Myc-dependent regulation of ribosomal RNA synthesis during
Drosophila development. Nat. Cell Biol. 2005, 7 (3), 295−302.
(60) Frixione, E. Recurring views on the structure and function of the
cytoskeleton: a 300-year epic. Cell Motil. Cytoskeleton 2000, 46 (2),
73−94.
(61) Eveleth, D. D., Jr.; Marsh, J. L. Sequence and expression of the
Cc gene, a member of the dopa decarboxylase gene cluster of
3356
DOI: 10.1021/acs.jproteome.6b00526
J. Proteome Res. 2016, 15, 3342−3357
Article
Journal of Proteome Research
haplolethal region of the Shaker gene complex of Drosophila. Genes
Dev. 1991, 5 (1), 132−40.
(80) Micas, A. F.; Ferreira, G. A.; Laure, H. J.; Rosa, J. C.; Bitondi, M.
M. Proteins of the integumentary system of the honeybee, Apis
mellifera. Arch Insect Biochem Physiol 2016, DOI: 10.1002/arch.21336.
(81) Jackson, V. N.; Cameron, J. M.; Zammit, V. A.; Price, N. T.
Sequencing and functional expression of the malonyl-CoA-sensitive
carnitine palmitoyltransferase from Drosophila melanogaster. Biochem.
J. 1999, 341 (Pt 3), 483−9.
(82) DeBerardinis, R. J.; Lum, J. J.; Thompson, C. B.
Phosphatidylinositol 3-kinase-dependent modulation of carnitine
palmitoyltransferase 1A expression regulates lipid metabolism during
hematopoietic cell growth. J. Biol. Chem. 2006, 281 (49), 37372−80.
(83) Middleton, B. The mitochondrial long-chain trifunctional
enzyme: 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase
and 3-oxoacyl-CoA thiolase. Biochem. Soc. Trans. 1994, 22 (2), 427−
31.
(84) Numa, S. s. Fatty Acid Metabolism and Its Regulation; Elsevier:
Amsterdam, 1984; Vol. XI, p 209.
(85) McNeil, M. E. A.; Schmidt, J. O. Other products of the hive. In
The Hive and the Honey Bee, 6th. ed.; Graham, J. M., Ed.; Dadant:
Hamilton, IL, 2015; pp 728−33.
3357
DOI: 10.1021/acs.jproteome.6b00526
J. Proteome Res. 2016, 15, 3342−3357