Proteomic Analysis of Honeybee (Apis mellifera L.) Pupae Head Development

Abstract

The honeybee pupae development influences its future adult condition as well as honey and royal jelly productions. However, the molecular mechanism that regulates honeybee pupae head metamorphosis is still poorly understood. To further our understand of the associated molecular mechanism, we investigated the protein change of the honeybee pupae head at 5 time-points using 2-D electrophoresis, mass spectrometry, bioinformatics, quantitative real-time polymerase chain reaction and Western blot analysis. Accordingly, 58 protein spots altered their expression across the 5 time points (13-20 days), of which 36 proteins involved in the head organogenesis were upregulated during early stages (13-17 days). However, 22 proteins involved in regulating the pupae head neuron and gland development were upregulated at later developmental stages (19-20 days). Also, the functional enrichment analysis further suggests that proteins related to carbohydrate metabolism and energy production, development, cytoskeleton and protein folding were highly involved in the generation of organs and development of honeybee pupal head. Furthermore, the constructed protein interaction network predicted 33 proteins acting as key nodes of honeybee pupae head growth of which 9 and 4 proteins were validated at gene and protein levels, respectively. In this study, we uncovered potential protein species involved in the formation of honeybee pupae head development along with their specific temporal requirements. This first proteomic result allows deeper understanding of the proteome profile changes during honeybee pupae head development and provides important potential candidate proteins for future reverse genetic research on honeybee pupae head development to improve the performance of related organs.

Full text

Proteomic Analysis of Honeybee (
Apis mellifera
L.) Pupae
Head Development
Aijuan Zheng
1,2.
, Jianke Li
1
*
.
, Desalegn Begna
1
, Yu Fang
1
, Mao Feng
1
, Feifei Song
1
1Key Laboratory of Pollinating Insect Biology, Ministry of Agriculture/Institute of Apicultural Research, Chinese Academy of Agricultural Science, Beijing, China, 2Feed
Research Institute, Chinese Academy of Agricultural Science, Beijing, China
Abstract
The honeybee pupae development influences its future adult condition as well as honey and royal jelly productions.
However, the molecular mechanism that regulates honeybee pupae head metamorphosis is still poorly understood. To
further our understand of the associated molecular mechanism, we investigated the protein change of the honeybee pupae
head at 5 time-points using 2-D electrophoresis, mass spectrometry, bioinformatics, quantitative real-time polymerase chain
reaction and Western blot analysis. Accordingly, 58 protein spots altered their expression across the 5 time points (13–20
days), of which 36 proteins involved in the head organogenesis were upregulated during early stages (13–17 days).
However, 22 proteins involved in regulating the pupae head neuron and gland development were upregulated at later
developmental stages (19–20 days). Also, the functional enrichment analysis further suggests that proteins related to
carbohydrate metabolism and energy production, development, cytoskeleton and protein folding were highly involved in
the generation of organs and development of honeybee pupal head. Furthermore, the constructed protein interaction
network predicted 33 proteins acting as key nodes of honeybee pupae head growth of which 9 and 4 proteins were
validated at gene and protein levels, respectively. In this study, we uncovered potential protein species involved in the
formation of honeybee pupae head development along with their specific temporal requirements. This first proteomic
result allows deeper understanding of the proteome profile changes during honeybee pupae head development and
provides important potential candidate proteins for future reverse genetic research on honeybee pupae head development
to improve the performance of related organs.
Citation: Zheng A, Li J, Begna D, Fang Y, Feng M, et al. (2011) Proteomic Analysis of Honeybee (Apis mellifera L.) Pupae Head Development. PLoS ONE 6(5):
e20428. doi:10.1371/journal.pone.0020428
Editor: Christos Samakovlis, Stockholm University, Sweden
Received February 12, 2011; Accepted April 27, 2011; Published May 26, 2011
Copyright: ß2011 Zheng 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.
Funding: This work was supported by the earmarked fund for Modern Agro-industry Technology Research System (CARS-45) and the National Natural Science
Foundation of China (No. 30972148). 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 interests exist.
* E-mail: apislijk@126.com
.These authors contributed equally to this work.
Introduction
The honeybee displays complete metamorphosis where an
individual passes through 4 developmental stages: egg, larva,
pupae and adulthood. After 3 days, the egg hatches into a feeding
stage called the larva. Six days later, the larva enters an
intermediate and inactive stage known as the prepupa sealed in
a cell by beeswax. After a few hours, internal changes begin that
transform the prepupa into a quiescent white pupa with 3 major
body regions that superficially look like those of an adult bee. With
gradual darkening, hairs and wings develop and after a few days
an adult bee emerges by chewing out of the wax cell capping [1,2].
The pupa stage is the longest post embryonic developmental
period of the honeybee. The temperature at which pupae are
raised influences the tasks and behavioral determination of the
adult bees [3]. It is reported that pupae weight increases with
honey production and pupae head weight increases with higher
royal jelly production [4]. Comparative biochemical analysis
between worker and queen heads has revealed that adults raised
under higher temperatures show higher probability to dance,
forage earlier, and more often are involved in more activities [3,5].
Ecdysteroid titer production levels peak earlier in queen than
worker pupae [6]. The head of honeybee is formed at the pupa
stage and consists of the brain and associated ganglia, hypopha-
ryngeal glands (HGs), mandibular glands, salivary glands, and
antennae, which contribute to neural, endocrine and/or exocrine
functions [7]. The head of the adult honeybee has been widely
investigated morphologically [8,9], biochemically [10] and
molecularly [11–15]. However, proteomic studies on honeybee
pupae at different developmental stages and even on sub-organs
are very limited. Recent completion of the honeybee (Apis mellifera
L.) genome sequence [16] has opened promising ways for detailed
investigations of honeybees using the proteomic approach. In this
study we investigate honeybee pupae head protein expression
profile changes at different developmental stages using the
proteomic approach to gain better understanding of molecular
factors involving in shaping the pupae head development.
Materials and Methods
Chemical Reagents
The following reagents were purchased: Urea, Tris-base, sodium
dodecyl sulfate (SDS), sodium bicarbonate, dithiothreitol (DTT),
iodoacetamide and bovine serum albumin (BSA) were purchased
from Sigma (St. Louis, MO, USA). Bio-lyte from Bio-Rad
(Hercules, CA, USA), acrylamide, N, N9-methylenebisacrylamide,
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ammonium persulfate (AP), N,N,N9,N9-tetramethylethylene di-
amine (TEMED), 3-[(3-cholamidopropyl) -dimethylammonio]-1-
propane sulfonate (CHAPS), glycerol, bromophenol blue, flamingo
fluorescent dye (Bio-Rad), coomassie brilliant blue (CBB) G-250. a-
cyano-4-hydroxycinnamic acid (CHCA) from Bruker Daltonics
(Billerica, Mass. USA), trypsin from Roche (Modified, Sequencing
Grade, Roche, Mannheim, Germany), and trifluoroacetic acid
(TFA) and acetonitrile from J. T. Baker (Phillipsburg, NJ, USA). All
the chemicals used for RNA isolation and real-time PCR were
purchased from Bio-Rad (Hercules, CA, USA). Other chemicals
used, but not specified here are noted in the text.
Biological Samples
Honeybee (Apis.mellifera lingustica) worker pupae were collected
on day 13, 15, 17, 19 and 20 from the apiary of Institute of
Apicultural Research, Chinese Academy of Agricultural Science in
May 2009. The exact age of the pupae were obtained by confining
the egg laying queen for 5 hours on a single wax comb frame
containing worker cells using a queen excluder. Subsequently, the
queen was removed and the frame containing the eggs was
maintained in the honeybee colony until the date of collection. A
total of 100 pupae heads were sampled for each time point based
on the compound eye pigmentation (Fig. 1) from 5 bee colonies
having similar conditions sourced and the heads were dissected
and stored at 280uC until analysis.
Protein Extraction and Two-dimensional Gel
Electrophoresis (2-DE)
Protein extraction was carried out as described previously [17].
Protein quantification was performed according to the method
developed by Bradford [18] using BSA as the standard and the
absorption was measured at 595 nm (Beckman, spectrophotom-
eter DU800).
A 450 mg protein sample was suspended in lysis buffer [8 M
urea, 2 M thiourea, 4% CHAPS, 20 mM Tris-base, 30 mM
DTT, 2% Bio-lyte pH 3–10] and then mixed with rehydration
buffer [8 M urea, 2% CHAPS, 0.001% bromophenol blue,
45 mM DTT, 0.2% Bio-lyte pH 3–10]. The mixture was loaded
on a 17 cm IPG strip (pH 3–10, linear, Bio-Rad). Isoelectric
focusing (IEF) was performed at 18uC according to manufacturer’s
instructions (Protean IEF Cell, Bio-Rad). Before SDS-PAGE, the
IPG strips were first equilibrated for 15 minutes in equilibration
buffer 1 [6 M urea, 0.375 M Tris-HCl (pH 8.8), 20% glycerol, 2%
Figure 1. Image of honeybee pupae in different positions at different developmental stages showing the occurrences of gradual
changes in phenotype and organogenesis as time passes. (A) and (B) represent corresponding whole pupal body and head development.
doi:10.1371/journal.pone.0020428.g001
Figure 2. Pattern of honeybee pupae head weight at different
developmental time points. Different letters (a, b, c) are significantly
different (p,0.05).
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SDS, 2% DTT] and then continued in equilibration buffer 2 [6 M
urea, 0.375 M Tris-HCl (pH 8.8), 20% glycerol, 2% SDS, 2.5%
iodoacetoamide] for another 15 minutes. Second dimension
electrophoresis, SDS-PAGE, was performed in a Protean II Xi
Cell (Bio-Rad) at 25 mA/gel for 6 hours.
Image Acquisition and Statistics Analysis
Gels were fixed overnight in 50% (v/v) ethanol with 10% (v/v)
acetic acid, washed in water, and stained with Flamingo
fluorescent dye (Bio-Rad) for image analysis and then further
dyed with CBB G-250 to visualize spots for mass spectrometry
(MS) analysis. Three independent biological replicates 2-DE gel
images were digitized with ImageScanner III (GE Healthcare) at
16 bit and 300 dpi resolution. Image filtration, background
subtraction, spot detection, spot matching, and quantitative
intensity (all the pixels making up the spot) analysis were
performed using PDQuest software (ver. 8.0.1, Bio-Rad). All gels
were matched with one of the selected reference gel. The match
analysis was performed in an automatic mode, and further manual
editing was performed to correct the mismatched and unmatched
spots. The expression level of a given protein spot was expressed in
terms of volume of the spot. To compare spot quantities between
gels accurately, the spot volumes were normalized as percentage of
the total volume of all of the spots in the gel. The means and
standard deviations from the triplicate experiments were calculat-
ed and the statistical significance of the expression level of the
protein and mRNA at differential time-stage were assessed with
one-way ANOVA (SPSS Version 16.0, SPSS Inc.), a Duncan’s
Multiple Range test was used to compare the difference between
means of the expression level at 5 time-point. An error probability
of p,0.05 was considered to be statistically significance of at least
1.5 fold changes.
Identification of Differentially Expressed Protein
Proteins showing significant expression were excised and
denatured, alkylated, trypsin digested as described previously
[11]. Matrix was prepared by dissolving a-cyano-4-hydroxycin-
namic acid (CHCA, Bruker Daltonics) in 50% acetonitrile/0.1%
Figure 3. 2-DE and CBB G-250 stained gels image showing differentially expressed protein spots profile obtained from different
developmental stages of honeybee pupae heads. Each identified protein spot is specified by number and indicated by arrow, where the spot
number with prefix ‘‘u’’ and ‘‘d’’ indicate up or down regulation.
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trifluoroacetic acid. Ten microliters of solution was added onto the
dried digests and vortexed for 30 min. A total of 1.5 mL of the
reconstituted in-gel digest sample was spotted initially on
Anchorchip target plate (600/384F, Bruker Daltonics), followed
by 0.5 mL of matrix solution. The dried sample on the target was
washed twice with 1 mL of 0.1% TFA, left for 30 seconds before
solvent removal. The digested peptide mixture was analyzed by an
Ultraflex II matrix assisted laser desorption ionization time of
flight/mass spectrometry mass spectrometer (MALDI-TOF MS)
(Bruker Daltonics) under the control of Flex Control 2.2 software
(Bruker Daltonics). MALDI-TOF spectra were recorded in the
positive ion reflector mode in a mass range from 700–4000 Da
and the ion acceleration voltage was 25 kV. Acquired mass spectra
were processed using the software Mascot distiller (Version 2.2,
default settings, Matrix Science) by default setting. Spectra were
calibrated by a protonated mass signal from a standard peptide
calibration mixture consisting of 8 peptides covering mass range
from 700 to 3100 with (Bruker, Billerica, MA Peptide Calibration
Standard 206196). Spectra originating from parallel protein
digestions were compared pairwise to discard common peaks
derived from trypsin autodigestion or from contamination with
keratins. The resulting peptide mass lists were used to search
against the nonredundant NCBI (NCBInr, release date, January
22, 2010) using MASCOT 2.2 (Matrix Science). Search
parameters were: Taxonomy: Apis mellifera; trypsin cleavage;
allow up to one missed cleavage; peptide mass tolerance 0.2 Da;
fixed modification: carbamidomethyl (C); variable modification:
oxidation (M). A total of 10,348,164 sequences and 3,529,470,745
residues in the database were actually searched.
When the identified peptides match to multiple members of a
protein family, or a protein appears under the same names and
accession number, the match was considered in terms of higher
Mascot score the putative function and differential patterns of
protein spots on 2-DE gels. Protein identifications were accepted if
the established probability was greater than 95% and contained at
least 2 identified peptides having maximum peptides coverage.
Bioinformatic Analysis
The expression profiles were performed using expression values
of protein spots at different developmental time point by
calculating average distances using cluster software (Gene cluster,
version 3.0).
Biological interaction networks (BIN) of the differentially
expressed proteins were analyzed using Pathway Studio. Hence,
experimental results were interpreted based on the context of
pathways, protein regulation networks, and protein interaction
maps in the Drosophila molecular networks database, which is
equipped with functional relationships from other scientific
literature. The applied filters included ‘‘all shortest paths between
selected entities.’’ The information received was narrowed down
to our protein list of interest, namely, those proteins whose
involvement and regulatory functions had been observed. Each
link was built with evidence from at least 3 publications. The
interactions between the imported proteins and all proteins stored
in the database were then identified. Protein entities which belong
to different functional groups were represented as different shapes
according to the default settings of the software as shown in the
legend.
Figure 4. Functional annotation and distributions of the differentially expressed proteins identified from honeybee pupae head at
different developmental time points.
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To enrich the identified proteins to specific functional terms, the
protein lists were analyzed using the ClueGo software and
applying the Drosophila database from the Gene Ontology database
(release date, January 10, 2010). Ontology selection as biological
process and enrichment analysis was done by right-side hyper-
geometric statistic test and its probability value was corrected by
the bonferroni method [19].
Validation of Differentially Expressed Proteins
To further verify the differential expression levels, quantitative
real-time PCR was run for proteins identified at day 13, 15, 17, 19
and 20. Based on the results of the gel-based comparison, specific
primers (Table S1) suited to simultaneously amplify various target
genes were designed according to the corresponding gene
sequences of the identified proteins and the available gene
information in the GenBank library by using the primer design
software (Beacon Designer 7.51, PREMIER Biosoft International
Palo Alto, CA). Total RNAs were prepared from the head on day
13, 15, 17, 19 and 20 using TRIzol reagent (Takara bio) and
cDNA synthesis was performed using TaKaRa RNA PCR Kit
(AMV) Ver.3.0 (Takara bio), according to the manufacturer’s
instruction. Real-time PCR was conducted using an iQ5
Multicolor Real-Time PCR Detection System (Bio-Rad). PCR
was performed in 25-ml reaction system containing 1 ml cDNA,
5 pmol forward and reverse primers, 12.5 ml SYBR Green
Supermix (Bio-Rad) and water. Fold-change was calculated using
the 2
2DDCt
method [20].
For Western blot, each of ald (spot d17), hsp60 (spot d9), Tcp-
1g(spot d13) and idh (spot u9) were subjected to 3 replication runs
and 4 mg of protein samples were loaded on each lane separated
by stacking (4%) and separating (12%) SDS-PAGE gels. To ensure
that the specific anti-ald, hsp60, Tcp-1gand Idh bands could be
detected, protein molecular marker was loaded when running the
gels. Gels were run at a voltage of 120 V for approximately 1.5 h
using Mini-Protein II Gel electrophoresis system (Bio-Rad
Laboratories Ltd.). Resolved proteins were transferred to a
nitrocellulose (NC) transfer membrane (0.2 mm pore size)
(Invitrogen) using the iBlot apparatus (Invitrogen, Carlsbad, CA).
Nonspecific binding was blocked with 5% (w/v) nonfat milk
powder in the Tris buffered saline (20 mM Tris-HCl, 150 mM
NaCl, pH 7.6) containing 0.1% (v/v) Tween-20 (TBS-T) at room
temperature for 1 h. The membranes were then incubated with
primary rabbit polyclonal anti-ald, hsp60, Tcp-1gand Idh
antibodies (Abcam, Massachusetts) at a dilution of 1:5000 in 2%
milk powder in TBS-T at 4uC overnight. Following three washes,
the membranes were further incubated with goat anti-rabbit IgG
conjugated with horseradish peroxidase (Pierce, Rochford, IL)
(1:10,000 in 2% milk powder in TBS-T), and were rolled for 1.5 h
at room temperature. At the end of this process, the NC
membranes were washed for 2 h, rolled at room temperature.
Immunoreactive protein bands were then visualized by enhanced
chemiluminescence detection (ECL, Pierce, Rochford, IL) re-
agents and quantified by densitometry using the Quantity-one
image analysis system (Bio-Rad Laboratories Ltd.). The human
anti-b-actin antibody (1: 5000, sigma) was detected simultaneously
as a loading control.
Results
In order to understand the developmental progresses, pictures
and weights of the pupae head were taken at 5 time-points (Fig. 1).
Accordingly, the pupae head weight between 13–17 days was
Figure 5. Functional comparisons of upregulated proteins between the early (13–17 days) and late (19–20 days) stage of honeybee
pupae head.
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significantly heavier than those at 19–20 days (Fig. 2). Subse-
quently protein extracts from pupae head were separated using 2-
DE and comparisons were done across 5 time points. As a result,
the molecular weight (Mr)andpIof pupae head proteins ranged
from 10.45 to 125.16 kDa and 3.7 to 9.56, respectively (Fig. 3).
The 2-DE image analysis revealed nearly 400 spots on each gel,
of which 85 spots showed differential expressions (.1.5 fold
change, p,0.05) and 59 of them were successfully identified by
MALDI-TOF MS (Table S2 and Fig. 3). The remaining
unidentified proteins spots could be attributed to their lower
abundance to produce enough spectra, or because the databases
search scores were not high enough (,95%) to yield unambig-
uous results. In this study, some protein spots were identified as
the same proteins, but appeared differently on 2-DE gels, most
likely due to post-translational modifications, such as phosphor-
ylation and possibly alternative splicing or proteolysis that results
in shifting of MrandpI.
Functional Classification of the Identified Proteins
The identified proteins were grouped into 11 functional classes
and proteins involved in carbohydrates metabolism and energy
production, development, cytoskeleton, protein folding and
protein biosythesis were found to be the major protein families
(Fig. 4). Interestingly, the proportions of functional classes (except
antioxidant and fatty acid metabolism proteins) indicated higher
representation from early to middle than the late developmental
stages. Specifically, nucleotide and amino acid metabolisms
proteins were expressed only during the early to mid develop-
mental stages (Fig. 5).
Hierarchical Cluster Analysis of Differentially Expressed
Proteins
The hierarchical cluster analysis of differentially expressed
proteins showed that 58 distinct proteins (excluding 1 protein with
unknown functions) were partaken in the expression intensity map
(Fig. 6). Generally, most of the protein spots behaved heteroge-
neously, but clustered under 2 large very homogenous expressional
pictures that were from the early to middle stages (day 13–17) and
the late developmental stage (day 19–20) with a shifting trend
inline with the pupae head developmental stage. From 36 protein
spots that were highly expressed during the early to middle stages,
proteins as carbohydrate metabolism and energy production,
development, protein biosythesis, cytoskeleton and protein folding
were recognized as major groups (Fig. 6). To this fact, there were 7
proteins spots involved in carbohydrate metabolism and energy
production (spots d14, d15, d17, d19, d20, d22 and d24), 6 in
development (spots d10, d11, d12, d13, d30 and d31), 6 in
biosythesis (spot d1–4, d5, d7), 5 in cytoskeleton (spots d25, d26,
d27, d29 and d32) and 4 in protein folding (spots d6, d8, d9 and
d28). In addition, there were 3 amino acid metabolism (spots d23,
d36 and d33), 3 molecular transporters (spots d16, d34 and d35)
and 2 nucleotide metabolism (spots d18, d21) that were
upregulated during the early to middle stage. On the other hand,
22 proteins were upregulated during the late pupae stage that
included 6 in development (spots u1, u3, u10, u11, u13 and u14), 5
in carbohydrate metabolism and energy production (spots u5, u6,
u9, u12, and u17), 4 in cytoskeleton (spots u8, u15, u21 and u23)
and 3 in proteins folding (spots u2, u4 and u22). The other
proteins upregulated during late developmental stages included
one involved in molecular transporter (spot u19); one related to
protein biosynthesis (spot u7), one associated with fatty acid
metabolism (spot u16) and one involved in antioxidant activity
protein (spot u20) (Fig. 6).
Functional Enrichment Analysis
Gene Ontology (GO) annotation provides 3 detailed and
structured terms that include molecular functions, biological
processes, and cellular components, which are currently widely
used in the analysis of large proteomic and genomic datasets.
Significantly overrepresented GO terms are examined to deter-
mine hypotheses for the biological events behind the data and
assist in providing a broad overview of the principal characteristics
of a proteome. Functional enrichment analysis was conducted
using the ClueGo software on all proteins that had altered their
expression across the 5 time-points. Hence, carbohydrate
metabolism and energy production, protein folding and cytoskel-
eton, development related proteins were significantly enriched
(Fig. 7).
Figure 6. Global expression intensity map (hierarchical clustering) showing upregulation (red) and downregulation (green) across
the ages of the pupae indicated on the top of each column with lists of functional proteins in the right column.
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Figure 7. Functional enrichment analysis of the differentially expressed proteins using the ClueGO software.
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Network Analysis
Pathway Studio analysis was employed to predict the
biological interaction network (BIN) with interactive relations
for the differentially expressed proteins. Accordingly, 33
proteins were recognized as key nodes with various relations
in the created BIN. The result from the established network
clearly indicated that carbohydrate metabolism and energy
production, development, protein folding and cytoskeleton were
the most linked protein families and protein categories and as
canbeseeninFig.8theupregulated species are color coded in
the legend.
Validation of Differentially Expressed Proteins
Among the 4 major groups (carbohydrate and energy
production, protein folding, development and cytoskeleton)
involved as key nodes in the BIN, 9 proteins were selected for
further validating the proteins differential expressions at mRNA
levels (Fig. 9) of which 4 of them were further confirmed by
Western blot analysis (Fig. 10). The selected proteins were, ald
(spot d17), pglym78 (spot u12), idh (spot u9), hsp60 (spot d9),
hsp83 (spot d6), hsc70-4 (spot u2), l(1)g0022 (spot d12), Tcp-1g
(spot d13) and tm2 (spot u21). Accordingly, 6 proteins showed
consistent mRNA expressions with the change patterns of their
corresponding proteins as in 2-DE gels. The 6 proteins that
showed consistent increased mRNA abundance were ald (spot
d17), hsp60 (spot d9), hsp90 (spot d6), l(1)g0022 (spot d12), tm2
(spot u21) and Tcp-1g(spot d13) and they were gene transcripts
from early to middle stage (Fig. 9). However, the transcripts of
genes of pglym (spot u12), idh (spot u9) and hsc70-4 (spot u2)
showed variations between the mRNA transcription and protein
abundance across the 5 time points and this might be due to
lack of a direct relationship between mRNA timing and protein
expressions and/or other regulatory mechanisms such as lack of
synchronization. The result of the Western blot analysis also
showed considerable expressional difference for ald (spot d17),
hsp60 (spot d9), Tcp-1g(spot d13) and idh (spot u9) and
the achieved differences were in line as in the 2-DE image
(Fig. 10).
Figure 8. Biological interaction network of the identified differentially expressed proteins from honeybee pupae head at the
different developmental stages. The big ellipse represents identified proteins in this experiment and each entity belongs to the category
described in the legend. The ‘‘Blue’’ and ‘‘Red’’ are the proteins upregulated during early to middle development stages (13–17 days) and during late
developmental stages (19–20 days), respectively.
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Discussion
In the life of the honeybee pupal stage follows the larval stage
and precedes adulthood. It is at the pupal stage that the adult
structures are formed and the larval structures are broken down.
The generation of organs inside the head mainly occurs at this
stage and hence, vital proteins have to be involved to support their
initiation, formation, and completion. Although it is known that
increased pupae weight is correlated with honey [21], and pupae
head weight with royal jelly production [4] these studies were
limited in morphological and phenotypic investigations, which
required a follow up study to uncover the molecular factors, which
cause these phenotypic phenomenon to occur. Accordingly, our
study reveals that proteins that are related to carbohydrate
metabolism and energy production, development, cytoskeleton
and protein folding are involved in accelerating the development
and metabolism of honeybee pupae head development in general
as well as salivary glands, hypopharyngeal glands and are involved
in restructuring of nervous system development, in particular.
Specifically, proteins involved in biosynthesis and metabolizing
amino acid were upregulated in the pupal head during early stages
of development (13–17 days) suggesting tremendous ongoing
physiological processes to ensure the formation, development and
structuring of important organs inside the pupae head. On the
other hand, the role of those proteins species that upregulated
during the late developmental stage (19–20 days) were found to
ensure the neuron development and to activate the functional
glands (Fig. 5, 6). Following this functional mission, most of the
differentially expressed proteins showed a shift in species in line
with developmental stages of the pupae head as a sign of task
accomplishment.
The development of salivary glands will help in the production
of enzymes, which will help with the honey ripening process (sugar
breakdown). Furthermore, the nervous system restructuring could
help in the development of the olfactory systems, which is
important in pollen and nectar collection by the forager bees and
ultimately contributes to improved honey production.
The significantly heavier weight of pupae heads during the
period between 13 to 17 days was positively related to the high
amount and varieties of upregulated proteins (36 of 58) suggesting
the biological success of fast growing pupae head depends on the
participations of high amounts and different varieties of proteins.
Carbohydrate metabolism and energy production proteins are
well known to play a major role in the process of developing
worker embryos, larvae and HGs [11,17,22]. Hence, the energy
metabolism proteins are required as a key metabolic fuel by the
worker bees for foraging flight [23] and as nutrients for neurons for
learning and memorization processes [24,25]. As well, their over
expressions in honeybee pupae head at early developmental stage
suggests high metabolic rates that demand high energy to ensure
the formation of important organs, head growth, neuron
development and metamorphosis. However, their typical late
developmental stage over-expressions might suggest their crucial
involvement in shaping and completion of full organ formation as
well as to equip them with physiological functions, like capabilities
of secreting royal jelly from HGs [11], acquire learning ability
from neuron systems, odorant from olfactory systems and other
basic tasks to be performed after emerging as an adult.
A group of development related proteins (spot d10, d11 and
u14) that regulate the pupae head development as well as the
development of bristle and hair in Drosophila [26,27] were
upregulated. Similarly, Tcp-1g(spot d13) and l(1)g0022 (spot
Figure 9. Transcript validation of differentially expressed proteins at different development periods. mRNA of different development
days are measured by quantitative real time PCR. Samples are normalized with reference gene (GAPDH) and with the expression abundant on day 13
as the reference sample. Error bar is standard deviation. Gene symbols indicating different genes refer to Table S2. Different letters (a, b, c) are
significantly different (p,0.05).
doi:10.1371/journal.pone.0020428.g009
Honeybee Pupal Head Proteome
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d12) proteins have been known in mitotic spindle organization
[28] and centriole replication [29]. Ran (spot d30) is involved in
regulating cell shape, cell adhesion and cell cycle [30,31]. Tctp
(spot d31) positively regulates cell size and eye growth [32]. Hence,
their elevated expressions from early to middle stage suggests their
involvement in regulating cell size, growth, cell bond and cell cycle
for the pupae head development as well as in the formations and
development of basic organs and their functionalities. For instance,
the nuclear encoded mitochondrial protein fork head (spot u14) is
known to have a biological process of salivary gland development;
ecdysone-mediated induction of salivary gland cell autophagic cell
death; and salivary gland morphogenesis [33]. And the late
developmental stage over-expressions of snap (spot u11) having a
function of synaptic transmission and compound eye morphogen-
esis [34] suggests its vital role in ensuring the formation of
functional compound eye. Also, ter94 (spot u1) has neuropil
function in the mushroom body and antennal glomeruli of adult
Drosophila head [35], crc (spot u3) with neuronal development,
olfactory system and odor-guided behavioral functions in Drosophila
and 14-3-3 zeta (spot u13) is known to assist in the process of
learning and long-term memory formation of the honeybee [7]. In
addition, the late stage over-expression of AnnIX (spot u10) is
likely to control cell apoptosis in the HG through managing
programmed cell death [36,37].
The major role of the storage proteins is to serve as a reservoir
for amino acids which will be utilized for tissue formation later
during the adult development [38]. Recently, hexamerins have
been reported acting as storage proteins for gonad development,
egg production, and support foraging activity [39]. Therefore, the
over-expressions of 6 forms of hexamerin 110 from the early to
middle stage of pupae head in this study is in line with the
investigations for Drosophila that showed increased expressions of
Figure 10. Western blot analysis of ald, hsp60, Tcp-1gand idh. y-axis represents relative expression level normalized by b-actin, x-axis
represents different development stages on day 13, 15, 17, 19 and 20, accordingly. Different letters (a, b, c) are significantly different (p,0.05).
doi:10.1371/journal.pone.0020428.g010
Honeybee Pupal Head Proteome
PLoS ONE | www.plosone.org 10 May 2011 | Volume 6 | Issue 5 | e20428

storage proteins during 3rd larval instar to pupae stage [40,41].
Thus, the heavier pupae head weight during the early to middle
stage is likely because of the higher protein storage that was
stored in the fat body of pupae and hence in the younger pupae
[42].
Protein folding is the physical process by which a polypeptide
folds from a random coil into a characteristic and functional three-
dimensional structure, which is essential for its functionality [43].
The currently identified heat shock protein (hsps) is induced in
honeybees either at high temperatures [44] or when the bee suffers
from pathogen infections [7,45–47]. However, because our
experiment was carried out under normal circumstances, its
upregulation serves only as molecular chaperones in living cells to
ensure that cell proteins function correctly [48,49] as was reported
for worker embryos [17], HGs [11], larvae [22], workers brain
[24,50], hemolymph [51] and venom gland [52].
Cytoskeleton proteins have dynamic structures helping to
maintain cells shape and play a vital role in both intracellular
transport and cellular division. Alterations of several cytoskeletal
protein expression as actin-binding, myosin, cuticle were
reported in pupae heads after bacterial challenge [7]. Upregula-
tions of actin 88F has been reported in the nurse bee brain in
relation to the olfactory system [24] and in the normal
embryogenesis of the worker bee and HGs [11,17]. Therefore,
the dynamic expressions of cytoskeleton proteins in this study
suggests a significant role in providing skeletal element to
maintain cell scaffold and to be involved in the processes of
organ formations and to equip the organs with properly
functioning facilities as in Drosophila [31,53–56].
Proteasomal proteins play an essential role in the degradation
pathway to supply amino acids for fresh protein synthesis. It was
further reported that they are also involve inthe processes of
honeybee HG gland development and royal jelly secretions [11].
In this study, the upregulations of 3 proteins related to the
metabolism of amino acid (spot d23, d36 and d33) at the early
developmental stage suggests their requirement as a nitrogen
building block in the fast growing glands residing in honeybee
pupae head.
Odorant-binding proteins (OBPs, spots d34, d35) are required
for recognition of chemical stimuli in olfactory system of insects
and reported in the forager honeybee antennae and larvae
developmental process [57,58]. The protein FKBP59 (spot d16) is
known to regulate calcium ion transport and detect light stimulus
in the visual perception of Drosophila [59]. The increased
expressions of these molecular transporter proteins in the young
pupae head indicate their vitalities as general carriers in the
developmental and physiological processes [60]. However, the
over-expressions of antdh (spot u19) in the old pupae head suggests
its possible preparations for the olfactory roles associated with
odor-based communication during the nurse bee stage develop-
ment [24]. In addition, the differential expressions of proteins as
rfabp (spot u16), antioxidant activity (spot u20) and pyd3 (spot
d18, d21) were in line with findings for the developing honeybee
larvae, embryos and HGs to metabolize fatty acid, nucleotides and
for removal of damage by reactive oxygen species [22,60,61].
Since the completion of honeybee genome sequence, RNA
interference has already been proved to be a successful tool for in
vivo studies of gene function and phenotypes [62,63]. The
matching validation results between the protein and the genes
provide information on the potential genes to manipulate
honeybee at the gene level to increase better understanding on
biological aspects and pave ways towards an effort to improve the
performance of important organs like HGs, salivary glands and
olfactory system residing in the head.
In conclusion, our global proteomic results provide an
overviewonthesystemichoneybeepupaeheaddevelopment
at the protein level. From the identified protein species and their
amounts, it is clear that the development of young pupae head
requires specific proteins to develop as well as to generate
associated organs residing. Young pupae head involves more
biosynthesis and metabolizing amino acids proteins than old
pupae head do. The old pupae head involves proteins which are
primarily related to the development and restructuring of organs
like neuron and olfactory systems. The bioinformatic analysis
also showed that proteins involved in metabolic energy,
regulation of development, cytoskeleton and protein folding
were the major contributors to pupae head development. Both
the predicted biological network and the validation results
helped us to detect important key node proteins for future target
functional analysis. Thus, further study will be required to
investigate what possible roles these proteins could play in
support of honeybee pupal head development by RNAi, or by
inhibiting the proteins’ functions by pharmacological approach-
es (e.g. hsp, proteasome and cytoskeletal inhibitors). To our
knowledge, this is the first proteomic report, which offers new
insight into honeybee pupae head development and provides
valuable information and expands our knowledge of the biology
of honeybees.
Supporting Information
Table S1 The primer sequences used for real-time PCR of the
differentially expressed genes in honeybee (Apis mellifera L.) pupae
head at different developmental stages.
(DOC)
Table S2 Identification of differentially expressed proteins in
honeybee (Apis mellifera L.) pupae head at different developmental
stages.
(DOC)
Author Contributions
Conceived and designed the experiments: JKL. Performed the experi-
ments: AJZ YF MF FFS. Analyzed the data: AJZ JKL YF MF FFS. Wrote
the paper: JKL AJZ DB.
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