Proteomic Analysis Reveals the Molecular Underpinnings of

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


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


Article


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


Article


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


Article


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


Article


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


Article


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


Article


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


Article


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


Article


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


Article


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


Article


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: [email protected]. 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


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


Article


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


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


Proteomic Analysis Reveals the Molecular Underpinnings of

Recommend Documents