Proteomic Analysis of Honey Bee Brain upon Ontogenetic and

Feb 9, 2009 - Department of Cell Biology, University of Brasılia, Brasılia, DF, Brazil, Genetic and Biochemistry Institute, Federal. University of Ube...
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Proteomic Analysis of Honey Bee Brain upon Ontogenetic and Behavioral Development Liudy Garcia,†,‡ Carlos H. Saraiva Garcia,‡ Luciana Karen Cala´bria,§ Gabriel Costa Nunes da Cruz,‡ Aniel Sa´nchez Puentes,| Sonia N. Ba´o,⊥ Wagner Fontes,‡ Carlos A. O. Ricart,‡ Foued Salmen Espindola,§ and Marcelo Valle de Sousa*,‡ Mass Spectrometry Group, Physics Department, CEADEN, Havana, Cuba, Brazilian Center for Protein Research, Department of Cell Biology, University of Brası´lia, Brası´lia, DF, Brazil, Genetic and Biochemistry Institute, Federal University of Uberlaˆndia, Uberlaˆndia, MG, Brazil, Department for Proteome Analysis, CIGB, Havana, Cuba, and Laboratory of Electron Microscopy, Department of Cell Biology, University of Brası´lia, Brası´lia, DF, Brazil Received September 30, 2008

The honey bee (Apis mellifera) is a social insect that shows complex and integrated behaviors. Its ability to read and respond to several sets of extrinsic and intrinsic signals is fundamental for the modulation of individual activities and social systems. For instance, A. mellifera behavior changes upon the ontogenetic differentiation from nurse to forager worker subcastes. In this work, brain proteomes of nurses and foragers were compared by two-dimensional gel electrophoresis within pH range of 4-7 in order to find proteins related to such an ontogenetic and behavioral development. Twenty differentially expressed proteins were detected by gel image computational analysis, and identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Nurse brain showed increased expression of major royal jelly proteins (MRJP1, MRJP2 and MRJP7), which are related to determination of castes during the honey bee larvae differentiation. Immunocytochemistry and electron microscopy showed that MRJP1 was localized in the cytoplasm of brain cells, seemingly along filaments of the cytoskeleton, in the antennal lobe, optical lobe and mushroom body. Also, MRJP1 was deposited on the rhabdom, a structure of the retinular cells, composed of numerous tubules. Such evidence suggests that MRJP1 could be associated to proteins of filamentous structures. MRJP1 was also found in intercellular spaces between cells in mushrooms bodies, indicating that it is a secreted protein. Other proteins implicated in protein synthesis and putative functions in the olfactory system were also upregulated in the nurse brain. Experienced foragers overexpressed proteins possibly involved in energy production, iron binding, metabolic signaling and neurotransmitter metabolism. Such differential expression of proteins may be related to ontogenetic and behavior changes in A. mellifera. Keywords: Apis mellifera • honey bee subcastes • brain proteome • two-dimentional gel electrophoresis • major royal jelly protein

1. Introduction The honey bee (Apis mellifera) is a social insect well-adapted to a wide range of environments and capable to perform multiple tasks and to present different behaviors.1,2 It has been used as an excellent model system in a variety of ethological and behavioral studies, such as navigation, social organization and learning.3-6 This insect has efficient nervous and sensorial systems which are responsible for their adaptive success.7 * To whom correspondence should be addressed. Marcelo Valle de Sousa, Brazilian Center for Protein Research, Laboratory of Biochemistry and Protein Chemistry, Department of Cell Biology, University of Brası´lia, 70910-900, Brazil. E-mail: [email protected]. Fax: + 55-61-32734608. † Physics Department, CEADEN. ‡ Brazilian Center for Protein Research, Department of Cell Biology, University of Brası´lia. § Federal University of Uberlaˆndia. | Department for Proteome Analysis, CIGB. ⊥ Laboratory of Electron Microscopy, Department of Cell Biology, University of Brası´lia.

1464 Journal of Proteome Research 2009, 8, 1464–1473 Published on Web 02/09/2009

Worker bees begin their adult life by performing tasks in the nest such as brood care (‘nursing’) and nest maintenance. The transition from nursing to foraging at about 2-3 weeks old involves changes in gene expression, neurochemistry, endocrine activity and brain structure.8 Older bees forage for nectar and pollen.9 Foraging involves learning and memorization of a feeding place, capacity of navigation and communication. On the other hand, the care inside the beehive needs sharpened odor recognition and pheromone perception.4,10 Honey bee age-related transition from hive work to foraging has been associated with an increase in the expression of the foraging (for) gene, which encodes a guanosine 3′,5′-monophosphate (cGMP)-dependent protein kinase (PKG).11 Studies on the neurophysiology and behavior of the honey bee have employed transcriptomic techniques such as expressed sequence tags determination and cDNA microarrays analysis to identify genes differentially regulated during caste and subcaste differentiation.12-14 Other studies demonstrated different pat10.1021/pr800823r CCC: $40.75

 2009 American Chemical Society

Proteomic Analysis of Honey Bee Brain terns of gene expression between forager and nurse subcastes as, for example, genes encoding mayor royal jelly proteins and metabolic enzymes,14-16 genes involved in cholinergic signaling,17 and genes involved in signal transduction, ion channeling, neurotransmitter transport, transcription and cell adhesion, suggesting plasticity and remodeling of neurocellular properties during aging and behavioral development in honey bees.18 The Honey Bee Genome Sequencing Consortium (HGSC) recently sequenced the honey bee A. mellifera genome (http:// www.hgsc.bcm.tmc.edu/projects/honeybee/). The 4.0 version assembly was released in March 2006 and published in October 2006.19 The complete and annotated honey bee genome and the knowledge gained from genomic contextual relationships offer important tools for comparative genomic and proteomic studies. Despite the importance of genomic and transcriptomic data, it is mainly at the proteomic level that one can grasp the real gene expressed products at their wide biochemical diversity and functionality, as mRNA abundance is not directly correlated to protein levels.20-22 Thus, proteomic analysis becomes necessary for achieving a more comprehensive view of the differential expression of bee brain proteins in response to ontogenetic and behavioral conditions. Using two-dimensional gel electrophoresis (2-DE) and mass spectrometry, we undertook a comparative proteomic analysis of A. mellifera brains from nurse and forager worker subcastes. Immunocytochemistry and electron microscopy were used for protein subcellular localization in the brain.

2. Materials and Methods 2.1. Insect Collection and Brain Dissection. A. mellifera adult worker subcastes (nurse and forager) were collected from colonies at Vereda Rosa Apiary (Brası´lia, Brazil). The nurse worker bees were identified by their anatomical structures (such as quantity of coats and state of the wings), their specific behavior (such as nursing behavior toward larvae) and by the presence of developed hypopharyngeal glands, which were removed during dissection. In relation to foragers, to ensure the selection of fully mature ones, only those carrying pollen were collected. Bees were anesthetized with chloroform, and brains were dissected and thoroughly washed for 2 to 3 s in ice-cold lysis buffer (7 M urea, 2 M thiourea, 1% DTT, 2% Triton X-100, and 0.5% Pharmalyte 3-10 or 4-7) containing a cocktail of protease inhibitors (cOmplete Mini-Protease Inhibitor Cocktail Tablets, Roche Diagnostics, Mannheim, Germany). After soaking with cold lysis buffer, brains were immediately immersed in liquid N2 and stored at -80 °C. 2.2. Sample Preparation. Experiments were conducted with samples prepared out of 10 pooled brains for each subcaste (nurse and forager) group. Brains were lysed by manual homogenization using a micropestle in a microcentrifuge tube in 200 µL of lysis buffer in ice, followed by incubation for 1 h at room temperature under shaking. Samples were then centrifuged at 13 000 rpm for 15 min at room temperature. The resulting supernatants were submitted to protein quantitation assay using 2D Quant kit (GE Healthcare, Uppsala, Sweden) and confirmed by amino acid analysis prior to 2-DE separation. 2.3. 2-DE. The two experimental groups (nurse and forager) were used for comparison. Three extracts (10 brains each) were prepared from each group with honey bees collected from a single colony, and two 2-DE gel replicates were run for each extract, yielding six gels per group. Also, two preparative gels were run for each extract. Optimized 2-DE was performed by

research articles combining isoelectrofocusing (IEF) using 18 cm IPG strips (GE Healthcare), pH range 4-7, for the first dimension and 10% T polyacrylamide SDS-PAGE for the second dimension. IPG strips were rehydrated for 12 h in 370 µL of lysis buffer supplemented with 10% (v/v) isopropanol containing 50 µg of proteins for analytical gels and 100 µg for preparative gels. IEF was carried out at 20 °C using an IPGphor II equipment (GE Healthcare) under the following conditions: 500 V (gradient) for 1 h, 1 000 V for 1 h and 8 000 V for 4 h and 30 min. Before the seconddimension step, the IPG gel strips were subjected to reduction and alkylation. Strips were soaked for 20 min in a solution containing 6 M urea, 30% glycerol, 2% SDS, and 125 mM DTT and for additional 20 min in the same buffer containing 300 mM acrylamide instead of DTT. SDS-PAGE was performed on 10% T polyacrylamide gels run on a Protean II system (BioRad, Hercules, CA) connected to a Multitemp II cooling bath (GE Healthcare). Electrophoresis was carried out at 25 mA constant current for 6 h at 20 °C. Proteins were visualized using a MS compatible silver staining procedure.23 2.4. Gel Image and Statistical Analyses. Acquisition of gel images was performed using a Sharp JX-330 scanner (Tokyo, Japan) at 300 dpi resolution. Digitalized images were imported by the software ImageMaster 2D Platinum version 5.0 (GE Healthcare) for analysis. Automatic spot detection under minimal user interference was applied to each gel followed by manual image edition when necessary. Automatic spot matching was carried out landmarked by 10 evenly distributed and well-defined spots. The amount of protein present in each spot was normalized with respect to the total amount applied in gel. The comparison between the two gel groups (nurse and forager) was based on the average percentage of the total spot volume per group. Data from spots displaying quantitative fold changes greater than 2 in their expression between the sample groups and present in at least five of the six replicate gels of each group were submitted to statistical analysis for rejection of null hypothesis of differential expression. Spots with low intensity or in doubtful zones had their data disregarded in our statistical analysis. For differential expression analysis, statistical significance was estimated through the Student’s t test available in the ImageMaster 2D Platinum software. Spots displaying statistically significant differential expression (p < 0.01) were selected for protein identification. 2.5. Protein Digestion and MS. Tryptic peptides from selected protein spots were obtained24 using sequencing grade modified trypsin (Promega, Madison, WI). Peptides were desalted and concentrated using ZipTips C18 (Millipore, Bedford, MA). Each sample (1 µL) was spotted onto the sample plate followed by 1 µL of matrix solution containing 10 mg/mL R-cyano-4-hydroxycinnamic acid in 0.1% trifluoroacetic acid (TFA) and 50% acetonitrile (ACN). The samples were allowed to dry prior to MALDI-TOF MS. The spectra were collected with a Reflex IV MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) in delayed extraction and reflector modes. The internal calibration was done using known trypsin autolysis and keratin peaks (842.50 and 1475.77, respectively). Peptide masses (MH+) were recorded in the range of 800-3000 Da. The software FLEXControl v. 1.1.0.0 (Bruker Daltonics) was used for spectra acquisition, while raw spectra processing was performed using XTOF v. 5.1.1 (Bruker Daltonics) for generation of peak lists. 2.6. Protein Identification. Peak lists were used for database search using BioTools v. 2.0 (Bruker Daltonics) linked to Mascot (http://www.matrixscience.com)25 against the NCBI protein Journal of Proteome Research • Vol. 8, No. 3, 2009 1465

research articles database (National Center for Biotechnology Information, Bethesda, MD). Monoisotopic masses of tryptic peptides were used to identify the proteins by Peptide Mass Fingerprinting (PMF). Error tolerance for peptide mass was lower than 100 ppm, and no restrictions were imposed on protein molecular mass or phylogenetic lineage. Searches that provided no significant scores were sent to a second turn with taxonomy restricted to other metazoa. Further search parameters were methionine oxidation as variable modification and propionamide cysteine (acrylamide alkylation) as fixed modification. Missed cleavages sites were set up to 0 in most of the cases. Hits were considered significant if the protein score exceeded the threshold score calculated by Mascot software assuming p-value < 0.05. 2.7. Immunocytochemistry and Electron Microscopy Analysis. Royal jelly (RJ) was provided by Apia´rios Girassol Ltd. (Uberlaˆndia, MG, Brazil). To prepare water soluble royal jelly proteins (WSRJP), RJ sample was homogenized in PBS, pH 8.0, containing 20 mM EDTA, and centrifuged at 10 000g for 10 min at 4 °C. The supernantant fraction was dialyzed in Tris-HCl buffer, pH 8.0, containing 0.5 mM EDTA. After dialysis, final supernatant was obtained by another centrifugation step. To challenge rabbits with WSRJP, antigen samples of 500 µg/mL of a mixture of WSRJP and Freund’s complete adjuvant (1:1), and subsequent 15-day reinforcement of 250 µg/mL were employed for generation of polyclonal antibodies. Animals were bled 3 days after the last injection. Serum was obtained by centrifugation at 3000 rpm for 3 min and stored at -80 °C. For purification of polyclonal antibodies for major royal jelly protein 1 (anti-MRJP1), 2 µg/µL WSRJP fraction was separated by SDS-PAGE 5-22% (300 V, 2 h) and stained (Coomassie Brilliant Blue R250, 1 h). MRJP1 bands were excised and electrotransferred onto a nitrocellulose membrane (300 V, overnight). Membranes were incubated in TBS-T (Tris-HCl, pH 7.4, 0.5% Tween 20) containing 5% of dried milk overnight at room temperature, prior to incubation with diluted serum at TBS (1:1) for 2 h. Subsequently, membranes were washed three times for 5 min with TBS-T, and anti-MRJP1 was eluted with 1.4% triethylamine (1 min) and transferred to vials containing 1 M Tris-HCl, pH 8.5, at room temperature. After elution, antibodies were dialyzed in TBS containing 0.1% sodium azide, quantified (Bradford’s method) and stored at -20 °C. The specificity of the antibodies was determined by immunoblotting after SDS-PAGE of nurse brain extract. Three regions of honey bee brains (antennal lobe, optical lobe and mushroom bodies) were dissected and fixed for 5 h at room temperature in a solution containing 0.5% glutaraldehyde, 4% paraformaldehyde, 0.2% picric acid in 5% sucrose and 5 mM calcium chloride in 0.1 M sodium cacodylate buffer, pH 7.2. After being rinsed in the same buffer overnight at 4 °C, free aldehyde groups were quenched with ammonium chloride in this buffer for 1 h at 4 °C. The material was stained en bloc with 2% uranyl acetate in 15% acetone for 2 h at 4 °C, subsequently dehydrated in acetone (30-100%), and embedded in LRGold resin. Ultrathin sections were collected on nickel grids and preincubated in phosphate saline buffer (PBS) containing 1.5% bovine albumin (PBS-BSA) and 0.01% Tween 20, and subsequently incubated for 1 h with anti-MRJP1, diluted 1:10 in PBSBSA. After rinsing in PBS-BSA, the grids were incubated for 1 h with labeled secondary antibody (anti-rabbit IgG, Au-conjugated, 10 nm, Sigma) diluted 1:20 in PBS-BSA. Finally, the grids were washed with PBS and distilled water, stained with uranyl 1466

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Garcia et al. acetate and lead citrate, and observed and photographed in a Jeol 1011 transmission electron microscope (Tokyo, Japan).

3. Results and Discussion 3.1. Protein Extraction and 2-DE. Different procedures were tested for honey bee brain protein extraction. Ultrasound, glass beads, and manual homogenization were used for cellular disruption. Best results were obtained using manual homogenization, which showed good protein recovery and integrity (data not shown). Moreover, it is a simpler procedure for protein extraction. Initial 2-DE experimental conditions were unable to focus proteins at basic pH regions of the gels when 3-10 IPG strips were used in the IEF step (Suppoprting Information Figure 1 A). Addition of 10% isopropanol to 2-DE sample buffer26 was necessary for better resolution of basic proteins (Supporting Information Figure 1B,C). For the second dimension of SDSPAGE, best resolution for high molecular mass polypeptides, which comprised the majority of spots in the 2-DE maps, was achieved under 10% T concentration (Supporting Information Figure 1C). Initial gel image computational analysis using wide pH range (3-10) 2-DE gels showed that most of the differentially expressed spots were concentrated in the acidic pH range (Supporting Information Figure 2). Therefore, IPG strips in the pH 4-7 range were used for further proteomic studies, since they provided better resolution (Supporting Information Figure 2). 3.2. Protein Identification. Twenty spots, corresponding to 16 distinct proteins, were shown to be differentially expressed in nurse and forager brains. Such proteins were identified by PMF (Table 1, Figure 1). Four spots were subcaste-specific, namely, spots 11 (major royal jelly protein 7, MRJP7) and 12 (MRJP2) in nurse (Figure 1) and spots 19 and 20 (alpha-glucosidases) in forager (Figure 1). The other 16 spots displayed different intensities between the subcastes. The differential expression of such 16 proteins were shown to be statistically significant by the Student’s t test (p < 0.01). A total of 14 proteins were identified with Mascot scores above 100 and sequence coverage higher than 24%, with the peptide mass tolerance 2.75 for n ) 12 were considered statistically significant

jelly,31-34 brain neuropeptides35 and, more recently, differences in the thorax muscle36 and whole-body protein profiles of the nest workers and the foragers.37,38 In the present work, comparative proteomic analysis of honey bee brains from nurse and forager subcastes was conducted. 3.3.1. Nurse Subcaste Up-Regulated Proteins. Three proteins detected and identified as up-regulated in nurse brain (MRJP1, MRJP2 and MRJP7) have also been previously detected in the secretion of nurse hypopharyngeal gland30 and royal jelly,31-34 as the secretion is also known. These proteins may be related to caste determination and social functions during the honey bee larvae differentiation as well as play an important role in honey bee nutrition.39 Gene expression of these royal jelly proteins in honey bee brain has been previously reported. For example, the gene of MRJP140 is expressed in a subset of kenyon cells in the mushroom bodies (MBs),41 suggesting a role for this protein in the central brain. Besides, MRJP2 and MRJP7 mRNAs were detected in wild-type worker brain tissue.42 Little is known about the function of these proteins in the honey bee brain. It

is possible that MRJPs could function in brain as reserve of amino acids for protein synthesis, specifically during the nurse to forager ontogenetic and behavioral development. To confirm the presence of MRJP1 in the honey bee brain, and to discard the possibility of being contamination from the hypopharyngeal gland content, immunolocalization experiments were performed in the three regions of the nurse brain. Anti-MRJP1 antiserum was specific as demonstrated in Supporting Information Figure 3. Electron microscopy showed labeling in antennal lobe (AL), optical lobe (OL) and MB (Figure 2). MRJP1 is located in the cytoplasm, while no labeling was found in mitochondria (Figure 2A,C). Actually, MRJP1 seems to be deposited along filaments of the cytoskeleton (Figure 2A,C). Also, MRJP1 is deposited on the rhabdom, a structure of the retinular cells, composed of numerous tubules (Figure 2E). Such evidence suggests that MRJP1 could be associated with proteins of filamentous structures. Images also showed that MRJP1 is found in intercellular spaces in MBs (Figure 3), indicating that it is a secreted protein. Such results suggests that MRJP1 might have extra and unsuspected functions Journal of Proteome Research • Vol. 8, No. 3, 2009 1467

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Figure 1. Comparative 2-DE gels (pI 4-7) of total protein extracts from honey bee brain. Nurse; forager. Amounts of 50 µg of protein were used. Arrows indicate proteins up-regulated in each subcaste that were identified by PMF as shown in Table 1. Zoomed boxes show regions with isoforms of differentially expressed proteins MRJP1 (spot 1) and alpha-glucosidase (spots 17 and 19). Fold changes for each differentially expressed protein spot are presented.

besides being just a possible reserve of amino acids for protein synthesis in the brain. Three other proteins that were up-regulated in nurse brainsproteins similar to calreticulin isoform 1, similar to TER94 isoform A isoform 1 and similar to AntDHsdo not have their function described in A. mellifera. Interestingly, homologous proteins from Drosophila probably play olfactory roles as discussed below. Calreticulin is a multifunctional Ca2+-binding molecular chaperone implicated in multiple cellular processes, including neuronal development.43,44 Calreticulin is also essential for integrin-mediated calcium signaling and cell adhesion.45 Other gene that may act in cell adhesion and also Ca2+-binding (BM40-SPARC) has high expression in nurse brain.46 In Drosophila, calreticulin plays a key role in olfactory system functions and odor-guided behavior, possibly by establishing its overall sensitivity to odorants.47 Behavioral reactions to naturally aversive odorants are compromised in Drosophila Crc mutants. These odor avoidance deficiency is not specific to one class of odorant and appear to be correlated to a change in odorant potency.47 Immunocytochemistry analysis demonstrated that TER94 is predominant in the nervous system (mushroom body and antennal glomeruli) of adult Drosophila.48 The antennal glomeruli comprise the terminations of the primary olfactory 1468

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neurons and dendrites of interneurons. Mushroom bodies apparently play a role in the processing and storage of chemosensory information, and are thought to be the site of olfactory learning.49 The high level of TER94 in these tissues may reflect the need for this protein in the developing growth cones and may help the targeting by catalyzing a membrane fusion event between TER and growth cone.48 Regarding AntDH, a short chain antennal dehydrogenase/reductase, Northern blots and in situ hybridization on cryosections analysis of heads and antennae of Drosophila showed that such protein expression was restricted to the third antennal segment, suggesting a role for AntDH in olfaction, maybe for odorant turnover.50 We also found that actin 88F is up-regulated in nurse brain. Previous report demonstrated a high concentration of F-actin in olfactory glomeruli in A. mellifera as well as in other insect and vertebrate species, indicating that this is an essential feature of such a synaptic complex most likely related to a high degree of synaptic and structural plasticity within the glomerular neuropil of primary olfactory centers.51,52 Furthermore, a striking long-term adult plasticity of F-actin rich glomeruli complexes in the olfactory and visual input regions of the mushroom calyx suggests that these synaptic changes might play a causal role in caste- and age-specific behavioral adaptations.52

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Figure 3. Detection of MRJP1 in intercellular space between mushroom bodies cells. Sample was inmunolabeled with antiMRJP1 and colloidal gold followed by electron microscopy. Abbreviations: ie, intercellular space; cm, cellular membrane. The bars on each picture represent 0.5 µm (left bars) and 0.2 µm (rigth bars).

Figure 2. Immunogold localization of MRJP1 in honey bee brain. Shown is electron microscopy images of three brain regions inmunolabeled with anti-MRJP1 and colloidal gold. (A) Mushroom bodies; (B) control; (C) antennal lobe; (D) control; (E) optical lobe; (F) control. The bars on each picture represent 0.5 µm (left bars) and 0.2 µm (rigth bars). Abbreviations: c, cytoplasm; m, mitochondria; r, rhabdom; d, desmosomes; g, pigment granules.

It is noteworthy that all the four proteins described above are potentially related to the olfactory system. Such proteins were found to be up-regulated in the nurse brain. As the environment inside the hive is dark, nurses should heavily rely on an odor-based communication to accomplish its behavior. Indeed, an interesting study recently showed the effects of honey bee chronological age and of social role on sensory sensitivity and associative olfactory learning performance.53 A reduction in olfactory acquisition function was related to social role, but not to chronological age. This decline occurred only in foragers with long foraging experience, but at the same time, the foragers showed less generalization of odors, which is indicative of more precise learning. Foragers that were reversed from foraging to nursing tasks, furthermore, did not show deficits in olfactory acquisition.53 Other four proteins up-regulated in nurse brain were protein similar to ERp60 CG8983-PA isoform A isoform 2, protein similar to T-complex chaperonin 5 CG8439-PA isoform A, protein similar to CG8351-PA isoform 1 and protein similar to

stubarista CG14792-PA isoform A. All of them act in protein folding during protein synthesis processes, except stubarista which act as a ribosomal protein. Besides, ERp60 belongs to protein disulfide isomerase (PDI) family, which interacts with calreticulin in glycoprotein folding cycle.54 Nurses need a high protein synthesis activity to develop the protein machinery necessary for the changes in brain structure that precede ontogenesis to foragers, which would justify the up-regulation of these proteins. 3.3.2. Forager Subcaste Up-Regulated Proteins. Foragers overexpressed alpha-glucosidase, transferrin, protein similar to glutamine synthetase 2 isoform C and protein similar to sarcoplasmic calcium-binding protein 2. Alpha-glucosidase, which converts sucrose, the primary component of nectar, into glucose and fructose, is expressed in several organs of A. mellifera.55-57 A purified alpha-glucosidase III was also able to efficiently hydrolyze maltose and maltotriose.58 Studies using microarrays and Northern blots showed alpha-glucosidase gene as up-regulated in experienced honey bee head when compared to naive ones.14 Honey bees accumulate glycogen in various body parts, including head.59 Alpha-glucosidase is also known as an enzyme involved in oligosaccharides and glycogen degradation, and products of carbohydrate metabolism are used not only as fuel to foraging flights,60 but also as nutrients for neurons in honey bee.61 Upregulation of alpha-glucosidase in brain could be related to higher energetic requirements associated to increased brain activity during learning and memorization processes that are triggered upon foraging. Journal of Proteome Research • Vol. 8, No. 3, 2009 1469

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Figure 4. Representative examples of MALDI-TOF mass spectra of tryptic digests from protein spots 18 and 21. Protein was identified as similar to glutamine synthetase 2, isoform C. Theoretical and experimental matched peptide masses between both spots are marked by small arrows. Different peptide mass appearing in mass spectrum of protein spot 18 was indicated by a large arrow.

Transferrin, an iron-binding protein, was up-regulated in forager brain, which coincides with previous reports that described, at transcriptional level, higher amounts of AmTRF (A. mellifera transferrin) mRNA in central brain neuropils and in pigmented eyes than in other body parts of foraging honey bee workers.62 Additionally, exposure of newly emerged bees to constant light for 24 h led to a 50% up-regulation of AmTRF message.62 Transferrin could then be important for the nervous system of foragers to deal with new visual inputs outside the hive. The high levels of AmTFR in the honey bee central nervous system could also be associated with a potential role as a component of a protection mechanism against oxidative stress by reactive oxygen species (ROS). Interestingly, recent results indicated that normal cellular redox in honey bee is crucial for olfactory processing, and chelation of iron prevents ROS-mediated oxidative stress.63 In insects, transferrin belongs to a multigene family,64 and presents other possible roles as an iron transport protein, an antibiotic agent, a vitellogenic inducer and a protein repressed by juvenile hormone.65 Another up-regulated protein in forager brain was identified as similar to the gene of Drosophila melanogaster sarcoplasmic calcium-binding protein 2 (dSCP2), whose product gave juvenile hormone diol kinase (JDHK) activity and showed high similarity to JHDK from Manduca sexta.66 Molecular modeling and crystal three-dimensional structure of these proteins revealed structural similarity to G-proteins and calcium binding proteins.66,67 Proteomics and in situ hybridization analysis revealed that JHDK expression was upregulated in MBs of forager honey bee brains.68 JHDK is an important enzyme involved in the juvenile hormone (JH) inactivation pathway. Higher levels of JHDK in forager brain 1470

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may be required for the regulation of JH titers. On its turn, JH induces changes in honey bee brain structures related to behavioral maturation and age polyethism.69 Sarcoplasmic calcium-binding protein 2, also known as calexcitin, has been related to associative memory by mobilization of intracellular Ca2+.70 It is possible that JHDK associates with Ca2+-signaling in A. mellifera MBs, fostering the high capacity of learning and memory of the honey bee.68 A recent study assessed the molecular basis of flight phenotypes in Drosophila flight defective mutants for inositol 1,4,5trisphosphate receptor (InsP3R), an intracellular Ca2+ release channel.71 For that, a microarray screen was done with RNA isolated from adult heads and thoraces. Down-regulation of several genes that affect the excitability of neurons and muscles was observed. Among these, the role of glutamine synthetase 2 was investigated further. This enzyme reduces glutamate levels at the synapse. Transcripts for the enzyme glutamine synthetase 2 (Gs2) were significantly reduced in Drosophila InsP3R mutants. This was accompanied by lower levels of glutamate, as well as by increased axonal branches and synapse number at the flight neuromuscular junction. A control of both glutamate levels and Gs2 expression by Ca2+ signals generated through the InsP3R through a cell nonautonomous mechanism was proposed. Levels of these molecules might be important for the maintenance of flight stability.71 A similar function for glutamine synthetase 2 could be proposed in the honey bee model. Foragers, which must have a complex motor behavior for performing foraging flights, showed overexpression of one of the enzyme species in relation to nurses according to our results, as a protein similar to glutamine synthetase 2 was present in two different spots (Figure 4), but only spot 18

Proteomic Analysis of Honey Bee Brain provided statistically significant differential expression between the subcastes. We additionally found that protein lethal(2) essential for life is up-regulated in forager brain. Previous report using Northern blot hybridization also showed that the gene which encodes that protein is up-regulated in forager head.14 This protein has unknown function in A. mellifera, but is a crystallin-like chaperonin, HSP20, encoded by l(2)efl (lethal 2 essential for life) gene in D. melanogaster and belonging to the group of hsp (heat shock protein) genes.72 Long-lived germ-line-less flies show increased production of Drosophila insulin-like peptides (dilps) and hypoglycemia but simultaneously exhibit several characteristics of insulin/IGF signaling impedance, as indicated by up-regulation of the Drosophila FOXO (dFOXO) target genes 4E-BP and l(2)efl and the insulin/IGF-binding protein IMP-L2, where dFOXO is known to be required for extended longevity.73 Perhaps protein lethal(2) essential for life might somehow be involved in regulating aging and lifespan in the honey bee. 4. Concluding Remarks. Our study demonstrated clear brain proteome differences between honey bee (A. mellifera) nurse and forager subcastes with distinct social roles. The differential expression of the identified proteins could be correlated to putative functions of each honey bee worker subcaste associated ontogenetic development or social roles. Such results are complementary to previous genomic and transcriptomic data. However, further research must be performed to achieve a scenario where the actual functions of such brain proteins can be precisely related to ontogeny and behavior. Studies at the genomic and transcriptomic levels, coupled with deeper proteomic analyses, are necessary to fully understand the differentiation mechanisms in A. mellifera. For example, new proteomic investigations with RNAi-modified brains and brain parts would be interesting, as well as interactome analyses of candidate proteins.

Acknowledgment. We would like to thank Dr. Lila Castellanos-Serra (CIGB, Havana, Cuba) for contributions to experimental procedures, Manoel Silva (Apiarios Vereda Rosa, Brasilia, Brasil) for insect collection, Juliana Souza da Silva (University of Brasilia) for electron microscopy analyses, Prof. Pedro Zanotta (University of Brasilia) for help with statistical analyses and Nuno M. Domingues (University of Brasilia) for technical assistance. This work was supported by postgraduate fellowship 190366/2005-2 from TWAS (The Academy of Sciences for the Developing World, Italy)/CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico, Brazil) to L.G.H, by research grant 480333/2004-1 from CNPq and by CT-INFRA fund from FINEP (Financiadora de Estudos e Projetos, Brazil). Supporting Information Available: Supplementary Figure 1, honey bee brain 2-DE gels in 3-10 pH range; Supplementary Figure 2, representative 2-DE gels (pI 3-10) of total protein extracts from honey bee brain; Supplementary Figure 3, specificity of anti-MRJP1 antibody. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Page, R. E., Jr.; Peng, C. Y. Aging and development in social insects with emphasis on the honey bee Apis mellifera L. Exp. Gerontol. 2001, 36 (4-6), 695–711. (2) Menzel, R.; Leboulle, G.; Eisenhardt, D. Small brains, bright minds. Cell 2006, 124 (2), 237–9.

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