Proteome of the head and thorax salivary glands in the Melipona quadrifasciata anthidioides Douglas Elias-Santos, Maria Fialho, Rui Vitorino, Leandro Oliveira, José Zanuncio, José Serrão

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Douglas Elias-Santos, Maria Fialho, Rui Vitorino, Leandro Oliveira, José Zanuncio, et al.. Proteome of the head and thorax salivary glands in the stingless bee Melipona quadrifasciata anthidioides . Apidologie, Springer Verlag, 2013, 44 (6), pp.684-698. ￿10.1007/s13592-013-0217-6￿. ￿hal-01201338￿

HAL Id: hal-01201338 https://hal.archives-ouvertes.fr/hal-01201338 Submitted on 17 Sep 2015

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie (2013) 44:684–698 Original article * INRA, DIB and Springer-Verlag France, 2013 DOI: 10.1007/s13592-013-0217-6

Proteome of the head and thorax salivary glands in the stingless bee Melipona quadrifasciata anthidioides

1 1 2 Douglas ELIAS-SANTOS , Maria do Carmo Q. FIALHO , Rui VITORINO , 1 3 1 Leandro L. OLIVEIRA , José C. ZANUNCIO , José Eduardo SERRÃO

1Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-000 Viçosa, Minas Gerais, Brazil 2Department of Chemistry, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal 3Departamento de Biologia , Universidade Federal de Viçosa, 36570-000 Viçosa, Minas Gerais, Brazil

Received 1 March 2013 – Revised 7 May 2013 – Accepted 23 May 2013

Abstract – The exocrine glands of social are related to the social communication, reproduction, and development of individuals. Eusocial bees have two types of salivary glands: the head salivary gland, which possibly functions in marking food sources, and the thorax salivary gland, which produces saliva. This study evaluated the major protein content of the head and thorax salivary glands of the stingless bee Melipona quadrifasciata anthidioides forager workers. The head salivary gland expresses 27 proteins in high quantity, including heat shock proteins, enzymes of the glycolysis pathway, gene regulation proteins, and an odorant- binding protein. The thorax salivary gland expresses 12 proteins, including heat shock proteins, cellular detoxification proteins, energy metabolism proteins, and proteins linked to environmental stress. The proteins identified in both the head and thorax salivary glands contribute to our understanding of their possible functions in stingless bees. behavior / odorant-binding protein / heat shock protein / / stress

1. INTRODUCTION and diversity in the body. The best- known functions of the exocrine glands are Social Hymenoptera are intriguing insects related to the communication, reproduction, and because of the complexity and organization of development of individuals (Cruz-Landim and their social lives. In bees, queens and workers Abdalla 2002). For communication, exocrine form distinct castes, each playing important glands release chemicals termed pheromones, roles within the colony (Michener 1974). More- which are a mix of compounds causing physi- over, in highly eusocial bees, there is a division of ological and/or behavioral responses in other labor between workers, which favors protection individuals of the same species (Free 1987). against natural enemies, increases resistance to The salivary glands of eusocial bees are environmental stress, and in resource storage classified into two types: head salivary glands (Roubik 1989). (labial glands) and thorax salivary glands. Almost all aspects of the social lives of Thorax salivary glands have secretory units that insects are linked to chemical signals produced open into an excretory duct toward the head and by exocrine glands that occur in high numbers fuses with the ducts of the head salivary glands (Cruz-Landim 1967). The secretory cells of the Corresponding author: J.E. Serrão, [email protected] thorax salivary glands of Scapitotrigona postica Manuscript editor: James Nieh (Meliponini), Xylocopa suspecta (Xylocopini), Proteome of salivary glands in M. quadrifasciata anthidioides 685 and Centris fuscata (Centridini) bees are rich in identify the main protein constituents of both rough endoplasmic reticulum (Cruz-Landim the head and thorax salivary glands in the 2009) and may be involved in the production stingless bee Melipona quadrifasciata anthi- of enzymes, such as proteases, lipases, and dioides workers to enhance the understanding of lactases, as reported for Apotrigona nebulata, the functions of these glands in social bees. Melipona becheeii, and S. postica (Arnold and Delage-Darchen 1978; Delage-Darchen et al. 2. MATERIAL AND METHODS 1979; Delage-Darchen and Darchen 1982; Costa and Cruz-Landim 2001). Nine proteins 2.1. Bees and salivary glands occur in both thorax salivary gland of Apis melifera workers and in stored royal jelly Forager workers of M. quadrifasciata anthidioides (Fujita et al. 2012). were obtained from three colonies kept in the apiary The head salivary glands occur only in the of the Federal University of Viçosa (20°45′ N, 42°52′ stingless bees Euglossini, Bombini, Meliponini, W), Viçosa state of Minas Gerais, Brazil. and Apini (Cruz-Landim 2009). These glands For head and thorax salivary gland extraction, 51 are derived from a secondary growth of the forager workers were collected from each colony, thorax salivary glands and are formed by totaling 153 bees. The bees were collected from close multicellular alveoli with a thin cuticle lining to the colony entrance when returning with corbicu- the gland lumen (Cruz-Landim 1967). Secretory lae loaded with grains. Bees were cryoanes- cells of the head salivary gland have features of thetized, dissected in phosphate-buffered saline, and cells that metabolize lipids in old bees, but in the head and thorax salivary glands were transferred young workers, the secretory cells have well- to 1 % protease inhibitor cocktail (Sigma, P27147), developed rough endoplasmic reticulum and homogenized in buffer [(7 M urea, 2 M thiourea, 4 % high concentrations of free ribosomes (Cruz- CHAPS, 2 % immobilized pH gradient (IPG) buffer, Landim 2009). A possible function of the head 40 mM dithiothreitol (DTT)], and centrifuged at salivary glands is to contribute to the worker’s 14,000×g for 30 min at 4 °C. To obtain soluble recruitment for foraging and trail marking proteins, only the supernatant was collected and (Jarau et al. 2004a). In the stingless bees stored at −80 °C until use. The amount of protein in Geotrigona subterrania and Geotrigona mom- the samples was determined according to the Brad- buca, head salivary glands and mandibular ford method using bovine serum albumin as a glands together play a role in marking food standard. sources and worker recruitment (Blum et al. 1970; Stangler et al. 2009). Moreover, in the 2.2. Two-dimensional gel electrophoresis orchid bee Euglossa viridissima, the head salivary glands play a role in the mechanism For isoelectric focusing of soluble proteins from of fragrance collection in plants (Eltz et al. the head and thorax salivary glands, immobiline 2007). The secretion of the head salivary glands DryStrip, pH 3–10 (7 cm, GE Healthcare), were is also related to wax manipulation for nest rehydrated in 150 μL of rehydration buffer (7 M urea, building (Heselhaus 1922), mandible lubrication 2 M thiourea, 4 % CHAPS, 2 % IPG buffer, 0.002 % (Simpson 1960), and resin manipulation (Santos Bromophenol blue) containing 100 μg of protein et al. 2009). In A. mellifera, head salivary from each sample for 24 h in five voltage steps (200- glands produce some proteins found in royal V step-n-hold for 1 h, 500-V step-n-hold for 1 h, jelly (Fujita et al. 2012). 1,000-V gradient for 1 h, 8,000-V gradient for 3 h, Considering that the head and thorax salivary and 8,000-V gradient for 4 h). The Ettan IPGphor glands of bees produce different substances that III® was used for focusing. The second dimension of have different functions as well as the ecolog- sodium dodecyl sulfate polyacrylamide gel electro- ical and economic importance and the risk of phoresis (SDS-PAGE) was performed using a Mini extinction of stingless bees, this study aimed to Protean IITM (Bio Rad) onto a 12 % polyacrylamide 686 D. Elias-Santos et al.

gel. The focused strips were equilibrated in buffer the mass range of 800–4,500 Da with about 1,500 (6 M urea, 75 mM Tris–HCl, 29.3 % glycerol, 2 % laser shots. For each sample spot, a data-dependent SDS, 0.002 % Bromophenol blue) containing 1 % acquisition method was created to select the four most DTT for 20 min under stirring. Following this, the intense peaks, excluding those from the matrix, trypsin buffer was replaced by the same buffer containing autolysis, or acrylamide, for subsequent MS/MS data 2.5 % iodoacetamide for 20 min in the dark. The acquisition. Mass spectra were internally calibrated with strips were then washed for 5 s in running buffer (3 % autodigested peaks of trypsin (MH+ =842.5, Tris–HCl, 14.4 % glycine, 1 % SDS, pH 8.8) and run 2,211.42 Da) allowing a mass accuracy >25 ppm. on 12 % SDS-PAGE gels at 200 V for 50 min. The gel was stained for 16 h in Coomassie G-250 solution 2.5. Protein identification (0.002 % Coomassie blue G-250, 10 % acetic acid, 50 % ethanol). To validate the data, three gels were The spectra were processed and analyzed using made for each gland. Then, the gels were digitalized the Global Protein Server Workstation (Applied and analyzed using Image Master 2D Platinum 7.0 Biosystems), which uses internal MASCOT software for spot detection and analysis. Each spot was (v.2.1.0, Matrix Science, London, UK) to search the assigned a different symbol for identification and peptide mass fingerprints and MS/MS data. The for further image analysis. Trembl non-redundant protein sequence database (January 18, 2011) was used for all searches under 2.3. Sample digestion All entries and the allowance for up to two missed tryptic cleavages. The peptide mass tolerance was The two-dimensional gel electrophoresis (2DE) 25 ppm and fragment ion mass tolerance was 0.3 Da. spots that were expressed more were manually Positive identifications were accepted up to 95 % of excised and the gel pieces were washed three times the confidence level. with 25 mM ammonium bicarbonate/50 % acetoni- The identified proteins were analyzed and associ- trile (ACN), one time with 100 % (ACN), and dried ated with their molecular functions, biological pro- in a SpeedVac (Thermo Savant). Then, 25 μLof cesses, and cellular components using the online tool 10 μg/mL sequence-grade modified porcine trypsin http://web.expasy.org/blast/ and the database http:// (Promega) in 25 mM ammonium bicarbonate was www.uniprot.org/. The results were related to the added to the dried gel pieces and the samples were biological roles of both the head and thorax salivary incubated overnight at 37 °C. Extraction of tryptic glands and to the different functions according to the peptides was performed by the addition of 10 % biological processes contained in the database. formic acid (FA)/50 % ACN three times and the peptides lyophilized in a SpeedVac (Thermo Savant). 3. RESULTS

2.4. Mass spectrometry analysis The concentrations of soluble proteins from each gland were 1.90±0.05 and 7.77±0.05 μg Tryptic peptides were resuspended in 10 μLofa per bee (N=153) in the head and thorax salivary 50 % ACN/0.1 % FA solution. The samples were glands, respectively, of M. quadrifasciata anthi- mixed (1:1) with a matrix consisting of a saturated dioides forager workers. In the 2DE maps, the solution of α-cyano-4-hydroxycinnamic acid pre- spot distribution of the head and thorax salivary pared in 50 % ACN/0.1 % FA. Aliquots of the glands showed different protein expression samples (0.5 μL) were spotted onto the matrix- patterns, evidencing a lower number of soluble assisted laser desorption/ionization (MALDI) sample proteins in the head salivary gland (198 spots) target plate. when compared with the thorax salivary gland Peptide mass spectra were obtained on a MALDI (225 spots) of M. quadrifasciata anthidioides time-of-flight (MALDI-TOF/TOF) mass spectrometer forager workers (Figure 1a, b). (4800 Proteomics Analyzer, ABSciex, Europe) in the The expressed proteins for the head and positive ion reflector mode. Spectra were obtained in thorax salivary glands of M. quadrifasciata Proteome of salivary glands in M. quadrifasciata anthidioides 687

Figure 1. Two-dimensional gel electrophoresis maps evidencing the identified spots using MALDI-TOF/TOF (arrows) in the head salivary gland (a) and in the thorax salivary gland (b)ofM. quadrifasciata anthidioides forager workers. anthidioides showed different isoelectric points head salivary gland maps. The analysis of three and molecular weights (Electronic supplemen- maps for the head salivary gland showed 60 tary material Table S1). Furthermore, there is an spot matches, while the thorax salivary gland enrichment of proteins with acidic pH in the showed 112 spot matches. The match between 688 D. Elias-Santos et al. the head and the thorax salivary gland maps stitutive proteins (proteasome subunit, actin 3, shows that 28 spots equate. Furthermore, there alpha-tubulin, actin/cofilin depolymerizing fac- is an enrichment of proteins with basic pH in tor homolog); environmental response proteins the gel map from the thorax salivary gland, and (beta-1 subunit of heat shock protein, CG7217- most of the spots has an estimated pI ranging PB); cellular detoxification proteins (glutathi- from 7.1 to 8.4 (Figure 1b). one-S-transferase, superoxide dismutase 1); and Although only a small part of the tran- proteins of unknown function (protein homolo- scriptome of the brain and abdomen has been gous to intra-flagellar transport 88). The pro- described in M. quadrifasciata in previous teins CG7217-PB and superoxide dismutase 1 studies, the genome is not completely evident correspond to spot 33. As observed, the 12 for this species and fault information (Woodard spots with pI ranging from 4.5 to 8.5 were et al. 2011). Thus, the identifications were based identified (Table II and Figures 1b and 2b). on homology to other species. From 42 excised spots, 26 spots were 4. DISCUSSION positively identified in the head salivary gland of M. quadrifasciata anthidioides forager work- The head salivary gland of M. quadrifasciata ers as belonging to several protein classes: anthidioides forager workers had a lower total α energy metabolic proteins (two -N-acetylga- protein content (1.90±0.05 μg) than the thorax lactosaminidase, fructose bisphosphate aldolase, salivary gland (7.77±0.05 μg), suggesting that protein similar to malic enzyme CG10120-PB, each gland synthesizes different compounds or arginine kinase, triosephosphate isomerase-like, that they have different intracellular protein glyceraldehyde-3-phosphate dehydrogenase, concentrations. As such, the secretory cells of phosphoglycerate mutase, CG1707-PA); pro- the head salivary glands in the honeybee A. teins of unknown function (AGAP001151-PA, mellifera have well-developed smooth endo- GM12998, similar to CG4300-PA, UPF0753 plasmic reticulum, and with the bee aging, protein sce4789, α-S1-casein, putative unchar- secretion accumulates in the cells and lumen acterized protein of Anopheles gambiae); con- of the gland (Silva de Morais 1978; Cruz- stitutive proteins (annexin IX CG5730-PC, 30S Landim 2009; Poiani and Cruz-Landim 2009). ribosomal protein S18, beta subunit of the E1 The head salivary gland cells of A. mellifera component from pyruvate dehydrogenase, ubiq- forager workers store lipids (Poiani and Cruz- uitin); environmental response proteins (heat Landim 2010); this explains the relevant ex- shock protein beta-1-like isoform 1, GG19234, pression of proteins similar to malic enzyme GG17772); genetic modulators (14-3-3 protein CG10120-PB, which plays a role in lipid zeta, windbeutel CG7225-PA, transcription co- metabolism (Figure 1a, spot 101c). However, repressor MIG3); and those with specific func- secretions from the thorax salivary gland are tions (odorant-binding protein). In some spots, aqueous (Simpson 1960; Cruz-Landim 1967), more than one protein was identified, e.g., spot which would explain the higher concentration 57—the 30S ribosomal protein S18 and of cytoplasmic soluble proteins in the thorax UPF0753 protein sce4789 (Figure 1a); spot salivary gland of M. quadrifasciata anthidioides 47—protein transcription co-repressor MIG3 forager workers. As observed in Figure 1b, most and the CG4300-PA-like protein (Table I and of the spots have basic pI, which gives basic Figures 1a and 2a). characteristics to the intracellular environment In the thorax salivary glands of M. quad- of the salivary gland of the thorax. In addition, rifasciata anthidioides forager workers, from 52 ultrastructural studies showed that secretory excised spots, 12 distinct proteins were identi- cells of the thorax salivary gland in forager fied and annotated to energy metabolic proteins workers of bees have abundant rough endoplas- (glyceraldehyde-3-phosphate dehydrogenase 2, mic reticulum and secretions accumulate into phosphoglycerate kinase, arginine kinase); con- cytoplasmic granules, suggesting a proteina- Table I. Proteins identified from the head salivary glands of M. quadrifasciata anthidioides forager workers.

Spota Protein (species)b Acession codeb Observed Calculated Observed End sequence (ion score)d MW/pIc mass mass peptided peptided

3C Alpha-N-acetylgalactosaminidase E2B637_9HYME 23/3.5 1,503.85 1,503.84 DIAQVVTTLFALGR (108) (Harpegnathos saltator) 1,631.95 1,631.94 KDIAQVVTTLFALGR (136) rtoeo aiaygad in glands salivary of Proteome 101C Arginine kinase (fragment) A1Y032_9HYME 40/6.61 2,368.13 2,368.14 DFGDVDTLGNLDPTGEFIVSTR (149) (Axestotrigona ferruginea) 72C GG17772 (Drosophila erecta) B3NWP9_DROER 15/5.5 1,596.75 1,596.74 DYGVLDEESGIPFR (91) 75C Ubiquitin, putative A2DM10_TRIVA 14/5.5 1,523.82 1,523.77 IKDKEGIPPDQQR (106) (Trichomonas vaginalis) 97C/5 Heat shock protein beta-1 E2A8U5_9HYME 29/7.1 1,653.84 1,653.82 FDVSQYTPEEIVVK (84) (Camponotus floridanus) 1,712.85 1,712.85 LGDFSVIDTEFSNIR (114) 1,923.02 1,923.02 LRFDVSQYTPEEIVVK (54) 113C Pyruvate dehydrogenase E1 component E2AI43_9HYME 39/7.3 1786.93 1786.91 VFILGEEVALYDGAYK (73) subunit beta, mitochondrial (Camponotus floridanus) M .

38C Phosphoglycerate mutase B4M5B8_DROVI 28/5.1 1,259.67 1,259.67 FDVAHTSLLTR (80) anthidioides quadrifasciata (Drosophila virilis) 1,307.71 1,307.63 YGEEKVKIWR (66) 48C GG19234 (Drosophila erecta) B3NVQ2_DROER 80/4.3 1,712.85 1,712.85 LGDFSVIDTEFSNIR (98) 74C Putative uncharacterized protein Q8T6R3_ANOGA 18/5.7 2,173.08 2,173.04 YTSKYDNINVDEILKSDR (85) (Anopheles gambiae) 23C Fructose-bisphosphate aldolase E2BI91_9HYME 45/4.3 2,098.12 2,098.12 IVPIVEPEILPDGDHDLAR (100) (Harpegnathos saltator) 14C Odorant-binding protein D6BJF5_BOMIG 16/4.5 1,652.78 1,652.79 YDNIDLDSILNSDR (115) (Bombus ignites) 2C Alpha-N-acetylgalactosaminidase E2B637_9HYME 24/3,2 1,503.85 1,503.85 DIAQVVTTLFALGR (96) (Harpegnathos saltator) 1,631.95 1,631.94 KDIAQVVTTLFALGR (10) 43 PREDICTED: similar to Annexin IX gi|66530527 49/7.9 1,771.90 1,772.01 GFGTDEQTIIDVLAHR (66) CG5730-PC,isoform C isoform 2 (Apis mellifera) 44 AGAP001151-PA (Anopheles gi|58396588 39/7.8 2,331.20 2,331.23 LGLALNFSVFYYEILNSPDR (81) gambiae str. PEST) 14-3-3 protein zeta (Aedes aegypti) 1433Z_AEDAE 2,303.20 2,303.21 LGLALNFSVFYYEILNSPDK (134) 689 690 Table I. (continued)

Spota Protein (species)b Acession codeb Observed Calculated Observed End sequence (ion score)d MW/pIc mass mass peptided peptided

45 GM12998 (Drosophila sechellia) gi|195358940 38/7.8 2,303.20 2,303.16 LGLALNFSVFYYEILNSPDK (130) 3,327.68 3,327.57 QAFDDAIAKLDTLNEDSYKDSTLIMQLLR (29) 47 PREDICTED: similar to CG4300-PA, gi|110750077 49/7.7 1,723.93 1,723.93 LLEYDIDKLVFEAR (100) isoform A isoform1 (Apis mellifera) 1,882.07 1,882.06 EIVILGGGDGGLLWELLK (89) 2,451.36 2,451.37 VAHTVLLDFTVPSNVIVDVEKR (110) Transcription corepressor MIG3 MIG3_YEAST 1,549.73 1,549.74 IMPSVNTGDMEISR (25) (Saccharomycescerevisiae) 1,705.84 1,705.84 RIMPSVNTGDMEISR (20) 49 Alpha-S1-casein (Bubalus bubalis) CASA1_BUBBU 18/7.7 39/7.7 1,267.70 1,267.70 YLGYLEQLLR (46) .EisSno tal. et Elias-Santos D. 52 PREDICTED: similar to CG1707-PA gi|66504768 24/6.8 1,867.00 1,867.02 SLPFYTEVLGMQLLQK (24) (Apis mellifera) 2,304.15 2,304.16 FSLYFLGYEDPKDIPTDKR (49) 56 PREDICTED: similar to malic enzyme gi|110761561 80/6.1 2,205.04 2,205.05 YGQNTLIQFEDFGNANAFR (158) CG10120-PB, isoform B isoform 1, 2,361.14 2,361.15 RYGQNTLIQFEDFGNANAFR (19) partial (Apis mellifera) 57 UPF0753 protein sce4789 Y4789_SORC5 42/5.9 1,329.66 1,329.75 IGDEDLEAALAGR (42) (Sorangium cellulosum) 30S ribosomal protein S18 gi|259500769 1,329.78 1,329.75 NQRMLTVAIKR (53) (Lactobacillus iners DSM13335) 59 Triosephosphate isomerase, TPIS_MAIZE 26/6.0 954.4832 954.4963 FFVGGNWK (63) cytosolic (Zea mays) 2,091.05 2,091.05 DWSNVVVAYEPVWAIGTGK (79) 63 PREDICTED: similar to windbeutel gi|110751310 30/4.7 1,519.79 1,519.79 NILHSFLYRDEL (87) CG7225-PA (Apis mellifera) 2,484.22 2,484.23 FDVAFPYGEKHEQYAQIAAATK (58) 65 Glyceraldehyde-3-phosphate G3P2_CAEBR 30/4.4 1,763.80 1,763.88 LISWYDNEFGYSNR (49) dehydrogenase (Caenorhabditis briggsae) a Spot code corresponds to the position marked on the gel (Figure 1a) b Protein name and accession code were derived from the UniProt protein knowledge base c Observed MW/pI (molecular weight/point isoelectric) were derived from the spot position in the gel (measured in kilodalton) d Calculated mass peptide and observed mass peptide (in dalton) from the peptide sequences analyzed Proteome of salivary glands in M. quadrifasciata anthidioides 691

Figure 2. Distribution of the 26 identified proteins according their possible role in the head salivary gland (a) and the 12 identified proteins according their possible role in the thorax salivary gland (b)ofM. quadrifasciata anthidioides forager workers. ceous nature of the gland secretion (Cruz- (Iovinella et al. 2011), a well-established Landim 2009). However, the morphological pheromone-producing gland (Michener 1974). and functional differences between the head Our results corroborate the proposed function of and thorax salivary glands are not found in all the head salivary gland in producing phero- bee species because head salivary glands are mones in the stingless bees recursa, A. present only in bees (Cruz-Landim 1967, 2009). mellifera, and S. postica (Jarau et al. 2004b; A significant part of the identified proteins Poiani and da Cruz-Landim 2009). Furthermore, was classified in ordorant-binding protein. In the head salivary glands of six species of fact, the expression of an odorant-binding Bombus (Bombini) showed the presence of protein proved an important role of the head volatile compounds involved in communication salivary gland in chemical communication between nest mates (Kubo and Ono 2010). among bees. Odorant molecules, such as pher- Head salivary gland extracts of the stingless omones, are hydrophobic and have low solubil- bees T. recursa and Trigona corvina indicate a ity in the cytoplasmic cell environment, which high probability of the compounds of this gland prevents intracellular transport. Thus, the trans- being involved in marking food sources (Jarau port of these molecules occurs when they bind et al. 2004b, 2010; Stangler et al. 2009). with a soluble protein, such as specific odorant- Odorant-binding protein 14 occurs in the head binding proteins, which have high solubility and salivary glands of nurse and forager workers of constant reversible capacity for the binding and A. mellifera (Furusawa et al. 2008; Fujita et al. dissociation of small molecules (Horst et al. 2012). Although, in the genus Melipona, the 2001). In wasps, odorant-binding proteins func- communication of food sources from foragers to tion as pheromone carriers, and their depletion the nest workers occurs by means of sounds prevents pheromone release (Pelosi et al. 2005). (Kerr and Esch 1965), an odorant substance Odorant-binding proteins were reported in the from the head salivary gland of M. quadrifas- mandibular glands of the honeybee A. mellifera ciata anthidioides may be used to mark the food 692 Table II. Proteins identified from the thorax salivary gland of M. quadrifasciata anthidioides forager workers.

Spota Protein (species)b Acession codeb Observed Calculated Observed End sequence (ion score)d MW/pIc mass mass peptided peptided

56T Proteasome subunit beta type E2BNX4_9HYME 32/7.1 1,790.96 1,790.96 DGVIIAADILGSYGSLAR (88) (Harpegnathos saltator) 18T Intraflagellar transport 88 homolog D4ACI9_RAT 101/8.2 852.4322 852.3956 RSGNYQK (79 protein total score) (Chlamydomonas sp.) 1,134.55 1,134.58 GSAFDPLGQSR 1,150.60 1,150.61 LDEALDSFLK 1,277.73 1,277.70 IMQNIGITFIK 1,289.70 1,289.74 NYSKAIKFYR 1,292.74 1,292.68 LKVNMGNIYLK .EisSno tal. et Elias-Santos D. 1,545.81 1,545.82 AALRGSAFDPLGQSR 154.5813 154.582 AALRGSAFDPLGQSR 1,604.70 1,604.73 SQAFQYYYESYR 1,621.85 1,621.77 FPENVECLRFLVR 1,635.78 1,635.80 EIGKCLLCGSTGPSDR 1,635.78 1,635.80 EIGKCLLCGSTGPSDR 1,810.93 1,810.94 LCTDIGLKEVQEYATK 2,233.15 2,233.09 LSAKLRALPGTDEPYESSGNK 2,327.07 2,327.15 EGSAGSDSGQNSSASSKSERLSAK 2,424.27 2,424.12 ASQYVELANDLEINKAITYLR 67T Arginine kinase (fragment) A1Y032_9HYME 48/7.5 2,368.13 2,368.15 DFGDVDTLGNLDPASEFIVSTR (121) (Axestotrigona ferruginea) 63T Heat shock protein beta-1 E2A8U5_9HYME 33/7.9 1,712.85 1,712.84 LGDFSVIDTEFSNIR (130) (Camponotus floridanus) 1,923.02 1,923.00 LRFDVSQYTPEEIVVK (79) 4T Glutathione S-transferase E2C1U9_9HYME 31/8.5 1,607.88 LTYFNITGLAEPIR (Harpegnathos saltator) 9 Actin-3 (Podocoryne carnea) ACT3_PODCA 51/4.0 1,515.75 1,515.76 IWHHTFYNELR (76) 1,790.89 1,790.91 SYELPDGQVITIGNER (95) 3,151.64 3,151.68 TTGIVLDSGDGVSHTVPIYEGYALPHAIIR (114) Table II. (continued)

Spota Protein (species)b Acession codeb Observed Calculated Observed End sequence (ion score)d MW/pIc mass mass peptided peptided

16 Tubulin alpha-1A chain (Homo sapiens) TBA1A_HUMAN 38/8.4 2,409.21 2,409.26 FDGALNVDLTEFQTNLVPYPR (99) 22 PREDICTED: similar to Cofilin/ gi|110751158 25/7.6 1,317.71 1,317.64 YVIFYIKDEK (68) actin-depolymerizing factor in glands salivary of Proteome homolog (protein D61, protein twinstar) 29 PREDICTED: similar to gi|110763826 53/5.6 2,042.00 2,042.00 TIVWNGPAGVFEFENFSK (153) phosphoglycerate kinaseisoform 1(Apis mellifera) 33 PREDICTED: similar to CG7217-PB, gi|110756016 20/6.7 2,571.34 2,571.34 MLADPAAQFTDALELSVDLPVLGGK (106) isoform B (Apis mellifera) 2,727.44 2,727.46 MLADPAAQFTDALELSVDLPVLGGKR (93) SOD1 (Apis mellifera ligustica) gi|38569378 2,309.14 2,309.11 HVGDLGNIEADASGVANVNITDK (110) 40 Glyceraldehyde-3-phosphate gi|307198667 30/4.5 2,667.38 2,667.39 VIHDNFEIVEGLMTTVHAITATQK (74) dehydrogenase 2 (Harpegnathos saltator) M . a Spot code corresponds to the position marked on the gel (Figure 1b) anthidioides quadrifasciata b Protein name and accession code were derived from the UniProt protein knowledge base c Observed MW/pI (molecular weight/point isoelectric) were derived from the spot position in the gel (measured in kilodalton) d Calculated mass peptide and observed mass peptide (in dalton) from the peptide sequences analyzed 693 694 D. Elias-Santos et al. source to aid the new forager bees in finding the phosphate to 3-phosphoglycerol phosphate; tri- correct food field. Thus, the odorant-binding osephosphate isomerase-like protein, which protein found (spot 14c) in the head salivary converts glyceraldehyde 3-phosphate to glycer- glands of M. quadrifasciata anthidioides may one phosphate; and arginine kinase, which has play a role as a carrier of chemical messages by an ATP-binding role (Boiteux and Hess 1974, controlling the sequestration or release of these 1981). The presence of proteins associated with compounds depending on the bee physiology energy metabolism in the head salivary gland of and behavior (Figure 3). M. quadrifasciata anthidioides corroborates The large number of proteins involved in previous findings where the secretory cells of energy metabolism suggests a high metabolic this gland in bees have the features of cells that rate in the head salivary gland in M. quad- play a role in lipid metabolism (Cruz-Landim rifasciata anthidioides. This is evidenced by the 2009) through the identification of proteins expression of fructose-bisphosphate aldolase, from lipid β-oxidation (Nelson and Cox 2008). which catalyzes fructose 1,6-bisphosphate Energy metabolism proteins such as cleavage into dihydroxyacetone phosphate and glyceraldehyde-3-phosphaste dehydrogenase 2, glyceraldehyde 3-phosphate during glycolysis phosphoglycerate kinase isoform 1 (Müller- (Midelfort et al. 1976). Furthermore, there is Dieckmann and Schulz 1995), and arginine also the presence of phosphoglycerate mutase, kinase identified in the thorax salivary gland which converts 3-phosphoglycerate to 2- of M. quadrifasciata anthidioides also suggest a phosphoglycerate in gluconeogenesis; α-N-ace- higher metabolism in this gland. The same may tylgalactosaminidase, which plays roles in car- be attributed to the homologous protein bohydrate breakdown; CG10120-PB, which has CG1707-PA of A. mellifera, which converts R- a similar role to the NADP-dependent malic S-lactoglutathione to glutathione and methyl- enzyme; glyceraldehyde-3-phosphate dehydro- glyoxal and supposedly interacts with CG9624, genase, which converts glyceraldehyde 3- which plays a similar role to pyruvate dehydro-

Figure 3. Schematic draw showing the possible action mechanism of odorant-binding protein in the head salivary gland of M. quadrifasciata anthidioides forager workers. Proteome of salivary glands in M. quadrifasciata anthidioides 695 genase in the glycolysis in cells of the head Thepresenceoftheβ-subunit protein of the salivary gland (Kerrien et al. 2012). proteasome, a cytoplasmic structure responsible for The presence of three heat shock proteins in the protein degradation (Alberts et al. 2008), may be head salivary gland of M. quadrifasciata anthi- due to the different developmental stages of the dioides—GG17772, GG19234 homologous to thorax salivary gland during the M. quadrifasciata proteins of Drosophila virilis (Diptera: Drosophi- anthidioides life span since secretory cells of this β lidae), and -1 heat shock protein homologous to gland have higher secretion activity in 15-day-old the ant Camponotus floridanus (Hymenoptera: workers, followed by a decrease in size and cell — Formicidae) suggests a high capacity for these degeneration (Silva de Morais 1978). Alternatively, cells to respond to environmental stimuli. Heat proteasome activity may suggest protein turnover shock proteins are chaperones that modulate the since thorax salivary glands have been suggested to structure and conformation of target proteins (Kim et al. 2007). In Drosophila hydea (Diptera: be multifunctional, releasing saliva for mouthpart Drosophilidae) larvae, these proteins are expressed lubrication and digestive enzymes (Costa and in response to heat and oxidative stresses (Ritossa Cruz-Landim 2001; Cruz-Landim 2009). 1962). Heat shock proteins have different functions Glutathione-S-transferases are the major detox- according to their family (Alberts et al. 2008), ification enzymes of insects, with an affinity for a acting as co-chaperones in specific processes of large range of endogenous and exogenous ligands interaction and function, regulating transcription (Sheehan et al. 2001). The presence of this protein factors, signaling components of phosphorylation in the thorax salivary gland of M. quadrifasciata routes, and protein kinase, and assembling com- anthidioides forager workers suggests a possible plex structures, such as centrosomes (Tower 2011). role for this gland in food detoxification as this In A. mellifera, heat shock protein cognate 3, 60- gland is directly involved in saliva formation (Cruz- kDa heat shock protein, heat shock protein cognate Landim 2009). Because glutathione-S-transferase 5, and heat shock protein 4 occur in the salivary is a hemolymph-circulating enzyme, one could glands (Fujita et al. 2010). The presence of heat suggest a contamination of the proteomic analyses shock proteins in both M. quadrifasciata anthi- from a hemolymph, but an overlap between plasma dioides and A. mellifera suggests the importance of and gland proteomes has been reported in insects these proteins in the salivary system of bees. (Paskewitz and Shi 2005; Celorio-Macera et al. Presumably, these heat shock proteins may be 2012). Analysis of the thorax salivary gland of A. involved in signaling arginine kinase protein, mellifera shows high concentrations of this protein which is also identified in the head salivary glands (Fujita et al. 2010). All together, these data suggest of M. quadrifasciata anthidioides. Thus, similar a similar role in both the species. Alternatively, the functions can be suggested for the thorax salivary occurrence of superoxide dismutase and gland of M. quadrifasciata anthidioides since it glutathione-S-transferase in the thorax salivary expresses isoform B of CG7217-PB and β-1 heat gland of M. quadrifasciata anthidioides might be shock protein. associated with the protection of the gland secretion The presence of superoxide dismutase 1 in the since these compounds protect venom toxins in thorax salivary gland of M. quadrifasciata anthi- bees (Peiren et al. 2008), wasps (Santos et al. 2010), and ants (Pinto et al. 2012). dioides suggests that the gland is involved in Annexin IX and windbeutel CG4225 are oxidative stress response since forager bees are expressed in the endoplasmic reticulum with frequently exposed to various environmental important roles in embryonic development during stresses (Nogueira-Neto 1997). The same may the dorsal–ventral axis determination of Drosoph- be said about the head salivary gland due to the ila melanogaster embryos (Konsolaki and Schup- presence of transcription co-repressor MIG3, a bach 1998;Bejaranoetal.2008). However, the DNA-binding transcriptional repressor involved presence of these proteins in the head salivary in response to toxic agents, such as ribonucleotide glands of M. quadrifasciata anthidioides adults reductase inhibitor (Dubacq et al. 2006). suggests participation in other metabolic func- 696 D. Elias-Santos et al. tions, which remain unknown. The occurrence of REFERENCES 14-3-3 zeta homologous protein and transcription co-repressor MIG3 suggests that the head salivary Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, gland cells of M. quadrifasciata anthidioides are K., Walter, P. (2008) Molecular biology of the cell. controlled by a significant transcriptional regula- Garland Science, New York tion because this protein is a well-characterized Arnold, G., Delage-Darchen, B. (1978) Nouvelles données sur l’équipement enzymatique des glandes signal translation regulator (Conklin et al. 1995; salivaires de l’ouvrière d’Apis mellifera (Hymenop- Dubacq et al. 2006). tera, ). Ann. Sci. Nat. Zool. 12, 401–422 Bejarano, F., Luque, C.M., Herranz, H., Sorrosal, G., 5. CONCLUSION Rafel, N., Pham, T.T., Milan, M. (2008) A gain-of- function suppressor screen for genes involved in dorsal–ventral boundary formation in the Drosoph- In conclusion, a 2DE map of the head and ila wing. Genetics 178, 307–323 thorax from M. quadrifasciata anthidioides Blum,M.,Crewe,R.M.,Kerr,W.E.,Keith,L.H., forager workers was defined, leading to the Garison, A.W., Walker, M.M. (1970) Citral in stingless bees, isolation and functions in trail- identification, for the first time, of more than 30 laying and robbing. J. Insect Physiol. 16, 1637–1648 distinct proteins. Considering the biological role Boiteux, A., Hess, B. (1974) Kinetics of glyceraldehyde- of the identified proteins in both the head and 3-phosphate dehydrogenase from yeast. Abstracts, thorax salivary glands, it can be suggested that 9th FEB S Meeting, Budapest, s2ai, 36. these glands have high activity, respond to Boiteux, A., Hess, B. (1981) Design of glycolysis. environmental stimuli, and have important roles Philos. Trans. R. Soc. B. 293,5–22 in physiological detoxification and food source Celorio-Macera, M.P., Sundmaln, S.M., Vogel, H., Rutishuer, D., Ytterberg, A.J., Zubarev, R.A., marking. These results contribute to our under- Niklas, J. (2012) Chemosensory proteins, major standing of their possible physiological and salivary factors in caterpillar mandibular glands. behavioral functions in bees. Nevertheless, Insect Biochem. Mol. Biol. 42, 796–805 future work for a deeper proteome analysis of Conklin, D.S., Galaktionov, K., Beach, D. (1995) 14-3- the head and thorax salivary glands is required. 3-Proteins associate with Cdc25-phosphatases. Proc. Natl. Acad. Sci. U.S.A. 92, 7892–7896 Costa, R.A.C., Cruz-Landim, C. (2001) Enzymes present ACKNOWLEDGMENTS in the toracic gland extracts from workers and males of Apis mellifera (Hymenoptera, Apidae). Sociobi- – We thank the Nucleus of Biomolecules of Universi- ology 37, 563 569 dade Federal de Viçosa, Brazil, and Mass Spectrometry Cruz-Landim, C. (1967) Estudo comparativo de algumas Center of Aveiro University, Portugal, for technical glândulas de abelhas (Hymenoptera: Apoidea) e respectivas implicações evolutivas. Arq. Zool São assistance. This research was supported by Brazilian Paulo 15, 177–290 research agencies CNPq, CAPES, and FAPEMIG. Cruz-Landim, C. (2009) Abelhas: Morfologia e função de sistemas. Editora UNESP, São Paulo Conflict of interest The authors declare any actual or potential competing interest. Cruz-Landim, C., Abdalla, F.C. (2002) Glândulas Exó- crinas das Abelhas. FUNPEC, Ribeirão Preto Delage-Darchen, B., Darchen, R. (1982) Les enzymes Protéome des glandes salivaires céphaliques et thoraciques digestives des glandes salivaires et de l’intestin ’ Melipona quadrifasciata de l abeille sans aiguillon moyen d’une abeille sociale du Mexique, Melipona anthidiodes beecheii (B). Ann. Sci. Nat. Zool. 4,91–96 Delage-Darchen, B., Talec, S.S., Darchen, R. (1979) Comportement / protéine de transport des odorants / Secrétion enzymatique des glandes salivaires et de protéine de choc thermique / Meliponini / Hymenoptera / l’intestin moyen d’une abeille sans dard Apotrigona stress / ouvrière nebulata (Sm.), (Hyménoptère, Apidés). Ann. Sci. Nat. Zool. 13, 261–267 Das Proteom der Kopf- und Thoraxspeicheldrüsen bei der Dubacq, C., Chevalier, A., Courbeyrette, R., Petat, C., Melipona quadrifasciata anthidioides stachellosen Biene Gidrol, X., Mann, C. (2006) Role of the iron mobilization and oxidative stress regulons in the Verhalten / Duftstoff bindendes Protein / genomic response of yeast to hydroxyurea. Mol. Hitzeschockprotein / Meliponini / Stress Genet. Genomics 275,114–124 Proteome of salivary glands in M. quadrifasciata anthidioides 697

Eltz, T., Zimmermann, Y., Haftmann, J., Twele, R., Kim, H.J., Hwang, N.R., Lee, K.J. (2007) Heat shock Francke, W., Quezada-Euan, J.J.G., Lunau, K. responses for understanding diseases of protein (2007) Enfleurage, lipid recycling and the origin of denaturation. Mol. Cells 23, 123–131 perfume collection in orchid bees. Proc. R. Soc. B – Konsolaki, M., Schupbach, T. (1998) Windbeutel, a gene Biol. Sci. 274, 2843 2848 required for dorsoventral patterning in Drosophila, Free, J.B. (1987) Pheromones of social bees. Chapman- encodes a protein that has homologies to vertebrate Hall, London proteins of the endoplasmic reticulum. Gene Dev. – Fujita, T., Kozuka-Hata, H., Ao-Kondo, H., Kunieda, T., 12, 120 131 Oyama, M., Kubo, T. (2012) Proteomic analysis of Kubo, R., Ono, M. (2010) Comparative analysis of volatile the royal jelly and characterization of the functions components from labial glands of male Japanese of its derivation glands in the honeybee. J. Proteome bumblebees (Bombus spp.). Entomol. Sci. 13,167–173 – Res 12, 404 411 Michener, C.D. (1974) The social behavior of bees: a Fujita, T., Kozuka-Hata, H., Uno, Y., Nishikori, K., comparative study. Havard University Press, Cambridge Morioka, M., Oyama, M., Kubo, T. (2010) Func- Midelfort, C.F., Gupta, R.K., Rose, I.A. (1976) Fructose- tional analysis of the honeybee (Apis mellifera L.) salivary system using proteomics. Biochem. Bioph 1,6-bisphosphate:isomeric composition, kinetics, Res. Commun. 397, 740–744 and substrate specificity for the aldolases. Biochem- istry 15, 2178–2185 Furusawa, T., Rakwal, R., Nam, H.W., Shibato, J., Agrawal, G.K., Kim, Y.S., Ogawa, Y., Yoshida, Y., Müller-Dieckmann, H.J., Schulz, G.E. (1995) Substrate Kouzuma, Y., Masuo, Y., Yonekura, M. (2008) specificity and assembly of the catalytic center Comprehensive royal jelly (RJ) proteomics using derived from two structures of ligated uridylate – one- and two-dimensional proteomics platforms kinase. J. Mol. Biol. 46, 522 530 reveals novel RJ proteins and potential phospho/ Nelson, D.E., Cox, M.M. (2008) Lehninger’s principles glycoproteins. J. Proteome Res. 7, 3194–3229 of biochemistry. W.H. Freeman, New York Heselhaus, F. (1922) Verwandter Formen. Zool Jahr Abt Nogueira-Neto, P. (1997) Vida e criação de abelhas Anat Ontog Thiere 43, 363–464 indígenas sem ferrão. Editora Nogueirapis, São Paulo Horst, R., Damberger, F., Luginbuhl, P., Guntert, P., Paskewitz, S.M., Shi, L. (2005) The hemolymph Peng, G., Nikonova, L., Leal, W.S., Wuthrich, K. proteome of Anopheles gambiae. Insect Biochem. (2001) NMR structure reveals intramolecular regula- Mol. Biol. 35, 815–824 tion mechanism for pheromone binding and release. Proc. Natl. Acad. Sci. U.S.A. 98, 14374–14379 Peiren, N., de Graaf, D.C., Vanrobaeys, F., Danneels, E.L., Devreese, B., Van Beeurnen, J., Jacobs, F.J. Iovinella, I., Dani, F.R., Niccolini, A., Sagona, S., (2008) Proteomic analysis of the worker Michelucci, E., Gazzano, A., Turillazzi, S., Felicioi, venom gland focusing on the mechanisms of A., Pelosi, P. (2011) Differential expression of protection against tissue damage. Toxicon 52,72–83 odorant-binding proteins in the mandibular glands of the honey bee according to caste and age. J. Pinto, J.R.A.D., Fox, E.G.P., Saidemberg, D.M., Santos, Proteome Res. 10, 3439–3449 L.D., Menegasso, A.R.S., Costa-Manso, E., Bueno, O.C., Pamla, M.S. (2012) Proteomic view of the Jarau, S., Hrncir, M., Ayasse, M., Schulz, C., Francke, venom from the fire ant Solenopsis invicta Buren. J. W., Zucchi, R., Barth, F.G. (2004a) A stingless bee Proteome Res. 11, 4643–4653 (Melipona seminigra) marks food sources with a pheromone from its claw retractor tendons. J. Chem. Pelosi, P., Calvello, M., Ban, L.P. (2005) Diversity of Ecol. 30, 793–804 odorant-binding proteins and chemosensory proteins in insects. Chem. Senses 30, 291–292 Jarau, S., Hrncir, M., Zucchi, R., Barth, F.G. (2004b) A stingless bee uses labial gland secretions for scent Poiani, S.B., Cruz-Landim, C. (2009) Cephalic salivary gland ultrastructure of worker and queen eusocial bees trail communication (Trigona recursa Smith 1863). – J. Comp. Physiol. A 190, 233–239 (Hymenoptera, Apidae). Anim. Biol. 59,299 311 Jarau, S., van Veen, J.W., Twele, R., Reichle, C., Poiani, S.B., Cruz-Landim, C. (2010) Morphological Gonzales, E.H., Aguilar, I., Francke, W., Ayasse, changes in the cephalic salivary glands of females and males of Apis mellifera and Scaptotrigona postica M. (2010) Workers make the queens in Melipona – bees: identification of geraniol as a caste determin- (Hymenoptera, Apidae). J. Biosci. 35,249 255 ing compound from labial glands of nurse bees. J. Ritossa, F. (1962) A new puffing pattern induced by Chem. Ecol. 36, 565–569 temperature shock and DNP in Drosophila. Experi- – Kerr, W.E., Esch, H. (1965) Comunicação entre abelhas entia 18, 571 573 sociais bresileiras e sua contribuição para o entendi- Roubik, D.W. (1989) Ecology and natural history of mento de sua evolução. Ciênc Cult 17, 529–537 tropical bees. Cambridge University Press, Cambridge Kerrien,S.,Aranda,B.,Breuza,L.,Bridge,A.,Broackes- Santos, C.G., Megiolaro, F.L., Serrão, J.E., Blochtein, B. Carter, F., et al. (2012) The IntAct molecular interaction (2009) Morphology of the head salivary and intra- database in 2012. Nucleic Acids Res. 40, 841–846 mandibular glands of the stingless bee Plebeia emerina 698 D. Elias-Santos et al.

(Hymenoptera: Meliponini) workers associated with Lep. durante a diferenciação pós-embrionária e ciclo propolis. Ann. Ent. Soc. Am. 102,137–143 secretor. Pap Avul Zool 31,251–281 Santos, L.D., Santos, K.S., Pinto, J.R.A., Dias, N.B., Simpson, J. (1960) The function of the salivary glands in Souza, B.M., Perales, J., Dumont, G.B., Castro, Apis mellifera. J. Insect Physiol. 4, 107–121 F.M., Kalil, J.E., Palma, M.S. (2010) Profiling the Stangler, E.S., Jarau, S., Hrncir, M., Zucchi, R., proteome of the venom from the social wasp Ayasse, M. (2009) Identification of trail phero- Polybia paulista: a cue to understand the envenom- – mone compounds from the labial glands of the ing mechanism. J. Proteome Res. 9, 3867 3877 stingless bee Geotrigona mombuca. Chemoecology Sheehan, D., Meade, G., Foley, V.M., Dowd, C.A. 19,13–19 (2001) Structure, function and evolution of glutathi- Tower, J. (2011) Heat shock proteins and Drosophila one transferases: implications for classification of aging. Exp. Gerontol. 46, 355–362 non-mammalian members of an ancient enzyme superfamily. Biochem. J. 360,1–16 Woodard, S.H., Fischman, B.J., Venkat, A., Hudson, M.E.,Varala,K.,Cameron,S.A.,Clark,A.G., Silva de Morais, R.L.M. (1978) Variações do conteúdo de Robinson, G.E. (2011) Genes involved in conver- DNA e volume nucleares nas glândulas salivares de gent evolution of eusociality in bees. Proc. Natl. operárias de Melipona quadrifasciata anthidioides Acad. Sci. U.S.A. 108, 7472–7477