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Zanuncio, José Serrão
To cite this version:
Proteome of the head and thorax salivary glands
in the stingless bee
Melipona quadrifasciata anthidioides
Douglas ELIAS-SANTOS1,Maria do Carmo Q. FIALHO1,Rui VITORINO2,
Leandro L. OLIVEIRA1,José C. ZANUNCIO3,José Eduardo SERRÃO1
1Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-000 Viçosa, Minas Gerais, Brazil 2
Department of Chemistry, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal 3
Departamento de Biologia Animal, 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 insects 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 / Hymenoptera / stress
1. INTRODUCTION
Social Hymenoptera are intriguing insects because of the complexity and organization of their social lives. In bees, queens and workers form distinct castes, each playing important roles within the colony (Michener 1974). More-over, in highly eusocial bees, there is a division of labor between workers, which favors protection against natural enemies, increases resistance to environmental stress, and in resource storage (Roubik1989).
Almost all aspects of the social lives of insects are linked to chemical signals produced by exocrine glands that occur in high numbers
and diversity in the insect body. The best-known functions of the exocrine glands are related to the communication, reproduction, and development of individuals (Cruz-Landim and Abdalla 2002). For communication, exocrine glands release chemicals termed pheromones, which are a mix of compounds causing physi-ological and/or behavioral responses in other individuals of the same species (Free1987).
The salivary glands of eusocial bees are classified into two types: head salivary glands (labial glands) and thorax salivary glands. Thorax salivary glands have secretory units that open into an excretory duct toward the head and fuses with the ducts of the head salivary glands (Cruz-Landim1967). The secretory cells of the thorax salivary glands of Scapitotrigona postica (Meliponini), Xylocopa suspecta (Xylocopini),
Corresponding author: J.E. Serrão, jeserrao@ufv.br Manuscript editor: James Nieh
of enzymes, such as proteases, lipases, and lactases, as reported for Apotrigona nebulata, Melipona becheeii, and S. postica (Arnold and Delage-Darchen 1978; Delage-Darchen et al.
1979; Delage-Darchen and Darchen 1982; Costa and Cruz-Landim 2001). Nine proteins occur in both thorax salivary gland of Apis melifera workers and in stored royal jelly (Fujita et al.2012).
The head salivary glands occur only in the stingless bees Euglossini, Bombini, Meliponini, and Apini (Cruz-Landim 2009). These glands are derived from a secondary growth of the thorax salivary glands and are formed by multicellular alveoli with a thin cuticle lining the gland lumen (Cruz-Landim1967). Secretory cells of the head salivary gland have features of cells that metabolize lipids in old bees, but in young workers, the secretory cells have well-developed rough endoplasmic reticulum and high concentrations of free ribosomes (Cruz-Landim 2009). A possible function of the head salivary glands is to contribute to the worker’s recruitment for foraging and trail marking (Jarau et al. 2004a). In the stingless bees Geotrigona subterrania and Geotrigona mom-buca, head salivary glands and mandibular glands together play a role in marking food sources and worker recruitment (Blum et al.
1970; Stangler et al. 2009). Moreover, in the orchid bee Euglossa viridissima, the head salivary glands play a role in the mechanism of fragrance collection in plants (Eltz et al.
2007). The secretion of the head salivary glands is also related to wax manipulation for nest building (Heselhaus1922), mandible lubrication (Simpson1960), and resin manipulation (Santos et al. 2009). In A. mellifera, head salivary glands produce some proteins found in royal jelly (Fujita et al.2012).
Considering that the head and thorax salivary glands of bees produce different substances that have different functions as well as the ecolog-ical and economic importance and the risk of extinction of stingless bees, this study aimed to
dioides workers to enhance the understanding of the functions of these glands in social bees.
2. MATERIAL AND METHODS 2.1. Bees and salivary glands
Forager workers of M. quadrifasciata anthidioides were obtained from three colonies kept in the apiary of the Federal University of Viçosa (20°45′ N, 42°52′ W), Viçosa state of Minas Gerais, Brazil.
For head and thorax salivary gland extraction, 51 forager workers were collected from each colony, totaling 153 bees. The bees were collected from close to the colony entrance when returning with corbicu-lae loaded with pollen grains. Bees were cryoanes-thetized, dissected in phosphate-buffered saline, and the head and thorax salivary glands were transferred to 1 % protease inhibitor cocktail (Sigma, P27147), homogenized in buffer [(7 M urea, 2 M thiourea, 4 % CHAPS, 2 % immobilized pH gradient (IPG) buffer, 40 mM dithiothreitol (DTT)], and centrifuged at 14,000×g for 30 min at 4 °C. To obtain soluble proteins, only the supernatant was collected and stored at−80 °C until use. The amount of protein in the samples was determined according to the Brad-ford method using bovine serum albumin as a standard.
2.2. Two-dimensional gel electrophoresis
gel. The focused strips were equilibrated in buffer (6 M urea, 75 mM Tris–HCl, 29.3 % glycerol, 2 % SDS, 0.002 % Bromophenol blue) containing 1 % DTT for 20 min under stirring. Following this, the buffer was replaced by the same buffer containing 2.5 % iodoacetamide for 20 min in the dark. The strips were then washed for 5 s in running buffer (3 % Tris–HCl, 14.4 % glycine, 1 % SDS, pH 8.8) and run on 12 % SDS-PAGE gels at 200 V for 50 min. The gel was stained for 16 h in Coomassie G-250 solution (0.002 % Coomassie blue G-250, 10 % acetic acid, 50 % ethanol). To validate the data, three gels were made for each gland. Then, the gels were digitalized and analyzed using Image Master 2D Platinum 7.0 for spot detection and analysis. Each spot was assigned a different symbol for identification and for further image analysis.
2.3. Sample digestion
The two-dimensional gel electrophoresis (2DE) spots that were expressed more were manually excised and the gel pieces were washed three times with 25 mM ammonium bicarbonate/50 % acetoni-trile (ACN), one time with 100 % (ACN), and dried in a SpeedVac (Thermo Savant). Then, 25 μL of 10 μg/mL sequence-grade modified porcine trypsin (Promega) in 25 mM ammonium bicarbonate was added to the dried gel pieces and the samples were incubated overnight at 37 °C. Extraction of tryptic peptides was performed by the addition of 10 % formic acid (FA)/50 % ACN three times and the peptides lyophilized in a SpeedVac (Thermo Savant).
2.4. Mass spectrometry analysis
Tryptic peptides were resuspended in 10 μL of a 50 % ACN/0.1 % FA solution. The samples were mixed (1:1) with a matrix consisting of a saturated solution of α-cyano-4-hydroxycinnamic acid pre-pared in 50 % ACN/0.1 % FA. Aliquots of the samples (0.5 μL) were spotted onto the matrix-assisted laser desorption/ionization (MALDI) sample target plate.
Peptide mass spectra were obtained on a MALDI time-of-flight (MALDI-TOF/TOF) mass spectrometer (4800 Proteomics Analyzer, ABSciex, Europe) in the positive ion reflector mode. Spectra were obtained in
the mass range of 800–4,500 Da with about 1,500 laser shots. For each sample spot, a data-dependent acquisition method was created to select the four most intense peaks, excluding those from the matrix, trypsin autolysis, or acrylamide, for subsequent MS/MS data acquisition. Mass spectra were internally calibrated with autodigested peaks of trypsin (MH+= 842.5, 2,211.42 Da) allowing a mass accuracy >25 ppm.
2.5. Protein identification
The spectra were processed and analyzed using the Global Protein Server Workstation (Applied Biosystems), which uses internal MASCOT software (v.2.1.0, Matrix Science, London, UK) to search the peptide mass fingerprints and MS/MS data. The Trembl non-redundant protein sequence database (January 18, 2011) was used for all searches under All entries and the allowance for up to two missed tryptic cleavages. The peptide mass tolerance was 25 ppm and fragment ion mass tolerance was 0.3 Da. Positive identifications were accepted up to 95 % of the confidence level.
The identified proteins were analyzed and associ-ated with their molecular functions, biological pro-cesses, and cellular components using the online tool
http://web.expasy.org/blast/ and the database http://
www.uniprot.org/. The results were related to the
biological roles of both the head and thorax salivary glands and to the different functions according to the biological processes contained in the database.
3. RESULTS
The concentrations of soluble proteins from each gland were 1.90±0.05 and 7.77±0.05 μg per bee (N=153) in the head and thorax salivary glands, respectively, of M. quadrifasciata anthi-dioides forager workers. In the 2DE maps, the spot distribution of the head and thorax salivary glands showed different protein expression patterns, evidencing a lower number of soluble proteins in the head salivary gland (198 spots) when compared with the thorax salivary gland (225 spots) of M. quadrifasciata anthidioides forager workers (Figure1a, b).
anthidioides showed different isoelectric points and molecular weights (Electronic supplemen-tary material Table S1). Furthermore, there is an enrichment of proteins with acidic pH in the
head salivary gland maps. The analysis of three maps for the head salivary gland showed 60 spot matches, while the thorax salivary gland showed 112 spot matches. The match between
the head and the thorax salivary gland maps shows that 28 spots equate. Furthermore, there is an enrichment of proteins with basic pH in the gel map from the thorax salivary gland, and most of the spots has an estimated pI ranging from 7.1 to 8.4 (Figure1b).
Although only a small part of the tran-scriptome of the brain and abdomen has been described in M. quadrifasciata in previous studies, the genome is not completely evident for this species and fault information (Woodard et al.2011). Thus, the identifications were based on homology to other species.
From 42 excised spots, 26 spots were positively identified in the head salivary gland of M. quadrifasciata anthidioides forager work-ers as belonging to several protein classes: energy metabolic proteins (two α-N-acetylga-lactosaminidase, fructose bisphosphate aldolase, protein similar to malic enzyme CG10120-PB, arginine kinase, triosephosphate isomerase-like, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase, CG1707-PA); pro-teins of unknown function (AGAP001151-PA, GM12998, similar to CG4300-PA, UPF0753 protein sce4789, α-S1-casein, putative unchar-acterized protein of Anopheles gambiae); con-stitutive proteins (annexin IX CG5730-PC, 30S ribosomal protein S18, beta subunit of the E1 component from pyruvate dehydrogenase, ubiq-uitin); environmental response proteins (heat shock protein beta-1-like isoform 1, GG19234, GG17772); genetic modulators (14-3-3 protein zeta, windbeutel CG7225-PA, transcription co-repressor MIG3); and those with specific func-tions (odorant-binding protein). In some spots, more than one protein was identified, e.g., spot 57—the 30S ribosomal protein S18 and UPF0753 protein sce4789 (Figure 1a); spot 47—protein transcription co-repressor MIG3 and the CG4300-PA-like protein (Table I and Figures1aand2a).
In the thorax salivary glands of M. quad-rifasciata anthidioides forager workers, from 52 excised spots, 12 distinct proteins were identi-fied and annotated to energy metabolic proteins (glyceraldehyde-3-phosphate dehydrogenase 2, phosphoglycerate kinase, arginine kinase);
con-stitutive proteins (proteasome subunit, actin 3, alpha-tubulin, actin/cofilin depolymerizing fac-tor homolog); environmental response proteins (beta-1 subunit of heat shock protein, CG7217-PB); cellular detoxification proteins (glutathi-one-S-transferase, superoxide dismutase 1); and proteins of unknown function (protein homolo-gous to intra-flagellar transport 88). The pro-teins CG7217-PB and superoxide dismutase 1 correspond to spot 33. As observed, the 12 spots with pI ranging from 4.5 to 8.5 were identified (Table IIand Figures1band2b).
4. DISCUSSION
proteina-T able I. Proteins identified from the head salivary glands of M. qua drifasciata anthidioides forager workers. Spot a Protein (species) b Acession code b Observed MW/p I c
Calculated mass peptide
d
Observed mass peptide
T able I. (continued) Spot a Protein (species) b Acession code b Observed MW/p I c
Calculated mass peptide
d
Observed mass peptide
ceous nature of the gland secretion (Cruz-Landim 2009). However, the morphological and functional differences between the head and thorax salivary glands are not found in all bee species because head salivary glands are present only in bees (Cruz-Landim1967,2009). A significant part of the identified proteins was classified in ordorant-binding protein. In fact, the expression of an odorant-binding protein proved an important role of the head salivary gland in chemical communication among bees. Odorant molecules, such as pher-omones, are hydrophobic and have low solubil-ity in the cytoplasmic cell environment, which prevents intracellular transport. Thus, the trans-port of these molecules occurs when they bind with a soluble protein, such as specific odorant-binding proteins, which have high solubility and constant reversible capacity for the binding and dissociation of small molecules (Horst et al.
2001). In wasps, odorant-binding proteins func-tion as pheromone carriers, and their deplefunc-tion prevents pheromone release (Pelosi et al.2005). Odorant-binding proteins were reported in the mandibular glands of the honeybee A. mellifera
(Iovinella et al. 2011), a well-established pheromone-producing gland (Michener 1974). Our results corroborate the proposed function of the head salivary gland in producing phero-mones in the stingless bees Trigona recursa, A. mellifera, and S. postica (Jarau et al. 2004b; Poiani and da Cruz-Landim2009). Furthermore, the head salivary glands of six species of Bombus (Bombini) showed the presence of volatile compounds involved in communication between nest mates (Kubo and Ono 2010). Head salivary gland extracts of the stingless bees T. recursa and Trigona corvina indicate a high probability of the compounds of this gland being involved in marking food sources (Jarau et al. 2004b, 2010; Stangler et al. 2009). Odorant-binding protein 14 occurs in the head salivary glands of nurse and forager workers of A. mellifera (Furusawa et al.2008; Fujita et al.
2012). Although, in the genus Melipona, the communication of food sources from foragers to the nest workers occurs by means of sounds (Kerr and Esch 1965), an odorant substance from the head salivary gland of M. quadrifas-ciata anthidioides may be used to mark the food
T able II. Proteins identified from the thorax salivary gland of M. quadrifasciata anthidioides forager workers. Spot a Protein (species) b Acession code b Observed MW/p I c
Calculated mass peptide
d
Observed mass pept
T able II. (continued) Spot a Protein (species) b Acession code b Observed MW/p I c
Calculated mass peptide
d
Observed mass pept
source to aid the new forager bees in finding the correct food field. Thus, the odorant-binding protein found (spot 14c) in the head salivary glands of M. quadrifasciata anthidioides may play a role as a carrier of chemical messages by controlling the sequestration or release of these compounds depending on the bee physiology and behavior (Figure3).
The large number of proteins involved in energy metabolism suggests a high metabolic rate in the head salivary gland in M. quad-rifasciata anthidioides. This is evidenced by the expression of fructose-bisphosphate aldolase, which catalyzes fructose 1,6-bisphosphate cleavage into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate during glycolysis (Midelfort et al. 1976). Furthermore, there is also the presence of phosphoglycerate mutase, which converts 3-phosphoglycerate to 2-phosphoglycerate in gluconeogenesis; α-N-ace-tylgalactosaminidase, which plays roles in car-bohydrate breakdown; CG10120-PB, which has a similar role to the NADP-dependent malic enzyme; glyceraldehyde-3-phosphate dehydro-genase, which converts glyceraldehyde
3-phosphate to 3-phosphoglycerol 3-phosphate; tri-osephosphate isomerase-like protein, which converts glyceraldehyde 3-phosphate to glycer-one phosphate; and arginine kinase, which has an ATP-binding role (Boiteux and Hess 1974,
1981). The presence of proteins associated with energy metabolism in the head salivary gland of M. quadrifasciata anthidioides corroborates previous findings where the secretory cells of this gland in bees have the features of cells that play a role in lipid metabolism (Cruz-Landim
2009) through the identification of proteins from lipidβ-oxidation (Nelson and Cox2008). Energy metabolism proteins such as glyceraldehyde-3-phosphaste dehydrogenase 2, phosphoglycerate kinase isoform 1 (Müller-Dieckmann and Schulz 1995), and arginine kinase identified in the thorax salivary gland of M. quadrifasciata anthidioides also suggest a higher metabolism in this gland. The same may be attributed to the homologous protein CG1707-PA of A. mellifera, which converts R-S-lactoglutathione to glutathione and methyl-glyoxal and supposedly interacts with CG9624, which plays a similar role to pyruvate
head salivary gland of M. quadrifasciata anthi-dioides—GG17772, GG19234 homologous to proteins of Drosophila virilis (Diptera: Drosophi-lidae), andβ-1 heat shock protein homologous to the ant Camponotus floridanus (Hymenoptera: Formicidae)—suggests a high capacity for these cells to respond to environmental stimuli. Heat shock proteins are chaperones that modulate the structure and conformation of target proteins (Kim et al. 2007). In Drosophila hydea (Diptera: Drosophilidae) larvae, these proteins are expressed in response to heat and oxidative stresses (Ritossa
1962). Heat shock proteins have different functions according to their family (Alberts et al. 2008), acting as co-chaperones in specific processes of interaction and function, regulating transcription factors, signaling components of phosphorylation routes, and protein kinase, and assembling com-plex structures, such as centrosomes (Tower2011). In A. mellifera, heat shock protein cognate 3, 60-kDa heat shock protein, heat shock protein cognate 5, and heat shock protein 4 occur in the salivary glands (Fujita et al.2010). The presence of heat shock proteins in both M. quadrifasciata anthi-dioides and A. mellifera suggests the importance of these proteins in the salivary system of bees. Presumably, these heat shock proteins may be involved in signaling arginine kinase protein, which is also identified in the head salivary glands of M. quadrifasciata anthidioides. Thus, similar functions can be suggested for the thorax salivary gland of M. quadrifasciata anthidioides since it expresses isoform B of CG7217-PB andβ-1 heat shock protein.
The presence of superoxide dismutase 1 in the thorax salivary gland of M. quadrifasciata anthi-dioides suggests that the gland is involved in oxidative stress response since forager bees are frequently exposed to various environmental stresses (Nogueira-Neto 1997). The same may be said about the head salivary gland due to the presence of transcription co-repressor MIG3, a DNA-binding transcriptional repressor involved in response to toxic agents, such as ribonucleotide reductase inhibitor (Dubacq et al.2006).
due to the different developmental stages of the thorax salivary gland during the M. quadrifasciata anthidioides life span since secretory cells of this gland have higher secretion activity in 15-day-old workers, followed by a decrease in size and cell degeneration (Silva de Morais1978). Alternatively, proteasome activity may suggest protein turnover since thorax salivary glands have been suggested to be multifunctional, releasing saliva for mouthpart lubrication and digestive enzymes (Costa and Cruz-Landim2001; Cruz-Landim2009).
Glutathione-S-transferases are the major detox-ification enzymes of insects, with an affinity for a large range of endogenous and exogenous ligands (Sheehan et al.2001). The presence of this protein in the thorax salivary gland of M. quadrifasciata anthidioides forager workers suggests a possible role for this gland in food detoxification as this gland is directly involved in saliva formation (Cruz-Landim 2009). Because glutathione-S-transferase is a hemolymph-circulating enzyme, one could suggest a contamination of the proteomic analyses from a hemolymph, but an overlap between plasma and gland proteomes has been reported in insects (Paskewitz and Shi 2005; Celorio-Macera et al.
2012). Analysis of the thorax salivary gland of A. mellifera shows high concentrations of this protein (Fujita et al.2010). All together, these data suggest a similar role in both the species. Alternatively, the occurrence of superoxide dismutase and glutathione-S-transferase in the thorax salivary gland of M. quadrifasciata anthidioides might be associated with the protection of the gland secretion since these compounds protect venom toxins in bees (Peiren et al.2008), wasps (Santos et al.2010), and ants (Pinto et al.2012).
func-tions, which remain unknown. The occurrence of 14-3-3 zeta homologous protein and transcription co-repressor MIG3 suggests that the head salivary gland cells of M. quadrifasciata anthidioides are controlled by a significant transcriptional regula-tion because this protein is a well-characterized signal translation regulator (Conklin et al.1995; Dubacq et al.2006).
5. CONCLUSION
In conclusion, a 2DE map of the head and thorax from M. quadrifasciata anthidioides forager workers was defined, leading to the identification, for the first time, of more than 30 distinct proteins. Considering the biological role of the identified proteins in both the head and thorax salivary glands, it can be suggested that these glands have high activity, respond to environmental stimuli, and have important roles in physiological detoxification and food source marking. These results contribute to our under-standing of their possible physiological and behavioral functions in bees. Nevertheless, future work for a deeper proteome analysis of the head and thorax salivary glands is required.
ACKNOWLEDGMENTS
We thank the Nucleus of Biomolecules of Universi-dade Federal de Viçosa, Brazil, and Mass Spectrometry Center of Aveiro University, Portugal, for technical assistance. This research was supported by Brazilian research agencies CNPq, CAPES, and FAPEMIG.
Conflict of interest The authors declare any actual or potential competing interest.
Protéome des glandes salivaires céphaliques et thoraciques de l’abeille sans aiguillon Melipona quadrifasciata anthidiodes
Comportement / protéine de transport des odorants / protéine de choc thermique / Meliponini / Hymenoptera / stress / ouvrière
Das Proteom der Kopf- und Thoraxspeicheldrüsen bei der stachellosen BieneMelipona quadrifasciata anthidioides Ve rhalten / Duftstoff bindende s Prot ein / Hitzeschockprotein / Meliponini / Stress
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