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Ultrastructural detection of lipids in the cephalic salivary glands of Apis mellifera and Scaptotrigona postica (Hymenoptera: Apidae) workers

ABSTRACT

Secretory cells of the cephalic salivary glands (CSGs) of eusocial bees produce and accumulate lipid-like secretion in the lumens of their alveoli. Correspondingly, secretory cells present typical ultrastructural features of lipid-compound producers. Previous work on bees has revealed inter-specific differences in the chemical composition of secretion, and the production mechanisms and secretory cycle of secretory cells. In this work a comparative analysis of the mechanisms of lipid storage in the CSGs of Apis mellifera (Linnaeus, 1758) and Scaptotrigona postica (Latreille, 1807) workers was carried out. The ultrastructural location of lipids was ascertained using imidazole-osmium (IO), using individuals in different stages of their life cycles. Lipid deposits were identified inside glandular cells and in the alveolar lumens in all individuals, but differences were observed between the species. The glandular cells of A. mellifera workers presented positive reactions to IO as droplets dispersed in the cytoplasm, as vesicles and in the channels formed by apical plasma membrane infolds. In S. postica , lipid compounds were detected inside the mitochondrial matrix and in smooth endoplasmic reticulum cisterns. In both species, forager workers exhibited the largest amounts of lipids stored in the alveolar lumen. The differences between the species are discussed, taking into account specific behavioral differences.

KEY WORDS:
Cytochemistry; excretory; imidazole-osmium; insect; salivary system

All insects possess exocrine glands of epidermal origin that are attached to their mouthparts. These glands are named according to the appendix to which they are connected. For instance, mandibular glands are connected to the mandible, and labial or salivary glands are connected to the labium. The salivary glands of insects generally have their secretory portion located in the thorax. In some species of bees, however, branches of the salivary glands can be found in the head. These glands, known as cephalic salivary (or labial) glands (CSGs), are fully developed and functional only in Apinae species (Cruz-Landim 1967Cruz-Landim C (1967) Estudos comparativos de algumas glândulas das abelhas (Hymenoptera, Apoidea) e respectivas implicações evolutivas. Arquivos de Zoologia 15(3): 177-290. doi: 10.11606/issn.2176-7793.v15i3p177-290
https://doi.org/10.11606/issn.2176-7793....
).

In eusocial bees, the products of the exocrine glands and their functions vary not only among the classes of colonial individuals, but also among their functional life phases, showing interesting adaptations within species, and great plasticity (Katzav-Gozansky et al. 1997Katzav-Gozansky T, Soroker V, Hefetz A (1997) Plasticity of caste-specific Dufour's gland secretion in the honey bee (Apis mellifera L.). Naturwissenschaften 84(6): 238-241. doi: 10.1007/s001140050386
https://doi.org/10.1007/s001140050386...
, Jarau et al. 2004Jarau S, Hrncir M, Zucchi R, Barth FG (2004) A Stingless Bee Uses Labial Gland Secretions for Scent Trail Communication (Trigona recursa Smith, 1863). Journal of Comparative Physiology A Neuroethology, Sensory, Neural, and Behavioral Physiology 190(3): 233-239. doi: 10.1007/s00359-003-0489-9
https://doi.org/10.1007/s00359-003-0489-...
, Santos et al. 2009Santos CG, Megiolaro F, Serrão JE, Blochtein B (2009) Morphology of the head salivary and intramandibular glands of the stingless bee Plebeia emerina (Friese) (Hymenoptera, Meliponini) workers associated with propolis. Annals of the Entomological Society of America 102(1): 137-143. doi: 10.1603/008.102.0115
https://doi.org/10.1603/008.102.0115...
). This plasticity is observed not only among different species, but also between the sexes and castes of the same species. For example, males of Bombus pratorum (Linnaeus, 1761) and Bombus lapidarius (Linnaeus, 1758) use the CSG secretion to mark flight routes and attract virgin queens (Bergman & Bergström 1997Bergman P, Bergström G (1997) Scent marking, scent origin, and species specificity in male premating behavior of two scandinavian bumblebees. Journal of Chemical Ecology 23(5): 1235-1251. doi: 10.1023/B:JOEC.0000006461.69512.33
https://doi.org/10.1023/B:JOEC.000000646...
), while females (queens and workers) of Bombus terrestris (Linnaeus, 1758) use CSG as a reproductive status signal (Amsalem et al. 2014Amsalem E, Kiefer J, Schulz S, Hefetz A (2014) The effect of caste and reproductive state on the chemistry of the cephalic labial glands secretion of Bombus terrestris . Journal of Chemical Ecology 40(8): 900-912. doi: 10.1007/s10886-014-0484-3
https://doi.org/10.1007/s10886-014-0484-...
). The function of the CSG secretion of A. mellifera is not clear, but it has been hypothesized to soften wax during nest construction (Heselhaus 1922Heselhaus F (1922) Die Hautchüsen der Apiden und Verwandter formen. Zoologische Jahrbücher Abteilung für Anatomie und Ontogenie der Tiere 43: 363-464.), and to lubricate mouthparts (Simpson 1960Simpson J (1960) The Functions of the Salivary Glands of Apis mellifera . Journal of Insect Physiology 4(2): 107-121. doi: 10.1016/0022-1910(60)90073-1
https://doi.org/10.1016/0022-1910(60)900...
). In meliponines, according to Jarau et al. (2004Jarau S, Hrncir M, Zucchi R, Barth FG (2004) A Stingless Bee Uses Labial Gland Secretions for Scent Trail Communication (Trigona recursa Smith, 1863). Journal of Comparative Physiology A Neuroethology, Sensory, Neural, and Behavioral Physiology 190(3): 233-239. doi: 10.1007/s00359-003-0489-9
https://doi.org/10.1007/s00359-003-0489-...
, 2006Jarau S, Schulz CM, Hrncir M, Francke W, Zucchi R, Barth FG, Ayasse M (2006) Hexyl decanoate, the first trail pheromone compound identified in a stingless bee, Trigona recursa . Journal of Chemical Ecology 32(7): 1555-1564. doi: 10.1007/s10886-006-9069-0
https://doi.org/10.1007/s10886-006-9069-...
, 2010Jarau S, Dambacher J, Twele R, Aguilar I, Francke W, Ayasse M (2010) The trial pheromone of a stingless bee, Trigona corvina (Hymenoptera, Apidae, Meliponini), varies between population. Chemical Senses 35(7): 593-601. doi: 10.1093/chemse/bjq057
https://doi.org/10.1093/chemse/bjq057...
), Schorkopf et al. (2007Schorkopf DLP, Jarau S, Francke W, Twele R, Zucchi R, Hrncir M, Schmidt VM, Ayasse M, Barth FG (2007) Spitting out information: Trigona bees deposit saliva to signal resource locations. Proceedings of the Royal Society of London. B Biological Sciences 274(1611): 895-898. doi: 10.1098/rspb.2006.3766
https://doi.org/10.1098/rspb.2006.3766...
) and Stangler et al. (2009Stangler ES, Jarau S, Hrncir M, Zucchi R, Ayasse M (2009) Identification of trail pheromone compounds from the labial glands of the stingless bee Geotrigona mombuca . Chemoecology 19: 13-19. doi: 10.1007/s00049-009-0003-0
https://doi.org/10.1007/s00049-009-0003-...
), the production of volatile components plays a role in communication through scent trails. Poiani et al. (2015Poiani SB, Morgan ED, Drijfhout FP, Cruz-Landim C (2015) Changes in the chemical profile of cephalic salivary glands of Scaptotrigona postica (Hymenoptera, Meliponini) workers are phase related. Journal of Experimental Biology 218: 2738-2744. doi: 10.1242/jeb.124180
https://doi.org/10.1242/jeb.124180...
), upon investigating the CSG secretion of a few species of bees, found oxygenated compounds in S. postica ; a mixture of oxygenated compounds and hydrocarbons in Melipona quadrifasciata (Lepeletier, 1836), and hydrocarbons in A. mellifera workers (Silvana Beani Poiani, unpublished data).

A typical colony of eusocial bees is composed of males and females (queen and workers). Males are present in the colony under some special conditions, and their function is to fertilize a new queen. Female members are divided into reproductive and partially non-reproductive castes. The queen is long-lived, and is responsible for egg production (reproduction). Workers are numerous, short-lived females, and are responsible for all services necessary to maintain the colony, such as brood care, building, attending the queen, guarding the entrance of the colony, and activities outside the nest (Snodgrass 1956Snodgrass RE (1956) Anatomy of the honey bee. New York, Vail-Ballow Press, 334p., Wilson 1971Wilson EO (1971) The insect societies. Cambridge, Belknap Press of Harvard University Press, 562p.). Workers of eusocial bees perform tasks according to their age, a phenomenon known as age polyethism or polyphenism. The development and functional cycles of several glands are directly related to the physiological state and life phase of the bee; in other words, the glands are activated when their secretion is necessary to perform tasks inside or outside the colony. A number of studies on the CSG of bees have investigated the chemical compounds at a specific phase of life or physiological status, for example: foragers (Jarau et al. 2004Jarau S, Hrncir M, Zucchi R, Barth FG (2004) A Stingless Bee Uses Labial Gland Secretions for Scent Trail Communication (Trigona recursa Smith, 1863). Journal of Comparative Physiology A Neuroethology, Sensory, Neural, and Behavioral Physiology 190(3): 233-239. doi: 10.1007/s00359-003-0489-9
https://doi.org/10.1007/s00359-003-0489-...
, Schorkopf et al. 2007Schorkopf DLP, Jarau S, Francke W, Twele R, Zucchi R, Hrncir M, Schmidt VM, Ayasse M, Barth FG (2007) Spitting out information: Trigona bees deposit saliva to signal resource locations. Proceedings of the Royal Society of London. B Biological Sciences 274(1611): 895-898. doi: 10.1098/rspb.2006.3766
https://doi.org/10.1098/rspb.2006.3766...
) or mature males (Bergman & Bergström 1997Bergman P, Bergström G (1997) Scent marking, scent origin, and species specificity in male premating behavior of two scandinavian bumblebees. Journal of Chemical Ecology 23(5): 1235-1251. doi: 10.1023/B:JOEC.0000006461.69512.33
https://doi.org/10.1023/B:JOEC.000000646...
). However, there have been no studies on how developed the CSGs are in different phases of the bee's life, or which changes occur on the morphology of these glands in different phases of the worker's life or physiological status.

We conducted a detailed morphological study of the CSGs of workers of A. mellifera and S. postica in different phases of their lives, in order to clarify the origin of the lipids stored in their CSGs. For that, we used a cytochemistry technique.

MATERIAL AND METHODS

Previous ultrastructural studies of bee's CSGs (Poiani & Cruz-Landim 2009Poiani SB, Cruz-Landim C (2009) Cephalic salivary gland ultrastructure of worker and queen eusocial bees (Hymenoptera, Apidae). Animal Biology 59(3): 299-311. doi: 10.1163/157075609X454935
https://doi.org/10.1163/157075609X454935...
) described the main features of gland cells. The cytochemical analysis presented here brings additional information about this gland.

In the ultrastructural cytochemical studies, we used CSGs from 10 newly emerged (NE) workers, 10 workers working in the brood comb area (CA), and 10 forager (FO) A. mellifera and S. postica workers. They were chosen based in the phase or task that they were performing in the colony at the moment they were collected. The specimens were collected in the apiary and meliponary maintained by the Institute of Biosciences of Universidade Estadual Paulista (UNESP) Rio Claro, SP, Brazil.

NE of both species were captured during their emergence from brood cells. CA of S. postica were collected while provisioning brood cells for the queen; and of A. mellifera , when feeding larvae. FO were collected when they returned from the field to the nest, with pollen on their legs.

Lipids were located using the imidazole-osmium (IO) method (Angermüller & Fahimi 1982Angermüller S, Fahimi DH (1982) Imidazole-buffered osmium tetroxide: an excellent stain for visualization of lipids in transmission electron microscopy. Histochemical Journal 14(5): 823-825. doi: 10.1007/BF01033631
https://doi.org/10.1007/BF01033631...
). All glands were dissected in insect physiologic solution and fixed for 2 hours in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4. Subsequently, the glands were washed twice in cacodylate buffer for 15 minutes per bath and in 0.1 M imidazole buffer, pH 7.5, for an additional 15 minutes. Post fixation was performed in 2% imidazole buffer containing 0.1 M osmium tetroxide, pH 7.5, for 30 minutes in the dark and at room temperature. The glands were washed twice in 0.1 M imidazole buffer, pH 7.5, next in phosphate-buffered saline (PBS), and finally in 10% ethanol, for 15 minutes per bath. The glands were dehydrated in increasing concentrations of acetone for 5 minutes each and embedded in Epon-Araldite resin. Resin polymerization was performed at 60°C for 24 hours. Thin sections were placed in copper grids and examined unstained in a Philips transmission electron microscope (TEM).

RESULTS

CSGs from NE, CA and FO workers treated with IO revealed several ultrastructural differences between the species and among the life phases.

Apis mellifera. The gland cells of A. mellifera NE workers exhibited smooth endoplasmic reticulum, medium electron density in the mitochondria, and nuclei with thick heterochromatin points. The alveolar space was narrow and contained small amounts of secretion. IO-highlighted droplets were scattered in the cytoplasm of the secretory cells and the secretion in the lumen (Figs. 1-2).

In CA workers, droplets were randomly dispersed in the cytoplasm (Fig. 3) as well as in heterogeneous granules positive for IO (Fig. 4). Most of the alveoli contained lumens loaded with IO-stained secretion (Figs. 3-4). The lumen of the alveoli of the CSG was lined by cuticle. In the glands of CA and FO, the gland cell apical plasma membrane was invaginated to form sub-cuticular channels, between which elongated mitochondria of medium electron density were present (Figs. 5-6).

In FO workers, IO positivity was found in the vesicles (Figs. 7-8) of the apical region and in the channels formed by the apical infolds (Fig. 6). The lumens of almost all alveoli were filled with IO-positive secretion.

Figures 1-4
Lipids, detected using imidazole-osmium, in cephalic salivary gland (CSG) cells of Apis mellifera workers. (1-2) Small droplets of lipid (arrows) dispersed in the cellular cytoplasm of a newly emerged worker (NE), mitochondria (m) of medium electron density and narrow alveolar lumens (l) containing scarce IO-positive secretion (s). (3-4) Gland cells from workers working in the brood comb area (CA). Note lipid droplets dispersed in the cytoplasm (arrows) (1C), heterogeneous granules (gr) (1D) and large amounts of lipid secretion (s) in the alveolar lumen (l). (c) Cuticle, (n) nuclei. Scale bars: 1, 2, 4 = 1 µm, 3 = 3 µm.

Figures 5-8
Imidazole-osmium preparations for lipid detection in gland cells from Apis mellifera workers. (5-6) Gland cells from worker working in the brood comb area (CA) (5) and forager (FO) (6) showing lipid droplets dispersed in the apical region (arrow), infolds (i) of the apical membrane forming channels flanked by mitochondria (m). (6) Note the presence of lipid droplets in the apical channels (arrows). (7-8) Osmium-imidazole-positive dots spread in the cytoplasm (arrows) and vesicles (ve) of forager cellular glands. (c) Cuticle, (s) secretion. Scale bars: 5 = 3 µm, 6 = 2 µm, 7-8 = 1 µm.

Scaptotrigona postica. Glandular cells from NE presented a low positive response to IO treatment, which appeared in the cytoplasmic vesicles and as a tiny, electron-dense lining of the alveolar luminal cuticle (Fig. 9). As noted in A. mellifera , the amount of IO-positive material increased in the CA glandular cells and alveolar lumen (Fig. 10), but showed a different pattern in FO cells, which exhibited few positive structures in the cells but the alveolar lumen filled with secretion.

In CA, some mitochondria appeared to react positively to IO, showing lipid content in the matrix. Additionally, the smooth endoplasmic reticulum cisterns contained positive material with the same electron density observed in the mitochondria and in the alveolar lumen (Fig. 10).

In FO workers, a few cells in the CSG were cuboidal (Fig. 11) and had large electron-dense droplets in the cytoplasm. However, the majority of cells were flat (Fig. 12) and, although some positive reaction to IO appeared in the cytoplasm of these cells, IO positivity was almost exclusively present in the alveolar lumen (Fig. 12). At this worker stage, glandular cells presented sparse organelles.

Figures 9-12
Detection of lipid by imidazole osmium (IO) in cells from the cephalic salivary glands of Scaptotrigona postica . (9) Gland cell from newly emerged (NE) and (10) worker working in the brood comb area (CA) showing vesicles containing heterogeneous material (vh) in the apical region. Mitochondria (m) are abundant and variable in electron density and shape. Note the droplets (arrow), smooth endoplasmic reticulum cisterns (rel) and some mitochondria showing IO-positive lipid content inside (ml). (11-12) Glandular cells from forager (FO) workers. (11) Cubic gland cells showing large lipid droplet (lip). (12) Gland cells flattened with drop-shaped lipid vesicles (lip) and heterogeneous vesicles (vh). Note the large alveolar lumen (l) accumulating secretion (s). (c) Cuticle, (m) mitochondrion, (n) nucleus, (s) secretion. Scale bars: 9 = 1 µm, 10, 12 = 2 µm, 11 = 3 µm.

DISCUSSION

Cellular morphology displays features that provide clues about cellular function. The CSG of bees has been extensively studied and the results of these studies have documented that these glands have secretory function (Snodgrass 1956Snodgrass RE (1956) Anatomy of the honey bee. New York, Vail-Ballow Press, 334p., Simpson 1960Simpson J (1960) The Functions of the Salivary Glands of Apis mellifera . Journal of Insect Physiology 4(2): 107-121. doi: 10.1016/0022-1910(60)90073-1
https://doi.org/10.1016/0022-1910(60)900...
, Santos et al. 2009Santos CG, Megiolaro F, Serrão JE, Blochtein B (2009) Morphology of the head salivary and intramandibular glands of the stingless bee Plebeia emerina (Friese) (Hymenoptera, Meliponini) workers associated with propolis. Annals of the Entomological Society of America 102(1): 137-143. doi: 10.1603/008.102.0115
https://doi.org/10.1603/008.102.0115...
). The morphological clues indicating cell secretory activity vary with the type of secreted product. Our cytochemistry results revealed the dynamics of producing and/or up-taking compounds by CSG cells and storing these compounds until they are used.

The glandular cells of workers and queens of A. mellifera and S. postica begin their secretory activity as soon as the adult emerges, and the resulting secretion is progressively accumulated in the gland's alveolar lumen as the bee ages (Poiani & Cruz-Landim, 2009Poiani SB, Cruz-Landim C (2009) Cephalic salivary gland ultrastructure of worker and queen eusocial bees (Hymenoptera, Apidae). Animal Biology 59(3): 299-311. doi: 10.1163/157075609X454935
https://doi.org/10.1163/157075609X454935...
, 2010aPoiani SB, Cruz-Landim C (2010a) Morphological changes in the cephalic salivary glands of females and males of Apis mellifera and Scaptotrigona postica (Hymenoptera, Apidae). Journal of Biosciences 35(2): 249-255. doi: 10.1007/s12038-010-0029-z
https://doi.org/10.1007/s12038-010-0029-...
, bPoiani SB, Cruz-Landim C (2010b) Changes in the size of cephalic salivary glands of Apis mellifera and Scaptotrigona postica (Hymenoptera: Apidae) queens and workers in different life phases. Zoologia 27(6): 961-964. doi: 10.1590/S1984-46702010000600018
https://doi.org/10.1590/S1984-4670201000...
, cPoiani SB, Cruz-Landim C (2010c) Cephalic salivary glands of two species of advanced eusocial bees (Hymenoptera: Apidae): morphology and secretion. Zoologia 27(6): 979-985. doi: 10.1590/S1984-46702010000600021
https://doi.org/10.1590/S1984-4670201000...
).

The results presented here have shown that the CSG of A. mellifera workers not only produces, but also intakes lipid compounds from hemolymph as the worker ages. This gland, then, has both secretory and excretory functions. It has been reported that some lipids present in the hemolymph might be excreted by exocrine glands (Hefetz et al. 1996Hefetz A, Taghizadeh T, Francke W (1996) The exocrinology of the Queen bumble bee Bombus terrestris (Hymenoptera: Apidae, Bombini). Zeitschrift für Naturforschung C 51(5-6): 409-422. doi: 10.1515/znc-1996-5-620
https://doi.org/10.1515/znc-1996-5-620...
, Meirelles et al. 2001Meirelles RMS, Silva ECM, Silva de Moraes RLM (2001) Lipid distribution in salivary glands of larvae and adult bees (Hymenoptera, Apidae). Cytobios 106: 57-66.). The material taken by the cells may be processed in their cytoplasm before being delivered, characterizing secretory activity; or it may just pass through it, characterizing excretion. Well-developed infoldings of the basal cell membrane found in CSGs (Poiani & Cruz-Landim 2009Poiani SB, Cruz-Landim C (2009) Cephalic salivary gland ultrastructure of worker and queen eusocial bees (Hymenoptera, Apidae). Animal Biology 59(3): 299-311. doi: 10.1163/157075609X454935
https://doi.org/10.1163/157075609X454935...
) increase the contact surface between the cell and its environment, and might be associated with excretion. Insect Malpighian tubules and the convoluted nephron tubules of vertebrates are both excretory organs and, as such, have cells with highly developed basal membrane infolds. In some insects, for instance Collembola, which do not have Malpighian tubules, the salivary glands have an excretory role (House & Ginsborg 1985House CR, Ginsborg BL (1985) Salivary gland, p. 195-224. In: Kerkut GS, Gilbert LJ (Eds.). Comparative Insect Physiology, Biochemistry and Pharmacology. Oxford, Pergamon Press.), and that function can be morphologically detected through the presence of very developed basal infolds of the gland cell membranes. The basal infolds of the cells are frequently continuous with the smooth endoplasmic reticulum. Materials uptake by the infolds are from exogenous origin and might circulate within the cellular reticular system, as seen in the Dufour glands of B. terrestris queens (Hefetz et al. 1996Hefetz A, Taghizadeh T, Francke W (1996) The exocrinology of the Queen bumble bee Bombus terrestris (Hymenoptera: Apidae, Bombini). Zeitschrift für Naturforschung C 51(5-6): 409-422. doi: 10.1515/znc-1996-5-620
https://doi.org/10.1515/znc-1996-5-620...
, Abdalla et al. 1999aAbdalla FC, Velthuis WWH, Cruz-Landim C, Duchateau MJ (1999a) Changes in the morphology and ultrastructure of the Dufour's gland during the life cycle of the bumble bee queen, Bombus terrestris L. (Hymenoptera: Bombini). Netherlands Journal of Zoology 49(4): 251-261. doi: 10.1163/156854299X00173
https://doi.org/10.1163/156854299X00173...
, bAbdalla FC, Velthuis WWH, Cruz-Landim C, Duchateau MJ (1999b) Secretory cycle of the Dufour's gland in workers of bumble bee Bombus terrestris (Hymenoptera: Bombini). Netherlands Journal of Zoology 49(3): 139-156. doi: 10.1163/156854299505650
https://doi.org/10.1163/156854299505650...
) and A. mellifera queens (Katzav-Gozansky et al. 2000Katzav-Gozansky T, Soroker V, Hefetz A (2000) Plasticity in caste-related exocrine secretion biosynthesis in the honey bee (Apis mellifera ). Journal of Insect Physiology 46(6): 993-998. doi: 10.1016/S0022-1910(99)00209-7
https://doi.org/10.1016/S0022-1910(99)00...
), and in the CSGs of A. mellifera (Poiani & Cruz-Landim 2009Poiani SB, Cruz-Landim C (2009) Cephalic salivary gland ultrastructure of worker and queen eusocial bees (Hymenoptera, Apidae). Animal Biology 59(3): 299-311. doi: 10.1163/157075609X454935
https://doi.org/10.1163/157075609X454935...
).

The IO treatment in CSG of A. mellifera workers has demonstrated the presence of lipids in the cytoplasm of CSG gland cells and in the alveolar lumens of NE workers, increasing continuously until the FO phase. Therefore, in this species, the gland cells continue their activity in foragers, in contrast with what happens in S. postica. The CSG cells present extensive infolds of the cellular apical membrane and IO-positive material could be detected within the channels formed by the infolds, as well in the smooth endoplasmic reticulum. That observation suggests that lipids absorbed from the hemolymph, through the basal plasma membrane infolds, reach the lumen by traveling through the endoplasmic reticulum until they are delivered to the lumen, where they mix with the gland's secretion. Apical membrane infolds mean an increased surface for secretion delivery and the presence of mitochondria located along the channels that are so formed indicates an active transport of content from the inner cell to the alveolar lumen. All these features confer an excretory function to the CSG gland of A. mellifera workers, besides a possible secretory role revealed by proteomic analysis (Fujita et al. 2010Fujita T, Kozuka-Hata H, Uno Y, Nishikori K, Morioka M, Oyama M, Kubo T (2010) Functional analysis of the honeybee (Apis mellifera L.) salivary system using proteomics. Biochemical and Biophysical Research Communications 397(4): 740-744. doi: 10.1016/j.bbrc.2010.06.023
https://doi.org/10.1016/j.bbrc.2010.06.0...
), which revealed enzymes related to lipids metabolism.

There are older reports about the use of the CSG secretion by A. mellifera workers (Heselhaus 1922Heselhaus F (1922) Die Hautchüsen der Apiden und Verwandter formen. Zoologische Jahrbücher Abteilung für Anatomie und Ontogenie der Tiere 43: 363-464., Simpson 1960Simpson J (1960) The Functions of the Salivary Glands of Apis mellifera . Journal of Insect Physiology 4(2): 107-121. doi: 10.1016/0022-1910(60)90073-1
https://doi.org/10.1016/0022-1910(60)900...
) and recent research has found a correspondence between the contents of the CSG and the chemical profile of the cuticle (Silvana Beani Poiani, unpublished data), suggesting that the CSG secretion may have these compounds exported to the body surface, where they act as recognition pheromones.

In contrast, the gland cells of S. postica do not exhibit specific morphological features that indicate excretory function. Therefore, it can be concluded that the lipids present in the alveoli lumen only originate through secretory gland activity. The CSG of S. postica produces lipid compounds during the NE and CA phases, storing it in the alveolar lumen until the worker becomes a forager and needs to use it. The IO treatment demonstrated the presence of small amounts of lipids in the cytoplasm and alveolar lumen of the CSG gland cells of NE workers. At the CA phase, the amount of lipids increased in the cytoplasm and alveolar lumen. These lipids were visualized as droplets in the cytoplasm and as a strongly IO-positive homogeneous material in the lumen. The presence of lipids into a network of smooth reticulum, vesicles and droplets present in the cytoplasm signals a secretory function. In FO, alveolar cells are flat, with almost no visible organelles. The alveolar lumen is full of secretion. The fact that the FO gland cells of S. postica were much more flattened and poor in organelles when compared to NE and CA indicates that the secretory activity is finished in FO phase, as proposed by Poiani & Cruz-Landim (2009Poiani SB, Cruz-Landim C (2009) Cephalic salivary gland ultrastructure of worker and queen eusocial bees (Hymenoptera, Apidae). Animal Biology 59(3): 299-311. doi: 10.1163/157075609X454935
https://doi.org/10.1163/157075609X454935...
). However, the alveoli of FO workers were still full with secretion, strongly suggesting that the secretion produced was not discharged until the forager phase. The CSG of S. postica synthesizes oxygenated compounds, especially esters which are phase-related (Poiani et al. 2015Poiani SB, Morgan ED, Drijfhout FP, Cruz-Landim C (2015) Changes in the chemical profile of cephalic salivary glands of Scaptotrigona postica (Hymenoptera, Meliponini) workers are phase related. Journal of Experimental Biology 218: 2738-2744. doi: 10.1242/jeb.124180
https://doi.org/10.1242/jeb.124180...
) and may be used in trail scents. In fact Jarau et al. (2004Jarau S, Hrncir M, Zucchi R, Barth FG (2004) A Stingless Bee Uses Labial Gland Secretions for Scent Trail Communication (Trigona recursa Smith, 1863). Journal of Comparative Physiology A Neuroethology, Sensory, Neural, and Behavioral Physiology 190(3): 233-239. doi: 10.1007/s00359-003-0489-9
https://doi.org/10.1007/s00359-003-0489-...
, 2006Jarau S, Schulz CM, Hrncir M, Francke W, Zucchi R, Barth FG, Ayasse M (2006) Hexyl decanoate, the first trail pheromone compound identified in a stingless bee, Trigona recursa . Journal of Chemical Ecology 32(7): 1555-1564. doi: 10.1007/s10886-006-9069-0
https://doi.org/10.1007/s10886-006-9069-...
, 2010Jarau S, Dambacher J, Twele R, Aguilar I, Francke W, Ayasse M (2010) The trial pheromone of a stingless bee, Trigona corvina (Hymenoptera, Apidae, Meliponini), varies between population. Chemical Senses 35(7): 593-601. doi: 10.1093/chemse/bjq057
https://doi.org/10.1093/chemse/bjq057...
), Schorkopf et al. (2007Schorkopf DLP, Jarau S, Francke W, Twele R, Zucchi R, Hrncir M, Schmidt VM, Ayasse M, Barth FG (2007) Spitting out information: Trigona bees deposit saliva to signal resource locations. Proceedings of the Royal Society of London. B Biological Sciences 274(1611): 895-898. doi: 10.1098/rspb.2006.3766
https://doi.org/10.1098/rspb.2006.3766...
) and Stangler et al. (2009Stangler ES, Jarau S, Hrncir M, Zucchi R, Ayasse M (2009) Identification of trail pheromone compounds from the labial glands of the stingless bee Geotrigona mombuca . Chemoecology 19: 13-19. doi: 10.1007/s00049-009-0003-0
https://doi.org/10.1007/s00049-009-0003-...
) attributed scent trail activity to these glands in forager meliponines, which use it as trail pheromone. Another function was attributed to the CSG of Plebeia emerina (Friese, 1900) in which the glandular secretion from middle-aged nurse workers is used to soften the propolis balls dispersed in the colony as a defense against invaders (Santos et al. 2009Santos CG, Megiolaro F, Serrão JE, Blochtein B (2009) Morphology of the head salivary and intramandibular glands of the stingless bee Plebeia emerina (Friese) (Hymenoptera, Meliponini) workers associated with propolis. Annals of the Entomological Society of America 102(1): 137-143. doi: 10.1603/008.102.0115
https://doi.org/10.1603/008.102.0115...
). Elias-Santos et al. (2013Elias-Santos D, Fialho MCQ, Vitorino R, Oliveira LL, Zanuncio JC, Serrão JE (2013) Proteome of the head and thorax salivar glands in the stingless bee Melipona quadrifasciata anthidioides . Apidologie 44(6): 684-698. doi: 10.1007/s13592-013-0217-6
https://doi.org/10.1007/s13592-013-0217-...
) identified enzymes related to lipid synthesis in the CSG of M. quadrifasciata anthidioides , which is consistent with the results of the present work.

In conclusion, the morphology of the CSG and its function differ between the species studied and among the life phase of the workers. The CSG has excretory and secretory functions in A. mellifera workers, while in S. postica it has only a secretory function.

ACKNOWLEDGEMENTS

We would like to thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the fellowship (Proc. 07/56682-1).

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Publication Dates

  • Publication in this collection
    2016

History

  • Received
    23 Nov 2015
  • Reviewed
    09 Jan 2016
  • Accepted
    07 Feb 2016
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