Cephalic salivary gland ultrastructure of worker and queen eusocial bees (Hymenoptera, Apidae)

© Koninklijke Brill NV, Leiden, 2009 DOI 10.1163/157075609X454935

Animal Biology 59 (2009) 299–311 brill.nl/ab

Cephalic salivary gland ultrastructure of worker and queen eusocial bees (Hymenoptera, Apidae)

Silvana Beani Poiani * and Carminda da Cruz-Landim

Department of Biology, UNESP - São Paulo State University, Rio Claro, SP, Brazil Av. 24A, no.1515, Bela Vista, CEP 13506-900

Abstract Eusocial bees present a pair of functional salivary glands in head, the cephalic salivary glands. Th ese glands from workers and queens of two eusocial bee species, Apis mellifera and Scaptotrigona postica , were exam-ined at diff erent life stages using routine transmission electron microscopy techniques to correlate mor-phology and gland functions. Ultrastructural features of worker and queen glands ducts and secretory units were descriptively compared between species. Th e duct cells present basal plasma membrane invagi-nations reaching the apical region. Intercellular space and invaginations contain material of similar elec-tron-density to the basal lamina, suggesting that substances might be directly absorbed from the hemolymph to the gland lumen. Th e secretory cells are rich in smooth endoplasmic reticulum, mitochon-dria, Golgi, and vesicles typical of lipid secretion. Secretory cells in S. postica become fl attened with age in contrast to A. mellifera , where cells remained cuboidal. Mitochondria are associated with secretory vesicles and may become lipid deposits. A possible role of worker and queen secretion is discussed, taking changes in caste gland morphology and their function in the colony into account. © Koninklijke Brill NV, Leiden, 2009

Keywords Apis mellifera; labial gland; lipid; mitochondria; pheromone; Scaptotrigona postica

Introduction

Th e mandibular, hypopharingeal and labial glands are the main parts of a bee’s salivary gland system. Th ey are all linked to mouth appendices from which they receive their names.

Th e labial glands are homologous with salivary glands of other insects and originate from larval salivary glands. Th erefore bee labial glands are known as true salivary glands. Th ese glands might display functions not related to food ingestion or digestion, al though secretion from bee labial glands is delivered on the tongue assuring contact with the ingested food.

*) Corresponding author; e-mail: [email protected]

300 S.B. Poiani and C. da Cruz-Landim / Animal Biology 59 (2009) 299–311

Salivary glands in all species of adult bees are pair structures located in the thorax, but in some species of eusocial bees from Apinae subfamily, there is also a functional pair of cephalic glands (Cruz-Landim, 1967 ).

Th e cephalic salivary glands originate during metamorphosis as outgrowings from the head portion of the thorax glands excretory duct (Cruz-Landim and Mello, 1967 ) and consist of alveolar secretory units (Cruz-Landim, 1967 ). Th e alveolar ducts join together to form collector ducts from whose fusion the excretory duct of each gland pair originates. Th e excretory ducts from the cephalic glands join to the thoracic ducts to form a common excretory duct which delivers secretion in the tongue. In Bombus and stingless bees (Meliponini) an enlargement exists where the thoracic and cephalic salivary glands ducts join; it is called the salivary pouch. In these cases the common excretory duct originates from the pouch (Cruz-Landim, 1967 ; Graf, 1968 ).

Although the secretory units of both the thoracic and the cephalic salivary glands have the same embryonic origin, their morphology and secretion quality are diff erent. Th e thoracic gland has tubular and the cephalic gland alveolar secretory units. Th oracic gland secretion is an aqueous, very diluted solution of proteins, containing digestive enzymes (Delage-Darchen et al., 1979; Simpson, 1960; Simpson et al., 1968 ) and cephalic gland secretion has an oily appearance. Heselhaus ( 1922 ) suggested its func-tion was to soften wax for manipulation during nest building. Simpson (1960) rejects this, arguing that not all bees with cephalic salivary glands build wax nests and suggests their function is mouth appendices lubrication. Jarau et al. ( 2004 ) and Schorcopf et al. (2007) demonstrated in Trigona recursa and Trigona spinipes respectively, that cephalic secretion is used in communicating food resource location by scent trail.

Most studies on cephalic salivary glands attempt to identify the chemical compo-sition of the secretion. Th ere are several works on Bombus species, mainly males. Th e present approach focuses on the morphological ultrastructural cell aspects of cephalic salivary glands in Apis mellifera and Scaptotrigona postica workers and queens , aiming to follow glandular development with bee activity changes to trace cell and secretion features capable of being correlated to their activities, and thus deduce secretion function.

Materials and methods

Material

Cephalic salivary glands were obtained from newly emerged (young), nurse (middle age) and forager (old) workers, and virgin and laying queens captured in colonies maintained in the Apiary of Instituto of Biociências of São Paulo State University (UNESP) – Rio Claro, SP, Brazil. Newly emerged workers were captured when leaving their brood cell, nurses while tending the brood, and the foragers when returning to the nest. Virgin queens from Apis mellifera were artifi cially produced.

S.B. Poiani and C. da Cruz-Landim / Animal Biology 59 (2009) 299–311 301

Methods

Glands were dissected from fi ve individuals of each phase and fi xed for two hours in 2.5% glutaraldehyde in 0.1M sodium cacodylate buff er pH 7.4. Post fi xation were done in 0.5% osmium tetroxide containing 0.8% potassium ferrocyanide in the same buff er during one hour. After washing in buff er, they were left for three hours in 2% tannic acid. After another wash, glands were stained with 1% uranyl acetate in 10% ethanol for two hours and dehydrated in acetone series.

Th ey were block imbedded in Epon-Araldite resin. Resin polymerization was in a stove at 60°C. Sections were placed in cupper grids, stained with uranyl acetate and lead citrate, and examined by Philips transmission electron microscope (TEM).

Results

Ducts and salivary pouch

Cross sections of the duct from the gland of S. postica showed the wall consisting of fl at cells in which the fusiform nucleus occupies almost all the cytoplasm. Some mitochon-dria and smooth endoplasmic reticulum (SER) can be seen. Th e cuticle lining lumen is thick with secretion stuck to it, which occupies almost all the lumen ( fi g. 1 ).

Th e salivary pouch in S. postica and the cephalic salivary gland ducts in A. mellifera consist of a layer of medium height epithelial cells with round or irregular nuclei, generally with one nucleolus. Th e apical cell surface is covered by a thin medium electron-dense cuticle containing electron-dense regions corresponding to cuticle thickenings ( fi gs 2 and 3 ). Th e cell’s plasma membrane presents apical invaginations, sometimes forming projections into the subcuticular space, similar to irregular micro-villi. In the base of these invaginations are some clear vesicles resulting from endocyto-sis. Th e basal plasma membrane presents invaginations that sometimes form loops. Intercellular spaces slightly enlarged and loops are fi lled with electron-dense material, similar to the basal lamina. Small vesicles arising from the invaginations extremities are full of the same electron-dense material. Cell contact is very sinuous, especially in the two apical thirds where the intercellular space is narrow and almost free of electron-dense material ( fi g. 2 ).

Cytoplasm from the salivary pouch and duct cells contains SER but is poor in organelles. Mitochondria are dispersed over the cytoplasm mainly accompanying basal plasma membrane invaginations. Th e mitochondria are irregular in shape with variable sizes and have disorganized cristae ( fi g. 3 ).

Secretory portion

Common features to workers and queens Apis mellifera. Th e apical surface of the secre-tory cells is covered by a thin non-sclerotized cuticle which continues into the ducts ( fi g. 4 ).


Figures 1-4. 1 – Gland duct cross section of S. postica . Note the thin basal lamina (bl) and thick luminal cuticle (c) with adhered secretion (s). 2 – Gland duct longitudinal section of A. mellifera . Note the medium height cells and electron-dense regions corresponding to cuticle reinforcements (cr). Th e thick basal lam-ina (bl) penetrates through invaginations (i) of the basal plasma membrane and forms endocytic vesicles in the extremities (ev). Th e very sinuous intercellular membrane (im) space is fi lled with electron-dense material (arrow). 3 – Salivary pouch of S. postica salivary cephalic gland. Detail of cell apical region show-ing the formation of a subcuticular space (su). Mitochondria (m) mainly in the apical region and accom-panying the intercellular membrane (im) showing disorganized cristae (arrow). 4 – General view of cell from the secretory portion of the cephalic salivary gland of A. mellifera showing the luminal cuticle (c), mitochondria (m), smooth endoplasmic reticulum (ser), nucleus (n) and active nucleolus (nu) with gran-ular (gr) peripheral and central fi brillar (fr) regions. cr = cuticle reinforcements; l = lumen; m = mitochon-dria; n = nucleus; tr = tracheae.


Basal plasma membrane invaginations form narrows canals which contain material with the same aspect and electron-density as the basal lamina. Th e loops seem to seg-regate cytoplasm portions ( fi gs 5 and 6 ). Th e intercellular spaces are open in the base and fi lled with electron-dense material from the basal lamina. Tracheae penetrate through the plasma membrane invaginations ( fi g. 5 ).

Th e SER is well developed and can present as tubules or small vesicles. Flat vesicles from the SER are frequently found accompanying lateral plasma membrane of cells. Th ese vesicles also contain electron-dense material. In this region, the SER originates from basal plasma membrane invaginations which form a true basal labyrinth ( fi g. 6 ). Th e rough endoplasmic reticulum (RER) is poorly developed but Golgi regions are common and appear as small groups of electron-lucent swollen lamellae which give it a vesicular aspect ( fi gs 5 and 7 ).

Th e mitochondria preferentially locate in the cell basal portion. Th ey are elongated with an electron-dense matrix and cristae parallel to their length ( fi g. 5 ). Th e gland cells present large central nuclei with more than one nucleolus, in which fi brilar and granular regions are sometimes distinguishable ( fi g. 4 ).

Specifi c features to workers and queens Apis mellifera. Th e alveolar cells are cubic or fl at in newly emerged workers and polymorphic mitochondria with electron-dense matrix along with other cell organelles can be seen in the cytoplasm. In nurse and forager workers and virgin and laying queens, the gland secretory cells are cubic. Mitochondria vary in size have electron-dense matrix and are abundantly dispersed overall the cyto-plasm ( fi gs 4 , 5 and 8 ). Low electron-density mitochondria can also be seen and they display disorganized cristae; this low electron-density material seems to be due to accu-mulation of secretion in their inter-membranes space ( fi gs 7 , 9 and 10 ).

Large electron-lucent vesicles or droplets found overall secretory cell cytoplasm from forager workers and laying queens were interpreted as secretion. In some cases the vesi-cles seemed to result from disorganized mitochondria ( fi gs 9 and 10 ) and in others from unchanged mitochondria; the latter containing membranous structures ( fi g. 9 ). Vesicles originating from mitochondrial disorganization are recognized by the presence of remaining cristae and lamellar content ( fi g. 10 ). Some are enveloped by just a membrane.

Virgin queens present giant round mitochondria in alveolar cells with few cristae, low electron-density matrix, and lipid-like content ( fi g. 11 ).

Scaptotrigona postica. Alveolar cells from cephalic salivary glands of newly emerged workers are cubic and display central round nuclei ( fi g. 12 ). Th e alveoli lumen is lined by a thin cuticle. Th e cell basal lamina is slender and of medium electron-density. Th e basal plasma membrane does not form invaginations diff erently in A. mellifera indi-viduals . In the cytoplasm, as well as SER, there are numerous ribosome groups, mito-chondria, and secretion deposits ( fi g. 12 ). Th e mitochondria present disorganized cristae and electron-lucent matrix. Th e secretion appears in two distinct types, one as granules with homogeneous medium electron-density content and the other as vacu-oles containing several small vesicles ( fi g. 13 ).


Mitochondria with medium electron-density matrix predominate and are through-out the cytoplasm ( fi g. 12 ). Some of these mitochondria have irregular shapes and are associated with low electron-dense granules. Some mitochondria associated to granules are so diff erent in shape that they can only be identifi ed by remaining cristae.

Some alveoli in nurse workers contain secretion and present fl attened epithelial cells similar to forager workers ( fi g. 14 ) while others are still empty and present cubic cells, morphologically similar to secretory cells of alveoli in newly emerged workers ( fi g. 12 ).

Figures 5-8. 5 – Basal portion of a secretory cell showing the basal lamina (bl), penetrating through cell membrane invaginations (i) and intercellular membrane space (im), in A. mellifera . Note the presence of elongated mitochondria (m) with longitudinal cristae and smooth endoplasmic reticulum (arrow). 6 – Basal labyrinth formed by plasma membrane invaginations in A. mellifera . Note the smooth endoplas-mic reticulum (arrow) and the endocytic vesicles (ev) that accompany the intercellular membranes (im). 7 – Changed mitochondria associated with secretion. Note the few cristae and enlarged intermembranous space (arrows). Note the presence of several Golgi regions vesicles (G). 8 – Secretory cell from forager worker showing the association of mitochondria (m), smooth endoplasmic reticulum (ser), and granules (gr). bl = basal lamina; i = membrane invagination; G = Golgi regions; m = mitochondria; tr = tracheae.


Figures 9-12. 9 and 10 – Mitochondrial changes related to secretion production in cephalic salivary gland of A. mellifera . In 9 note the association between mitochondria (m) and vesicles containing membranous material (mv) and in 10 the apparent accumulation of secretion in the mitochondria (arrows). 11 – Cephalic salivary gland cell from a A. mellifera virgin queen showing large mitochondria (m) with few cristae, apparently accumulating lipid. Note very electron-dense secretion (s) in the alveolus lumen. 12 – General view of salivary cephalic gland from newly emerged workers of S. postica . Note the ribosome groups (r), nucleus (n), nucleolus (nu) and cuticle (c) lining the alveolus lumen (l) which con-tains secretion (s). bl = basal lamina; cv = dense coated vesicles; m = mitochondria; tr = tracheae.

Alveoli secretory cells full with secretion have few mitochondria and many free ribos-omes. Th e cell nuclei are elongated or rounded and occupy almost all the cytoplasm ( fi g. 14 ).

Alveolar cells from laying queens are cubic and have some ultrastructural diff erences to workers. Th e SER consists of narrow canals that have swollen in some regions form-ing spaces full of fl occulent material ( fi g. 15 ). Large vacuoles store electron-dense material with an acicular aspect ( fi g. 16 ).


Figures 13-16. 13 – Gland cell from a S. postica newly emerged worker. Secretion in the enlarged lumen as smooth endoplasmic reticulum (arrow), homogeneous granules (gr 1 ) and droplets containing vesicles (gr 2 ). 14 – Flat gland cells in forager workers and alveolar lumen with secretion (s). 15 and 16 – Laying queen gland of S. postica . In 15 note the widening of the smooth endoplasmic reticulum (ser) that accu-mulates fl occulent material inside (arrows). In 16 observe large deposits of acicular material (am) in cyto-plasm vacuoles. bl = thin basal lamina; mv = vesicle containing membranous structure; n = nucleus.

Discussion

A. mellifera cephalic salivary gland ducts and the salivary pouch of S. postica both have walls formed from medium height cells while gland duct cells in S. postica are fl at. Pouch and duct cell cytoplasm in both species is poor in organelles and not secretory, as already observed by Silva de Moraes and Cruz-Landim ( 1975 ). Nevertheless, the


presence of material with texture and electron-density equal to the basal lamina, inside the basal plasma membrane invaginations and basal intercellular spaces, suggests that material might be captured from the hemolymph and transported to the lumen through these cells. A similar function was suggested in Calliphora larva gland duct cells by Oschman and Berridge (1970) and in bees by Meirelles et al. ( 2001 ). According to Meirelles et al. ( 2001 ) absorbed material is of a lipid nature, which explains the high electron-density of the basal lamina, and might, at least in part, be used in the compo-sition of the epicuticle from the cuticle lining the duct lumen. An excretory function is common to certain glands and their ducts and may be so in this case.

Another function of duct cells is suggested by the clear small vesicles originating from apical plasma membrane invaginations that might means reabsorption of material from the lumen (Cruz-Landim, 1994 ). Th erefore, the duct cell may absorb material of a lipid nature from the hemolymph and deliver it in the lumen and in the other direc-tion, absorb non-lipid material from the lumen and return it to the hemolymph. Th is latter function is also common to the salivary pouch as suggested by cell morphology.

Extensive invaginations of the basal plasma membrane are present in glandular secretory cells at all phases of A. mellifera workers and queens but absent in S. postica. Th e canals formed by these invaginations contain similar electron-dense material to the basal lamina and invaginations are accompanied by fl at SER vesicles and mitochon-dria. Th is morphology suggests that, as with ducts, substances are being captured from the hemolymph and used in secretion synthesis, or are transported through cell endo-plasmic reticulum to the lumen. Also the SER is involved with the lipid synthesis (King and Akai, 1982 ) which explains its ample development in these cells. Th e absence of basal invaginations in gland cells of S. postica might imply the lack or little absorp-tive function to these cells.

Glandular cells which produced pheromones generally present well developed SER and Golgi regions in the cytoplasm with mitochondria and inclusions consisting of lamellar bodies, corresponding to lipids (Noirot and Quennedey, 1974; Billen, 1991 ; Billen and Morgan, 1998 ). Such cell characteristics were found in A. mellifera and S. postica .

Lipids absorbed from the hemolymph might acquire a diff erent composition by being modifi ed by the cell or might cross the cell without suff ering any modifi cation. Th e existence of open intercellular spaces containing electron-dense material indicates the possibility that a proportion of the lipids found in gland secretion are from the hemolymph and unmodifi ed, therefore not secreted by the gland cells. A similar condi-tion was observed in the Dufour gland of Bombus terrestris queens (Hefetz et al., 1996 ; Abdalla et al., 1999a , 1999b ) and A. mellifera (Katzav-Gozansky et al., 2000 ).

Mitochondria suff er changes related to secretory cell functional stage, which change with an individual’s age or colony activity. Changes in mitochondria and their associa-tion to cellular secretion are common in lipid secreting cells (Billen, 1991 ) and are due to their role in lipid synthesis. Th e outer mitochondrial membrane is porous in con-trast to the inner one which is impermeable to most small molecules and ions includ-ing H + protons (Junqueira and Carneiro, 2000 ; Lehninger et al., 2000). Th e observation that mitochondria with low electron-dense matrix in secretory cells of both bee species


were associated with secretion is the morphological expression of this mitochondrial function. Since the inner mitochondrial membrane was seen to be disrupted in the organelles involved in secretion, they are no longer impermeable to lipid molecules from the cytosol and thus might become a depository of this material. It is also known that some fatty acids can enter the mitochondria where they are broken in order to enter the Krebs cycle and be used in energy production (De Roberts and Hib, 2001 ). However disruption of the inner mitochondrial membrane compromises mitochondria oxidative capacity in such a way that fatty acids can accumulate in the matrix and lipid synthesis enzymes may enter through the outer membrane and generate them.

Th e association between mitochondria and lipids in insects was fi rst described by Ranade ( 1933 ), and later by Ratcliff e and King ( 1969 ) and Boissin ( 1970 ). Ratcliff e and King ( 1969 ) reported the transformation of mitochondria in residual bodies or autophagic vacuoles before originating lipid droplets. Caetano et al. ( 2002 ) showed mitochondria storage lipids in post-pharyngeal gland cells of Dinoponera australis (Formicidae), describing these structures as mitochondria derivatives. A. mellifera and S. postica did not present any intermediary structures like autophagic vacuoles or residual bodies, but it is obvious that mitochondria undergo changes and accumulate lipids.

Changes in S. postica worker cephalic salivary glands are more marked than in A. mellifera : the alveoli lumen became wide and cells changed from cubic to fl at due to pressure from secretion accumulation in the lumen. Secretion starts to accumulate in the cell cytoplasm of very young bees quickly followed by morphological evidence of secretion delivery into the lumen. Secretion consists of vesicles with homogeneous membranous content, similar to A. mellifera and typically lipid in nature. Th is secre-tion is only produced in young workers, and from the nurse phase onwards, the gland just stores secretion in the alveoli lumen while in A. mellifera secretion production continues throughout worker life. S. postica laying queen gland cells present large vacuoles containing electron-dense material apparently resulting from the fusion of small droplets.

Th e chemical composition of cephalic salivary glands secretion has been studied in A. mellifera (Arnold and Delage-Darchen, 1978 ), Apotrigona nebulata (Delage-Darchen et al., 1979), Melipona beecheii (Delage-Darchen and Darchen, 1982) and Scaptotrigona mexicana (Delage-Darchen et al., 1982 ) however no function was suggested for it. In Chalicodoma sicola bees, secretion from this gland consists of hydrocarbons that are used to make brood cells impermeable (Kronenberge and Hefetz, 1984). In Bombus (Rhodobombus) mesomelas the gland is smaller and the small amount of secretion cor-responds to cuticular hydrocarbons (Terzo et al., 2005 ). According to Arnold et al. ( 1996 ) the same hydrocarbons found in A. mellifera worker salivary glands are found in the body cuticle.

If cephalic salivary gland secretion is eventually spread over the body with the legs or passed to nestmates by trophalaxis, it may serve as individual recognition and colony integration. In Bombus cryptarum and Bombus magnus the gland secretes hydrocarbons which are species specifi c acting as recognition signals between species (Bertsch et al., 2005 ); a similar function might also be true of A. mellifera and S. postica secretion.


It might also be used to aid tasks such as material collection by foragers, as this kind of individual has more stored secretion. Th is type of function was attributed to salivary gland secretion in Plebeia emerina by Blochtein ( 2006 ), specifi cally in propolis manip-ulation in the colony by middle aged workers. Another function attributed to this secretion is as trail making pheromones as seen in Trigona recursa and Trigona spinipes by Jarau et al. ( 2004 ) and Schorkopf et al. ( 2007 ), respectively.

A. mellifera queens maintain their reproductive hegemony in the colony through the pheromone produced by mandibular glands action. It is possible that cephalic salivary gland secretion has a complementary role. Velthuis and Van-Es ( 1964 ) observed that even in the absence of a mandibular gland, the A. mellifera queen remains attractive to workers suggesting sources other than mandibular glands for the queen secretions. In S. postica queen secretion from the mandibular gland does not have the same role as in A. mellifera and therefore cephalic salivary secretion might have a recognition role in its place.

Th e conclusion drawn from the present observations is that the morphology of cephalic salivary gland secretory cells in both studied species is compatible with secre-tion of a lipid nature and that secretion quantities stored are higher in older workers and queens, suggesting that there is more need for it in worker foraging activities and laying queens. Th e possibility that it serves as nestmate and laying queen recognition for workers is still an open question.

Acknowledgements

Th e authors acknowledge fi nancial support from FAPESP, CAPES, CNPq and the anonymous referees that contributed to the article improvement.

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Cephalic salivary gland ultrastructure of worker and queen eusocial bees (Hymenoptera, Apidae)

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