Synthesis and secretion of beeswax in honeybees Hr Hepburn, Rtf Bernard, Bc Davidson, Wj Muller, P Lloyd, Sp Kurstjens, Sl Vincent To cite this version: Hr Hepburn, Rtf Bernard, Bc Davidson, Wj Muller, P Lloyd, et al.. Synthesis and secretion of beeswax in honeybees. Apidologie, Springer Verlag, 1991, 22 (1), pp.21-36. hal-00890889 HAL Id: hal-00890889 https://hal.archives-ouvertes.fr/hal-00890889 Submitted on 1 Jan 1991 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. Original article Synthesis and secretion of beeswax in honeybees HR Hepburn RTF Bernard BC Davidson WJ Muller P Lloyd SP Kurstjens SL Vincent 1 Rhodes University, Department of Zoology and Entomology, Grahamstown 6140; 2 University of the Witwatersrand, Department of Medical Biochemistry; 3 University of the Witwatersrand, Department of Physiology, Johannesburg, 2193, South Africa (Received 1 July 1990; accepted 15 November 1990) Summary — The ultrastructure of the cells of the wax gland complex in honeybee workers was studied in relation to the synthesis and secretion of beeswax. The hydrocarbon and fatty acid pro- files of epidermal cells and oenocytes were determined in relation to the ages of the bees. Smooth endoplasmic reticulum (SER) is absent from both epidermis and adipocytes from adult emergence until the end of wax secretion. The oenocytes are rich in SER. The hydrocarbon and fatty acid con- tent of the oenocytes, averaged for age, closely matches that of newly secreted wax. That the oe- nocytes are the probable source of the hydrocarbon fraction of beeswax is consistent with histo- chemical and autoradiographic data for honeybees and with biosynthetic data from other insects. The cyclical changes of organelles and chemical composition of the wax gland complex closely coin- cide with measured, age-related rates of wax secretion in honeybee workers. wax secretion / wax synthesis / wax gland / ultrastructure / chemical composition INTRODUCTION Beeswax is a complex mixture (Tul- loch, 1980) produced by tissues in the ab- domen of the bee (Dumas and Edwards, tasks within the division of Many labour of 1843; Piek, 1961). The work of Piek worker honeybees are closely associated (1961, 1964) showed that acetic acid is with of activi- age-related cycles glandular very probably taken up by the oenocytes ty (Ribbands, 1953; Winston, 1987). His- and that acetate is used for the synthesis studies of wax secretion tological suggest of hydrocarbons. This interpretation was that production begins when the worker is strengthened by further studies of micro- slightly less than 1 wk old, peaks at = 2 wk somal preparations from the worker abdo- and then wanes (Rösch, 1927; Freuden- men (Blomquist and Ries, 1979; Lambre- stein, 1960; Boehm, 1965; Hepburn et al, mont and Wykle, 1979). Although the 1984). Of the tissues within the wax gland oenocytes may play a major role in the complex, the epidermis and the oenocytes synthesis of wax, the synthetic capacity of in particular have been implicated as cen- individual cell types within the wax gland tral to the synthesis and secretion of wax complex and their possible contribution to (Rösch, 1927; Reimann, 1952; Boehm, wax production have remained unex- 1965). plored. * Correspondence and reprints Searches for the means by which wax eters were converted to volumes, assuming that synthesized within the abdomen actually the cells were spherical. reaches the surface of the animal suggest that it passes through the pore canal sys- tem of the cuticle (Locke, 1961; Locke and Electron microscopy Huie, 1980). Yet the means by which the precursors are transported from as yet un- Bees were anaesthetized on ice, and fixative established points of origin has remained (2.5% glutaraldehyde in 0.2 M cacodylate buffer elusive. We conducted studies of wax syn- (pH 7.1)) injected into the haemocoel (Locke and After 10 the fat and thesis and secretion in honeybees to spe- Huie, 1980). min, body cifically identify sites for the of hy- overlying epidermis were excised and further origin fixed by immersion in the above fixative for 4 h drocarbon and acids within the wax fatty at room temperature. After primary fixation the gland complex and to establish the neces- tissues were washed in the buffer, secondarily sary ultrastructural correlates of this activi- fixed in 1.0% buffered osmium tetroxide, dehy- ty and of their transport. Equally important, drated and embedded in a Taab/Araldite resin we measured the actual rates of wax se- mixture. Ultrathin sections (silver/gold) were stained with acetate (Watson, 1958) and cretion in bees of different ages to assess uranyl lead citrate and examined us- how well chemical of the tis- (Reynolds, 1963) composition ing a Jeol JEMXII transmission electron micro- sues and of ultrastructural cycles change scope. with the of wax corresponded cycles pro- Volume densities (volume of the component duction within this stage of the division of related to the volume of the containing cell) of labour. the wax gland organelles were calculated using the point count method (Weibel and Bolender, 1973). A grid of 99 squares, each 9 mm2, was MATERIALS AND METHODS drawn on the screen of the electron microscope onto which the image (29 000X) of the tissue was superimposed. At least 10 oenocytes and 10 adipocytes from 3 wax glands per age group Animals were used for the point count analysis. Newly emerged adult bees (Apis mellifera ca- pensis) were marked by painting the thorax, Chemical analyses placed in hives, and subsequently sampled at = 72-h over a use in intervals 25-d period for sub- Wax gland cells used for fatty acid and hydro- and chemical studies. sequent microscopical carbon analysis were harvested as follows. Live bees were flash-frozen in liquid nitrogen and stored at -70 °C until processed. Tissue was ob- Light microscopy tained after thawing by dissection of the abdo- men in a phosphate buffered saline solution. Deep freezing followed by saline rehydration at Bees were anaesthetized on ice and the fat room temperature caused only the adipocytes to body dissected out under saline (Miller and burst. The remaining intact oenocytes in the for- James, 1976). Fat bodies were stained for 2 mer fat body layer were collected with a micropi- min in 0.2% methylene blue and viewed on a pette and the purity of the cell type (exclusion of cavity slide with a light microscope. The vol- adipocytes) confirmed by microscopy. After re- umes of adipocytes and oenocytes were calcu- moval of the inner tissues, only those epidermal lated from light microscope preparations. The cells underlaying wax mirrors were scraped free diameters of 10 adipocytes and 10 oenocytes and similarly harvested. Epidermal cells and oe- from each of 5 bees from each age group were nocytes were separately spun at 200 g for 15 measured with an ocular micrometer. The diam- min to obtain pellets. The cell pellets were extracted with chloro- day the volume density of these tightly form:methanol (2:1 v/v), the extracts washed packed tubules is high (table I). Similarly, with saline and reduced under vacuum (0.9%) there is a large increase in whole oenocyte Half of each was (Floch et al, 1957). sample volume as noted by used for and the remain- (table I) previously hydrocarbon analysis Boehm The relative volumes of the der used to prepare fatty acid esters (FAME) us- (1965). ing the method of Moscatelli (1972). The FAME oenocytes and the SER remain elevated were separated using a 10% SP2330 6 m x throughout the secretory phase (table I, fig 3 mm ID column in a Varian 3400 GC and were 3). By d 18, both oenocytes and SER be- quantitated using a Varian 4270 integrator. The gin to decrease (table I) with the simultane- were 2 m x 0.2 hydrocarbons separated using a ous appearance of primary lysosomes and mm ID QVI column with the same GC/integrator autolytic vacuoles. Lipid and protein drop- system. Samples of scale and comb wax were lets were never observed in the extracted in parallel with the cell samples and oenocytes and other cellular showed no processed in the same way. organelles evident cyclical changes associated with wax synthesis (table I). Wax secretion The adipocytes are characterized by an extensive plasma membrane reticular sys- tem, numerous mitochondria, peroxisomes Wax secretion was measured in worker bees and RER, and a few small bodies from queenright colonies of ≈ 10 000 bees hived Golgi (fig in 5-framed nuclei, 3 frames of which contained 4). SER is notably absent from adult emer- brood and food stores. The bees were heavily gence through foraging. Massive lipid fed sugar syrup (25-50 g sucrose/l water) ad lib- droplets occupy ≈ 60% of cell’s cytoplasm itum to stimulate comb building. Newly emerged in the young worker but decrease substan- at the bees were marked for age and introduced tially over the next few days (table II). Like rate of 150 bees/hive every 3 d into each of 7 colonies. On d 21 the colonies contained a spec- trum of age groups that were 3, 6, 9, 12, 15, 18 and 21 d old. All of these bees were then har- vested and their wax scales removed and weighed on a Cahn C-32 microbalance.