Synthesis and secretion of beeswax in honeybees Hr Hepburn, Rtf Bernard, Bc Davidson, Wj Muller, P Lloyd, Sp Kurstjens, Sl Vincent

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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￿

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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. Such measurements were made over 2 yr and very large samples of each age group were eventual- ly obtained as follows. Bee group age is fol- lowed by sample size for that age in parenthe- ses : 3 (1360), 6 (1543), 9 (1635), 12 (2003), 15 (1973), 18 (1552), and 21 (1230).

RESULTS

Ultrastructure

The epidermis of the wax mirror is under- lain by a fat body layer, comprising oeno- cytes and adipocytes (figs 1, 2). In the oe- nocytes of the newly emerged worker bee SER is barely discernible, but by the fourth the oenocytes, the adipocytes also in- stores are notably large and the plasma crease in volume prior to wax synthesis membrane reticular system is well devel- (table II). During wax synthesis, glycogen oped. As synthesis wanes, lipid droplets in- (fig 5). There are many hemidesmosomes between each adipocyte and its basal lami- na (fig 5). In places where adjacent adipo- cytes are < 50 nm apart, they are joined by desmosomes and gap junctions (fig 5 in- sert). The basal laminae of neighbouring oenocytes are separated by a gap of ≈ 150 nm, and like the adipocytes, are attached to their basal laminae by hemidesmo- somes. No junctions were seen to link any 2 oenocytes. Similarly, where oenocyte abuts adipocyte, only hemidesmosomes are present (figs 6, 7). The gap between adipocyte and oenocyte is = 210 nm. Al- though the cells of the fat body become closely applied to the basal lamina of the epidermis, particularly during the period of synthesis and secretion (cf Hepburn, 1986), the epidermal cells and cells of the fat body are not bound together by any form of junction but remain separated by their basal laminae. crease in size while the other organelles either remain unchanged or show small decreases in size (table II). Hydrocarbons and fatty acids Within the fat body adjacent adipocytes are separated by a gap of = 250 nm which The hydrocarbons and fatty acids of the is filled with material of the basal lamina epidermis and oenocytes were analyzed in bees of the same ages as those used in dominated by the C25 and C27 groups, and the ultrastructural studies. There was an a decrease in the C33 fraction of the oe- increase in the saturated hydrocarbons nocytes in relation to age (table III). The (table IV). Notable differences include an increase in the C29 pool while C25 and C27 remained about the same. Among the un- saturated groups there was a marked re- duction in 35:1 in the epidermis in relation to age (table IV). The fatty acid profiles of the oenocytes and epidermal cells in relation to age are given in tables V and VI respectively. While the total pool of saturated fatty acids in the oenocytes remained much the same there were notable increases in 12:0 and decreases in 16:0 and 24:0 in relation to age (table V). Only minor changes oc- curred in the unsaturated fatty acid pool. The epidermal cells showed even fewer changes in fatty acid composition in rela- tionship to age (table VI). Values for scale wax are based on sam- ples of scales that were harvested over several years in other age-related experi- ments. Thus, these values represent the already averaged content of thousands of individual scales taken from as many bees between 3 and 21 d of age. (An internal control run established no differences be- tween samples of scale wax that were freshly secreted or were 2 yr old.) Because of the necessity to pool wax scale sam- ples, the data of tables III to VI are re- expressed as total averages for direct comparison with scale wax in tables VII and VIII. The former provide insight into the metabolic activities of the wax gland tissues on an age-related basis, the latter allow comparisons of average product con- tent. In the case of hydrocarbons, the aver- age content of scale wax shows a 50% re- duction in the saturated C25 group com- pared with the 2 wax gland tissues (table VII). Also, the C33 hydrocarbons of the oe- saturated groups increased at the expense nocytes are far less than those of either of the unsaturated groups, particularly in epidermis or scale wax. The other hydro- the case of 33:1. The trends for the epider- carbons are much the same for tissue and mal cells are similar, but on a smaller scale scale wax (table VII). In the case of the fat- ty acids, there are large differences be- Wax secretion tween the short chain groups (C12 and C14) and the long ones (C24-C28) for the The amounts of wax borne on average by tissues and the scale wax. There are also worker bees of different ages are shown in notable differences between tissue and figure 8. Paired comparisons of different scale wax among the unsaturated fatty ac- age groups showed that not all age groups ids (table VIII). differed significantly. It is nonetheless (Hepburn and Muller, 1988). Moreover, on harvest, one cannot tell whether an individ- ual honeybee with no or only little wax on it is in this state because it either did not se- crete any wax or had recently removed its scales and added them to the comb- building in progress. Consequently, one cannot relate any specific amount of wax back to a defined zero time. However, the general trend of the data is highly signifi- cant and fully supported by the one-way analysis of variance. Thus, the amount of wax borne per bee is significantly (P < 0.05) affected by the age class of the bee.

DISCUSSION worth commenting on the magnitude of the standard deviation. It requires between 24 and 48 h for any particular honeybee work- When the adult worker bee emerges from er to produce a moderate-sized wax scale its cell the cuticle of the wax mirror is = 3 μm thick, and unlike other regions of the burn, 1986) and these details have been exoskeleton (Menzel et al, 1969), remains reconfirmed in this study. The epidermis this size. Its basic ultrastructure has al- lacks both dermal glands (Schnelle, 1923) ready been described (Locke, 1961; Hep- and, more importantly, smooth endoplas- mic reticulum (SER). The latter is absent Hepburn and Muller, 1988), and which is during peak wax secretion (Sanford and considered to be indispensable for lipogen- Dietz, 1976) and, in fact, from the entire esis. On the other hand, the adipocyte is post-ecdysial life of the worker bee. The the primary site of intermediary metabolism idea that the epidermis has no role in the in insects (Downer, 1985; Keeley, 1985), actual synthesis of beeswax is not new and the large quantities of lipid, protein (Holz, 1878). Indeed, it is now a general and glycogen in the adipocytes associated principle that SER is essential to lipid bio- with the wax gland support this generaliza- synthesis in insects (Dean et al, 1985; tion. The early mobilization of lipid from the Keeley, 1985; Sedlak, 1985) and other ani- adipocyte (table II) suggests that it might mals (Alberts et al, 1983; Hall, 1984). The produce beeswax precursors. However, major role of the epidermis in the produc- the absence of communicating junctions tion of wax appears to be the development between adipocytes and oenocytes, and of an elaborate of small system transport the fact that adipocyte lipid reserves are tubules (Reimann, 1952; Locke, 1961; depleted prior to both the maximal devel- Hepburn, 1986). opment of the oenocyte SER and wax pro- The most notable and dynamic feature duction mitigate against this possibility. of the oenocyte is the abundant SER Likewise, at maximal wax production the whose rise and fall are synchronized with lipid content of the adipocyte is more or measured periods of secretion (figure 8; less constant. Finally, paraffins are synthe- sized by oenocytes and triglycerides by The general trends in the hydrocarbon the adipocytes of locusts (Diehl, 1973), profiles of the oenocytes include an age- and in beetles, lipid oxidation proceeds in related increase in the saturated compo- oenocytes after they have taken up lipid nents (table III) reflecting considerable syn- droplets through the PMRS from adipocy- thetic activity. By comparison, the epider- tes (Romer et al, 1974). Collectively these mal hydrocarbons show more modest observations do not support an adipocyte changes in relation to the ages of the bees origin for beeswax lipids. (table IV); these are pronounced among the more minor the unsaturated Although the fine structure of the wax groups, We believe that the mirror cuticle and its wax tubules compounds. hydrocar- transport bons of the reflect have now been visualized epidermis oenocyte- (cf Hepburn, derived material in transit because the 1986) there remains the problem of the epi- dermis lacks SER and its age-related physical transport of beeswax precursors, changes in hydrocarbons are not syn- a precise answer to which eludes us. How- chronized with the cycle of secretion. ever, Kurstjens et al (1990) recently report- There is an apparent discrepancy between ed a partial characterisation of the proteins the and low content of the oe- of beeswax scales and comb in which high C25 C35 nocytes vis-à-vis wax scales; but it could some 17 fractions were separated. Two of be that is formed from and these fractions have been implicated in C33 C25 C27 outside of the oenocytes. wax precursor transport on the grounds that their molecular weight distributions The fatty acid profiles of the epidermal closely approximate those of known hon- cells lacked evident patterns of change consistent with the of the bees or eybee apolipophorins. Thus it was sug- ageing gested that hydrocarbons and fatty acid with the cycle of wax synthesis and secre- tion VI - precursors of beeswax might be synthe- (table excepting C18). By contrast, differences the acid sized in the oenocytes (now strongly sup- large among fatty ported in the present work) and then trans- pools of the oenocytes relate both to the ported through the haemolymph to the ages of the bees and to the cycle of syn- surface of the insect in the form of primary thesis (table V) and secretion (fig 8). Like- the for ac- or modified apolipophorins (Kurstjens et al, wise, ’average’ composition fatty 1990). ids in oenocytes more closely mirrors scale wax composition than does that of the epi- Before comparing the chemical content dermis (table VIII). The presence of and in- of the wax gland tissues with that of scale crease of C12 in the oenocytes coupled to wax, it is important to note that the hydro- its absence from epidermis and scale wax carbon and acid content of the A m fatty (tables V, VI and VIII) further suggests that capensis comb wax (tables VII and VIII) the oenocytes perform chain elongation re- are virtually identical to those of its sister actions. The decrease in C16 and C24 over race, A m scutellata Tulloch reported by time in the is also consistent (1980); results which lend confidence to oenocytes with synthesis and subsequent export. the analysis presented here. The nature and origins of the large differences be- tween scale and comb wax VII and (tables CONCLUSIONS VIII) are post-secretory phenomena and have been treated in detail elsewhere (Kurstjens et al, 1985; Davidson and Hep- Both epidermal cells and adipocytes lack burn, 1986; Hepburn and Kurstjens, 1988). SER, an organelle essential to lipogenesis in insects, from emergence of the adult, pocytes semblent servir de source d’éner- through the cycle of wax synthesis and se- gie pour la synthèse et la secrétion. cretion and on to behavior. The foraging Les fractions hydrocarbure et acide gras oenocytes are the only cells of the wax des adipocytes et des oenocytes ont été with SER and whose devel- gland complex analysées en fonction de l’âge des opmental fate closely matches periods of abeilles. La composition et le rapport des 2 wax I and Unlike synthesis (tables II; fig 8). groupes de composés suivent étroitement those of the epidermis, the hydrocarbon ceux de la cire fraîchement secrétée. Il and acid of isolated fatty profiles oenocy- semble que les oenocytes soient le siège tes share much in common with newly syn- primaire de la synthèse des hydrocarbures thesized scale wax. That the oenocytes et des acides gras de la cire et que ces are the probable source of beeswax hydro- précurseurs de la cire soient transportés carbons is the close supported by cyclical par des lipophorines à travers l’épiderme changes in ultrastructure that coincide with jusqu’à la surface de la cuticule des miroirs of secretion of beeswax age-related cycles à cire. Les grandes différences de compo- by worker honeybees. These interpreta- sition chimique entre la cire fraîchement tions are consistent with the histochemical secrétée et la cire des rayons sont des data of Reimann (1952), the autoradio- phénomènes postérieurs associés à la graphic studies of Piek (1964) and studies malaxation de la cire pendant la construc- of hydrocarbon synthesis in other insects tion. by Diehl (1973, 1975). On a déterminé la quantité de cire pro- duite par les abeilles en fonction de leur et les résultats montrent est Résumé — et sécrétion de la âge qu’elle y Synthèse liée. La courbe caracté- cire chez l’abeille (Apis mellifera L). significativement ristique de la sécrétion cirière suit étroite- L’étude a porté sur : ment celle des changements de l’ultra- - les modifications ultrastructurales des or- structure et de l’histologie des glandes ci- ganites des glandes cirières; rières. Parmi tous les tissus des glandes - les modifications des spectres d’hydro- cirières de l’abeille, seuls les oenocytes carbures et d’acides gras des cellules des présentent les caractères nécessaires, glandes cirières et de la cire et dans leur ultrastructure et leurs modifica-

- la secrétion réelle de la cire en fonction tions temporelles, pour être le siège de la de l’âge des ouvrières. secrétion des hydrocarbures et des acides gras de la cire. Il n’existe pas de reticulum endoplasmi- essentiel à la que lisse, organite synthèse cire / secrétion ci- des dans ni dans les / synthèse / glande lipides, l’épiderme, rière / ultrastructure chi- des cirières chez les / composition adipocytes glandes mique ouvrières adultes. Néanmoins, les oeno- cytes sont extrêmement riches en reticu- lum endoplasmique lisse et le développe- Zusammenfassung — Synthese und ment dans le temps de cet organite suit Abscheidung von Wachs bei den Honig- étroitement celui de la sécrétion cirière. La bienen. Es wurden die Veränderungen der fonction primaire de l’épiderme dans la sé- Ultrastruktur der Organellen in den wachs- crétion cirière est l’élaboration des canali- absondernden Zellen, Veränderungen in cules pour le transport de la cire. Les adi- den Kohlenwasserstoff- und Fettsäure- Profilen der Wachsdrüsenzellen, des struktur-Veränderungen des Wachs- Wachses und des umittelbar abgesonder- drüsenkomplexes überein. Von allen ten Wachses in Bezug auf das Alter der Gewebebestandteilen des Wachsdrüsen- Honigbienen bestimmt. Ein endoplasmati- komplexes der Arbeitsbienen zeigen nur sches Retikulum, die für die Lipidsynthese die Oenozyten die Ausstattung in ihrer entscheidende Organelle, kommt weder in Ultrastruktur und ihren zeitlichen der Epidermis noch in den Adipozyten des Veränderungen die nötigen Voraussetzun- Wachsdrüsenkomplexes der adulten Ar- gen als Sekretionsquelle für die Kohlen- beitsbiene vor. Es sind jedoch die Oenozy- wasserstoff- und Fettsäureanteile des Bie- ten extrem reich an endoplasmatischem nenwachses. Retikulum und die Entwicklung dieser Or- ganelle über die Zeit stimmt sehr gut mit Wachsabscheidung / Wachssynthese / der Wachsabscheidung überein. 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