HAL Id: hal-00890889https://hal.archives-ouvertes.fr/hal-00890889

Submitted on 1 Jan 1991

HAL is a multi-disciplinary open accessarchive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come fromteaching and research institutions in France orabroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, estdestinée au dépôt et à la diffusion de documentsscientifiques de niveau recherche, publiés ou non,émanant des établissements d’enseignement et derecherche français ou étrangers, des laboratoirespublics ou privés.

Synthesis and secretion of beeswax in honeybeesHr 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 beeswaxin honeybees. Apidologie, Springer Verlag, 1991, 22 (1), pp.21-36. �hal-00890889�

Original article

Synthesis and secretion of beeswax in honeybees

HR Hepburn RTF Bernard BC Davidson WJ MullerP 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 wasstudied 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. Smoothendoplasmic reticulum (SER) is absent from both epidermis and adipocytes from adult emergenceuntil 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

Many tasks within the division of labour ofworker honeybees are closely associatedwith age-related cycles of glandular activi-ty (Ribbands, 1953; Winston, 1987). His-tological studies of wax secretion suggestthat production begins when the worker isslightly less than 1 wk old, peaks at = 2 wkand then wanes (Rösch, 1927; Freuden-stein, 1960; Boehm, 1965; Hepburn et al,1984). Of the tissues within the wax glandcomplex, the epidermis and the oenocytesin particular have been implicated as cen-tral to the synthesis and secretion of wax(Rösch, 1927; Reimann, 1952; Boehm,1965).

Beeswax is a complex mixture (Tul-loch, 1980) produced by tissues in the ab-domen of the bee (Dumas and Edwards,1843; Piek, 1961). The work of Piek

(1961, 1964) showed that acetic acid is

very probably taken up by the oenocytesand that acetate is used for the synthesisof hydrocarbons. This interpretation wasstrengthened by further studies of micro-somal preparations from the worker abdo-men (Blomquist and Ries, 1979; Lambre-mont and Wykle, 1979). Although theoenocytes may play a major role in the

synthesis of wax, the synthetic capacity ofindividual cell types within the wax glandcomplex and their possible contribution towax production have remained unex-

plored.

*

Correspondence and reprints

Searches for the means by which waxsynthesized within the abdomen actuallyreaches the surface of the animal suggestthat it passes through the pore canal sys-tem of the cuticle (Locke, 1961; Locke andHuie, 1980). Yet the means by which theprecursors are transported from as yet un-established points of origin has remainedelusive. We conducted studies of wax syn-thesis and secretion in honeybees to spe-cifically identify sites for the origin of hy-drocarbon and fatty acids within the waxgland complex and to establish the neces-sary ultrastructural correlates of this activi-

ty and of their transport. Equally important,we measured the actual rates of wax se-cretion in bees of different ages to assesshow well chemical composition of the tis-sues and cycles of ultrastructural changecorresponded with the cycles of wax pro-duction within this stage of the division oflabour.

MATERIALS AND METHODS

Animals

Newly emerged adult bees (Apis mellifera ca-pensis) were marked by painting the thorax,placed in hives, and subsequently sampled at =72-h intervals over a 25-d period for use in sub-sequent microscopical and chemical studies.

Light microscopy

Bees were anaesthetized on ice and the fatbody dissected out under saline (Miller andJames, 1976). Fat bodies were stained for 2min in 0.2% methylene blue and viewed on acavity slide with a light microscope. The vol-umes of adipocytes and oenocytes were calcu-lated from light microscope preparations. Thediameters of 10 adipocytes and 10 oenocytesfrom each of 5 bees from each age group weremeasured with an ocular micrometer. The diam-

eters were converted to volumes, assuming thatthe cells were spherical.

Electron microscopy

Bees were anaesthetized on ice, and fixative(2.5% glutaraldehyde in 0.2 M cacodylate buffer(pH 7.1)) injected into the haemocoel (Lockeand Huie, 1980). After 10 min, the fat body andoverlying epidermis were excised and furtherfixed by immersion in the above fixative for 4 hat room temperature. After primary fixation thetissues were washed in the buffer, secondarilyfixed in 1.0% buffered osmium tetroxide, dehy-drated and embedded in a Taab/Araldite resinmixture. Ultrathin sections (silver/gold) werestained with uranyl acetate (Watson, 1958) andlead citrate (Reynolds, 1963) and examined us-ing a Jeol JEMXII transmission electron micro-scope.

Volume densities (volume of the componentrelated to the volume of the containing cell) ofthe wax gland organelles were calculated usingthe point count method (Weibel and Bolender,1973). A grid of 99 squares, each 9 mm2, wasdrawn on the screen of the electron microscopeonto which the image (29 000X) of the tissuewas superimposed. At least 10 oenocytes and10 adipocytes from 3 wax glands per age groupwere used for the point count analysis.

Chemical analyses

Wax gland cells used for fatty acid and hydro-carbon analysis were harvested as follows. Livebees were flash-frozen in liquid nitrogen andstored at -70 °C until processed. Tissue was ob-tained after thawing by dissection of the abdo-men in a phosphate buffered saline solution.Deep freezing followed by saline rehydration atroom temperature caused only the adipocytes toburst. The remaining intact oenocytes in the for-mer fat body layer were collected with a micropi-pette and the purity of the cell type (exclusion ofadipocytes) confirmed by microscopy. After re-moval of the inner tissues, only those epidermalcells underlaying wax mirrors were scraped freeand similarly harvested. Epidermal cells and oe-nocytes were separately spun at 200 g for 15min to obtain pellets.

The cell pellets were extracted with chloro-form:methanol (2:1 v/v), the extracts washedwith saline (0.9%) and reduced under vacuum(Floch et al, 1957). Half of each sample wasused for hydrocarbon analysis and the remain-der used to prepare fatty acid esters (FAME) us-ing the method of Moscatelli (1972). The FAMEwere separated using a 10% SP2330 6 m x3 mm ID column in a Varian 3400 GC and were

quantitated using a Varian 4270 integrator. Thehydrocarbons were separated using a 2 m x 0.2mm ID QVI column with the same GC/integratorsystem. Samples of scale and comb wax wereextracted in parallel with the cell samples andprocessed in the same way.

Wax secretion

Wax secretion was measured in worker bees

from queenright colonies of ≈ 10 000 bees hivedin 5-framed nuclei, 3 frames of which containedbrood and food stores. The bees were heavilyfed sugar syrup (25-50 g sucrose/l water) ad lib-itum to stimulate comb building. Newly emergedbees were marked for age and introduced at therate 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. Suchmeasurements were made over 2 yr and verylarge 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 beeSER is barely discernible, but by the fourth

day the volume density of these tightlypacked tubules is high (table I). Similarly,there is a large increase in whole oenocytevolume (table I) as previously noted byBoehm (1965). The relative volumes of theoenocytes and the SER remain elevated

throughout the secretory phase (table I, fig3). By d 18, both oenocytes and SER be-gin to decrease (table I) with the simultane-ous appearance of primary lysosomes andautolytic vacuoles. Lipid and protein drop-lets were never observed in the oenocytesand other cellular organelles showed noevident cyclical changes associated withwax synthesis (table I).

The adipocytes are characterized by anextensive plasma membrane reticular sys-tem, numerous mitochondria, peroxisomesand RER, and a few small Golgi bodies (fig4). SER is notably absent from adult emer-gence through foraging. Massive lipiddroplets occupy ≈ 60% of cell’s cytoplasmin the young worker but decrease substan-

tially over the next few days (table II). Like

the oenocytes, the adipocytes also in-crease in volume prior to wax synthesis(table II). During wax synthesis, glycogen

stores are notably large and the plasmamembrane reticular system is well devel-

oped. As synthesis wanes, lipid droplets in-

crease in size while the other organelleseither remain unchanged or show smalldecreases in size (table II).

Within the fat body adjacent adipocytesare separated by a gap of = 250 nm whichis filled with material of the basal lamina

(fig 5). There are many hemidesmosomesbetween each adipocyte and its basal lami-na (fig 5). In places where adjacent adipo-cytes are < 50 nm apart, they are joined bydesmosomes and gap junctions (fig 5 in-sert). The basal laminae of neighbouringoenocytes are separated by a gap of &ap; 150nm, and like the adipocytes, are attachedto their basal laminae by hemidesmo-somes. No junctions were seen to link any2 oenocytes. Similarly, where oenocyteabuts adipocyte, only hemidesmosomesare present (figs 6, 7). The gap betweenadipocyte and oenocyte is = 210 nm. Al-though the cells of the fat body becomeclosely applied to the basal lamina of theepidermis, particularly during the period ofsynthesis and secretion (cf Hepburn,1986), the epidermal cells and cells of thefat body are not bound together by anyform of junction but remain separated bytheir basal laminae.

Hydrocarbons and fatty acids

The hydrocarbons and fatty acids of theepidermis and oenocytes were analyzed in

bees of the same ages as those used inthe ultrastructural studies. There was anincrease in the saturated hydrocarbons

dominated by the C25 and C27 groups, anda decrease in the C33 fraction of the oe-

nocytes in relation to age (table III). The

saturated groups increased at the expenseof the unsaturated groups, particularly in

the case of 33:1. The trends for the epider-mal cells are similar, but on a smaller scale

(table IV). Notable differences include anincrease in the C29 pool while C25 and C27remained about the same. Among the un-saturated groups there was a marked re-duction in 35:1 in the epidermis in relationto age (table IV).

The fatty acid profiles of the oenocytesand epidermal cells in relation to age aregiven in tables V and VI respectively.While the total pool of saturated fatty acidsin the oenocytes remained much the samethere were notable increases in 12:0 anddecreases 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 fewerchanges 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 overseveral years in other age-related experi-ments. Thus, these values represent thealready averaged content of thousands ofindividual scales taken from as many beesbetween 3 and 21 d of age. (An internalcontrol run established no differences be-tween samples of scale wax that werefreshly secreted or were 2 yr old.) Becauseof 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 VIIand VIII. The former provide insight intothe metabolic activities of the wax glandtissues on an age-related basis, the latterallow 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 (tableVII). Also, the C33 hydrocarbons of the oe-nocytes are far less than those of either

epidermis or scale wax. The other hydro-carbons are much the same for tissue andscale wax (table VII). In the case of the fat-

ty acids, there are large differences be-tween the short chain groups (C12 andC14) and the long ones (C24-C28) for thetissues and the scale wax. There are also

notable differences between tissue andscale wax among the unsaturated fatty ac-ids (table VIII).

Wax secretion

The amounts of wax borne on average byworker bees of different ages are shown in

figure 8. Paired comparisons of differentage groups showed that not all age groupsdiffered significantly. It is nonetheless

worth commenting on the magnitude of thestandard deviation. It requires between 24and 48 h for any particular honeybee work-er to produce a moderate-sized wax scale

(Hepburn and Muller, 1988). Moreover, onharvest, one cannot tell whether an individ-ual honeybee with no or only little wax on itis in this state because it either did not se-crete any wax or had recently removed itsscales and added them to the comb-

building in progress. Consequently, onecannot relate any specific amount of waxback to a defined zero time. However, thegeneral trend of the data is highly signifi-cant and fully supported by the one-wayanalysis of variance. Thus, the amount ofwax borne per bee is significantly (P <

0.05) affected by the age class of the bee.

DISCUSSION

When the adult worker bee emerges fromits cell the cuticle of the wax mirror is = 3

&mu;m thick, and unlike other regions of theexoskeleton (Menzel et al, 1969), remainsthis size. Its basic ultrastructure has al-

ready been described (Locke, 1961; Hep-

burn, 1986) and these details have beenreconfirmed in this study. The epidermislacks both dermal glands (Schnelle, 1923)and, more importantly, smooth endoplas-

mic reticulum (SER). The latter is absentduring peak wax secretion (Sanford andDietz, 1976) and, in fact, from the entirepost-ecdysial life of the worker bee. Theidea that the epidermis has no role in theactual synthesis of beeswax is not new

(Holz, 1878). Indeed, it is now a generalprinciple that SER is essential to lipid bio-synthesis in insects (Dean et al, 1985;Keeley, 1985; Sedlak, 1985) and other ani-mals (Alberts et al, 1983; Hall, 1984). Themajor role of the epidermis in the produc-tion of wax appears to be the developmentof an elaborate system of small transporttubules (Reimann, 1952; Locke, 1961;Hepburn, 1986).

The most notable and dynamic featureof the oenocyte is the abundant SERwhose rise and fall are synchronized withmeasured periods of secretion (figure 8;

Hepburn and Muller, 1988), and which isconsidered to be indispensable for lipogen-esis. On the other hand, the adipocyte isthe primary site of intermediary metabolismin insects (Downer, 1985; Keeley, 1985),and the large quantities of lipid, proteinand glycogen in the adipocytes associatedwith the wax gland support this generaliza-tion. The early mobilization of lipid from theadipocyte (table II) suggests that it mightproduce beeswax precursors. However,the absence of communicating junctionsbetween adipocytes and oenocytes, andthe fact that adipocyte lipid reserves aredepleted prior to both the maximal devel-opment of the oenocyte SER and wax pro-duction mitigate against this possibility.Likewise, at maximal wax production thelipid content of the adipocyte is more orless constant. Finally, paraffins are synthe-

sized by oenocytes and triglycerides bythe adipocytes of locusts (Diehl, 1973),and in beetles, lipid oxidation proceeds inoenocytes after they have taken up lipiddroplets through the PMRS from adipocy-tes (Romer et al, 1974). Collectively theseobservations do not support an adipocyteorigin for beeswax lipids.

Although the fine structure of the waxmirror cuticle and its wax transport tubuleshave now been visualized (cf Hepburn,1986) there remains the problem of thephysical transport of beeswax precursors,a precise answer to which eludes us. How-ever, Kurstjens et al (1990) recently report-ed a partial characterisation of the proteinsof beeswax scales and comb in whichsome 17 fractions were separated. Two ofthese fractions have been implicated inwax precursor transport on the groundsthat their molecular weight distributionsclosely approximate those of known hon-eybee apolipophorins. Thus it was sug-gested that hydrocarbons and fatty acidprecursors of beeswax might be synthe-sized in the oenocytes (now strongly sup-ported in the present work) and then trans-ported through the haemolymph to thesurface of the insect in the form of primaryor modified apolipophorins (Kurstjens et al,1990).

Before comparing the chemical contentof the wax gland tissues with that of scalewax, it is important to note that the hydro-carbon and fatty acid content of the A mcapensis comb wax (tables VII and VIII)are virtually identical to those of its sisterrace, A m scutellata reported by Tulloch(1980); results which lend confidence tothe analysis presented here. The natureand origins of the large differences be-tween scale and comb wax (tables VII andVIII) are post-secretory phenomena andhave been treated in detail elsewhere

(Kurstjens et al, 1985; Davidson and Hep-burn, 1986; Hepburn and Kurstjens, 1988).

The general trends in the hydrocarbonprofiles of the oenocytes include an age-related increase in the saturated compo-nents (table III) reflecting considerable syn-thetic activity. By comparison, the epider-mal hydrocarbons show more modestchanges in relation to the ages of the bees(table IV); these are pronounced amongthe more minor groups, the unsaturated

compounds. We believe that the hydrocar-bons of the epidermis reflect oenocyte-derived material in transit because the epi-dermis lacks SER and its age-relatedchanges in hydrocarbons are not syn-chronized with the cycle of secretion.There is an apparent discrepancy betweenthe high C25 and low C35 content of the oe-

nocytes vis-à-vis wax scales; but it couldbe that C33 is formed from C25 and C27outside of the oenocytes.

The fatty acid profiles of the epidermalcells lacked evident patterns of changeconsistent with the ageing of the bees orwith the cycle of wax synthesis and secre-tion (table VI - excepting C18). By contrast,large differences among the fatty acidpools of the oenocytes relate both to theages of the bees and to the cycle of syn-thesis (table V) and secretion (fig 8). Like-wise, the ’average’ composition for fatty ac-ids in oenocytes more closely mirrors scalewax composition than does that of the epi-dermis (table VIII). The presence of and in-crease of C12 in the oenocytes coupled toits absence from epidermis and scale wax(tables V, VI and VIII) further suggests thatthe oenocytes perform chain elongation re-actions. The decrease in C16 and C24 over

time in the oenocytes is also consistentwith synthesis and subsequent export.

CONCLUSIONS

Both epidermal cells and adipocytes lackSER, an organelle essential to lipogenesis

in insects, from emergence of the adult,through the cycle of wax synthesis and se-cretion and on to foraging behavior. Theoenocytes are the only cells of the waxgland complex with SER and whose devel-opmental fate closely matches periods ofwax synthesis (tables I and II; fig 8). Unlikethose of the epidermis, the hydrocarbonand fatty acid profiles of isolated oenocy-tes share much in common with newly syn-thesized scale wax. That the oenocytesare the probable source of beeswax hydro-carbons is supported by the close cyclicalchanges in ultrastructure that coincide withage-related cycles of secretion of beeswaxby worker honeybees. These interpreta-tions are consistent with the histochemicaldata of Reimann (1952), the autoradio-graphic studies of Piek (1964) and studiesof hydrocarbon synthesis in other insectsby Diehl (1973, 1975).

Résumé &mdash; Synthèse et sécrétion de lacire chez l’abeille (Apis mellifera L).L’étude a porté sur :- les modifications ultrastructurales des or-

ganites des glandes cirières;- les modifications des spectres d’hydro-carbures et d’acides gras des cellules des

glandes cirières et de la cire et- la secrétion réelle de la cire en fonctionde l’âge des ouvrières.

Il n’existe pas de reticulum endoplasmi-que lisse, organite essentiel à la synthèsedes lipides, dans l’épiderme, ni dans les

adipocytes des glandes cirières chez lesouvrières adultes. Néanmoins, les oeno-cytes sont extrêmement riches en reticu-lum endoplasmique lisse et le développe-ment dans le temps de cet organite suitétroitement celui de la sécrétion cirière. Lafonction primaire de l’épiderme dans la sé-crétion cirière est l’élaboration des canali-cules pour le transport de la cire. Les adi-

pocytes semblent servir de source d’éner-gie pour la synthèse et la secrétion.

Les fractions hydrocarbure et acide grasdes adipocytes et des oenocytes ont étéanalysées en fonction de l’âge desabeilles. La composition et le rapport des 2groupes de composés suivent étroitementceux de la cire fraîchement secrétée. Il

semble que les oenocytes soient le siègeprimaire de la synthèse des hydrocarbureset des acides gras de la cire et que ces

précurseurs de la cire soient transportéspar des lipophorines à travers l’épidermejusqu’à la surface de la cuticule des miroirsà cire. Les grandes différences de compo-sition chimique entre la cire fraîchementsecrétée et la cire des rayons sont des

phénomènes postérieurs associés à lamalaxation de la cire pendant la construc-tion.

On a déterminé la quantité de cire pro-duite par les abeilles en fonction de leur

âge et les résultats montrent qu’elle y estsignificativement liée. La courbe caracté-ristique de la sécrétion cirière suit étroite-ment celle des changements de l’ultra-structure et de l’histologie des glandes ci-rières. Parmi tous les tissus des glandescirières de l’abeille, seuls les oenocytesprésentent les caractères nécessaires,dans leur ultrastructure et leurs modifica-tions temporelles, pour être le siège de lasecrétion des hydrocarbures et des acidesgras de la cire.

cire / secrétion / synthèse / glande ci-rière / ultrastructure / composition chi-mique

Zusammenfassung &mdash; Synthese undAbscheidung von Wachs bei den Honig-bienen. Es wurden die Veränderungen derUltrastruktur der Organellen in den wachs-absondernden Zellen, Veränderungen inden Kohlenwasserstoff- und Fettsäure-

Profilen der Wachsdrüsenzellen, desWachses und des umittelbar abgesonder-ten Wachses in Bezug auf das Alter derHonigbienen bestimmt. Ein endoplasmati-sches Retikulum, die für die Lipidsyntheseentscheidende Organelle, kommt weder inder Epidermis noch in den Adipozyten desWachsdrüsenkomplexes der adulten Ar-beitsbiene vor. Es sind jedoch die Oenozy-ten extrem reich an endoplasmatischemRetikulum und die Entwicklung dieser Or-ganelle über die Zeit stimmt sehr gut mitder Wachsabscheidung überein. Die

primäre Funktion der Epidermis für dieWachsabscheidung ist die Ausbildung derKanälchen für den Wachstransport. DieAdipozyten scheinen als Energiequelle fürdie Synthese und Absonderung zu dienen.

Die Kohlenwasserstoff- und Fettsäure-fraktion der Adipozyten und Oenozytenwurden in Bezug auf das Alter der Bienenanalysiert. In Zusammensetzung und demVerhältnis der beiden Stoffgruppen be-steht eine gro&szlig;e Überreinstimmung mitfrisch abgesondertem Bienenwachs. Esscheint so zu sein, da&szlig; die Oenozyten dieprimäre Synthese-Stelle der Kohlenwas-serstoff- und Fettsäure-Anteile am Bienen-wachs sind und da&szlig; diese Wachs-Vorstufen durch die Epidermis zur

Oberfläche der Wachsspiegel transportiertwerden. Bedeutende chemische Unter-schiede zwischen frisch abgesondertemBienenwachs und dem Wachs der Wabensind nachträgliche Erscheinungen, die alsFolge der Bearbeitung des Wachseswährend des Bauens auftreten.

Es wurde die Wachsmenge bestimmt,die von Bienen verschiedenen Alters

produziert wird, und es konnte gezeigtwerden, da&szlig; eine signifikante Alters-

abhängigkeit besteht. Die charakteristi-

sche Kurve der Wachsabscheidung in

Bezug auf das Bienenalter stimmt sehreng mit den histologischen und Ultra-

struktur-Veränderungen des Wachs-

drüsenkomplexes überein. Von allenGewebebestandteilen des Wachsdrüsen-

komplexes der Arbeitsbienen zeigen nurdie Oenozyten die Ausstattung in ihrerUltrastruktur und ihren zeitlichen

Veränderungen die nötigen Voraussetzun-gen als Sekretionsquelle für die Kohlen-wasserstoff- und Fettsäureanteile des Bie-nenwachses.

Wachsabscheidung / Wachssynthese /Wachsdrüse / Ultrastruktur / chemi-sche Zusammensetzung

REFERENCES

Alberts B, Bray D, Lewis J, Raff M, Roberts K,Watson JD (1983) Molecular Biology of theCell. Garland, NY

Blomquist GJ, Ries MK (1979) The enzymaticsynthesis of wax esters by a microsomalpreparation from the honeybee, Apis mellife-ra L. Insect Biochem 9, 183-188

Boehm B (1965) Beziehungen zwischen Fettkör-per, Oenocyten und Wachsdrüsentwicklungbei Apis mellifica L. Z Zellforsch Mikrosk Anat

65, 74-115Davidson BC, Hepburn HR (1986) Transforma-

tions of the acylglycerols in comb construc-tion by honeybees. Naturwissenschaften159, 73-74

Dean RL, Locke M, Collins JV (1985) Structureof the fat body. In: Comprehensive InsectPhysiology, Biochemistry and Pharmacology(Kerkut GA, Gilbert LI, eds) Pergamon, Ox-ford, vol 3

Diehl PA (1973) Paraffin synthesis in the oeno-cytes of the desert locust. Nature (Lond) 243,468-470

Diehl PA (1975) Synthesis and release of hydro-carbons by the oenocytes of the desert lo-cust, Schistocerca gregaria. J Insect Physiol21, 1237-1246

Downer RGH (1985) Lipid metabolism. In: Com-prehensive Insect Physiology, Biochemistry

and Pharmacology (Kerkut GA, Gilbert LI,eds) Pergamon, Oxford, vol 10

Dumas JB, Edwards HM (1843) Remarques surla production de la cire. Ann Sci Nat (Paris)20, 1-8

Folch J, Lees M, Sloane-Stanley GM (1957) Asimple method for the isolation and purifica-tion of total lipids from animal tissues. J BiolChem 226, 497-501

Freudenstein H (1960) Einfluss der Pollennah-rung auf des Bauvermögen, die Wachsdrü-sen und den Fettkörper der Honigbiene (Apismellifera L). Zool Jahrb Allg Zool PhysiolTiere 69, 95-124

Hall PF (1984) Cellular organization for steroido-genesis. Int Rev Cytol 86, 53-95

Hepburn HR (1986) Honeybees and Wax.

Springer, HeidelbergHepburn HR, Kurstjens SP (1988) The combs of

honeybees as composite materials. Apidolo-gie 19, 25-36

Hepburn HR, Muller WJ (1988) Wax secretion inhoneybees. Thermal integrity of the festoon.Naturwissenschaften 75, 628-629

Hepburn HR, Hugo JJ, Mitchel D, Nijland MJM,Scrimgeor (1984) On the energetic costs ofwax production by the African honeybee,Apis mellifera adansonii. S Afr J Sci 80, 363-368

Holz H (1978) Das Organ der Wachsbildung. Bi-enen-Ztg 34, 183-184

Keeley IL (1985) Physiology and biochemistry ofthe fat body. In: Comprehensive Insect Physi-ology, Biochemistry and Pharmacology (Ker-kut GA, Gilbert LI, eds) Pergamon, Oxford,vol 3

Kurstjens SP, McClain E, Hepburn HR (1990)The proteins of beeswax. Naturwissenschaf-ten 77, 34-35

Kurstjens SP, Hepburn HR, Schoening FRL,Davidson BC (1985) The conversion of waxscales into comb wax by African honeybees.J Comp Physiol B156, 95-102

Lambremont EM, Wykle RL (1979) Wax synthe-sis by an enzyme system from the honeybee. Comp Biochem Physiol 63B, 131-135

Locke M (1961) Pore canals and related struc-tures in insect cuticle. J Biophys Biochem Cy-tol 10, 589-618

Locke M, Huie F (1980) Ultrastructure methodsin cuticle research. In: Cuticle Techniques inArthropods (Miller TA, ed) Springer-Verlag,Berlin

Menzel R, Wladarz G, Lindauer M (1969) Ta-gesperiodisches Ablagerungen in der Endo-kutikula der Honigbiene. Biol Zentrabl 88, 61-67

Miller T, James J (1976) Chemical sensitivity ofthe hyperneural nerve muscle preparation ofthe American cockroach. J Insect Physiol 22,981-988

Moscatelli EA (1972) Methanolysis of cerebro-sides with boron trifluoride-methanol. Lipids7, 268-271

Piek T (1961) Synthesis of wax in the honey bee(Apis mellifera L) Proc K Ned Akad Wet SerC Biol Med Sc 64, 648-654

Piek T (1964) Synthesis of wax in the honeybee(Apis mellifera L). J Insect Physiol 10, 563-572

Reimann K (1952) Neue Untersuchungen überdie Wachsdrüse der Honigbiene. Zool JahrbAbt Anat Ont 72, 147-188

Reynolds ES (1963) The use of lead citrate ofhigh pH as an electron-opaque stain in elec-tron microscopy. J Cell Biol 17, 208

Ribbands CR (1953) The Behaviour and SocialLife of Honeybees. Bee Research Associa-tion, London

Romer F, Emmerich H, Nowock J (1974) Bio-synthesis of ecdysones in isolated prothorac-ic glands and oenocytes of Tenebrio molitorin vitro. J Insect Physiol 20, 1975-1987

Rösch GA (1927) Uber die Bautätigkeit im Bie-nenvolk und das Alter der Baubienen. Weiter-er Beitrag sur Frage nach der Arbeitsteilungim Bienenstaat. Z Vgl Physiol 6, 265-298

Sanford MT, Dietz A (1976) The fine structure ofthe wax gland of the honeybee (Apis mellife-ra L). Apidologie 7, 197-207

Schnelle H (1923) Über den feineren Bau desFettkorpers der Honigbiene. Zool Anz 57,172-179

Sedlak BJ (1985) Structure of endocrine glands.In: Comprehensive Insect Physiology, Bio-

chemistry and Pharmacology (Kerkut GA,Gilbert LI, eds), Pergamon, Oxford, vol 7

Tulloch AP (1980) Beeswax - composition andanalysis. Bee World 61, 47-62

Watson ML (1958) Staining of tissue sectionsfor electron microscopy with heavy metals.J Biophys Biochem Cytol 4, 475-478

Weibel ER, Bolender RP (1973) Stereologicaltechniques for electron microscopic mor-phometry. In: Principles and Techniques ofElectron Microscopy: Vol 3, Biological Appli-cations (Hayat MA, ed) Van Nostrand-Reinhold, Princeton

Winston M (1987) The Biology of the HoneyBee. Harvard, Cambridge