Original article

Patterns of larval food productionby hypopharyngeal glandsin adult worker honey bees

D Knecht, HH Kaatz

Lehrstuhl Entwicklungsphysiologie, Zoologisches Institut der Universität Tübingen,Auf der Morgenstelle 28, D-7400 Tübingen, FRG

(Received 30 May 1990; accepted 15 June 1990)

Summary — Ultrastructural changes of the hypopharyngeal gland cells were analyzed duringimaginal development of worker honey bees (Apis mellifera L). The rough endoplasmic reticulum in-creases immediately after emergence, reaches a maximum during the nursing phase and decreasesin field bees. Accordingly, high rates of protein synthesis were measured in nurse bees and low pro-tein production in foragers. Secretion reservoirs are formed within the intracellular ductules. Theyare surrounded by a sheath of numerous microvilli. Larval food proteins are secreted into the reser-voirs and stored as demonstrated immunocytochemically. Even in foraging bees, small amounts oflarval food proteins are stored, indicating the flexibility of the worker to potentially react to changingcolonial and environmental conditions.

Apis mellifera / hypopharyngeal gland / protein synthesis / ultrastructure / immunocytochem-istry / ontogenesis

INTRODUCTION

The basic studies of Anna Maurizio poin-ted to the significance of regular pollensupply for maximum morphogenesis andfunction of the hypopharyngeal glands in

queenright worker honey bees. She de-monstrated that pollen not only promotesthe initial growth of the glands in adultbees but also promotes the maximum pro-duction of honey processing enzymes(Maurizio 1954, 1959, 1961, 1962a). Alack of pollen in the young bee’s diet de-

lays the normal functions of the glands. If,later on, a worker is fed on pollen, theglands are capable of growing completelyand develop regular functions (Maurizio,

1962b), attaining a normal size in thenurse phase during which mainly larvalfood proteins are produced (Halberstadt,1980; Takenaka and Kaatz, 1987). Whenthe workers become foragers, the glandsdecrease considerably in volume and in-stead secrete enzymes like invertase

(Simpson et al, 1968; Halberstadt, 1980).The functional and morphological transi-tion from nursing to foraging bee is control-led by juvenile hormone (Jaycox, 1976;Robinson, 1987).

Despite a number of ultrastructural stu-dies (Beams et al, 1959; Cruz-Landim andHadek, 1969; Halberstadt, 1970), functio-nal aspects of the glandular structureshave rarely been investigated. Thus, it is

unknown as to where larval food proteinsand secretory enzymes are synthesizedand stored and if changes related to ageand function occur. Therefore, we analy-zed these questions in the course of thelifespan of adult worker bees.

MATERIALS AND METHODS

The experiments were carried out from Maythrough August 1989 using Carniolan hybrids(Apis mellifera L) from our apiary. Newly emer-ged workers were colour-marked and kept in

queenright colonies until the experiment.

Electron microscopy

The hypopharyngeal glands were fixed in gluta-raldehyde (3%, v/v), 0.125 mol·l-1 CaCl2, 0.18 mol·l-1 MgSO4, 0.064 mol·l-1 s-collidine (stemsolution: 0.4 mol·l-1 s-collidine: 5.34 ml s-

collidine, 18 ml (1 mol·l-1) HCl per 100 ml) for2 h at 4°C, washed in distilled water and post-fixed in osmiumtetroxide (3.3%, w/v). They weredehydrated in ethanol and exposed to propy-leneoxide which was slowly replaced by Eponresin solution (32 g Epon 812, 16.032 g 2-

dodecenyl succinic acid anhydride, 17.36 g me-thylnadic anhydride, 0.6-0.8 g 2,4,6,-Tris(dime-thyl-amino-methyl)phenol). Polymerization las-ted 2 d at 60°C. Uranylacetate and lead citratestained ultrathin sections were studied using aZeiss EM 9.

Antiserum specificityfor larval food proteins

The antiserum was raised in rabbits injectedwith royal jelly (0.2 ml) and Freund’s completeadjuvant. After separation by SDS-

polyacrylamide gel electrophoresis on gradientslab gels of 5-20% acrylamide (Laemmli, 1970),specificity was analyzed by immunoblotting(Towbin et al, 1979) using homogenates of fatbody and labial glands (for control purposes), as

well as homogenates of hypopharyngeal glandsand worker jelly of 1-2-d-old larvae as test

samples. The nitrocellulose sheets were incuba-ted with a quenching solution (3% bovine serumalbumin in 10 mmol·l-1 tris buffered saline) for1 h at 37°C. Diluted antiserum (1:3000) was ap-plied overnight at 4°C, followed by the peroxi-dase conjugated antiserum against rabbit immu-noglobulin G (1:2000, 2 h). Finally the sheetswere stained in 4-chloronaphtol (0.04% w/v,13.3% ethanol, 67 mmol·l-1 Tris-HCl, 0.015%H2O2) for 20-30 min at room temperature.

Immunocytochemistry

The hypopharyngeal glands were fixed inBouin’s solution for 24 h at 4°C, dehydrated inethanol and embedded in glycol methacrylateresin (HistoresinTM, LKB). Sections (1.5 μm)were cut with a microtome 1140/Autocut (Jung,Heidelberg), air-dried, mounted on microscopicslides and stained with the PAP method (Stern-berger, 1979). Larval food proteins were detec-ted with the antiserum against royal jelly diluted1:500 and incubated for 24 h at room tempera-ture. The second antibody (1:100 dilution) andthe third, peroxidase conjugated antibody(1:200), were each incubated at 37°C for 1 h.Larval food proteins were rendered visible with a4-chloro-1-naphthol-solution (0.03% (w/v) dissol-ved in ethanol (final concentration: 1%), 0.03%H2O2 in 50 mmol·l-1 Tris buffered saline, pH 7.6)for 20 min.

Protein synthesis

Synthesis of water-soluble proteins in the hypo-pharyngeal glands was determined by an injec-tion of 74 kBq 35S-methionine/2 μl into the he-mocoel of 10 workers per age group with 2

replicates. The 45 min of incubation proved tobe linear for tracer incorporation. The dissectedglands were homogenized in phosphate buffe-red saline (150 mmol·l-1 NaCl, 40 mmol·l-1phosphate, 10 mmol·l-1 methionine, pH 6.70),centrifuged (10 min, 10 000 g) and the tracer in-corporation into proteins of the supernatant wasmeasured according to Kaatz et al (1985).

RESULTS

Rough endoplasmatic reticulumand protein synthesis

In the pharate adult worker the rough en-doplasmatic reticulum (RER) of hypopha-ryngeal gland cells is scarcely developedand spheroidally arranged (fig 1 a). Duringthe first few days after emergence theRER increases rapidly (fig 1b) until at d 8most of the space between the secretionreservoirs is filled by well-developed lamel-lar stacks of RER (fig 1c). In foragers theRER decreases and only parts remain (fig1d). This pattern of ultrastructural develop-ment corresponds well to the total proteinsynthesis monitored in the hypopharyngealglands in vivo (fig 2). Until d 4 after emer-gence protein synthesis increases morethan 20-fold. During the nursing phase, es-pecially from 8-16 d of age, the highestrates of protein synthesis are observed inthe hypopharyngeal glands. After d 20,protein synthesis declines drastically bymore than 90% within 4 d and remains at alow level in field bees.

Secretion storageand transport apparatus

Each gland cell is equipped with a twistedexporting ductule called an intracellularductule (fig 5bc). However, a cell mem-brane separates the ductule from glandcell cytoplasm, indicating extracellular rath-er than intracellular structures. In addition,the gland cell membrane is folded to a

great extent and surrounds the ductulewith a sheath of numerous microvilli (fig3a-c). The ductule is lined by a cuticlewhich consists of a thin and fenestrated

epicuticle and a more extensive, fibrillarendocuticle. Secreted material is found in

secretion reservoirs which are formed in

the space between the gland cell mem-brane and endocuticle of the exportingductule (fig 3b). Thus, the secretion reser-voir and the exporting ductule represent amorphological unit providing storage andtransport of the secretions.

During adult development, the glandularultrastructure changes in a typical way.The ductule and the microvilli persistthroughout a worker’s life, whereas the vol-ume of the secretion reservoirs changes.In the pharate adult worker, the latter islimited to a small space between the endo-cuticle and gland cell membrane and is stillfree of any secretions (fig 3a). During thefollowing days the space is filled up withsecretional products, reaching a maximumat d 8 (fig 3b). This level persists through-out the hive bee period. Later on, in thefield bee stage, the reservoirs shrink grad-ually until only a small space is left. How-

ever, in most foragers examined, the ex-porting ductules are still filled withsecretions (fig 3c). Obviously these chang-es in the volume of the secretion reservoirsare the prime cause for the volume altera-tions of the whole glands.

Localization of larval food proteins

The antiserum raised against royal jellyproteins specifically recognizes all the po-lypeptides in worker jelly as well as thecorresponding secretory proteins within thehypopharyngeal glands. In control tissuesno reacting antigens could be detected (fig4), confirming the specificity of the antise-rum.

Using this antiserum for immunocyto-chemical staining within the hypopharyn-geal gland cells, the secretion reservoirsand also the exporting ducts are clearlymarked (fig 5a-c). For the first time, thestorage of larval food proteins is evidenced

within the secretion reservoirs. Since thesame picture was obtained in all the aciniof any nurse bees examined, it is apparentthat no specialization in synthesis and sto-rage exists. Moreover, the intensity of thestain changes in relation to a worker’s age.No stain is visible in glandular sections ofnewly eclosed workers (data not shown).However in newly emerged workers, a

weak signal occurs within the exportingducts and the small secretion reservoirs

(fig 5a). The intensity of the stain reachesits maximum at d 8 (fig 5b). Secretion re-servoirs have increased in size and are

strongly marked. Surprisingly, the hypo-pharyngeal glands of foraging beescontain small amounts of larval food pro-teins (fig 5c). Obviously, the glands containlarval food proteins throughout the lifespanof imaginal bees in varying quantities.

DISCUSSION

The terms intracellular ductule, extracellu-lar duct, and collecting duct have beenused for the 3 exporting structures of thehypopharyngeal glands (Soudek, 1927;Kratky, 1931; Beams et al, 1959; Halber-

stadt, 1970). The intracellular ductule hasheretofore been regarded as an integralpart of each gland cell and as separatefrom the secretion reservoir. We suggestthat the secretion reservoir and the expor-ting ductule form a functional unit for sto-rage and transport of the secretion mate-rial. This unit is separated from the glandcell by a cell membrane. Therefore theterm intracellular ductule appears to be

misleading. The secretion reservoirs areextensions of the space between the endo-cuticle and gland cell membrane. They aresurrounded by a sheath of microvilli proba-bly involved in exocytosis of the gland cellsecretions. Thus, storage of larval food

proteins in the secretion reservoirs is com-bined with the export of the secretionsacross the fibrillar endocuticle and the fe-nestrated epicuticle along the ductule.

The secretory cells of the hypopharyn-geal gland are equipped with a well deve-loped rough endoplasmatic reticulum

(RER), typical of protein-secreting cells.The degree of development of RER in thegland cells (fig 1) as well as the temporalpattern of total protein synthesis (fig 2)match the 3 known phases of glandularsize development (Hassanein, 1952; Mau-rizio, 1954). Gland activity is still low in

newly emerged bees, which have rathersmall glands containing as yet little RER.Within the first days following emergence,as the glands grow and the RER in-

creases, total protein synthesis rises rapid-ly, continuing until full size and RER deve-lopment are reached, thus providing highsynthetic activity for brood rearing in the

nursing phase. After bees become fora-

gers, this protein production declines rapid-ly. Obviously, the extent of the RER is cor-related with the rate of protein synthesis inthe hive bee period of imaginal worker de-velopment.We have shown that the secretion re-

servoirs of summer bees do indeed store

the larval food proteins (fig 5a-c), a hypo-thesis already assumed by Halberstadt

(1980). During the hive bee phase, larvalfood protein production makes up the mainproportion of newly synthesized proteins,amounting to 80% of the net synthesis(Takenaka and Kaatz, 1987). Consequent-ly, the volume of the secretion reservoirsreflects the amount of produced and sto-red larval food proteins and gives rise tothe changing size of the acini. For this rea-son as commonly practiced, the size of thehypopharyngeal glands can be used to ea-

sily estimate the glandular activity and phy-siological status of the summer bee (Mauri-zio, 1954; Rutz et al, 1976). In spite of

contradictory data (Brouwers, 1982), thevolume of the glands is recommended forsuch an estimation of the physiological sta-tus of a worker honey bee. However, itmust be taken into account that the above-mentioned typical developmental patternsof hypopharyngeal gland activities can beinfluenced by colony conditions as well asby seasonal changes. Maurizio (1954,1962b) stated that pollen supply is requi-

red for the initial growth of the glands. Fur-thermore, the presence of brood is essen-tial for the maintenance of glandular size(Free, 1961) and larval food production(Huang and Otis, 1989). In caged bees,normal gland development is inhibited

(Crailsheim and Stolberg, 1989). Winter

bees, although with hypertrophied acini,show a low rate of protein synthesis (Brou-wers, 1982).

Changes in glandular volume and rateof protein synthesis characterize the 2 dis-tinct major functional phases of the glandsduring imaginal normogenesis (Halber-stadt, 1980): production of larval food andhoney processing enzymes. Both phasesevidently overlap to some extent. This isindicated by the occurrence of smallamounts of larval food protein in the secre-tion reservoirs of foragers as well as by the

detection of newly synthesized larval foodprotein in foraging workers (Takenaka andKaatz, 1987). Maurizio (1959, 1961,1962a, b) has already shown that there isa maximum of hydrolyzing sugar-invertingenzymes in old foraging bees, but thatsome activity is already detectable in

young bees with well-developed glands, aswell as in winter bees.

The simultaneous occurrence of honeyprocessing enzymes and larval food in thehypopharyngeal gland requires their cyto-logical compartmentalization. We haveshown that larval food proteins are storedin the secretion reservoirs throughout thenurse phase. This means that the hypo-pharyngeal glands are not compartmentali-zed into different functional parts, but intodifferent secretory compartments withinthe same gland cell, as hypothesized byWetzig (1964), Cruz-Landim and Hadek

(1969), Ortiz-Picon and Diaz-Flores

(1972). However, neither the storage sitesfor sugar-inverting enzymes nor any selec-tive secretory mechanism, which must ne-cessarily exist, have been studied yet.

The partially overlapping functional

phases and the persistence of the structu-ral apparatus for production and storage oflarval food protein in the hypopharyngealglands throughout the imaginal life of theworker seem to be, respectively, functionaland morphological characteristics of the

flexibility within the colonial division of la-bour. Caste evolution (Engels, 1990) mayhave been guided by conflicting forces thatpromote ergonomic efficiency on the onehand and flexibility responding to environ-mental challenges on the other. The nur-sing capacity of the worker bee is obvious-ly an example of a highly flexible system.Its adaptability is especially apparent in thecase of colony manipulations (Rösch,1930) in which the larval food productioncould, for nursing purposes, be reactivatedin old foraging bees.

ACKNOWLEDGMENTS

We are grateful to W Engels for critically readingthe manuscript and C Bardele, K Eisler and HSchoppmann for support in the electron micro-scopy.

Résumé — Modalités de production dela nourriture larvaire par les glandes hy-popharyngiennes chez les ouvrièresd’abeilles adultes. Outre les enzymespour l’élaboration du miel, les glandes hy-popharyngiennes des ouvrières produisentune part importante de la nourriture lar-

vaire. On a étudié au niveau de l’ultrastruc-

ture, chez des ouvrières adultes de divers

âges, les modifications des systèmes desynthèse et de stockage des glandes nour-ricières au cours du développement nor-mal. On a ensuite déterminé in vivo l’activi-té de synthèse des cellules glandulairespar injection d’un traceur (35S-méthionine)et localisé par immunocytochimie les pro-téines larvaires qu’elles produisent.

La quantité de reticulum endoplasmiquebrut dans les cellules glandulaires (fig 1)varie en fonction de l’âge. Elle augmentejuste après l’émergence (fig 1 a, b), atteintun maximum au stade nourrice puis dimi-nue et reste à un niveau bas chez les buti-neuses (fig 1 d). Ce mode de développe-ment se reflète dans les quantités deprotéines synthétisées par les glandes hy-popharyngiennes (fig 2). Au cours des 4premiers j de l’ontogenèse, la synthèseprotéique augmente de plus de 20 fois.

C’est à la phase nourrice que l’on observeles taux les plus élevés et ce n’est quechez les ouvrières, qui sont âgées de 20 j,que la synthèse protéique diminue forte-ment, de plus de 90%.

Chaque glande cellulaire d’un acinusest pourvue d’un système de stockagepropre et d’un canal d’évacuation (fig 3a-c). La lumière du canal d’évacuation est li-

mitée par une épicuticule fine et poreuse

et une endocuticule fibrillaire. Les secré-tions glandulaires sont stockées dans unréservoir qui occupe l’espace entre lamembrane riche en microvillosités des cel-lules glandulaires et l’endocuticule. Le ré-servoir et le canalicule qui en sort sont sé-parés de la cellule glandulaire par une

membrane cellulaire commune et désignésun peu à tort dans la littérature par canali-cules intracellulaires. Ils forment à l’évi-dence une unité fonctionnelle. Les réser-voirs à sécrétions subissent des

modifications importantes de volume aucours du développement imaginal. Ils sontencore petits chez les ouvrières au stadenymphe (fig 3a), grossissent au cours desjours suivants pour atteindre un maximumau 8e j (fig 3b). Quand les ouvrières de-viennent butineuses, les réservoirs à sé-crétions se rétrécissent (fig 3c). Les modifi-cations typiques en fonction de l’âge duvolume des glandes hypopharyngiennesglobales au cours de l’ontogenèse sont

principalement dues aux modifications devolume des réservoirs à secrétions.

Les protéines de la nourriture larvaire

sont décelables dans les canalicules d’éva-cuation et les réservoirs à secrétions (fig 5a-c) par immunocytochimie à l’aide d’unantisérum spécifique anti gelée royale (fig4). Pour la première fois on a pu confirmerl’hypothèse selon laquelle les protéines dela nourriture larvaire étaient stockées dansles réservoirs à secrétions. L’intensité de la

coloration immunocytochimique dépend del’âge des ouvrières. On peut ainsi déjà re-connaître un léger marquage des réser-voirs à secrétions chez les ouvrières ré-cemment écloses (fig 5a). Il augmente defaçon continue jusqu’au 8e j où il est maxi-

mal (fig 5b). Même chez les butineuses lesprotéines de la nourriture larvaire sont pré-sentes, avec un marquage beaucoup plusfaible bien sûr (fig 5c).

Manifestement, les 2 principales fonc-tions des glandes nourricières, production

de nourriture larvaire et production d’en-zymes, ne s’excluent pas mutuellement,mais s’exercent en partie parallèlement.La présence de nourriture larvaire jusqu’austade butineuse contribue sans aucun

doute à ce que la colonie d’abeilles puisseréagir de façon flexible à des exigences desoin au couvain et s’adapter rapidementaux conditions fluctuantes du milieu. Ceci

explique aussi les anciennes observationsselon lesquelles des butineuses peuventredevenir nourrices en cas de besoin.

Apis mellifera / glande hypopharyn-gienne / synthèse protéique / ultrastruc-ture / immunocytochimie / ontogenèse

Zusammenfassung — AltersabhängigeMuster der Larvenfuttersaftproduktionin den Hypopharynxdrüsen adulter Ho-nigbienenarbeiterinnen. Die Hypopha-rynxdrüse der Arbeiter in produziert we-sentliche Anteile des Larvenfuttersaftes,außerdem Enzyme für die Honigbereitung.Bei imaginalen Arbeiterinnen unterschiedli-chen Alters wurde untersucht, wie sich

Synthese- und Speicherapparat der Futter-saftdrüsen während der Normalentwik-

klung ultrastrukturell verändern. Fernerwurde die Syntheseaktivität der Drüsenzel-len mit Tracerinjektionen in vivo bestimmtund die von ihnen produzierten Larvenfut-tersaftproteine immunocytochemisch loka-lisiert.

Die Menge an rauhem endoplasmati-schen Retikulum in den Drüsenzellen (Abb1) verändert sich altersabhängig. So steigtder Gehalt an rauhem endoplasmatischenRetikulum nach dem Schlüpfen an (Abb1 a, b), erreicht ein Maximum im Ammen-alter (Abb 1c) und sinkt bei Sammlerinnenauf ein konstant niedriges Niveau ab (Abb1d). Dieses Entwicklungsmuster spiegeltsich in der in vivo-Proteinsyntheseleistungder Hypopharynxdrüse wider (Abb 2). ImVerlauf der ersten vier Tage der Ontoge-

nese steigt die Proteinsynthese um mehrals das 20fache an. In der Ammenphasekönnen die höchsten Proteinsyntheseratenbeobachtet werden. Erst bei Sammlerin-

nen, die älter als 20 Tage sind, vermindertsich die Proteinsynthese drastisch um

mehr als 90%.

Jede Drüsenzelle eines Acinus ist miteinem eigenen Speicherapparat und Aus-führgang ausgestattet (Abb 3a-c). DasLumen des Ausführgangs wird von einerdünnen, porösen Epicuticula und einer fi-

brillären Endocuticula begrenzt. Die Drü-sensekrete werden im Sekretspeicher ge-lagert, der den Raum zwischen derMikrovillireichen Zellmembran der Drüsen-zelle und der Endocuticula einnimmt. Se-

kretspeicher und ausführendes Kanälchensind von der Drüsenzelle durch eine ge-meinsame Zellmembran abgegrenzt undwerden in der Literatur etwas irreführendals intrazelluläres Kanälchen bezeichnet.Offenbar bilden sie eine funktionelle Ein-heit. Die Sekretspeicher unterliegen im

Verlauf der imaginalen Ontogenese erheb-lichen Volumenveränderungen. Sie sindbei pharat adulten Arbeiterinnen noch klein(Abb 3a) und vergrößern sich während dernächsten Tage der Entwicklung, bis sie einMaximum am 8. Tag erreichen (Abb 3b).Wenn die Arbeiterinnen als Sammlerinnen

tätig werden, schrumpfen ihre Sekretspei-cher (Abb 3c). Die typischen altersabhäng-igen Volumenveränderungen der gesam-ten Hypopharynxdrüse während der adul-ten Ontogenese werden offenbar in ersterLinie durch die Volumenänderungen derSekretspeicher verursacht.

Larvenfuttersaftproteine sind mit einemspezifischen Antiserum gegen Gelée

Royale (Abb 4) immunocytochemisch in

den ausführenden Kanälchen und den Se-

kretspeichern nachweisbar (Abb 5a, b).Damit konnte erstmalig die Vermutung be-legt werden, daß Larvenfuttersaftproteinein den Sekretspeichern gelagert werden.

Die Intensität der immunocytochemischenFärbung hängt vom Alter der Arbeiterin ab.So läßt sich bereits bei frisch geschlüpftenArbeiterinnen eine erste schwache Markie-

rung in den Sekretspeichern erkennen

(Abb 5a). Sie nimmt kontinuierlich zu, bisein Maximum um den 8. Tag erreicht ist

(Abb 5b). Selbst bei Flugbienen kommenFuttersaftproteine vor, allerdings ist dieNachweisreaktion deutlich schwächer (Abb5c).

Offensichtlich schließen sich beide

Hauptfunktionen der Futtersaftdrüse, Fut-tersaft- und Enzymproduktion, nicht gegen-seitig aus, sondern werden teilweisenebeneinander ausgeübt. Das Vorhanden-sein von Larvenfuttersaft bis in die Flugbie-nenphase hinein trägt ohne Zweifel dazubei, daß das Bienenvolk flexibel auf Pfle-

geerfordernisse im Brutgeschäft reagierenund sich damit wechselnden Umweltbedin-

gungen rasch anpassen kann. Dies erklärtauch die alten Beobachtungen, daßSammlerinnen bei entsprechendem Bedarfim Volk wieder als Ammenbienen tätigwerden können.

Apis mellifera / Hypopharynxdrüse /

Proteinsynthese / Ultrastruktur / Immu-nocytochemie / Ontogenese

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