Review

Reciprocal interactions between honeybeesand combs in the integration of some colony

functions in Apis mellifera L.*

H.R. Hepburn

Department of Zoology and Entomology, Rhodes University, Grahamstown 6140,South Africa

(Received 11 June 1997; accepted 28 October 1997)

Abstract - Recent advances in studies of the reciprocal interactions between honeybees andtheir combs are reviewed. Wax secretion is age-related, varies with season, is unaffected by thequeen, juvenile hormone or the corpora allata but is enhanced in swarming. Comb building isenhanced by the queen. Nest structure can be explained as a self-organization process as can thepatterns of brood, honey and pollen. The comb and its contents provide gross information to thecolony as to crowding and space which affect brood rearing, energy consumption and combbuilding. Significant chemical and physical changes occur in the wax during comb buildingand during its subsequent use. Comb mediates pheromonal cues for cell capping, repairs and queencell construction, nectar forage, colony defense and colony odor. Mechanically, the combstransmit vibrational signals in the waggle dance and recruitment of new foragers. © Inra/DIB/AGIB/Elsevier, Paris

honeybee / combs / pheromones / sound / self-organization

1. INTRODUCTION

The exquisite structure of the combs ofhoneybees reflects something of the com-plex behavior needed to have made them.But far from static, the combs constitute adynamic system that is reciprocally inter-active with honeybees. In consequence,

both undergo continual changes in elab-oration and organization as the cycle ofthe colony and its nest unfolds. As moreknowledge about bees accumulates thenotion of ’reciprocal interactions’ betweenthe bees and their combs becomes increas-

ingly obvious. Sometimes these interac-tions are simultaneous, other times they

* In memoriam: R. DarchenTel.: (27) 461 318098; fax: (27) 461 24377; e-mail: zohh@warthog.ru.ac.za

may be primarily ’bee-to-nest’ or ’nest-to-bee’. Unraveling these intricate patternsand processes sometimes seems

intractable. However, fresh insights intoold problems always renew impetus in thecenturies-old quest of comprehending hon-eybees in a predictive way.

This review is admittedly unbalanced inseveral ways. There is no focus on the dualfunctions of comb in brood rearing andfor storage, nor allusion to the problems ofallocation of drone and worker cells. Nor,is the problem of maximizing reproduc-tive output with a minimum investmentin wax considered. It simply reports recentdevelopments on ’comb-bee’ interactionssince this topic was last comprehensivelyreviewed (Hepburn, 1986). These specif-ically include the dynamics of comb build-ing, the material properties of the combs,the regulation of comb building and cycleof wax secretion, and how the contents ofthe combs become distributed. Greater

developments have occurred in areaswhere combs form a basis for chemicaland acoustical communications and theseare stressed.

2. COMB BUILDING

2.1. Nest cavity

All of the cavity-dwelling honeybeeshave extremely wide tolerances on theacceptability of a potential nest site interms of volume; but the European racesprefer a volume of about 40 L (Seeley andMorse, 1976) while the African races pre-fer a volume of some 20 L as measured

directly in nature (McNally and Schnei-der, 1996) or in choice preference tests(Berg, 1996). In any event, the nest cavityimposes constraints and the colony is abso-lutely bound in three dimensions by itsparticular conformation. As the combsincrease in number and size they must bear

correspondingly larger loads of brood andstores as well as accommodate the ther-mal and respiratory needs of the colony.

Additional insights are to be gainedabout cavity filling from experimentalstudies of transitional hive design wherethe main objective is to eliminate all butthe top bar of a conventional hive frame,but at the same time to avoid comb attach-ments to the cavity wall that interfere withbeekeeping practice. Interesting resultsthat bear on this problem come from sucha study by Budathoki and Free (1986). Ifone regards their manipulated frame side-bars as surrogate cavity walls, then as theslope of the cavity changes so does theprinciple of attachment. The greater theangle of departure is from the vertical, thelesser the degree of attachment. Similarly,with greater slope, the distance from thecomb base to the attachment site increases.Least comb is attached to a continuallycurved surface.

2.2. Comb development

Primed for comb construction in a newnest cavity, workers often begin to buildsimultaneously at several different sites.But the orientation of the bees in the fes-toons form a parallel, flexible scaffoldingthat predetermines the coordinates of fur-ther construction (Darchen, 1968). Thewhole process of development of the three-dimensional structure can be interpretedas a self-organizing system of bee-bee andbee-comb interactions (Belic et al., 1986).This model has captured the essential fea-tures of comb construction and by-passesthe need for any dependency on an intrin-sic blue-print in honeybees (figure 1).

While the midwalls of neighboringcombs may be far from parallel, the dis-tances between them eventually reflect thebee space tolerances typical of the raceconstructing them. Analyses of these con-structions show that the building toler-

ances of honeybees are wide and thatmany steps in construction are indepen-dent of one another (Hepburn, 1983,1986). However, once two or three combshave begun to be built, the relative posi-tions of new combs have effectively beendetermined (figure 1). To achieve paral-lelism the bees adjust cell length and intro-duce irregular cell bases and if thesemanipulations are not adequate to meettheir tolerances, additional combs may beinserted to this end (Hepburn and Whif-fler, 1991). All that is required is the back-to-back proprioception of two workerswho can estimate the length of a straightline segment.

3. MATERIAL PROPERTIESOF COMBS

3.1. Mechano-chemical changes

Order of magnitude differences in themechanical properties of wax scales and

comb wax and their texture-adjusted filmsclearly indicate that the process of combbuilding involves chemical modification ofthe waxes (Kurstjens et al., 1985). Fur-ther analysis of the protein fractionrevealed some 17 bands in the elec-

trophoretograms, some unique to each wax(scale and comb) and others shared (Kurst-jens et al., 1990). Two inferences weremade from the data: two fractions com-mon to both waxes are of similar molecu-lar weight to other insect lipophorins andthey may be gland-to-surface transportproteins. In the mastication of wax scalesadditional protein is added, presumablylipases, because comb has a higher monoa-cylglycerol content than the diaclyglyc-eride-richer wax scales (Davidson andHepburn, 1986). The latter effect is toincrease the degree of saturated bonds incomb thus contributing to better stiffness(Kurstjens et al., 1990).

Since lipolytic enzymes added to thewax by the bees require an aqueousmedium, a source of moisture needs to bepresent in the wax medium. Given an aver-

age relative humidity in a hive of aboutRH-50 (Simpson, 1961), moisture is avail-able as a by-product of respiration, ther-moregulation and dehydration of nectar.Also required is a means to deliver thismoisture into the wax. Recently, Donhoweand Fennema (1992) demonstrated thatthe water vapor permeance of beeswaxfilms is sufficient to deliver 1.7 g water

per kg wax into the comb structure. Theyfurther pointed out that although beeswaxis primarily hydrophobic, the esters,hydroxyl groups of free alcohols and thecarboxyl groups of free fatty acids arehydrophilic. Physical effects of wax hydra-tion would include matrix swelling andan increase in the diffusion coefficient ofthe wax (Donhowe and Fennema, 1992).

The modifications of comb perfor-mance by the presence of proteins andwater can now be related to the material

properties of combs as they evolve in thenest (Hepburn and Kurstjens, 1988). Inthe course of its development new combwax is an isotropic plastic whose mechan-ical properties depend heavily on temper-ature. In time, generations of larvae intro-duce silk into the waxen structure in arandom alignment to achieve equal prop-erties in all directions. Thus, with use thecomb becomes a fiber-reinforced com-

posite material which exhibits propertiesentirely different from the individual com-ponents. The addition of silk greatlyimproves the load-carrying capacity of thecombs (Hepburn and Kurstjens, 1988).Although not a theoretically ideal stiffplate structure (Nachtigall and Kresling,1992), the mature comb is nonetheless aremarkable compromise between technicalconstruction and the biological purposes itserves. A flow diagram for the conversionof wax scales into comb is shown in figu-re 2.

Finally, some recent discoveries aboutcomb chemistry have been made but theirsignificance is not yet apparent. Puleo(1991) summarized the details of the

minor constituents of beeswax and listedsome 117 compounds derived from propo-lis and also commonly found in combwax. Of these, 41 are specifically associ-ated with wax aroma. Similar reports oncompounds derived from propolis haveappeared (Seifert and Haslinger, 1991;Tomas-Barberan et al., 1993). Of consid-erable interest is that Brand-Garnys andSprenger (1988) analyzed the simple estersand hydrocarbons of beeswax using highresolution gas chromatography and foundthat 16 subspecies of A. mellifera couldbe unequivocally recognized by theirchemical signatures. Circumstantial evi-dence suggests that some of these com-

ponents may help define different dancearenas (cf below).

3.2. Thermal properties

Another extremely interesting aspectof the bee-comb relationship is thatbetween a thermoregulated temperatureof about 35 °C and the workability of thewax. Over the range of temperaturesencountered in different areas of the nestthe amount of energy required to work thewax into comb at 35 °C is half that at25 °C which is an energetic savings (Hep-bum, 1986). At new comb building sites,often away from the main cluster espe-cially as the nest expands to severalcombs, the festoons provide some of theirown ambience of warmth at about 35 °Cbecause the temperature differencebetween a festoon and the main cluster

may be some 5 °C (Hepburn and Muller,1988).

The thermal properties of combs inrelation to colonial heat balance, part ofthe entire energetic equation of a colony,were explored by Southwick (1985). Hemeasured the thermal conductivity of iso-lated comb and found that while the combs

provide insulation, the clustering behaviorof the bees actually increases the insulat-

ing effectiveness of the combs. When thereis a compact layer of bees on the combs,conductivity can be reduced by an order ofmagnitude. Given the conductivity of waxcombs and bees clustered on and between

them, insulation of the combs is the com-bined effect of combs and the behavior ofthe bees themselves (Southwick, 1985).

4. REGULATION OF COMBBUILDING

4.1. Seasonality

It is well established experimentallythat newly settled swarms are prodigious

comb builders (Lee and Winston, 1985;Hepburn, 1986), but in a temporo-spatialframework, comb building only reachesparity with other wax working (cappingand repairing) at the height of the colonygrowth cycle (Muller and Hepburn, 1992).Comb building is conducted in differentareas of the nest by many individuals,some clustered in festoons others not,while other wax works are often the effortsof individual bees (Lindauer, 1952).Changing ratios of what work and whereit is carried out can be assessed by fol-lowing the raw wax in a colony with thechanging seasons.

Muller and Hepburn (1992) found thatin the course of a year as much wax isfound in festoon bees as in bees elsewhere

among the combs but seasonal picturesare quite different (figure 3). It appearsthat wax-bearing bees can be found in theright places at the appropriate times. Thewax bees shift from one area of the nest to

another, for example, with heavy nectarflows for honey cell capping or to areasrequiring brood capping. This ensures aclose correspondence between comb area’needs’ and the presence of bees with waxscales. Although not all would agree (Fer-gusson and Winston, 1988) the distributionof these wax bees is largely predicated onan underlying age-based cycle of glandu-lar secretion (Hepburn et al., 1991).

4.2. The effects of storage space

That strong nectar flows fuel comb

building is an old principle of beekeepingand a proposed explanation for the rela-tionship was formulated by Butler (1954).He argued that the greater the influx ofnectar into the colony, the longer the housebees must retain it. This, of course,requires the right combination of avail-able storage space and ratio of foragers tohouse bees. Serving as distended reser-voirs over time, these bees assimilate some

of the nectar sugar and become stimulatedto secrete wax. This sensible idea has

proven far easier to appreciate than to test.Using queenright colonies in which combavailable for nectar storage was experi-mentally reduced or entirely eliminated,a correlation between engorgement of the

honey stomach and wax secretion wasobtained (Hepburn and Magnuson, 1988).

This experiment did not distinguishbetween physical distension of the honeystomach and the time such a bee mightspend in trying to disgorge and store thenectar. Nonetheless the observation is indi-

rectly supported by experiments in whicheither the deprivation of combs (Fergussonand Winston, 1988) or lack of sufficientstorage space (Seeley, 1995) both lead toincreased foraging, accelerated wax secre-tion and, ultimately, comb building. Col-lectively, the experimental data lead to asimple feedback system: forager dancingeffectively recruits more nectar-foragers;when this nectar is difficult to off-load, aspecial tremble or stop dance is performedwhich inhibits further recruitment (See-ley, 1992; Nieh, 1993).

During comb building there are con-comitant changes in population size, den-sity, nectar and pollen influx which reflecthoneybee-comb interactions. Of these,Harbo (1988) examined the relationshipbetween colony size, brood productionand combs for colonies that were totallyequalized. He found that those colonieswhich had produced the largest amountof comb had also produced the largestnumber of brood and adult workers. To

separate queen from comb effects, a sec-ond experiment was performed using largeand small combs as the variables of inter-est. Comb effects were significant (queensnot) and small combs resulted in reducedbrood production.

But there is more to a colony in a cav-ity, and the variables richer than had beenassessed. Harbo (1993) extended his find-ings to assess the effects of nest cavity

(hive) volume on growth and productivityby adjusting the population density againstvolume. In winter, crowded bees con-sumed less honey per bee and reared lessbrood than the less crowded colonies. Dur-

ing the flows of spring through autumn,the crowded colonies produced morehoney but less brood than the less crowdedones. In another experiment, comb effectswere tested against space effects. Bothaffected brood rearing and honey produc-tion. Colonies with combless extra spaceproduced less honey and more brood thanthose with the same amount of comb butless space (Harbo, 1993). These resultscomplement those of Taranov (1959) andSzabo (1977) who had shown that broodproduction and comb construction are notcompetitive activities: the exclusion ofone activity does not accelerate the other.

4.3. Nectar and pollen

With an age-related division of labormechanism for wax established, one mustconsider the development of wholecolonies in a seasonal and interseasonalcontext. It is a common observation thatthe comb is built in response to colonyneed especially given a strong nectar flow.For example, Seeley (1995) reports theresults of some interesting and pertinentexperiments. In observation hives in whichthe manipulated variable was the amountof nectar on hand, comb building was ini-tiated during an artificial flow when thehoney storage cells were nearly full. Dura-tion of the comb building activity was afunction of both intensity and duration offlow. This routine principle of commer-

cial beekeeping has now acquired addi-tional experimental support (Seeley, 1995).

The fundamental importance of pollenas a source of protein for wax secretionhas long been established [cf. referencesin Hepburn (1986)]. However, bee-combinteractions with respect to pollen havebeen little studied. Camazine (1993)reported on a series of experiments to elu-cidate how a colony might regulate pollenforage: 1) direct assessment of pollenstocks by individual foragers; or 2) indi-rectly in which one group, house bees,might make such a determination andsomehow inform pollen foragers accord-ingly. In one experiment pollen supple-mentation led to a reduction in pollen for-age; another showed that foragers do notrequire direct contact with pollen stores toadjust their foraging. Subsequent experi-ments to assess the role of pollen odor wereconflated by brood odor. So while the basicapproach remains viable, this problem mustbe solved in future analyses.

4.4. The queen

Other indirect evidence for the Butlermechanism comes from studies of swarm-

ing and absconding. Comparisons of work-ers from natural swarms, heavily engorgedwith nectar, with those of settled coloniesdemonstrated that more swarm bees borewax (not amount of wax per bee) thantheir settled counterparts. It also transpiredthat there were no measurable differencesin the pheromonal bouquets of themandibular glands of the relevant queens(a decade ago the general explanatorypanacea for queen-worker interactions)which cast doubt on their efficacy in stim-ulating wax secretion (Hepburn and Whif-fler, 1988). Likewise, in abscondingcolonies, just as many bees bore wax whenheaded by free-running queens as thoselacking physical access to her presencebecause of a cage of double-gauze (Hep-burn, 1988).

Thus, the possible importance of thequeen for wax secretion needed to beaddressed systematically. This wasachieved in a series of experimentsemploying various queen states: queen-less, mated and virgin queens as well asdead ones to obtain a pheromonal spec-trum. Experimental manipulationsincluded surgical ablation and occlusion ofthe mandibular and tergal abdominalglands, respectively, and the use of divi-sion boards to restrict the access of dividedcolonies to different parts of the queen’sbody. At the end of the trials the amount ofwax recoverable from festoon bees didnot significantly differ: the physical andchemical queen had no measurable effecton wax secretion but did on comb building(Whiffler and Hepburn, 1991a). That asmuch as half of the standing stock of waxscales is distributed among non-festoonbees was only discovered somewhat later(Muller and Hepburn, 1992).

4.5. The cycle of wax secretion

These experiments had been performedin different seasons with varying field con-ditions as well as races of bees, so morecontrolled information on wax secretionwas needed. First, using bees of preciselyknown age, it was shown that secretionwas a continuous process, there was nodiel rhythm (Hepburn and Muller, 1988).This led to a 2-year study of wax secre-tion in queenright colonies from whichthe wax scales of some 11 000 bees wererecovered and weighed. Wax secretionwas parabolically cyclic and related toage: secretion begins at about 3 days afteremergence, reaches a peak between 9 and12 days and wanes between 18 and 21days (Hepburn et al., 1991). This thenfilled the gap between the histologicalobservations of Rösch (1927) and thephysiology of the secretory cycles.

The cyclical changes of cellularorganelles and the chemical compositionof beeswax precursors found in the

haemolymph (Francis et al., 1989) andgland tissues closely coincide with mea-sured, age-related rates of wax secretion(Hepburn et al., 1991). Attempts to alterthis cycle by increasing or decreasing theamounts of juvenile hormone and or theaddition or removal of the corpora allatahad no measurable effect on the onset ofwax secretion, its duration or the amountof wax actually produced for this agecohort (Muller and Hepburn, 1994). Thuswax secretion is one of the division oflabor activities for a certain age cohort,but may vary with the proportions of sub-ordinate and dominant workers

(Hillesheim et al., 1989). This coincidenceof physiology and behavior parallels otherpolyethisms such as colony defense (Whif-fler et al., 1988; Breed et al., 1990) andbrood care (Liu, 1989; Crailsheim andStolberg, 1989): all are predictable activ-ities correlated with age and cycles ofglandular function.

5. DISTRIBUTIONOF COMB CONTENTS

It has been tacitly assumed for centuriesthat the patterns observable in the arrange-ment of nest contents is in some mysteri-ous way ’in the nature of bees’. However,the casual observation that pollen andhoney are regularly deposited in emptycells within the brood area during the day,only to be removed to their ’proper’ placesduring the night, led to an especially sem-inal paper on pattern formation in combuse in honeybees by Camazine (1991). Heconducted experiments to validate one oftwo mutually exclusive hypotheses: 1) ablueprint or template in which patternsdevelop in some pre-ordained and speci-fied way intrinsic to bees; or 2) a self-organization mechanism in which patternemerges spontaneously from the dynamic

interactions among the processes of plac-ing and then displacing the relevant nestelements.

In a series of classically simple obser-vations and experiments Camazine (1991)noted that the brood pattern is initiated bythe laying habits of the queen who musttake into account the presence of nearbybrood and, perhaps, the comb boundaries.This given, the queen lays eggs and thebees deposit both nectar and pollen hap-hazardly among the combs in the firstinstance. Possibly informed by the pres-ence of young nurse bees, the queen doesnot lay eggs outside of the nascent broodarea but continually searches for emptycells near other eggs or brood. Cells in the

brood area filled with honey or pollen arepreferentially emptied of their contents.This is experimentally shown by a fre-quency distribution for cell emptying fromthe brood area. It is a function of distanceto the nearest brood cell. Brood cells so

emptied of nectar and pollen are thenfound by the queen who lays in them, andso the pattern develops (figure 4).

Camazine (1991) and colleagues thenproceeded to develop a computer simula-tion model to establish pattern-formingrules as estimated from the actual experi-ments. Using the empirical events fromthe observation hive as the parameter val-ues, he was able to reveal the interactingprocesses that contribute to pattern for-mation. The simulation also produced thefinal pattern observed in the observationhive and confirms the interpretation ofpattern formation. The model and the self-

organization hypothesis appear extremelyrobust and parsimonious. This idea hasbeen further analyzed mathematically byJenkins et al. (1992) who derived rate con-stants for the removal and redeposition ofhoney and pollen in order to achieve theircharacteristic bands and positions abovethe brood areas (figure 4).

6. COMBS ANDCOMMUNICATION

6.1. Queen cells

While there is substantial evidence thatwax secretion is not influenced by the sta-tus or quality of the queen, comb build-ing and queen cell construction are dif-ferent matters. The construction of queencells is contextually quite different fromthat of combs, but in both the role of the

queen is significant. There are two impor-tant observations to consider. First, Boch(1979) showed that when a closed queencell was given to a small queenless colony,further queen cell construction was greatlyreduced in comparison to control colonies.Second, Fell and Morse (1984) demon-strated a rapid decline in the number ofnew queen cells built following construc-tion of those cells found on the first dayafter queen loss.

Unfortunately, yet other experimentswhich implied a pheromonal role for the

queen in emergency queen cell construc-tion led to conflicting results [cf. discus-sion and references in Whiffler and Hep-bum (1991a)]. To resolve these problems,these authors measured the effects of

queen state on the inhibition of emergencyqueen cell construction exactly as had beencarried out for comb building using free-running, caged and division board queens(Whiffler and Hepburn, 1991b). Greatestinhibition of queen cells was achieved incolonies with free-running queens andleast in queenless ones. For the former,queen inhibition diminished with increas-

ing impairment of pheromonal functionthrough removal of the mandibular glandsand occlusion of the abdominal tergiteglands. Nonetheless, there was markedvariation within and between treatment

groups just as had been found in otherstudies (Fell and Morse, 1984; Winstonet al., 1989; Kaminski et al., 1990) andfor which there was no obvious explana-tion.

It is now evident that the inhibition of

emergency queen cell construction is notan all-or-nothing phenomenon but iseffected as a series of graded responses(Hepburn et al., 1988; Engels et al., 1993).All of the experimental data on this prob-lem can now be accommodated within an

interpretation that the relevant pheromonalscent of the queen can be recovered not

only from the site of secretion but fromall parts of her body, including the legs(Winston et al., 1989; Slessor et al., 1990;Engels et al., 1993). In this view inhibi-tion of emergency queen cell constructionis primarily mediated by pheromones ofthe mandibular gland spread over thewhole surface of the queen as originallysupposed by Butler (1954), as well as bycontributions from the tarsal and abdom-inal tergite glands (Lensky and Cassier,1992).

There is also an element of the combinvolved in the inhibition of swarmingqueen cell construction. Lensky and

Slabeski (1981) added footprint (or tarsalgland) pheromone to the inhibitory effect.They demonstrated that in populous andcrowded queenright colonies that swarmcells are constructed at the bottom edges ofthe combs because the queen does notmove to the edges of the nest. Hence combedges lack footprint pheromone. Whenthey placed a combination of mandibulargland and tarsal gland extracts on the bot-tom edges of the combs swarm queen cellconstruction was inhibited. Similarly, if afresh comb surface is provided, unmarkedby pheromones of the queen, queen cellconstruction will proceed. Under emer-gency conditions, the majority of queencells are constructed in the center of thebrood area based on cells containing eggsor larvae (Lensky, pers. comm.). This alsoexplains the fact that there is no measur-able change in the pheromonal bouquetof the mandibular glands of queens whosecolonies are preparing to swarm (Seeleyand Fell, 1981). In this case comb wax isthe intermediate in the transmission of the

inhibitory pheromones.In this discussion the chemical aspects

of the queens’ pheromones have beenemphasized. Due to the abundance andcomplexity of these bouquets, the signif-icance of individual and/or permutations ofcompounds has not been precisely deter-mined. It should also be remembered thatthe control of queen cup and swarmingcell construction is complex and dependson both ecological (temperature, avail-ability of nectar) and biological factorssuch as worker density (Lensky andCassier, 1992).

6.2. Cell capping and repairs

In studies on the chemical ecology ofbrood pheromones, Le Conte et al. ( 1990)demonstrated that a series of only fourmethyl esters (part of a normal

pheromonal cocktail produced by queens,

workers and drones) is sufficient to inducethe capping of cells containing mature lar-vae. These compounds are multifunctionaland differing ratios modulate the feedingbehavior of worker bees or lead to cell

capping (Trouiller et al., 1991; Trouiller,1993; Le Conte et al., 1995a). In the caseof queen cells, the developmental progressof the larvae is constantly signaled by theester ratios. This was confirmed by usingsubstitutes doped with the pheromones(Le Conte et al., 1995b). Following cap-ping of the doped dummy queens theircontinued acceptance in the colonydepended on their ability to emit the cor-rect esters. Presumably, this also explainsthe inhibition of queen cell constructionin the experiment of Boch (1979).

There remains the vexing problem ofsignal specificity. At any given time a sub-stantial portion of the larval populationcould be emitting capping signals. Thesemight only indicate that there is an areaof brood comb requiring the attention ofhouse bees to perform capping. Goetz andKoeniger (1992) reasoned that if cappingdepends entirely on pheromones thenbrood cells should be capped accordingto larval age. Alternatively if capping canbe advanced by decreasing cell length, ordelayed by increasing it, then larval size inrelation to cell depth could be important intriggering capping behavior. They exper-imentally modified larval distance fromthe cell opening by artificially increasingor decreasing cell depth. Their resultsseemed to show that the worker bees

responded according to the distanceparameter and not age (Goetz andKoeniger, 1992).

Subsequently, Le Conte et al. (1994)revisited this problem. Having previouslyshown that workers will cap cells con-

taining paraffin dummies doped with theester blend of old larvae (Le Conte et al.,1990) they then proceeded to a more thor-ough analysis of pheromonal (ester) con-centration, the position of the larvae in the

cells, the effects of ester blends on workercapping and then as pertains to queen cells.Their results showed that capping activ-ity depended on ester concentrations whichsuggest that the presence of a larva is

pheromonally mediated. The position ofthe larva influences capping, possibly byaffecting the head-space of pheromoneavailable to stimulate workers. In tests ofblend discrimination, dummies doped withthe young larval blend (ethyl esters) werenot capped, dummies with the old larvalblend (methyl esters) induced capping.

Le Conte et al. (1994) conducted furthertests on queen cell construction and cap-ping in dequeened colonies in which theeffects of both larval position andpheromonal blend were measured. Great-est queen cell construction occurred whendummies were doped with the young lar-val blend and positioned at the bottom ofthe cells. Thus, for both the capping ofworker larvae and queen cell constructionthe results of Le Conte et al. (1994) indi-cate that workers can and do discriminatebetween young and old larvae, the bou-quets of their respective ester blends andthe positions of the larvae in their cells.The combined results of Goetz and

Koeniger (1992) and those of Le Conte etal. (1990, 1994) are not mutually exclu-sive in principle; rather, more experimen-tation is needed to establish their possibleinterrelationships.

The honeybee approach to obstaclesand necessary repairs to the combs havebeen thoroughly documented by Darchen(1968; and references therein). An inter-esting idea that Darchen had but appar-ently never tested, was that once com-pleted cells had been put into use theywere primed or painted with some propo-lis- or varnish-like material which inhib-ited bees from further modifying them. Ifa piece of comb is excised experimentally,the bees soon repair it, the interpretationbeing that the inhibitory substance wasremoved. Chauvin (1992) recently

reported that such a propolis-like materialoccurs on the outer edges of cells and onlythere. So here too a chemical cue is pro-vided by the combs which direct the activ-ities of worker bees.

6.3. Colony odor

Another way in which the bees andtheir combs interact is in the generationand recognition of a colony odor, an ideafrom a previous century brought to fruitionby Kalmus and Ribbands (1952). A fewyears later Juska (1978) contributed furtherto the identification of nest-mate recog-nition cues by demonstrating that foot-print or trail scents are deposited on thecombs from the tarsal glands as the queenmoves about. That the scent persists forsome time led to the conclusion that it is anadditional way of communicating queen-rightness. That the combs might acquireand then dispense the chemical signatureof the queen is a parsimonious way of pro-ducing a general signal. Here, as in inhi-bition of queen cell construction on thebottom edges of the comb, the integrativesignal is mediated through the combs.

The idea of colony odor was pursuedwith this view by Breed et al. (1988a) whodiscovered that colony odor (their ’hiveeffect’) is acquired very soon after adulteclosion and, in cross-fostering experi-ments, the effect of colony odor com-pletely masked genetic differencesbetween the bees. The mechanism for this result was pursued in a second work(Breed et al., 1988b) when comb-condi-tioned bees were introduced to otherrelated but comb-naive bees, the formerwere rejected; when the receiving beeswere comb-conditioned to the same combsource as the introduced bees the latterwere accepted. Whatever the origin of thebees, bees conditioned to the same combsource were more readily acceptable thanbees between comb-type transfers.

Although there was no specific controlfor individual bee odors as such in theBreed experiments, it is reasonable toassume that they would have been over-ridden when different bees conditioned tothe same comb source were found to be

mutually acceptable. It is pertinent to recallhere that Michelsen et al. (1989) alwayshad to ’condition’ their mechanical danc-

ing bees to the hive odor of the test colonyin order to stave off attacks on the motor-ized dancers. Because beeswax is a chem-ical potpourri, Breed et al. (1988b)repeated the work with a neutral paraffinwax to which scent was added or not andthe same kinds of results were obtainedas had been with the beeswax.

It remains uncertain, of course, whetherthe observed effects derive from geneticdifferences in chemistry of comb sourceand/or the possible adsorption of envi-ronmental odors onto the waxes. The two

possibilities are certainly not mutuallyexclusive. A common mechanism couldwell be the interaction between shared

pools of adsorbed chemical volatiles fromthe epicuticle of the bee as well as thecomb. This kind of mechanism would

explain how flower volatiles that changewith changing plant forage become incor-porated into the colony system of chang-ing colony odor.

Comb wax mediates other forms of

honeybee behavior as well. For example,Fergusson and Free (1981) found that theodor of comb alone was sufficient stimu-lus for workers to release scent from theNasanov gland, a common source of clus-ter-inducing pheromone. This is consis-tent with the fact that honeybees preferold combs to new ones, possibly becausethey are dosed with footprint pheromone(Free, 1987). Comb volatiles also stimulateincreased nectar foraging (Rinderer andHagstad, 1984; and references therein),but bioassays of oxygenated combvolatiles were equivocal (Blum et al.,1988). A final case is that colonies given

large amounts of empty comb attack tar-gets twice as fast and often as colonieswith little empty comb (Collins andRinderer, 1985). Presumably, a surfeit ofcomb volatiles signals a shortage ofreserves and the advantage of protectingthem against predators/robbers.

6.4. Communication of sounds

Signals or cues arising from the combsinclude mechanical as well as chemicalinformation. Indeed, following the dis-covery of the dance language, its mode ofactual transmission long remained enig-matic. When potential recruits attend adancing forager, they periodically emitvibrations against the combs which elicita response from the dancer to give theemitter a sample of her nectar (Michelsenet al., 1986a; Sandeman et al., 1996). Sim-ilar vibrations, the tremble or stop dance,are made by returning foragers who havedifficulty in off-loading nectar to housebees. The signal here may be used as anegative feedback way of reducing fur-ther forager recruitment (Seeley, 1992;Kirchner, 1993; Nieh, 1993) and/or as ameans of recruiting more nectar-receiverbees (Seeley, 1992). Finally, queen pip-ing is mediated through vibration of thecombs (Michelsen et al., 1986b). This andthe wing vibrations of dancers are per-ceived by Johnston’s organ.

Other bee-comb interactions of a recip-rocal nature involve the transmission ofvibrations (sounds) for communication.One of the events that occurs among bees

tending a dancing forager is that periodi-cally a worker presses her thorax downon the comb to vibrate it. This often elic-its a response from the dancer to give theemitter a sample of nectar (Michelsen etal., 1986a). The tremble or stop dance(mentioned previously) works acousticallyin the same way (Kirchner, 1993). A thirdexample is that of queen piping in which

the quacking element emitted by virginqueens still in their cells is transmitted

through the wax (Michelsen et al., 1986b).In all of these examples, transmission ofvibrations or sounds through a mediumdepends on the density, elasticity and tem-perature of the substrate. For practical pur-poses of the honeybee nest, temperatureand density can be taken as constants, sothat the greater the stiffness, the higherthe speed of transmission. Because soundintensity decreases with the density of themedium and with distance, an essentiallyanhydrous, low density wax is the idealsolid biological medium for sound trans-mission.

Kirchner (1993) indicated some impor-tant differences in communication bysounds and pheromones. Vibrational sig-nals allow for the speedier transmissionof a signal, a temporal coding element,and localization of the message throughrapid attenuation of the signal.Pheromones and other chemical signalsare pervasive in distribution and becausethey follow the laws of diffusion there is along time constant for the life of the signal(Kirchner, 1993). A perfect example ofthis effect lies in colony odor and theintranest transmission of queenrightness.Naumann et al. (1992) have recentlyshown that some of the queen’s mandibu-lar gland pheromonal components secretedon her surface are acquired by workers ofthe retinue. Workers having contact withretinue bees acquire the signal themselves.However the signal is also spread in foot-prints from the passage of the queen andworkers onto the combs in which it dif-fuses and is slowly but eventually lostfrom the system (Naumann et al., 1992).

6.5. The waggle dance

Having seen that the delivery of foragefrom the field and the further recruitmentof foragers are associated with the trans-

mission of sound through the comb, onecan ask if there are specific sites for wag-gle dances. Tautz (1996) discovered thatforagers that danced on empty areas ofcomb recruited three times as many dancefollowers as bees that danced on areas of

capped brood. Clearly the recruitment pro-cess could be enhanced if some particu-lar site on the comb was mutually recog-nizable to both dancers and dancefollowers. Indeed, dancing bees are com-monly found on the lower comb near theentrance of observation hives (Tautz,1996).

The existence of specific dance siteson combs was very recently demonstratedby Tautz and Lindauer (1997) in a mostelegant way with a two-frame observationhive modified to have the entrance half

way up the side. After the bees weretrained to a non-scented sugar solution ina feeder nearby, the location of dancingbees was noted for several days. Foragerswere then marked and allowed to visit thefeeders and return and dance in the hive.After a while the feeders were closed andall of the bees were shaken out of the hiveand the positions of the two combsswitched: upper comb became bottomcomb and vice-versa. The bees were thenallowed back into the hive, the feedersreopened and the sites of the dancersrecorded. All of the dances performed byall 20 of the marked dancers on the combswere recorded before and after switchingthe comb positions during seven suchexchanges in subsequent days. In the totalseries of seven such comb switches in

nearly 90 % of 365 dance episodes, thedancers went to that site on the specificcomb on which the dance had first been

established, and irrespective of the positionof the comb in the hive.

Tautz and Lindauer (1997) consideredthe possibility of the bees using spatialcues but noted that after combs wereswitched the bees also walked over thecombs until they located the site of the

first dance before recommencing dancing.They suggested that the rediscovery of aspecific dance site would be most parsi-moniously explained by a chemical marker(footprint pheromone) that would allowsite reinforcement throughout the day butwhich would fade at night thus allowingthe location of a dance site to change withchanging conditions in the nest. By thesame token, many of the recently discov-ered volatiles (cf. mechano-chemicalchanges above) could serve as cues fordance recruits, if they too seek the samedance site after comb switching. Thiscould hold important implications for theanalysis of patriline performance in thedivision of labor.

7. CONCLUDING REMARKS

The material properties of the combprovide both the structure necessary toaccommodate the colony as well as amulti-faceted medium for chemical andmechanical communication. Through theirmanipulation of wax and then comb, thebees are provided with a system of cuesof varying time constants which in turnmodify their behavior. In this context, ther-moregulation has been evolutionarily cru-cial in obtaining the best performance ofthe comb materials and also the enzymesystems of the bees themselves. At thesame time, new discoveries of the uniquechemical signatures in the waxes of dif-ferent subspecies and the roles of chemi-cals in queen cell construction, cell cap-ping and the waggle dance of honeybeesmay have opened a Pandora’s box ofunexpected factorial complexity.

Nonetheless, we have progressed farenough to see that virtually the whole ofthe comb building process and the pat-terns that emerge from its use can both be

explained parsimoniously and predictivelyby self-organization. This at least elimi-nates having to impose demands on the

neurophysiological capacity or even thegenome of honeybees for which there isno evidence. Honeybee society operatesin three physical dimensions which varywith the activities of the bees. The entire

system is in a constant state of flux in afourth dimension, time. This is particu-larly evident in the many reciprocal inter-actions between bees and their combs asboth are born, grow and mature. Furtheranalysis of this n-dimensionality mightwell profit from the mathematical powerof virtual reality techniques.

ACKNOWLEDGEMENTS

I thank J.R. Harbo, Y. Le Conte, Y. Lenskyand W.J. Muller for kindly reviewing this workin manuscript form.

Résumé - Interactions réciproquesentre les abeilles mellifères et les rayonsdans l’intégration de certaines fonctionsde la colonie chez Apis mellifera L. Cetarticle résume les progrès récents dansl’étude des interactions réciproques entreles abeilles mellifères et leurs rayons dansla mesure où elles concernent l’intégra-tion des fonctions de la colonie. Le choixdu volume de la cavité du nid varie d’unerace à l’autre, mais une fois les abeillesinstallées, la construction des rayons quis’ensuit peut être considérée comme un« processus d’autoorganisation »(figure 1), dans lequel les dimensions de laconstruction et les écarts admissibles peu-vent simplement découler de la proprio-ception des abeilles en position dos-à-dos.

Au cours de la construction des rayons,les écailles de cire subissent des modifi-cations physiques et chimiques par addi-tion d’enzymes, qui changent les liaisonschimiques et renforcent ainsi les rayons(figure 2). Dans le nid terminé la présencecombinée des abeilles et des rayonsconduit à une isolation thermique plus

grande que celle fournie par chacune desparties.

La construction des rayons est réguléepar la saison. Les abeilles cirières sont

présentes dans tout le nid et pas seulementdans les chaines cirières. Les interactionsnid-abeille qui affectent la constructioncomprennent le volume de la cavité,l’espace disponible pour le stockage, lataille de la population, l’élevage du cou-vain et le flux de nectar (figure 3).

Une plus grande tendance à la construc-tion est associée à la présence d’une reineet existe aussi pendant l’essaimage et ladésertion. La secrétion de cire proprementdite est un cycle lié à l’âge au sein de ladivision du travail et n’est pas affectée parla reine.

La façon la plus économe d’expliquer larépartition de l’utilisation des rayons entremiel, pollen et nid à couvain est de consi-dérer qu’il s’agit d’un processus d’auto-organisation, qui ne présuppose aucun pland’ensemble dans le cerveau de l’abeille.Le processus a été modélisé mathémati-

quement à partir de données réelles et s’estrévélé avoir une valeur prédictive repo-sant sur quelques règles simples (figure 4).

Les rayons jouent un rôle très particu-lier dans la communication au sein de lacolonie. La présence de phéromonesroyales influe sur le déclenchement de laconstruction de cellules royales, sur lemoment et le lieu où elles vont êtreconstruites. De la même façon, l’opercu-lation du couvain semble être comman-dée par la combinaison d’une phéromonede couvain et de variables physiques tellesque le rapport entre la taille de la larve etcelle de la cellule. Le travail de réparationdes rayons est stimulé par l’absence d’unesubstance inhibitrice sur les bords des cel-lules. L’odeur de la colonie est commu-

niquée par la cire des rayons et influenceégalement la défense de la colonie. Quele « plancher de danse » semble être définichimiquement est une découverte d’ungrand intérêt.

On a montré récemment que les rayonsfaisaient partie intégrante de la commu-nication sonore par les vibrations, qui sontessentielles pour le recrutement des buti-neuses lors de la danse frétillante. De lamême façon, le chant des reines agit partransmission des vibrations par le rayon.La cire est un milieu idéal et elle permet latransmission rapide de messages codés.Par ailleurs les phéromones obéissent auxlois de la diffusion avec une constante de

temps longue.Il existe entre les abeilles et leurs nids

tout un ensemble d’interactions réci-

proques qui varient dans le temps plus oumoins rapidement. Puisqu’il devient deplus en plus difficile de suivre simultané-ment l’action des variables connues, nous

pourrions tirer profit à l’avenir des tech-niques mathématiques, utilisées dans laconstruction des « réalités virtuelles »,pour étudier les abeilles. © Inra/DIB/

AGIB/Elsevier, Paris

Apis mellifera / auto-organisation /rayon / phéromone / communicationsonore

Zusammenfassung - Die Rolle wech-selseitiger Beziehungen zwischen

Honigbienen und Waben bei der Inte-gration einiger Koloniefunktionen vonApis mellifera. Dieser Artikel faßt neueErkenntnisse über die wechselseitigenBeziehungen zwischen Honigbienen undihren Waben zusammen, soweit diese fürdie Integration der Koloniefunktionen vonBedeutung sind. Verschiedene Bienen-rassen unterscheiden sich in der Auswahlder Nisthöhlen. Sobald die Bienen sich

niedergelassen haben, kann das darauf-folgende Bauverhalten als ein selbstorga-nisierender Prozess verstanden werden

(figure 1), bei dem die Baumaße und diedabei eingehaltenen Toleranzen in einfa-cher Weise von der Propriorezeption vonBienen in Rücken - an Rücken - Position

abgeleitet werden kann.

Während des Wabenbaus werden die

Wachsplättchen physikalisch und che-misch durch Zufügen von Enzymen modi-fiziert. Diese verändern die chemischen

Bindungen und versteifen hierdurch dieWaben (figure 2). In dem fertigen Nest-bau bewirken die Bienen in Verbindungmit den Waben eine höhere thermischeIsolation als jede der Komponenten fürsich.

Der Wabenbau unterliegt einer jahres-zeitlichen Steuerung. Hierbei treten wachs-erzeugende Bienen nicht nur bei den Bau-bienen, sondern überall im Nest auf. DieAusscheidung von Wachs ist ein alters-abhängiger Prozess innerhalb des Musterstemporärer Arbeitsteilung. Dieser wirdvon der Anwesenheit einer Königin nichtbeeinflusst.

Einflußgrößen, die vom Nest auf dasBauverhalten der Bienen wirken, umfassendas Volumen der Nisthöhle, den vorhan-denen Speicherplatz, die Populationsgröße,die Brutaufzucht und den Nektarfluss

(figure 3). Die Neigung zum Wabenbauwird durch die Anwesenheit einer Köni-

gin, durch Schwärmen und den Auszugaus der Nisthöhle verstärkt.

Die sparsamste Erklärung der Vertei-lung der Wabennutzung bei der Speiche-rung von Honig und Nektar und derAnlage von Brutzellen ist ein selbstorga-nisierender Prozess, der keinen Gesamt-plan im Gehirn der Honigbiene voraus-setzt. Dieser Prozess konnte unter

Annahme weniger einfacher, auf realenUntersuchungsergebnissen basierenderRegeln mathematisch als voraussagungs-fähiges Modell simuliert werden (figure 4).

Die Waben spielen in der Kommuni-kation innerhalb des Bienenvolkes einebesondere Rolle. Die Anwesenheit von

Königinnenpheromon beeinflußt, ob, wound zu welcher Zeit Königinnenzellenangelegt werden. Ebenso scheint die Ver-deckelung der Brutzellen durch die Kom-bination eines Brutpheromons mit physi-kalischen Variablen wie dem Verhältnis

der Größe der Larven zur Zellgrößegesteuert zu werden. Die Reparatur derWaben wird durch die Abwesenheit inhi-bitorischer Substanzen an den Zellenrän-dem stimuliert. Der Wabenduft wird durchdas Wabenwachs übertragen und beein-flußt auch das Verteidigungsverhalten. Esist eine interessante Entdeckung, daß der’Tanzboden’ der Sammelbienen chemischdefiniert zu sein scheint.

Kürzlich konnte gezeigt werden, daßdie Waben integraler Bestandteil derVibrationskommunikation bei der Anwer-

bung von Sammlerinnen während desSchwänzeltanzes sind. In ähnlicher Weise

erfolgt auch die Übertragung des Quakensder Königinnen durch die Waben. Wachsist hierbei ein ideales Medium zur Wei-

terleitung von Vibrationen. Durch diesekönnen zeitlich codierte Nachrichtenschnell übertragen werden. Im Gegensatzzu Pheromonen, die eine lange Zeitkon-stante haben, unterliegen Vibrationen einerraschen Abschwächung.

Zwischen Honigbienen und ihrenNestern bestehen eine Vielzahl rezipro-ker Beziehungen, die sich über die Zeit inunterschiedlichen Geschwindigkeiten ver-ändern. Da die gleichzeitige Verfolgungdes Einflusses der bekannten Variablenzunehmend schwieriger wird, werden wirin Zukunft wahrscheinlich von der

Anwendung der in der Konstruktion ’vir-tueller Realitäten’ verwendeten mathe-matischen Techniken auf das Studium der

Honigbienen profitieren. © Inra/DIB/

AGIB/Elsevier, Paris

Apis naellifera / Waben / Pheromone /Schall / Selbstorganisation

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