Influence of Social Environment on Genetically Based Subfamily Signature in the Honeybee

Journal of Chemical Ecology, Vol. 26, No. 10, 2000

0098-0331/ 00/ 1000-2321$18.00/ 0 2000 Plenum Publishing Corporation

2321

INFLUENCE OF SOCIAL ENVIRONMENT ONGENETICALLY BASED SUBFAMILY SIGNATURE

IN THE HONEYBEE

GERARD ARNOLD,1,* BRIGITTE QUENET,2

and CLAUDINE MASSON1

1Neurobiologie Experimentale et Theorie des Systemes complexes, CNRS UPR 908116 rue Claude Bernard, 75231 Paris cedex 05, France

2Neurobiologie Experimentale et Theorie des Systemes complexes, CNRS UPR 9081, ESPCI10 rue Vauquelin, 75005 Paris, France

(Received May 25, 1999; accepted May 30, 2000)

Abstract—In honeybees, the cuticular hydrocarbon profiles are partly geneti-cally based and differ between subfamilies, which suggests that they mightbe used by the workers as labels for subfamily recognition. This ability couldpotentially form the basis for nepotistic conflicts between subfamilies thatwould be detrimental to the inclusive fitness of the colony. Here we have com-pared the subfamily hydrocarbon profiles of 5-day-old workers maintained inisolation with those kept in their parental colony. We demonstrate that thecuticular hydrocarbon profiles tend to be less distant between most subfami-lies within the hive compared with those held in isolation. The main conse-quence of this partial homogenization of the majority of subfamily signaturesmay result in a reduction of the number of recognizable subfamilies in thecolony. Nevertheless, a few subfamilies retain very distinct cuticular hydro-carbon profiles.

Key Words—Honeybee, Apis mellifera, kin recognition, cuticular hydro-carbons, chemical signature, genetic relatedness, subfamilies, nepotism.

INTRODUCTION

Within honeybee colonies, polyandry leads to the presence of 7–20 subfamilies(Estoup et al., 1994). The worker with an ability to discriminate between super-sisters (coefficient of relationship r c 0.75) and half-sisters (r c 0.25) should havea large fitness advantage with respect to nepotistic behavior, but this ability, ifexpressed, would form the basis for conflicts between the workers of the various

*Present address: Laboratoire PGE, CNRS, BP1, 91198 Gif.sur.Yvette, France.

ARNOLD, QUENET, AND MASSON2322

subfamilies. This should be detrimental to the overall colony (Moritz and South-wick, 1992; Ratnieks and Reeve, 1992). Thus, the colony may benefit by reducingor eliminating information about subfamily discrimination (Keller, 1997).

A subfamily level of recognition is thought to exist in honeybees (Page etal., 1991; Getz and Smith, 1983), but it has been demonstrated only for artificialcolonies consisting of two subfamilies. In such colonies, recruits have a strong ten-dency to follow dances performed by a member of their own subfamily (Oldroyd etal., 1991), and workers groom and feed a larger number of supersisters (Frumhoffand Schneider, 1987). Even in the cases for which there is a statistically significanttrend for workers to favor their supersisters, this preference generally is expressedweakly (Breed et al., 1994). In order to explain this weak preference, these authorssuggested that the worker bees may simply be incapable of producing or perceiv-ing subfamily recognition signals on a fine enough scale to discriminate amongtheir sisters. Alternatively, Keller (1997) suggested that kin discrimination mightbe selectively disfavored by scrambling recognition labels. This could be achieved,for example, by transferring odors between colony members.

In social insects, cuticular hydrocarbons are involved in kin recognition atdifferent levels including species, colony, and caste levels (Howard, 1993; Nel-son and Blomquist, 1995). In honeybees, the cuticle contains a mixture of differ-ent hydrocarbons that present quantitative variations among individuals (Franciset al., 1989; Page et al., 1991). Using a behavioral bioassay, Getz and Smith(1987) showed that workers do indeed perceive and respond to C23 and C25

alkanes and discriminate between mixtures of them as long as their proportionsare relatively dissimilar.

In previous work, we demonstrated that cuticular hydrocarbon profiles of5-day-old workers from a normal colony differ significantly among subfamiliesdepending upon rearing conditions; isolated, in groups of 10 in an incubator, ormatured in their parental colony (Arnold et al., 1996). In that study, we positedthat isolated bees have a cuticular hydrocarbon composition of purely geneticorigin, unlike both the grouped bees and the hive bees where hydrocarbons maybe influenced from contact with other workers (and for the hive bees, from theenvironment). From our earlier results with isolated bees, we concluded that thecuticular hydrocarbon profiles are genetically based (Arnold et al., 1996). Sincethe cuticular hydrocarbon profiles of the hive bees differ significantly betweenthe subfamilies, we concluded that they may be used as labels for subfamilyrecognition by workers in the hive. Therefore, in 5-day-old hive bees, the modi-fications of hydrocarbon profiles by environmentally acquired cues and/ or nest-mate contact were not sufficient to completely homogenize subfamily profiles.

In the present paper, we have focused the statistical analysis of our data(Arnold et al., 1996) on the trends of homogenization of the hydrocarbon pro-files under hive conditions and the influence of the social environment. We havecompared the subfamily cuticular hydrocarbon profiles of hive bees, which we

HONEYBEE SUBFAMILY SIGNATURE 2323

believe to have both genetic and environmental origins, and those of the isolatedbees, which we believe to have a purely genetic origin. We show that duringdevelopment of socially reared workers, subfamily signatures tend to converge(although they do not become completely homogeneous), among the subfami-lies; a few subfamilies retain distinct profiles.

METHODS AND MATERIALS

Insects

As previously described (Arnold et al., 1996), emerging workers of Apismellifera mellifera were collected from one colony and were maintained undertwo different conditions, either isolated (N c 200) or in a hive (N c 200), untilthe age of 5 days.

The hive bees were marked at emergence with a dot of paint on the thoraxand maintained in their parental colony. The isolated bees were placed in 50-mlpolypropylene tubes and held in an incubator at 338C. This temperature corre-sponds to the mean temperature of the brood area of the colony where honeybeeworkers stay during the first five days of their life. Isolated bees were fed withcandy (mixture of honey and sugar) containing 5% pollen. The hive bees werefed with the honey and pollen contained in the combs.

In total, 117 isolated bees and 117 hive bees were individually analyzed fortheir genetic relatedness and hydrocarbon composition of their cuticule.

Assessment of Genetic Relatedness in the Colony

Workers were assigned to their respective subfamily based upon two highlyvariable microsatellite loci (A76, A107) (Estoup et al., 1994), genetic markersunrelated to cuticle hydrocarbons.

DNA was extracted according to Garnery et al. (1990). PCR reaction wasconducted with a 10 ml solution containing 5–10 ng DNA template; 400 nMof each primer; 75 mM each of dGTP, dCTP, and dTTP; 6 mM dATP, 0.7mCi[35S]dATP, 1–1.2 mM MgCl2, 20 mg/ ml bovine serum albumin, 1× Promegareaction buffer, and 0.4 unit of Promega Taq polymerase. After a denaturing stepof 3 min at 948C, samples were processed through 30 cycles consisting of 30sec at 948C, 30 sec at 58–608C, and 30 sec at 728C. The last elongation stepwas lengthened to 10 min. A sample of 2 ml of each reaction was run on 6%polyacrylamide sequence gels.

Among the 16 subfamilies identified in the complete sample, three (numbers2, 5, and 8) were composed of too few workers—less than three individuals inone of the two rearing conditions—and were discarded. In the 13 remainingsubfamilies, the number of isolated bees was 107 and the number of hive beeswas 108.


Chemical Analysis

Cuticular hydrocarbons were extracted for 5 min in 1 ml of pentane. Theywere analyzed by gas chromatography (Girdel 300) on a 30-m nonpolar capillarycolumn and constituents identified by mass spectrometry (Nermag R 10-10-CGC-MS). Data on percentage composition were obtained by integration of theareas under the peaks of the chromatograms by means of a data jet integratorSP 4600. For each individual, the cuticular hydrocarbon profile consists of therelative proportions of all the hydrocarbons.

Statistical Analysis Methods

Global Analysis of Effect of Rearing Conditions. The data were organizedas a N × n matrix, M, of general term m(i, u, R), a, formed by the profiles of all thestudied bees. R represents a rearing condition and can be replaced by I for theisolated bees, or by H for the hive bees; u represents a subfamily and belongs to{1, 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16}, the labels of the S c 13 subfamiliesconsidered in the analysis; i represents an individual, its values belong to {1,. . . , Nu, R}, with Nu, R, the number of bees in subfamily u and rearing conditionR. The index a represents the hydrocarbon, a ∈ {1, . . . , 22}; m(i, u, R), a, and isthe relative proportion of hydrocarbon a in the profile of bee i from subfamilyu in rearing condition R. N, the number of rows of M, is the total number ofbees; NI is the number of isolated bees and NH , the number of hive bees.

N c NI + NH c16

∑u c 1

u � {2, 5, 8}

N(u, I ) +16

∑u c 1

u � {2, 5, 8}

N(u, H ) c 107 + 108 c 215

The number of its columns, n, is the number of hydrocarbons, which equals22.

In order to study the global modifications induced by the rearing condition,we first applied a factorial correspondence analysis (Escofier and Pages, 1982)(Unistat 7.0) to the whole data matrix M.

Test of Homogenization of Subfamily Hydrocarbon Profiles. From its N(u, R)

individuals (approximately 8 individuals per subfamily), each of the S subfami-lies has been characterized by a subfamily hydrocarbon profile for each rearingcondition R c I or R c H, which is the mean profile m(u, R) of its bees, and by thecorresponding covariance matrix C(u, R). The mean profile is a vector of dimen-sion 22, with a general term ma

(u, R):


ma(u, R) c 1

N(u, R)

N(u, R)

∑i c 1

m(i, u, R), a

The covariance matrix is of dimension 22 × 22, with a general term

ca, b(u, R) : c

a, b(u, R) c 1

N(u, R) − 1

N(u, R)

∑i c 1

(m(i, u, R), a − ma(u, R)) . (m(i, u, R), b − m

b(u, R))

Thus, each subfamily u can be assigned four parameters: two vectors mu, I

and mu, H , and two matrices Cu, I and Cu, H . The 22 diagonal terms of Cu, R arethe intrasubfamily variances of each hydrocarbon in rearing conditions R.

In order to point out a possible homogenization of the subfamily hydrocar-bon profiles in a hive, we used for each hydrocarbon a an F test to comparethe intersubfamily variations in the isolated bees with the intersubfamily vari-ations in the hive bees. For each hydrocarbon a and each rearing condition R,we computed a mean vector ma

R and the corresponding intersubfamily varianceva

R, according to:

maR c 1

S

16

∑u c 1

u � {2, 5, 8}

ma(u, R) and va

R c 1S − 1

16

∑u c 1

u � {2, 5, 8}

(ma(u, R) − ma

R)2

Effects of Rearing Conditions on Mahalanobis Distances Between Subfam-ily Hydrocarbon Profiles. In order to know if this possible homogenization uni-formly affects the hydrocarbon profiles of all the subfamilies, we applied ananalysis that takes into account both the mean hydrocarbon profile of each sub-family and its dispersion. This is done through the measurement of distancesbetween the subfamilies in the multidimensional space of the hydrocarbons, forthe isolated bees (I ) on one hand, and for the hive bees (H ) on the other hand.

A classical way to estimate the distance between populations is to com-pute the so-called Mahalanobis distance (Mahalanobis, 1948), which takes intoaccount the mean covariance matrix of the population. The mean covariancematrices CR, R c I or H, have been computed as follows (Manly, 1994):

CR c 1NR

16

∑u c 1

u � {2, 5, 8}

N(u, R) . C(u, R)


The Mahalanobis distance between two subfamilies u and v in the rearingcondition R is defined as:

du, vR c

g(m(u, R) − m(v, R)) . CR . (m(u, R) − m(v, R))T

where the superscript T stands for the transposition of the vector.We defined a parameter able to bring information about the differences in

the relative position of the subfamilies to one another, in the 22-dimensionalspace, between the isolated condition and the hive condition. We used the relativedifference of the previous distances, ddu, v, simply defined according to:

ddu, v c (du, vI − du, v

H )du, v

I

This index represents the general term of a matrix DD of dimension 13× 13. If ddu, v is positive, it means that the subfamilies u and v are relativelycloser when the bees are taken from the hive than when they are isolated; ddu, v

is an index that indicates the convergence of the cuticular hydrocarbon profiles.When ddu, v is negative, it means that the subfamilies u and v are relativelymore different, i.e., that cuticular hydrocarbon profiles diverge. Matrix DD issymmetric, since it is derived from the distance matrices. All the diagonal termsof DD are zeros. As this matrix can be used to describe the rearrangement of thesubfamilies when the bees are in the hive, it can be advantageous to reorganizeit by reordering its lines and columns in order of decreasing values of ddu; themean convergence index:

ddu c 1S

16

∑vc 1

v� {2, 5, 8}

ddu, v

Such a reorganization of the matrix does not affect its symmetry, and as theaverage value of each line or column represents the mean difference of distanceddu of subfamily u with the other subfamilies, its first lines and columns cor-respond to the subfamilies that are the most centered when in hive. We calledthis newly organized matrix DD′.

RESULTS

Global Analysis of Effect of Rearing Conditions. The factorial correspon-dence analysis shows a clear separation of the two types of rearing conditions


FIG. 1. Projections of individuals (worker bees, in A) and variables (hydrocarbons, in B)on the principal plane of a factorial correspondence analysis (UNISTAT). The two graphsare extracted from a single coprojection, they are separated here for clarity. Hive bees andisolated bees are denoted by open squares and open circles, respectively. Hydrocarbonsare identified by the number of C atoms (Me indicates a methylated compound).


I and H, and points out the compounds that are the most characteristic of eachcondition (Figure 1). For instance, the heavy compounds C31 and C33 appearsuperimposed on the hive bees’ cluster, while the methylated compounds forma group on the isolated bees’ cluster.

In this factorial plane, hive bees and isolated bees are not only in verydistinct clusters, but isolated bees form a more diffuse cloud than hive bees,the latter condition suggests a greater variability of hydrocarbon concentrationin isolated bees, and therefore, a global homogenization of subfamily chemicalsignatures in the hive bees.

Test of Homogenization of Subfamily Hydrocarbon Profiles. The F tests3

applied to compare the intersubfamily variances vaI and va

H show that amongthe 22 values of a (the hydrocarbon concentrations), 19 present a significantlylower value for hive rearing than for isolated conditions, [P(va

H c vaI vs va

H < vaI ) <

0.05]. An order of magnitude of decrease for intersubfamily variation is given bythe mean value of the variances: vI c 1.68 and vH c 0.36, where vR c 1/ n ∑n

a c 1va

R. These results show clearly that the subfamily signatures are significantlycloser when the bees are in a hive than when they are isolated.

Effects of Rearing Conditions on Distances Between Subfamily Hydrocar-bon Profiles. In order to know if the homogenization of the hydrocarbon pro-files affects all subfamilies uniformly, we analyzed the matrix DD′ containingthe differences of the Mahalanobis distances computed and ranked as indicatedin Methods and Materials (Table 1). A correct interpretation of this measure-ment requires some care. Each matrix of distances for I and H, respectively,contains distances that are ‘normalized’ since they are computed with the met-rics introduced by the matrices CI and CH . Two subfamilies u and v could haveappeared to be closer when isolated than in the hive condition if the comparisonwas based on distances computed with the same metric (euclidean metric forinstance). When a term ddu, v of DD′ is positive, it means that, independentlyof the global “hive” effect, the subfamilies u and v are even closer when in ahive than when isolated. If ddu, v is negative, it means that the subfamilies arerelatively farther apart, even if the hive effect also applies to them.

From this analysis, some subfamilies (1, 4, 11, 12, and 16) stand out, as theyappear to be more “at the center of the hydrocarbon multidimensional cloud”when in hive than when isolated (Figure 2), i.e., all of them are closer to anyother subfamily (except subfamily 9, which appears to be farther from all thesubfamilies). We can conclude from the analysis of matrix DD′ that hive homog-enization does not affect the different subfamilies uniformly. In particular, sub-family 9, which has the most distinct profile (Arnold et al., 1996), is the sub-family which is set apart from all the others. This observation suggests that there

3Because the variances compared here are deduced from mean values by application of the centrallimit theorem, the F test is valid, even if the data are not normally distributed.


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FIG. 2. Characteristic distances between each subfamily (labelled on the graph), and thesubfamilies of a reference group (circled): in isolated bees (x axis) and in hive bees ( yaxis). A fitted exponential model is plotted with confidence limits at 95%. Its equation isy c exp(a + bx), with a c −0.43 ± 0.11 and b c 0.46 ± 0.03). The hydrocarbon profilesof subfamilies 3, 7, and 10 converge to the mean profile defined by the reference group,unlike those of subfamilies 6, 13, and 9, which diverge. This is especially important forsubfamily 9, whose characteristic distance to the reference group increases by 26% inthe hive condition.

may be a relationship between a characteristic distance in hive and a character-istic distance in the isolated conditions.

In order to identify such a relationship, we first need to define these “char-acteristic distances.” For this purpose, let us consider subfamilies that exhibitthis property of being centered when in hive to form a “reference group” for themeasurement of a “characteristic distance:” the generic letter to indicate such asubfamily will be g. Now, we can define, for each rearing condition R, a char-acteristic distance of every subfamily u to the members of the reference group{g}:

du, {g}R c 1

5 ∑g c 1, 4, 11, 12, 16

du, gR

Figure 2 shows the evolution of du, {g}R as a function of du, {g}

I . This functionis a rapidly increasing one, and it is best fitted by an exponential expression. Inother words, if, globally, the hydrocarbon profiles appear to be closer to oneanother for the hive bees than for bees maintained in isolation, the distancesbetween the subfamilies have not all been reduced, nor have they even been


kept constant. Figure 2 reveals a convergence of the closest subfamilies and arelative divergence of the more distant ones.

DISCUSSION

The cuticular signature has both endogenous and exogenous components.The exogenous component arises from chemicals acquired directly from theenvironment. The endogenous component is controlled from metabolic processesthat are influenced by both genetic and environmental factors (Getz and Page,1991). Here we addressed the question of how much each of these two factorscontribute during the ontogeny of the hydrocarbon profiles.

Our results show clearly that in 5-day-old bees, the cuticular signaturesof the subfamilies are significantly closer when the bees are reared in the hivethan when they are isolated. Thus, a partial homogenization of the hydrocarbonprofiles takes place under the influence of the social environment.

Because the environment of the hive (comb wax, pollen, honey) is com-mon for all the workers, it could not be involved substantially in differentialchanges of the hydrocarbon profiles, which show two opposite trends amongthe subfamilies: most converge while the more distant ones continue to diverge.The nestmates, which form the social environment, should be common for allthe workers, provided that the distribution of the subfamilies within the nest isuniform (i.e., if all of their workers remain or move in every part of the hive inthe same way). This is not the case in a colony artificially composed of only twosubfamilies, where workers exhibited significant spatial heterogeneity within thenest (Calderone and Page, 1988), owing either to task specialization or to sub-family recognition (Oldroyd et al., 1994). The spatial distribution of the workersin a normal colony composed of numerous subfamilies is unknown and shouldbe analyzed in order to determine if the differential modifications of the hydro-carbon profiles are related to any spatial heterogeneity of the subfamilies.

Interestingly, in hive bees there is convergence of the subfamily hydrocar-bon profiles that are closest in the isolated bees, and a relative divergence ofthe more distant ones. We hypothesize that workers of subfamilies that have aclose cuticular hydrocarbon profile have a tendency to reciprocally partake inmore trophallaxis, allogrooming, and physical contact than workers of subfami-lies bearing more different chemical signatures. This might cause an active or apassive transfer of the hydrocarbons between the workers and therefore increasethe similarity between the cuticular hydrocarbon profiles.

In ants, hydrocarbons are transferred between nestmates, mainly by thepostpharyngeal glands of the workers. In Cataglyphis iberica, for example, allsatellite nests of a colony have a common colonial odor (Dahbi and Lenoir,1998). Chemical plasticity has been demonstrated in the case of artificially mixed


colonies of ants, where the two species became chemically more closely relatedafter qualitative and quantitative changes in the hydrocarbon profile of eachspecies, either by an active synthesis and/ or an active transfer (Bagneres et al.,1991; Bonavita et al., 1996).

Despite the trend towards homogenization demonstrated here, the cuticularhydrocarbon profiles are still different in 5-day-old hive bees, but less so thanin isolated bees (Arnold et al., 1996). The statistical evidence does not implythat all the workers of a colony are able to discriminate their subfamilies. In thecase of subfamilies sharing close profiles, the workers could probably poorly,or not at all, discriminate others from their own subfamily. The main conse-quence could result in a reduction of the number of recognizable subfamilies inthe colony. Although polyandry could make workers behave nepotistically bydiscriminating and favoring supersisters over half-sisters, we conjecture that aninherent limitation operates in the intracolonial discrimination system of work-ers. This mechanism might have evolved in order to prevent conflicts from beingexpressed, and therefore to facilitate the evolution of highly cooperative behavior(Ratnieks, 1991).

Conversely, subfamilies bearing distinct profiles should have a better dis-criminatory ability with respect to each other, and this could induce their work-ers to behave nepotistically. If there is only a small number of such subfamilies,this could explain why nepotistic tendencies in general are expressed weakly ina colony (Breed et al., 1994), even during choice of a new queen (Tilley andOldroyd, 1997).

Acknowledgments—We thank Benoıt De Schepper and Christian Malosse for chemical analy-sis; Gerard Dreyfus, Jean Francois Ferveur, and David Horn for valuable comments; and Neil Emeryfor help in improving the English of the manuscript.

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BONAVITA-COUGOURDAN, A., RIVIERE, G., PROVOST, E., BAGNERES, A. G., ROUX, M., DUSTICIER,G., and CLEMENT, J. L. 1996. Selective adaptation of the cuticular hydrocarbon profiles of theslave-making ants Polyergus rufescens and their Formica rufibarbis and F. cunicularia slaves.Comp. Biochem. Physiol. 113B:313–329.

BREED, M. D., WELCH, C. K., and CRUZ, R. 1994. Kin discrimination within honey bee colonies:an analysis of the evidence. Behav. Proc. 33:25–40.

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Influence of Social Environment on Genetically Based Subfamily Signature in the Honeybee