Kin recognition and virgin queen acceptance by worker honey bees (Apis mellifera L.)

Anim. Behav., 1986, 34, 1061 1069

Kin recognition and virgin queen acceptance by worker honey bees (Apis mellifera L.)

ROBERT E. PAGE, JR*$ & ERIC H. ERICKSON, JRt *Department of Entomology, University of Wisconsin, Madison, WI53706, U.S.A.

tBee Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Department of Entomology, University of Wisconsin, Madison, I4/1 53706, U.S.A.

Abstract. Virgin honey bee queens provide genetic cues that can be used by workers to assess genetic relationships. We found that groups of caged workers, taken directly from colonies containing a functional queen and two patrilineal subfamilies, could distinguish among queens of different degrees of kinship. The first virgin queen that an individual worker encountered was assessed by that worker on the basis of her kinship similarity to the individual interacting worker or her similarity to the caged group. When a second virgin queen was introduced, she was assessed by workers based on her similarity to the first virgin queen. The outcome of the ensuing fight to the death among the two introduced queens was dependent upon their relationship to each other. The resident queen had a strong advantage when the virgin queens were from different subfamilies but the outcome was nearly even when they were patrilineal sisters.

The ability of workers in social insect societies to distinguish among nestmate and non-nestmate kin is a central issue in sociobiology (Crozier & Dix 1979; H611dobler & Michener 1980; Breed & Bekoff 1981; Getz 1981, 1982; Michener 1982; Blaustein 1983; Holmes & Sherman 1983; Lacy & Sherman 1983). This is a consequence of the importance of nestmate and kin recognition systems in demon- strating the role of kin selection in shaping social evolution (Hamilton 1964a, b, 1972). Kin selection theory predicts that altruistic acts should be directed preferentially toward individuals that are more closely related and helps explain the frequent occurrence of extreme altruism found in the social Hymenoptera as a function of higher genetic relationships among nestmates, a consequence of haplodiploidy. The high genetic relationships among sibling nestmates are maintained, however, only under conditions of single insemination of female reproductives (or multiple insemination with sperm clumping or precedence), single queen nests, or inbreeding (see Page & Metcalf 1982).

Honey bees provide both a challenge to the study of kin selection and a test of the function of kin selection in social evolution. This is a consequence of the extreme expression of polyandry by honey

:~ Present address: Department of Entomology, The Ohio State University, Botany and Zoology Building, Room 103, 1735 Neil Avenue, Columbus, OH 43210, U.S.A.

bee queens leading to a large number of patrilineal subfamilies within a nest and, consequently, a high variance and a low average genetic relationship among worker nestmates (Page & Metcalf 1982; Laidlaw & Page 1984; Page et al. 1984). Evidence that workers act preferentially toward more closely related reproductive individuals (virgin queens and drones) during reproductive events within the colony would be a clear demonstration of the importance of kin selection. However, most pub- lished studies to date have addressed worker- worker interactions or the ability of workers to recognize individual queens.

Recent studies have shown that worker honey bees have the ability to distinguish among nestmate workers on the basis of probable genetic factors (Breed 1983; Getz & Smith 1983). Breed et al. (1985) have shown that workers maintained in groups of mixed genotypes recognize and behave differently toward their own genotype when interacting with other workers. Breed (1981) and Boch & Morse (1982) showed that workers can learn to discriminate an individual queen and can discriminate this queen among other queens that are related or unrelated. Getz et al. (1982) demonstrated an uneven segregation of patrilines of workers during colony fission, suggesting an ability of workers to identify and interact preferentially with patrilineal subfamily members within a colony. However, they suggest that their results may only demonstrate

1061

1062 Animal Behaviour, 34, 4

genetic variability in response to the fissioning stimulus.

To date, we are aware of four studies that directly address kin recognition in the context of reproduction. We (Page & Erickson 1984) demonstrated that honey bee workers have the ability to distinguish among groups of queen larvae and preferentially rear closely related nestmate larvae (G = 0.75, pedigree coefficient of relationship: Cro- zier 1970; Pamilo & Crozier 1982) over less related non-nestmate larvae (G=0.25 or 0.31). We have also demonstrated (unpublished data) that workers discriminate less when the genetic relationships of nestmate and non-nestmate larvae are reduced. Noonan (1985) has verified these results in the context of single colonies with two patrilines. She showed that workers discriminate and preferentially care for patrilineal sister queen larvae. Breed et al. (1984) found no evidence for brood discrimination on the basis of genotypic similarity with adult workers in cross-fostering experiments using queenless colonies and adult and brood honey bees of presumably low genetic relatedness.

In this study, we test the abilities of workers to discriminate virgin queens of varying genetic relationships. We present evidence that workers

behave differently toward more closely related reproductive individuals.

M A T E R I A L S AND M E T H O D S

Three experiments were conducted during the summer of 1984 to test the ability of honey bee workers from colonies consisting of two patrilines to discriminate virgin queens based upon the genetic relationship of the virgin queens to the workers. Eight colonies were established as sources of virgin queens and workers for these experiments (see Fig. 1). Queens of these colonies were sisters derived from a queen mother that was homozygous for the recessive mutant gene, cordovan (cd). When homozygous, this gene produces a brown-coloured integument on body areas that are normally black (+ , wild type). The queen mother was mated by instrumental insemination to a single unrelated drone, hemizygous for the mutant marker. All daughter queens were, therefore, homozygous cordovan, with a pedigree coefficient of relationship G=0.75.

Newly-emerged drones were chosen from the brood combs of two drone colonies (one selected

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A B C D

Figure 1. Pedigree for queens and workers used for this study. Explanation of the crosses is provided in the text. Solid lines represent eggs; dashed lines represent sperm. Roman numerals at left margin represent generations. Queens from generation II were queen mothers for workers and virgin queens (generation III) used for behavioural assays. Pedigree coefficients of relationship for individuals are: Gx.v=0"75; GA.A=GB.B=Gc.c=GD.D=0"75; GA.c=GB.D=0"44; GA.B = Gc.D = 0"25; GA.D = Gs-c = 0" 19.

Page & Erickson: Kin recognition in honey bees 1063

for cordovan and one for wild type) and aged circa 12 days in a holding colony until sexually mature. All cordovan drones were brothers, as were wild type drones. Each of the eight daughter queens was mated by instrumental insemination to two different, unrelated drones, one cordovan, and one wild type. Queens were placed into small colony units containing six combs, where they began laying eggs. After about 6 weeks, each colony consisted of two subfamilies of workers, each derived from a different drone father and with a distinctive phenotypic marker: cordovan, or wild type. Members o f the same subfamily had a coefficient of relationship G = 0'75 (super-sisters; see Laidlaw 1974 for use of this terminology for haplodiploid genetics) with G = 0.25 (half-sisters) between subfamilies. Genetic relationships of individuals between colonies are given in the caption for Fig. 1.

Virgin queens were then raised from a single colony at a time by grafting young larvae from worker cells located on the brood combs into queen cells in a single unrelated colony that had been manipulated to stimulate workers to rear queens. Up to 40 queens from a single colony were raised at a time using this method. Before emergence of the adult virgin queens (queens emerged about 11-12 days following the graft), the queen cells were transferred into clean individual glass vials, provi- sioned with a small amount of sugar candy, and placed in an incubator at 34~ 50~0 R H until emergence. Sugar candy was always from a single, homogeneous source. Virgin queens were allowed to age in the vials for up to 48 h after emerging. Queens within any set of trials were of approxima- tely the same age. The above procedure provided

equal treatment of all test queens of a given colony and the complete isolation of workers and virgin queens until tested.

One to three samples o f adult workers were taken at 2-3-week intervals f rom seven of the eight colonies in order to determine the frequencies of the two subfamilies. For each period, samples of adults were taken by randomly vacuuming workers directly off the combs. In addition, a single brood comb, containing sealed brood and emerging adult workers, was removed from the colony, placed in a cage, and stored in an incubator while adult workers emerged. Workers from each sample were then segregated by phenotype into subfamilies and counted. Samples of adults from the colony and samples of emerging adults from the brood combs were then combined in order to estimate the frequencies of the two subfamilies within in each colony. The eighth colony (no. 3, Table I) deve- loped a severe case o f American foul brood disease and was removed from the experiment without sampling the workers; however, it was used as a souce of workers for some assays for experiment 2.

Experiment 1

Sister workers were collected at random from comb areas adjacent to brood in the source colony of the virgin queens and combined to form groups of 10 super-sisters each. Workers were collected without anaesthetic or handling by vacuuming them directly into 0.135-1itre cardboard cups with wire screen covers. These cups also served as the observation arenas for the behavioural assays, Equal numbers of cups of wild type and cordovan

Table I. Subfamily composition of colonies used, the colony source of the queens and/or workers, and the number of trials in experiments 1 and 2 that were conducted

No. trials No. trials Subfamily experiment 1 experiment 2

Colony No. of no. samples cd/cd cd/+ Total %cd/cd Queens Workers Queens Workers

1 3 1114 370 1484 75 0 0 10 0 2 2 521 466 987 53 22 22 0 21 3 0 - - - - - - 0 0 0 19 4 2 596 272 868 67 58 58 13 0 5 2 396 233 629 63 0 0 19 23 6 3 776 125 901 86 20 20 21 0 7 3 645 571 1216 47 69 69 0 0 8 1 331 103 434 76 8 8 0 0


subfamilies (usually five of each) were collected for each set of assays and taken directly into the laboratory. Behavioural assays began within 15 rain of collection of the last group of 10 workers and were usually completed within 2 h; therefore, groups of workers were out of their respective colonies for 15-120 rain before use.

Virgin queens that had emerged in glass vials were selected and paired with groups of workers according to genetic relationship: super-sisters (same father) or half-sisters (different fathers). An individual queen was introduced into an observation arena (cup) containing 10 workers by inverting the vial containing the queen over a hole in the side of the arena. Behaviour of the queen and workers was observed for 5 min after which a clean vial of sugar syrup (necessary food) was inverted over the cage. Most agonistic behaviour occurs within 5 rain of queen introduction (see Breed 1983). All sugar syrup solution came from a single homogenous source for any set of trials. Each queen was used for one assay, as were groups of workers. Observation arenas were used once and discarded.

Five-minute observations of behaviour were scored for an individual trial on the basis of the behaviour of the queen and any single worker within the group as follows.

(1) Workers bit the queen for a period of time sufficient for the observer to see the mandibles of the worker engaged on the queen.

(2) Workers fed the queen. (3) The queen fed the workers. (4) Workers stung and/or bit each other. (5) The queen stung and/or bit the workers. (6) The queen was alive and being fed and

groomed 30 min after introduction to the workers. The numbers of such interactions per trial were

not counted.

Experiment 2

Experiment 2 was identical to experiment 1 except that workers and virgin queens came from different colonies. This test differed from experiment 1 with respect to the degree of genetic similarity between the workers in the arena and the virgin queen that was introduced (see Fig. 1).

Experiment 3

Arenas containing a queen and workers from experiment 1 were paired according to the genetic

relationship of the queens to each other, the queens to the workers in their respective arenas, and the workers in the different arenas to each other. The queen to be introduced was removed from one arena and placed into the other arena with the resident queen and her workers. In order to identify the individual queens, one wing tip was clipped on either the resident or introduced queen before introduction, except in some cases where phenotypic markers were used for identification. All introductions of the second queen into the resident arena took place after 30 min and within 6 h of the initial queen introduction from experiment 1; most second introductions were within 2 h.

Behaviour of workers toward both queens was observed for 5 min and recorded as follows.

(1) Workers bit the introduced queen. (2) Workers bit the resident queen. (3) Workers stung and/or bit each other. (4) The resident queen stung and/or bit the

workers. (5) The introduced queen stung and/or bit the

workers. Fighting between the queens was also observed during this period and queen survivorship in each arena was recorded for up to 8 h. Both queens were scored as alive and discarded after this time.

Chi-squared contingency table analyses were conducted on the data (Sokal & Rohlf 1969).

R E S U L T S AND D I S C U S S I O N

The typical behaviour of virgin queens upon introduction into the arena consisted of cessation of motion, a bowing of the body exposing a maximum body surface area, the lifting of the legs (most often the mesothoracic pair) and the express- ing of a clear fluid from between open mandibles. This behaviour (and the clear fluid) was also observed in a virgin queen that was emerging from her cell within a vial, isolated from workers. Workers usually groomed the virgin queen, took the clear substance, and fed the queen. Occasio- nally a queen received what has been termed a bite (Breed 1981, 1983) from one or more of the workers.

Our results show that there are genetic components associated with cues perceived by workers during recognition and discrimination at the time of acceptance of virgin queens. Super-sister groups of workers from colonies consisting of two subfam-

Page & Erickson: Kin recognition in honey b.ees 1065

Table 1I. Number of virgin queen introductions from experiment 1 in which queens were fed, workers were fed, and queens were bitten as a function of the average genetic relationship of the virgin queen to the workers in the observation arena

Yes No Total

Queen fed by workers Super-sister 41 51 92 Z2=2.03 NS Half-sister 47 38 85 df= 1 Total 88 89 177

Worker fed by queen Super-sister 42 50 92 )~2=0.20 NS Half-sister 36 49 85 df= 1 Total 78 99 177

Queen bitten Super-sister 21 71 92 Z 2= 10-48" Half-sister 39 46 85 df= 1 Total 60 117 177

* P=0.01; NS: not significant at the 0.05 level.

Table IlL The number of introductions from experiment 2 in which the queen was bitten, as a function of the genetic relationship of the queen to the workers

Queen bitten

Yes No Total

G=0.44 11 21 32 Z2=0"38NS G=0.19 13 18 31 df=l Total 24 39 63

NS: not significant at the 0.05 level.

ilies (experiment 1) discr iminated between super- sister and half-sister virgin queens. The probabi l i ty of half-sister queens being bi t ten upon in t roduc t ion was twice tha t of the super-sisters (Table II). However , there were no subfamilial differences wi th respect to the frequency workers fed queens or queens fed workers. Of 177 int roduct ions , only two queens were no t accepted after 30 min. Both queens were half-sisters of the workers in their respective arenas and b o t h were s tung to death. W o r k e r pheno type (wild type or cordovan) was indepen-

dent of bi t ing behav iour 0 ( = 0 . 1 9 , df= 1, NS). In exper iment 2, there were no statistically

significant differences between the frequency of

bites for queens o f the two genetic re la t ionships (Table III). However, the results are in the same direction as those o f exper iment 1 and, in combina- t ion with exper iment 1, define a significant l inear relat ionship between degree of genetic re la t ionship of workers to virgin queens and reject ion as a funct ion of bi t ing behav iour (Fig. 2). Feeding behaviour was the same as for exper iment 1. Wi th 63 int roduct ions , only 1 queen was no t accepted after 30 min. She was related to the workers by G = 0 - 1 9 and was s tung to death.

Our results differ significantly f rom those of Breed (1981) in t ha t the workers in his study used the last queen with which they had con tac t as the referent dur ing recogni t ion and acceptance of the subsequent ly in t roduced queen. Worke r s in our study made a de te rmina t ion of the first virgin queen consistent with pheno type ma tch ing with themselves or o ther members of the group as referents, or consis tent with the recogni t ion allele hypothesis (for discussion see Blaustein 1983; Holmes & She rman 1983; Lacy & She rman 1983). The expected result f rom our exper iment 1 if the last queen (the queen mothe r ) was the referent, was equal t rea tment of virgin queens of b o t h subfamilies represented, since they were equally related to the queen and were assumed to have had equal recognit ion cues.

Virgin queen recogni t ion was dist inct f rom worker nes tmate recogni t ion dur ing our study as is demons t ra ted by the following. (1) Workers bit or

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o I I I 0 0.25 0.50 0.75

GENETIC RELATIONSHIP

Figure 2. Linear regression of proportion of queens introduced that were bitten by one or more workers in the arena as a function of the pedigree coefficient of the relationship of the virgin queen to the workers. Circles represent data for individual colonies (P < 0'05, t-test of the linear component based on the angular transformed data); squares represent weighted averages for each genetic relationship classification.


stung other workers much less than they did virgin queens. Ou t of 177 in t roduct ions for exper iment 1, we observed only four occasions of worke r -worke r agonistic behav iour (based on bi t ing behaviour) among super-sisters, compared with 21 episodes of agonistic behav iour against super-sister virgin queens. Dur ing exper iment 3, we observed six occurrences of workers bi t ing or st inging o ther workers compared to 12 occurrences of workers bit ing the accepted resident queen, even though there were 10 times more workers present. (2) Mos t in t roduced queens were fed and g roomed by the workers.

The workers in our s tudy rapidly learned to recognize the first virgin queen in t roduced in exper iment 1 and used her as the ma jo r referent (or a ma jo r c o m p o n e n t of the recogni t ion template) for recognit ion of the second in t roduced queen (Breed 1981 provided an elegant demons t r a t ion of the ability of workers to discriminate queens). A second in t roduced queen (experiment 3) was more likely to be bi t ten by workers than a resident queen (Table IV), regardless of her re la t ionship to the resident queen, the resident workers (Table V), her own workers, or the relat ionship of her workers to the resident workers or the resident queen (Table

Table IV. The number of introductions of virgin queens from experiment 3 in which the resident and/ or the introduced queen were bitten by workerst

Queen bitten

Yes No Total

Queens super-sisters Resident 5 45 50 )~2=8.39,, Introduced 17 33 50 df= l Total 22 78 100

Queens hal~sisters Resident 7 24 31 Z2= 6.80** Introduced 17 14 31 df= l Total 24 38 62

R x C x A independence: G = 20.90"*, df=4 A • C independence: G=5.17"; df= I R • C independence: G= 15.16'*; df= 1

t Cases where queens were super-sisters and half- sisters are shown as separate arrays. A three-way G-test of independence (Sokal & Rohlf 1969) is provided where: R = row, C = column and A = ar- ray.

* P < 0 . 0 5 ; ** P < 0 . 0 1 .

Table V. The number of introductions of virgin queens in which workers bit the resident and/or the introduced queen (separate arrays are a function of the relation of the queens to the resident workers)

Queen bitten

Yes No Total

Super-sisters Resident 6 35 41 Z2=5.14 * Introduced 14 24 38 df= 1 Total 20 59 79

Half-sisters Resident 6 34 40 )~2= 9.57"* Introduced 20 23 43 df= 1 Total 26 57 83

R x C x A independence: G = 16.16"*, df= 4 A x C independence: G =0-721 NS, df= 1 R x C independence: G = 15.16"*, df= 1

Separate arrays are a function of the relation of the queens to the resident workers. * P<0.05; ** P<0.01; NS: not significant at

the 0.05 level.

VI). Resident queens, unlike the initial in t roduc- t ion in exper iment 1, were b i t ten equally by super- sister and half-sister workers ()~2= 0.00 ys, df= 1). Both the resident queen and the in t roduced queen of a given trial were more likely to be bi t ten when they were half-sisters than super-sisters (Table IV) but the difference was greater for the second in t roduced queen. We must emphasize, however, t ha t in t roduced queens tha t were half-sisters of the workers received more bites than super-sisters, a l though the difference was not statistically significant.

There were no significant differences between resident and in t roduced queen survivorship when the queens were super-sisters (Table VII). How- ever, the resident queen had a 1.7:1 advantage when the queens were half-sisters, perhaps suggesting kin recogni t ion and preferential t rea tment of super-sister virgins by virgin queens. Queen fights were highly variable in dura t ion and intensity. Frequently, one or bo th queens defaecated (or released some volatile substance) on each other or on the b o t t o m of the arena, causing appa ren t avoidance behav iour by the workers. This substance merits addi t ional investigation.

Workers do not appear to affect the outcome of


Table VI. The number of introductions in which the introduced queen was bitten by the resident workers as a function of the genetic relationship of the introduced queen to her own workers, the introduced queen's workers to the resident queen, and the introduced queen's workers to the resident workers

Queen bitten

Yes No Total

Relationship to own workers Super-sister 23 23 46 )~2 = 2.81 NS Half-sister 11 24 35 dr= 1 Total 34 47 81

Relationship of introduced queen's workers to resident queen

Super-sister 18 24 42 Z2 =0.03 NS Half-sister 16 23 39 d f - 1 Total 34 47 81

Relationship of introduced queen's workers to resident workers

Super-sister 16 18 34 Z 2 = 0.62 Ns Half-sister 18 29 47 df= I Total 34 47 81

NS: not significant at the 0.05 level.

Table VII. The number of resident and introduced queens surviving after 8 h in experiment 3

the queen dominance struggle in any obvious way. However, we cannot discount the possibility that they do affect the outcome by preferentially feeding queens or by aggression, since we did not make regular observations of the arenas after the initial 5 min. In 81 independent trials there were no observations of workers stinging either queen. However, a single worker occasionally engaged in prolonged biting (usually on the wing of the introduced queen) resulting in an apparent disadvantage to the individual queen during her dominance fight. This behaviour is difficult to interpret because of the artificiality of the arena environment. Frequently the queen responded to the prolonged biting by folding her legs and becoming motionless. The biting worker then carried the queen around the cage. This behaviour could be interpreted as queen removal or displacement as opposed to directed aggression.

Both the resident and the introduced queen were more likely to survive 8 h when they were half- sisters of the workers (Table VIII). Perhaps an interaction of cues of half-sister workers and a foreign virgin queen resulted in a greater search and fight stimulus for an individual queen, One measure o f search and fight stimulus may be the frequency with which queens bite and/or sting workers in the presence o f another queen. How- ever, our data are not sufficient to demonstra te this because only 21 of the 162 queens f rom experiment

Queen alive

Yes No Total

Table VIII. The number of queens surviving after 8 h from experiment 3 as a function of the relationship of the virgin queens to the resident workers

Super-sisters Resident 30 19 49 Z 2 =0.40 NS Introduced 29 20 49 df= 1 Total 59 39 98

Half-sisters Resident 26 6 32 Z 2 = 8.21 * Introduced 15 17 32 df= 1 Total 41 23 64

R x C x A independence: G = 8-76 (P < 0.10), df=4 A x C independence: G=0-24 NS, df= I R x C independence: G=3.78 (P<0.I0), df= l

Separate arrays are a function of the relationship of the virgin queens to each other. * P < 0.01; NS: not significant at the 0.05 level.

Queen alive

Yes No Total

Super-sisters Resident 25 14 39 X2=3.30 (P<0.10) Introduced 17 22 39 df= 1 Total 42 36 78

Half-sisters Resident 31 11 42 )C2=0'89 NS Introduced 27 15 42 df= 1 Total 58 26 84

R x C x A independence: G = 8-19 (P < 0-10), df= 4 A x C independence: G = 3.97", df= l R x C independence: G = 3.78 (P < 0.10), dr= 1

* P < 0.05; NS: not significant at the 0.05 level.


3 responded agonistically toward workers; 12 of these were half-sisters to the workers in the arena. There were nine observations of resident queen agonistic behaviour toward workers. It is also possible that these results are spurious.

The net result from experiment 3 is that resident queens had an overall significant advantage that was greatest when they were in arenas with half- sister workers and half-sister queens, whereas the introduced queens did best in arenas with half- sister workers and super-sister queens. Even though the above survivorship results are highly significant statistically, we accept them with cau- tion. There was considerable unexplained variability from day to day in the genetic relationships of survivors of queen contests and the proportion of contests that were resolved within 8 h; this suggests a need for further research.

There was no significant effect of the wing-clip marking method on the outcome of queen contests (Z2=0.152 Ns, dr= 1).

C O N C L U S I O N

Our data clearly demonstrate the potential for workers to discriminate among virgin queens of different subfamilies and preferentially to accept them on the basis of genetic similarity to themselves. The recognition cues of the queens appear to be associated with genotype; the recognition mechanisms could be innate (e.g. recognition alleles), self-imprinting or self-learning, or based on learning cues of the group. Whatever the mechanism (or mechanisms), we must accept one of two alternatives: (1) workers recognized the first virgin queen as being like themselves, individually (by either phenotype matching or by recognition alleles), or (2) workers recognized her based on her phenotypic similarity to the group of super-sister workers in the arena. Alternative 1 is supported by the findings of Breed et al. (1985). If alternative 2 is correct, then workers must readily change their recognition template to the genetically determined phenotypic cues of individuals in their immediate surroundings, a mechanism of questionable value for nestmate or kin recognition. A variation of the 'odd bee' experiment of Buckle & Greenberg (1981), or the genotypic mix experiment of Breed et al. (1985) is necessary to determine whether the group or the individual is serving as the referent during introduction of the first queen.

The significance of the two apparently conflict- ing recognition mechanisms operating is unclear. Recognition of the first virgin queen could be nestmate recognition as a defence against nest parasitism by unrelated virgin queens from other nests, or simply intruder rejection. If the latter, then we must ask why workers have the ability to change the referent template so rapidly, since the group composition of the arenas was very different from that of the source colony (Table I). If recognition of the first queen is based on kin recognition with each worker using herself as the referent, then we must ask what possible significance such behaviour can have in light of the observation that workers readily accept the virgin queen regardless of relationship, learn her labels, and use her as the referent for additional introductions. We cannot, of course, rule out the possibility that some of our results are artefacts of our experimental design.

Use of the resident virgin queen as a recognition template may be a consequence of colony level selection. Workers may tend to prefer the first queen to emerge because, all things being equal, she is the most likely to succeed to the nest. The first queen to emerge frequently cuts holes in the cells containing other virgin queens and stings them before they emerge (see Butler 1975 for a discussion of this behaviour). Under our test conditions, queen conflict sometimes results in injury to both queens. With an average of 17 different subfamilies of workers present in a colony (Adams et al. 1977; see also Page & Metcalf 1982; Laidlaw & Page 1984), the risk of ending up without a succeeding queen, resulting in colony failure, may be high if each subfamily is treating its own super-sister virgin queen preferentially. Furthermore, the likeli- hood is low that each subfamily will end up with a representative virgin queen, considering the small numbers of virgins reared (Page & Erickson, unpublished data), resulting in relatively low potential for kin selection acting on queen acceptance (Page & Metcalf 1982).

The above hypothesis is supported by some evidence. (1) It is likely that natural selection for a decreased queen development time, based on a first-queen-to-emerge advantage for nest succes- sion, is responsible for the observed difference in development times of queens (16 days) and workers (21 days), which is significant considering the larger size of queens. (2) In a separate study using small queenless colony units (Page & Erickson, unpublished data) we found that only nine out of 32 (28%)


uni ts al lowed more than one queen to emerge; the rest were destroyed in their cells. These small units p roduced an average of 5.2 queen cells each.

The results presented show the potent ia l for kin recogni t ion th rough genetic labelling and preferential t r ea tmen t of virgin queens t h r ough label recognit ion, bu t they do not demons t ra te t ha t such behav iour occurs in a na tura l context. Our results, as well as those of others, may be a consequence of the simplistic test env i ronment we provide, bu t they clearly po in t to the need for cont inuing research in this area.

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(Received 22 February 1985; revised 10 July 1985; MS. number: ,14492)

Documents

Kin recognition and virgin queen acceptance by worker honey bees (Apis mellifera L.)