When do honey bee guards reject their former nestmates after swarming?

Insectes soc. 49 (2002) 56 61 0020-1812/02/010056-06 $ 1.50+0.20/0 Birkhäuser Verlag, Basel, 2002 Insectes Sociaux Research article When do honey bee guards reject their former nestmates after swarming? M. Beekman 1, 4, J.N.M. Calis 2, B.P. Oldroyd 3 and F.L.W. Ratnieks 1 1 Laboratory of Apiculture and Social Insects, Department of Animal and Plant Sciences, Sheffield University, Sheffield S10 2TN, United Kingdom 2 Laboratory of Entomology, Wageningen University, POB 8031, 6700 EH Wageningen, the Netherlands 3 School of Biological Sciences, University of Sydney, A12, Sydney NSW, Australia 4 Author for correspondence and present address: Schools of Biological Sciences & Mathematics and Statistics, University of Sydney, A12, Sydney NSW, Australia. e-mail: mbeekman@bio.usyd.edu.au Received 25 April 2001; revised 3 August 2001; accepted 29 October 2001. Summary. Honey bees (Apis mellifera L.) reproduce by swarming wherein the mother queen leaves the nest with approximately two thirds of her workers (the prime swarm). Several daughter queens are raised in the original nest, and these start to emerge shortly after the first swarm departs. One or several of these daughter queens may then leave the original colony sequentially with smaller afterswarms. Here we study the change in acceptance of former nestmates after colony reproduction using free-flying honey bee colonies. We used a total of four colonies each of which we divided to make four new colonies: two artificial swarms (the offspring colonies) and two colonies that mimic established ( old ) colonies after swarming. The way the original (mother) colonies were divided allowed us to determine the relative importance of wax comb on cue divergence. Half of the divisions contained the original wax combs from the mother colony whereas the other divisions were not provided with comb requiring them to construct their own. We then tested the acceptance of former nestmates by introducing foragers at the hive entrances and observing the behaviour of guard bees. Our results did not show a consistent change in acceptance of former nestmates after swarming. In two out of four replicates, workers originating from the colonies that contained the original wax combs were rejected by guards from the initially comb-less colonies. This suggests that comb wax plays an important role in nestmate recognition. However, the remaining two replicates did not show a response; all former nesmates were still accepted two weeks after artificial swarming. Key words: honey bees, Apis mellifera, swarming, nestmate recognition. Introduction Intraspecific recognition occurs in many contexts and ranges from recognition of self to recognition of a potential mating partner (see Sherman et al. (1997) for a review). In social insects one major recognition context is discrimination between nestmates and non-nestmates. This context is important because in many species intruder non-nestmate conspecifics can cause harm during territorial disputes (eg African weaver ants Oecophylla longinoda: Hölldobler and Wilson, 1978), by cannibalism (Formica polyctena: Driessen et al., 1984) or slave capture (Myrmecocystus mimicus: Hölldobler, 1981). In the honey bee Apis mellifera non-nestmates rob stored honey and this can kill the victim colony (Winston, 1987). In the honey bee, guard bees are primarily responsible for preventing robbing (Butler and Free, 1952). Guard bees patrol the nest entrance, inspect entering bees, and exclude non-nestmates (Butler and Free, 1952) using odour cues (Breed et al., 1992). Downs and Ratnieks (1999) studied the acceptance of bees when these bees were unrelated to the guard bees but fostered in the guard bees colony. The results showed that workers that shared the same adult environment as a guard were equally likely to be accepted irrespective of whether they were related to the guard or not. This shows that guards use recognition cues that entering workers have acquired from the adult nest environment when discriminating between nestmates and non-nestmates. Downs and Ratniek s study also showed that recognition cues are not only acquired during the adult stage, but can be lost and reacquired during adult life. The most likely explanation is that bees acquire odours from the wax combs and thus that cues used in nestmate recognition are environmental. However, there could still be a genetic component, but which is acquired from the common nest environment rather than being derived from the worker s own genotype (Breed et al., 1995a).

Insectes soc. Vol. 49, 2002 Research article 57 In this study we investigated the changes in nestmate recognition after reproductive swarming. Honey bees reproduce by swarming wherein the mother-queen leaves the colony with approximately two thirds of the workers while one or several daughter queens are reared in the established ( old ) colony. Swarms often nest within 100 200 m of the old colony (Jaycox and Parise, 1980; Jaycox and Parise, 1981; Schmidt, 1995; Schneider, 1995) and this may lead to competition between the newly founded (swarm) colony and old colony. Therefore, it is expected that cue divergence should occur and former nestmates will be rejected at some time after swarming. Because relatedness between the old and newly founded (offspring) colonies is initially high, guards can only use environmental cues to discriminate between nestmates and former nestmates after swarming. Previous work (Breed et al., 1998) has shown that the rejection of former nestmates coincides with the production of wax in the offspring colony. Breed et al. (1998) used foragers that were kept in cardboard cups for 24 72 h with pieces of comb that were newly built by the swarm. These foragers were then brought into contact with guards from the original ( old ) colony. The results of these laboratory tests showed that under these conditions guards started to reject their former nestmates (i.e. bees from the swarm) three to seven days after swarming. How do we explain such a rapid divergence of nestmate recognition cues? Bees in a swarm are initially of the same genetic mixture as those in the old colony. If nestmate recognition cues are based on wax volatiles, it could be that odours picked up during foraging are incorporated into newly-built wax making the wax of the newly founded swarm colony different from the old wax present in the mother colony. However, floral oils do not appear to have an effect on the acceptance rate of intruder bees (Downs et al., 2000) thus making it unlikely that the foraging environment has a large influence on the odour of the wax. However, other odours, like odours of larval faeces, can also be incorporated in wax or the smell of wax may simply differ when wax ages, thus changing the odour even if the wax is built by the same bees. The aim of this study was to investigate changes in the acceptance of former nestmates following reproductive swarming. We used free-flying colonies, actual guards at hive entrances and workers that developed naturally in their natal colony. In this way the workers had the opportunity to acquire a natural suite of recognition cues and not only those cues derived from wax. We used colonies that had built their own combs and then created artificial swarms by moving the mother-queen into a new (comb-less) hive with a portion of her workers (analogous to the prime swarm). We made a second comb-less colony by introducing a new (daughter) queen, thus representing an after-swarm. These two colonies had to build their own wax combs and represent the offspring colonies. We then divided the original combs into two and moved them into new hives each with workers and an emerging daughter-queen thus mimicking two recently swarmed old colonies. Although this is an unnatural situation because normally there is only one colony with the old combs with brood, we did this so we could determine the effect of the presence of the same wax comb on nestmate recognition. During a period of 13 days we then tested the acceptance of bees transferred from the old colonies into the newly founded (offspring) colonies and vice versa to determine when former nestmates are no longer accepted by guard bees. We predicted the following. If guard bees use cues that are solely or predominantly heritable, one would expect that former nestmates would not be rejected for 3 4 weeks after swarming. Rejection would only occur after brood produced by the daughter queen matured to forager age, thereby creating genetic differences between the guards and intruders. Our experiment lasted 13 days and thus ended well before this genetic divergence could have occurred. Thus if genetically based cues are used for nestmate recognition, all former nestmates should be equally acceptable to guards of all divided colonies and one would not expect rejection of former nestmates to occur in our study. On the other hand, if guards use environmentally based cues, cue divergence should occur much faster. If comb wax plays a pivotal role in nestmate recognition, as suggested by previous authors (Breed et al., 1988, 1995a, b, 1998) we would expect that guards of the colonies that comprised combs from the mother colony (the old colonies) should continue to accept each other s foragers after swarming. Similarly, workers originating from the colonies that had to build their own wax (the offspring colonies) would not reject each other. However, workers originating from colonies with the old wax combs would be expected to be rejected by guards of the colonies that build their own wax combs as soon as new wax has been constructed and vice versa. Materials and methods Study colonies Four honey bee (Apis mellifera) colonies of mixed European race were studied. Due to frequent beekeeper manipulations, domestic honey bee colonies rarely contain combs made by the bees present in the colony. To obtain colonies that only contained combs made by the bees in that colony we manipulated our colonies so that all existing combs were replaced by newly-built ones. To do this we sequentially removed the existing combs and replaced them with empty frames in which bees built their own new combs, in the two months before data were collected. This manipulation was necessary to avoid the possibility of cue contamination by the use of wax combs made by other colonies (Breed et al., 1988; Downs and Ratnieks; 1999). This procedure also closely matched natural conditions were bees only have wax comb built by their own colony. The study was conducted twice, once in August 2000 in Laren, the Netherlands (trial 1) and once in January 2001 at Pearl Beach, Australia (trial 2). Both experiments were conducted in mid summer. Each trial used 2 colonies. Experimental design Prior to splitting, each mother (original) colony had approximately 20,000 40,000 bees. Each mother colony was divided into 4 colonies (Fig. 1). Two of the divided colonies had 5 combs from the mother colony (colonies A and B; the old colonies). The other two colonies (colonies C and D; the offspring colonies) had to build their own combs. One of these comb-less colonies, equivalent to a prime swarm, was

58 M. Beekman et al. Nestmate recognition in honey bees as rejection. When the introduced bee was inspected (licked and antennated by guards) and allowed to enter the nest, or remained on the entrance for five minutes without being rejected, this was classed as acceptance. The hives had a standard long bottom board that extended approximately 5 cm beyond the hive entrance to facilitate bee introductions and guard observations. Each colony received 5 foragers from each of the other colonies per day. In addition, 5 nestmates (NM) and 5 non-nestmates (NN: originating from an unrelated colony) were introduced, resulting in a total of 20 (trial 1) or 25 (trial 2) introductions per colony per day. One (trial 1) or 3 (trial 2) days prior to dividing the mother colony and on the day the artificial swarms were made (day 0), 10 nestmates and 10 non-nestmates were introduced to determine the levels of nestmate and non-nestmate acceptance. All bioassays were conducted blind, so that the observer was unaware of the origin of the introduced bees. A laboratory study (Breed et al., 1998) has shown that the occurrence of cue divergence coincided with the building of new wax by the swarms. Bees will start to build new combs as soon as they are in a comb-less hive although it will take a few days before they actually have some new comb built. In our study the trials lasted 11 (trial 1) and 13 (trial 2) days after the mother colonies were divided to ensure that the bees had built a sufficient amount of wax to show an effect of wax odour on cue divergence. Data analysis Figure 1. How each mother colony was divided. Colonies A and B represent the old colonies; colonies C and D the offspring colonies. Bees originating from the same division (nestmates) plus bees from the other three divisions (former nestmates) were introduced (arrows with bee icons) into each colony after dividing (only bees introduced and originating from colony A are shown here). Virgin queens were grafted from the mother colonies and reared in an unrelated colony. No combs means that the colony had to build its own combs. Note that in trial 1 the C colonies did not exist given the mother-queen (colony D). The other three colonies were given virgin daughter queens of the mother queen. These virgin queens had been reared in separate queen rearing colonies from larvae grafted from the 4 appropriate mother (original) colony using standard techniques, and then matured after emergence for 8 days in the queen-rearing colony in cages. After division, colonies were moved to a new apiary to prevent the bees from leaving their hives in search of their original colony. These new apiaries were approximately 7 (the Netherlands) and 90 km (Australia) away from the original apiaries. In trial 1 the hives that contained no combs and a virgin daughterqueen (C colonies), were rapidly abandoned by the workers. Therefore, trial 1 had only 3 colonies per replicate. To prevent this happening again in trial 2, we caged all the virgin queens for 3 days after introduction. At the end of the experiment the colonies were opened and checked for the presence of a laying queen and brood, and to estimate the amount of comb built (for the C and D colonies only) and the number of adult bees. Bioassays Acceptance of introduced bees by guards was measured using a standard bioassay (Downs and Ratnieks, 1999) in which returning foragers are collected, placed in a vial and chilled in ice and then placed at the entrance of a bee hive. Before being placed at the hive entrance the introduced bees were allowed to warm up so that they could walk but not fly. The behaviour of guards towards the introduced bee was then observed. When the introduced bee was bitten or stung this was classed Acceptances of non-nestmates and nestmates by guards prior to colony division were compared using chi square tests. Differences in rates of acceptance among the colonies after making the artificial swarms were compared using two-tailed Fisher exact tests rather than chi square tests because of the low numbers of introduced bees per colony. Because of multiple comparisons, Bonferroni corrections were applied (Rice, 1989) for an overall level of significance of 5%. Results Acceptance of nestmates and unrelated non-nestmates Table 1 shows that prior to the experimental manipulation, all mother colonies were able to discriminate between nestmates (NM) and non-nestmates (NN). Across both trials, 91% of nestmates and 25% of non-nestmates were accepted before the colonies were divided. The difference in acceptance between nestmates and non-nestmates was statistically significant for all four colonies (chi square tests, P 0.001). However, after division discrimination between nestmates and non-nestmates was not as strong: in trial 1 78% of nestmates and 59% of non-nestmates were accepted, in trial 2 76% of nestmates and 56% of non-nestmates (data pooled). Although in only 4 of the 14 colonies the difference between nestmate and non-nestmate acceptance was statistically significant (Table 2, chi square test), in each colony more nestmates than non-nestmates were accepted. Acceptance of former nestmates We were interested in the change in rates of acceptance of former nestmates after artificial swarming. To determine whether the rate of acceptance changed during the course of

Insectes soc. Vol. 49, 2002 Research article 59 Table 1. Proportions of nestmates (NM) and unrelated non-nestmates (NN) accepted by entrance guards before dividing the mother colonies. Assessments were made 1 day prior to colony division (day 1, trial 1), 3 days prior to colony division (day 3; trial 2) and on the day the divisions were made (day 0). The number of bees introduced are given in parentheses. The difference in acceptance between nestmates and nonnestmates was significantly different for all colonies (chi square test, P 0.001) Colony Origin Day Proportion of bees accepted (n) Trial 1 1 NM 1 1.0 (10) 0 1.0 (10) NN 1 0.5 (8) 0 0.1 (10) 2 NM 1 0.8 (10) 0 1.0 (10) NN 1 0.3 (10) 0 0.4 (10) Pooled: NM 0.9 NN 0.3 Trial 2 3 NM 3 0.8 (10) 0 0.8 (10) NN 3 0.2 (6) 0 0.3 (10) 4 NM 3 1.0 (9) 0 0.9 (10) NN 3 0.2 (9) 0 0.0 (9) Pooled: NM 0.9 NN 0.2 the experiment, we pooled the data from the first two days after dividing and compared this with the data of the last two days the trials were performed (days 10 and 11 for trial 1, days 12 and 13 for trial 2). This was done for each of the 14 colonies. In all four replicates there was no significant difference in the acceptance rates of bees originating from the A colonies by guards from the B colonies (both with half of the original combs) and vice versa (two-tailed Fisher exact test, P 0.05, Table 3). This analysis shows that workers from the A and B colonies had not diverged in terms of their accep- tance rates during the course of the experiment. Therefore, from these colonies data were pooled in subsequent analyses. Similarly, bees originating from the C colonies (virgin queen, no combs) were accepted by the D colonies (mother queen, no combs) and vice versa and therefore these data were also pooled (only for colonies 3 and 4 because colonies 1 and 2 did not have a C colony; Table 3). In artificial swarms derived from colonies 1 and 3, the rate of acceptance of foragers from the natal comb colonies (A and B) into non-natal comb colonies (C and/or D) declined over time suggesting that the cues used by bees for nestmate recognition diverged during the experimental period. No such change was evident in the swarms derived from colonies 2 and 4 (Fig. 2, Table 4). Although we used a twotailed Fisher exact test to test for statistically significant differences (see Table 4), we have plotted regression lines in Figure 2 to show the change in acceptance. Status of the colonies at end of experiment The status of the colonies (number of combs built, number of bees present, presence of young queen and whether she was laying) were assessed at the end of the experiment (Table 5). All colonies were of approximately equal size except colony 1D which had twice as many bees compared with the other colonies (Table 5). This was probably because bees from the original fourth division in trial 1 (C: no combs and a new queen) joined this colony after dividing. Colony 4B (trial 2) lost its queen during the experiment as indicated by the presence of emergency queen cells. Colony 4C (trial 2) lost its queen immediately after the colony was made. To keep the bees in this colony, artificial queen pheromone containing 9ODA (Bee Boost) was added before the bioassay was started. Discussion The aim of our study was to investigate changes in acceptance of former nestmates after colony division by swarming and the effect of the origin of wax comb on this. Our results Trial 1 Proportion accepted: Trial 2 Proportion accepted: Colony 1 NM NN P Colony 3 NM NN P A 0.71 (34) 0.63 (35) 0.49 A 0.79 (43) 0.64 (39) 0.13 B 0.71 (34) 0.61 (33) 0.39 B 0.77 (43) 0.32 (44) 0.00 D 0.60 (35) 0.46 (35) 0.05 C 0.74 (43) 0.67 (45) 0.43 D 0.66 (44) 0.48 (40) 0.09 Colony 2 Colony 4 A 0.77 (35) 0.74 (35) 0.78 A 0.93 (44) 0.60 (45) 0.00 B 0.94 (34) 0.74 (35) 0.02 B 0.71 (45) 0.60 (40) 0.28 D 0.89 (35) 0.37 (35) 0.00 C 0.69 (45) 0.60 (45) 0.38 D 0.75 (40) 0.58 (38) 0.11 pooled 0.78 0.59 0.00 pooled 0.76 0.56 0.00 Table 2. Proportion of nestmates (NM) and unrelated non-nestmates (NN) accepted by guard bees after artificial swarming. Data from all days following swarming have been pooled (11 days for trial 1, 13 days for trial 2). The number of bees introduced are given in parentheses. P-values are based on chi square tests

60 M. Beekman et al. Nestmate recognition in honey bees Table 3. Comparison of acceptance rates of bees originating from the A (C) colonies by the B (D) colonies at the beginning (first two days pooled) and the end (last two days pooled) of the experimental period. Colonies A and B each contained half of the combs from the mother colony, colonies C and D had to build their own combs. The number of bees introduced are given in parentheses. P-values are based on twotailed Fisher exact tests Colony A into B B into A C into D D into C P P P P 1 1.00 (20) 0.37 (20) 2 0.13 (20) 1.00 (19) 3 0.71 (20) 0.71 (20) 0.01 (24) 1.00 (20) 4 1.00 (20) 1.00 (20) 1.00 (20) 0.58 (20) Table 4. Acceptance rates of bees originating from the combined A+B colonies (with old combs) by the combined (C+)D colonies (with new combs) at the beginning (first two days pooled) and the end (last two days pooled) of the experimental period. The number of bees introduced are given in parentheses. Shaded cells are statistically significant results (two-tailed Fisher s exact test, a after Bonferonni correction: 0.006). Acceptance rates are presented in Figure 2 Colony A+B into D D into A+B A+B into C+D C+D into A+B P P P P 1 0.000 (40) 0.020 (40) 2 1.000 (40) 0.040 (38) 3 0.003 (81) 0.002 (80) 4 0.134 (80) 1.000 (78) Table 5. Status of the colonies at the end of the experiment number of number of young queen young queen Colony combs built bees present? laying? 1A 4500 y n 1B 4500 y y 1D 6 9000 old queen 2A 4500 y y 2B 4500 y n 2D 3 4500 old queen 3A 4500 y y 3B 4500 y y 3C 3 4500 y y 3D 3 4500 old queen 4A 4500 y y 4B 4500 n* 4C 3 4500 n** 4D 3 4500 old queen Figure 2. Acceptance of bees originating from colonies A+B (with old combs) introduced into colony D (for colonies 1 and 2, where there was no C colony) or into colonies C+D (with newly build combs) (for colonies 3 and 4). Lines represent the linear regression to show the change in response (dotted for A+B into (C+)D; undotted for (C+)D into A+B) * emergency queen cells present. ** Bee Boost added before start of bioassays. suggest that comb wax plays an important role in developing nestmate recognition cues. In artificial swarms derived from colonies 1 and 3, workers originating from the offspring colonies C and D (i.e. the colonies that had built their own wax combs) were rejected by guards from the old colonies A and B which contained the old combs. In addition, workers originating from colonies 3C and 3D were rejected by guards from colonies 3A and 3B (Table 4). This indicates that the presence of the same wax affects acceptance by guard bees. However, this did not always occur: swarm colonies derived from colonies 2 and 4 did not show any change at all, i.e. all former nestmates were accepted by guards. The discrimination between nestmates and unrelated non-nestmates was less pronounced after the colonies were divided

Insectes soc. Vol. 49, 2002 Research article 61 (Table 2). This may have been due to the decreased colony size after dividing and could explain why we did not find a change in 2 of the 4 replicates. It is not surprising that comb wax plays an important role in nestmate recognition. Both honey bee comb wax (Hepburn, 1986; Tulloch, 1980) and the honey bee cuticle surface (Page et al., 1991) are composed primarily of hydrocarbons and this facilitates rapid cue transfer from wax to cuticular surface (Breed et al., 1995b). Comb wax is also a plausible source of colony recognition cues because the chemical composition of wax is genetically variable (Breed et al., 1995b). This means that the chemical composition of comb wax varies among colonies and workers that acquire cues from wax will be uniformly labelled with a colony-specific cue. However, our results from colonies 1 and 3 suggest that differences in wax odour are not solely determined by the genotype of the bees that produce it. Our results of 2 of 4 replicates where guards rejected their former nestmates when they originated from colonies that did not contain the same wax combs, suggest that other odours, for instance odours acquired during foraging or odour of larval faeces, are also incorporated into wax and these odours can be used as nestmate recognition cues. Alternatively, the odour of wax may change as it ages, resulting in old wax containing different odours compared with new wax, even if the bees that built the wax are genetically similar. Using environmental cues instead of heritable cues enables honey bee colonies to adapt quickly to changing conditions. However, when environmentally acquired differences between individuals are low (Greenberg, 1979) or minimised artificially (Breed et al., 1985), heritable cues can be used to discriminate between nestmates and non-nestmates. 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