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Plasticity of worker reproductive strategies in Bombus terrestris: lessons from artificial mixed-species colonies
ANIMAL BEHAVIOUR, 2006, 72, 1417e1425 doi:10.1016/j.anbehav.2006.05.008
Plasticity of worker reproductive strategies in Bombus terrestris: lessons from artificial mixed-species colonies CEDR IC A L A UX *, A BRA H AM H EF ETZ † & P IERR E J AI SS ON*
*Laboratoire d’Ethologie Expe´rimentale et Compare´e, Universite´ Paris 13 yG. S. Wise Faculty of Life Sciences, Department of Zoology, Tel Aviv University (Received 15 November 2005; initial acceptance 1 March 2006; final acceptance 12 May 2006; published online 16 October 2006; MS. number: A10300)
We used the experimental paradigm of artificial mixed colonies of two phylogenetically related bumblebee species to analyse the dynamics of the reproductive skew in societies of Bombus terrestris. Artificial mixedspecies colonies were set up by introducing callow B. terrestris workers either into a queenright (QR) or a queenless (QL) colony of B. lapidarius. The introduced B. terrestris workers were well integrated into their host B. lapidarius colony and displayed nesting activities that did not differ from those of the resident B. lapidarius workers. However, the introduced B. terrestris workers did show a different reproductive behaviour. While B. lapidarius workers did not develop ovaries in a B. lapidarius QR colony but did so in a B. lapidarius QL group, adopted B. terrestris workers in a B. lapidarius QR colony developed ovaries as if they were under QL conditions. These results indicate that, in mixed-species colonies, B. terrestris workers are irresponsive to the queen’s inhibitive action on ovary development. In QL homospecific and heterospecific predominately B. terrestris mixed-worker colonies (1Bl þ 5Bt), reproduction was dominated by a single B. terrestris worker, whereas in QR B. lapidarius or QL equally mixed-worker colonies (3Bl þ 3Bt), almost all B. terrestris workers developed ovaries. We suggest that in the presence of enough heterospecific workers, B. terrestris workers behave as parasites. This last finding suggests that worker reproduction in B. terrestris is highly plastic and that the experimental paradigm of artificial mixed colonies may provide new insights into the evolution of social parasitism in this taxon. Ó 2006 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Reproductive skew is a hallmark of insect societies, where female reproduction is highly biased in favour of one or more individuals, the queens, whereas nonreproductive individuals, the workers, are helpers. In many species, however, workers are not irreversibly sterile and remain able to lay viable eggs if they are orphaned, or at certain stages of colony development. Reproductive plasticity in workers is highly contextual and depends on the underlying genetic and social structures of the colony (Bourke & Franks 1995; Crozier & Pamilo 1996). Moreover, it is governed by two seemingly opposed selection pressures: selfishness, driven by combined individual and kin selection; and cooperativeness, driven by
Correspondence and present address: C. Alaux, Department of Entomology, University of Illinois, Urbana, IL 61801, U.S.A. (email: [email protected]). A. Hefetz is at the G. S. Wise Faculty of Life Sciences, Department of Zoology, Tel Aviv University, 69978 Tel Aviv, Israel. P. Jaisson is at the Laboratoire d’Ethologie Expe´ rimentale et Compare´e, CNRS UMR 7153, Universite´ Paris 13, 93430 Villetaneuse, France. 0003e 3472/06/$30.00/0
combined kin- and colony-level selections. Thus, unravelling the ultimate and proximate mechanisms for the evolution and maintenance of such a reproductive skew offers a major topic of interest. Caste-specific pheromones, particularly queen pheromones, are believed to regulate reproductive skew in social insects. They may operate either by actively inhibiting worker ovary development (Butler & Fairey 1963; Ho¨lldobler & Bartz 1985; Hoover et al. 2003) or by acting as an honest fertility signal (Keller & Nonacs 1993; Endler et al. 2004; Dietemann et al. 2005). In the latter case, workers are hypothesized to use all available information to adjust their behaviour in a way that will maximize their fitness. This further explains why worker sterility is plastic and conditional. Workers, therefore, are selected to enhance overall colony growth and refrain from reproducing as long as the genetic gain through inclusive fitness surpasses that obtained through direct fitness. It is consequently adaptive for both workers and the queen to evolve mechanisms for queen recognition, namely queen-specific signals, which may not necessarily be species specific.
1417 Ó 2006 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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ANIMAL BEHAVIOUR, 72, 6
The bumblebee Bombus terrestris has become a model system for testing both ultimate and proximate processes related to reproductive social conflicts (van Honk et al. 1981; Duchateau & Velthuis 1988; Ro¨seler & van Honk 1990; Bloch et al. 1996; Bloch 1999; Bloch & Hefetz 1999; Alaux et al. 2004a, 2005; Lopez-Vaamonde et al. 2004a). Colonies of B. terrestris are annual with one, singly inseminated queen (Estoup et al. 1995; Schmid-Hempel & Schmid-Hempel 2000), creating a potential conflict between the queen and workers over male production. Colony life cycle follows two main phases (Alford 1975). The first, the social phase, is characterized by cooperation, which maximizes ergonomic growth and gyne production as well as reproductive self-restraint among workers, despite their potential to lay viable haploid eggs (Alaux et al. 2004a). The regulation of worker reproduction is mediated by the perception of nonvolatile queen pheromones via direct antennal contacts. Workers also establish a dominance hierarchy among themselves during this phase (van Honk & Hogeweg 1981; van Doorn & Heringa 1986). However, this reproductive dominance hierarchy among workers is highly flexible and can change throughout colony development. The second phase marks the breakdown of social structure and is characterized by an accelerated competition between the queen and the workers over reproduction and among workers for access to reproduction (Bloch & Hefetz 1999). Queeneworker conflict over male production in the bumblebee B. terrestris is thus well evidenced. Recent molecular studies, however, have revealed that despite the overt competition between the queen and her workers over male production during the competition phase, the queen seems to produce over 95% of the adult males (Alaux et al. 2004b; Lopez-Vaamonde et al. 2004b). The queen is able to successfully outcompete the workers owing to her higher reproductive output, and greater aggression and oophagy capacity. Worker reproduction seems to succeed only when the queen dies prematurely, suggesting that workers may in fact execute the queen in order to reproduce (Bourke 1994; Alaux et al. 2004b). Finally, the selfish interest of workers may be expressed when they invade an alien conspecific colony, where they can unrestrainedly lay eggs (Lopez-Vaamonde et al. 2004b). All these insights into queeneworker conflict in B. terrestris have raised the hypothesis that worker reproduction is highly plastic and may depend on the social circumstances. Workers are thus able to show two opposite phenotypes, from cooperation to selfishness, depending on the dominant selection level. However, unravelling the proximate mechanisms that result in either worker sterility or their fertility is sometimes complicated under natural conditions. Thus, the use of mixed-species groups might be considered as potentially revealing, since each species may express its preprogrammed traits, whether kin-selected or not, independently of colony-level selection. Moreover, it enables investigation of the level of reproductive plasticity of workers as well as examination of the effects of social environment on reproductive plasticity. For this purpose, we used a system of mixed-species colonies to unravel the mechanisms and flexibility level of
the reproductive strategies that may be shown by worker B. terrestris, but that remain hidden from the researcher under normal colony conditions (Jaisson 2005). We created mixed-species colonies and groups using B. terrestris and B. lapidarius, two phylogenetically closely related species (Kawakita et al. 2004). Although reproductive skew and queeneworker conflict have been less studied in B. lapidarius (Free et al. 1969), their phylogenetic proximity and similarity of life histories suggested that their social behaviour might also be comparable. Thus, we first tested whether worker B. lapidarius refrain from reproduction under queenright (QR), but not queenless (QL) conditions, and whether the queen has a pheromone regulating worker reproduction. We then investigated whether worker B. terrestris can integrate into a B. lapidarius colony, and whether reproductive strategy of B. terrestris workers changed in heterospecific colonies compared to homospecific colonies. Finally, we created QL mixed-species groups to further probe into worker reproductive strategies.
MATERIAL AND METHODS
Bumblebee Rearing Queens of B. lapidarius were collected in the field near Paris in early spring 2003 and 2004. They were allowed to found a colony in the laboratory by placing each one in a wooden box (17.5 26 15 cm) kept in the dark at 28 2 C and 50% relative humidity. They were fed ad libitum with sugar syrup and fresh pollen. The experiments started once these colonies had reached 10 workers (i.e. about 1 week after the first worker emergence). Observations were performed under red light through a glass covering the nestbox. Colonies of B. terrestris were obtained either from a commercial supplier (GTICO SARL, Foissy sur Vannes, France) or from our laboratory stock (colonies reared from queens obtained from commercial colonies). All colonies were reared under the same standard conditions.
Regulation of Worker Reproduction in Homospecific B. lapidarius Colonies To test whether queens of B. lapidarius produce a volatile pheromone that inhibits worker reproduction, we split nests into two compartments separated by a doublemesh (1-cm-wide) screen. One compartment included all colony workers and the queen (QRC) while the second compartment contained five callow (1-day-old) nestmate workers (QLC). No physical contact was possible between these separated workers and the rest of the colony, including the queen. QL groups of five callow workers housed separately served as control. Both the QL and the QLC were supplied with young cocoons (collected from young donor colonies) as a substrate for cell construction (van Doorn 1987). We recorded the occurrence of newly sealed egg-cells in the QLC and QL colonies, and compared ovary development (mean length of the terminal oocytes) between workers from the QLC, QR and QL colonies. Control workers, originating from nonmanipulated QR
ALAUX ET AL.: WORKER REPRODUCTIVE PLASTICITY IN BEES
colonies 20 days after the first worker emergence, were also frozen for determination of ovary development (at this colony age workers have had the time required to develop ovaries; Free et al. 1969).
honey pots, feeding larvae, brood incubation, enlargement of wax cover around the brood, threatening behaviour (buzzing), and overt aggression.
Regulation of Worker Reproduction in Mixed-species Colonies
Introduction of B. terrestris Callow Workers into B. lapidarius QR Colonies
Queenright artificial mixed-species colonies
To test whether a putative B. lapidarius queen pheromone inhibits ovary development in adopted young callow B. terrestris workers, we introduced five B. terrestris workers that had been individually marked using numbered tags (Opalith Pla¨ttchen, Friedrich Wienold, Germany) into a QR B. lapidarius colony (N ¼ 17). The introduced B. terrestris were callow workers that had been allowed to emerge in homospecific QL colonies to prevent contact with the conspecific queen. Five control callow B. lapidarius workers were equally marked and then reintroduced into their colony. In those colonies in which there were insufficient B. lapidarius callow workers at the start of experiment, callow workers from other colonies were introduced. To verify whether the introduced B. terrestris workers had fully integrated into their host colony, we performed scan-sampling behavioural observations on seven randomly selected QR mixed colonies every 2 min for 30 min twice a day (mornings and afternoons during days 2e6). We noted 16 behavioural acts related to worker tasks (Cameron 1989): foraging, guarding (at nest entrance or on the brood, antennae raised), patrolling (rapid movements), nest inspection, fanning, immobility (antennae lowered), self-grooming, scraping wax (workers recycle the wax of empty cocoons), anchoring (building of wax area), excavation of empty cocoons, working on
Egg laying by B. terrestris workers introduced into QR B. lapidarius colonies (N ¼ 17) was estimated from the seventh day following introduction (the time needed for ovary development in QL callow workers), by counting the number of eggs in the newly sealed egg-cells. Since B. lapidarius queens lay a significantly greater number of eggs per cell than do B. terrestris workers at their first egg laying in QL colonies (Table 1), we could discern to which species the laid eggs belonged. To ascertain that these were B. terrestris eggs, a sample (from 2 to 4 eggs of each colony’s egg-cup) was transferred into small QL B. terrestris colonies containing young nonreproductive workers and reared to adulthood. All the emerged adults were B. terrestris males. Workerequeen interactions were estimated by monitoring the number of workerequeen antennal contacts of the marked individuals (5 B. terrestris workers þ 5 B. lapidarius workers for each observed colony, N ¼ 5) daily for 30 min (15 min morning and afternoon), from the second day of worker introduction until the first oviposition by B. terrestris workers occurred. The experiment ended when the first egg laying by the adopted B. terrestris was observed (B. lapidarius workers never laid eggs under these conditions, see Results). Thereafter, all marked bees were removed and dissected to determine ovary development. The five groups in
Table 1. Comparison of the means (ManneWhitney U test) and ranges (c2 test) for the number and size of eggs laid by B. terrestris and B. lapidarius queens and workers in the different experimental situations MeanSD number Range of number of eggs/cell of eggs/cell B. lapidarius queen QL B. terrestris workers* QR mixed B. terrestris workers
8.91.17, N¼16 4.051.40, N¼20 3.901.30, N¼17
7e12 2e7 2e6
QL B. lapidarius workers 3Blþ3Bt colonies 1Blþ5Bt colonies
B. lapidarius queen versus QL B. terrestris workers B. lapidarius queen versus QR mixed B. terrestris workers QL B. terrestris workers versus QR mixed B. terrestris workers QL QL QL QL QL
B. B. B. B. B.
terrestris workers versus QL B. lapidarius workers terrestris workers versus 3Blþ3Bt colonies terrestris workers versus 1Blþ5Bt colonies lapidarius workers versus 3Blþ3Bt colonies lapidarius workers versus 1Blþ5Bt colonies
MeanSD egg size (mm)
Range of egg size
3.100.11, N¼54
2.75e3.25
2.850.1, N¼55 3.150.11, N¼78 3.110.08, N¼57
2.67e3.08 2.92e3.42 2.92e3.33
ManneWhitney U test Mean number of eggs/cell U¼1, P<0.001 U¼0, P<0.001 U¼170, P¼1
c2 test Range of number of eggs/cell c2¼33.3, P<0.001 c2¼33, P<0.001 c2¼3.2, P¼0.73
Mean egg size U¼176.5, P< 0.001 U¼1628.5, P<0.05 U¼1445, P¼0.69 U¼90, P<0.001 U¼96, P<0.001
Range of egg size c2¼71.3, P<0.001 c2¼10.63, P¼0.22 c2¼4.1, P¼0.77 c2¼106.9, P<0.001 c2¼80.24, P<0.001
QL: queenless colonies; QR: queenright colonies. *Egg characteristics of QL B. terrestris workers correspond to their first oviposition.
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which some of the introduced B. terrestris (N ¼ 4, 11% of introduced workers) and B. lapidarius workers (N ¼ 2, 6% of introduced workers) died during the experiment were excluded from this analysis.
Queenless artificial mixed-species colonies We created two types of QL mixed colonies to investigate the plasticity of worker reproduction under these conditions and determine the role of the number of heterospecific workers in the reproductive plasticity. The first type comprised three B. lapidarius and three B. terrestris workers (called QL 3Bl þ 3Bt, N ¼ 20) and the second was composed of one B. lapidarius and five B. terrestris workers (called QL 1Bl þ 5Bt, N ¼ 12). Control groups consisted of either five B. terrestris (N ¼ 12) or five B. lapidarius workers (N ¼ 12), each group of which was housed separately. Once the first egg-cell was constructed all workers were frozen for later dissection. Worker oviposition in homospecific control colonies showed that egg size of B. lapidarius was significantly smaller than that of B. terrestris (Table 1). Egg size, therefore, served as a reliable measure for determining egg species identity. Behavioural observations were performed on a randomly chosen sample of each of the QL mixed (3Bl þ 3Bt, N ¼ 8; 1Bl þ 5Bt, N ¼ 8), homospecific B. terrestris (N ¼ 9) and homospecific B. lapidarius (N ¼ 8) colonies. They were performed twice a day (10 min in the morning þ 10 min in the afternoon) during days 2e7, focusing on workere worker aggression (biting, grappling and head butting, see Bloch et al. 1996 for details).
Statistics We used a factorial correspondence analysis (FCA) on the complete behavioural repertoire (number of items for each recorded task) applied to the marked bees (Spad 3.1 software, Spadsoft, Paris, France). Bees that died during the behavioural experiment were not taken into account in the FCA. Permutation tests by stratum (StatXact 3.1, Cytel Software, Cambridge, Massachusetts, U.S.A.) were performed for B. lapidarius (N ¼ 33) and B. terrestris workers (N ¼ 31) on the resulting coordinates of each individual in the FCA (root 1 and 2), taking into account their mixed-group origin. This enabled us to consider intercolonial variation. To analyse how ovary development was distributed among workers, we first determined whether there was any significant variation between groups within each treatment (Table 2). Next, we categorized their development into five stages representing equal ovary development range for each species (B. lapidarius: stage 1: 0e0.54 mm; stage 2: 0.55e1.08 mm; stage 3: 1.09e1.62 mm; stage 4: 1.63e2.16 mm; stage 5: 2.17e2.7 mm; B. terrestris: stage 1: 0e0.75 mm; stage 2: 0.76e1.5 mm; stage 3: 1.51e2.25 mm; stage 4: 2.26e3 mm; stage 5: 3.01e3.75 mm), and used a KolmogoroveSmirnov test to determine whether the resulting distributions deviated from a normal distribution (Statistica 6.1 software, Statsoft, Tulsa, Oklahoma, U.S.A.). In groups that showed
Table 2. Results of KruskaleWallis tests on the mean size of worker ovary development under the different social regimes
QLC homospecific B. lapidarius QL B. terrestris workers QL B. lapidarius workers QR mixed B. terrestris workers QR mixed B. lapidarius workers QL B. terrestris workers (3Blþ3Bt) QL B. lapidarius workers (3Blþ3Bt) QL B. terrestris workers (1Blþ5Bt)
H
df
P
1.49 3.84 6.40 13.54 10.14 13.98 16.24 1.37
8, 45 11, 60 11, 60 11, 60 1, 60 19, 60 19, 60 11, 60
0.992 0.974 0.845 0.259 0.517 0.78 0.64 0.99
QLC: queenless compartment of nest; QL: queenless colonies; QR: queenright colonies.
a normal distribution of ovary development, reproduction was considered as not skewed. RESULTS
Regulation of Worker Reproduction in Homospecific B. lapidarius Colonies The average time to first oviposition by B. lapidarius workers after group establishment in the QLC of the homospecific colonies was not significantly different from that in the control QL colonies (ManneWhitney U test: U ¼ 45, N1 ¼ 9, N2 ¼ 12, P ¼ 0.522; Table 3). Ovary development in workers from both QL (N ¼ 60) and the QLC colonies (N ¼ 45) was significantly greater than that of workers from nonmanipulated QR colonies (N ¼ 150) (KruskalleWallis test: H2,269 ¼ 116.16, P < 0.001; Siegele Tukey post hoc test: QL/QLC: P ¼ 0.612; QR/QL and QR/ QLC: P < 0.001; Table 3). This demonstrates that if a queen pheromone does affect worker reproduction, it is nonvolatile as in B. terrestris (Alaux et al. 2004a).
Social Integration of B. terrestris Workers Adopted by B. lapidarius Colonies In-nest behaviour of the introduced B. terrestris workers (N ¼ 31) was compared to that of the marked and reintroduced B. lapidarius workers (N ¼ 33) during days 2e6 following introduction (FCA on all observed individuals and considering all the behaviours outlined above; Fig. 1). The first two roots revealed that individuals of both species created overlapping clouds and did not differ significantly according to their behavioural repertoire (permutation test: root 1: P ¼ 0.116; root 2: P ¼ 0.602). Thus, at least with respect to the measured parameters, worker B. terrestris showed an integration into their host B. lapidarius colony.
Regulation of Worker Reproduction in Mixed-species Colonies Queenright artificial mixed-species colonies In the queenright mixed colonies (N ¼ 17) on a mean SD of 8.06 1 days after B. terrestris workers were
ALAUX ET AL.: WORKER REPRODUCTIVE PLASTICITY IN BEES
Table 3. Reproductive timing and ovary development (mean SD) of callow workers from the different colony types
Colony type QR homospecific B. lapidarius QLC homospecific B. lapidarius QL homospecific B. lapidarius QL homospecific B. terrestris QR mixed colonies QL mixed colonies (3Blþ3Bt) QL mixed colonies (1Blþ5Bt)
Worker type
Ovary development (mm)
N workers
Marked B. lapidarius Introduced B. terrestris B. lapidarius B. terrestris B. lapidarius B. terrestris
0.150.42 1.570.57 1.360.83 0.871.12 0.160.36 2.130.82 1.180.84 2.060.87 0.80.8 1.071.13
150 45 60 60 60 60 60 60 12 60
Days until egg laying by the first worker
N colonies 6 9 12 12 17
d 8.441.17 8.081.04 7.580.62 d 8.061 7.80.6
20 12
d 7.580.76
QR: queenright colonies; QLC: queenless compartment of nest; QL: queenless colonies.
B. lapidarius colonies was greater than that of workers kept in homospecific QL colonies (ManneWhitney U test: U ¼ 743.5, N1 ¼ 60, N2 ¼ 60, P < 0.001; Table 3). This difference resulted from the fact that in homospecific QL colonies only one bee had fully developed ovaries, whereas in the QR mixed colonies, all the adopted B. terrestris workers had developed ovaries (Fig. 2a). Thus, ovary development was not skewed as expected from the normal reproductive strategy shown by QL B. terrestris workers.
Queenless artificial mixed-species colonies Egg size was used to compare the pattern of worker reproduction for B. terrestris and B. lapidarius in QL mixedspecies groups. The mean egg size in mixed-species QL groups 1Bl þ 5Bt differed significantly from those of QL B. lapidarius colonies but not from those of the QL B. terrestris colonies (Table 1). In mixed-species QL groups 3Bl þ 3Bt, egg size differed significantly from both QL homospecific colonies. However, in QL colonies 3Bl þ 3Bt, egg size was closer to that of QL B. terrestris colonies than to that of QL B. lapidarius colonies. Moreover, the range 1 B. lapidarius workers B. terrestris workers Root 2 (13.5%)
introduced we observed some egg-cells containing fewer eggs than expected from those normally laid by B. lapidarius queens (Table 1). Egg numbers in these cells were not significantly different from those of QL B. terrestris colonies, indicating that they might have originated from B. terrestris workers. To verify this assumption, we reared a subset of these eggs in a young QL colony composed of nonreproducing B. terrestris workers. All the brood developed exclusively into B. terrestris males. It thus seems that the adopted B. terrestris workers had started to oviposit after the expected physiological time-lag required for ovary development in QL callow workers, and that the B. lapidarius queen had failed to inhibit heterospecific worker reproduction. The time required to achieve reproduction for B. terrestris workers did not differ significantly between social regimes (KruskalleWallis test: H5,82 ¼ 5.99, P ¼ 0.306; Table 3). In contrast, resident B. lapidarius workers in these colonies, whether reintroduced or nonmanipulated, did not reproduce in the presence of the queen, whereas they started to lay eggs after a mean SD of 8.08 1.04 days when reared in QL colonies (Table 3). Behavioural observations on the B. terrestris bees revealed that they had a significantly higher rate of contact with the B. lapidarius queen than the B. lapidarius workers had with their mother queen (1.23 0.25, N ¼ 25 versus 0.68 0.4 antennal contacts per worker per 30 min, N ¼ 25; ManneWhitney U test: U ¼ 148.5, P < 0.01). Moreover, antennal contact rates between B. terrestris workers and the B. lapidarius queen were not significantly different from those shown by prospective reproductive B. terrestris workers with their own queen, but were higher than the rate shown by nonreproductive workers (data in Alaux et al. 2004a; ManneWhitney U test: U ¼ 699.5, N1 ¼ 25, N2 ¼ 60, P ¼ 0.626 and U ¼ 1082.5, N1 ¼ 25, N3 ¼ 312, P < 0.001, respectively). Worker B. lapidarius from QR mixed colonies or from QR homospecific colonies had undeveloped ovaries, as opposed to QL workers, which had significantly larger terminal oocytes (ManneWhitney U test: QR mixed versus QR homospecific workers: U ¼ 4292, N1 ¼ 60, N2 ¼ 150, P ¼ 0.601; QR mixed versus QL workers: U ¼ 417.5, N1 ¼ 60, N3 ¼ 60, P < 0.001; Table 3). Interestingly, the average ovary development of B. terrestris workers in QR
0.5
0
–0.5
–1 –1
–0.5
0 Root 1 (20.6%)
0.5
1
Figure 1. Distribution of workers of B. lapidarius (N ¼ 33 workers) and B. terrestris (N ¼ 31) colonies as a function of their behavioural pattern obtained from scan sampling. Each point represents an individual on the first two axes of the factorial correspondence analysis of behavioural data. Workers that died before the end of the observations were not considered for analysis.
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ANIMAL BEHAVIOUR, 72, 6
40 (a) B. terrestris
QR mixed QL mixed (3Bl+3Bt) QL mixed (1Bl+5Bt) QL
35
D=0.16, P=0.081 D=0.151, P=0.117 D=0.225, P<0.01 D=0.262, P<0.001
30 25 20 15 10 5 Worker number
1422
0
Stage 1
Stage 2
Stage 3
Stage 4
Stage 5
60 (b) B. lapidarius
QR mixed D=0.456, P<0.001 QL mixed (3Bl+3Bt) D=0.168, P=0.061 QL D=0.103, P=0.515
50
40
30
20
10
0
Stage 1
Stage 2
Stage 3
Stage 4
Stage 5
Ovary development (mm) Figure 2. Ovary development distribution of B. terrestris and B. lapidarius workers from the different mixed and homospecific colonies (N ¼ 60 workers for each condition). (a) B. terrestris workers. (b) B. lapidarius workers. D and P values based on KolmogoroveSmirnov test of normality are reported for each condition. A significant P value means that the ovary development distribution was skewed.
of egg sizes in QL mixed colonies (3Bl þ 3Bt and 1Bl þ 5Bt) was significantly different from that of QL B. lapidarius colonies but not from that of QL B. terrestris colonies. Thus, all the eggs oviposited could be attributed to B. terrestris workers in QL mixed colonies 1Bl þ 5Bt. Although some of the eggs in QL mixed colonies 3Bl þ 3Bt could have been laid by B. lapidarius workers, it is very likely that most of them originated from B. terrestris workers. Ovary development of B. terrestris workers within the mixed-species colonies revealed two distribution patterns (Fig. 2a). When they were either adopted by a QR B. lapidarius colony (QR mixed) or reared with an equal number of B. lapidarius workers (QL mixed 3Bl þ 3Bt), most of
them had developed ovaries and mean oocyte size distribution did not differ significantly from normal distribution. In contrast, when reared either in pure B. terrestris QL groups or in the mixed colonies where they were the majority (QL 1Bl þ 5Bt), ovary development was highly skewed, with only one dominant bee with developed ovaries. The same analysis for B. lapidarius revealed that in both the pure QL colonies and the mixed-species group (QL mixed 3Bl þ 3Bt) most workers had similar nonskewed ovary development (Fig. 2b). In contrast, workers from the QR colonies did not develop ovaries. Ovary development in the single B. lapidarius workers from the 1Bl þ 5Bt QL mixed colonies was scattered (range
ALAUX ET AL.: WORKER REPRODUCTIVE PLASTICITY IN BEES
0e1.99 mm) and had a low mean SD oocyte length (0.8 0.8 mm, N ¼ 12; Table 3). Ovary development was in concordance with the results of the behavioural observations. In the homospecific QL colonies of B. terrestris, agonistic behaviour was performed by a single worker, and in B. lapidarius colonies the dominant worker displayed 85.7 11.5% of the aggression. In the QL mixed 3Bl þ 3Bt colonies (N ¼ 8 colonies), intraspecific aggression reached 21 8.8% for B. terrestris and 10.85 9.2% for B. lapidarius of total aggression (each of them displayed by only one individual). Interspecific aggression in these groups constituted 68.15 11.4% of all aggression, of which 90.5 14.2% was performed by a single B. terrestris worker and 9.5 14.2% was displayed by a single B. lapidarius worker in each colony. In the QL mixed 1Bl þ 5Bt colonies, aggression was exclusively displayed by B. terrestris workers. Thus, the reproductive plasticity shown by B. terrestris workers changed accordingly to the composition of mixed colonies. The low reproductive skew observed between B. terrestris workers was not due to the simple detection of heterospecific workers but rather to their number.
DISCUSSION Worker reproduction is governed by two selection processes. Individual selection drives workers to behave selfishly since they gain maximum fitness by rearing sons (Bourke & Franks 1995; Crozier & Pamilo 1996). Such selfishness may cause considerable colony deterioration to the point that optimal rearing of sexuals is hampered. However, colony-level selection can nullify this selfishness, resulting in worker sterility. In bumblebees, colonylevel selection is very powerful since genetic gain from future gynes is greater than that from future sons, forcing workers to ensure that gyne production has been initiated before they attempt to reproduce (Alaux et al. 2005). Thus, worker reproduction is conditional and dependent on social context (see Introduction). We constructed mixedspecies colonies and groups of two phylogenetically related bumblebees, B. terrestris and B. lapidarius, to assess the level of this reproductive plasticity and the mechanisms implied. This kind of experimental paradigm has been used successfully in ants to examine the nature of both the template and label involved in nestmate recognition (reviewed by Lenoir et al. 2001; Errard et al. 2005; Jaisson 2005). We first established that B. lapidarius workers do not reproduce under queenright conditions and that, if this inhibition is pheromonally mediated, the pheromone used is nonvolatile. Thus, worker B. lapidarius seem to behave like B. terrestris workers, in which direct antennal contact with the queen is required for complete reproductive inhibition to occur (Alaux et al. 2004a). Bombus terrestris workers were observed to be integrated into the queenright colonies of B. lapidarius into which they had been introduced: they received no aggression from the resident bees and behaved as if they were in their native colony. This was confirmed by the factorial analysis applied on multiple behaviours, which showed that
worker B. terrestris contributed as much to B. lapidarius colony maintenance and growth, including brood care, as did the conspecific workers. In contrast to the resident B. lapidarius workers, however, they did not refrain from reproducing and behaved as if queenless by developing ovaries within about 8 days (i.e. the time necessary for a dominant worker to develop ovaries under queenless conditions; Alaux et al. 2004a). We exclude the possibility of an artefact related to the introduction event, because introduced B. lapidarius, whether resident or alien, did not develop ovaries in the presence of the queen. We also exclude the possibility that the lack of inhibition under our experimental conditions was due to insufficient contacts between the queen and B. terrestris workers (assuming, for example, that B. lapidarius queens produced a nonvolatile pheromone that might have an interspecific inhibitory effect). The B. lapidarius queens were in fact antennated at rates that were not different from those performed by prospective reproductive workers towards their mother B. terrestris queens before the competition phase (Alaux et al. 2004a). We conclude that the B. lapidarius queen pheromone is ineffective on B. terrestris workers, whether or not they perceive it. However, because the introduced B. terrestris workers did not behave entirely as alien intruders, it is possible that two pheromones are involved: a queen attractant/recognition that might be non-species-specific (Vienne et al. 1998) and a species-specific ovary-inhibiting pheromone. In wasps, contrasting with our results, Ishay et al. (1986) showed that reproductive Vespula germanica workers reared in a colony of Vespa orientalis were inhibited, suggesting the role of a non-species-specific pheromone or of inhibitory physical aggression. Our findings also offer new insights regarding the evolution of queen control or queen signal (Keller & Nonacs 1993). The complexity (West-Eberhard 1981) as well as the species diversity of queen pheromones may reflect the evolution of an unstable arms race between the two castes with regard to inhibition of worker reproduction. On the other hand, an honest queen signal is quite stable and thus queen pheromones should vary at a lower rate between species. The lack of control of the queen B. lapidarius over the reproduction of B. terrestris workers tends to favour the first hypothesis. Perhaps the most striking result obtained from the artificial mixed colonies was the change in the reproductive strategy of B. terrestris workers. Generally, queenless workers of B. terrestris compete aggressively for reproduction, a conflict that is solved by the worker dominance hierarchy in which only one worker in a group develops ovaries (Bloch et al. 1996; Alaux et al. 2004a). Workere worker inhibition is also apparent when callow workers are introduced into a queenright colony that is well into the competition phase (Bloch & Hefetz 1999). In contrast, in our mixed-species groups, all the introduced B. terrestris had similar ovary development, whether they constituted a minority group (only 5 workers introduced into a whole queenright B. lapidarius colony) or were in groups with an equal number of heterospecific workers (3Bl þ 3Bt). In both cases it appears that the B. terrestris workers did not compete among themselves but developed ovaries
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ANIMAL BEHAVIOUR, 72, 6
independently of each other, at their own developmental rates. One possible explanation is that, being a minority group in a large host colony, B. terrestris workers did not encounter each other at sufficient rates to support dominance hierarchy establishment. This, however, cannot explain why all three B. terrestris workers developed ovaries when housed in small mixed groups with equal numbers of worker B. lapidarius, in contrast with the dominance hierarchy established between three B. terrestris workers housed together (Bloch et al. 1996). Alternatively, we suggest that the change in reproductive strategy by B. terrestris workers might represent exploitation, to the extent of parasitism, of heterospecific workers. This conclusion agrees with the result obtained in groups in which B. terrestris constituted the majority and in which a clear dominance hierarchy was established. This is also consistent with an earlier report that B. terrestris workers show intraspecific social parasitism by laying male eggs in conspecific colonies (Lopez-Vaamonde et al. 2004b). Mixed-species colonies could also occur through queen usurpation of pre-emergent homo- and/or heterospecific host colonies. Queens generally usurp another nest during the foundation phase, but can also do so in very young colonies composed of the first batch of workers (Alford 1975). In this case, the patterns of nest usurpation (i.e. choice of heterospecific or homospecific hosts) would be constrained by whether or not usurper queens can inhibit worker reproduction in host species. That would explain why nest usurpation involving two subgenera has never been found (Hobbs 1965). These results demonstrate that worker B. terrestris have a plastic and context-dependent reproductive strategy. In homospecific colonies and queenless groups, workers compete aggressively between themselves for access to reproduction. The conflict is solved by establishing reproductive dominance, which eventually results in group stability and allows the successful rearing of brood. Kinselected interests are in this case surpassed by colony-level selection because success in brood rearing requires worker cooperation; the subordinate workers benefit because they gain inclusive fitness from rearing nephews. However, when B. terrestris workers were housed in heterospecific colonies they seemed to switch to interspecific competition, so brood rearing may be fully supported by the heterospecific workers, and since the host queen is incapable of controlling their reproduction, the B. terrestris workers may be able to fully express their selfish interest to rear sons. Their behaviour strikingly resembles that of the parasitic clone of Apis mellifera capensis workers, which activate their ovaries in queenright host colonies of Apis mellifera scutellata (Martin et al. 2002; Neumann & Moritz 2002), as well as that of the anarchistic honeybee (a phenotype of Apis mellifera), where some workers display a rare phenotype by developing ovaries despite the queen’s presence (Oldroyd et al. 1994; Hoover et al. 2005). Another remarkable observation was the lack of a clear reproductive dominance between B. lapidarius workers whether in homospecific or mixed-species colonies compared to B. terrestris workers. This pattern of reproduction raises an interesting question. Is the lack of reproductive dominance due to an incomplete control of the
dominant worker or to a share of reproduction between individuals? The results presented here demonstrate the advantages of the mixed-species colonies paradigm. It not only discloses selfish behaviour that may be hidden when observing a homospecific group, but it also may shed light on the evolution of social parasitism: for example, how Psithyrus overcomes hostequeen inhibition and inhibits ovary development of host workers (Fisher 1984; Vergara et al. 2003). Acknowledgments This work was funded by a J. and M. L. Dufrenoy grant (Acade´mie d’Agriculture de France) to C.A. We are grateful to Paul Devienne for technical assistance, to Rumsa€ıs Blatrix and two anonymous referees for helpful comments, and to Naomi Paz for her editorial assistance. The experiments comply with the current laws of France.
References Alaux, C., Jaisson, P. & Hefetz, A. 2004a. Queen influence on worker reproduction in bumblebees (Bombus terrestris) colonies. Insectes Sociaux, 51, 287e293. Alaux, C., Savarit, F., Jaisson, P. & Hefetz, A. 2004b. Does the queen win it all? Queeneworker conflict over male production in the bumblebee, Bombus terrestris. Naturwissenschaften, 91, 400e403. Alaux, C., Jaisson, P. & Hefetz, A. 2005. Reproductive decisionmaking in semelparous colonies of the bumblebee Bombus terrestris. Behavioral Ecology and Sociobiology, 59, 270e277. Alford, D. V. 1975. Bumblebees. London: DavisePoynter. Bloch, G. 1999. Regulation of queeneworker conflict in bumblebee (Bombus terrestris) colonies. Proceedings of the Royal Society of London, Series B, 266, 2465e2469. Bloch, G. & Hefetz, A. 1999. Regulation of reproduction by dominant workers in bumblebee (Bombus terrestris) queenright colonies. Behavioral Ecology and Sociobiology, 45, 125e135. Bloch, G., Borst, D. W., Huang, Z. Y., Robinson, G. E. & Hefetz, A. 1996. Effects of social conditions on juvenile hormone mediated reproductive development in Bombus terrestris workers. Physiological Entomology, 21, 257e267. Bourke, A. F. G. 1994. Worker matricide in social bees and wasps. Journal of Theoretical Biology, 157, 292. Bourke, A. F. G. & Franks, N. R. 1995. Social Evolution in Ants. Princeton, New Jersey: Princeton University Press. Butler, C. G. & Fairey, E. M. 1963. The role of the queen in preventing oogenesis in worker honey bees. Journal of Apiculture Research, 2, 14e18. Cameron, S. A. 1989. Temporal patterns of division of labor among workers in the primitively eusocial bumble bee Bombus griseocollis (Hymenoptera: Apidae). Ethology, 80, 137e151. Crozier, R. H. & Pamilo, P. 1996. Evolution of Social Insect Colonies. Oxford: Oxford University Press. ¨ lldobler, B. 2005. Role of the Dietemann, V., Peeters, C. & Ho queen in regulating reproduction in the bulldog ant Myrmecia gulosa: control or signalling? Animal Behaviour, 69, 777e784. van Doorn, A. 1987. Investigations into the regulation of dominance behaviour and the division of labour in bumblebee colonies (Bombus terrestris). Netherlands Journal of Zoology, 37, 255e276.
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van Doorn, A. & Heringa, J. 1986. The ontogeny of a dominance hierarchy in colonies of the bumblebee Bombus terrestris (Hymenoptera, Apidae). Insectes Sociaux, 33, 3e25. Duchateau, M. J. & Velthuis, H. H. W. 1988. Development and reproductive strategies in Bombus terrestris colonies. Behaviour, 107, 186e207. Endler, A., Liebig, J., Schmitt, T., Parker, J. E., Jones, G. R., ¨ lldobler, B. 2004. Surface hydrocarbons of Schreier, P. & Ho queen eggs regulate worker reproduction in a social insect. Proceedings of the National Academy of Sciences, U.S.A., 101, 2945e2950. Errard, C., Hefetz, A. & Jaisson, P. 2005. Social discrimination tuning in ants: template formation and chemical similarity. Behavioral Ecology and Sociobiology, 59, 353e363. Estoup, A., Scholl, A., Pouvreau, A. & Solignac, M. 1995. Monoandry and polyandry in bumble bees (Hymenoptera; Bombinae) as evidenced by highly variable microsatellites. Molecular Ecology, 4, 89e93. Fisher, R. M. 1984. Dominance by a bumble bee social parasite (Psithyrus citrinus) over workers of its host (Bombus impatiens). Animal Behaviour, 32, 304e305. Free, J. B., Weinberg, I. & Whiten, A. 1969. The egg-eating behaviour of Bombus lapidarius L. Behaviour, 35, 313e317. Hobbs, G. A. 1965. Ecology of species of Bombus Latr. (Hymenoptera: Apidae) in southern Alberta: III. Subgenus Cullumonobombus. Canadian Entomologist, 97, 1293e1302. ¨ lldobler, B. & Bartz, S. H. 1985. Sociobiology of reproduction in Ho ants. In: Experimental Behavioral Ecology and Sociobiology (Ed. by B. Ho ¨ lldobler & M. Lindauer), pp. 237e258. Sunderland, Massachusetts: Sinauer. van Honk, C. G. J. & Hogeweg, P. 1981. The ontogeny of the social structure in a captive Bombus terrestris colony. Behavioral Ecology and Sociobiology, 9, 111e119. ¨ seler, P. F., Velthuis, H. H. W. & Hoogeven, van Honk, C. G. J., Ro J. C. 1981. Factors influencing the egg laying of workers in a captive Bombus terrestris colony. Behavioral Ecology and Sociobiology, 9, 9e14. Hoover, S. E. R., Keeling, C. I., Winston, M. L. & Slessor, K. N. 2003. The effect of queen pheromones on worker honey bee ovary development. Naturwissenschaften, 90, 477e480. Hoover, S. E. R., Winston, M. L. & Oldroyd, B. P. 2005. Retinue attraction and ovary activation: responses of wild type and anarchistic honey bees (Apis mellifera) to queen and brood pheromones. Behavioral Ecology and Sociobiology, 59, 278e284. Ishay, J., Rosenzweig, E. & Pechhaker, H. 1986. Comb building by worker groups of Vespa crabo L., V. orientalis L. and Paravespula germanica Fabr. (Hymenoptera: Vespinae). Monitore Zoologico Italiano, 20, 31e51.
Jaisson, P. 2005. Kinship and fellowship in ants and social wasps. In: Kin Recognition. 2nd edn (Ed. by P. G. Hepper), pp. 60e93. Cambridge: Cambridge University Press. Kawakita, A., Sota, T., Ito, M., Ascher, J. S., Tanaka, H., Kato, M. & Roubikf, D. W. 2004. Phylogeny, historical biogeography, and character evolution in bumble bees (Bombus: Apidae) based on simultaneous analysis of three nuclear gene sequences. Molecular Phylogenetics and Evolution, 31, 799e804. Keller, L. & Nonacs, P. 1993. The role of queen pheromones in social insects: queen control or queen signal? Animal Behaviour, 45, 787e794. Lenoir, A., D’Ettorre, P., Errard, C. & Hefetz, A. 2001. Chemical ecology and social parasitism in ants. Annual Review of Entomology, 46, 573e599. Lopez-Vaamonde, C., Koning, J. W., Jordan, W. C. & Bourke, A. F. G. 2004a. A test of information use by reproductive bumblebee workers. Animal Behaviour, 68, 611e618. Lopez-Vaamonde, C., Koning, J. W., Brown, R. M., Jordan, W. C. & Bourke, A. F. G. 2004b. Social parasitism by male-producing reproductive workers in a eusocial insect. Nature, 430, 557e560. Martin, S. J., Beekman, M., Wossler, T. C. & Ratnieks, F. L. W. 2002. Parasitic Cape honeybee workers, Apis mellifera capensis, evade policing. Nature, 415, 163e165. Neumann, P. & Moritz, R. F. A. 2002. The Cape honeybee phenomenon: the sympatric evolution of a social parasite in real time? Behavioral Ecology and Sociobiology, 52, 271e281. Oldroyd, B. P., Smolenski, A. J., Cornuet, J.-M. & Crozier, R. H. 1994. Anarchy in the beehive: a failure of worker policing in Apis mellifera. Nature, 371, 749. ¨ seler, P. F. & van Honk, C. G. J. 1990. Castes and reproduction in Ro bumblebees. In: Social Insects: an Evolutionary Approach to Castes and Reproduction (Ed. by W. Engels), pp. 147e166. Berlin: Springer-Verlag. Schmid-Hempel, R. & Schmid-Hempel, P. 2000. Female mating frequencies in Bombus sp. from central Europe. Insectes Sociaux, 47, 36e41. ¨ der, S., Almanza, M. T. & Wittmann, D. Vergara, C. H., Schro 2003. Suppression of ovarian development of Bombus terrestris workers by B. terrestris queens, Psithyrus vestalis and Psithyrus bohemicus females. Apidologie, 34, 563e568. Vienne, C., Errard, C. & Lenoir, A. 1998. Influence of the queen on worker behaviour and queen recognition behaviour in ants. Ethology, 104, 431e446. West-Eberhard, M. J. 1981. Intragroup selection and the evolution of insect societies. In: Natural Selection and Social Behaviour (Ed. by R. D. Alexander & D. W. Tinkle), pp. 3e17. New York: Chiron Press.
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Plasticity of worker reproductive strategies in Bombus terrestris: lessons from artificial mixed-species colonies CEDR IC A L A UX *, A BRA H AM H EF ETZ † & P IERR E J AI SS ON*
*Laboratoire d’Ethologie Expe´rimentale et Compare´e, Universite´ Paris 13 yG. S. Wise Faculty of Life Sciences, Department of Zoology, Tel Aviv University (Received 15 November 2005; initial acceptance 1 March 2006; final acceptance 12 May 2006; published online 16 October 2006; MS. number: A10300)
We used the experimental paradigm of artificial mixed colonies of two phylogenetically related bumblebee species to analyse the dynamics of the reproductive skew in societies of Bombus terrestris. Artificial mixedspecies colonies were set up by introducing callow B. terrestris workers either into a queenright (QR) or a queenless (QL) colony of B. lapidarius. The introduced B. terrestris workers were well integrated into their host B. lapidarius colony and displayed nesting activities that did not differ from those of the resident B. lapidarius workers. However, the introduced B. terrestris workers did show a different reproductive behaviour. While B. lapidarius workers did not develop ovaries in a B. lapidarius QR colony but did so in a B. lapidarius QL group, adopted B. terrestris workers in a B. lapidarius QR colony developed ovaries as if they were under QL conditions. These results indicate that, in mixed-species colonies, B. terrestris workers are irresponsive to the queen’s inhibitive action on ovary development. In QL homospecific and heterospecific predominately B. terrestris mixed-worker colonies (1Bl þ 5Bt), reproduction was dominated by a single B. terrestris worker, whereas in QR B. lapidarius or QL equally mixed-worker colonies (3Bl þ 3Bt), almost all B. terrestris workers developed ovaries. We suggest that in the presence of enough heterospecific workers, B. terrestris workers behave as parasites. This last finding suggests that worker reproduction in B. terrestris is highly plastic and that the experimental paradigm of artificial mixed colonies may provide new insights into the evolution of social parasitism in this taxon. Ó 2006 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Reproductive skew is a hallmark of insect societies, where female reproduction is highly biased in favour of one or more individuals, the queens, whereas nonreproductive individuals, the workers, are helpers. In many species, however, workers are not irreversibly sterile and remain able to lay viable eggs if they are orphaned, or at certain stages of colony development. Reproductive plasticity in workers is highly contextual and depends on the underlying genetic and social structures of the colony (Bourke & Franks 1995; Crozier & Pamilo 1996). Moreover, it is governed by two seemingly opposed selection pressures: selfishness, driven by combined individual and kin selection; and cooperativeness, driven by
Correspondence and present address: C. Alaux, Department of Entomology, University of Illinois, Urbana, IL 61801, U.S.A. (email: [email protected]). A. Hefetz is at the G. S. Wise Faculty of Life Sciences, Department of Zoology, Tel Aviv University, 69978 Tel Aviv, Israel. P. Jaisson is at the Laboratoire d’Ethologie Expe´ rimentale et Compare´e, CNRS UMR 7153, Universite´ Paris 13, 93430 Villetaneuse, France. 0003e 3472/06/$30.00/0
combined kin- and colony-level selections. Thus, unravelling the ultimate and proximate mechanisms for the evolution and maintenance of such a reproductive skew offers a major topic of interest. Caste-specific pheromones, particularly queen pheromones, are believed to regulate reproductive skew in social insects. They may operate either by actively inhibiting worker ovary development (Butler & Fairey 1963; Ho¨lldobler & Bartz 1985; Hoover et al. 2003) or by acting as an honest fertility signal (Keller & Nonacs 1993; Endler et al. 2004; Dietemann et al. 2005). In the latter case, workers are hypothesized to use all available information to adjust their behaviour in a way that will maximize their fitness. This further explains why worker sterility is plastic and conditional. Workers, therefore, are selected to enhance overall colony growth and refrain from reproducing as long as the genetic gain through inclusive fitness surpasses that obtained through direct fitness. It is consequently adaptive for both workers and the queen to evolve mechanisms for queen recognition, namely queen-specific signals, which may not necessarily be species specific.
1417 Ó 2006 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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ANIMAL BEHAVIOUR, 72, 6
The bumblebee Bombus terrestris has become a model system for testing both ultimate and proximate processes related to reproductive social conflicts (van Honk et al. 1981; Duchateau & Velthuis 1988; Ro¨seler & van Honk 1990; Bloch et al. 1996; Bloch 1999; Bloch & Hefetz 1999; Alaux et al. 2004a, 2005; Lopez-Vaamonde et al. 2004a). Colonies of B. terrestris are annual with one, singly inseminated queen (Estoup et al. 1995; Schmid-Hempel & Schmid-Hempel 2000), creating a potential conflict between the queen and workers over male production. Colony life cycle follows two main phases (Alford 1975). The first, the social phase, is characterized by cooperation, which maximizes ergonomic growth and gyne production as well as reproductive self-restraint among workers, despite their potential to lay viable haploid eggs (Alaux et al. 2004a). The regulation of worker reproduction is mediated by the perception of nonvolatile queen pheromones via direct antennal contacts. Workers also establish a dominance hierarchy among themselves during this phase (van Honk & Hogeweg 1981; van Doorn & Heringa 1986). However, this reproductive dominance hierarchy among workers is highly flexible and can change throughout colony development. The second phase marks the breakdown of social structure and is characterized by an accelerated competition between the queen and the workers over reproduction and among workers for access to reproduction (Bloch & Hefetz 1999). Queeneworker conflict over male production in the bumblebee B. terrestris is thus well evidenced. Recent molecular studies, however, have revealed that despite the overt competition between the queen and her workers over male production during the competition phase, the queen seems to produce over 95% of the adult males (Alaux et al. 2004b; Lopez-Vaamonde et al. 2004b). The queen is able to successfully outcompete the workers owing to her higher reproductive output, and greater aggression and oophagy capacity. Worker reproduction seems to succeed only when the queen dies prematurely, suggesting that workers may in fact execute the queen in order to reproduce (Bourke 1994; Alaux et al. 2004b). Finally, the selfish interest of workers may be expressed when they invade an alien conspecific colony, where they can unrestrainedly lay eggs (Lopez-Vaamonde et al. 2004b). All these insights into queeneworker conflict in B. terrestris have raised the hypothesis that worker reproduction is highly plastic and may depend on the social circumstances. Workers are thus able to show two opposite phenotypes, from cooperation to selfishness, depending on the dominant selection level. However, unravelling the proximate mechanisms that result in either worker sterility or their fertility is sometimes complicated under natural conditions. Thus, the use of mixed-species groups might be considered as potentially revealing, since each species may express its preprogrammed traits, whether kin-selected or not, independently of colony-level selection. Moreover, it enables investigation of the level of reproductive plasticity of workers as well as examination of the effects of social environment on reproductive plasticity. For this purpose, we used a system of mixed-species colonies to unravel the mechanisms and flexibility level of
the reproductive strategies that may be shown by worker B. terrestris, but that remain hidden from the researcher under normal colony conditions (Jaisson 2005). We created mixed-species colonies and groups using B. terrestris and B. lapidarius, two phylogenetically closely related species (Kawakita et al. 2004). Although reproductive skew and queeneworker conflict have been less studied in B. lapidarius (Free et al. 1969), their phylogenetic proximity and similarity of life histories suggested that their social behaviour might also be comparable. Thus, we first tested whether worker B. lapidarius refrain from reproduction under queenright (QR), but not queenless (QL) conditions, and whether the queen has a pheromone regulating worker reproduction. We then investigated whether worker B. terrestris can integrate into a B. lapidarius colony, and whether reproductive strategy of B. terrestris workers changed in heterospecific colonies compared to homospecific colonies. Finally, we created QL mixed-species groups to further probe into worker reproductive strategies.
MATERIAL AND METHODS
Bumblebee Rearing Queens of B. lapidarius were collected in the field near Paris in early spring 2003 and 2004. They were allowed to found a colony in the laboratory by placing each one in a wooden box (17.5 26 15 cm) kept in the dark at 28 2 C and 50% relative humidity. They were fed ad libitum with sugar syrup and fresh pollen. The experiments started once these colonies had reached 10 workers (i.e. about 1 week after the first worker emergence). Observations were performed under red light through a glass covering the nestbox. Colonies of B. terrestris were obtained either from a commercial supplier (GTICO SARL, Foissy sur Vannes, France) or from our laboratory stock (colonies reared from queens obtained from commercial colonies). All colonies were reared under the same standard conditions.
Regulation of Worker Reproduction in Homospecific B. lapidarius Colonies To test whether queens of B. lapidarius produce a volatile pheromone that inhibits worker reproduction, we split nests into two compartments separated by a doublemesh (1-cm-wide) screen. One compartment included all colony workers and the queen (QRC) while the second compartment contained five callow (1-day-old) nestmate workers (QLC). No physical contact was possible between these separated workers and the rest of the colony, including the queen. QL groups of five callow workers housed separately served as control. Both the QL and the QLC were supplied with young cocoons (collected from young donor colonies) as a substrate for cell construction (van Doorn 1987). We recorded the occurrence of newly sealed egg-cells in the QLC and QL colonies, and compared ovary development (mean length of the terminal oocytes) between workers from the QLC, QR and QL colonies. Control workers, originating from nonmanipulated QR
ALAUX ET AL.: WORKER REPRODUCTIVE PLASTICITY IN BEES
colonies 20 days after the first worker emergence, were also frozen for determination of ovary development (at this colony age workers have had the time required to develop ovaries; Free et al. 1969).
honey pots, feeding larvae, brood incubation, enlargement of wax cover around the brood, threatening behaviour (buzzing), and overt aggression.
Regulation of Worker Reproduction in Mixed-species Colonies
Introduction of B. terrestris Callow Workers into B. lapidarius QR Colonies
Queenright artificial mixed-species colonies
To test whether a putative B. lapidarius queen pheromone inhibits ovary development in adopted young callow B. terrestris workers, we introduced five B. terrestris workers that had been individually marked using numbered tags (Opalith Pla¨ttchen, Friedrich Wienold, Germany) into a QR B. lapidarius colony (N ¼ 17). The introduced B. terrestris were callow workers that had been allowed to emerge in homospecific QL colonies to prevent contact with the conspecific queen. Five control callow B. lapidarius workers were equally marked and then reintroduced into their colony. In those colonies in which there were insufficient B. lapidarius callow workers at the start of experiment, callow workers from other colonies were introduced. To verify whether the introduced B. terrestris workers had fully integrated into their host colony, we performed scan-sampling behavioural observations on seven randomly selected QR mixed colonies every 2 min for 30 min twice a day (mornings and afternoons during days 2e6). We noted 16 behavioural acts related to worker tasks (Cameron 1989): foraging, guarding (at nest entrance or on the brood, antennae raised), patrolling (rapid movements), nest inspection, fanning, immobility (antennae lowered), self-grooming, scraping wax (workers recycle the wax of empty cocoons), anchoring (building of wax area), excavation of empty cocoons, working on
Egg laying by B. terrestris workers introduced into QR B. lapidarius colonies (N ¼ 17) was estimated from the seventh day following introduction (the time needed for ovary development in QL callow workers), by counting the number of eggs in the newly sealed egg-cells. Since B. lapidarius queens lay a significantly greater number of eggs per cell than do B. terrestris workers at their first egg laying in QL colonies (Table 1), we could discern to which species the laid eggs belonged. To ascertain that these were B. terrestris eggs, a sample (from 2 to 4 eggs of each colony’s egg-cup) was transferred into small QL B. terrestris colonies containing young nonreproductive workers and reared to adulthood. All the emerged adults were B. terrestris males. Workerequeen interactions were estimated by monitoring the number of workerequeen antennal contacts of the marked individuals (5 B. terrestris workers þ 5 B. lapidarius workers for each observed colony, N ¼ 5) daily for 30 min (15 min morning and afternoon), from the second day of worker introduction until the first oviposition by B. terrestris workers occurred. The experiment ended when the first egg laying by the adopted B. terrestris was observed (B. lapidarius workers never laid eggs under these conditions, see Results). Thereafter, all marked bees were removed and dissected to determine ovary development. The five groups in
Table 1. Comparison of the means (ManneWhitney U test) and ranges (c2 test) for the number and size of eggs laid by B. terrestris and B. lapidarius queens and workers in the different experimental situations MeanSD number Range of number of eggs/cell of eggs/cell B. lapidarius queen QL B. terrestris workers* QR mixed B. terrestris workers
8.91.17, N¼16 4.051.40, N¼20 3.901.30, N¼17
7e12 2e7 2e6
QL B. lapidarius workers 3Blþ3Bt colonies 1Blþ5Bt colonies
B. lapidarius queen versus QL B. terrestris workers B. lapidarius queen versus QR mixed B. terrestris workers QL B. terrestris workers versus QR mixed B. terrestris workers QL QL QL QL QL
B. B. B. B. B.
terrestris workers versus QL B. lapidarius workers terrestris workers versus 3Blþ3Bt colonies terrestris workers versus 1Blþ5Bt colonies lapidarius workers versus 3Blþ3Bt colonies lapidarius workers versus 1Blþ5Bt colonies
MeanSD egg size (mm)
Range of egg size
3.100.11, N¼54
2.75e3.25
2.850.1, N¼55 3.150.11, N¼78 3.110.08, N¼57
2.67e3.08 2.92e3.42 2.92e3.33
ManneWhitney U test Mean number of eggs/cell U¼1, P<0.001 U¼0, P<0.001 U¼170, P¼1
c2 test Range of number of eggs/cell c2¼33.3, P<0.001 c2¼33, P<0.001 c2¼3.2, P¼0.73
Mean egg size U¼176.5, P< 0.001 U¼1628.5, P<0.05 U¼1445, P¼0.69 U¼90, P<0.001 U¼96, P<0.001
Range of egg size c2¼71.3, P<0.001 c2¼10.63, P¼0.22 c2¼4.1, P¼0.77 c2¼106.9, P<0.001 c2¼80.24, P<0.001
QL: queenless colonies; QR: queenright colonies. *Egg characteristics of QL B. terrestris workers correspond to their first oviposition.
1419
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ANIMAL BEHAVIOUR, 72, 6
which some of the introduced B. terrestris (N ¼ 4, 11% of introduced workers) and B. lapidarius workers (N ¼ 2, 6% of introduced workers) died during the experiment were excluded from this analysis.
Queenless artificial mixed-species colonies We created two types of QL mixed colonies to investigate the plasticity of worker reproduction under these conditions and determine the role of the number of heterospecific workers in the reproductive plasticity. The first type comprised three B. lapidarius and three B. terrestris workers (called QL 3Bl þ 3Bt, N ¼ 20) and the second was composed of one B. lapidarius and five B. terrestris workers (called QL 1Bl þ 5Bt, N ¼ 12). Control groups consisted of either five B. terrestris (N ¼ 12) or five B. lapidarius workers (N ¼ 12), each group of which was housed separately. Once the first egg-cell was constructed all workers were frozen for later dissection. Worker oviposition in homospecific control colonies showed that egg size of B. lapidarius was significantly smaller than that of B. terrestris (Table 1). Egg size, therefore, served as a reliable measure for determining egg species identity. Behavioural observations were performed on a randomly chosen sample of each of the QL mixed (3Bl þ 3Bt, N ¼ 8; 1Bl þ 5Bt, N ¼ 8), homospecific B. terrestris (N ¼ 9) and homospecific B. lapidarius (N ¼ 8) colonies. They were performed twice a day (10 min in the morning þ 10 min in the afternoon) during days 2e7, focusing on workere worker aggression (biting, grappling and head butting, see Bloch et al. 1996 for details).
Statistics We used a factorial correspondence analysis (FCA) on the complete behavioural repertoire (number of items for each recorded task) applied to the marked bees (Spad 3.1 software, Spadsoft, Paris, France). Bees that died during the behavioural experiment were not taken into account in the FCA. Permutation tests by stratum (StatXact 3.1, Cytel Software, Cambridge, Massachusetts, U.S.A.) were performed for B. lapidarius (N ¼ 33) and B. terrestris workers (N ¼ 31) on the resulting coordinates of each individual in the FCA (root 1 and 2), taking into account their mixed-group origin. This enabled us to consider intercolonial variation. To analyse how ovary development was distributed among workers, we first determined whether there was any significant variation between groups within each treatment (Table 2). Next, we categorized their development into five stages representing equal ovary development range for each species (B. lapidarius: stage 1: 0e0.54 mm; stage 2: 0.55e1.08 mm; stage 3: 1.09e1.62 mm; stage 4: 1.63e2.16 mm; stage 5: 2.17e2.7 mm; B. terrestris: stage 1: 0e0.75 mm; stage 2: 0.76e1.5 mm; stage 3: 1.51e2.25 mm; stage 4: 2.26e3 mm; stage 5: 3.01e3.75 mm), and used a KolmogoroveSmirnov test to determine whether the resulting distributions deviated from a normal distribution (Statistica 6.1 software, Statsoft, Tulsa, Oklahoma, U.S.A.). In groups that showed
Table 2. Results of KruskaleWallis tests on the mean size of worker ovary development under the different social regimes
QLC homospecific B. lapidarius QL B. terrestris workers QL B. lapidarius workers QR mixed B. terrestris workers QR mixed B. lapidarius workers QL B. terrestris workers (3Blþ3Bt) QL B. lapidarius workers (3Blþ3Bt) QL B. terrestris workers (1Blþ5Bt)
H
df
P
1.49 3.84 6.40 13.54 10.14 13.98 16.24 1.37
8, 45 11, 60 11, 60 11, 60 1, 60 19, 60 19, 60 11, 60
0.992 0.974 0.845 0.259 0.517 0.78 0.64 0.99
QLC: queenless compartment of nest; QL: queenless colonies; QR: queenright colonies.
a normal distribution of ovary development, reproduction was considered as not skewed. RESULTS
Regulation of Worker Reproduction in Homospecific B. lapidarius Colonies The average time to first oviposition by B. lapidarius workers after group establishment in the QLC of the homospecific colonies was not significantly different from that in the control QL colonies (ManneWhitney U test: U ¼ 45, N1 ¼ 9, N2 ¼ 12, P ¼ 0.522; Table 3). Ovary development in workers from both QL (N ¼ 60) and the QLC colonies (N ¼ 45) was significantly greater than that of workers from nonmanipulated QR colonies (N ¼ 150) (KruskalleWallis test: H2,269 ¼ 116.16, P < 0.001; Siegele Tukey post hoc test: QL/QLC: P ¼ 0.612; QR/QL and QR/ QLC: P < 0.001; Table 3). This demonstrates that if a queen pheromone does affect worker reproduction, it is nonvolatile as in B. terrestris (Alaux et al. 2004a).
Social Integration of B. terrestris Workers Adopted by B. lapidarius Colonies In-nest behaviour of the introduced B. terrestris workers (N ¼ 31) was compared to that of the marked and reintroduced B. lapidarius workers (N ¼ 33) during days 2e6 following introduction (FCA on all observed individuals and considering all the behaviours outlined above; Fig. 1). The first two roots revealed that individuals of both species created overlapping clouds and did not differ significantly according to their behavioural repertoire (permutation test: root 1: P ¼ 0.116; root 2: P ¼ 0.602). Thus, at least with respect to the measured parameters, worker B. terrestris showed an integration into their host B. lapidarius colony.
Regulation of Worker Reproduction in Mixed-species Colonies Queenright artificial mixed-species colonies In the queenright mixed colonies (N ¼ 17) on a mean SD of 8.06 1 days after B. terrestris workers were
ALAUX ET AL.: WORKER REPRODUCTIVE PLASTICITY IN BEES
Table 3. Reproductive timing and ovary development (mean SD) of callow workers from the different colony types
Colony type QR homospecific B. lapidarius QLC homospecific B. lapidarius QL homospecific B. lapidarius QL homospecific B. terrestris QR mixed colonies QL mixed colonies (3Blþ3Bt) QL mixed colonies (1Blþ5Bt)
Worker type
Ovary development (mm)
N workers
Marked B. lapidarius Introduced B. terrestris B. lapidarius B. terrestris B. lapidarius B. terrestris
0.150.42 1.570.57 1.360.83 0.871.12 0.160.36 2.130.82 1.180.84 2.060.87 0.80.8 1.071.13
150 45 60 60 60 60 60 60 12 60
Days until egg laying by the first worker
N colonies 6 9 12 12 17
d 8.441.17 8.081.04 7.580.62 d 8.061 7.80.6
20 12
d 7.580.76
QR: queenright colonies; QLC: queenless compartment of nest; QL: queenless colonies.
B. lapidarius colonies was greater than that of workers kept in homospecific QL colonies (ManneWhitney U test: U ¼ 743.5, N1 ¼ 60, N2 ¼ 60, P < 0.001; Table 3). This difference resulted from the fact that in homospecific QL colonies only one bee had fully developed ovaries, whereas in the QR mixed colonies, all the adopted B. terrestris workers had developed ovaries (Fig. 2a). Thus, ovary development was not skewed as expected from the normal reproductive strategy shown by QL B. terrestris workers.
Queenless artificial mixed-species colonies Egg size was used to compare the pattern of worker reproduction for B. terrestris and B. lapidarius in QL mixedspecies groups. The mean egg size in mixed-species QL groups 1Bl þ 5Bt differed significantly from those of QL B. lapidarius colonies but not from those of the QL B. terrestris colonies (Table 1). In mixed-species QL groups 3Bl þ 3Bt, egg size differed significantly from both QL homospecific colonies. However, in QL colonies 3Bl þ 3Bt, egg size was closer to that of QL B. terrestris colonies than to that of QL B. lapidarius colonies. Moreover, the range 1 B. lapidarius workers B. terrestris workers Root 2 (13.5%)
introduced we observed some egg-cells containing fewer eggs than expected from those normally laid by B. lapidarius queens (Table 1). Egg numbers in these cells were not significantly different from those of QL B. terrestris colonies, indicating that they might have originated from B. terrestris workers. To verify this assumption, we reared a subset of these eggs in a young QL colony composed of nonreproducing B. terrestris workers. All the brood developed exclusively into B. terrestris males. It thus seems that the adopted B. terrestris workers had started to oviposit after the expected physiological time-lag required for ovary development in QL callow workers, and that the B. lapidarius queen had failed to inhibit heterospecific worker reproduction. The time required to achieve reproduction for B. terrestris workers did not differ significantly between social regimes (KruskalleWallis test: H5,82 ¼ 5.99, P ¼ 0.306; Table 3). In contrast, resident B. lapidarius workers in these colonies, whether reintroduced or nonmanipulated, did not reproduce in the presence of the queen, whereas they started to lay eggs after a mean SD of 8.08 1.04 days when reared in QL colonies (Table 3). Behavioural observations on the B. terrestris bees revealed that they had a significantly higher rate of contact with the B. lapidarius queen than the B. lapidarius workers had with their mother queen (1.23 0.25, N ¼ 25 versus 0.68 0.4 antennal contacts per worker per 30 min, N ¼ 25; ManneWhitney U test: U ¼ 148.5, P < 0.01). Moreover, antennal contact rates between B. terrestris workers and the B. lapidarius queen were not significantly different from those shown by prospective reproductive B. terrestris workers with their own queen, but were higher than the rate shown by nonreproductive workers (data in Alaux et al. 2004a; ManneWhitney U test: U ¼ 699.5, N1 ¼ 25, N2 ¼ 60, P ¼ 0.626 and U ¼ 1082.5, N1 ¼ 25, N3 ¼ 312, P < 0.001, respectively). Worker B. lapidarius from QR mixed colonies or from QR homospecific colonies had undeveloped ovaries, as opposed to QL workers, which had significantly larger terminal oocytes (ManneWhitney U test: QR mixed versus QR homospecific workers: U ¼ 4292, N1 ¼ 60, N2 ¼ 150, P ¼ 0.601; QR mixed versus QL workers: U ¼ 417.5, N1 ¼ 60, N3 ¼ 60, P < 0.001; Table 3). Interestingly, the average ovary development of B. terrestris workers in QR
0.5
0
–0.5
–1 –1
–0.5
0 Root 1 (20.6%)
0.5
1
Figure 1. Distribution of workers of B. lapidarius (N ¼ 33 workers) and B. terrestris (N ¼ 31) colonies as a function of their behavioural pattern obtained from scan sampling. Each point represents an individual on the first two axes of the factorial correspondence analysis of behavioural data. Workers that died before the end of the observations were not considered for analysis.
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ANIMAL BEHAVIOUR, 72, 6
40 (a) B. terrestris
QR mixed QL mixed (3Bl+3Bt) QL mixed (1Bl+5Bt) QL
35
D=0.16, P=0.081 D=0.151, P=0.117 D=0.225, P<0.01 D=0.262, P<0.001
30 25 20 15 10 5 Worker number
1422
0
Stage 1
Stage 2
Stage 3
Stage 4
Stage 5
60 (b) B. lapidarius
QR mixed D=0.456, P<0.001 QL mixed (3Bl+3Bt) D=0.168, P=0.061 QL D=0.103, P=0.515
50
40
30
20
10
0
Stage 1
Stage 2
Stage 3
Stage 4
Stage 5
Ovary development (mm) Figure 2. Ovary development distribution of B. terrestris and B. lapidarius workers from the different mixed and homospecific colonies (N ¼ 60 workers for each condition). (a) B. terrestris workers. (b) B. lapidarius workers. D and P values based on KolmogoroveSmirnov test of normality are reported for each condition. A significant P value means that the ovary development distribution was skewed.
of egg sizes in QL mixed colonies (3Bl þ 3Bt and 1Bl þ 5Bt) was significantly different from that of QL B. lapidarius colonies but not from that of QL B. terrestris colonies. Thus, all the eggs oviposited could be attributed to B. terrestris workers in QL mixed colonies 1Bl þ 5Bt. Although some of the eggs in QL mixed colonies 3Bl þ 3Bt could have been laid by B. lapidarius workers, it is very likely that most of them originated from B. terrestris workers. Ovary development of B. terrestris workers within the mixed-species colonies revealed two distribution patterns (Fig. 2a). When they were either adopted by a QR B. lapidarius colony (QR mixed) or reared with an equal number of B. lapidarius workers (QL mixed 3Bl þ 3Bt), most of
them had developed ovaries and mean oocyte size distribution did not differ significantly from normal distribution. In contrast, when reared either in pure B. terrestris QL groups or in the mixed colonies where they were the majority (QL 1Bl þ 5Bt), ovary development was highly skewed, with only one dominant bee with developed ovaries. The same analysis for B. lapidarius revealed that in both the pure QL colonies and the mixed-species group (QL mixed 3Bl þ 3Bt) most workers had similar nonskewed ovary development (Fig. 2b). In contrast, workers from the QR colonies did not develop ovaries. Ovary development in the single B. lapidarius workers from the 1Bl þ 5Bt QL mixed colonies was scattered (range
ALAUX ET AL.: WORKER REPRODUCTIVE PLASTICITY IN BEES
0e1.99 mm) and had a low mean SD oocyte length (0.8 0.8 mm, N ¼ 12; Table 3). Ovary development was in concordance with the results of the behavioural observations. In the homospecific QL colonies of B. terrestris, agonistic behaviour was performed by a single worker, and in B. lapidarius colonies the dominant worker displayed 85.7 11.5% of the aggression. In the QL mixed 3Bl þ 3Bt colonies (N ¼ 8 colonies), intraspecific aggression reached 21 8.8% for B. terrestris and 10.85 9.2% for B. lapidarius of total aggression (each of them displayed by only one individual). Interspecific aggression in these groups constituted 68.15 11.4% of all aggression, of which 90.5 14.2% was performed by a single B. terrestris worker and 9.5 14.2% was displayed by a single B. lapidarius worker in each colony. In the QL mixed 1Bl þ 5Bt colonies, aggression was exclusively displayed by B. terrestris workers. Thus, the reproductive plasticity shown by B. terrestris workers changed accordingly to the composition of mixed colonies. The low reproductive skew observed between B. terrestris workers was not due to the simple detection of heterospecific workers but rather to their number.
DISCUSSION Worker reproduction is governed by two selection processes. Individual selection drives workers to behave selfishly since they gain maximum fitness by rearing sons (Bourke & Franks 1995; Crozier & Pamilo 1996). Such selfishness may cause considerable colony deterioration to the point that optimal rearing of sexuals is hampered. However, colony-level selection can nullify this selfishness, resulting in worker sterility. In bumblebees, colonylevel selection is very powerful since genetic gain from future gynes is greater than that from future sons, forcing workers to ensure that gyne production has been initiated before they attempt to reproduce (Alaux et al. 2005). Thus, worker reproduction is conditional and dependent on social context (see Introduction). We constructed mixedspecies colonies and groups of two phylogenetically related bumblebees, B. terrestris and B. lapidarius, to assess the level of this reproductive plasticity and the mechanisms implied. This kind of experimental paradigm has been used successfully in ants to examine the nature of both the template and label involved in nestmate recognition (reviewed by Lenoir et al. 2001; Errard et al. 2005; Jaisson 2005). We first established that B. lapidarius workers do not reproduce under queenright conditions and that, if this inhibition is pheromonally mediated, the pheromone used is nonvolatile. Thus, worker B. lapidarius seem to behave like B. terrestris workers, in which direct antennal contact with the queen is required for complete reproductive inhibition to occur (Alaux et al. 2004a). Bombus terrestris workers were observed to be integrated into the queenright colonies of B. lapidarius into which they had been introduced: they received no aggression from the resident bees and behaved as if they were in their native colony. This was confirmed by the factorial analysis applied on multiple behaviours, which showed that
worker B. terrestris contributed as much to B. lapidarius colony maintenance and growth, including brood care, as did the conspecific workers. In contrast to the resident B. lapidarius workers, however, they did not refrain from reproducing and behaved as if queenless by developing ovaries within about 8 days (i.e. the time necessary for a dominant worker to develop ovaries under queenless conditions; Alaux et al. 2004a). We exclude the possibility of an artefact related to the introduction event, because introduced B. lapidarius, whether resident or alien, did not develop ovaries in the presence of the queen. We also exclude the possibility that the lack of inhibition under our experimental conditions was due to insufficient contacts between the queen and B. terrestris workers (assuming, for example, that B. lapidarius queens produced a nonvolatile pheromone that might have an interspecific inhibitory effect). The B. lapidarius queens were in fact antennated at rates that were not different from those performed by prospective reproductive workers towards their mother B. terrestris queens before the competition phase (Alaux et al. 2004a). We conclude that the B. lapidarius queen pheromone is ineffective on B. terrestris workers, whether or not they perceive it. However, because the introduced B. terrestris workers did not behave entirely as alien intruders, it is possible that two pheromones are involved: a queen attractant/recognition that might be non-species-specific (Vienne et al. 1998) and a species-specific ovary-inhibiting pheromone. In wasps, contrasting with our results, Ishay et al. (1986) showed that reproductive Vespula germanica workers reared in a colony of Vespa orientalis were inhibited, suggesting the role of a non-species-specific pheromone or of inhibitory physical aggression. Our findings also offer new insights regarding the evolution of queen control or queen signal (Keller & Nonacs 1993). The complexity (West-Eberhard 1981) as well as the species diversity of queen pheromones may reflect the evolution of an unstable arms race between the two castes with regard to inhibition of worker reproduction. On the other hand, an honest queen signal is quite stable and thus queen pheromones should vary at a lower rate between species. The lack of control of the queen B. lapidarius over the reproduction of B. terrestris workers tends to favour the first hypothesis. Perhaps the most striking result obtained from the artificial mixed colonies was the change in the reproductive strategy of B. terrestris workers. Generally, queenless workers of B. terrestris compete aggressively for reproduction, a conflict that is solved by the worker dominance hierarchy in which only one worker in a group develops ovaries (Bloch et al. 1996; Alaux et al. 2004a). Workere worker inhibition is also apparent when callow workers are introduced into a queenright colony that is well into the competition phase (Bloch & Hefetz 1999). In contrast, in our mixed-species groups, all the introduced B. terrestris had similar ovary development, whether they constituted a minority group (only 5 workers introduced into a whole queenright B. lapidarius colony) or were in groups with an equal number of heterospecific workers (3Bl þ 3Bt). In both cases it appears that the B. terrestris workers did not compete among themselves but developed ovaries
1423
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ANIMAL BEHAVIOUR, 72, 6
independently of each other, at their own developmental rates. One possible explanation is that, being a minority group in a large host colony, B. terrestris workers did not encounter each other at sufficient rates to support dominance hierarchy establishment. This, however, cannot explain why all three B. terrestris workers developed ovaries when housed in small mixed groups with equal numbers of worker B. lapidarius, in contrast with the dominance hierarchy established between three B. terrestris workers housed together (Bloch et al. 1996). Alternatively, we suggest that the change in reproductive strategy by B. terrestris workers might represent exploitation, to the extent of parasitism, of heterospecific workers. This conclusion agrees with the result obtained in groups in which B. terrestris constituted the majority and in which a clear dominance hierarchy was established. This is also consistent with an earlier report that B. terrestris workers show intraspecific social parasitism by laying male eggs in conspecific colonies (Lopez-Vaamonde et al. 2004b). Mixed-species colonies could also occur through queen usurpation of pre-emergent homo- and/or heterospecific host colonies. Queens generally usurp another nest during the foundation phase, but can also do so in very young colonies composed of the first batch of workers (Alford 1975). In this case, the patterns of nest usurpation (i.e. choice of heterospecific or homospecific hosts) would be constrained by whether or not usurper queens can inhibit worker reproduction in host species. That would explain why nest usurpation involving two subgenera has never been found (Hobbs 1965). These results demonstrate that worker B. terrestris have a plastic and context-dependent reproductive strategy. In homospecific colonies and queenless groups, workers compete aggressively between themselves for access to reproduction. The conflict is solved by establishing reproductive dominance, which eventually results in group stability and allows the successful rearing of brood. Kinselected interests are in this case surpassed by colony-level selection because success in brood rearing requires worker cooperation; the subordinate workers benefit because they gain inclusive fitness from rearing nephews. However, when B. terrestris workers were housed in heterospecific colonies they seemed to switch to interspecific competition, so brood rearing may be fully supported by the heterospecific workers, and since the host queen is incapable of controlling their reproduction, the B. terrestris workers may be able to fully express their selfish interest to rear sons. Their behaviour strikingly resembles that of the parasitic clone of Apis mellifera capensis workers, which activate their ovaries in queenright host colonies of Apis mellifera scutellata (Martin et al. 2002; Neumann & Moritz 2002), as well as that of the anarchistic honeybee (a phenotype of Apis mellifera), where some workers display a rare phenotype by developing ovaries despite the queen’s presence (Oldroyd et al. 1994; Hoover et al. 2005). Another remarkable observation was the lack of a clear reproductive dominance between B. lapidarius workers whether in homospecific or mixed-species colonies compared to B. terrestris workers. This pattern of reproduction raises an interesting question. Is the lack of reproductive dominance due to an incomplete control of the
dominant worker or to a share of reproduction between individuals? The results presented here demonstrate the advantages of the mixed-species colonies paradigm. It not only discloses selfish behaviour that may be hidden when observing a homospecific group, but it also may shed light on the evolution of social parasitism: for example, how Psithyrus overcomes hostequeen inhibition and inhibits ovary development of host workers (Fisher 1984; Vergara et al. 2003). Acknowledgments This work was funded by a J. and M. L. Dufrenoy grant (Acade´mie d’Agriculture de France) to C.A. We are grateful to Paul Devienne for technical assistance, to Rumsa€ıs Blatrix and two anonymous referees for helpful comments, and to Naomi Paz for her editorial assistance. The experiments comply with the current laws of France.
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