Behav Ecol Sociobiol (2001) 50:366–370 DOI 10.1007/s002650100369
O R I G I N A L A RT I C L E
B. P. Oldroyd · T. C. Wossler · F. L. W. Ratnieks
Regulation of ovary activation in worker honey-bees (Apis mellifera): larval signal production and adult response thresholds differ between anarchistic and wild-type bees Received: 21 November 2000 / Revised: 22 March 2001 / Accepted: 5 April 2001 / Published online: 18 May 2001 © Springer-Verlag 2001
Abstract One-day-old anarchistic (selected for successful worker reproduction) and wild-type honey-bee workers were introduced into queenright colonies of honeybees of two treatments. In treatment 1, all eggs and larvae were offspring of queens from an anarchistic line. In treatment 2, all eggs and larvae were offspring of wildtype queens. In both treatments, adult workers were wild type. This experimental arrangement was used to test the importance of larval genotype on ovary activation in young adult workers. After 12 days, the introduced bees were dissected to determine the frequency of ovary activation. In those colonies provided with wild-type brood, 0% of introduced wild-type bees and 16% of anarchistic bees had activated ovaries. In those colonies provided with anarchistic brood, 13% of introduced wild-type bees and 41% of anarchistic bees had activated ovaries. These results strongly support the hypothesis that selection for high levels of worker reproduction in anarchistic stocks has reduced the amount or composition of brood pheromones produced by larvae that normally signal workers to refrain from reproduction. They also suggest that anarchistic workers have a higher threshold for these signals than wild-type bees. Keywords Apis mellifera · Worker policing · Anarchy · Brood pheromones · Honest signals
Communicated by T. Seeley B.P. Oldroyd (✉) School of Biological Sciences A12, University of Sydney 2006, Australia e-mail:
[email protected] Fax: +61-2-93514771 T.C. Wossler Department of Zoology and Entomology, University of Pretoria, Pretoria, 0002, South Africa F.L.W. Ratnieks Laboratory of Apiculture and Social Insects, Department of Animal and Plant Sciences, Sheffield University, Sheffield, S10 2TN, UK
Introduction Honey-bee workers have ovaries but are rarely fertile. In a colony with a queen and larvae, only about 1 worker in 10,000 has fully activated ovaries (Ratnieks 1993). However, in queenless colonies, about 10% of the workers will have fully formed eggs in their ovaries, and many eggs are laid (Velthuis 1970). Honey-bee workers cannot mate but because of arrhenotokous parthenogenesis, these unfertilized eggs give rise to fully functional males (Cook 1993). Workers are related to sons by 0.5, but to sons of the queen by 0.25 (Ratnieks 1988; Visscher 1998). A worker therefore benefits from laying eggs. Nevertheless, few workers in queenright colonies have active ovaries, and only one male in a thousand is the son of a worker (Visscher 1989, 1996). The evolutionary reason for worker sterility is that although workers are more related to their sons than to those of the queen, they are even less related to those of half-sister workers (r=0.125) (Starr 1979; Woyciechowski and Lomnicki 1987; Ratnieks 1988). Because of the high levels of polyandry found in honey-bees (Palmer and Oldroyd 2000), the majority of nestmates encountered by workers are half-sisters not full-sisters (Laidlaw and Page 1984). Therefore, on average, workers are much more related to the male offspring of the queen than to the male offspring of other workers (average relatedness to worker’s sons=0.15 for a paternity frequency of 10). This sets the stage for the evolution of worker policing. That is, workers mutually enforce the sterility of their nestmates (Ratnieks 1988). One well-characterized mechanism is the killing of worker-laid eggs. This is based on the ability of workers to identify queen-laid eggs (Ratnieks and Visscher 1989), probably because they are marked with a queen-produced pheromone placed on queen-laid eggs as they pass over the sting sheath (Ratnieks 1995). Worker-laid eggs are thought to lack this pheromone and are therefore eaten by workers (Ratnieks and Visscher 1989). Another potential mechanism is aggression towards workers with activated ovaries (Visscher and Dukas 1995).
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Once efficient worker policing mechanisms have evolved, the reproductive benefits of egg laying within queenright colonies are very low. This selectively favors self-restraint: workers refrain from personal reproduction in the presence of honest signals indicating the presence of queen or brood (Seeley 1985; Keller and Nonacs 1993). However, worker reproduction in queenless colonies remains beneficial (Ratnieks 1988), so complete sterility has not evolved (Robinson et al. 1990; Page and Robinson 1994). Fifth-instar larvae appear to provide the main signals that prevent ovary activation in workers (Trouiller et al. 1991; Arnold et al. 1994; Mohammedi et al. 1998). Signals that inhibit worker reproduction are also thought to arise from the tergal glands of queens (Wossler and Crewe 1999a, 1999b). In a colony in which worker reproduction is rare, a worker that can cause the colony to rear her eggs would have an enormous reproductive advantage (Ratnieks 1988). Such ‘anarchistic’ (Oldroyd et al. 1994) genotypes are probably prevented from spreading within populations by widespread costs of worker reproduction leading to colony-level selection for new policing mechanisms that thwart any new anarchistic genotypes that may arise (Montague and Oldroyd 1998; Oldroyd and Osborne 1999). Colonies in which worker reproduction is at high frequency are non-viable (Barron et al. 2001). Thus an evolutionary tension exists between individual-level selection for personal reproduction and colony-level selection for worker sterility (Hamilton 1964), in which the equilibrium condition appears to favour effective policing (Oldroyd and Osborne 1999; Barron et al. 2001). In 1994, Oldroyd et al. identified a queenright colony in which many males were sons of workers not the queen. A second such colony was found in 1996 (Montague and Oldroyd 1998), and descendants of this queen comprise the ‘anarchistic’ line maintained by the University of Sydney (Oldroyd and Osborne 1999), in which 5–18% of the workers have activated ovaries (Oldroyd and Osborne 1999; Barron and Oldroyd 2001), and the workers frequently lay eggs (Oldroyd et al. 1999) which are policed at lower frequency than eggs from queenless workers (Oldroyd and Ratnieks 2000). In this paper, we use the term ‘anarchistic’ to describe bees or colonies from this line, i.e. ‘anarchistic’ is a description of genotype. Barron and Oldroyd (2001) introduced 1-day-old workers of both wild-type and anarchistic stocks into both queenless and queenright anarchistic and wild-type foster colonies. Dissection of these workers when 12 days old showed that anarchistic workers were more likely than wild-type workers to have activated ovaries, whatever the host. This indicates that anarchistic workers have a higher threshold for the inhibitory signals produced by queens and brood. Queens and brood of anarchistic colonies are also likely to produce signals that are dissimilar to those of wild-type queens and brood because anarchistic workers are more likely to activate their ovaries in an anarchistic than in a wild-type host colony (Oldroyd et al. 1999; Barron and Oldroyd 2001)
Barron and Oldroyd’s (2001) experiment confounded the effects of adult worker, queen and brood genotype on worker ovary activation. In this experiment, we investigated the effects of one of these components, brood genotype, on rates of ovary activation in introduced anarchistic and wild-type bees in isolation. We show that brood genotype is a critical factor in the regulation of worker ovary activation, independent of queen and adult worker genotype.
Methods Experiments were conducted in Sheffield, UK, in May 2000 using bees from three anarchistic (A) and three wild-type (WT) source colonies housed in standard Langstroth hive equipment. The queens heading these colonies had been imported from Australia, and had been introduced the previous season. This allowed complete replacement of workers. The A queens were the results of five generations of selection for the anarchistic syndrome (Oldroyd and Osborne 1999) and were closely related but not sisters. The WT queens were standard Australian commercial stock of Italian lineage and were probably half-sisters. To establish the experimental colonies (see Fig. 1), the three WT colonies were divided into two smaller colonies (E a and E b) such that each had five combs of bees and three combs of brood. These six divided colonies were each furnished with a vertical division of queen excluder material so that the introduced queen could be confined to two combs on one side of the division with the original brood combs on the other. A new queen (Q) of standard Australian commercial Italian stock was then introduced into each of these six colonies using an introduction cage (Laidlaw and Eckert 1962). The introduced queens were naturally mated sisters, and unrelated to the WT queens. After 4 days, the new Q queens were released from their cages onto the two side combs where they began laying. After a further 3 days, the side combs, now containing eggs from the new queens, were removed and discarded. This procedure was repeated every
Fig. 1 Experimental set up. For each of three replicates, ten-comb wild-type colonies were split into two five-frame halves. New wild-type queens were introduced into confined chambers of the two new colonies (E a and E b), and any eggs they laid were removed before hatching. Brood combs containing eggs from an anarchistic (A) or a wild-type (S) colony were added to the colonies every 3 days
368 3 days to ensure that the experimental worker bees were never exposed to larvae or pupae of the Q queens. The Q queens were used to ensure that each E colony had an equivalent wild-type laying queen so that queen pheromones did not differ between the experimental treatments. On the day the six E colonies were set up, the three original WT and the three A queens were introduced to new storage colonies (S and A respectively; see Fig. 1) where they laid eggs. On each of the days that the eggs were removed from the six E colonies, a comb containing eggs (and occasionally very young larvae) was added to each. The three E a colonies received eggs from one of the A queens and the E b colonies received eggs from their WT mother now in colony S (Fig. 1). The queens of the colonies (A and S) providing eggs were confined to two combs by queen excluders, and new combs were provided for them to lay in when the combs of eggs were removed. The number of eggs and larvae provided to the E colonies was approximately equal in all cases, and the rearing conditions for eggs were similar. The experiment was terminated before any introduced eggs emerged as adults. To summarise, these manipulations resulted in two experimental colonies E a and E b, each replicated three times, as follows: ● ●
treatment 1 (E a colonies): WT adult workers; A larvae and eggs; WT queen unrelated to workers or brood. treatment 2 (E b colonies): WT adult workers; WT larvae and eggs; WT queen unrelated to workers or brood.
On the 8th day, we temporarily removed one or two sealed brood combs containing emerging brood from the E colonies and placed them in an incubator at 35 C and high humidity so that emerging workers could be captured. A brood (maintained in nursery colonies) was also emerged in this way. On the 9th day, 1-dayold workers of the A colonies (A1, A2 and A3) and two of the WT colonies (E1 and E2) that had emerged from the combs were marked with coloured poster pens (uniPosca; Mitsubishi Pencil Co.) to indicate their colony of origin. These 1-day-old workers were then divided into six equal lots and introduced to each of the E colonies. Due to a lack of bees, the two E1 colonies were not provided with bees from the A3 mother. Marking continued for a further 2 days. WT brood combs were then returned to their E colonies. In total, between 50 and 100 workers from each E and A colony were introduced into each of the E colonies. Twelve days after introducing the first 1-day-old workers (21 days after the colonies were divided), we caught all marked bees and dissected them to determine the extent of ovary activation (Ratnieks 1993). We classified ovary development as either non-active (no distinct ova could be seen) or active (at least one distinct ovum present in the ovarioles).
Results
Eight days after the experiment was set up, all the original queen’s eggs and larvae in the main section of the hives (i.e. the side that was isolated from the introduced queen) had developed into pupae and were sealed by the bees. Thus the larvae from the original queens were no longer contributing to the production of brood pheromones. Because pupal cells are sealed over in honey-bee colonies, and because most brood pheromones come from fifth-instar larvae (Trouiller et al. 1991), the majority and perhaps all brood pheromones present in our E colonies likely arose from larvae that we experimentally moved into them. If, however, sealed pupae do produce some brood pheromones that are transferred to adult workers, this would have acted to reduce, not increase, differences between treatments. In summary, our manipulations resulted in three pairs of colonies. The two colonies of each pair (E a and E b) had similar WT adult workers, and similar WT sister queens. They differed only in the genotype (A or WT) of the eggs and larvae. This allowed us to test the affects of immature-brood genotype on rates of ovary activation.
The type of larvae to which the introduced workers were exposed had a highly significant effect on the probability of ovary activation (Table 1, Fig. 2). When exposed to WT larvae, WT workers failed to develop their ovaries (Fig. 2). However, 12.6% of WT workers had developed oocytes when exposed to A larvae. Introduced A workers showed the same trend, but the probability of ovary activation was significantly greater than in WT workers exposed to either larval type (χ2=41.4, df=1, P
