Worker reproductive parasitism and drift in the western honeybee Apis mellifera

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1 Behav Ecol Sociobiol (2010) 64: DOI /s ORIGINAL PAPER Worker reproductive parasitism and drift in the western honeybee Apis mellifera Nadine C. Chapman & Madeleine Beekman & Benjamin P. Oldroyd Received: 10 August 2009 / Revised: 5 September 2009 / Accepted: 7 September 2009 / Published online: 18 September 2009 # Springer-Verlag 2009 Abstract When a honeybee (Apis spp.) colony loses its queen and is unable to rear a new one, some of the workers activate their ovaries and produce eggs. When a colony has a queen (i.e., it is queenright) almost all worker-laid eggs are eaten, but when hopelessly queenless, the workers become more tolerant of worker-laid eggs and rear some of them to adult drones. This increased tolerance renders a queenless colony vulnerable to worker reproductive parasitism, wherein unrelated workers enter the colony and lay eggs. Here, we show that the proportion of unrelated (non-natal) workers significantly decreases after an Apis mellifera colony becomes queenless. The remaining nonnatal workers are as likely to have activated ovaries as natal workers, yet they produce more eggs than natal workers, resulting in significantly higher reproductive success for non-natal workers. In a second experiment, we provided queenless and queenright workers with a choice to remain in their own colony or to join a queenless or queenright colony nearby. The experiment was set up such that worker movement was unlikely to be due to simple orientation errors. Very few workers joined another colony, and there was no preference for workers to drift into or out of queenless or queenright colonies, in accordance with the proportion of non-natal workers declining significantly after becoming queenless in the first experiment. Communicated by O. Rueppell N. C. Chapman (*) : M. Beekman : B. P. Oldroyd Behaviour and Genetics of Social Insects Laboratory, School of Biological Sciences A12, University of Sydney, Sydney 2006, Australia nadine.chapman@bio.usyd.edu.au Keywords Worker reproductive parasitism. Intraspecific parasitism. Conspecific parasitism. Egg dumping. Worker laying. Worker drift. Apis mellifera Introduction Some reproductive systems are vulnerable to infiltration by cheater individuals that lay eggs in the nest of a conspecific, where the eggs may be reared. Such parasitism is widespread in birds (Yom-Tov 1980, 2001) and is also known in a variety of insects (Tallamy 2005) including the lace bug Gargaphia solani (Tallamy and Denno 1982), the tree hopper Polyglypta dispar (Eberhard 1986), the burying beetle Anecrophorus vespilloides (Muller et al. 1990), various solitary bees (e.g., Fabre 1914; Eickwort 1975), the social aphid Pemphigus abesinymphae (Abbot et al. 2001), and some solitary wasps (e.g., Hansell 1987; Field 1989). Conspecific or intraspecific parasitic reproductive behavior, in which individuals lay eggs in the nest of another, is also known in eusocial insects (Field 1992; Brockmann 1993; Beekman and Oldroyd 2008). Examples include several species of honeybee (e.g., Lundie 1954; Nanork et al. 2005, 2007), sweat bees (Kukuk and May 1991; Soro et al. 2009), the bumblebee Bombus terrestris (Birmingham et al. 2004; Lopez-Vaamonde et al. 2004), ants (Heinze and Keller 2000), and some wasps (e.g., Gamboa 1978; Klahn 1988). Functional sterility in eusocial insects is thought to be enforced due to inclusive fitness benefits (e.g., Ratnieks 1988; Foster et al. 2006). Honeybee queens mate multiply (Palmer and Oldroyd 2000), and workers are therefore more closely related to the male offspring of the queen than they are on average to the male offspring of their fellow

2 420 Behav Ecol Sociobiol (2010) 64: workers, which are primarily half sisters. As a result, workers can increase their inclusive fitness by preventing their sister workers from reproducing (Ratnieks 1988; Ratnieks and Visscher 1989; Ratnieks et al. 2006). Honeybee workers can apparently distinguish worker-laid eggs from queen-laid eggs by an as-yet unidentified mechanism (Martin et al. 2005a, b), and due to hypothesized inclusive fitness benefits, workers use this ability to prevent worker reproduction by selectively removing ( policing ) worker-laid eggs (Ratnieks and Visscher 1989; Ratnieks 1993, 1995; Visscher 1996; Wenseleers et al. 2004b; Beekman and Oldroyd 2005). Highly efficient policing of worker-laid eggs can potentially lead to the evolution of acquiescence in which workers do not activate their ovaries in the presence of a queen and brood (Wenseleers et al. 2004a). The combined forces of coercion and acquiescence are apparently potent, for workers with activated ovaries have never been found in the presence of the queen in the red dwarf honeybee Apis florea (Halling et al. 2001) orthegianthoneybeeapis dorsata (Wattanachaiyingcharoen et al. 2002). In the Asian hive bee Apis cerana, up to 5% of random-aged queenright workers may have active ovaries (Oldroyd et al. 2001; Nanorketal.2007), but no males have been found to be the offspring of workers (Oldroyd et al. 2001). In wild-type colonies of temperate Apis mellifera with a queen, less than one in 10,000 workers have activated ovaries (Ratnieks 1993), and less than 0.1% of drone offspring may be attributed to workers (Visscher 1989). Despite widespread functional worker sterility in the presence of the queen, the honeybee s reproductive system is potentially vulnerable to exploitation by workers that use the brood-rearing capacities of a colony for personal reproduction. In the artificially selected anarchistic line maintained at Sydney University, workers produce many male offspring even in the presence of the queen (Oldroyd et al. 1994; Montague and Oldroyd 1998; Oldroyd and Osborne 1999). Workers of the Cape honeybee (Apis mellifera capensis) are capable of producing clonal offspring via thelytoky in the presence of the queen (e.g., Onions 1912; Anderson 1963; Beekman et al. 2009). If A. mellifera capensis workers find themselves in a colony of another subspecies, they often activate their ovaries and lay many eggs that produce a new generation of yet more parasitizing A. mellifera capensis workers (reviewed in Allsopp 1993; Neumann and Hepburn 2002; Neumann and Moritz 2002; Beekman et al. 2008; Beekman and Oldroyd 2008). During reproductive swarming events, A. mellifera capensis workers frequently lay in queen cells; they may even enter other colonies and lay eggs in queen cells there (Jordan et al. 2008). When a honeybee colony becomes queenless and is unable to raise a new queen (i.e., it is hopelessly queenless), some of the workers must activate their ovaries and lay eggs in order for the colony to produce a final cohort of reproductive drones before ultimately perishing (Page and Erickson 1988; Robinson et al. 1990; Martin et al. 2004). For these eggs to mature, policing of worker-laid eggs must be curtailed (Miller and Ratnieks 2001; Nanork et al. 2006). However, the cessation of worker-policing renders a hopelessly queenless colony vulnerable to worker reproductive parasitism by workers from other colonies which enter the nest and lay eggs there. Indeed, hopelessly queenless colonies of A. florea (Nanork et al. 2005) and A. cerana (Nanork et al. 2007) are parasitized by non-natal workers, and these workers have disproportionately higher reproductive success per individual than natal workers. Non-natal workers have been found in queenright colonies of all honeybee species that have been investigated thus far (e.g., Moritz et al. 1995; Paar et al. 2002; Pfeiffer and Crailsheim 1998; Neumann et al. 2000; Jensen et al. 2005; Nanork et al. 2005, 2007). Wild honeybee colonies are known to aggregate (Underwood 1990; Oldroyd et al. 1995, 1997b, 2000; Paar et al. 2002; Rinderer et al. 2002; Baum et al. 2005, 2008; Wattanachaiyingcharoen et al. 2008), and it is thought that non-natal workers often drift into nearby colonies due to orientation errors (Rauschmayer 1928; Free 1958). Drifting is particularly common in A. mellifera, and due to the often short distances between colonies kept in apiaries, up to 40% of the workers in a colony may be from other colonies (e.g., Pfeiffer and Crailsheim 1998). Drift of workers between colonies is influenced by apiary layout (placement and orientation of colonies in relation to other colonies, especially in rows), with drift into nearby colonies in the same orientation being more likely than to those that face a different direction (Free 1958; Free and Spencer-Booth 1961; Jay 1965, 1966a, b, 1968; Pfeiffer and Crailsheim 1998). The predominant direction of movement of workers between queenless and queenright colonies varies between studies. Delaplane and Harbo (1987) found that queenless and queenright A. mellifera colonies attract drifted workers equally. Free and Spencer-Booth (1961) reported that significantly more workers drifted from queenright to queenless colonies than from queenless to queenright colonies. Furthermore, although they did not provide a statistical test, their data suggest that there was less drift between queenright colonies than between queenless colonies. Neumann et al. (2001) found that A. mellifera capensis dispersers (long-range drifters) from queenright colonies were found significantly more often in queenless colonies, and those from queenless colonies were significantly more often found in queenright colonies. Choice experiments in A. florea found that workers from hopelessly queenless colonies are significantly more likely to leave their colony than workers from queenright colonies,

3 Behav Ecol Sociobiol (2010) 64: and these workers are significantly more likely to join other hopelessly queenless colonies than queenright ones (Chapman et al. 2009a). Thus, there is considerable variation between studies on the direction of movement of workers between queenless and queenright colonies. Here, we investigate whether hopelessly queenless colonies of A. mellifera are parasitized by workers from other colonies, and if so, if these unrelated workers achieve disproportionately greater reproductive success than natal workers by testing the hypotheses that: 1. The proportion of non-natal workers in queenless A. mellifera colonies does not change from the proportion that is found when the colonies were queenright. 2. The proportion of non-natal offspring produced in queenless A. mellifera colonies is the same as the proportion of non-natal workers present in the colony (i.e., they have equal reproductive success as natal workers). 3. Non-natal workers are equally likely to have active ovaries as natal workers. In a second experiment, we measure the extent of drift of workers to and from hopelessly queenless and queenright A. mellifera colonies to test the hypotheses that: 4. Queenless workers are as likely as queenright workers to join another colony. 5. Queenless and queenright colonies are as likely to acquire workers from other colonies. 6. Workers that remain in their own colony have equal rates of ovary activation as drifted workers that joined the colony. Materials and methods Experiment 1: worker reproductive parasitism in queenless A. mellifera colonies The experiment was conducted at an apiary situated in Richmond, NSW. Trial 1 began on 27th September, 2006 (colonies 1 3). Trial 2 began on 8th January, 2008 (colonies 4 and 5). Trial 3 began on 5th March, 2008 (colony 6). Trial 2 behaved differently from the other two trials and is treated separately (see below). It is known that the rate at which workers move between colonies is influenced by season (Free 1958; Pfeiffer and Crailsheim 1998; but see Jay 1965). Thus, while the proportion of nonnestmate workers present in our colonies may have differed between trials due to environmental factors (Free 1958; Free and Spencer-Booth 1961; Jay 1965, 1966a, b, 1968; Pfeiffer and Crailsheim 1998), the null hypotheses that natal and non-natal workers should respond equally to queenlessness, activate their ovaries to an equal extent, and produce offspring in proportion to their incidence in the colony are not affected by any such variation. There were more than 100 colonies within a 2.5-km range of the experimental apiary. These colonies provided multiple sources of workers that could potentially move into our queenless colonies. The study colonies were headed by naturally mated queens of commercial Italian origin. Each colony comprised 30,000 40,000 bees, housed in two-story Langstroth hives. Colonies remained in situ throughout the experiment and were spaced at least 1.5 m apart. All colonies of a trial were manipulated on the same day. We removed the queen from the colonies and collected a random sample of approximately 200 workers using a handheld vacuum cleaner, at the time of queen removal, taking workers haphazardly from throughout the colony. These random samples were then genotyped (trials 1 and 3) to enable us to determine the proportion of non-natal workers present in the colonies before experimental manipulation. Queen cells were removed 1 week after queen removal. After 5 weeks of queenlessness (trials 1 and 3), we sampled approximately 500 workers using the above method and collected approximately 200 drone eggs from each colony. All drone eggs were the offspring of workers, as drones were sampled more than 24 days after queen removal, which is the time taken for a drone to mature from egg to adult (Winston 1987). We dissected workers (Oldroyd et al. 2001) and classified eggs of any size as active and ovaries without eggs as inactive. We selected approximately 200 workers with inactive ovaries and all workers with active ovaries for genetic analysis and genotyped the drone brood to determine the proportion of each sample that was non-natal. DNA was extracted using the Chelex method (Walsh et al. 1991; Oldroyd et al. 1997a). Samples were amplified at microsatellite loci Am5, Am8, Am14, Am46, Am56, and Am61 (Solignac et al. 2003) and analyzed on a 3130xl Genetic Analyzer (Applied Biosystems, California). Resultant data files were analyzed using GENEMAPPER software (Applied Biosystems), and maternal alleles were assigned at each locus based on the majority of workers having a particular allele (in which case we inferred the queen was homozygous at this locus) or having one of two alleles (indicating that the queen was heterozygous at the locus). Non-natal workers were identified due to them not carrying a consensus queen allele at one or more loci. Paternal alleles for each locus were then inferred by subtraction and individual workers assigned to patrilines based on their deduced genotypes. We assigned drone eggs as the offspring of a worker patriline based on sharing the same alleles as that patriline. A drone that carried alleles incompatible with being the offspring of any patriline present in the colony was regarded as being the offspring of a non-natal worker. All

4 422 Behav Ecol Sociobiol (2010) 64: workers and drone eggs that were identified as being of nonnatal origin were genotyped a second time to confirm their non-natal status. The microsatellite loci were highly polymorphic (Table 1); thus, the power to detect non-natal workers was high. We performed χ 2 tests to compare the proportion of nonnatal workers present before and after a colony was made queenless, the relative reproductive success of natal and non-natal workers, and the rate of ovary activation in natal and non-natal workers. A non-natal worker may be erroneously identified as a natal worker if she carries a queen allele at each locus examined. This is given by Q p i1þp i2 2 where p i1 and p i2 are the i frequencies of the queen s two alleles at the ith locus (Neumann et al. 1999; Paar et al. 2002). A son of a non-natal worker can be erroneously classified as the son of a natal worker if he fortuitously carries a resident allele (an allele carried by the queen or one of her mates) at all loci. This probability is Q p i, where i p i is the frequency of the male s resident allele at the ith locus. Because we did not have population-wide allele frequencies, we used the array of alleles in the workers sampled to obtain an approximation. A son of a natal worker can be erroneously classified as the son of a non-natal worker if his mother s patriline was not sampled among the workers. The more rare a patriline is, the greater the possibility that it will not be sampled. The probability of not sampling a patriline of proportion k is (1 k) n, where n is the number of workers sampled (Foster et al. 1999). We sampled workers per colony. This sample size means that the probability of nondetection of a rare worker patriline, of frequency 1% for example, due to it not being sampled, is low (P<0.01). The possibility of nondetection of a subfamily because two fathering males shared the same genotype is not relevant to the detection of the sons of non-natal workers. No eggs were found after 5 weeks of queenlessness in trial 2 (colonies 4 and 5). We continued to check these two colonies weekly for eggs. After the colonies had been queenless for 9 weeks without producing any eggs we Table 1 Number of alleles and heterozygosity for each of the microsatellite loci used in experiment 1 Loci Number of alleles Heterozygosity Am Am Am Am Am Am Average collected a sample of 201 workers from each colony and dissected their ovaries to determine if they had been activated. Experiment 2: the effects of queenlessness on worker drift in A. mellifera Each replicate utilized four colonies headed by naturally mated queens of commercial Italian origin. Each of the colonies comprised 30,000 40,000 workers. There were at least 100 colonies within 2 km of the apiary. Replicate 1 commenced on 6th September 2006, replicate 2 on 4th October 2006, and replicate 3 on 13th November For each replicate, we moved four A. mellifera colonies housed in two-story Langstroth hives to a field 0.5 km from the apiary at the University of Western Sydney, Richmond Campus. We arranged the colonies in a 5 5 m square with the entrances facing out from the center. This arrangement minimizes orientation errors (Free 1958; Jay 1966a, b, 1968). Colonies were left for 3 days before further manipulation. We placed combs of emerging workers from eight to ten colonies not used as discriminator colonies in a 35 C incubator overnight. Over 2 days, we marked 2,360 workers for each colony for replicate 1, 2,500 for replicate 2, and 2,080 for replicate 3 on the thorax with nontoxic paint (Posca Paint Pens, Mitsubishi Pencil Co., Japan) using a different color for each colony. The origin of the newly emerged workers was randomized to control for genotypic effects on the likelihood of drifting. We then dequeened two of the colonies such that each colony had the choice of two equidistant colonies from itself, one colony with the same queenstate and one of the opposite queenstate (Fig. 1). There was also one colony on the diagonal slightly further away which had the same queenstate as the colony in question. Using this design, twice as many workers would be expected to move to a colony of the opposite queenstate due to them having twice as many options for that state, though it must be acknowledged that the colony on the diagonal was slightly further away than the other two colonies. Nonetheless, a worker had the choice of at least one colony of each queenstate within the experimental setup. We introduced the marked bees into their host colonies by spraying them with sugar solution to encourage acceptance of the marked workers. Queen cells were removed from the queenless colonies 1 week later. Three weeks after the workers were added to the colonies, we collected approximately 100 marked resident workers from each colony (marked with the color corresponding to that of their colony) and collected all the drifted workers (having a color different from that of the colony they were found in), noting which colony they had drifted from. We scored the ovaries of all the collected workers as above. We then performed χ 2 tests to test the hypotheses that workers from queenright and queenless host colonies are equally

5 Behav Ecol Sociobiol (2010) 64: Fig. 1 Layout of experimental colonies in experiment 2, showing the absolute number of workers that drifted between colonies. Circles with a cross in the center are queenless likely to move to other colonies, that queenright and queenless colonies are equally likely to acquire workers from other colonies, and that drifted workers are equally likely as nondrifted workers to have activated ovaries. Results Replicate 1 Replicate 2 Replicate 3 Experiment 1: worker reproductive parasitism in queenless A. mellifera colonies The proportion of non-natal workers decreased significantly in all colonies from an average of 7.4% when queenright to 0.5% after the colonies were made hopelessly queenless (colony 1: # 2 1 ¼ 28:17, P<0.001; colony 2: #2 1 ¼ 21:25, P< 0.001; colony 3: # 2 1 ¼ 29:95, P <0.001; colony 6: ¼ 97:90, P<0.001; # 2 1 ¼ 14:82, P<0.001; pooled data: #2 1 Table 2). We performed a χ 2 test of heterogeneity (Sokal and Rohlf 1995) and found that there was no significant heterogeneity between the colonies in the extent of the decline (# 2 3 ¼ 3:71, P=0.29) Under queenless conditions, non-natal workers had greater reproductive success per capita than natal workers in three of the four colonies (colony 1: # 2 1 ¼ 39:08, P< 0.001; colony 2: # 2 1 ¼ 48:10, P <0.001; colony 3: # 2 1 ¼ 0:92, P=0.34; colony 6: #2 1 ¼ 5:29, P=0.020; Table 2). There was significant heterogeneity in the relative reproductive success of natal and non-natal workers among the four colonies (# 2 3 ¼ 8:58, P=0.035). Overall, non-natal workers formed 0.5% of the worker population after queenlessness, but these non-natal workers were responsible for 7.7% of the eggs. This was significantly greater than the reproductive success of natal workers (all colonies combined: # 2 1 ¼ 101:97, P<0.001). The probability that a non-natal worker was erroneously identified as a natal worker was for colony 1; for colony 2; for colony 3, and for colony 6. Thus, this is a negligible source of error. The average and approximate probability of a nonnatal-derived male having the same genotype as a natalderived male and being misclassified as such is 0.052±SE for colony 1; 0.012±0.002 for colony 2; ± for colony 3; and ± for colony 6. Thus, it is likely that some drone eggs were classified as being natal when they should have been classified as non-natal, and the rate of parasitism was slightly underestimated. Non-natal workers did not differ significantly from natal workers in their rates of ovary activation (pooled # 2 1 ¼ 0:07, P=0.80; Fig. 1). However, because only four of the 921 genotyped queenless workers with active ovaries were nonnatal and only four of 768 genotyped queenless workers with inactive ovaries were non-natal, there was insufficient power to detect a difference in ovary activation rates between natal and non-natal workers should such a difference exist. In trial 2, neither colony showed evidence of worker-laid eggs after 9 weeks of being hopelessly queenless. Nonetheless, workers had activated ovaries in both colonies (colony 4, 50.7%; colony 5, 57.7%). There were adequate stores of pollen and honey in the colonies, suggesting that lack of food cannot explain the absence of worker-produced eggs. There was empty comb available in which the workers could lay. Experiment 2: the effects of queenlessness on worker drift in A. mellifera Assuming that most marked workers were successfully introduced into the experimental colonies, only % of marked workers per replicate moved into another colony within the experiment (Fig. 1). We tested the null hypothesis that queenless and queenright colonies had equal probability of acquiring drifted

6 424 Behav Ecol Sociobiol (2010) 64: Table 2 Ovary activation rate and reproductive success of natal and non-natal workers in experiment 1 Colony Pooled Before queen removal Number of workers genotyped % non-natal workers present After queen removal Number of workers genotyped % non-natal workers present % genotyped non-natal workers with active ovaries % genotyped natal workers with active ovaries Number of offspring genotyped % non-natal-derived offspring workers. We found that in one of the three replicates and when all replicates were combined, queenless and queenright colonies acquired similar proportions of drifted workers (replicate 1: # 2 1 ¼ 0:18, P=0.67; pooled: #2 1 ¼ 0:13, P=0.72; Fig. 1). However, queenright colonies gained more drifted workers than queenless colonies in replicate 2 (# 2 1 ¼ 4:55, P=0.033), while in replicate 3 queenless colonies gained more drifted workers than queenright colonies (# 2 1 ¼ 6:25, P=0.012). Unsurprisingly, there was significant heterogeneity between the replicates (# 2 2 ¼ 10:85, P=0.004). We then tested the null hypothesis that drifted workers had equal probability of originating from queenright and queenless colonies. We found that workers from queenless and queenright colonies are equally likely to join another colony (replicate 1: # 2 1 ¼ 1:6, P=0.21; replicate 2: #2 1 ¼ 0:73, P= 0.39; replicate 3: # 2 1 ¼ 0:25, P=0.62; pooled: #2 1 ¼ 0:28, P= 0.60; Fig. 1). There was not significant heterogeneity between replicates (# 2 2 ¼ 2:30, P=0.32). Drifted workers were as likely to have active ovaries as workers that stayed in their host nest (resident workers) in the three replicates (replicate 1: # 2 1 ¼ 0:43, P=0.52; replicate 2: # 2 1 ¼ 0:94, P=0.33; replicate 3: #2 1 ¼ 0:28, P=0.60; Table 3) and in the pooled replicates (# 2 1 ¼ 0:65, P=0.42). There was no significant heterogeneity between the replicates (# 2 2 ¼ 0:99, P=0.61). Discussion This study shows that worker reproductive parasitism occurs in hopelessly queenless colonies of the Western honeybee. After an A. mellifera colony becomes hopelessly queenless, the proportion of non-natal workers drops by an order of magnitude; however, in most colonies, the nonnatal workers which remain have disproportionately higher reproductive success per capita than natal workers. Relatively high reproductive success of non-natal workers in queenless colonies has now been shown to occur in A. florea (Nanork et al. 2005), A. cerana (Nanork et al. 2007), and wild-type A. mellifera (this study). This suggests that worker reproductive parasitism of queenless nests is a widespread phenomenon in the genus Apis. In A. florea, an open-nesting species, queenless colonies show very high levels of worker reproductive parasitism with up to 36% of drone eggs being the offspring of non-natal workers (Nanork et al. 2005). In A. cerana, a cavity-nesting species, rates of reproductive parasitism are lower ( % Nanork et al. 2006), but even this level of parasitism likely represents a significant fitness cost. The extent of worker reproductive parasitism in queenless A. mellifera colonies ( %, this study) is similar to that seen in A. cerana. While the proportion of non-natal workers significantly increases in A. florea after queen loss (from 2% to 4.5%, Nanork et al. 2005), the proportion of non-natal workers significantly decreases in A. cerana (from 4.3% to 1.8%, Nanork et al. 2007) and A. mellifera (from 7.3% to 0.5%, this study). It has been proposed that cavity-nesting species (i.e., A. mellifera and A. cerana) have stronger defenses against worker reproductive parasitism due to their ability to guard the entrance to their colony and prevent non-natal workers from entering (Nanork et al. 2007; Chapman et al. 2008). Indeed, guards of queenless A. mellifera colonies Table 3 Proportion of drifted and resident workers with active ovaries in experiment 2 Replicate Pooled Number of drifted workers dissected % drifted workers with active ovaries Number of resident workers dissected % resident workers with active ovaries

7 Behav Ecol Sociobiol (2010) 64: that have not commenced laying eggs reject significantly more non-nestmate workers than nestmate workers, while guards in queenright colonies did not make this distinction (Chapman et al. 2009a). However, this phenomenon has not been observed in A. cerana (Chapman et al. 2008). Increased guarding should result in the proportion of non-nestmate workers declining after a colony is made queenless, as we have seen here. Thus, our current study gives additional support for the hypothesis that queenless colonies of cavity-nesting honeybees have heightened vigilance against non-natal workers as a defense against worker reproductive parasitism. While it is possible that existing non-natal workers are also removed, we consider this a less likely mechanism. Non-natal workers adopt their host colony s odor shortly after acceptance (Breed et al. 2004, 1998; Downs and Ratnieks 1999), and thus it is difficult to see how non-natal individuals could be identified after having gained entrance to the colony (Chapman et al. 2008). Choice experiments in A. florea have revealed that workers from hopelessly queenless colonies are more likely to drift and that they preferentially drift into other queenless colonies rather than into queenright colonies (Chapman et al. 2009b). Here, we have shown that A. mellifera does not show this behavior. No pattern could be detected in the preference of queenright and queenless workers to move into either queenright or queenless colonies. Earlier studies on worker drift between queenless and queenright A. mellifera colonies have produced mixed results (Free and Spencer-Booth 1961; Delaplane and Harbo 1987; Neumann et al. 2001). It is clear, however, that the phenomenon found in A. florea in which large numbers of workers drift out of queenless colonies and into other queenless colonies (529 out of 549 drifted workers; Chapman et al. 2009b) is not found in A. mellifera. What is the likelihood of a potential parasite coming across a queenless colony to parasitize? Queenlessness is common among managed bees due to the frequent manipulation of colonies and the failure of queens to return from mating flights (Perez-Sato et al. 2008), with perhaps 2 3% of colonies being queenless at any one time (B.P.O., personal observation). In the wild, queenlessness may be less common than in managed A. mellifera, but in tropical species where migration and predation is frequent, there may be similar rates of queenlessness (Oldroyd and Wongsiri 2006). Thus, even without active search for queenless nests, there is some chance that drifted workers may encounter queenless nests that are vulnerable to worker reproductive parasitism. How do non-natal workers achieve higher reproductive success than natal workers in A. florea, A. cerana, and A. mellifera? Non-natal workers of A. cerana (Nanork et al. 2007) and A. florea (Nanork et al. 2005) are significantly more likely to have active ovaries than natal workers, and it is expected that proportionally they would produce more eggs. However, in this study, non-natal workers of A. mellifera were no more likely to have active ovaries than natal workers. We suspect that in fact non-natal workers do have higher rates of ovary activation than natal works in A. mellifera but that our low sample size was insufficient to detect the difference. Alternatively, non-natal workers may have more ovarioles than natal workers, thus enabling them to produce more eggs at a time. The finding of two queenless colonies without eggs after an extended period of queenlessness is unusual. Production of drones which have the potential to mate with a queen before the colony dies is the only reproductive option for a hopelessly queenless colony. Since workers with active ovaries were found in these colonies, we can assume that policing of worker-laid eggs was never switched off. This phenomenon has been reported previously (Châline et al. 2004); however, in that case worker-laid eggs were observed in the colonies. The lack of worker-laid eggs cannot be due to poor nutrition, as the colonies had ample stores of pollen and nectar. Acknowledgments We wish to thank our beekeeper Michael Duncan, our lab managers Julie Lim and Marcus McHale for their assistance, and Gretchen Wheen for allowing us to conduct experiments on her property. We also thank Jerome Buhl, James Makinson, Nathan Lo, Ros Gloag, and Peter Oxley for helping to mark the bees. We thank the members of the Genetics and Behaviour of Social Insects Laboratory, University of Sydney, Andrew Bourke, and two anonymous reviewers for their helpful comments on the manuscript. These experiments were performed according to the laws of Australia. The study was funded by an Australian Research Council grant to M. Beekman and B.P. Oldroyd. References Abbot P, Withgott JH, Moran NA (2001) Genetic conflict and conditional altruism in social aphid colonies. Proc Nat Acad Sci USA 98: Allsopp MH (1993) Summarized overview of the capensis problem. S Afr Bee J 65: Anderson RH (1963) The laying worker in the Cape honeybee Apis mellifera capensis. J Apicult Res 2:85 92 Baum KA, Rubink WL, Pinto MA, Coulson RN (2005) Spatial and temporal distribution and nest site characteristics of feral honey bee (Hymenoptera: Apidae) colonies in a coastal prairie landscape. 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