J Ethol (2010) 28:61–66 DOI 10.1007/s10164-009-0155-y

ARTICLE

Male production by non-natal workers in the bumblebee, Bombus deuteronymus (Hymenoptera: Apidae) Jun-ichi Takahashi Æ Stephen J. Martin Æ Masao Ono Æ Isamu Shimizu

Received: 22 January 2008 / Accepted: 17 February 2009 / Published online: 24 March 2009 Ó Japan Ethological Society and Springer 2009

Abstract Social insect societies are considered to be composed of many extremely cooperative individuals. While workers are traditionally believed to behave altruistically, recent studies have revealed behaviors that are more selfish. One such example is intraspecific social parasitism, where workers invade conspecific colonies and produce male offspring that are reared by unrelated host workers. Such intraspecific parasitism has been reported in honeybees (Apis cerana, and A. florea) and ‘‘semi-wild’’ bumblebee colonies of Bombus terrestris. Here we report on intraspecific social parasitism by workers in ‘‘wild’’ colonies of the bumblebee B. deuteronymus. Three of the 11 B. deuteronymus colonies studied were invaded by nonnatal workers, of which 75% became reproductive and produced 19% of the adult males. The invading non-natal workers produced significantly more males than resident natal workers and the non-natal brood was not discriminated against by the natal workers.

J.-i. Takahashi (&)
I. Shimizu Center for Ecological Research, Kyoto University, Otsu, Shiga 520-2113, Japan e-mail: [email protected]; [email protected] J.-i. Takahashi Laboratory of Systematic Entomology, Department of Ecology and Systematics, Hokkaido University, Sapporo 060-8589, Japan J.-i. Takahashi
M. Ono Honeybee Science Research Center, Tamagawa University, Tokyo 194-8610, Japan S. J. Martin Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK

Keywords Bombus deuteronymus
Bumblebee
Social parasitism
Social Hymenoptera
Worker reproduction

Introduction Genetic relatedness between workers is a central parameter for testing social behavior based on kin selection (Crozier and Page 1985; Crozier and Pamilo 1996; Boomsma and Ratnieks 1996). The mating frequency of the queen is one of the key determinants of the colony kin structure in monogynous eusocial Hymenoptera (Hughes et al. 2008). Due to haplodiploid sex determinism the relatedness of a worker to the males of a half sister (0.125) is lower than the relatedness to queen-produced males (0.25), and worker reproduction should be inhibited by mutual policing, thus allowing the queens to monopolize male production. Therefore, in monogynous species worker policing is predicted to evolve more readily when queens are polyandrous rather than monandrous (Woyciechowski and Lomnicki 1987; Ratnieks 1988). Although many social wasps and ants consist of monogynous and monandrous colonies, workers still police worker-laid eggs in queen-right colonies (Foster and Ratnieks 2001; Wenseleers and Ratnieks 2006); for example, in the ant Diacamma sp., Kikuta and Tsuji (1999) first demonstrated that worker-produced males were inhibited by worker policing even in monogynous and monandrous colonies. Bumblebee (Bombus) colonies are typically founded by a single queen that has been mated once or occasionally twice (Estoup et al. 1995; Schmid-Hempel and SchmidHempel 2000; Cnaani et al. 2002; Brown et al. 2002; Payne et al. 2003; Takahashi et al. 2008a, b). Furthermore, the lack of worker reproduction in queen-right colonies has

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been reported in four Bumblebee species headed by singly mated queens [B. terrestris (Lopez-Vaamonde et al. 2004, 2007), B. impatiens (Cnaani et al. 2002), B. hypnorum (Brown et al. 2003), and B. ignitus (Takahashi et al. 2008b)]. Drifting behavior, where foraging workers move between intraspecific colonies, has long been observed in social insects, especially in honeybees (Apis mellifera) where colonies are often kept at high densities in close proximity to each other; for example, up to 40% of the workers in a honeybee colony may not have been born there (Pfeiffer and Crailsheim 1998), i.e., they are nonnatal bees that have drifted from nearby colonies. In B. oxidentalis, Birmingham et al. (2004) reported that drifted workers tend to leave small colonies and seek out more mature ones that have larger populations and food stores. In this species maintained in greenhouses up to 28% of the workers in a colony were non-natal (Birmingham and Winston 2004). Genetic investigations have now identified non-natal, i.e., drifted, workers in colonies of bumblebees (Paxton et al. 2001; Lopez-Vaamonde et al. 2004), vespinae wasps (Foster et al. 1999, 2000; Takahashi et al. 2002), and honeybees (Paar et al. 2002; Nanork et al. 2005, 2007). Furthermore, large-scale drifting in Polistes wasps has been demonstrated using radiotagging technology (Sumner et al. 2007). Despite drifting being commonplace, non-natal worker-derived males are rarely detected in queen-right colonies since in majority of social insects male production is monopolized by the queen (Wenseleers and Ratnieks 2006). However, workers in queenless colonies of many social insect species can activate their ovaries and produce a final batch of workerderived males (Brown et al. 2003; Takahashi et al. 2008b; Visscher 1996; Nanork et al. 2007). Until recently, drifting was considered accidental and not detrimental to the colony. However, studies of queenless colonies of B. terrestris (Lopez-Vaamonde et al. 2004, 2007), A. cerana (Nanork et al. 2007), and A. florea (Nanork et al. 2005) have found a high proportion of the final batch of worker-derived males are in fact produced by non-natal workers. This suggests that this ‘‘drifting’’ phenomenon is actually a form of intraspecific social parasitism and this behavior may be much more widespread than previously thought (Beekman and Oldroyd 2008). The aim of this study was to investigate using DNA microsatellite genotyping whether intraspecific social parasitism occurs in a wild population of the Japanese bumblebee, B. deuteronymus. This species nests above ground in grassy tufts (Sakagami and Katayama 1977; Katayama et al. 1993) and so may be more vulnerable to invasion by social parasites than the underground nests of B. terrestris. Therefore, we determined the maternity of workers and males in wild B. deuteronymus colonies.

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Materials and methods Sampling and molecular analysis Between 1999 and 2003, 11 entire colonies of B. deuteronymus were collected during late August from grasslands near the Toyohira river in Misumai, Sapporo city, Hokkaido, Japan. All nests were located within a 15 km2 area. During August the production of gynes (new queens) and males was occurring. From each colony ca. 80% of the workers were dissected and their ovarian state categorized as: undeveloped (nonactive ovaries) or fully developed (active ovaries containing mature oocytes). To determine the origin of workers and the source of any adult males, i.e., males produced by the queen, resident worker (natal) or drifted worker (non-natal), we genotyped all workers with fully developed ovaries, plus 15 workers with undeveloped ovaries and 15 males from each colony. To determine worker–worker relatedness and paternity frequency, 15 female pupae were genotyped from each colony. Template DNA was extracted from individuals by boiling the macerated antenna tissue in 250 ll 5% Chelex (Bio-Rad) resin (Walsh et al. 1991). Microsatellite DNA analyses was performed using eight microsatellite primers (B10, B11, B96, B100, B121, B124, B126, and B131) (Estoup et al. 1995). Polymerase chain reaction (PCR) was performed in a total volume of 15 ll containing 1.0 ll template DNA, 0.2 lM primer, 1.2 ll deoxynucleotide triphosphate (dNTP) mixture (250 lM), 1.5 ll 109 reaction buffer, 1.5 mM MgCl2, and 0.05 units Taq DNA polymerase (TaKaRa). All PCR reactions were performed under the following conditions: following denaturation at 94°C for 3 min, the samples were subjected to 30 cycles of 94°C for 30 s, 46–54°C for 30 s (Estoup et al. 1995), and 72°C for 30 s. The forward primer of each marker was 50 -end-labeled with a fluorescent phosphoramidite. The PCR products were visualized by ABI 3100 Genetic Analyzer using 500 LIZTM as an internal size standard. The fragments were analyzed using ABI GeneScan software (version 3.7) and ABI Genotyper DNA fragment analysis software (version 3.7). In order to obtain reproducibility of genotypes, analysis from PCR to genotyping was repeated three times per non-natal worker and male. Colony kin structure analysis We estimated the regression relatedness among the workers (b), inbreeding coefficient (F), and allele frequencies using Relatedness 4.2 (Goodnight and Queller 1994). Pedigree estimates of relatedness among workers (r) were made by inspecting worker genotypes across the

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63

eight loci for each colony (Boomsma and Ratnieks 1996) assuming outbreeding (Schmid-Hempel and SchmidHempel 2000). Workers’ sons are only detected if: (a) the queen and her mate have different alleles and (b) they inherit the worker’s paternal allele. With fair meiosis, the paternal allele is transmitted with a probability of 50%. Even if worker genotypes are informative, 50% of workers’ sons cannot be distinguished from queens’ sons at this locus. The total number of assignable males in the ith colony (pj) can be calculated as follows: Pj ¼

n X

pi ð1  0:5li Þ;

ð1Þ

Results Variation at microsatellite loci In order to determine the heterozygosity and the number of alleles at the selected loci, a total of 183 adult females and 165 males of B. deuteronymus were successfully genotyped. For each colony, the eight loci exhibited adequate levels of polymorphism with two to seven alleles per locus (Table 1). Based on an examination of the male genotypes, there was no evidence of a null allele in this species. The expected heterozygosities (HE) for the polymorphic loci ranged from 0.51 to 0.74.

1

where n is the number of patrilines in the colony, pi is the proportional representation of the ith patriline, and li is the number of informative loci analyzed at the ith patriline. The expected number of males that are derived from workers (the total number of assignable males Na) in a P sample can be calculated as Na = (PjNj), where Nj is the number of males analyzed for the jth colony. If workers produce x proportion of the males, then the probability of not sampling any worker-produced males is ð1  xÞNa (Foster et al. 2000). The genotype of the mother queen was inferred from the genotypes of 15 female pupae. A non-natal worker was one that possessed a genotype that could not be derived from that of the inferred queen genotype. The probability of not sampling a patriline of proportion k is (1 - k)n, where n is the number of workers sampled, according to Foster et al. (1999). The probability of not sampling worker from a rare male patriline was kept to acceptable levels by the analysis of 15 female pupae. In this study, the nonsampling probability of 10% patriline is 0.2 for 15 females. It is possibly to confuse a non-natal worker with a natal worker if she carries at least one of the queen’s alleles at all loci. The probability of misclassifying a non-natal worker was calculated according to Nanork et al. (2007). This probability can be calculated by sum of the frequency of the queen’s first allele and the frequency of the queen’s second allele at each locus. If a B. deuteronymus queen is singly mated, as most Bombus species are (Schmid-Hempel and Schmid-Hempel 2000), worker-produced males can inherit an allele from either the queen or her mate (father of worker). Under this assumption a male was classified as being produced by a non-natal worker, if it did not share an allele with the queen or the worker consensus genotype. Again it is possible to misclassify males if the genotype of the queen’s mate was not detected from the natal workers.

Colony kin structure Each B. deuteronymus queen was inseminated by a single male and in all 11 colonies adult males were produced during the reproductive period (late August). Inbreeding coefficients in all colonies were not significantly different from zero at any locus, as is expected in a random mating system (F = 0.018). These results demonstrate that, under conditions of random mating, B. deuteronymus colonies are genetically monandrous and monogynous. The mean regression relatedness (r) among workers in the 11 B. deuteronymus colonies was high (0.74 ± 0.016 standard deviation, SD) and close to the expected value (0.75) for an outbreeding monogynous and monandrous species. The dissection of ca. 80% of workers from each colony revealed at least one worker with fully developed ovaries in 10 of the 11 colonies (Table 2). Subsequent genotyping revealed that in seven colonies worker-produced males were present. In one queen-right and two queenless colonies, four non-natal workers were detected, three of which had fully developed ovaries (Table 2). Furthermore, we also detected 11 males in these three colonies produced by

Table 1 Number of alleles (NA), and expected (HE) and observed (HO) heterozygosity for eight microsatellite loci in Bombus deuteronymus workers Locus

n

NA

HE

HO

B10

183

3

0.59

0.65

B11

183

5

0.65

0.75

B96

183

4

0.54

0.60

B100

183

3

0.52

0.56

B121

183

4

0.52

0.59

B124

183

7

0.74

0.78

B126

183

4

0.62

0.65

B131 Mean

183 183

2 4.0

0.51 0.59

0.55 0.64

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Table 2 Collection date, queen condition, and number of workers, males, and new queens in 11 Bombus deuteronymus colonies Colony no.

Collection date

Queen condition

Number of workers

Female pupa

Number of males

New queen

Worker

Adult

Pupa 19

9901

1999, Aug 26

Present

34

6

1

22

0001

2000, Aug 24

Present

38

22

0

79

3

0102

2001, Aug 25

Present

22

9

4

15

16

0202

2002, Aug 28

Present

50

24

1

60

5

0301

2003, Aug 27

Present

37

19

3

38

18

9902

1999, Aug 26

Absent

27

8

3

28

20

9903 9904

1999, Aug 26 1999, Aug 26

Absent Absent

32 44

16 18

0 2

18 33

17 20

0003

2000, Aug 24

Absent

23

21

0

38

6

0101

2001, Aug 25

Absent

18

3

0

35

1

0201

2002, Aug 28

Absent

28

7

0

33

3

Table 3 Numbers and percentages (%) where appropriate of natal and non-natal individuals and probability of misclassifying a non-natal worker in 11 Bombus deuteronymus colonies Colony Number of Number of workers Number of the 15 no. dissected with developed genotyped workers worker ovaries with undeveloped ovaries

Number of genotyped workers with fully developed ovaries

Number of genotyped worker-derived males

Natal worker (%)

Non-natal Natal worker worker (%) (%)

Non-natal Queen worker (%) (%)

Natal worker (%)

Probability of misclassifying a non-natal worker

Non-natal worker (%)

9901

33

2

15 (100)

–

1 (50)

1 (50)

12 (80.0) –

3 (20.0)

0.0032

0001

30

1

15 (100)

–

1 (100)

–

15 (100)

–

0.0055

0102

22

2

15 (100)

–

2 (100)

–

14 (66.7) 1 (33.3) –

0.0009

0202

40

1

15 (100)

–

1 (100)

–

15 (100)

–

0.0012

0301

30

3

15 (100)

–

3 (100)

–

7 (46.7) 8 (53.3) –

0.0043

9902

26

3

15 (100)

–

2 (66.7) 1 (33.3)

6 (40.0) 4 (26.7) 5 (33.3)

0.0056

9903

30

2

15 (100)

–

2 (100)

–

9 (60.0) 6 (40.0) –

0.0054

9904

40

3

15 (100)

–

3 (100)

–

12 (80.0) 3 (20.0) –

0003

22

5

14 (93.3) 1 (6.7)

4 (80.0) 1 (20.0)

0101

17

1

15 (100)

–

1 (100)

–

15 (100)

–

–

0.0021

0201

25

0

15 (100)

–

–

–

15 (100)

–

–

0.0049

non-natal workers. The probability of misclassifying a nonnatal worker was averaged at 0.0036 across all colonies (Table 3). This indicates that neither natal nor non-natal broods were discriminated against by the large number of natal workers in these colonies. No significant difference was found in the number of workers with active ovaries among queenless and queen-right colonies (Wilcoxon ranksum test Z = -0.46, P = 0.65). In the three colonies invaded by non-natal workers they produced 19% of the males, 17% of males were produced by natal-workers, and the remaining 64% were queen-produced males. This resulted in non-natal workers producing 20 times more males per individual than natal workers in these three

123

– –

9 (60.0) 3 (20.0) 3 (20.0)

0.0043 0.0029

colonies. Therefore, on average, non-natal workers have significantly higher (Fisher’s exact test P \ 0.001) reproductive success than do non-natal workers.

Discussion Our study is the first to demonstrate successful intraspecific social parasitism by workers in wild bumblebee colonies. Although social parasitism by non-natal workers has been previously detected in B. terrestris (Lopez-Vaamonde et al. 2004), these colonies were maintained under experimental conditions at higher densities than occur in the

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field. However, the invading workers were believed to originate from nearby wild colonies. In this and previous studies (Beekman and Oldroyd 2008) the number of nonnatal worker-derived males was greater in queenless than in queen-right colonies. It is possible that queenless colonies may be more susceptible to invading workers due to a relaxation of the nestmate recognition system (Jay 1965). A key finding of this study is the high proportion of reproductive non-natal workers that we found in three of the colonies and that non-natal brood is not discriminated against by the large number of natal workers. In B. deuteronymus colonies, an average non-natal worker produces nearly 20 times more males per individual than does a natal worker. This reproductive dominance of non-natal workers by producing a high proportion of the male brood has also been found in B. terrestris (Lopez-Vaamonde et al. 2004), A. florea (Nanork et al. 2005), A. cerana (Nanork et al. 2007), and the cape honeybee A. mellifera capensis (Martin et al. 2002). This last situation is similar to that found in the sweat bee Lasioglossum malachurum, where non-natal workers, but not natal workers, can lay fertilized eggs in the presence of the queen that then successfully develop into gynes (Paxton et al. 2002). Furthermore, our results indicate that B. deuteronymus queens characteristically mate with a single male, and so worker relatedness is high (0.74), which is consistent with previous research suggesting that the majority of European, North American, and Japanese bumblebee species have singly inseminated queens (Estoup et al. 1995; SchmidHempel and Schmid-Hempel 2000; Paxton et al. 2001; Brown et al. 2002, 2003; Payne et al. 2003; Takahashi et al. 2008a, b). In the present study, male production in queen-right B. deuteronymus colonies is dominated by the queen, but we did observed a small proportion (16%) of worker-derived males in the queen-right colonies, as has been previously found in the bumblebees B. terrestris (Bourke and Ratnieks 1991; Alaux et al. 2004), B. melanopygus (Owen and Plowright 1982), and B. hypnorum (Paxton et al. 2001). In contrast, the six queenless colonies that are favored by workers exhibited a higher production (27%) of worker-derived males (Table 3). Although reproductive bumblebee workers can be recognized by several unique behavioral characteristics (Asada and Ono 2000; Lopez-Vaamonde et al. 2004) the brood of both nonnatal workers appears not to be discriminated against. This may be because, in queenless colonies, the policing of worker-laid eggs by other workers is absent or weak (Miller and Ratnieks 2001) as was observed in B. terrestris (Lopez-Vaamonde et al. 2004, 2007) and B. ignitus (Takahashi et al. 2008a). Acknowledgments This study was supported by a Japan Society for the Promotion of Science research fellowship to J.T. and the PRO

65 NATURA 2008 fund for bumblebee conservation. This research was financially supported in part by the Global COE Program A06 to Kyoto University.

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