Insectes soc. 47 (2000) 333– 336 0020-1812/00/040333-04 $ 1.50+0.20/0 © Birkhäuser Verlag, Basel, 2000
Insectes Sociaux
Research article
Worker reproduction in Austroplebeia australis Friese (Hymenoptera, Apidae, Meliponini) P.M. Drumond 1, B.P. Oldroyd 2 and K. Osborne 2 1 2
Laboratório de Abelhas, Instituto de Biociências, Universidade de São Paulo, CEP 05508-900, São Paulo, SP, Brazil, e-mail:
[email protected] School of Biological Sciences, Macleay Building A12, University of Sydney, NSW 2006, Australia
Received 6 April 1999; revised 5 April and 23 May 1999; accepted 8 June 2000.
Summary. Two DNA microsatellite markers were used to assess the effective number of mating and the maternity of males in two colonies of Austroplebeia australis (Hymenoptera, Meliponini). In both colonies, the worker genotypes indicated that a single male had inseminated the queens, and the male genotypes indicated that the queens had produced the majority of the males sampled. These results are contrary to the widespread idea that workers would reproduce more readily in monandrous colonies. Possibly, alternative mechanisms such as queen policing, which hinders worker reproduction, might be evolved within this stingless bee taxon.
phic eggs either in A. australis or in A. symei. Nevertheless, no reproductive worker egg was recorded in circa three months of methodical observation (Drumond et al., 1999). As conclusion, the study suggested that reproductive worker eggs might be absent or occur only at a specific period during the development of colonies of Austroplebeia. To investigate these hypotheses, we have collected males in different periods of the year in two colonies of A. australis. The probable mother of these males was then assessed by using two DNA microsatellite markers initially developed for the Brazilian stingless bee Melipona bicolor (Peters et al., 1998).
Key words: Austroplebeia, worker reproduction, mating, stingless bees, Meliponini.
Material and methods
Introduction Although the presence of several workers with mature ovaries is a common event in queenright colonies of stingless bees (Sakagami, 1982), very little is known with regards to worker reproduction in this taxon. The studies so far available suggest that in some species of stingless bees, workers can lay two different kinds of eggs: “reproductive worker eggs” (that develop into males) and “trophic worker eggs” (that are eaten either by the queen or by the workers) (Sommeijer and Bruijn, 1994). At the present, actual worker reproduction (laying of eggs that develop into males) has been confirmed for Melipona favosa (Sommeijer et al., 1999), Melipona quadrifasciata (Sakagami, 1982), Melipona subnitida (Contel and Kerr, 1976; Koedam et al., 1999), Plebeia droryana (Machado et al., 1984) and Scaptotrigona postica (Beig, 1972; Bego, 1982). In Austroplebeia, behavioural observations demonstrated that workers were able to develop ovaries and produce tro-
Two A. australis colonies from Duaringa, QLD (23 : 43 S, 149 : 40 E) were set in a glasshouse located at The University of Sydney, NSW, in April 1997. Both colonies (C1 and C2) had an external access tube, which allowed the workers to forage freely outside the glasshouse. From May 1997 to June 1998, at least once a week, we spent approximately 60 minutes in front of each colony in order to verify the presence of the queen (marked with a small dot on the thorax), males and new brood cells. Callow males were thus collected, killed by freezing and stored at – 70°C. To infer the genotype of the queens, adult workers were collected from each colony, fixed in ethanol and stored at – 20°C. DNA was then extracted by using standard techniques based on Wash et al. (1991). Primers Mbi259AAG and Mbi278AAG (Peters et al., 1998) were utilised for the DNA amplification. The reverse sequence of each primer was fluorescently labelled with HEX (Gibco BRL) to allow visualisation during the electrophoresis. PCR reactions were conducted in 20 µl volumes using 5 µl of template DNA, 1 mM Mg+, 100 µM of each dNTP and 400 mM of primers. Mbi259AAG was annealed at 53°C and Mbi278AAG at 56°C for 35 cycles. PCR products were run on denaturing 6% polyacrylamide gels using a GS 2000 automated DNA fragment analyser (Corbett Research, Australia). The results were analysed using the software Onedscan (Scanalytics, a division of CSPI, USA). We also used the Macintosh computer program Relatedness 4.2c to estimate genetic relatedness and the standard errors (http://gsoft.smu.edu/Gsoft.html), as defined in Queller and Goodnight (1989).
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Worker reproduction in Austroplebeia australis
Results In colony C1, 87 males were sampled mostly during the winter (20 males in June, 3 in July, 59 in August and 5 in September 1997). In colony C2, although males were seen practically throughout the period of study, the DNA analyses were made on 94 males sampled in three different periods of the year (31 males in May 1997, 31 in January and 32 in June 1998). In the end of June 1998, it was noticed that the queen in colony C2 had stopped ovipositing, in spite of being still present in the colony. At that time, the workers ceased brood cell construction and began to destroy newly sealed cells. The marked queen was last seen in the beginning of August. On the same day, a virgin queen was also seen running across the damaged brood area. A few months later, the colony died without any queen. Analyses of 27 workers of colony C1 revealed two alleles at each locus, whereas analyses of 28 workers of colony C2 revealed three alleles (Tables 1 and 2). For the two colonies analysed, relatedness among workers from the same colony was very close to the 0.75 expected under single mating (Hamilton, 1964) (C1: relatedness = 0.743 ± 0.002; C2: relatedness = 0.829 ± 0.005; Population F = 0.231, as given by Relatedness 4.2c). Based on the above assumption, we generated the expected genotype frequencies among males assuming first that a
monandrous queen was the mother of all males, and then under the assumption that workers produced all males (Tables 1 and 2). In colony C1, the observed distribution of males does not differ significantly from that expected if the queen was the sole mother of males ( c 2 = 6.8, d.f. = 3, P > 0.05) but it differs significantly from that expected if the workers were the exclusive mothers of the males ( c 2 = 72.4, d.f. = 3, P < 0.001). This strongly suggests that in colony C1, the queen was the mother of all or nearly all of the males sampled. In colony C2, it is clear that individuals other than the primary queen produced males (Table 2). The queen could not have produced five individuals of genotype L/S, L/R and K/R as they carry non-queen alleles. If we exclude these five individuals (because of zero expected values), the observed ratio of male genotypes is close to that expected if the queen was the sole mother of the males ( c 2 = 3.9, d.f. = 3, P > 0.25). On the other hand, the observed distribution of males differs strongly from that expected if the workers were solely responsible for male production ( c 2 = 262.2, d.f. = 8, P > 0.001). This strongly indicates that the queen laid the vast majority of the male eggs, with just a few additional individuals contributing to the production of male eggs. As we spent time observing the colony during sample collections, we believe that the presence of another functional queen would not have gone unnoticed, and that these individuals were workers.
Table 1. Genotypes of queens, workers and males in Austroplebeia australis – colony C1 Assumption
A single queen of genotype AB/CD mating to one male of genotype B/C*
Worker genotypes
AB/CC AB/CD BB/CC BB/CD
Number
Male genotypes
Predicted
Observed
6.75 6.75 6.75 6.75
05 07 05 10
A/C A/D B/C B/D
Number predicted
Number observed
Assuming worker maternity
Assuming queen maternity
16.31 5.43 48.93 16.31
21.75 21.75 21.75 21.75
16 20 19 32
* Size alleles: A = 186 bp, B = 180 bp (locus Mbi259AAG); C = 120 bp, D = 132 bp (locus Mbi278AAG).
Table 2. Genotypes of queens, workers and males in Austroplebeia australis – colony C2 Assumption
A single queen of genotype JK/ST mating to one male of genotype L/R*
Worker genotypes
JL/SR JL/TR KL/TR KL/SR
Number
Male genotypes
Predicted
Observed
7.0 7.0 7.0 7.0
10 04 08 06
J/S J/T K/S K/T J/R L/T L/S K/R L/R
Number predicted
Number observed
Assuming worker maternity
Assuming queen maternity
5.87 5.87 5.87 5.87 11.75 11.75 11.75 11.75 23.50
22.75 22.75 22.75 22.75 0.0 0.0 0.0 0.0 0.0
* Size alleles: J = 188 bp, K = 193 bp, L = 200 bp (locus Mbi259AAG); R = 124 bp, S = 130 bp, T = 136 bp (locus Mbi278AAG).
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Discussion This study suggests that in the two colonies of A. australis analysed, the queens are monandrous and that males arise primarily from queen-laid eggs. This, to some extent, disagrees with some predictions based on the kin selection theory (Hamilton, 1964; Crozier and Pamilo, 1996). In monandrous taxa, worker-worker and queen-worker conflicts over male production are expected as workers remain much more related to their own eggs (G = 0.5), than to those of other workers (G = 0.375) or to those of the queen (G = 0.25). Thus, the prediction would be that, in monandrous stingless bee species (Peters et al., 1999, but also see Paxton et al., 1999), workers would contribute to the majority of male eggs. Therefore, when this does not occur, as in the two colonies of A. australis analysed in our study, we could presume that some kind of queen policing behaviour for the control of worker sterility might be occurring within the taxon. The occurrence of this queen policing may be indeed why the oviposition ritual in Austroplebeia exhibits some level of elaboration (Drumond et al., 1999), as this might represent a way by which queens can assure reproductive dominance over workers (Sommeijer et al., 1984; Crespi, 1992; Sommeijer and Van Buren, 1992; Drumond et al., 1999). This possible queen dominance over workers, nevertheless, can be flawed, as some workers of the colony C2 were definitely able to produce their own sons. Crespi (1992) proposed that species without worker oviposition in queenright colonies would be expected to present the simplest oviposition ritual, since there is no queenworker or worker-worker conflict. This seems to be true for Frieseomelitta, where workers do not develop ovaries even when queenless (Cunha et al., 1986). On the other hand, in Melipona, Plebeia and Scaptotrigona, species whose workers produce some or most of the males, the oviposition ritual is characterised by some elaborate queen-worker interactions (Zucchi, 1993). Although the oviposition ritual of A. australis is not very elaborate, it is also characterised by some striking behaviours such as “workers moving their heads forwards and backwards in front of the queen” or “workers raising the first legs when the queen comes closer” (Drumond et al., 1999). Therefore, even though workers in Austroplebeia contribute very little to male production, they still represent a source of conflict for the queen as they have developed ovaries in queenright colonies (Drumond et al., 1999). Sommeijer et al. (1984) hypothesised that seasonal changes in colony populations may be related to levels of worker reproduction in colonies of Melipona. This could also be the case in Austroplebeia. This aspect, however, was not precisely measured in this study, although both colonies of A. australis were apparently in good condition, with adequate supplies of food and large number of workers. Besides, it would even be possible that the presence of virgin queens could weaken the queen control over worker reproduction and contribute to the appearance of worker-laid males. Finally, it was also noticeable the fact that the two colonies of A. australis, set at the same condition, had exhibited two distinct periods of male production. Though it is generally held that
Research article
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male production in stingless bees is not restricted to particular seasons (Sakagami, 1982), it is possible that the placement of A. australis nests outside their natural distribution area could have interfered in the results obtained. As so far there is no study that has systematically investigated the factors mentioned above, we recommend that future studies of male production involve not only a higher number of nests studied under different conditions but also frequent sampling throughout the whole season.
Acknowledgments We thank J. Paar, K. Clarke, K. Palmer, K. Wilkes, L. Halling, M. Cox and the reviewers for their constructive comments on the manuscript. Thanks also to M. Henshaw, Y. Chzn and Z. Doan for the technical support. This research was supported by an Australian Research grant to B.P. Oldroyd. P.M. Drumond was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (Fapesp – Brazil).
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