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Journal
Apidologie
DOI http://dx.doi.org/10.1051/apido/2010031
Published online 02 June 2010

© INRA/DIB-AGIB/EDP Sciences, 2010

1. INTRODUCTION

South Africa is home to two subspecies of honeybee, the African bee Apis mellifera scutellata (hereafter scutellata), and the Cape honeybee, A. m. capensis (hereafter capensis). Capensis is confined to the southern part of the Western and Eastern Cape, whereas scutellata is found throughout the rest of South Africa and countries to its north (Hepburn and Radloff, 1998). The two subspecies interact within a hybrid zone, producing (when queens mate with males of both subspecies) mixed colonies comprising both pure and hybrid workers (reviewed in Beekman et al., 2008). Capensis is unique in that it is the only subspecies or species of Apis in which workers are capable of thelytokous parthenogenesis, the production of diploid offspring without mating (Onions, 1912; Verma and Ruttner, 1983). This ability allows it to become a lethal social parasite of scutellata colonies, as capensis workers can enter a scutellata colony, activate their ovaries and lay eggs which in turn develop into reproductive adults (Martin et al., 2002; Beekman et al., 2008). Reproductive workers do not participate in normal hive duties, causing colony collapse if they become too numerous (Martin et al., 2002).

Scutellata was introduced into Brazil in 1956. Descendents of this introduction, known as the Africanized honeybee (AHB), have subsequently spread throughout the tropical and subtropical regions of the Americas (Spivak et al., 1991). The invasiveness of AHB in the Americas led to concerns that the scutellata population from which AHB descended would eventually overrun the capensis population (Anderson, 1980). Conversely, anthropogenic introductions of capensis into scutellata’s native range have led to the death of thousands of scutellata colonies due to capensis parasitism (Allsopp, 1992). Yet, despite the competitive adaptations of both subspecies the hybrid zone separating capensis and scutellata appears to be stable (Hepburn and Crewe, 1991). It has been postulated that south of the zone, capensis’s social parasitism gives it a selective advantage, while in the north, scutellata has an advantage due to its high reproductive rate (Beekman et al., 2008).

In this study, we examined whether scutellata patrilines are over-represented in the offspring of both capensis and scutellata queens inseminated with equal numbers of drones of both subspecies. It was recently suggested that sperm of AHB males has an advantage over sperm from non-AHB males; when queens were artificially inseminated with sperm from both AHB and European drones, more workers were sired by AHB males compared with European males in months 2, 3 and 4 after inseminations (DeGrandi-Hoffman et al., 2003). If scutellata is able to out-compete capensis via sperm competition, then this may be one factor that gives scutellata a reproductive advantage in the northern part of the hybrid zone (Beekman et al., 2008).

2. MATERIALS AND METHODS

2.1. Queen-rearing

Capensis colonies used in this study were unselected colonies typical of those found around Stellenbosch, Western Cape (33°56′ S, 18°51′ E). Scutellata colonies were obtained from Douglas, Northern Cape (26° 01′ S, 29° 22′ E). Stellenbosch is well south of the hybrid zone whereas Douglas is well north of the zone within scutellata’s native range (Hepburn and Crewe, 1991). To reduce the likelihood of social parasites destroying our experimental colonies, the scutellata colonies were moved to an apiary separate from the capensis colonies near Stellenbosch. Queens were reared in late September and early October 2008. Queen-cells were harvested from the colonies nine days after grafting and emerged in an incubator at 35 °C. Upon emergence, the queens’ wings were clipped and individually stored for genetic analysis. Newly emerged queens were placed with 20–30 newly emerged attendant bees until insemination.

2.2. Instrumental insemination of queens and collection of workers

Three scutellata and three capensis queens were artificially inseminated (Laidlaw, 1978) between five and nine days after emergence. For each queen we used semen from five capensis and five scutellata drones. Semen was collected alternately from drones of the two different subspecies. Once all semen was collected in the capillary, the queen was anaesthetised with CO2 and inseminated. Even though we did not measure the exact volumes used, we took care that a similar amount of semen was used for each queen. We then introduced the queens into 5-frame scutellata colonies with scutellata workers and brood to maximise acceptance of the inseminated queens. As there are no diagnostic markers that distinguish capensis and scutellata subspecies (Franck et al., 2001), we kept the drones used for genetic analysis so that we could determine the father of the workers sampled.

As soon as queen-produced brood was about to eclose, we collected either pupae or freshly emerged workers from each colony. Thereafter we collected emerging brood at monthly intervals for three months. We collected approximately 100 workers from each of the six colonies at each sampling date. Previous findings suggest that if there is an effect of sperm competition it is apparent by the third month post insemination (DeGrandi-Hoffman et al., 2003). Therefore, sampling ended after the fourth month.

2.3. Genetic analyses

DNA was obtained from the queen (wingtips), the fathering drones and workers and pupae (2–3 legs) from each colony using a high salt extraction method (Aljanabi and Martinez, 1997). The fathering drones were screened with seven Apis mellifera microsatellite markers used in previous parentage studies: Am005, Am006, Am008, Am046, Am052, Am059 and Am061 (Solignac et al., 2003). For colony C1, one microsatellite marker was sufficient for distinguishing capensis and scutellata patrilines (Am061). For the other colonies, duplex polymerase chain reactions were required (Colonies S1 and C2: Am008/Am061, Colonies S2 and S3: Am008/Am059, Colony C3: Am046/Am061).

PCR product (0.4 μL) from each multiplex reaction was added to 10 μL formamide and 100 nL LIZ DNA size standard (Applied Biosystems). Samples were run on a 3130xl Genetic Analyser (Applied Biosystems) with capillary length 36 cm and injection time of 15 s at 1200 V for 41 min. Results were analysed using Genemapper 3.7 (Applied Biosystems) and the patriline (capensis or scutellata) of each individual was determined.

2.4. Statistical analyses

We calculated the proportion of workers sired by scutellata drones produced in each month by each queen. We used contingency tests on the number of workers sired by capensis and scutellata fathers to test for change in this proportion each month by each queen and to compare the total number of scutellata-patrilines produced by each queen subspecies. We further tested if colonies within subspecies show the same directional change using χ2-tests of heterogeneity.

3. RESULTS

We found a high degree of variability among the colonies in the number of workers sired by scutellata drones (Tab. I and Fig. 1). The number of scutellata-patriline individuals changed significantly over time in 3 of 6 colonies (Tab. I and Fig. 1). Sperm use within queen subspecies was variable (Tab. I and Fig. 1). There was a significant effect of queen genotype on the number of scutellata derived workers, with scutellata queens producing a significantly higher number of scutellata patriline workers than capensis queens (data pooled per queen subspecies, , P < 0.001) (Fig. 1).

Table I

χ2 tests of the change in number of workers sired by scutellata drones over time (months 1–4, apart from colony S2 which lost its queen during the fourth month before samples could be collected) per colony (S1–S3: colonies headed by scutellata queens, C1–C3: colonies headed by capensis queens). See Figure 1 for a graphical representation of the data. A significant bias towards use of sperm of a particular subspecies was detected in colonies S3, C1 and C3 (see Fig. 1 for the direction of change). To determine if colonies within subspecies show the same directional change, we performed heterogeneity tests. Heterogeneity χ2 were calculated by taking the absolute value of the difference of ‘χ2 of Total’ and the ‘Total of χ2’. p-values were calculated from the χ2-distribution of the χ2-values and degrees of freedom. Within queen genotypes there was a significant heterogeneity among colonies.

thumbnail Figure 1

Proportion of individuals sired by scutellata drones produced in each month by scutellata (S1–S3) and capensis queens (C1–C3). A significant bias towards use of sperm of a particular genotype was detected in colonies S3, C1 and C3 (see Tab. I). Numbers above bars represent the total number of bees successfully genotyped for that sampling date. When pooled across queen genotype scutellata queens produced a significantly higher proportion of scutellata patriline workers than capensis queens (, P < 0.001).

4. DISCUSSION

DeGrandi-Hoffman et al. (2003) found a significant increase in the number of workers sired by scutellata drones in the second, third and fourth month after insemination and concluded that this was evidence for sperm competition. Contrary to their results we found no directional increase in the contribution of scutellata drones. If anything, our results seem to suggest that the number of scutellata-produced offspring decreases over time, especially in capensis queens (Fig. 1). Although the number of spermatozoa produced by capensis (8.9 ± 1.1million, Buys, 1990) and AHB (9.2 ± 1.8 million, Rinderer et al., 1985) drones are similar (no data are available for scutellata in South Africa), the variance among drones is enormous within subspecies and even breeder lines (Koeniger et al., 2005). Our colonies varied significantly in the number of scutellata workers produced over time, and this may possibly be explained by differences in spermatozoa numbers among drones. We note however, that the number of spermatozoa produced by drones is not directly correlated with paternity frequency as spermatozoa numbers are significantly lower in AHB drones compared with European drones (Rinderer et al., 1985). Nonetheless in the study by DeGrandi-Hoffman et al. (2003) AHB drones sired disproportionally more workers than European drones.

Interestingly, our results also show that capensis queens produced more capensis patriline offspring, while scutellata queens produced more scutellata patriline offspring despite the presence of sperm of both subspecies in the queens’ spermatheca (Fig. 1). Such an effect of queen subspecies was absent in the study of DeGrandi-Hoffman et al. (2003). Our results suggest that scutellata sperm may be disadvantaged in capensis queens. A similar ‘same subspecies advantage’ has been reported in mating swarms of mixed honeybee subspecies where queens produced more offspring sired by drones of their own subspecies (Koeniger et al., 1989). The mechanisms that lead to assortative paternity are unknown, but in our study we can exclude any effect of female mate choice (Baer, 2005). Assortative paternity in our study could have arisen either via cryptic female choice prior to fertilization, or the preferential rearing of pure subspecies’ offspring. The latter would lead to an increase over time of workers sired by drones of the same subspecies as the queen, consistent with our results when the data are pooled across queen subspecies. However, given the small colony-level sample size and the significant variation among colonies within the same subspecies, we recommend that the conclusion that offspring of each subspecies are overrepresented in hybrid colonies headed by queens of the same subspecies be viewed with caution. A more thorough study is required. Should it be confirmed however, then this would be a significant factor in the capensis-scutellata hybrid zone dynamics and in the stability of the hybrid zone (Beekman et al., 2008).

Acknowledgments

We thank Christian Fransman for his help in the field. Marcus McHale, Julie Lim and Rute Brito assisted with genetic analysis. Thank you to three anonymous reviewers whose comments improved the manuscript. B.P.O. and M.B. are supported by the Australian Research Council. M.B. further acknowledges support from the University of Sydney. T.C.W. is supported by the National Research foundation.

References

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All Tables

Table I

χ2 tests of the change in number of workers sired by scutellata drones over time (months 1–4, apart from colony S2 which lost its queen during the fourth month before samples could be collected) per colony (S1–S3: colonies headed by scutellata queens, C1–C3: colonies headed by capensis queens). See Figure 1 for a graphical representation of the data. A significant bias towards use of sperm of a particular subspecies was detected in colonies S3, C1 and C3 (see Fig. 1 for the direction of change). To determine if colonies within subspecies show the same directional change, we performed heterogeneity tests. Heterogeneity χ2 were calculated by taking the absolute value of the difference of ‘χ2 of Total’ and the ‘Total of χ2’. p-values were calculated from the χ2-distribution of the χ2-values and degrees of freedom. Within queen genotypes there was a significant heterogeneity among colonies.

All Figures

thumbnail Figure 1

Proportion of individuals sired by scutellata drones produced in each month by scutellata (S1–S3) and capensis queens (C1–C3). A significant bias towards use of sperm of a particular genotype was detected in colonies S3, C1 and C3 (see Tab. I). Numbers above bars represent the total number of bees successfully genotyped for that sampling date. When pooled across queen genotype scutellata queens produced a significantly higher proportion of scutellata patriline workers than capensis queens (, P < 0.001).

In the text