Multiple Mating
Mating Behaviour
Evolution of Multiple Mating
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Mating Frequency in Honey Bees...
Another Inbreeding Compensation

The degree of multiple mating (polyandry) is always high im Apis Mellifera, but varies between the different subspecies, with A. m. larmarckii having the lowest mean observed numbers of mating at 5.0 and A. m. capensis the highest at 34.0 (Francket al. 2000). The dark European honey bee, A. m. mellifera has intermediate mating frequencies averaging 16.5 (Estoup et al. 1994; Kryger and Moritz 1997).

Virgin queens normally fly 2-3 km, and drones further (Ruttner and Ruttner 1966; Böttcher 1975). The maximum recorded mating distancebetween a queen’s hive and those of her mates is 17 km (Winston 1987).

No studies have investigated the distribution of mating distances, so that it is unknown how exceptional this distance is.Several new studies of honey bees in Europehave shown that most A. m. mellifera populationsare threatened by hybridisation and introgressionwith other introduced honey bee subspecies (Garnery et al. 1998a, 1998b; Jensen et al. 2005).For the conservation of remaining A. m. mellifera populations it is thus important to gain information on mating distances and isolation at a localscale. The aim of the present study was to determine mating distances and isolation in Hope Valley and Edale in order to plan future conservationand controlled breeding activities. To do this weused DNA microsatellite markers to determine thefathers of the worker progeny of queens that mated naturally in these valleys.Materials and methodsExperimental designThe basic design was for virgin queens and drones to make mating flights from hives at differentlocations in the two semi-isolated valleys, so that a distribution of mating distances could be obtainedby paternity analysis of worker progeny using DNA microsatellites. The paternity analysis used data on the genotypes of the worker progeny, the genotypes of their mothers (the mated test queens)and the genotypes of their possible fathers (the drone producing queens) (Figure 2). Male Hymenoptera are haploid and are produced by arrhenotokous parthenogenesis. As a result, the summation of offspring drone genotypes can be used to determine their mother queen’s diploid genotype (Figure 2). Experimental apiaries and their positioning Six experimental apiaries were established; three in Hope Valley and three in Edale. In Hope Valley Figure 1. Map of Edale and Hope Valley, with the approximate areas indicated by dotted-line envelopes. Virgin queens flew from mating nucleus hives in six apiaries (•) and mated with drones from the same six apiaries and from hives belonging to local beekeepers(n). Villages are indicated by open circles and names. the apiaries were approximately 4–6 km apart and placed in the west (Losehill Hall), centre (Platt’s farm) and east (Stoke's farm) of the valley, respectively (Figure 1). In Edale the apiaries were approximately 2.5 km apart with the centrally located apiary at Edale Mill being about 2.5 km from the other two (Barber Booth to the west and Carr House to the east) and from the Losehill Hall apiary in Hope Valley. Within Edale matingscould be achieved by level flying along the valley,whereas Losehill, a mountain ridge of c. 500 m, separates Lose hill Hall from Edale. Since Edale is connected to Hope Valley at the village Hope (see Figure 1), bees from Edale could still meet and mate with bees from Losehill Hall by level flying along that route, but would have to fly further.Each apiary contained 12 queen mating colonies and two drone producing colonies. In addition, two more apiaries were set up in Hope Valley each with just two drone-producing hives. Drone producing and queen mating hive set up Drone production was stimulated in the experimental apiaries by giving colonies frames of drone comb and sugar syrup. The numbers of drones Figure 2. Transmission of genes in the experimental setup. The drone mother queens contributed their gametes to the worker progeny of the test queen via matings of their haploid sons. The genotype of each drone mother queen was deduced from the genotypes of pooled tissue from c. 10–20 drones per colony. The genotype of each mated test queen was deduced from her worker progeny. Subsequently the workers were assigned to a drone mother queen through paternity analysis. reared in each drone producing colony were estimated by photographing the drone comb approximately 4 weeks before mating took place.Samples of 10–20 drone pupae were taken from each colony for genetic analysis to determine the genotype of the drone mother queen.Queen mating hives were set up with 4–6 frames of bees and brood but without any males or a queen. A marked virgin queen was then introduced into each mating hive using a mailing cage. The virgins were reared using standard queen rearing procedures (Laidlaw and Page 1997). All queens were sisters, reared from the same breeder colony, except for a few that developed as emergency queens from worker cells. The virgin queenswere released from their cages at approximately 1 week of age and allowed to mate. Approximately 6 weeks later, worker pupae or larvae were sampled from each colony with a successfully mated queen and used for genetic analyses. Local colonies Beekeepers in the Hope Valley allowed us to inspect their colonies for drone production approximately 4 weeks before the experimental matings took place. Drone production was observed in 25 of the colonies inspected. Samples of 4–20 drone pupae were taken from all these colonies for genotyping and paternity assignment. The local beekeepers are organised in a club and cooperated fully with us, so that we believe to have a fairly complete sample of colonies in Hope Valley. We also obtained DNA of a single workerbee from all the colonies to estimate the background allele frequencies in the Valley. These estimates were applied for statistical inferences of the likelihood of deduced queen genotypes and for paternity analysis. Genetic Analysis Drone and worker samples Equal amounts of tissue were taken from each drone from a given colony (a single leg from pupae or an equal amount of larval tissue) and DNA was extracted from the pooled tissue sample with the DNeasy tissue kit (QIAGEN, Inc., Santa Clara,California). Seventeen microsatellite loci (A7, A8,A24, A28, A43, A88, A113, Ap33, Ap36, Ap43,B124, A14, A76, A79, Ap218, Ap85, Ac11) were amplified and analysed for each of the 37 drone mother colonies according to standard procedures (Baudry et al. 1998; Solignac et al. 2003). The combination of the eleven most variable loci (Table 1) produced multilocus genotypes that could uniquely identify drones from each drone mother colony. DNA was also extracted from 16 immature workers from each mated test queen using the ChelexÒ extraction technique (Walsh et al. 1991). The same 11 microsatellite loci were amplified and analysed for each of these workers. Paternity analysis Genotypes of each of the mated test queens were deduced from the genotypes of their 16 worker offspring using MateSoft Version 1.0 b (Moilanenet al. 2004), which analyse male-haplodiploid mating systems based on the expression of co-dominant genetic markers, such as DNA micro-satellites. Because honey bee queens mate multiply, MateSoft's "broad deduction" method was chosen. MateSoft calculates the weighted probabilities of all possible queen genotypes, based on the observed allele frequencies in the population. At any locus the queen genotype may be determined unambiguously or there may be several alternative possibilities. When the analysis indicated several possible queen genotypes, we only used the loci where the weighted probabilities of the most likely genotype were above 0.80. Worker offspring were assigned to drone mother queens using the likelihood-based method inCervus 2.0 (Marshall et al. 1998), a softwarepackage which performs large-scale parentage analysis in diplo-diploid mating systems using co-dominant genetic markers. To overcome complications due to haplodiploidy, worker progeny were assigned to drone mother queens rather than to the father drones themselves, because drones can be regarded as the flying gametes of a queen, each drone producing clonal sperm. We assumed a genotyping error rate across all loci and individu-als of 1% and a sampling coverage of 95% of the candidate parents (drone mother queens), thus allowing for the possibility that there were additional colonies we did not know about. Statistical confidence limits of the most likely parentalassignments were obtained from the difference in the log likelihood of the most likely and the secondmost likely parent compared to a test statistic produced in a simulation model. Paternity assignment of particular worker offspring to a particular drone mother queen was, however, only accepted if it involved at most two mismatches between the putative drone mother queen (father), the mated test queen (mother) and the offspring genotypes. Worker progeny from each mated test queen that were assigned to the same drone mother queen might originate from either the same drone or from brother drones. By subtracting the mated test queen's own genotype we were able to group sibling offspring into patrilines (Figure 2). We thus estimated the observed mating frequency of each mated queen by the number of patrilines detected in her offspring sample. The effective mating frequency of each mated test queen was calculatedwith a correction for finite sample size and unequalpaternal contributions (paternity skew) (Pamilo1993; Boomsma and Ratnieks 1996).me¼ ðn À 1Þ= nXki¼1y2iÀ 1!where k is the number of patrilines observed, yiis the observed proportion of the ith patriline, and nis the number of workers genotyped. Results Accuracy of paternity analysis Our genetic analyses had great power. The probability of justifiably excluding a single randomly chosen unrelated drone mother queen from parentage at one or more loci was 99.75 when only data on the genotypes of the offspring worker and candidate parent (drone mother queen) were used. This rose to 99.99 when the deduced genotypes of the mated test queen were also used (Table 1). 595(90.7%) of the genotyped offspring were assignedto the experimental or beekeeper-owned dronemother queens from Edale and Hope Valley with aconfidence of P>0.80, (548 with P>0.95). 61worker offspring (9.3%) could not be assigned toany of the known drone mother queens, and arethus most likely to have fathers from non-sampledhives belonging to beekeepers in the Hope Valleyor from drones that had flown in from outside.Table 1. The 11 DNA microsatellite markers used for paternity analysis and the locus-specific MgCl2concentration, annealingtemperature (Ta), number and size range of the alleles, expected heterozygosity (He), average exclusion probabilities for a single parent(Exclusion 1) and a second parent when the first parent is known (Exclusion 2)Locus[MgCl2]Ta(C)No. allelesSize range(bp)HeExclusion 1Exclusion 2A7a1.2 mM558105–1260.6010.2110.386A113b1.2 mM5511200–2360.4290.1050.266Ap43c1.2 mM556134–1490.6380.2200.368A85d1.5 mM557190–2020.7400.3480.530B124a1.5 mM5513216–2500.8870.6270.772A76a1.2 mM6024209–3150.8960.6600.795A79c1.2 mM60991–1180.5150.1460.308Ap36c1.2 mM5511141–1690.8600.6010.752Ap33c1.2 mM5515223–2570.8760.5540.716Ac11d1.5 mM5510111–1290.7810.4040.582A14d1.5 mM5515216–2560.8160.4790.651Mean=11.7Combined=0.99785Combined=0.99996Average exclusion probabilities were obtained by summing individual exclusion probabilities across all combinations of genotypes,weighted by genotype frequencies. The combined exclusion probabilities across all loci represent the average probability of excluding asingle randomly chosen unrelated individual from parentage.Data are based on 734 individuals. The markers used were obtained from:aEstoup et al. 1994;bEstoup et al. (1995);cBaudry et al.(1998) anddSolignac et al. (2003).532 -------------------------------------------------------------------------------- Page 7 Non-assignable offspring were observed in 28 ofthe 41 mated test queens.Mating frequency and mating distancesThe 16 worker progeny from each of the 41 matedqueens gave an average of 10.2 (SE±2.02) fathersdetected per queen. The lowest recorded numberof detected fathers was 5 and the highest 14 (Fig-ure 3). As a result, the entire analysis is based on418 confirmed matings in total. There was nosignificant difference between the mean number ofobserved matings per apiary in an unbalancedanalysis of variance (F5,35=0.12, P=0.987). Themean estimated effective paternity per queen was17.2 (SE±10.9). We defined mating distance as thedistance between the position of the drone pro-ducing hive and the position of the mating hivehosting the mated queen. The maximum matingdistance was approximately 15 km, which wasobserved in one queen. Most of the offspringresulted from mating distances of 7.5 km or less(Figure 4). Approximate one fifth of the matingsoccurred between queens and drones that origi-nated from the same apiary.Mating locationsMore than half of the matings (53.8%) in thethree apiaries in Edale took place with dronesproduced in Edale and two thirds (66.4%) of thematings in the three apiaries in Hope Valley tookplace with drones produced in Hope Valley(Table 2). Overall, approximately 80% of allmatings of Edale queens took place with dronesproduced in Edale and the two immediatelyFigure 3. Distribution of observed number of matings of test queens based on genetic analysis of 16 worker offspring per queen. Thecurve is a fitted binominal distribution.Figure 4. Distributions of mating distances. The cumulative distribution of mating distances is plotted as the curve, whereas the barsindicate the separate percentages of matings obtained at specific mating distances. Ninety percent of the offspring resulted from matingdistances of 7.5 km or less and half of the offspring from mating distances of 2.5 km or less (see dotted lines).533 -------------------------------------------------------------------------------- Page 8 adjacent Hope Valley locations, Losehill Hall(9%) and Hope (16%). This shows that Edale isrelatively but not fully isolated. In addition, theproportion of matings in Edale that could not beassigned to a known drone mother queensincreased from zero in the most westerly apiary,Barber Booth, to 13% when moving east in thedirection of Hope.A higher proportion of matings from HopeValley (19%) could not be assigned to knowndrone mother queens compared to the matings inEdale (7%) (Table 2). The two Edale apiaries inthe middle (Edale Mill) and west (Barber Booth)both had queens that only mated with drones fromEdale hives, whereas all queens from the easternEdale apiary, Carr House, had mated with at leastone drone originating outside Edale. In the threeHope Valley apiaries some of the queens hadmated with drones originating from Edale, espe-cially at the western most apiary Losehill Hallwhere two thirds of the queens had mated with atleast one drone from Edale. This suggests thatgene flow occurs both ways between the two val-leys and that drones are able to fly over Losehill,the mountain directly between the Losehill Hallapiary and the Edale Mill apiary.DiscussionThe high resolution of the genetic markers and thelarge sample size of matings imply that our resultspresent a clear picture of mating distances in twoadjacent valleys. We were able to show that queensand drones from the two valleys do mate. The 11microsatellite loci provided the necessary power todetermine the origin of most of the drone fathersand to distinguish father drones that were brothers.The results were in good agreement with previousstudies of mating frequency and mating distance inhoney bees.PolyandryPolyandry is the rule in honey bees and the numberof matings per queen is high. Several papers havediscussed the evolutionary aspects of polyandry inhoney bees and other social insects (e.g. Boomsmaand Ratnieks 1996; Palmer and Oldroyd 2000). Thecurrent view is that polyandry increases the fitnessof a queen through increased genetic variabilityamong her worker offspring. Advantages of in-creased intra-colonial genetic variability may beimprovements in social organisation and toleranceto environmental changes including pathogens.Division of labour and reproduction greatlyreduces the effective population sizes of social in-sects like honey bees, because a very small numberof individuals produce all the offspring, while thelarge majority are non-reproducing workers thathelp their mother to raise siblings and maintain thecolony (Crozier 1979). The number of colonies in agiven honeybee population is therefore much clo-ser to the effective population size than the actualnumbers of bees. However, when queens are matedto multiple males, the effective population sizeincreases considerably (Crozier and Page 1985), soTable 2. Proportion of matings according to location (see Figure 1) of the drone mother coloniesDrone mother colony locationsQueen mating apiaries in EdaleAll EdaleBarber BoothEdale MillCarr HouseEdale60.00%54.29%49.14%53.87%Hope Valley40.00%40.00%37.93%39.11%Edale, Hope, Losehill Hall85.88%81.43%71.55%78.60%Unknown0.00%5.71%12.93%7.01%Drone mother colony locationsQueen mating apiaries in Hope ValleyAll Hope ValleyLosehil HallPlatt’s farmStoke’s farmEdale19.75%7.14%10.34%14.50%Hope Valley61.73%78.57%62.07%66.40%Unknown18.52%14.29%27.59%19.10%534 -------------------------------------------------------------------------------- Page 9 that high queen-mating frequencies are desirable inhoney bee conservation.Our estimate of queens mating with17.2 dronesis in close agreement with previous estimates forA. m. mellifera (on average 16.5) (Estoup et al.1994; Kryger and Moritz 1997) and indicates thatmating was normal. The mating frequency in ourstudy might be slight overestimations due to therather small number of offspring sampled, espe-cially for colonies with high numbers of observedpatrilines (Tarpy and Nielsen 2002). The locationof apiaries can also have a significant effect onmating frequencies. In A. m. carnica the effectivemating frequency was higher in a mainland apiarycompared to an island apiary, where high windspeeds and relative low temperatures (15–20 °C)prevail during the mating flights (Neumann et al.1999). It remains to be explored, however, whethernative A. m. mellifera that have adapted to suchharsh environments for thousands of years mightperform better than A. m. carnica, which evolvedin a continental environment.Reproductive isolationA considerable recent research effort has focusedon locating and characterising the remainingpopulations of native black bees in Europe(Franck et al. 1998; Garnery et al. 1998a, 1998b;Jensen et al. 2005). Now that these efforts arebecoming successful, maintaining stocks of nativeblack bees becomes relevant. In some countriescontrolled breeding for certified bee breeders takesplace on small islands. In Denmark, for example,such locations have to be approved every year bythe Danish Plant Directorate (Anonymus 1993).However, island-based isolated mating stations arenot practical in all countries, and controlledbreeding is often achieved by creating matingstations isolated by distance or topography such asmountain valleys (Ruttner 1988b), so that detailedinformation on mating distances becomes impor-tant. In the present study the mating distanceswere mostly below 8 km. Peer and Farrrar (1956)observed mating distances of 9–10 km by usingcordovan queens and cordovan drones eventhough wild type drones were abundantly avail-able at shorter distances. (Cordovan is a single-locus recessive body-colour marker). The maximalmating distance recorded in our study wasapproximately 15 km, which is in accord withother biological observations (Klatt 1929, 1932;Peer 1957).Edale, which was free of honey bee coloniesprior to our experiment, is semi-isolated in terms ofhoney bee mating. As expected, the geographicallymost isolated apiary, Barber Booth in westernEdale, which is surrounded on three sides byinhospitable mountains and moorlands, was themost isolated location in terms of honey bee mat-ing. An increasing proportion of matings to dronesoriginating from outside Edale was observed fur-ther down the valley in the direction of Hope, whereseveral local beekeepers live. Edale queens alsomated with drones from Losehill Hall, and viceversa, showing that Losehill Mountain does notprovide complete reproductive isolation. Queens,drones or both were apparently able to fly over oraround this 500 m high mountain (but rising only c.300 m from the valley bottoms), since quite manycolonies contained offspring of fathers from bothsides of Losehill. This corroborates a study byRuttner (1976) that used a colour mutant andmarked drones to show that they were able toovercome differences in altitude of 500 m or moreand return to their hives. This indicates that evenconsiderable differences in altitude are not sufficientto provide complete mating isolation, and thatother factors such as the overall topography of theterrain and the local climate also play a role.About one fifth of the queen matings in HopeValley were to drones from unknown drone mo-ther queens suggesting that significant gene flowfrom the surrounding areas is unavoidable even infairly well isolated valleys. However, the actualproportion of outside matings is almost certainlylower as the limited number of drones in some ofthe samples from beekeeper-owned colonies mayhave resulted in undetected alleles in their mothers.This implies that some of the unassigned offspringmight in fact have been offspring of these dronemothers. A recent method of genotyping livequeens from small pieces of wing tip (c. 2 mm2)(Châline et al. 2004) would eliminate problemswith undetected queen alleles and could be used todetermine the genotypes of drone mother queensand mated test queens directly instead of having toinfer them from samples of progeny. In addition,there may have been some beekeeper-owned col-onies that we were not aware during our sampling.535 -------------------------------------------------------------------------------- Page 10 Conservation implicationsOver the last century the number of beekeepers inNW Europe and thus the number of honey beecolonies, in particular A. m. mellifera, hasdecreased significantly. Wild colonies are rarebecause very few old hollow trees have remainedstanding in modern landscapes and because Var-roa mite infections tend to be fatal for untreatedcolonies. The maintenance of populations of na-tive black honey bees thus relies on active coop-eration with beekeepers, as all beekeepers in acertain area need to be comply with keeping nativehoney bees only to maximize the success of con-servation efforts. A case in point illustrating suchan active cooperative conservation effort is therequeening program of the honey bee populationin Hope Valley that was initiated by BIBBA.Our present results show that the effectivemating distance is similar to the size of the HopeValley, confirming that this area is a reasonablelocation for maintaining a panmictic and relativelypure population of black honey bees. It will benecessary, however, to continue the conservationprogrA. m. melliferae in the entire area of the valley and tofurther reduce hybridisation with imported bees,such as the yellow Italian bee A. m. ligustica, ifnecessary by requeening.Several studies have investigated the effect ofcommercial honeybees on the native fauna ofother, annual and often solitary bees. RecentlyForup and Memmott (2005) showed a negativeassociation between bumblebee and honeybeeabundance, but no apparent effect of honeybeedensity on bumblebee diversity. So, although theremight be competition between honeybees andother bees, the ultimate effect of these interactionsare as yet unclear. However, it is prudent to alsotake the presence of other endangered bees andnon-bee pollinators into consideration whendesigning new potential A. m. mellifera reserves.Conservation and improvement of native hon-ey bee populations is a challenge due to the spec-tacular open-mating system of honey bees. Theinformation obtained in the present study is,therefore, important for evaluating the status andimprovement of both new and existing reserves fornative honey bees. More specifically it appears thatHope Valley and Edale can play complementaryroles in the conservation of A. m. mellifera inBritain through, respectively, their suitability formaintaining a large breeding population, and thepossibility for controlled matings.AcknowledgementsABJ, KAP, and NC were funded by the EU(FW5-ENV) research network ‘Beekeeping andApis Biodiversity in Europe’ (BABE) (contractEVK2-CT-2000-00068). We thank the local (EastMidlands) branch of BIBBA (Bee Improvementand Bee Breeders Association) and beekeepers andlandowners in Edale and Hope Valley for theirassistance during the bee sampling and for pro-viding land to set up apiaries.ReferencesAnonymus (1993) Danish law of beekeeping No 11, article 14.Baudry E, Solignac M, Garnery L, Gries M, Cornuet J-M,Koeniger N (1998) Relatedness among honeybees (Apismellifera) of a drone congregation. Proc. R. Soc. Lond. B.,265, 2009–2014.Boomsma JJ, Ratnieks FLW (1996) Paternity in eusocialHymenoptera. Phil. Trans. R. Soc. Lond. 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Bienenkunden, 13, 243–247.Kryger P, Laidlaw H, Page RE (1997) Queen Rearing and BeeBreeding, Wicwas Press, Chesire, CT.Marshall TC, Slate J, Kruuk LEB, Pemberton JM (1998) Sta-tistical confidence for likelihood-based paternity inference innatural populations. Mol. Ecol., 7, 639–655.Maul V, Hähnle A (1994) Morphometric studies with purebredstock of Apis mellifera carnica Pollmann from Hessen.Apidologie, 25, 19–132.Moilanen A, Sundström L, Pedersen JS (2004) MATESOFT: Aprogram for deducing parental genotypes and estimatingmating system statistics in haplodiploid species. Mol. Ecol.Notes, 4, 795–797.Neumann P, Moritz RFA, van Praagh J (1999) Queen matingfrequency in different types of honey bee mating apiaries.J. Api. Res., 38, 11–18.Olden JD, Poff NL, Douglas MR, Douglas ME, Fausch KD(2004) Ecological and evolutionary consequences of biotichomogenisation. Trends Ecol. Evol., 19, 18–24.Palmer KA, Oldroyd BP (2000) Evolution of multiple mating inthe genus Apis. 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Bienenforsch.,8, 332–354.Solignac M, Vautrin D, Loiseau A, Mougel F, Baudry E,Estoup A, Garnery L, Haberl M, Cornuet JM (2003) Fivehundred and fifty microsatellite markers for the study of thehoneybee (Apis mellifera L.) genome. Mol. Ecol. Notes, 3,307–311.Tarpy DR, Nielsen DI (2002) Sampling error, effective pater-nity, and estimating the genetic structure of honey bee col-onies (Hymenoptera: Apidae). Ann. Entomol. Soc. Am., 95,513–528.Walsh PS, Metzger DA, Higuchi R (1991) Chelex-100 asa medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques, 10,506–513.Winston ML (1987) The Biology of Honey bee, Harvard Uni-versity Press, Cambridge, MassachusettsWoyke J (1964) Cause of repeated mating flights by queenhoneybees J. Api. Res., 3, 17–23.537 Multiple mating and sperm usage in the honeybee Dr. Helge Schlüns School of Tropical Biology, James Cook University The Western honeybee (Apis mellifera L.) has an extremely polyandrous mating system. In general honeybee queens mate with at least ten drones, but even more than forty matings were detected. Queens often take multiple nuptial flights. The cost of multiple nuptial flights was studied in relation to potential benefits. The mating frequency of naturally mated queens was analysed using DNA fingerprinting. Queens that were restricted to one nuptial flight, but wanted to take an additional flight, had significantly fewer matings than queens which started oviposition after a single nuptial flight. Moreover, the sperm number stored in the spermatheca significantly increased with the number of matings. Presumably, queens adjust their nuptial flight frequency according to the mating success of the previous nuptial flights. Furthermore, the findings suggest that a certain number of matings is required to fill the spermatheca to its storage capacity. The average sperm number per copulation or nuptial flight that a queen receives depends on the sperm numbers produced by the drones. Yet, drones might differ in sperm numbers for several reasons. Wing lengths (size indicator) and sperm numbers of small and large drones were compared. Small drones produce significantly fewer spermatozoa than normally sized drones. The rearing investment per spermatozoon is lower for small than for normally sized drones because small drones produce more spermatozoa in relation to their body weight. Since colonies usually produce large drones, the enhanced investment must be outweighed by a mating advantage of large drones. The varying sperm numbers of drones can have an impact on their individual fitness. But in addition, the pattern of sperm utilization by the queen affects the drones fitness. Thus, the consequences of sperm utilization for the fitness of the queen’s mates were studied using DNA-fingerprinting. The impact of the insemination sequence and the amount of semen on the sperm utilization were analysed. The data show no significant effect of the insemination sequence but a strong impact of the semen volume of a drone on the frequency of his worker offspring in the colony. This effect was not linear and the patriline frequencies of the drones contributing larger semen volumes are disproportionately enhanced. If these observations are also valid for natural matings, drone honeybees should maximise the number of sperm but not apply specific mating tactics to be first or last male in a mating sequence.


Schlüns, H., Moritz, R. F. A., Neumann, P., Kryger, P. & Koeniger, G. (2005) Multiple nuptial flights, sperm transfer and the evolution of extreme polyandry in honeybee queens. Animal Behaviour, 70, 125-131.

Schlüns, H., Koeniger, G., Koeniger, N. & Moritz, R. F. A. (2004) Sperm utilization pattern in the honeybee (Apis mellifera). Behavioral Ecology and Sociobiology, 56, 458-463.

Schlüns, H., Schlüns, E. A., van Praagh, J. & Moritz, R. F. A. (2003) Sperm numbers in drone honeybees (Apis mellifera) depend on body size. Apidologie, 34, 577-584.

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