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Testing reliability of a potential island mating apiary using DNA microsatellites Peter Neumann, Job P. van Praagh, Robin F.A. Moritz, Jost H. Dustmann To cite this version: Peter Neumann, Job P. van Praagh, Robin F.A. Moritz, Jost H. Dustmann. Testing reliability of a potential island mating apiary using DNA microsatellites. Apidologie, Springer Verlag, 1999, 30 (4), pp.257-276. hal-00891583 HAL Id: hal-00891583 https://hal.archives-ouvertes.fr/hal-00891583 Submitted on 1 Jan 1999 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Original article Testing reliability of a potential island mating apiary using DNA microsatellites Peter Neumanna Job P. van Praagh Robin F.A. Moritza Jost H. Dustmann a Martin-Luther-Universität Halle-Wittenberg, Fachgebiet Molekulare Ökologie, Institut für Zoologie, Kröllwitzerstr. 44, 06099 Halle/Saale, Germany b Department of Zoology and Entomology, Rhodes University, Grahamstown 61440, South Africa c Niedersächsisches Landesinstitut für Bienenkunde Celle, Wehlstr. 4a, 29223 Celle, Germany (Received 25 July 1998; revised 21 November 1998; accepted 12 May 1999) Abstract - Twenty-four virgin sister queens were kept for 21 days in mating nuclei on the drone-free island Baltrum to test the reliability of a potential mating area. On each of the neighbouring islands Nordemey and Langeoog (750 m and 2 km away) 12 sister queens were kept with drones. Workers from colonies with island-mated queens (Baltrum n = 11, Langeoog n = 7 and Nordemey n = 6) were genotyped with four DNA microsatellite loci (n = 996) to estimate queen mating frequency. No differences in queen mating frequency were observed between Langeoog and Nordemey. However, the level of polyandry on Baltrum was significantly lower than on the neighbouring islands, indicating that mating conditions were much more difficult. The standard genetic distance and differences in allele frequencies between the populations were determined to estimate putative origins of the drones. In this study, 43.7 % of the identified drone fathers did not descend from any of the queens on the adja- cent islands. They were most likely from mainland colonies at least 5.4 km (3 km across open water) away, showing that the combination of distances over open water and over dry land is important in explaining the mating behaviour of honeybee queens. © Inra/DIB/AGIB/Elsevier, Paris Apis mellifera / DNA microsatellite / island / mating control / polyandry 1. INTRODUCTION around islands have a negative impact on the orientation of honeybee workers during Islands are routinely used as mating api- their flights [15]. Thus, islands have been aries to achieve controlled matings of virgin claimed to be ideal places because queens queens [34]. The large areas of open water and drones are not expected to cross open * Correspondence and reprints E-mail: [email protected] water during their mating flights. However, derived from worker offspring and only bee breeders have repeatedly reported worker samples are needed to evaluate the uncontrolled matings even on these safe number of matings of the queen. The num- island mating areas. Recent studies of queen ber of queens which have mismated and the honeybee mating behaviour on drone-free number of times these queens mated with islands strongly support these observations unselected drones can be precisely deter- because they reveal that queens returned mined. from their nuptial flights with a mating sign even during high tide [33]. In this project we tested the reliability of a new on the island So far, the of potential mating apiary reliability mating apiaries of Baltrum has been tested with- (Germany) using virgin queens using virgin queens and DNA microsatellites. out drone colonies [5, 10, 17-19, 21, 27, 31], displacement experiments of drones [5, 11, 20] and marker phenotypes such as dif- ferent races [2, 28] or mutants such as cor- 2. MATERIALS AND METHODS dovan [29]. Whereas some islands seem to provide controlled matings (e.g. [5, 20]) 2.1. Experimental design others apparently do not ([29] among oth- ers). A key factor is the distance between island and mainland. Successful mating Virgin sister queens (n = 48) were reared in summer 1995. Twenty-four of them were kept flights of virgin queens of more than 10 km in mating nuclei on the island of Baltrum which across water have never been open reported is free of other honeybee colonies. No foraging [10, 21, 30, 31]. Virgin queens were able workers could be observed before the experi- to cross at least 1 km or less across open ments and no drones could be attracted using a water [ 19, 29]. There are also reports on lure [14] during normal drone flight activity [25]. matings 7-8 km across open water [17, 18]. The distances from the Baltrum apiary towards Other authors [5, 20] could find no evi- the next available drone-producing colonies on the islands and Lan- dence of such long mating distances across neighbouring Norderney and on the mainland are in water for several North Sea islands. geoog (figure 1) given open table I. On the neighbouring island mating api- it seems as if distinct local charac- Thus, aries Langeoog and Norderney 12 queens each teristics of an island are also mating apiary were kept in the vicinity of 15 (Norderney) or important. 10 (Langeoog) drone-producing sister-queen In addition to the problems resulting from colonies (figure 1). No other bee apiaries are known on these islands. All queens were unusual test conditions [29] also the cor- virgin allowed to mate freely during a period of 21 days. dovan test may suffer from the pitfall of the Each queen was able to absolve mating flights marker with phenotypes interfering honey- at the age of 7 days onwards. The queens of the bee behaviour as shown for workers [13]. drone colonies on Norderney were daughters of The recent advance in honeybee DNA a single mother queen instrumentally insemi- microsatellite technology [8, 9] allows for a nated using mixed semen [22] of drones from genetical control of the reliability of mat- three sister-queen colonies. This results in a max- ing apiaries without interfering with honey- imum number of seven alleles per locus in the bee behaviour and routine bee breeding worker offspring on this island. On Langeoog a maximum number of 12 alleles per locus was practice. DNA microsatellites can be used to possible. Sealed worker brood samples (n = 50 assess the number of in precisely patrilines per queen) were taken from colonies with mated a DNA tests can be honeybee colony [9]. queens and raised isolated in an incubator. easily incorporated in the routine procedure Recently emerged workers were immediately at mating apiaries. The genotypes of the stored in 96 % ethanol at -15 °C until DNA mother queen and her drone mates can be extraction. We used four DNA microsatellites which were developed by Estoup et al. [8, 9]. Multi- plex PCR was performed using two pairs of loci (A43/B 124, A76/A 107) and the standard proto- cols given in Estoup et al. [8, 9]. Amplification products were electrophoresed on 6 % poly- acrylamide sequencing gels for 5.5 h (A76/A107) or 5 h (A43/B 124) with M13mp18 control DNA sequencing reactions run on the same gel as size standards. Microsatellite alleles were scored as fragment lengths in base pairs. 2.3. Genotype analysis 2.2. DNA isolation and number of matings and microsatellite analysis The genotypes of the mother queens and the DNA was phenol extracted from single work- father drones were derived from the genotypes of ers (n = 40 per colony) following routine proto- the sampled workers. The queen was assumed cols [1] with the following changes: to be homozygous when an allele was present in worker of the The was con- 1) workers were incubated with agitation in every colony. queen insect Ringer solution (127 mM NaCl, 1.5 mM sidered to be heterozygous when every worker CaCl2, 5 mM KCl, pH 7.4 with NaOH) for 1 h at carried one of two alleles. The paternal alleles RT before extraction; were those not carried by the queen. We used the putative genotype of the mother queen to 2) single worker thoraces were homogenised exclude additional allele combinations. If multi- in 400 of DNA extraction buffer mM μL (100 ple queen genotypes were possible at a given 100 mM Tris-HCl 10 mM NaCl, (pH 8.0), NaCl, locus we chose, as a rule, the allele combination 0.1 % SDS); yielding the lowest number of observed matings 3) DNA was resuspended in 30 μL DDH2O. (n0). In case a drone’s genotype could not be unambiguously determined owing to heterozy- bility that two genes, one drawn randomly from gosity of the queen at that locus, we assumed an population 1 and the other from population 2, equal possibility for yielding one or the other are identical. This set was calculated for each of queen allele for calculating the allele frequen- the four loci. Then, the average for all loci was cies of the drone populations. calculated in each of the three cases: (J1, J2, J12). 2.4.4. 2.4. Data Improved Bonferroni analysis procedure for χ2-tests 2.4.1. Number of estimated matings (k) We calculated a χ2-test for each allele at each locus to evaluate if the alleles shown the As a result of finite sample sizes the number by drones which had mated with the queens on Bal- of observed patrilines may severely underesti- and have a common mate the actual number of subfamilies.

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