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Apidologie30 (1999)251-276 Paris O Inra/DIB/AGIB/Elsevier.
Original article
Testingreliability of a potentialislandmating apiary usingDNA microsatellites Peter Neumannu'b,Job P. van Praaghc,Robin F.A. Moritzux, Jost H. Dustmannc a Martin-Luther-UniversitätHalle-Wittenberg,FachgebietMolekulareÖkologie, Institut für Zoologie, Kröllwitzerstr. 44, 06099Halle/Saale,Germany b Departmentof Zoology and Entomology,RhodesUniversity,Grahamstown6,l440,SouthAfrica c Niedersächsisches Landesinstitutfür BienenkundeCelle, Wehlstr.4a,29223 Celle, Germany (Received25 July 1998;revised2l November 1998;accepted12May 1999)
Abstract - Twenty-four virgin sisterqueenswere kept for 2l days in mating nuclei on the drone-free islandBaltrum to testthe reliability of a potentialmating area.On eachof the neighbouringislands Norderneyand Langeoog(750 m and 2 km away) l2 sisterqueenswere kept with drones.Workers fiom cofonieswith island-matedqueens(Baltrum n= ll, Langeoogn=7 and Norderneyn = 6) were genotypedwith four DNA microsatelliteloci (n = 996) to estimatequeenmating frequency.No ditferencesin queen mating frequency were observedbetweenLangeoog and Norderney. However, the level of polyandry on Baltrum was significantly lower than on the neighbouringislands,indicating that matingconditionswere much more difficult. The standardgeneticdistanceand differencesin allele frequenciesbetweenthe populationswere determinedto estimateputativeorigins of the drones.In this study, 43.1 Voof the identified drone fathersdid not descendfrom any of the queenson the adjacentislands.They were most likely from mainlandcoloniesat least5.4 km (3 km acrossopenwater) away, showingthat the combinationof distancesover openwater and over dry land is importantin explainingthe mating behaviourof honeybeequeens.O Inra/DIB/AGIB/Elsevier,Paris Apis melffera / DNA microsatellite / island / mating control / polyandry
I.INTRODUCTION Islandsare routinelyusedas mating apiariesto achievecontrolledmatingsof virgin queens[34]. The large areasofopen water * Correspondence and reprints E-mail:
[email protected]
around islandshave a negativeimpact on the orientation of honeybeeworkers during their flights [5]. Thus, islandshave been claimedto be ideal placesbecausequeens and drones are not expectedto cross open
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waterduringtheir matingflights. However, b e e b r e e d e r sh a v e r e p e a t e d l yr e p o r t e d uncontrolledmatingseven on thesesafe islandmatingareas.Recentstudiesof queen honeybeemating behaviouron drone-free islandsstronglysupporttheseobservations becausethey revealthat queensreturned from their nuptialflights with a mating sign evenduringhigh tide [33]. So far, the reliability of mating apiaries has been testedusing virgin queenswitho u t d r o n ec o l o n i e s[ 5 , 1 0 , 1 1 - 1 9 , 2 1 , 2 1 , 311,displacement experimentsof drones[5, I 1, 201and markerphenotypessuchas differentraces12,281or mutantssuchas cordovan [291.Whereassomeislandsseemto provide controlledmatings(e.g. [5, 20]) othersapparentlydo not ([29] among others). A key factor is the distancebetween i s l a n da n d m a i n l a n d .S u c c e s s f um l ating flights of virgin queensof more than l0 km acrossopen water have never beenreported [ 1 0 , 2 1 , 3 0 , 3 1 ] .V i r g i n q u e e n sw e r ea b l e to crossat least I km or less acrossopen water [9, 29]. There are also reportson matings7-8 km acrossopenwater [17, 18]. Other authors[5, 20] could find no evidenceof suchlong matingdistancesacross open water for severalNorth Sea islands. Thus, it seemsas if distinctlocal characteristicsof an islandmating apiary are also lmponant.
derived from worker offspring and only worker samples are needed to evaluate the number of matings of the queen. The number of queens which have mismated and the number of times these queens mated with unselected drones can be oreciselv determined. ln this project we tested the reliability of a potential new mating apiary on the island o f B a l t r u m t G e r m a n y )u s i n gv i r g i n q u e e n \ and DNA microsatellites.
2. MATERIALS 2.1. Experimental
AND METHODS design
Virgin sisterqueens(n = 48) were rearedin summer 1995.Twenty-tbur of them were kept in matingnuclei on the islandof Baltrum which is free of other honeybeecolonies.No fbraging workers could be observedbefbre the experimentsand no dronescould be attractedusing a lure [4] duringnormaldronetlight activity [25]. The distancesfiom the Baltrum apiary lowards the next availabledrone-producingcolonieson the neighbouringislands Norderney and Langeoogandon the mainland(figurel) aregivenin table L On the neighbouringislandmating apiariesLangeoogand Norderney 12 queenseach were kept in the vicinity of l5 (Norderney)or l0 (Langeoog)drone-producingsister-queen colonies(figure l). No other bee apiariesare In additionto the problemsresultingfrom unusualtest conditions[29] also the cor- known on theseislands.All virgin queenswere allowed to matelreely during a period of 2l days. dovan testmay suffer from the pitfall of the Each queenwas able to absolvemating flights markerphenotypesinterferingwith honey- at the ageof 7 days onwards.The queensof the beebehaviouras shownfor workers[13]. dronecolonieson Norderneywere daughtersof T h e r e c e n t a d v a n c ei n h o n e y b e eD N A a singlemotherqueeninstrumentallyinsemimicrosatellitetechnology[8, 9] allowsfor a nated using mixed semen[22] of dronesfrom colonies.This resultsin a maxgeneticalcontrol of the reliability of mat- threesister-queen imum number of sevenallelesper locusin the ing apiarieswithout interf'eringwith honeybee behaviourand routine bee breeding worker offspring on this island.On Langeooga maximum numberof l2 allelesper locus was practice.DNA microsatellitescan be usedto possible.Sealedworker brood samples(n = 50 preciselyassessthe numberof patrilinesin per queen)weretakenfiom colonieswith mated a honeybeecolony [9]. DNA testscan be q u e e n sa n d r a i s e di s o l a t e di n a n i n c u b a t o r . easilyincorporatedin the routineprocedure Recentlyemergedworkerswere immediately at mating apiaries.The genotypesof the storedin 96 c/oethanol at -15 'C until DNA mother queenand her drone matescan be extraction.
Tcsting reliability of a potentialisland mating apiary
259
'2km' Norderney
Figure l. Map of the apiarylocationson the islandsLangeoog(A), Baltrum (B) and Norderney(C) and on the neighbouringmainland(1 5).
Table I. Distancesfrom the Baltrum coloniesto the next availabledronessources. Baltrum
Mainland Langeoog Norderney
Total
Open water
5.4km 7 . 8k m 13.6 km
3km 1 . 7k m 700m
2.2. DNA isolation and microsatellite analysis DNA was phenol extractedfrom singleworkcrs (n = 40 per colony)lollowingroutineprotocols I I ] with the following changes: 1) workers were incubatedwith agitationin insectRingersolution(127 mM NaCl, 1.5mM CaCl,,5 mM KCl, pH 7.4 with NaOH) for t h at RT beforeextraction: 2) singleworker thoraceswere homogenised in 400 pL of DNA extractionbuffer (100 mM NaCl, 100mM Tris-HCl (pH 8.0), 10mM NaCl, 0.1 7cSDS); 3) DNA was resuspended in 30 pL DDH,O.
We used four DNA microsatelliteswhich were developedby Estoup et al. [8, 9]. Multiplex PCR was performedusingtwo pairsof loci (A4318124,AT6lAlO7) and the standardprotocols given in Estoupet al. [8, 9]. Amplification products were electrophoresedon 6 c/apolyacrylamidesequencinggels for 5.5 h (A76lA 107) or 5 h (A43l8124)with Ml3mpl8 controlDNA sequencingreactionsrun on the samegel as size standards.Microsatellitealleleswere scoredas fragmentlengthsin basepairs.
2.3. Genotype analysis and number of matings The genotypesof the motherqueensand the father droneswere derivedfiom the genotypesof the sampledworkers. The queenwas assumed to be homozygouswhen an allelewas presentin everyworker of the colony.The queenwas consideredto be heterozygouswhen every worker carried one of two alleles.The paternalalleles were those not carried by the queen.We used the putative genotype of the mother queen to excludeadditionalallelecombinations.lf multiple queengenotypeswere possibleat a given locus we chose,as a rule, the allelecombination yielding the lowestnumberof observedmatings (nn).In casea drone'sgenotypecould not be
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P. Neumannet al
unambiguouslydeterminedowing to heterozygosity of the quecnat that locus,we assumedan equalpossibilityfbr yielding one or the other queenallele for calculatingthe allelefiequencics of the dronepopulations.
2. 4. 4. I mprov ed B onfe rro ni
2.4.Data analysis
procedurefor f -tests
2.4.1. Number of estimated matings (k) As a rcsult of finite samplesizesthe number of observedpatrilines may severelyunderestimate the actual number of subfamilies.Therefore, wc estimatedthe numbcrof patrilinesin an infinite sampletbllowing Cornuetand Aries [zl]:
I
t
r\".1
E ( k t = k - l * - l t - ; JI K' )
L
trr
wherc E(k) is the cxpectednumbcrof patrilines in thecolony,A is the numberof equallyfiequent patrilinesand n is the samplesize. We followed Oldroyd et al. [26] and numerically evaluatedk by substitutingE(k) with our observednumberof matings(no)and the worker samplesizesfbr n.
2.4.2.Numberof effectivemales(m) The uvcrageinlrücolonialrclatedncss C was estimatedaccordingto Estor-rp et al. [91.Then, the numberof effectivemales(m") was calculatedfollowing Chcvaletand Cornuet[3]: l l 'l ^ : :
bility that two gencs,one drawn randomlyfrom population 1 and the other from population 2, are identical.This setwas calculatedfbr cachof the tbur loci. Then, the averagefbr all loci was calculatedin eachof the threecases:(Jp J,, J D).
2
r)r
4G-l
whererr" is thenumherol effectivcmulesan.lC is the averageintracolonialrelatedness.
We calculateda X2-testfor eachalleleat each locus to evaluateif the alleles shown bv the droneswhichhadmatedwith theoueenrun Bultrum. NorderneyanclLangeooghive a common allelepool or not. Sincethe high numberof alleles at the usedmicrosatelliteloci mav causesisnificant diff'erencesonly by chance,we usedin improvedBonferoni procedure[16,32) to adjust thc significancelevels.We alsousedthis proced u r e t o c o m b i n et h e d e p e n d e n t e s tr e s u l t s becausethe tcst statisticcannotbe split up into independenttest statisticsL\6,321. For n test statistics, Qr 42.,....,4,,and fbr 0, as a continuously distributedslatisticfor testingthe null hypothesisHo.,versusthe alternativehypothesisH,.,(i = l, .....,n) theoverallhypothcsis H. is rejectcdil for al leustone i:
P14< d1i1
(4)
wherep(i) are the orderedp-valuesfbr 12-tests, a(l) is the significancelevel fbr thc subhyporhesisH,. For eachH, q,, was calculatedas follows: q (-s) u... (/) n-i+l wirh a = 0.05.
2.5. Putative origin of Baltrum worker bees' fathers
2.4.3. Genetic distance We usedthe standardgeneticdistanceof [23]:
D=-rr +L ,IJIJZ
{.r)
with
J:Zpi
,t, =lti
, a n dJ 1=, \ w ,
where -/, is the probability that two ran 0.05).The allele sourceof sexuallymature droneswas at frequenciesfor all testedmicrosatelliteloci least5.4 km away. are given in table 1V. Our results of the The Baltrum queensshoweda signifiare shownin table V. We found for X2-tests all testeddronepopulationswith the excep- cantlysmallernumberof matingscompared 2.6. Comparisons of queen mating frequencies
262
P. Neumannet al
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< ? 0,05),wasdaraufhindeutet,daBdie Paarungsbedingungen auf Baltrum erschwert s i n d . D i e D r o h n e n ,m i t d e n e ns i c h d i e BaltrumKöniginnengepaarthaben,konnten aufgrundihrer Allele nicht von den Nachbarinselnstammen.43,7Vcder identifizierten Drohnenstammennicht von den Drohnenvölkernder benachbartenInseln. Sie kamen am wahrscheinlichsten vom Festland. das am weitestenüber offenesWasser entf'erntlag (Tubelle 1). Es kann nicht a u s g e s c h l o s s ewne r d e n ,d a B 5 0 , 7 7 o d e r Drohnen von Langeoogstammen.Diese Drohnen zeigtenjedoch signifikant unterschiedlicheAllelfrequenzen zu den Drohnen,mit denensichdie LangeoogKöniginnen gepaart haben (Tubelle V'). Yrer D r o h n e nw i e s e na u f b e i d e nB e l e g s t e l l e n vorkommendeAllele auf und konntenvon keiner Herkunft ausgeschlossen werden. Signifikantunterschiedliche Alleltiequenzen (TobelleI zeigtendie DrohnenPopulationenvon Langeoogund Norderneyund die Patrilinienvon Baltrum.Diesdeutetdaraufhin, daB PaarungenzwischenDrohnen v o n L a n g e o o go d e r N o r d e r n e yu n d d e n Königinnenvon Baltrumunwahrscheinlich sind.Eine hohe senetische Distanzwurde
275
zwischenden Drohnenvon Norderneyund Baltrum gefunden(Tabelle V1),was ebent'allszeigt,daBPaarungen mit Drohnenvon Norderneyam unwahrscheinlichsten sind, obwohl die Entfemung über off'enesWasser am geringstenist. DieseErgebnissedeuten daraufhin,daB eine Kombination aus Entfernungenüber Wasserund über Land zu den nächstenDrohnenvölkernfür dasPaarungsverhalten der BaltrumKöniginnenvon Bedeutungist (Tabelle1). Ob sich Königinnen unter regulärenBedingungenauf eineretabliertenBelegstelleBaltrum,d.h. mit einer ausreichenden Anzahl von Drohnenvölkern.auchmit unselektiertenDrohnen paaren,bleibt off-en,da dasAufstellen unbegatteterKöniginnen ohne Drohnenvölkernicht tür die Einschätzung der Zrxer1ässigkeiteiner Belegstelleunter Routinebedingungengeeignetist. UnsereErgebnisse demonstrierenjedoch eindeutig,dap auf Baltrum kontrollierlePaarungennicht garantiert werden können.EntwederDrohnen oder Königinnen oder beide Geschlechter sind in der Lage, bei ihren Paarungsflügen gröpereStreckenofTenenWasserszu überq u e r e nW . i r e m p f e h l e tnl u h e re i n eÜ b e r prüfungder Sicherheitvon Inselbelegstellen, die wenigerals 8 km vom Festlandentfernt liegen.O lnra/DIB/AGIB/Elsevier,Paris Apismelkfera / Insel / DNA-Microsatelliten / Paarungskontrolle / Polyandrie
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