Lisinlr the combination ol the tr,l'o. ','anaDle ioci iiescrihred hcre and the (]-iocus. :1. : .i I r'"c ale able to ciiscrirlinatc severai patrilines ol tlrc honelibc-c colonv.
Apidologie29 ( 1998)255 263 O lnra/DlB/AGIB/Elscvier. Paris
25-5
Original article
A simple,non-radioactiveDNA fingerprinting method for identifying patrilines in honeybeecolonies Martin Beyea*,PeterNeumanna, JanaSchmitzoväb, JaroslavKlaudinyb,St"funAlbertb,JozefSim.itht, MariusFelderc.Robin F.A. Moritza " l n s t i t u t l ' ü r M t r l e k u l l r e ( ) k o l r r - g i e .M u r t i n L u t h c r - U n i v e r s i t i i t H i r l l e . K r i i l l w i t z c r S t r . 4 , 1 .0 6 0 9 9 H a l l e . G e r r n a n v l ' l . a b o r a t c t r ro, f C e n e t i c E n g i n e e r i n - e .l u . s t i t L r t eo f C h e m i s t r y . S l o v a k A c a c l c r l y o f S c i c n c c s . D i r b r a v s k a c e s t a 9 . S K 8 , 1 23 8 B r a t i s l a v ä . S l o v a k i a ' L B G e n e t i k . U n i v c r s i t a c t K a i s e r s l a u t e r n .P o s t f a c h 3 0 . + 9 .D - 6 7 6 - 5 3K a i s c r s l a u t c n r .G e r m a n l
(Rcccivecl 6 October-1997:acceptecl I I Decernber1997)
Abstract Prinrcrswere derivedflanking a rniclosatcllite motil'of the clonedZ-locus.The PCR productof the Z-locLrswas variablcin sizeand up to four alleleswerefbund in a sarnple ol l I wurkerswithin onc colony. Using the corlbinationof thlee loci. the Z. the Q (both linkeclto thc sex locus)and a rcyal.jcllvproteingene(RJP57-l)we were ableto discrinrinate fivc patlilinesin the 1l worker sample.Using the '"vcllcstablishcd rniclosatcllite tcchuolo-rr1. howcvet.sevenand six patrilincscould be identitied.The techniquemay cnablclabolatories which iack an isotopefacilitl,and ccluippedwith onlv a PCIRthcr-rnocvclel and agarosegel apparatusto studv thc polvanclrous mating s1'stcmofthc honer'tree in rr rarietv o1'diflerent contexts.O Inra/DIB/AGlIl/Elsevier. Paris fingerprinting / patriline / rnating / honel'beeI Apis melliJbraI l?CR
I. IN'I'RODUCTION
T h c r r u l t i p l en r a t i n cs v s t L - nr er s u i t si n i i gcncticsubtarnilrstrurciure of r honevbee arr-siredhv thesanre T h c n u n r b e ro f m a t i n g so f h o n e v b e e colonr':super-sislers quecnsciln \rArvclranraticall,r l r , . l r ef a t h e r .i v h i l e h a l f - s i s t e r : tiorn six up h a p i o i cC 'fhis t o 2 8 i n n a t u l apl o p u l a t i o ni is0 " i 6 . i 8 l . irai,cilillerenfdronclitther:. llruscr.,
256
Martin Beye et al
largeintracolonialgeneticvariance,which is of substantialinterestfor both evolutionaryandbehavioralgenetics16,11,201. In recentyearsa wide rangeof different DNA techniqueshave beenestablished for honeybeesto identify the subfamily structureof coloniessuchas multilocus DNA fingerprinting[5, 15] and RAPD (randomamplified polymorphic) marker technologyI I , l2]. More recentlymicrosatellitetechnologyhas beendeveloped for honeybeesand provedto be a powerful tool for exactly determiningthe number of patrilinesand the intracolonialrelatedness[0]. However,this techniqueis time consumingandrequiressophisticated equipmentsuch as a radioactiveisotope laboratoryand largepolyacrylamidegels. Here we presenta very simpleand fast methodfor identifying patrilinesof the honeybee,which doesnot require radionucleotidelabelling.We usedsequence specificprimersof the Q-locus[13], the Z- locus [3, 4l and the locusof royaljelly proteinRJP57-l !, 141,which produce distinct and highly variableDNA products in the PCR reaction.The obtainedDNA fragmentshavesufficientsizedifferences to easily be distinguishedin agaroseor smallpolyacrylamidegel electrophoresis.
2. MATERIALS AND METHODS 2.1.Bee samples workersweretakenfrom Adult honeybee theouterframesof two coloniesA andB (l I workersand7 droneseach)at theapiaryof the r i e n e n z u ct G h te r m a nt iyn B u y r i s c hLe. A .l - ü B J u n e1 9 9 5 .
2.2. DNA isolation fromsingleworDNA wasphenolextracted kersfbllowingroutineprotocols [2] withsome minorchanges: in insectringer l) workerswereincubated (121mM NaCl,1.5mM CaCl2, 5 mM solution
KCl, pH 7.4 with NaOH) fbr at room temperaturebeforeextraction; 2) worker thoraceswere homogenizedin 400 prl- of DNA extraction buff-er(100 mM N a C l , 1 0 0m M T r i s - H C l ( p H 8 . 0 ) , 1 0 m M NaCl.0. l7o SDS)I in 30 prl-ddH2O. 3) DNA was resuspended
2.3. Microsatellite fingerprinting W e u s e da s e t o f D N A - n - r i c r o s a t e l l i t e s which wasdevelopedby Estoupet al. [9]. Multiplex PCR was pertbrmedusing two pairs of loci (A43-B124,A16-A107)andthe standard protocolsofEstoupet al. [9, l0].
2.4. Non-radioactive fi ngerprinting: the Z, Q and RIP 57-1 locus The 1.7kb clonedZ-locus fragment[3, 4l was sequencedusing an automatedsequencer following the manufäctor'sinstructions.Primers(figure 1c) were designedwhich flanked an approximately 130 bp long microsatellite (TTTC)' motif in this fragment.The overall lengthof the resultingPCR productwas about 650 bp, which can be resolvedin an agarose gel. The primersfor PCR amplificationof the RJP 57-1 locusweredesignedfrom the cDNA of RJP 57-1 [1]. The primerswere sequence derived from region Rl (nucleotides1261 to 1 2 8 4 ) a n d R 2 ( n u c l e o t i d e s1 6 4 0 t o 1 6 6 6 ) (figure lb). The primers (figure lc) and the PCR conditionsfor the Q-locushavebeenpreviously describedI I 3]. PCR amplificationsof Z. Q and RJP57-| werecaniedout as described in tuble L
2.5. Electrophoresis and determination
of genotYPes
Non-radioactiveamplificationproducts were separatedon 3 7, agarosegels (Ultra Pure DNA grade,BioRad Laboratories)at 7 V/cm or on small 8 7o polyacrylamidegels for 2 h at 200 V with a 100 bp ladder as size standard. Each electrophoresiswas performedfollowing the routineprotocolsof Sambrooket al. [2 I l. Radioactiveamplification products were electrophorizedon 6 7opolyacrylamidesequen-
A simple,non-radioactiveDNA fingerprintingmethod
251
a. Z locus primers ZI
5'.AGCCGACTAATATAATTTC-3'
22
5'-GGAAAGAGGGTTATTATAC-3'
b. RJP 57-l locus primers RI
5'-TGTAGATGACTTAATGAGAAACAC-3'
R2
5'-ATGTAATTTTGAAGAATGATGAACTTG-3'
c. Q- locusprimers 5'-AGTC'CAGCCAGCTACTGAGAG-3' Ql Q2
5'-AGTGCAGCCACGTGCCTGAAT-3'
Figure l. Primersusedin the non-radioactivefingerprintingdesignedfrom sequences of the Z, RPJ57-land Q-locus.
Table I. PCR conditionsof the Z, Q and RJP57-I primers. MgCl, pflmer
Z primers 1 . 5m M 400nM
Q primers 1 . 5m M 400nM
RJP57-lprimers 2.5mM 500nM
numberof cycles step I step2 step3 step4 step5
3 min 94'C 30s 94'C 45s49"C 1 min 72 "C l0 min72"C
41 3 min 94 "C I min94'C I min 55 'C 2 mrn72 "C l0 min72 "C
30 3 min94 "C 3 0s 9 4 ' C 3 0s 5 4 ' C I m i n7 2 ' C l 0 m i n7 2 ' C
cing gels for 5.5 h (A76lA107)or 5 h (443/8124) togetherwith M l3mp18 control DNA sequencingreactionsas sizestandardon the samegel. Alleles were scoredas fragment lengthsin basepairs.
3. RESULTS AND DISCUSSION The PCR fragmentof the Z-locus [4] provedto be polymorphicevenin the progeny of a single queen(Jigure2a). Four alleleswere detectedin a sampleof I I
workers of colony B, three DNA length polymorphismrangingfrom 610 up to 670 'null 'null alleles' bp and a allele'.These occur if PCR productsare lacking at the testedlocustl, l9l. The proteinof the cDNA cloneRIP57-1 on its C-terminus, a 100 [1, 14]possesses a m i n oa c i dr e s i d u el o n gr e p e t i t i v ree g i o n consistingof a 2O-foldrepeatedsequence motif XQNXX. Amplification of this r e g i o n r e v e a l e dl e n g t hp o l y m o r p h i s m (Albert et a1.,unpublisheddata).Four and
2513
Martin Beycet al
A colony B
1521 18 3 19 r2M32 20 7 26 34 M130
il-
B colony A
drones M4
5
6 7 9 t 2 1 3 2 0 2 7 3 2 3 9M 3 0 1 5 1 7 2 8
Figure 2'a. Polvacrvlarnide tel showin-ttheZ-locusPCRproductof I I uolkersandtu'o drones of'colonl'ts.The Z locusPC-'R productshorveda hi-shvaliabilitvwithin onecolony:threelerrgth polvnlorphrsrn anclone 'null allele'(seetext).The fragrnent sizeof thc allcleswere:670.6.10.610 'null bp and allcle'.The allclelen,sthrvls deternrined in cornpalison to a 100bp DNA laddclM as a sizcstandard(GIBCO BRL. a 6-50bp bancl(above7(X)bp bancl)is inclicated b1-an arrowl. b . A g a r - o s e - us ch lo u ' i n u t h e R J P 5l kTr c u s P C R p r o d u c t olfl u t x ' k e l s a n d f i r L r r c l r o n c s o l ' c o k r n y A. Thc RJP-57l locusPCRproductis hi-uhl1, r'ariableu'ithinonccokrnvancllbur dif'fbrent allclcs 5 .1 0 .+ ( 1 5h p r e , r u l dh e i t l e r t t i l i c dT.h e r L l l c l lee t t . r : u r - l ( r { -) . l . 1 + t hr r . J c t t ' r ' r r r i nier d 1ü , r n r p i r l i \ ot n {, a l(X) bp DNA laddelNl as a sizc standard(GIBCO BRL. the 600 bp bandis inclicatecl by an arrow).
two allclcswerc found in the I I worker p r o r c n i e so f ' c o l o n v A ( . t ' ' i g u r2eä ) a n c l colonyB. respectir'ely. rangingfiom -10-5 up to 460 bp in size.
patrilinesol tlrc honelibc-ccolonv. Aithough Q and Z are two iinkeclrnarkcrs(both linked to the ser locusl. this clclesnot necessariiv intluencc iite ,r,letection of pan'ilines. The characteristic corlbination of aileiesat L i s i n l rt h e c o m b i n a t i o no l t h e t r , l ' o irll three threc ioci iQ. ü.. RJP.i7-i . tthie ![ t ','anaDle ioci iiescrihred hcreandthe(]-iocus r.vasusedio deten"nineihc nurnbelol'tätfi:1. : .i I r'"cale ableto ciiscrirlinatc severai lines. Wc conrDüreci thc por"er oi' ihis tcch-
A simple,non-radioactiveDNA fingerprintingmethod
nique to the numberof patrilinesdetermined by four informative loci microsatellitefingerprinting [0] using fbur informativeloci (476, A107, Bl24 and 443'-tablesII and IItt.
Five patrilinesweredetectedin colony A using the non-radioactivefingerprint technique,but sevenpatrilineswere identified using the microsatellitetechnique (tobleIIa). Using the informationof both
Table II. Informativegenotypesof the threeloci Z Q RJP57-I of colony A and B. The genotype of the queenwas determinedby the haploid drone offspring. Only informative bands('father alleles') different to thoseof the queenare listed in the worker progeny.Some 'father alleles' cannot be unambiguouslydeterminedas they eitherbe identicalto one or two ofthe queen'salleles or representa 'null allele'. The resultingpatrilines(a-e) were comparedto the microsatellite technology(= ms technology)( I 7). Combining both data setsan overall numberof nine patrilineswas identified(I IX). (nd = missingvalue). (bp) Genotype (bp) Genotype (bp) Patrilines Patrilines Sample Genotype by Overallnumber RJP57-llocus Z-locus mstechnologyofpatrilines Q-locus a. Colony A Queengenotype4051460
Worker t) 7 27 12 l-)
u
435 420
100t0
nd 600 600
560/0 700 700 100 nd
4 600 20 -)z
39
700 435 420
600 600
I I 2 II 2 II III 3 IV 3 4 V 4 VI a 5 VII b 6 VIII IX c 1 ines 9 patrilines 5 patrilines 7 patril a a a b c d
I I
b. Colony B
genotype 4051460 Queen Worker 3 2l l5 l8 26 32 l2 l9 34 7 20
700/700
6t0/0
680 680 nd
640 640 670 670
a a
l l
b b
2 2
C
J
670 670
b d
3 1
e
4
600 600
610 610 610
b 5 b 5 b 6 5 patrilines6 patrilines
I I II II III IV v VI VII VII VIII 8 patrilines
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A simple,non-radioactiveDNA fingerprintingmethod
techniques, nine patrilineswereidentified. In colony B the resolutionwas similar: five patrilines were detectedusing the s i n g l el o c u sd a t ao f Q , Z a n d R J P 5 7 I- , whereassix patrilineswere found in the microsatellitefingerprinting (table I I b). The resultingoverallnumberof patrilines usingthe informationof all loci waseight. The numberof patrilinesdetermined by the non-radioactive fingerprintingwas less than the number of patrilines found in microsatellitefingerprinting.The averagenumberof allelesper locuswas 3.3 (colony A) and 3 (colony B) in the nonradioactivefingerprintingwhile the average allele numberin the microsatellite f i n g e r p r i n t i n gw a s s l i g h t l y h i g h e r :4 . 7 allelesper locus in colony A and 3.7 in colony B. The highernumberof patrilines found in the microsatellitefingerprinting could be explainedby the higher number of allelesdetectedper locus and by using a 4th locus in the microsatellitefingerprinting approach.A higherresolutionof patrilineswas obtainedusingthe combined dataof both techniques.
tionalhighlyvariableloci (e.g.STSprimers(sequence-tagged site)derivedfrom RAPDmarkers) becomeavailable. ACKNOWLEDGMENT This study was financially supportedby the DFC and the ScientificGrant Agency of Ministry of Educationof Slovak Republic and the SlovakAcademyof Sciences(2/1098/95)and the BMWB. We thank RosemarieHoffmann for technicalassistanceand Dieter Mautz for providing bee samples.
R6sum6 - Une m6thode d'analyse simple et non radioactive d'empreinte g6n6tique pour identifier les lign6es paternelles des colonies d'abeilles. La techniquedesmicrosatellites a 6t6 d6velopp6echez I'abeille(Apis mellferaL.) p o u r d d t e r m i n e rl e n o m b r e e x a c t d e lign6espaternelleset le degr6de parent6 entremembresde la mömecolonie.Nous a v o n sm i s a u p o i n t u n e t e c h n i q u eq u i , contrairementä celle desmicrosatellites, In many cases,the resolutionof the ne n6cessite pasI'utilisationde radioisonon-radioactivetechniquemight be suf- topeset avonscompar6les deux m6thodes ficient to resolvequestionsrelatedto the q u a n t ä l e u r p r 6 c i s i o n . D e s a m o r c e s matingfrequency.The procedurefor deter- (figure I a) ont 6t6 d6velopp6esä partir de mining patrilinesis a relativelysimple, r d g i o n ss i t u 6 e sd e p a r t e t d ' a u t r ed ' u n fast and cheapmethod comparedto the motif microsatellitedu locus Z et on a more labour-intensiveand complextech- trouv6par PCRjusqu'ä quatreallölesdifnique of microsatellitetechnologyusing fdrentsdansun 6chantillonde 11 ouvriöres radioactiveor fluorescentlabelledDNA provenantde la colonieB \[igure 2ul. A fragmentsand large sequencinggels. In l ' a i d e d ' u n f r a g m e n tR J P 5 7 - 1q, u i r e n our laboratorywe found that the analysis ferme un motif codantpour une prot6ine of the PCR fragmentswith just agarose de gel6eroyale,la PCR a permis de mettre g e l s w a s a b o u t t w i c e a s f a s t a s l a r g e en dvidencejusqu'ä quatreallölesdiff6PAGEs (which did not include the expo- rentsdansun 6chantillonde I I ouvriöres suretime of films) and reducesthe coststo de la colonieA (figure2b).En combinant two thirds.This techniquemay alsoenable les trois locusZ, Q et RJP57-I (tableautS, laboratories withjust an agarosegel appa- cinq lign6espatemellesont 6t6 misesen ratusand a PCR thermocyclerto study the 6videncedansla colonieA et cinq dans polyandrousmating systemof the honey- la colonie B (tableau 11,6chantillonsde beein a varietyof differentcontexts.This 11 individuspar colonie).Parla technique non-radioactivetechniquemay become d e s m i c r o s a t e l l i t e s( t a b l e a u I I I ) , o n a even more powerful, if primers of addi- trouv6un nombrede pöresl6görementplus
262
Martin Beye et al.
6lev6,'.7et 6 respectivement (tableau II). Cette techniquepourraitpermettreaux laboratoiresde petite taille, 6quip6sseulementd'un thermocycleurPCR et d'une cuve d'dlectrophorösepour gel d'agarose, d'6tudier le systömede reproductionpolyandrede l'abeille. O Inra/DIB/AGIB/Elsevier, Paris Apis mellifura / empreinte g6n6tique / lignde paternelle / PCR / accouplement
Zusammenfassung - Eine einfache, nicht-radioaktive DNA Fingerprintmethode zur Bestimmung der Patrilinienanzahl in Bienenvölkern. Es wurden Primer (Abb. 1a\ aus flankierenden BereicheneinesMicrosatellitenmotivsdes Z-Locus entwickelt und etabliert.Bis zu 4 Allele des Z-Fragmentswurden in einer Stichprobe von I I Arbeiterinnen einer Kolonie in der PCR (Polymerasen-Ketten-Reaktion)gefunden (Abb. 2a). Mit Hilfe des RJP57-l Fragments,das ein Motiv einesGel6e-RoyalProteinsenthält, konntenbis zu 4 Allele in der Stichprobe nachgewiesenwerden (Abb. 2b). Durch die Kombination der 3 Loci, Z, Q und RIP57-1) (Tabelle0 konnten 5 Patrilinien in einer Stichprobevon I I Individuen nachgewiesenwerden (Tabelle 1I). Die Anzahl, der durch die MikrosatellitenTechnologie(Tabelle 1fl nachgewiesenen Väter,war mit 6 und 7 (TabelleID in der gewähltenStichprobenur unwesentlich höher. Diese Technik könnte selbst kleinerenLaboratorienermöglichen,die nur mit einem PCR-Thermocyclerund einer Agarose-Gel-Kammerausgestattet sind, das polyandrischePaarungssystem der Honigbienezu studieren.O Inra/DIB/AGIB/Elsevier.Paris
Honigbiene / Begattung I Patrilinie / Fingerprint / PCR
REFERENCES tll
Alberr S., Klaudiny L., Simüth J., Newly dis, covered featuresof the updatedsequenceof royal jelly protein RJP57-1,longer repetitive region on C-terminus and homolgy Io Drosophila melanogaslar yellow protein, J. Apic. R e s . 3 5( 1 9 9 6 )6 3 - 6 8 .
[2]
Beye M., RaederU., Rapid DNA preparation from beesand % GC fractionation, Bio. Techniques 14 (1993) 372-311.
[3]
B e y e M . , E p p l e nC . , M o r i t z R . F . A . ,S e x l i n kage in the honey bee Apis melliferaL. detected by multilocusDNA fingerprinting,Natur, wissenschaften8l (1994) 460462.
t4l
Beye M., Crozier R.H., Crozier Y.C., Moritz R.F.A., Mapping the sex locus of the honey bee (Apis melliftra), Naturwissenschafren83 0996)424426.
[5]
BlanchetotA., Genetic relatednessin honeybees as establishedby DNA fingerprinting, J . H e r e d .8 2 ( 1 9 9 1 )3 9 1 - 3 9 6 .
[6]
B r e e d M . D . , B e n n e t8 . , K i n r e c o g n i t i o ni n h i g h l y e u s o c i a li n s e c t s ,i n : F l e t c h e rD . J . C . , MichenerC.D. (Eds.),Kin Recognitionin Animals, Wiley, Chichester,1987,pp. 243-285.
[7]
Callen D.E., Thampson A.D., Shen Y., Philips H.A., RichardsR.I-, Mulley J.C., SutherlandG.R.,Incidenceand origin of'null' alleles in the (AC)n microsatellitemarkers, Am. J. Hum. Genet.52 (1993)922-927.
t8l
Dreller C., Fondrk M.K., PageR.E., Genetic variability affects thc behavior in a feral honeybee colony, Naturwissenschaften82 (1995) 243245.
[9]
Estoup A., Solignac M., Harry M., Cornuet J.M., Characterisationof (GT)n and (CT)n microsatellitesin two insect species'.Apis melliftra and Bombus terrestris, Nucleic Acids R e s .2 l ( 1 9 9 3 )1 4 2 1 - 1 4 3 1 .
[0]
Estoup A., Solignac M., Cornuet J.-M., Precise assessment of the numbcr of oartilines and gencticrelatedness in honeyheecJlonies.Proc. R. Soc.Lond. B 258 (1994) lJ .
I l] Fondrk M.K., PageR.E., Hunt G.J.,Parernity analysisof worker honeybeesusing random amplified polymorphic DNA, Naturwissenschaften80 ( I 993) 226,231. [2]
Hunt G.J., Page R.E., Patternsof inheritance with RAPD molecular markersreveal novel typesof polymorphismin the honcy bee,Theor. A p p l . G c n e t . 8 5( 1 9 9 3 )l 5 2 0 .
! 31 Hunt G.J.,PageR.E., Linkage analysisof sex detcrmination in the honey bec (Apis mellif e r u L . ) , M o l . G e n .G e n c t . 2 4 .({1 9 9 4 )5 1 2 - 5 l f l .
A simple,non-radioactive DNA fingerprintingmethod
I l . l l K l a u d i n yJ . .H a n e sJ . , K u l i l a j o v äJ . . A l b e r tS . . S i m i r t hJ . . M o l e c u l a rc l o n i n g o f t w o c D N A s tiom hcad of the nursehoneybee(Api.smelli I e r u L . ) c o d i n g l i r r r c l a t c dp r o t e i n so f r o y a l j e l l y .J . A p i c .R c s .3 3 ( 1 9 9 4 )1 0 - 5l 1 l . l l . 5 l M o r i t z R . F . A . ,M c u s c l M . S . . H a b e r lM . . O l i gonucleotideDNA fi ngerprintingdiscrirninatcs s u p e ra n d h a l i - s i s t e r si n h o n c y b c cc o l o n i e s \ A p i , sm e l l i l e r u L . ) . N a t u r r i ' i s s e n s c h a l i e7n8 ,l991) 1221.124. ! 6 1 M o r i t z R . F . A . .K r y g c r P . . K o c n i g e rG . . K o e n i g e rN . . E s t o u pA . . T i n g e kS . .H i g h d e g r e eo l ' ptrlyandrf in Apis dorxtttt queensdetectcdby n r i c r o s a t e l l i ti e , a r i a b i l i t vB. c h a r ' .E c o l . S o c i o b i o l .3 7 ( 1 9 9 5 )3 5 7 - 3 6 3 . I l 7 I M o r i t zR . F . A . .K r y g c rP . .A l l s o p pM . H . . C o m petitionlin royaltyin bees.Nature38'1( I 996)3 l.
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l l S l N c u m a n nP . . T h c g e n o t y p i cc o m p o s i t i o no f h o n e y b e ec o l o n i e s .P h . D . t h e s i s . M . artinL u t h e r U n i v e r s i t äH r a l l e .H a l l e .1 9 9 8 . | 9 1 O l d r o y dB . P . ,S m o l e n s kAi . J . ,C o r n u e tJ . M . . W o n g s i r i S . , E s t o u pA . . R i n d e r e rT . E . . C r o zier R.H..Levelsof polyandryand intracolonial gcnctic rclationshipsin Apls dorsutu (Htnte nrrytent:Apidue).Ann. Entrmol.Sm. Arner.89 ( |996)276-283. l 2 0 l R o b i n s o nG . E . .P a g cR . E . .G c n c t i cd e t c n n i n a tion of guardingand undertakingin honeybee c o l o n i e sN . a t u r e3 3 3 ( | 9 8 u ) 3 5 6 - 3 5 8 . [ 2 1] S a m b r o o kJ . . F r i t s c hE . F . .M a n i a t i sT . . M o l e cular Cloning - A LaboratoryHandbook.2nd e d . . C l o l d .S p r i n gH a r b o u rl . a b o r a t o r vP r c s s . Ncw York. l9ti9.
