Pertanika 13(2), 189·194 (1990)

Identification of Nasonov Pheromones and the Effectsof Synthetic Pheromones on the Clustering Activity

of the Asiatic Honeybee (APis Cerana)

NORHANI ABDUlLAH, ASIAH HAMZAH,JOHAR! RAMLI and MAKHDZIR MARDANI

Depmtment ofBiochemist?y and Microbiology1Department ofPlant ProtectionUniversiti PC11anian Malaysia

43400 UPM Serdang, Selangur Dm,,1 Ehsan, Mala)'sia

Key words: honey bees, A. cerana, Nasonov pheromones, clustering activity.

ABSTRAK

Lebah maduApis cerana jenis pek"'ja telah diambil daripada beberapa koloni untuk kajian pencilian rembesanNasonov dan aktiviti perkelompokan ),ang diuji dengan beberapa jenis komponen feromon perkelompokansintetik. Ekslraksi umbesan kelenjar Nasonov dilakukan dengan tiga kaedah iaitu - kaedah pencelupanabdomen, kaedah pengasingan kelenjar dan kaedah ekstraksi menggunakan pecagari. Analisis feromondilakukan dengan Kromatografi Cecair Gas. KompOlum-komponen Nasonov ),ang dikesan ialah (E) -sitml,(Z)-sitral dan geraniol. Ragaimanajlun han)'a (E) -sitml dan geraniol dapat dinilai kepekatann),a. Kaedahpencelupan abdomen mengesan 20.0 ug (E)-sitml per lebah manakala kaedah pengasingan helenjar han)'amengesan 3.8 ug per lebah. Geraniol pula han)'a dapat dikesan dengan kaedah pengasingan kelenjar denganparas 2.5 ug per lebah. (Z)-sitml han)'a dapat dikesan, tetapi kepekatann),a tersangat rendah untuk di­anggarkan. Kajian perkelompokan pula dijalankan dalam sebuah hilik tertutup pada sulm bilik. Komponensintetik seperti asid 9-okso-2-desenoik (90DA), campumn (E), (Z)-sitml, geraniol, nerol dan famesol diujiseear"a berasingan aiau seea-ra berkombinasi. Setiap komponen atau kombinasi campuran dijerap pada kertasturas dan digantung di dalam silindlff bert~buk )'ang dipasang lampu di dalamn)'a. Lima silindlff digunakanuntuk setiap ujian. Di antam komponen sintetik ),angdiuji, campumn (E), (Z) -sitml didapati palingberkesanuntuk menmik lebah pek"ja supa),a membentuk kelompo".

ABSTRACT

Worker bees from several Apis cerana colonies were sampled for identification ofNasonov secretions and theirclustering activity in 1°esponse to different synthetic components of the swarming pheromones. Extraction ofNasonov gland secretion was canied (mt try three methods - Abdomen Dippingmethod, Excised Gland method andS)'ringe Extmction method - and its anal),sis was canied out b), Gas Liquid Chromatograph),. Nasonovcomponents delected were (E)-citml, (Z)-citml and g~ranioL However, onl), (E)-citml and geraniol werequantified. The Abdoment Dipping method detected 20.0 ug per bee oj (E) -citral, whereas the Excised Glandmethod detected onl), 3.8 ug. Geraniol was on I)' detected try the latter technique at 2.5 ng Plff bee. (Z) -citral wasdetected in t·race amounts. The clustering experiments were conducted in a confined darkened room at roomtemperat1m. Chemical components, consisting of 9-oxo-2-decenoic acid designated as 9ODA, (E), (Z)-citral,geraniol, nerol andfarnesol, were tested either singly or in vmious combinations. Each component or mixtures ofthese componenls were adsorbed on afilterpaperplaced in alighted peifomted ca,.dboard cannister. Five cannislenwere used in each tlial. Among the synthetic compon""ts tested, the (E), (Z) -citral was[oltnd to be most effectivein attracting w01*er bees to fonn a clustero

NORHA~I ABDULLO\H, ASIAH HAMZAH, JOHARI RAMLI AND wlAKHDZIR i\V\RDAN

INTRODUCTION

The Nasonov gland, situated between the ter­gites of the 6th and 7th abdominal segmentsof the honeybee, secretes Nasonov phe­romones. In the Nasonov gland of thehoneybee Apis mellifera, the Nasonovpheromone components were identified as (E)and (Z)-citral, neroI, geraniol, nerolic acid,geranic acid and (E,E)-farnesol, In1: 1:1:100:75: 12:50 proportions, respectively(Free et al. 1981). It was found that the Nasonovpheromone secretions of A. mellifera elicitedresponses such as clustering activity duringswarming and foraging at artifical feeding ofsugar syrup (Morse and Boch 1971; Mautz etal. 1972; Free and Williams 1970). It was alsoshown by Free et al. (1983) that Nasonovpheromone lures, singly, in concert or with thequeen substance (9-oxo-2-decenoic acid,designated as 90DA) could result in clusteringactivities.

AlJis arana, a common spesies in the Indo­Malayan region, has not been studied in termsof the components of the Nasonov glandsecretions and their clustering or swarmingeffects. This study was intended to identify thenatural components of Nasonov gland sec­retions and the effects of synthetic chemicalsconsisting of 9-oxo-2-decenoic acid, (£'), (2)­citral, geraniol, nerol and farnesol on theswarming activity of A. cerana.

MATERI.A.LS AND METHODS

Chemical components used to reseluble natu­rally occurring pheromonas were pure (E), (2)­citral (Koch Light), 95% farnesol (mixture ofisomers, Aldrich), 95% geraniol (Sigma), 95%nerol (Tokyo Kasei) and 90DA (from JA.Pickett). These chemical components were usedwithout further purification.

Capturing of Worker BeesAbout 700-1000 bees were captured from anumber of wild bee colonies in the vicinity ofcoconut holdings at Universiti PertanianMalaysia. Bees from one colony were used forone trial. They were captured directly from thehives and placed in a box (40x40x40 em) filledwith a wire gauze for ventilation. Allexperiments on clustering behaviour were done

after the captured bees were settled and calmedinside the box for approximately two hours.Some worker bees ,vere kept aside for theextraction of the Nasonov pheromones. Theextraction of gland secretions was catTied outon the same day.

Analyses of Nasonov Gland Secretions1. Extraction of Nasonov Gland SecretionThe following methods were employed forextraction of secretions of the Nasonov glandbetween the 6th and 7th abdominal tergites ofthe honey bee.

a. Syringe Extraction MethodTo obtain a sample of the Nasonov gland, thelive bee (immobilized by placing it in the coldat 4°C for 3-5 min) was held between theforefinger and thumb with the Nasonov grooveexposed. The tip of the needle of a 10 III syringecontaining 2 ~l hexane was placed between the6th and 7th dorsal abdominal tergites and thehexane was expelled from the syringe onto theNasonov gland groove. The hexane expelledwas immediately withdrawn back into thesyringe. About 0.4 ~l of hexane solution wasrecovered and analysed by gas liquidchromatography (GC).

b. Abdomen Dipping MethodWhen the A. cerana workers were captured andreleased in the experimental room, it wasobserved that many of thein raised theirabdomen and exposed their Nasonov glandsand fanned them with their wings. Such workerswere caught by the abdomen using a forcepand the lower part that contained the Nasonovgland was shaken in 0.5 ml hexane. Ten beeswere used for each 0.5 ml hexane and 1.5 ~l

was directly analysed by CC.

c. Dispersing Excised Gland MethodFor determinig the absolute amounts ofpheromones present in the Nasonov gland, thebees were killed and the 6th and 7th tergitesof the abdomen containing the Nasonov glandwas dissected and placed in 0.5 ml hexane. Tenglands were isolated and shaken in 1 mlhexane, and 1.5 ~l solution was immediatelyanalysed by CC.

190 PERTANIKA VOL 13 NO.2, 1990

IDENTIFICATION OF NASONOV PHERO"ONES AND THE EFFECTS OF SYNTHETIC PHEROMONES

2. Identification oj Pheromone 17)' GasChwmatography

Nasonov pheromones of A. cerana were analysedby Gas Chromatograph (HP 5840 fitted withan FID detector). Two prepacked glass columns(SupeJco, 2.5% Carbowax 20M on ChromosorbG-HP 80-100 mesh, 6' x 1/4") were used forlhe analyses with nitrogen as the carrier gaswith a flow rate of 20 rnl/rnin. The oventClnperatures were programmed to changefrom 50 to 200°C at IO°C/min. A standardmixture of 8 mg (E), (Z)-eitral, 4 mg nerol, 4mg geraniol and 4 mg farnesol per m) hexanewas used as the reference.

Effects oj Synthetic Pheromones on the Clustering• Activity oj A. cerana

Clustcling response to the synthetic pheromonecomponents were conducted in an experi­mental room measuring 3.3x2.7x3.1 m. Theroom was darkened by plastering black paperover the glass windows as preliminaryexperiments showed that bees were attractedto the blight daylight. Cardboard containersin the form of cannisters measuring 24 em inlenght and 8 em in diameter, perforated withholes (3 mm wide and 1.5 em apart) were usedto hold the synthetic pheromone components.They were hung in a straight line on a wire atabout 50 em apart. The cannisters were lightedwith 5 watt bulbs (which was placed in eachcannister) while the rest of the room was indarkness. This was necessary because in earlierexperimenLS it was observed that bees wouldconverge for light at the glass windows. Thelighted cannisters would attract bees equallyand any differences would be the results of thevarious chemicals used. The lighted cannisterswould also facilitate observation of the bees inthe darkroom. The temperature and relativehumidity of the room was about 28°C and 60%,respectively. The posilions of each cannistercontaining synthetic components of theNasol1ov pheromone was randomly changed forevery experiment and its replicate. The samecannisters were used for all trials after a weekof aeration.

Each pheromone was tested singly or inconcert and the amount used for studying theclustering activity was 60 l1g, 30 l1g, 1.5 mg, 3

mg and 1.5 mg for (E), (Z)-citral, nerol, farnesol,geraniol and 90DA, respectively. These levelswere 15 times higher than those used byFerguson et al. (l979) in their study on thec1usteling activity of A. melliJera. Higher quantitywas necessary since preliminary experimentsshowed that lower levels were not effective withA. cerana.

The synthetic pherOlnone componentsdissolved in hexane was adsorbed onto. filterpaper (Whatman No.1) and placed in eachcannister. The cannisters were then covered bywrapping a piece of paper around them. Thecovered cannisters were then hung in the room.Mter 30 min the cover over each cannister "\idS

removed and 700-1000 worker bees wereimmediately released into the room. Onecannister was always left untreated (control)for each experiment.

Each experiment was repeated at least twiceon different days using a fresh batch of workerbees from a different colony. The number ofbees which had clustered on each cannisterwas counted at 5 min intervals for I hour.

RESULTS AND DISCUSSION

Detection oj PheromonesTable I shows the Nasonov pheromonesdetected and their concentrations accordingto the methods used to extract them from theworker bees. Gas chromatograph peaks cor­responding to (E)-ei tral, (Z)-eitral and geraniolwere detected from samples obtained by theSyringe Extraction procedure. Other extractionprocedures showed the presence of only (E)­citra! and geraniol. For (E)-eitral, the amountdetected in the glands by the AbdomenDipping method gave a higher value (20 l1gper bee) than the Excised Gland method (3.8ug per bee). The amount of geraniol detectedby the Excised Gland method was 2.5 l1g perbee. Pickett et al. (1980), using the 'DispersingExcised Gland' method, reported that A.mellifem excreted 0.02 and 1.80 l1g per bee of(E)-eitral and geraniol, respectively. In thepresent study. the other components of

asonov gland produced by A. mellifera (Freeet at. 1981) were not detected in A. cerana. Thefailure to detect their presence could possiblybe due to their minute amounts and also to

PERTANlKA VOl.. 13 NO.2, 1990 191

NORHANI ABDULLAH, ASIAH HA~'IZAH,JOHARI RAM:U AND H'\KHDZIR l\lARDA.1~

TABLE IComponents of Nasonov gland secretions and their concentration

Extracting Methods Pheromonesdetected

Ilg per bee

A.cerana A. melliJema

Syl-ingc Extraction

Abdomen DippingDispersing ExcisedGland

(E)-citral(Z)-cilTalGeraniol(lIj-citral(lIj-citralGeraniol

not quantifiednot quantifiednot quantified

20.03.82.5

0.021.80

"Pickett el aL 1980.

60so10

oo

.2

20 30 40

TIME (minutes)

Fig. J: CLuJfeli11g activit)1 oj A. cerana worker bees oujJheromones tested singly.

the highest. However the cluster was not stableafter 40 min. The other Nasonov pheromonesviz, nerol, geraniol and famesol were noteffective when compared to (E), (Z)-citral.Geraniol and 9DDA were able to attract only afew number of bees.

When the cannisters were treated with90DA in combination with either geraniol,nerol, famesol or (E), (Z)-ciu<ll, bees were foundto be attracted to all these mixtures (Fig. 2).Again, the clusters did not seem to be stableafter 40 min. When 90DA was tested in concert,vith (E), (Z)-citral, geraniol, nerol and famesol,its presence was not essential (Fig.)), but thecluster on the cannister containing CGFN +

1

I.t NEROli CITRAlo 90DA

.8 o GERANIOLI CONTROL

.6%0

~~

0~ ."0~~

Clusle>ing Aclivity of A. ceranaThe amount of pheromones used was 15 xhigher than that used for A. mellifera (Fergusonet at. 1979) as lower concentrations were foundineffective.

Since the number of bees collected for eachtrial varied within the range of 700 to 1000,the number of bees attracted to each cannisterat each obselvation was normalized before thedata could be presented. This was done bygiving to the maximum number of beesattracted to a particular cannister for each triala value of one. All other counts of bees attractedto each ca.nnister in the trial were compared tothis maximum number and calculated as aproportion of the maximum. In this clusteringactivity study. up to 30% difference in responseto a particular treattnent was obseIVed betweenduplicates.

Fig. 1 shows the relative number of beesthat landed on the cannister containing either(E), (Z)-citral, geraniol, nerol or 90DA. The first15 min after their release could be consideredas an adapatation period where the bees justcrawled around and made short flights. Thebees then began to fly and clustered on thecannisters. After 30 min, the number of beeson the cannister containing (EJ, (Z)-citral was

the fact that they are only produced whenrequired (Pickett el al. 1981).

It would have been better if the t\'w honeybee species could be studied at the same time.However, this is not possible since A. AlIeUi/erais not available in this country.

192 PERTANIKA VOL. 13 NO.2, 1990

IDENTIFICnrOI" OF NASONOV PHEROMOI"ES AND THE EFFECTS OF SYNTHETIC PHEROMONES

60503020JOo~L,-~4--.,-J~;;:;;:t:"*'~W

oTIME (minutes)

h~. -I: nil' '1Jf'f"llilf'liI'.\j oj (1':). (Z)·titml in dllstn1ngactivit)' oJA. ccrana workrr Urss.

(20·60 lIg) of (E), (Z)-citral showed no effectsof citral concentrations on the number of beessettling on the cannisters.

The results indicate that (E), (Z)-cirral playsan important role in attracting bees, but theother components (nerol, geraniol, farnesoland 90DA) are also required for the cluster tobe more stable. With A. meufJem, Free et aL(1981) also observed the elTectiveness of citralin clustering activity when compared to geraniolor farnesol. 90DA was also not effective inclustering activity of A. cerana. It could bepossible that other factors are required forcluster formation as Ferguson et aL (1979) ob­served pure queen factor was rather ineffectivecompared to a crushed queen's head. Nerol,as similarly observed in A. melli/era (Free et at.1981), was also not effective.

The effectiveness of (E),(Zj-citral against acombination of 90DA, geraniol, nerol andfarnesol needs to be mentioned again as theresults seemed to be in contrast to the findingsof Ferguson et al. (1979) where they observeda combination of 90DA and the other com­ponents to be more effective than the individualcomponent. Since (E)-cilfal was detected insubstantial amounts, (up to 20 lIg/bee) it couldbe the key factor in initiating cluster formationin A. cemna. The fact that higher concentrationwas required for its activity was congruous toits high production in the gland. However, the

1

• 9ODA+C

• CGFND CGFN+90DA

.8 0 (ITRAt.I CONTR{l.

.6z0~

~

:;'0 .,~~

.2

60

60

50

50

3020

20 30 '0T1ME (mHlutesJ

10

10

o W~~'::::::!.=~-=t=l=o=t='o--f-~o

0!---1~~9=±-t--+--r---'-+--Ho

.2

6900A, CGFN

DCGFNt90DA,8 I CONTROL

90DA seemed to be more stable. However, (E),(Z).-eitral was observed to be more effectivewhen tested against this combination (Fig. 4),but as observed earlier the cluster was not stableafter 40 min. Experiments with different levels

JUI£ (minutes>

Fig. ): nit, ('lint oj C)UJJA UII ((Iof,'ling (uliul')' oj A.ccrana worker bees.

!'Ig. 2: (.lI/.\fl'lIlI~ 1111/1 111.\ II) .\. «'1;\1101 wOr/It'J'IJFf'.\ Oil

uml()us combhwliol1S oj ph('romOtles.Legend:90DA 9-oxo-2-derellOic acid

C (E), (Z)·cilmlN lIerolG geraniolF {anl('sol

("I ill l/lh""/lIl'//! !i.![I1/('\/

PERTANlKA VOL. 13 NO.2, 1990 193

NORHN'\[I ABDULLAH, ASIAH HAMZAH, JOHARl RAMLI AND ~IAKHDZIR ~IARD.,",\[

clustering activity was tested in a confined spaceand the results mayor may not be the sameunder field conditions.

ACKNO~DGEMENTS

The authors are grateful to J.A. Pickett fromthe Rothamsted Experimental Station forproviding the queen substance (90DA),International Development Research (IDRC)for the research grant and Universiti PertanianMalaysia for the facilities.

REFERENCESFERGUSON, A.W., JB. FREE, JA, PICKEIT and M.

WINDER. 1979. Techniques for Studying HoneyBee Pheromones Involved in Clustering, andExperiments on the Effect of Nasonov andQueen Pheromones. Physiol. Entomol. 4:339-344.

FREE,j.B., and LB. WILLL-\MS. 1970. Exposure uftheNasanov Gland by Honey Bees (A. mellifera)

Collecting Water. Behaviour 37:286--290.

Free, JB. AW. FERGUSON, and JA. PICKEIT. 1981.Evaluation of the Various Components of theNasonov Pheromones Used by Clustering Honey

Bees. Physiol. Enlomol. 6:263-268.

FREE, JB" AW. FERGUSON andJA. PICKETr. 1983. ASynthetic Pheromone Lure to Induce WorkerHoneybees to Consume Water and ArtificialForage. j. APic. Res, 22(4) :224-228.

MAUTZ, D., R. BaCH and R.A MORSE. 1972. QueenFinding by Swarming Honey Bees. Ann. Entomol.Soc. Am. 65:440-443.

MORSE, RA. and R. BaCH. 1971. Pheromone Concertin Swarming Honey Bees (Hymenoptera,Apidae). Ann. Entomol. Soc. Am. 64:1414-1417.

PICKEn, JA., I.H. WILLIAMS, A.P. MARTIN and M.e.S?>.IITH. (1980). Nasonov Pheromone of the Ho­ney Bee, A. mellifera L. (Hymenoptera, Apidae)Part 1. Chemical Characterization.). Chern. Eml.

6(2): 425-34.

PICKETI, J.A., LH. ·WILLIAMS, M.e. Si\HTH, and AP.MARTIN. (198]). Nasono\' Pheromone of theHoney Bee A. melli/era L. (Hymenoptera,Apidae) Part III. Regulation of PheromoneComposition and Production. J. Chern. Ecol.7(3):543-554.

(Received 23 March, 1989)

194 PERTANIK-\ VOL. l~ NO. 2. l~lO