Age-related patterns of volatile cephalic constituents in queens of the neotropical postica Latr (, ) E Engels, W Engels, G Lübke, W Schröder, W Francke

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E Engels, W Engels, G Lübke, W Schröder, W Francke. Age-related patterns of volatile cephalic constituents in queens of the neotropical stingless bee Scaptotrigona postica Latr (Hymenoptera, Apidae). Apidologie, Springer Verlag, 1993, 24 (6), pp.539-548. ￿hal-00891128￿

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Age-related patterns of volatile cephalic constituents in queens of the neotropical stingless bee Scaptotrigona postica Latr (Hymenoptera, Apidae)

E Engels W Engels G Lübke W Schröder W Francke

1 Zoologisches Institut der Universität, Auf der Morgenstelle 28, D -72076 Tübingen; 2 Institut für Organische Chemie und Biochemie der Universität, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany

(Received 18 February 1993; accepted 30 April 1993 )

Summary — In heads of queens of the neotropical stingless bee Scaptotrigona postica identifica- tion and quantification of 68 volatile compounds was carried out by individual GC/MS analysis. Hy- drocarbons, , esters, carboxylic acids, methylketones and lactones were found in distinct on- togenetic patterns. In all young queens secondary alcohols constitute 60-80% of the cephalic secretions. In receptive virgins 10-12 d after emergence the odour pattern contains = 7% methyl- ketones. Only in recently mated queens at the age of 12-14 d did the amount of unsaturated buty- rates reach = 20%. In old egg-laying queens carboxylic acids (> 80%) are the main components, while Z-9-tricosene forms the major component in the neutral fraction. The presumable functions of the queen pheromone in stingless bees have been discussed in relation to reproductive biology and colony cycle. stingless bee / reproduction / queen cephalic volatile / age-related variation / pheromone / Scaptotrigona postica

INTRODUCTION similar in composition independent of age (Free, 1987; De Hazan et al, 1989), in In the highly eusocial bees and stingless bees 2 largely different patterns stingless bees, the study of mating biology of volatile compounds exist in young and has determined conformity in many re- old queens (Engels et al, 1987). Evidently spects but also distinct differences as re- the odour of young queens induces lek gards certain details (Engels, 1988; Engels formation by males close to nest entranc- and Imperatriz-Fonseca, 1990). Whereas es where they may wait a long time for a in honey bees queen pheromones are virgin queen (Kerr et al, 1962; Engels and Engels, 1984). A young queen usually un- al cells containing queen pupae were collected dertakes several very short orientation from strong colonies and placed in mini-nuclei. of was checked loops before she leaves for a nuptial flight Emergence gynes daily. Young were introduced into small col- which lasts 10 min or less. virgins free-flying only Single onies. These nuclei were is since the mating inspected mating highly probable queen every 2nd d. Before the queens were dissected, returns with a which she her- mating sign their attractivity to drones was tested in the mat- self removes and which takes up much ing box bioassay (Engels and Engels, 1988). time and and ac- (Engels Engels, 1988), For individual GC/MS analysis samples were cording to sperm counts in vesicles taken from queens of known age and life histo- compared with the spermatheca (Kerr et ry. The head of a queen was removed and al, 1962). Based on this particular premat- immediately put into a small vial containing ing behavior, a mating box laboratory bio- = 200 μl pentane (Merck Uvasol). After concen- tration GC/MS of the extracts were assay was developed which showed Scap- analyses carried out via a HP 5890 to a VG 70/ to be most coupled totrigona postica gynes 250 SE (Ayasse et al, 1990, 1993; Engels et al, attractive to drones at an age of 10-14 1990). and particularly at 12 d (Engels and En- gels, 1988), corresponding well to the real mating age. Eighty-five volatile com- RESULTS pounds could be identified in the head of queen bees (Engels et al, 1990). Accord- to the differences found in and ing young Incipient nest development old queens, "copies" of the corresponding bouquets were prepared using synthetic compounds. In field bioassays, dummies The mating nuclei were equipped with ce- impregnated with these "artificial head" rumen and food stores, as found in an in- blends were exposed to drone aggrega- cipient filial nest. As there was no worker tions. The drones’ reaction clearly indicat- traffic for transportation of material from a ed that they could recognize the scent of a maternal colony, the nuclei had to be sup- young queen (Engels et al, 1990). This plied with fermented and syrup confirmed our assumption that in stingless every 2nd d. The workers usually built bees, as in honey bees, cephalic volatiles some pots and stored honey. The en- are important components of the female trance of a newly established colony con- sex pheromone. Whether ontogenetic pat- taining a young gyne was always closed terns in head volatiles might distinctively for 6-8 d by a bulb. The workers did not label a receptive virgin by a bouquet spe- forage during this period. Only a few d cific for the mating age was investigated in prior to the nuptial flight of the virgin was the present study. In addition, a brief re- the entrance slightly reopened. port on the development of incipient S After mating of the young queen, sever- postica colonies containing young queens al changes in worker behavior could be ob- in the premating, mating and postmating served. The nurse bees consumed increas- phase respectively has been presented. ing amounts of pollen and constructed a small brood nest covered with an involu- crum. 1 or 2 cells were built MATERIALS AND METHODS Initially only and provisioned. Older bees began to for- 3-4 d after the Colonies of S postica were kept in the Depart- age pollen. Usually nuptial ment of Genetics meliponary, USP Campus at flight the queen was seen for the first time Ribeirão Preto, . March through April, roy- on the small brood nest, and subsequently started ovipositioning. Although at this Age-related patterns of cephalic stage a young queen is already to some volatiles and queen attractivity extent physogastric she at first lays only 3- 6 but this rate eggs/d, increases rapidly. Young and old queens differ considerably Normally 4 or 5 d after the start of egg lay- in the constitution of their cephalic volatile ing, ≈ 50 operculated brood cells are bouquets (fig 1). The percentage of 68 vol- found. At 25 d of age the full physogastric atiles was determined in the individual pen- status of an intensely laying queen is at- tane extracts of heads of 13 queens via tained, and the brood nest consists of 1 quantitative GC. All identified compounds 000-2 000 cells. This stimulates the work- contributing to at least 0.01 % of the spec- ers to construct an entrance funnel and to trum were considered. These were 17 al- guard the nest. Forager traffic is then so cohols, 5 ketones, 29 hydrocarbons, 8 es- heavy that reinforcement of such colonies ters and 9 carboxylic acids (table I). once a wk becomes necessary, until the According to the proportions of the com- first young workers emerge after ≈ 50 d. pounds, the individual odour patterns could

easily be grouped into 4 classes corre- tric queens. The drones in the mating box, sponding to age groups. The queens however, copulated with one of the egg- belonging to these groups were sampled laying queens who had just been taken 0- 5, 10-12, 12-14 and 20-100 d after from a large colony. emergence respectively, representing the physiological status of premating gynes, receptive virgins, mated young queens Compound patterns correlated and egg laying, physogastric old queens with the queen’s physiological status (fig 2). This status was confirmed by the results of the mating box test. The premat- ing gynes were not attractive for the In all young queens collected < 15 d after drones. One of these solicited food from emergence, the secondary alcohols repre- the males in the mating box. Only the re- sented predominant volatiles in the cephal- ceptive virgins received immediate copula- ic extracts (fig 2). In 10-14-d-old queens, tion attempts from the drones. One was the 2-alkanols constituted 75% of the bou- made when she had just left the nest for quet. In contrast, in the old egg-laying the mating flight. The already mated young queens the amount of alkanols was re- queens were much less attractive, and the duced to < 5%. Instead carboxylic acids same was the case for the old physogas- were found to be the main compounds, av- eraging ≈ 80%. In newly emerged gynes, only during the brief mating period at ≈ 12 the carboxylic acid content was ≈ 30%. In d after emergence is a virgin highly attrac- the cephalic extracts of receptive and high- tive to the males (Engels and Engels, ly attractive virgins, a mean content of 7% 1988). However, already a few d prior to methyl ketones was calculated. In all other the short nuptial flight drones form a lek groups, the percentage of ketones was aggregation close to the colony (Kerr et al, much less. Butyrates of straight-chain un- 1962; Engels and Engels, 1984). In a re- saturated primary alcohols were found to ceptive virgin, evidently the highly volatile contribute ≈ 20% to the bouquet only in secondary alcohols comprising 60-80% of young but already mated queens. The 7% the cephalic bouquet in young queens are mean hydrocarbon content present in the involved in mate attraction, as in field tests head extracts of old physogastric queens using a dummy impregnated with the 4 was high compared to that in younger indi- major 2-alkanols, drones in an aggregation viduals. were found to be attracted (Engels et al, 1990). After a successful mating flight, the young queen becomes much less attrac- DISCUSSION tive to the drones (Engels and Engels, 1988). She still has to be accepted by the The worker’s closure of the entrance to an colony (Engels and Imperatriz-Fonseca, incipient nest containing a gyne not yet 1990). Only the egg-laying and physogas- ready for mating may prevent the prema- tric queen attains a stable dominant royal ture formation of a drone aggregation. This status (Engels, 1986). "closed-door state" of a filial nest might In stingless bees, the queen apparently also prevent other gynes from the maternal contributes important information to nest or nests from colony neighbouring entering odour. Only in the event that a virgin is and the resident The bulb displacing virgin. present in a colony is the formation of a constructed colonies to close by incipient drone aggregation released. Since after a the entrance resembled the sealing ob- successful mating flight this lek assembly served cold during long-lasting rainy peri- in the vicinity of a filial nest soon disinte- ods. grates (Engels and Engels, 1984), the nest The data on cephalic volatile composi- odour must change, signalling to the males tion in individual S postica queens clearly that they can no longer expect to find a vir- indicate not only pronounced differences in gin. the odour of and old bouquet young Another question is that of a possible but also queens (Engels et al, 1987, 1990), negative label regarding a recently mated a distinct that age-related pattern gradually queen. Is there a post-copulation message in the first 2 wk changes, particular during indicating to the drones that this young after This raises emergence. questions queen is no longer receptive? In the primi- concerning the reasons for such ontoge- tively eusocial halictine bee Lasioglossum netic patterns in adult queen cephalic vola- malachurum, the volatile composition of tiles with respect to stingless bee repro- the Dufour’s gland secretion changes sig- ductive biology. nificantly within 1 d after mating (Ayasse In contrast to honey bees (Velthuis, et al, 1993). It is interesting to note that the 1985), stingless bee queens are not al- butyrates found in high proportions only in ways attractive to workers, which pursue such S postica queens show some structu- and often kill a young gyne (Engels and ral relationship to certain acetates which Imperatriz-Fonseca, 1990). In S postica are typical sex pheromones in Lepidopte- ran females (Arn et al, 1992). Under natu- versity of São Paulo, Campus Ribeirão Preto. ral conditions the mated queen will not We appreciate the facilities kindly provided by undertake another nuptial flight, and there- our colleagues, ZLP Simões and LS Gonçalves. The work was supported by DFG grants fore will not meet more ma- normally any (Schwerpunkt Chemische Ökologie En 89/11 ture drones which in stingless bees colo- and Fr 507/8). nies always leave the maternal colony and act as and vagabonds (Engels Engels, Résumé — en fonction de In the box bi- Analyse l’âge 1984; Engels, 1987). mating des constituants volatils des drones confronted with a glandes oassay recently chez les reines de mated showed little interest céphaliques Scapto- young queen postica, mélipone néotropicale. (Engels and Engels, 1984; confirmed by On a déterminé par chromatographie en the biotest results reported here). phase gazeuse (fig 1 ) la concentration de The post-mating transition of the young 68 composés volatils (fig 2) provenant queen to the status of an accepted egg- d’extraits au pentane de têtes de reines de laying head of the colony (Engels and Im- Scaptotrigona postica. Une série de 13 rei- has a peratriz-Fonseca, 1990) prominant nes d’âge connu, élevées dans un rucher influence on the workers’ behaviour. Con- expérimental à l’université de Sâo Paulo à struction and of brood cells as provisioning Ribeirão Preto, a été analysée. Les résul- well as laying of alimentary eggs is stimu- tats montrent des différences entre les lated in young nurse bees, whereas colony pourcentages des divers composés. Ces defense and foraging is intensified in old différences peuvent être considérées field bees (Engels et al, 1979). In the hon- comme un profil ontogénétique qui permet ey bee, a similar stimulation, and in addi- de séparer nettement les jeunes reines tion retinue formation (which is not ob- avant, au moment de l’accouplement et served in a comparable manner in après, ainsi que les vieilles reines pondeu- stingless bees), is released by a blend of ses (fig 2). Chez les jeunes reines les al- major components present in the queen’s cools secondaires dominent, formant 60 à mandibular gland secretions (Slessor et al, 80% du bouquet. Chez les vieilles reines 1988, 1990). Whether in physogastric S physogastriques ils ne représentent plus postica queens cephalic volatiles repre- que 5%, alors que les acides carboxyli- sent this dominance signal function, per- ques représentent plus de 80%. Le Z-9- haps in combination with the secretions tricosène est le composé principal de la from the large abdominal tergal glands in fraction neutre de cette classe d’âge. Les stingless bee queens(Cruz-Landim et al, reines vierges réceptives se caractérisent 1980), still has to be investigated. In the par un bouquet odorant typique de l’âge de head extracts of old and egg-laying 10-12 j et qui se compose de 5 à 10% de queens, Z-9-tricosene constitutes the main méthylcétone. Seules les jeunes reines qui hydrocarbon; this compound is wide- viennent de s’accoupler présentent un spread among and is also known pourcentage relativement élevé d’esters as a sex pheromone component in female (20% de butyrates insaturés). Le dévelop- house flies (Adams, 1986). pement du nid chez cette espèce de méli- pone est brièvement décrit. On discute des fonctions possibles de la phéromone roya- ACKNOWLEDGMENTS le en rapport avec la biologie de la repro- duction et le cycle de la colonie, qui pré- This study was carried out in cooperation with sentent des différences par rapport à the Departments of Genetics and Biology, Uni- l’abeille domestique. Meliponinae / Scaptotrigona postica / dem von Honigbienen Bekannten aufge- reproduction / glande céphalique / phé- zeigt werden. romone / composé volatil / effet âge Stachellose Bienen / Fortpflanzung / Kö- nigin-Kopfduftstoffe / Ontogenetische Zusammenfassung — Alterskorrelierte Pheromon-Muster Muster in der Zusammensetzung flüch- tiger Inhaltsstoffe von Kopfdrüsen bei Königinnen der neotropischen stachel- REFERENCES losen Biene Scaptotrigona postica. In den Köpfen einzelner Königinnen der neo- Adams T S (1986) Effect of different female pro- tropischen stachellosen Biene Scaptotrigo- duced pheromone components on male courtship behavior in the house fly, Musca na wurde mittels postica Gaschromatogra- domestica. Adv Invertebr Reprod 4, 297-304 phie (Abb 1) die Konzentration von 68 Arn Tóth Priesner E List of sex Inhaltsstoffen in Pentanextrak- H, N, (1992) flüchtigen pheromones of Lepidoptera and related at- ten von Einzelköpfen bestimmt (Abb 2). tractants. In: Int Organ Biol Control (IUBC). Die Analysenergebnisse einer Serie von INRA, Montfavet, 2nd, 179 pp 13 altersbekannten Königinnen, die in Bra- Ayasse M, Engels W, Hefetz A, Lübke G, silien auf einem Versuchsbienenstand der Francke W (1990) Ontogenetic patterns in Universität von São Paulo in Ribeirão amounts and proportions of Dufour’s gland Preto gehalten wurden, ergaben charakte- volatile secretions in virgin and nesting ristische Unterschiede in den Anteilen der queens of Lasioglossum malachurum (Hyme- Sie lassen sich als noptera: Halictidae). Z Naturforsch 45c, 709- Einzelkomponenten. 714 ontogenetisches Muster interpretieren, auf- M, W, Hefetz A, J, Lübke dessen vor, wäh- Ayasse Engels Tengö grund Jungköniginnen G, Francke W (1993) Ontogenetic patterns of rend und nach dem sowie Paarungsalter volatiles identified in Dufour’s gland extracts eierlegende Altköniginnen sich klar unter- from queens and workers of the primitively scheiden (Abb 2). Bei Jungköniginnen do- eusocial halictine bee, Lasioglossum mala- minieren sekundäre Alkohole mit Anteilen churum (Hymenoptera: Halictidae). Insectes von 60-80% am Bouquet. Bei physogastri- Soc 40, 41-58 schen Altköniginnen machen diese nur Cruz-Landim C da, Höfling M C A, Imperatriz- V L and mandibular noch unter 5% aus, Carbonsäuren dage- Fonseca (1980) Tergal in queens of Paratrigona subnuda gen über 80%. Z-9-Tricosen ist die glands in der Neutralfraktion (Moure) (Hymenoptera: Apidae). Morphology Hauptkomponente and associated behaviour. Naturalia 5, 121- dieser Altersstufe. Paarungsbereite Jung- 133 zeichnen sich durch ein für das königinnen Engels E, Engels W (1984) Drohnen- Alter von 10-12 Tagen typisches Duftmus- Ansammlungen bei Nestern der stachellosen ter aus, das 5-10% Methylketone enthält. Biene Scaptotrigona postica. Apidologie 15, Bereits begattete Jungköniginnen hinge- 315-328 gen weisen einen relativ hohen Anteil von Engels E, Engels W (1988) Age-dependent Estern auf, nämlich etwa 20%. queen attractiveness for drones and mating in the bee, In einem kurzen Abriß wird die Nestent- stingless Scaptotrigona postica. J Apic Res 27, 3-8 wicklung bei dieser Art stachelloser Bienen W, Francke, W Do beschrieben. Funktionen des Kö- Engels E, Engels (1987) Mögliche stingless bees have two complexes of queen nigin-Pheromons werden in Zusammen- substances? In: Chemistry and Biology of hang mit der Fortpflanzung und dem Nest- Social Insects (Eder J, Rembold H, eds) Ver- zyklus diskutiert, wobei Unterschiede zu lag Peperny, Munich, 710-711 Engels W (1986) The concept of chemical com- tion to reproduction in the stingless bee, munication in as realized in social Scaptotrigona postica (Hymenoptera: Api- bee reproduction. Adv Invertebr Reprod 4, dae: Trigonini). Entomol Gen 15, 91-101 285-296 Free J B (1987) Pheromones of Social Bees. Engels W (1987) Pheromones and reproduction Chapman and Hall, London, 218 pp in Brazilian stingless bees. Mem Inst Oswal- De Hazan M, Lensky J, Cassier P (1989) Effects do Cruz 35-45 82, suppl III, of queen honeybee (Apis mellifera L) ageing Engels W (1988) Fortpflanzungsstrategien bei on her attractiveness to workers. Comp Bio- Bienen. Reproductive strategies in bees. chem Physiol 93A, 777-783 Verh Dtsch Zool Ges 81, 155-167 Kerr W E, Zucchi R, Nakadaira J T, Butolo J E Engels W, Imperatriz-Fonseca VL (1990) Caste (1962) Reproduction in the social bees (Hy- development, reproductive strategies and menoptera: Apidae). J NY Entomol Soc 70, control of fertility in honey bees and stingless 265-276 bees. In: Social Insects, an Evolutionary Ap- Slessor K N, Kaminski L A, King G G S, Borden proach to Castes and Reproduction (Engels J H, Winston M L (1988) Semiochemical ba- W, ed) Springer Verlag, Berlin, 167-230 sis of the retinue response to queen honey Engels W, Engels E, Lotz G, Dröscher R, Witt- bees. Nature 332, 354-356 mann H (1979) Nestbiologie der stachello- Slessor K N, Kaminski L A, King G G S, Winston sen Biene Scaptotrigona postica. Hochschul- M L (1990) Semiochemicals of the honey bee unterrichts-Farb-Ton-Film 16 mm, IWF-Nr C queen mandibular glands. J Chem Ecol 16, 1351, Göttingen 851-860 Engels W, Engels E, Lübke G, Schröder W, Velthuis H H W (1985) The honey bee queen Francke W (1990) Volatile cephalic secre- and the social organization of her colony. tions of drones, queens and workers in rela- Fortschr Zool 31, 343-357