Original article 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 &mdash; 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, alcohols, 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 honey 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 drone 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 &mu;l 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 pollen 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, Brazil. 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, &ap; 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 &ap; 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 &ap; 80%. In newly emerged gynes, only during the brief mating period at &ap; 12 the carboxylic acid content was &ap; 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 &ap; 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).