J Chem Ecol (2011) 37:1255–1262 DOI 10.1007/s10886-011-0034-1

Queen Volatiles as a Modulator of Tetragonisca angustula Drone Behavior Macario M. Fierro & Leopoldo Cruz-López & Daniel Sánchez & Rogel Villanueva-Gutiérrez & Remy Vandame

Received: 3 June 2011 / Revised: 30 August 2011 / Accepted: 2 October 2011 / Published online: 12 November 2011 # Springer Science+Business Media, LLC 2011

Abstract Tetragonisca angustula mating occurs during the virgin queen nuptial flight, usually in the presence of a drone congregation area (DCA). The presence of virgin queen pheromone is considered the trigger for DCA establishment, although this has not been demonstrated experimentally. We established meliponaries, in different habitats, with T. angustula virgin queens during the main drone reproduction period. Eight DCAs were observed in urban areas, and all established outside or near colonies containing at least one virgin queen. The accumulation of drones in the DCAs occurred from 08:00 to 18:00 h and over 3–35 days. The number of drones in DCAs ranged from 60 to 2,000. In field trials, drones were attracted to virgin queens and also, unexpectedly, to physogastric queens. Volatiles collected from both virgin and physogastric queens elicited strong electoantennogram (EAG) responses from drones. Virgin and physogastric queen volatiles were qualitatively similar, but quantitatively different, in chemical composition. The queen’s abdomen was the principal source of these compounds. Isopropyl hexanoate (IPH), the most abundant compound in virgin queen volatiles and one of the most M. M. Fierro : L. Cruz-López (*) : D. Sánchez El Colegio de la Frontera Sur, Unidad Tapachula, Carretera Antiguo Aeropuerto Km 2.5, Tapachula, Chiapas, Mexico CP 30700 e-mail: [email protected] R. Villanueva-Gutiérrez El Colegio de la Frontera Sur, Unidad Chetumal, Avenida Centenario Km 5.5, Chetumal, Quintana Roo, Mexico CP 77014 R. Vandame El Colegio de la Frontera Sur, Unidad San Cristóbal, Carretera Panamericana y Periférico Sur s/n, Barrio María Auxiliadora, San Cristóbal de Las Casas, Chiapas, Mexico CP 29290

abundant in physogastric queen volatiles, was identified as one of the compounds that elicited EAG responses and was demonstrated to attract drones in a field test. Key Words Stingless bees . Tetragonisca angustula . Electroantennography . Drone congregation area . Isopropyl hexanoate . Mating behavior . Apidae . Meliponinae

Introduction Stingless bees (Hymenoptera, Apidae, Meliponinae) are natural pollinators in the tropics, as well as the major taxonomic group of highly eusocial bees of the family Apidae (Michener, 2000). Unlike colony reproduction in Apinae, mating in Tetragonisca angustula is a slow process, involving a virgin queen leaving the mother colony, either in a swarm or unaccompanied, and establishing a new nest where fertilization occurs (Kerr et al., 1962; Veen Van and Sommeijer, 2000a). The typical mating process, following in-nest development of virgin queens, is characterized by the formation of drone congregation areas (DCA) (Ferreira, 1994). The bioethology of DCAs has been well studied in honey bees, Apis mellifera. In honey bees, drones from many colonies aggregate in specific places to mate with virgin queens in midair (Loper et al., 1992). Drones are attracted by a blend of queen pheromone, especially 9-keto-2-(E)-decenoic acid (9-ODA); Slessor et al., 1988). These locations are constant over years, even when no queen is present (Baudry et al., 1998). In stingless bees, DCAs also are a common, but not well studied, phenomenon. Unlike honey bees, most congregations of stingless bee drones occur with drones perched on objects, often directly outside of colonies in which queen supersedure, or swarm settlement, has taken place and from which,

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consequently, a young queen will take off for nuptial flight (Engels and Engels, 1984, 1988; Engels, 1987; Roubik, 1990; Sommeijer and De Bruijn, 2004). Pheromones of virgin queens are believed to trigger DCA formation (Sommeijer and de Bruijn, 1995; Imperatriz-Fonseca et al., 1998; Veen Van and Sommeijer, 2000a), although definitive experimental proof has yet to demonstrate this. Tetragonisca angustula DCAs have been observed near nests (Sakagami, 1982; Engels and Engels, 1988; Veen Van and Sommeijer, 2000a; Galindo-López and Bernhard-Kraus, 2009), but rarely elsewhere in the field. Consequently, little is known about how mating occurs (Imperatriz-Fonseca et al., 1998; Veen Van and Sommeijer, 2000b). In order to learn more about T. angustula mating behavior, we established meliponaries with virgin queens in different habitats during the main male-production period (Velthuis et al., 2005). Curiously, after establishing these meliponaries, and while preparing new colonies to raise additional virgin queens, drones from a DCA located about 7 m away were attracted to a single physogastric (i.e., fertilized) queen previously removed from a queen-right colony (unpublished data). This led us to wonder whether queen volatiles were responsible for drone attraction. Until now, there have been only two reports on drone attraction in stingless bees. The first was with Scaptotrigona postica (Engels et al., 1990), in which an artificial mixture of secondary alcohols (present in cephalic volatiles found in virgin queens) was shown to attract drones in an aggregation in the field. The second, with S. mexicana (Verdugo-Dardon et al., 2011), used both field and laboratory experiments, and showed that drones differentiated between virgin and physogastric queen volatiles originating from the head. Virgin queen cephalic extract elicited strong drone antennal and behavioral responses. In this study, we observed T. angustula drone assembling behavior, and examined drone behavioral and electrophysiological responses to natural and synthetic volatiles of virgin and physogastric queens. We also recorded the relative abundances of volatiles in regard to queen reproductive status, and located the source of volatiles in the queen body.

and 92° 16″ W) and ECOSUR (14°53′ N and 92°17′ W), with five and seven colonies, respectively, 2 km apart. Each colony contained at least one recently emerged virgin queen. Meliponaries were established by Dec 2009, with the exception of ECOSUR, which was established in Dec 2010 at the El Colegio de la Frontera Sur (ECOSUR) facility. Most of the work reported here used colonies at the urban ECOSUR site.

Methods and Materials

Electrophysiology Gas chromatography-electroantennographic detection (GC-EAD) analyses were performed using SPME volatile collections of living queens and queen body parts, as well as 2 μl of synthetic isopropyl hexanoate (IPH; 2 mg/ml). Antennae from drones caught from a DCA, or workers (nurse and forager) from established colonies, were carefully removed, and the base was inserted into a reference glass capillary electrode, previously filled with saline solution (Malo et al., 2004). The distal end of an antenna was inserted into the tip of the glass recording capillary electrode. The signal generated by

Study Site and Meliponaries Experiments were carried out in the Soconusco region, Chiapas, Mexico, from Dec. 2009 to March 2011. Four T. angustula meliponaries were established in two sites, eight km apart. Site 1) agroforestry cacao plantation, with two meliponaries, Izapa (14°54′N and 92°11′W) and Medio Monte (14°53′ N and 92°11′ W), with seven colonies in each site, 2.8 km apart; Site 2) Urban area with two meliponaries, Downtown (14° 54″ N

Bee Colonies and Recording All colonies in the meliponaries were removed from feral nests and established in wooden boxes (30×20×10 cm) from which the queen was removed. The queenless colonies contained 500–1,000 workers, few drones, several combs with emerging broods, at least one mature queen cell, and sufficient food stores. The meliponaries were observed daily for drone congregations. After the first DCA was formed, we checked every 2 h between 08:00 and 18:00 h, and recorded the number of drones. Ten bait hives in each site, located to attract swarms of T. angustula, were hung 1–15 m from the occupied hive. These bait hives were checked daily to determine whether new colonies had settled in them (Engels and Imperatriz-Fonseca, 1990). Collection of Volatiles and Sample Preparation Individual virgin (1–5 d-old; N=6) and physogastric (N=17) queens (both types taken directly from the nest and assayed immediately), as well as individual heads, thoraces, and abdomens from queens (N=3), were placed in 20 ml Erlenmeyer flasks with an aluminum foil lid. Queen bees were left in flasks for 30 min before volatile collection. Volatiles were collected from the flasks for 30 min. at ambient temperature using a Solid Phase Micro-Extraction method (SPME) fiber (65 mm polydimethylsiloxane divinylbenzene; Supelco, Bellefonte, PA, USA). For extracts, an individual intact queen (virgin or physogastric), or dissected body parts (head, abdomen, and thorax), was crushed in 400 μl of dichloromethane. Extracts were concentrated to 100 μ1 (queens and abdomen) or 50 μl (head and thorax) per queen equivalent by a nitrogen stream, and stored at −20°C until used for analysis or bioassays.

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an antenna was passed through a high-impedance amplifier (Syntech NL 1200, Hilversum, The Netherlands) and displayed on a monitor using Syntech software for processing GC-EAD signals. One drone or worker antenna constituted one sample (N=10). Chemical Analyses To identify EAD-active compounds, volatiles and extracts were analyzed on a Varian Star model 3400 CX GC (Palo Alto, CA, USA). A DB-5 column (30 m×0.25 mm ID) was temperature programmed from 50°C (held for 2 min) to 250°C at 15°C min−1. The injection port temperature was held at 200°C. The GC was coupled to a Varian Saturn 4D mass spectrometer and integrated data system. Ionization was carried out by electron impact at 70 eV, 230°C. Mass spectral identifications of the compounds were confirmed by comparison of retention times and mass spectra with those of synthetic standards. The relative amount of each compound was calculated from peak area, while the relative percentage of a component was calculated relative to the sum of all peak areas. Preparation of Esters Esters were prepared by heating the corresponding alcohol (10 μl), carboxylic acid (5 μl), and acetic acid (2 μl) together with a drop of concentrated sulfuric acid in a Keele microreactor (Attygalle and Morgan, 1986) for 12 h at 100°C. The reaction was neutralized with NaHCO3, and esters extracted with hexane (200 μl). IPH was synthesized from isopropyl alcohol (Merck, Mexico, S.A., chemical purity 99.7%) and hexanoic acid (Sigma-Aldrich, Toluca, Mexico, chemical purity 99%); butyl hexanoate (BH) from butanol (Productos Quimicos, Monterrey, Mexico, chemical purity 99.4%) and hexanoic acid; hexyl hexanoate (HH) from hexanol (SigmaAldrich, Toluca, Mexico, chemical purity 98%) and hexanoic acid. Behavioral Bioassays To test the attractiveness of samples from both virgin and physogastric queens, bioassays were conducted in the ECOSUR bee yard. Live physogastric queens (N=5), in a wire cage on a 1.5-m-high tripod, and a 5 mm diam. cotton ball in another wire cage (used as positive and negative controls, respectively) were exposed three times, each 1 min., daily around noon, with breaks of 30 min between exposures. These tests were conducted over 5 d at a distance of 1, 3, 5, 7, 10, 15, and 20 m from DCA 3 (containing ~2,000 drones, estimated by counting the number of males that visited the cages). Virgin queens (N=3), 3–7 d-old were exposed on 3 different days, as described above, to DCAs 3 and 6 (each with ~2,000 males). A short distance, two-choice bioassay was carried out to test the attractiveness of parts of the queen body (head,

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thorax, and abdomen; N=3), as well as queen extract, and IPH alone or combined with BH and HH. A queen, a part of the queen body, a cotton ball impregnated with one queen equivalent, IPH (0.4 mg) alone, or a blend of IPH (0.4 mg), BH (0.2 mg) and HH (0.2 mg), was presented on a stand (0.5 m from DCA 7 with ~150 drones), along with a blank cotton ball (control), around noon for 5 min. A drone’s response was classified as “attract” when it flew upwind toward and within 5 cm of a sample; “touch” when it had brief contact with a cotton ball or a wire cage; and “mount” when it attempted to copulate. To avoid habituation, cotton balls and wire cages were changed after each test. Each queen was used only once per day. Statistical Analysis The significance of drone attractiveness to both queen statuses at different distances, as well as the drone responses to the queen body parts and synthetic blend or alone, were calculated using the Wilcoxon T test with continuity correction. This method is contained in the software R Development Core Team (2010). A significance level of 5% was used.

Results Drone Congregation Areas A total of eight T. angustula DCAs were observed during the experiment. Two established in the “Downtown” site and six in the “ECOSUR” site, both in urban areas. Three DCAs had more than 2,000 males during the days of highest assemblage. DCAs remained in place for up to 35 d (Table 1). The DCAs formed on different substrates, such as a clay tile, a wooden planter with some bees on overhanging vegetation, or a hive, but always close to (~0.5 m) or beside colonies or bait hives with a recently established swarm containing a virgin queen. The mean number of drones arriving the first day was 190 (±147). Males arrived daily at the congregation site from 08:00 to 18:00 h, with peak arrival time between 11:00 and 14:00 h. No DCA was observed at any of the two (Izapa and Medio Monte) meliponaries in the agroforest location. Electrophysiology Queen volatiles, collected by SPME from either virgin or physogastric queens, elicited strong signals from T. angustula drone antennae (Fig. 1a and b, respectively), but not from females (nurses and foraging workers, data not shown). At least one EAD-active compound was apparent (retention time, 6.7 min) from both types of queens. This compound was present in the abdomen of queens. Antennal responses to extracts of intact queen or dissected abdomens elicited a weaker signal than the SPME collections. Although traces of the active

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Table 1 Establishment of Drone Congregation Areas (DCA) of Tetragonisca angustula at urban areas in the Soconusco Region, Chiapas, Mexico DCA

Arrival date

Total drones first day

# Drones largest assembly

Days to largest assembly

*Substrate used for assembly

DCA duration (days)

1 2 3 4 5 6 7 8

12/12/20091 02/20/20101 01/23/20112 01/31/20112 02/06/20112 02/11/20112 02/15/20112 03/19/20112

200 80 150 80 150 500 300 60

500 250 2,400 500 800 2,200 2,000 60

3 3 3 5 2 24 13 1

†Hive Wood planter †Hive †Hive ‡Bait hive ‡Bait hive ‡Clay tile †Hive

5 4 19 5 4 34 35 3

Key: †Colonies containing virgin queens; ‡ Bait hive with new colony with virgin queen; 1 DCAs in downtown site; 2 DCAs in ECOSUR site; *Substrate with same letter mean same location

compound were found in the thorax and head, no EAD responses to volatiles collected from these parts were obtained. Synthetic IPH elicited a discernible EAD response from drone (Fig. 1c), but not female, antennae (data not shown). Chemical Analyses Typical gas chromatograms of SPME volatile collections from live virgin and physogastric T. angustula queens are presented in Fig. 1a and b, respec-

tively. Three major peaks were identified: IPH [retention time: 6.7 min., major m/z (relative abundance,%): 43(100), 56(47), 84(41), 99(44), 117(50)]; BH [retention time: 8.3 min., m/z 43(60), 56(100), 71(29), 99(78), 117(57)], and HH [retention time: 10.3 min., m/z 43(100), 60(40), 71 (20), 99(46), 117(22)]. Chemical analyses of the two types of queens were similar qualitatively, but differed quantitatively, with virgin queens characterized by an abundance of IPH, a small amount of BH, and a trace of HH, while physogastric queens had relatively large amounts of IPH and HH, and a small amount of BH (Table 2). Interestingly, young, recently mated queens (2–4 wk-old) had relative amounts of HH in between the other two types (Table 2). The composition in the abdomen of virgin queens was similar to that for live queens, whereas the thorax and head had only traces of IPH and HH, as well as several compounds not found in the abdomen (Table 2).

Table 2 Relative abundance* of compounds in intact virgin and physogastric Tetragonisca angustula queens (±SD) and their body parts

Fig. 1 Coupled gas chromatographic (with flame ionization detection; FID) and electroantennographic (EAD) responses of Tetragonisca angustula males to virgin (a) and physogastric queen (b) volatiles, or to a blend of synthetic compounds (c). Peaks: 1=isopropyl hexanoate, 2=butyl hexanoate, 3=hexyl hexanoate

Queen status

Peak 1** (IPH)

Peak 2 (BH)

Peak 3 (HH)

Virgin (N=6) Young phys (N=4)1 Physogastric (N=13) Abdomen (N=3)2 Thorax (N=3)2 Head (N=3)2

44.2 (±16.4) 37.4 (±16)

7.8 (±3.2) 5.3 (±2.7)

6.6 (±6.5) 12.9 (±4.4)

38.9 (±9.9)

8.1 (±2.4)

26.2 (±6.4)

22.4 (±13.9)

3.53 (±4)

8.6 (±4.7)

8.3 (±8.1)† Traces

1.7 (±1)† Not found

1 (±0.9)† Not found

Keys: *According to area peak size; **Electroantennogram-active compound; 1 Young physogastric queen 4–6 wk-old; 2 from virgin queens; †possible contamination from abdomen. IPH isopropyl hexanoate; BH butyl hexanoate; HH hexyl hexanoate

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Drone Behavioral Bioassay with Living Queens and Synthetic Compounds Drones were attracted to live virgin and physogastric queens over various distances. Both virgin and physogastric queens were detected by drones when slowly brought closer to the DCA (~2,000 males), with attraction increasing the closer the queens were exposed to the DCA (Fig. 2). At 20 m from the DCA, no males were attracted to queens. Of the three body parts tested in the bioassay, the greatest number of responses occurred to the abdomen of a virgin queen, with fewer males responding to the thorax and none to the head (Table 3). The attract, touch, and mount responses to the abdomen and thorax were greater than the corresponding responses to the control. Only a few seconds after exposing the cage containing a queen’s abdomen, drones flew upwind and touched or attempted to mount it, with fairly consistent responses recorded over each of the 5 min of the bioassay (Table 3). One equivalent of virgin queen extract attracted a significant number of males (relative to the control), of which only two (not different from that to the control) touched the cotton ball containing the extract. Relatively high numbers of drones were attracted to synthetic IPH, but few (three, not different from the control) of these actually touched the cotton ball. Similar responses were obtained to the mixture of IPH, BH and HH. The blank (control) did not attract any drones (Table 3).

Discussion Tetragonisca angustula male aggregations are common, usually establishing during the reproductive stage of colonies. However, it is rare to find wild T. angustula DCAs, and

Fig. 2 Numbers of drones, from large congregations of ~2,000 males, attracted to virgin (N=9) (light bars), and physogastric (N=15) (dark bars) Tetragonisca angustula queens. Queens were exposed for 1 min at different distances from the drone congregations. Same letter of the same case, among drone responses at different distances, indicate no statistical difference (P>0.05; Wilcoxon T test with continuity correction)

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consequently, not much is known about how mating occurs (Imperatriz-Fonseca et al., 1998; Veen Van and Sommeijer, 2000b). In our study, we found two T. angustula DCAs established in the “Downtown” meliponary and six DCAs in the “ECOSUR” meliponary, both urban areas. All DCAs were established during the main drone production period, as observed in other stingless bee species (Velthuis et al., 2005). The DCAs were found close to nests containing a virgin queen and located on a variety of substrates, as known for several stingless bee species (Sakagami, 1982; Engels and Engels, 1988; Veen Van and Sommeijer, 2000a; Sommeijer and De Bruijn, 2004; Galindo-López and Bernhard-Kraus, 2009). These results suggest that the presence of at least one virgin queen is a primary element that triggers the onset of DCA formation (Sommeijer and de Bruijn, 1995; ImperatrizFonseca et al., 1998; Veen Van and Sommeijer, 2000a). However, it seems that it is not the only factor, as males still aggregated at the same site even after the queen was fertilized. We speculate that T. angustula drones may leave “markings” that are used in the orientation of other drones toward a given site, as observed in S. mexicana. In this species, once a site is selected, the aggregation remains in the same place regardless of the queen’s status (virgin, physogastric, or even queenless) (Galindo-López and Bernhard-Kraus, 2009). We observed no DCAs formed in the agroforest meliponaries, suggesting that the density of feral nests, as well as spatial distribution, plays a crucial role in the formation of aggregations. Tetragonisca angustula uses a broad diversity of substrates for nesting, and is thus well suited to urban environments (Noll et al., 1993; Sousa et al., 2002). In our study areas, T. angustula was three times more abundant in urban areas, and was distributed relatively homogenously, than in the agroforest areas, where it has a more random spatial distribution (Fierro et al., in press).

0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 4 5 3 6 1 0 0 0 0 0 0 1 0 2 0 7 5 13 8 3

** No drones responded (attract, touch, or mount) to the control

* Response different (P<0.05) from that to the control

IPH=isopropyl hexanoate (IPH); synthetic blend ¼ IPH þ butyl hexanoate þ hexyl hexanoateðHHÞ

0 0 0 0 0 0 1 0 1 0 5 3 4 5 2 3 2 2 3 2 8 5 6 8 4 17 21 13 18 19 25 33 28 27 31 1 2 3 4 5

8 12 5 9 12

Attract* (min)

Touch*

Mount*

Attract*

Touch*

Mount*

0 0 0 0 0

3 2 4 4 5

Touch Attract* Touch Attract* Attract*

Touch

Mount

Synthetic (IPH) Queen extract Head Thorax Abdomen Time

Table 3 Tetragonisca angustula drone responses to virgin queen body parts, whole queen extract, or synthetic chemicals

Mount

Synthetic blend

Mount

Control**

J Chem Ecol (2011) 37:1255–1262 Cotton ball

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With the exception of DCA 8, all our drone aggregations were formed by greater than several hundred males. Such large numbers of drones should ensure that queens mate with an almost panmictic sample and therefore avoid inbreeding (Baudry et al., 1998). The stay of drones at a DCA generally lasts only a few days, with males disappearing after the queen has been fertilized (Veen Van and Sommeijer, 2000a). However, three of the six DCAs we found (Table 1), and remained in the same place for 19–35 d. This behavior is similar in DCAs observed in meliponaries of S. mexicana, in which DCAs remained in the same place for 24 d (Galindo-López and Bernhard-Kraus, 2009). This long stay may occur because of the intensive handling of bee colonies within a meliponary (preparing new colonies to raise new virgin queens). The range of attraction (1–20 m) of drones we observed in our bioassay is consistent with that of other stingless bee species (Engels and Engels, 1988; Sommeijer and De Bruijn, 2004) and congruent with the successful formation of DCAs seen in urban areas. The efficiency of queen-male chemical communication is amplified by male-male communication, and the uniform spatial distribution of nests in urban areas favors DCA formation (Fierro et al., in press). Curiously, when we were preparing colonies to raise new virgin queens, drones from a DCA (>2,000 males) were attracted to a single physogastric queen removed from a queenright colony and placed in a wire cage (unpublished data). This attraction was similar to that observed to virgin queens, and may indicate that polyandry occurs in this species, as reported for several other species of solitary and social Hymenoptera (Page and Metcalf, 1982; Page, 1986). Queens of highly eusocial bees mate only during a short period at the beginning of their reproductive lives. For example, queens of Apis species copulate with an average of 23 drones, during one or more mating flights, and then start oviposition (Adams et al., 1977). In contrast, stingless bee queens have long been thought to mate only once, e.g., M. quadrifasciata (Kerr, 1969; Silva et al., 1972; Peters et al., 1999). However, mating in stingless bees has been studied in only a small number of species (Lopes et al., 2003), and evidence of attraction to physogastric queen volatiles (Engels and Engels, 1988; Campos and Melo, 1990) may indicate that queens of some species do mate more than once. Both virgin and physogastric queens released the same three esters, but in different relative amounts. Engels et al. (1990) and Verdugo-Dardon et al. (2011) found a similar result in cephalic chemistry between virgin and physogastric queens in both S. postica and S. mexicana. In our study, the most obvious difference between virgin and physogastric queens was the relative amount of HH. This compound had elevated levels in recently mated queens. In S. mexicana queens, HH became the most abundant com-

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pound once a queen was fertilized and began egg laying (Grajales-Conesa et al., 2007). Thus HH levels may be a marker for a queen’s reproductive state, and perhaps may be involved in regulating colony organization and worker behavior. Further work is needed to test this. While HH has previously been found in S. mexicana, the other two compounds have not been found in other stingless bees. Butyl hexanoate is used as a sex pheromone in Megalotomus quinquespinosus (Heroptera, Alydidae, Alydinae), and as a kairomone in Cydia pomonella and Grapholita molesta (Lepidoptera, Tortricidae), (El-Sayed, 2011). Isopropyl hexanoate has been found in extracts of sexually mature Drosophila putrida (Diptera, Drosophilidae) males, while in D. recens it is a sex pheromone (Jaenike et al., 1992). Isopropyl hexanoate is widely distributed in flower essences where its evolutionary function is to attract pollinators (Dobson and Bergström, 2000). Although T. angustula males showed strong attraction to IPH, either alone or in combination with BH and HH, males exhibited little touching. Since isolated abdomens both attracted and elicited males to mount, this suggests that there may be other close-range olfactory stimuli involved in eliciting more complete behavior. Similar results have been found in Apidae. For example, with the bumblebee, Bombus terrestris, a blend of synthetic virgin queen pheromone elicited male attraction but did not provoke “mounting” (Krieger et al., 2006). In honey bee queen pheromone studies, 9-ODA (a queen sex pheromone component) attracted males (Butler and Fairey, 1964), but this compound alone was less attractive than an intact queen (Slessor et al., 1988). In S. postica, a mixture of four synthetic secondary alcohols, placed on a dummy, elicited only occasional “touching” when presented to a drone congregation (Engels et al., 1990). The presence of additional behaviorally active compounds produced by queens should be investigated. We have shown that volatiles from both virgin and physogastric T. angustula queens attract and influence the behavior of drones. This attraction is caused, at least in part, by IPH released from the abdomen of a queen. However, further research is needed to elucidate any biological function of the presence of this compound in T. angustula physogastric queens, as well as its possible effect as a queen pheromone in the reproduction of the highly eusocial Meliponini. Acknowledgments We thank Dr. Edi Malo for advice on GC-EAD, Antonio Santiesteban and Bernardino Diaz for technical assistance, and Dr. Gerald Loper for assistance in the English translation. This work was made possible through a doctoral scholarship (57515) granted by El Consejo Nacional de Ciencia y Tecnología (CONACYT). Finally, we thank the Research and Postgraduate Division of the Universidad Autónoma de Chiapas (UNACH) for permission and support.

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