Cephalic and Dufour gland secretions of Scaptotrigona mexicana queens: Chemical composition and biological activity Julieta Grajales-Conesa, Julio C. Rojas, Miguel Guzmán-Díaz, Manuel Rincón-Rabanales, Leopoldo Cruz-López To cite this version: Julieta Grajales-Conesa, Julio C. Rojas, Miguel Guzmán-Díaz, Manuel Rincón-Rabanales, Leopoldo Cruz-López. Cephalic and Dufour gland secretions of Scaptotrigona mexicana queens: Chemical composition and biological activity. Apidologie, Springer Verlag, 2007, 38 (1), pp.38-46. hal-00892233 HAL Id: hal-00892233 https://hal.archives-ouvertes.fr/hal-00892233 Submitted on 1 Jan 2007 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie 38 (2007) 38–46 38 c INRA/DIB-AGIB/ EDP Sciences, 2007 DOI: 10.1051/apido:2006052 Original article Cephalic and Dufour gland secretions of Scaptotrigona mexicana queens: Chemical composition and biological activity* Julieta G-Ca, Julio C. Ra, Miguel G´ -D´a, Manuel R´ -Rb, Leopoldo C-L´ a a El Colegio de la Frontera Sur, Unidad Tapachula. Carr. Ant. Aeropuerto, Km.2.5, AP. 36. CP 30700, Tapachula, Chiapas, Mexico b Universidad Autónoma de Chiapas, Departamento de Biotecnología, Tapachula, Chiapas, Mexico Received 21 November 2005 – Revised 10 May 2006 – Accepted 31 May 2006 Abstract – Cephalic and Dufour gland secretions of virgin and physogastric queens of Scaptotrigona mex- icana were analyzed by GC-MS. Worker responses to extracts and selected compounds identified in these secretions were evaluated in experimental hives. Cephalic secretions of virgin queens and 1-month-old physogastric queens are constituted of 2-ketones, 2-alcohols and esters, while the cephalic secretions of 1- year-old physogastric queens are constituted of 2-alcohols and esters. Dufour gland secretions from virgin queens are mainly constituted of 2-alcohols, ketones, hydrocarbons and esters, while Dufour gland secre- tions from physogastric queens are mainly constituted of esters, where hexyl hexanoate is the major com- pound. Workers were significantly attracted to natural extracts and synthetic blends of selected compounds of the 1-year-old physogastric queen cephalic secretion as well as to a blend of enantiomeric 2-alcohols. Dufour gland secretion was not attractive to workers. Scaptotrigona mexicana / queens / cephalic secretions / Dufour gland / stingless bees / Apidae 1. INTRODUCTION retinue behavior (Winston, 1993). In contrast, little is known about the chemical composi- The stingless bees species of the Neotrop- tion and role of queen substances in sting- ical areas show an astonishingly great diver- less bees (Engels et al., 1987, 1988, 1990, sity and many aspects of their biology, ecology 1993, 1997; Abdalla et al., 2004; Gracioli- and behavior are so far unknown. Stingless Vitti et al., 2004). For instance, there is no bees, like all other eusocial bees, use differ- evidence so far that queen pheromones guide ent ways of communication inside and outside or induce queen retinue (Engels et al., 1990) the nest, but so far most studies have focused or inhibit the ovary development of work- on visual, auditory and chemical communi- ers. It is clear that physogastric queens use chemical communication to organize work in- cation (Michener, 1974; Nieh, 1999). One of ff the most advanced eusocial bees is the west- side the colony, caste di erentiation and nest ern honeybee, Apis mellifera. In this species, homeostasis (Nogueira-Neto, 1997). Also vir- queen substances are involved in the inhibition gin queens use a sexual pheromone to attract of queen rearing and swarming, the preven- drones (Engels et al., 1990). Behavioral obser- tion of worker ovary development and queen vations in Scaptotrigona mexicana, a species widely distributed in Mexico showed that Corresponding author: L. Cruz-López, workers of queenless colonies are attracted [email protected] to physogastric queens, whereas the work- ers in queenright colonies are not attracted * Manuscript editor: Klaus Hartfelder Article published by EDP Sciences and available at http://www.edpsciences.org/apido or http://dx.doi.org/10.1051/apido:2006052 Queen secretions of S. mexicana 39 to physogastric queens (Grajales et al., 2005). 30 m, 0.25 mm i.d., 0.25 µm, J&W Scientific) This previous study, however, did not identify using helium as carrier gas (275.8 KPa) at 65 ◦C. the chemical composition or glandular origin Identification of the natural compounds was done of the pheromones involved in queen-worker by comparison of retention times and mass spectral interaction in S. mexicana, and many ques- data with those of synthetic compounds. Tentative tions concerning this topic remain to be in- identification of unknown compounds was carried vestigated. In this study we chemically iden- out using the NIST 2002 spectral library or mass tified the cephalic and Dufour gland secretions spectral data reported in literature. The relative in virgin and physogastric queens of S. mex- percentage of the components was calculated from icana and evaluated the behavioral responses the sum of the recorded peaks. Quantification of of worker to these substances. the glandular secretion was performed by using tridecane (1 µg/sample) as an internal standard. Synthetic compounds were obtained from Fluka 2. MATERIALS AND METHODS and Sigma-Aldrich (Toluca, Mexico). The purity of these compounds was >95% by GC. Hexyl 2.1. Bees hexanoate and hexyl butanoate were prepared according to Attygalle and Morgan (1986). One-year-old (n = 12) and 1-month-old physo- gastric queens (n = 10), as well as virgin queens of different ages (n = 15) of S. mexicana were col- lected from different hives of the meliponary of El 2.4. Behavioral tests Colegio de la Frontera Sur (ECOSUR) located in ◦ ◦ Cacahoatan (14 59’N, 92 10’W, altitude 480 m), The bioassays were carried out in the meliponary Chiapas from January to February, 2005. Immedi- of ECOSUR in a special room designed to exclude ately after collection, queens were transferred to ◦ all possible external noises and odors. The tests 2 mL glass vials and placed at –20 C for 10 min were performed in 15 hives (30 × 20 cm) based on to immobilize them and avoid the emptying of their the model of Engels et al. (1987), and were subdi- glands at the moment of dissection. vided into a brood nest and a storage part, connected only by two narrow tunnels to avoid rapid odor ex- change. The hives were prepared in November 2004 2.2. Sample preparation to obtain strong colonies in March 2005 (hives with The heads of the queens were carefully removed an at least four months old physogastric queen). The and the Dufour glands were dissected in distilled nest boxes were covered with a plexiglass plate for water with two pairs of fine forceps under a stere- the experiments. Three days before the tests, the omicroscope. Individually and separately, the heads queens were removed from the hives, because in and the Dufour glands were crushed in 2 mL of hex- preliminary observations workers of this condition ane, and then concentrated with nitrogen to 100 µL. showed a higher response to queens (Grajales et al., The secretion samples were stored at –20 ◦Cforfur- 2005). The reserves were removed from the storage ther analysis and bioassays. area to clear the arena. Behavioral tests were per- formed from March to August, 2005. Two differ- ent experiments were carried out at different times; 2.3. Chemical analysis (a) in the first one, the extracts of one queen equiv- alent of cephalic and Dufour gland secretions of Gas chromatography-mass spectrometry of physogastric and virgin queens were evaluated; and cephalic and Dufour gland secretions was carried (b) in the second one, the effect of natural secretions out with a Varian CP-3800 (Palo Alto, CA) was compared with synthetic blends of compounds chromatograph linked to a Varian Saturn 2200 identified from cephalic secretions. The synthetic mass spectrometer (Palo Alto, CA). The samples blends used in the experiments were prepared ac- were analyzed using a non polar capillary column cording to the natural proportions of each compo- (DB-5MS, 30 m × 0.25 mm i.d. 0.25 µm, J&W Sci- nent from the extract using hexane as a solvent; entific). The oven was programmed from 50 ◦Cto (a) a blend representing one head equivalent of se- 280 ◦Cat15◦C/min. The carrier gas was helium at lected components identified in 1-year-old physo- 1mL/min. Chiral separations were carried out with gastric queens (5 µL of 29% 2-heptanol, 9% decane, a cyclodextrin-based capillary column (Cyclosil-B, 2% limonene, 55% 2-nonanol, 2% hexyl hexanoate 40 J. Grajales-Conesa et al. Table I. Mean (±SE) composition (%) of the cephalic volatiles of S. mexicana queens; virgin (VQ), 1-month old physogastric (PQ1m) and 1-year old physogastric (PQ1y). Peak Compound VQ (n = 11) PQ1m (n = 6) PQ1y (n = 10) 1 2-Heptanone 0.8 ± 0.19 (t) ( - ) 2 2-Heptanol 15 ± 3.18 17 ± 4.48 29 ± 4.87 3 Decane 2 ± 1.04 ( - ) 9 ± 2.03 4 Limonene (-) (-) (t) 5 Nonen-2-ol (t) ( - ) ( - ) 6 2-Nonanone 0.5 ± 0.13 2 ± 1.15 ( - ) 7 Isomerofpeak5 (t) (-) (-) 8 2-Nonanol 62 ± 4.35 64 ± 2.23 55 ± 4.88 9 Hexyl butanoate (t) 1 ± 0.31 (t) 10 Isomer of peak 11 0.5 ± 0.11 1 ± 0.22 2 ± 0.49 11 Undecen-2-ol 17 ± 2.38 6 ± 1.59 ( - ) 12 2-Undecanol 2 ± 0.41 8 ± 5.36 5 ± 3.03 13 Hexyl hexanoate ( - ) (t) 0.5 ± 0.11 (t) = traces, ( - ) = chemical compound not found.