Mandibular gland component analysis in the head extracts of Apis cerana and Apis nigrocincta Christopher Keeling, Gard Otis, Soesilawati Hadisoesilo, Keith Slessor To cite this version: Christopher Keeling, Gard Otis, Soesilawati Hadisoesilo, Keith Slessor. Mandibular gland component analysis in the head extracts of Apis cerana and Apis nigrocincta. Apidologie, Springer Verlag, 2001, 32 (3), pp.243-252. 10.1051/apido:2001126. hal-00891687 HAL Id: hal-00891687 https://hal.archives-ouvertes.fr/hal-00891687 Submitted on 1 Jan 2001 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 32 (2001) 243–252 243 © INRA/DIB-AGIB/EDP Sciences, 2001 Original article Mandibular gland component analysis in the head extracts of Apis cerana and Apis nigrocincta Christopher I. KEELINGa*, Gard W. OTISb, Soesilawati HADISOESILOc, Keith N. SLESSORa a Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, V5A 1S6, Canada b Department of Environmental Biology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada c Pusat Litbang Hutan dan Konservasi Alam, Jl. Gunung Batu No. 5, Bogor, Indonesia (Received 5 September 2000; revised 26 February 2001; accepted 28 February 2001) Abstract – Head extracts of workers and mated queens of the closely related species of Apis cerana and A. nigrocincta from Sulawesi, Indonesia were quantitatively analyzed by gas chromatography- mass spectrometry for several mandibular gland components. The amounts of many compounds were significantly different between species for both queens and workers. Quantities of 10 of the 16 compounds quantified in queen bees differed significantly between the two species. Of the three known mandibular gland retinue pheromone components in A. cerana queens [(E)-9-oxodec-2-enoic acid (9-ODA), (E)-9-hydroxydec-2-enoic acid (9-HDA), and methyl p-hydroxybenzoate (HOB)], the amounts of 9-HDA and HOB were significantly different between species. Quantities of 6 of the 11 compounds quantified in worker bees differed significantly between the two species. This quantitative analysis supports the hypothesis that A. cerana and A. nigrocincta are indeed separate species. Apis cerana / Apis nigrocincta / honey bee / mandibular gland / pheromone 1. INTRODUCTION from “The Celebes” (Sulawesi), Apis nigrocincta Fabr. Smith, 1861. However, It has long been known that cavity-nest- most reports about honey bees from this ing honey bees inhabit Sulawesi and the sur- region have presumed that the cavity-nest- rounding islands of Indonesia. Maa (1953) ing bees there are forms of Apis cerana recognized a unique species of honey bee Fabr., the common hive bee that ranges over * Correspondence and reprints E-mail: [email protected] 244 C.I. Keeling et al. most of Asia. Recently two distinct popu- (Plettner et al., 1996; Plettner et al., 1998). lations of cavity-nesting honey bees were In mated queens, compounds functionalized verified in Sulawesi (Hadisoesilo et al., at the penultimate (ω-1) position of the chain 1995). Although sympatric in at least two [(E)-9-oxodec-2-enoic acid (9-ODA) and areas of Sulawesi, they do not appear to the two enantiomers of (E)-9-hydroxydec-2- hybridize, suggesting they are distinct enoic acid (9-HDA)] predominate. In work- species. ers, compounds functionalized at the termi- nal (ω) position [10-hydroxydecanoic acid Morphometric analyses have indicated (10-HDAA) and (E)-10-hydroxydec-2-enoic that the smaller, darker morph corresponds acid (10-HDA)] predominate. The mandibu- to A. cerana and the larger morph with yel- lar glands of A. mellifera queens also pro- lowish clypeus and legs corresponds to duce several aromatic compounds of which A. nigrocincta (Hadisoesilo et al., 1995; methyl p-hydroxybenzoate (HOB) and Damus and Otis, 1997). These species also 4-hydroxy-3-methoxyphenylethanol (HVA) have different drone cell cappings. It is well account for the retinue attraction of the documented that A. cerana drone cells are queen mandibular glands when combined sealed with a wax capping underlain by a with 9-ODA and 9-HDA (Slessor et al., hard conical cocoon structure that contains a 1988). These compounds comprise the central pore (Ruttner, 1988; Boecking et al., queen mandibular pheromone for retinue 1999). In contrast, sealed A. nigrocincta attraction in A. mellifera but do not account drone cells have only a thin wax capping for all of the retinue attraction or chemical and lack the hardened cap with pore (Hadis- communication attributed to the queen oesilo and Otis, 1998). The timing of the (Slessor et al., 1998; Winston and Slessor, mating flights also differs between the two 1998; Keeling et al., 2000a). species, with flights of A. cerana drones preceding and only slightly overlapping Unfortunately, the queen mandibular those of A. nigrocincta drones (Hadisoesilo pheromone of A. cerana has not been so well and Otis, 1996), thereby affecting almost characterized. It is known that HVA is absent complete reproductive isolation between the from queen mandibular glands and does not populations. These differences indicate that increase retinue attraction of A. cerana work- they are two distinct species and that we ers when added to the other three com- might expect to find other differences. pounds (Plettner et al., 1997). There has The chemical composition of the been no analysis of the mandibular gland mandibular glands of both honey bee queens composition of A. nigrocincta. In this study, and workers have previously been analyzed we quantitatively analyzed head extracts for as a method to compare different species several mandibular gland components to (Plettner et al., 1997). In the queen, these reveal any differences between the mandibu- glands produce pheromone messages that lar gland compositions of these closely elicit the retinue of workers, attract drones related species. on mating flights, and are believed to control The compounds quantified in this study several aspects of colony functioning (Win- include those previously quantified in other ston and Slessor, 1998). The functions of studies (Plettner et al., 1997; Keeling et al., the components in the worker mandibular 2000b) as well as some of the compounds glands are attributed to food preservation recently identified in A. mellifera queen and larval nutrition (Winston, 1987). mandibular glands (Engels et al., 1997; In A. mellifera L., both female castes pro- Matsuyama et al., 1997; Keeling and duce distinctive blends of compounds in Slessor, unpublished observations). Apart their mandibular glands with functionalized from those mentioned above, the compounds aliphatic acids predominating in both castes quantified have not been reported to be Analysis of A. cerana and A. nigrocincta 245 pheromone components in any honey bee University. Upon arrival, samples were species and may only represent differences stored at –20 °C until extracted. in biosynthetic pathways of the different species and castes of honey bees. Most of the aliphatic acids are biosynthetically linked 2.2. Extraction and analysis to the major queen and worker produced acids (Plettner et al., 1996; Plettner et al., Although all compounds analyzed orig- 1998). The biosynthetic pathways of the inate within the mandibular glands, the aromatic compounds HOB and HVA have whole head was extracted because the ana- not been reported but the other aromatic lytes are leached from the intact mandibular compounds quantified in this study are glands into the rest of the head by the ship- potentially linked biosynthetically to these ping solvent (Plettner et al., 1997). Bee pheromone components. As we learn more heads were crushed in the shipping solvent about the mandibular gland biochemistry of and extracted with additional methanol. honey bees and test compounds for biolog- Extracts were centrifuged briefly to remove ical activity in honey bee species other than solids and then stored at –20 °C until ana- A. mellifera, some of these compounds may lyzed. be of importance in Apis semiochemistry. Extracts were then analyzed as their trimethylsilyl derivatives by gas chro- matography-mass spectrometry in a man- 2. MATERIALS AND METHODS ner similar to Keeling et al. (2000b). Fifteen of the compounds quantified were obtained 2.1. Collection of specimens commercially as follows: 3-hydroxyoctanoic acid (3-HOAA), methyl p-hydroxybenzoate Bees from both wild and managed (HOB), 3-hydroxydecanoic acid (3-HDAA), colonies were collected in September and 4-hydroxy-3-methoxyphenylethanol (HVA), October of 1998 in Sulawesi, Indonesia. A. 10-hydroxydecanoic acid (10-HDAA) nigrocincta were collected from Bobo and (E)-coniferyl alcohol, (E)-ferulic acid, Rahmat, near Palu, in Central Sulawesi and 4-hydroxy-3-methoxyacetophenone, decane- Air Mandidi and Kauditan, near Manado, dioic acid (C10:0 DA), and (E)-4-hydrox- in Northern Sulawesi. A. cerana were col- ycinnamic acid from Sigma-Aldrich Canada lected from Bobo, Rahmat, and Lolu, in Inc. (Oakville, ON, Canada); 4-hydroxy- Central Sulawesi. A forager, queen, and acetophenone and 4-hydroxybenzoic