Caractérisation de la phéromone des butineuses, l’Oléate d’Ethyle transmission

Distribution of the Forager Pheromone (Ethyl Oleate) in the Honey Bee Colony Résumé : Les butineuses émettent une phéromone, l’EO, qui permet l’autorégulation du développement comportemental des ouvrières (nourrices) de la colonie par contact. Un nombre important de butineuses inhibe le développement comportemental des nourrices et inversement, un faible nombre de butineuses accélère la maturation des nourrices. Des facteurs internes et externes au nid peuvent moduler les besoins en butineuses, et donc le ratio nourrices/butineuses. Nous avons voulu savoir si une possible fluctuation de l’EO pourrait être une réponse à des contraintes environnementales et des modifications du nid pour ajuster le ratio nourrices/butineuses. Nous avons également clarifié le mode de transfert de cette phéromone entre butineuses et nourrices. Une étude de la quantité d’EO de différentes parties des butineuses, la tête, le thorax, l’abdomen, la cuticule, les pelotes de pollen, le nectar et le jabot a montré que le pollen des butineuses est un vecteur de l’EO ainsi que la cuticule des butineuses. Parallèlement, pour étudier la dynamique de l’EO, des butineuses de pollen et de nectar ainsi que des nourrices ont été prélevées chaque mois durant deux années à chaque période de floraison (Mai à Septembre) dans trois ruches génétiquement différentes. Une augmentation de production du taux d’EO a été observée durant la période estivale. L’origine génétique des abeilles ne semble pas impliquée, alors que les conditions environnementales semblent jouer un rôle majeur. Les contacts cuticulaires ou cuticule-antenne entre butineuses et nourrices sont nécessaires à la transmission de la phéromone inhibitrice et le pollen peut servir de stockage de la molécule, mais être également un vecteur lors de son ingestion et de son contact avec les abeilles nourrices. La dynamique de production de l’EO montre une fluctuation en réponse aux contraintes environnementales. Il est nécessaire de savoir si les nourrices adaptent leur sensibilité à la phéromone et ajustent leurs développements comportementaux en fonction des fluctuations d’EO.

EO chemical analysis All samples were prepared in a solution A of 1.9 ml of iso-hexane with the addition of 100 µl of two internal standard solutions at 10 ng/µl (arachidic acid methyl ester and methyl heptadecanoate, Sigma-Aldrich, France). Total EO amount was analyzed on different samples (pool of 5 bees, load of pollen…). Each sample was crushed with a glass rod during 2 min at 4°C in the solution A and centrifuged for 20 min at 4°C (2,500 g). The cuticular extracts were prepared by rinsing 5 bees in the solution A for 1 min at 4°C. Then the supernatant was collected and applied to a silica column (silica gel 60, particle size 40–63 µm, 230–400 mesh). The first fraction was eluted in 3 ml of a solvent mixture (98.5% iso-hexane, 1.5% diethyl ether). Then, the second fraction containing the EO was eluted in 3 ml of a second solvent mixture (94% iso-hexane, 6% diethyl ether). 1 ml of this fraction was concentrated to 10 µl under nitrogen stream and 1 µl was injected into a gas chromatograph (2014, Shimadzu, Japan) equipped with a split-splitless inlet, a flame ionization detector and a capillary column Omegawax 100 (10 m x 0.10 mm, 0.10 µm film thickness). The samples were injected in split mode. Hydrogen was used as carrier gas with a column flow of 0.52 based on retention times of EO synthetic compound (Sigma-Aldrich, France) and by comparison of internal standard area respectively using gas chromatography solution program (Shimadzu, Japan). The EO confirmation was done by a mass spectrometer (CP2010, Shimadzu, Japan) operated in the electron impact mode at 70 eV with continuous scans (every 0.2 sec) from a mass to charge ratio (m/z) of 70 to 400.

EO transmission (forgers to hive bees) To gain insight into the mode of transfer of EO among bees in a colony, we measured EO levels in different honey bee parts: head, thorax, abdomen, cuticle, but also in the food transfer to nurse by the forager: the nectar and the pollen. Foragers used in the different experiments came from a typical field colony headed by a naturally mated queen. Foragers were caught by closing the hive entrance and immediately frozen at -20°C; before EO analysis. Each sample was analyzed for total EO levels with the methods described above. First, to know the distribution of EO in foragers, we analysed EO levels in the 3 parts of foragers: head, thorax and abdomen. As a control we also measured the EO level of intact foragers. Ten groups of 5 bees were dissected for three lots of 10 samples of each honey bee sections (body parts) and 10 groups of 5 bees were kept unchanged (control). Before analyzing the EO the different samples were weighed in order to have the mean amount of EO per sections and also per mg of each section. Then we studied EO on the cuticle of honey bee. Ten groups of 5 foragers were analysed for total EO amount as a control and cuticular extracts (rinse the cuticle) were made on 10 groups of 5 foragers to know the percentage of EO found on the cuticle. Also the quantity of EO in nectar of foragers was examined. The EO level in the nectar sampled directly from the forager crop and the nectar regurgitated by the forager were quantified on two different groups because foragers could add secretions to the nectar from the hypopharyngeal gland during trophallaxis (Simpson, 1960; Simpson et al., 1968). The abdomens of 6 groups of 5 foragers were dissected, an incision was made in order to clear the crop, and then with a syringe the crop content was sampled. The crop envelope was analyzed separately. In a second group of bees (6×5 foragers), nectar regurgitation was recovered by applying pressure on the abdomen with a forceps and the regurgitated nectar was collected with a microcapillary pipette and put in a 2mL vial. A third group of foragers (6×5) was analysed for the total amount of EO as a control. In a final step the pollen load of foragers was considered as a potential transfer of EO between nurse and forager. Pollen is in contact with the forager cuticle before the formation of a pollen load and foragers add oral fluids during pollen handling (Winston, 1987). The pollen could be a way to distribute EO in the colony.