Biosynthesis and Seasonal Variation of Ethyl Oleate, a Primer Pheromone of the Honey Bee (Apis mellifera L.)

Transcription

1 Biosynthesis and Seasonal Variation of Ethyl leate, a Primer Pheromone of the Honey Bee (Apis mellifera L.) Carlos Castillo 1,3, Hao Chen 1, Carolyn Graves 1, Alban Maisonnasse 2, Yves Le Conte 2 and Erika Plettner 1 1 epartment of Chemistry, Simon Fraser University, Burnaby, BC., Canada 2 Laboratoire Biologie et Protection de l Abeille, Institut National de la Recherche Agronomique, Avignon, France 3 National Bee iagnostic Centre, Grande Prairie Regional College, Grande Prairie, AB, Canada

2 Honey Bee (Apis mellifera) Social insect living in colonies Queen: 1 rones: 2 to 3,000 Workers: 60,000 Pollination, hive products Caste polyphenism Age polyethism Larvae and eggs Worker Queen rone 2

3 evelopment of a worker bee Eclosion Larval molts Pupation Emergence H H H H H H Ecdysteroid Juvenile Hormone (JH) Queen attendants Nursing Nectar processing Foraging JH 3

4 Ratio of foragers to in-hive workers : Faster transition (E)-β-cymene Slower transition Ethyl leate 1 day old 10 days old Cohort Colony 15 days old Maisonnasse et al. 2009, Apidologie 40, Leoncini et al. 2004, PNAS 101,

5 1) ATP 2) CoASH R H Pheromone: Ethyl leate, hypothesis + EtH H 2 phosphate 1 stepwise condensation R SCoA 2 direct condensation hydrolysis 2 3 hydrolysis Enzymes: 1. FAS modules/coa ester synthases 2. Carboxylesterases, ester synthases 3. α/β hydrolases (e.g. lipases) R= H 2 H 2 EtH CoASH R Et 1) What are the sources of the activated oleyl precursor and of the ethanol, 2) Where is E produced, and which gene(s) is/are involved? 3) What is the seasonal variation of E biosynthesis? 5

6 Tissue perfusion: location of ester synthase activity Head Thorax Abdomen X ( 4)R C 2 C 3 R 9 0 H H ( 4)R R C 2 C X = H X = S N H R R X = 6

7 Source from flowers Where does the ethanol come from? H H H H H H Feeding and vapour exposure experiments ( 4)R C 2 C 3 H ( 4)R 9 R 4 C 2C 3 5 7

8 Feeding and vapour exposure experiments Table 1. etection of labeled E (ng/bee) extracted from the honey crop and exoskeleton of worker bees fed and exposed to vapors of labeled substrates. Substrate 1 Treatment 2 Honey crop 3 Exoskeleton 4 F-fed F-vapor N-fed N-vapor F-fed F-vapor N-fed N-vapor d 6 -EtH F f :F v 72 ± ± ± ± N f :N v ± ± ± ± 1.9 F f :N v ± ± 7.5 N f :F v ± ± F v :N v ± ± 14.6 d 9 -E F f :N f :F v :N v N N N N 132 ± ± ± ± 20 1: Substrate added to syrup to feed bees: d 6 -EtH (10%) or d 9 -E (50 µg.ml -1 ). 2: Treatments: F f : Forager fed, F v : Forager vapor, N f : Nurse fed, N v : Nurse vapor. 3: Honey crop: dissected from treated bees. 4: Exoskeleton: washed from treated bees. N: Not etected. - : not evaluated. ata is the average of at least 3 replicates ± SE. 8

9 Identification of candidate genes for the biosynthesis of ethyl oleate In silico analysis (BLAST) of the honeybee genome Alpha/Beta Hydrolases Long Chain Acyl CoA Synthases Fatty Acid Transport Proteins esaturases Cloning and sequencing of target genes Quantitative expression analysis In situ hybridization Recombinant protein expression In vitro activity assays The Honeybee Genome Sequencing Consortium. Nature 443, 931 (2006) 9

10 Real time PCR expression analysis of target genes Table 2. Relative expression analysis of target genes among worker bees: 1 day old, 15 day old & 15 day old-e treated. Sample α/β-3 LC-ACS FA FATP Head 1 day vs. 15 day 1 day vs. 15 day-e 15 day vs. 15 day-e U (0. 0) (1.8)* (2.2)** (1.9)** (2.4)** Thorax 1 day vs. 15 day 1 day vs. 15 day-e 15 day vs. 15 day-e (12.3)** (20.6)** (0.6)* (2.2)* (2.8)** (0.6)* Abdomen 1 day vs. 15 day (2.6)* 1 day vs. 15 day-e (3.3)** (2.0)* (10.9)** 15 day vs. 15 day-e (4.2)** αβ: alpha-beta hydrolase, ACS: acyl CoA synthase, LC-ACS: long chain-acs, FA: fatty acid desaturase, FATP: fatty acid transport protein. : up-regulated, : down-regulated Between brackets is the mean factor change of the gene rounded to the first decimal. *: PH < 0.05; **: PH <

11 Where is ethyl oleate biosynthesized in the bee? Four gene candidates: α/β-3, LC-ACS, FA, FATP. In situ hybridization (old nurses) RPL13a α/β hydrolase 3 LC-ACS FA FATP 11

12 In situ hybridization (forager thorax) CSG MB eye CA TSG Ant AL Pr HC α/β hydrolase 3 12

13 In situ activity assay (Forager sections) CSG MB C, F eye CA TSG E, H 2-Naphthyl leate EtH Hydrogen Peroxidase Horse Radish Peroxidase Ant Pr AL HC, G 13 13

14 The ester synthase: the product of the αβ-3 gene? E produced in the HC & exuded FPLC to exoskeleton mrna detected in the esophagus Transesterification occurs in the esophagus αβ-3 gene has a signal peptide E not detected in honey or bee regurgitate SS PAGE ka Activity F-7/8 F-14/15 F-23/24 F-42/43 F-57/58 Mix-F Regurg. E (ng) ± ± 2.9 N 6 ± ± 32 14

15 Peptide sequencing of honey bee regurgitate fraction Table 3. Proteins identified in the peptides isolated from the F-23/24 peak Protein XP_ escription Alpha glucosidase NP_ Major royal jelly protein 6 XP_ Similar to protein yellow precursor NP_ Major royal jelly protein 5 XP_ Choline dehydrogenase. Similar to CG9518-PA NP_ Glucose oxidase XP_ Similar to thioredoxin peroxidase 1 CG1633-PA XP_ Esterase lipase XP_ XP_ XP_ Prophenoloxidase-activating factor Similar to peroxiredoxin 2540 CG11765-PA Similar to Glutathione S Transferase S1 CG8938-PA NP_ dorant binding protein 21 15

16 Activity Assays of α/β-3 and EsLi Recombinant Proteins Recombinant α/β-3 Protein α/β-3 enzyme kinetics analysis 16

17 In vitro activity of recombinant α/β-3 and EsLi proteins 17

18 In vitro activity of recombinant α/β-3 and EsLi proteins 18

19 Seasonal changes of ethyl oleate (E) titers in honey bees A a ng of E / bee b 63.7 b A ng of E / Bee 21.7 b 56.4 a 68.1 a Nurses Pollen Foragers B 400 C 400 a ng of E / bee b c a a c b c b b b b b b b May June July August September 2008 Nurses Pollen Foragers ng of E / bee a a bc b b ab b bc b ab ab c ab a b May June July August September 2009 Nurses Pollen Foragers Nectar Foragers 19

20 Reactions followed in the experiments with caged bees and in vitro with honey crop fluids A. R' A H (Present in regurgitate ) d 6 -EtH ( ) GB11403 and GB13365 R' d 5 -E B. R R R H 2 unknown lipase R H d 4 -A ( ) GB11403 and GB13365 R d 9 -E d 4-3 TG R' = R = 20

21 E production by caged honey bees fed with syrup + d 6 -EtH, in the winter of

22 Effects of Methoprene on the biosynthesis of E in caged honey bee workers, 2011 Early spring Summer 22

23 Effects of Methoprene on the E biosynthetic activity of honey crop fluid from worker bees Early spring Summer 23

24 Seasonal variation in the E biosynthetic activity of honey bee crop fluid 24

25 Ethyl oleate biosynthesis and distribution in the colony 1) Esophagus & honey crop: A H + H secreted ester synthase E + H 2 N ALCHL WILL BE PERMITTE T BEES UNER 19 AYS F AGE 2) R R H R H lipase R Rapid E biosynthesis in the honey crop and transport to the bee s cuticle surface. 25

26 Age Polyethism in Honeybees Queen attendants Queen: QMP * cleaner Young brood: (E)-β-ocimene A. B. C. * Receivers Pollen forager: E (low) E EtH EtH E Nurses Activities Cleaning cells ld brood: BEP * Tending the queen Nursing Pollen forager: E (low) * Nectar forager: E (high) Nurse-to-forager transition * Receiving: interaction with foragers, first flights * EtH Foraging Nectar forager: E (high) * JH III & E exudation Age (d) Location Brood area Storage area utside of the nest Castillo et al.,

27 Conclusions Both nurses and foragers are capable of synthesize E. α/β 3 and EsLi synthesize E, express in the inner epithelium of the esophagus and/or secrete into the regurgitate. E is produced and accumulated in the honey crop and exuded via the bee cuticle. Methoprene, a JH analog, increases the biosynthesis and translocation of E into the cuticle The biosynthesis of E follows the growing season of the colony, reaching peaks in the summer. E is the product of a non-oxidative metabolism of ethanol. 27

28 Acknowledgments Funding: SFU: Erika Plettner Hao Chen Carolyn Graves Collaborations: Yves Le Conte Alban Maisonnasse Wolfgang Rӧssler James Watmough Human Frontier Science Program 28