Identification of Trail Following and Alarm Pheromones of Lasius Flavus Using Bioassay-Directed Fractionation

The identity of trail following pheromones secreted by the ant Lasius flavus were investigated using bioassay-directed fractionation techniques. Extracts of whole bodies were fractionated by high performance liquid chromatography and fractions tested for trail following activity in circular bioassays. Active fractions were analysed by gas chromatography- mass spectrometry (GC-MS) and structures of potential pheromones were confirmed by comparison with synthetic standards. The pheromones 2,6-dimethyl-5-heptenol (DMH) and mellein were identified in active fractions from extracts of whole body and headless ants respectively. Targeted analyses revealed that DMH was detected in the heads of worker ants at a concentration (mean ± SD) of 1.12 ± 0.58 ng per head. Mellein (stereoisomer not identified) was detected at 5.56 ± 1.4 pg per hindgut which was the lowest concentration of this compound detected in an ant species. In comparative bioassays of commercially available standards, (R)-mellein resulted in trail following activity at 0.01 pg per cm which is the lowest known detectable concentration of a pheromone by an ant, whereas a stereoisomeric mixture of DMH was 100-fold less active. These pheromones were tested for alarm activity by measurement of the mandible opening response in L. flavus worker ants. Standard DMH was found to cause a heightened level of aggression in at concentrations of ≥ 1 pg/l, indicating a likely function of an alarm pheromone. The response to (R)-mellein at the same concentrations was always lower, in keeping with its primary function of a trail following pheromone. This work reveals that bioassay directed fractionation can be a useful approach to identify the different behavioural pheromones in a social insect.