SUMMARYThe surface of insects is covered by a complex mixture of cuticular hydrocarbons (CHCs) to prevent desiccation. In many species these lipids also have communicative functions, but often it is unknown which components are crucial for the behavioural response. Furthermore, it is often ignored that polar lipids also occur on the insectsʼ cuticle and might interact with CHCs. In the parasitic wasp Lariophagus distinguendus, CHCs function as a contact sex pheromone eliciting wing-fanning in males. Interestingly, not only females but also newly emerged males have the pheromone, resulting regularly in homosexual courtship. However, males deactivate the pheromone within the first two days after emergence. This deactivation is accompanied by the disappearance of 3-methylheptacosane (3-MeC27) and some minor components from the CHC profile of males. Here we show that 3-MeC27 is a key component of the contact sex pheromone which, however, triggers courtship behaviour only if an olfactory background of other cuticular lipids is present. Males responded to (S)-3-MeC27 enantioselectively when applied to filter paper but on three-dimensional dummies both enantiomers were behaviourally active, suggesting that physical stimuli also play a role in sexual communication of the wasps. Finally, we report that triacylglycerides (TAGs) are also essential components of the pheromone, and present evidence that TAGs actually occur on the cuticle of L. distinguendus. Our data provide novel insights into the semiochemical function of cuticular lipids by showing that the bioactivity of CHCs may be influenced by the stereochemistry and a synergetic interaction with long time ignored TAGs.

Solvent extraction of bioactive molecules from glands, tissues, or whole organisms is a common first step in chemoecological studies. Co-extraction of a surplus of high boiling materials such as triacylglycerides (TAGs) and other lipids with higher molecular weight might hamper the identification of volatile or medium-volatile semiochemicals by high resolution chromatographic and spectroscopic techniques. Therefore, effective clean-up procedures are needed to separate potential semiochemicals from the accompanying materials. Size exclusion high performance liquid chromatography (SE-HPLC), a technique often disregarded by chemoecologists, has proved to be a rapid and efficient clean-up method for complex crude extracts. We demonstrated that TAGs can be baseline separated from typical semiochemicals within less than 10 min on a porous gel stationary phase based on highly cross-linked polystyrene/divinylbenzene. We applied the method as a rapid one-step clean-up procedure for the analysis of juvenile hormone III in insect hemolymph by gas chromatography-mass spectrometry. We furthermore introduced some recent application examples on insect pheromones to demonstrate that SE-HPLC is not only an effective method for the purification of crude extracts, but can as well be used as a first fractionation step for the bioassay-guided identification of behavior modifying natural products. SE-HPLC can be well operated with low-boiling solvents such as dichloromethane, and results in fraction volumes of typically less than one ml, which decreases the danger of losing volatile analytes during subsequent concentration steps.