Detection of pheromone biosynthetic and degradative enzymes in vitro.

Highly sensitive, luminescent assays have been developed to measure enzyme activities involved in the metabolism of a major class of insect pheromones which includes fatty aldehydes, alcohols, and their acetate esters. These assays have been applied to measure the in vitro biosynthesis and degradation of the sex pheromone (trans:cis-11-tetradecenal, 96:4) of the eastern spruce budworm, Choristoneura fumiferana. Three activities were detected on analyses of extracts of the female moths: (a) an esterase that hydrolyzes both the cis and trans isomers of 11-tetradecenyl acetate, (b) an oxidase that converts fatty alcohols to aldehydes in the presence of O2, and (c) an NAD-dependent aldehyde dehydrogenase. The coupled luminescent response of bacterial luciferase to long chain aldehydes was used to measure rates of reaction as low as 0.1 pmol/min since only low amounts of material can be analyzed. Specific activities of these enzymes were higher in the pheromone producing gland than in other parts of the moth, implicating these enzymes, and the oxidase in particular, in the pathway of pheromone biosynthesis. The pathway was supported in vivo by demonstrating that topical application of 3H-labeled tetradecanyl acetate onto the insect gland resulted in the formation of [3H]tetradecanol and [3H]tetradecanoic acid, thus providing evidence that all three enzymes were functional in the living insects.