Insect Biochem. Vol. 17, No. 1, pp. 53-59, 1987 0020-1790/87 $3.00+0.00 Printed in Great Britain. All rights reserved Copyright © 1987 PergamonJournals Ltd BIOSYNTHESIS OF THE ACETATE ESTER PRECURSOR OF THE SPRUCE BUDWORM SEX PHEROMONE BY AN ACETYL CoA: FATTY ALCOHOL ACETYLTRANSFERASE DAVID MORSE* and EDWARD MEIGHEN~" Department of Biochemistry, McGill University, 3655 Drummond Street, Montreal, Quebec H3G 1Y6, Canada (Received 24 December 1985; revised and accepted 23 April 1986) Abstract--The biosynthesis of l l-tetradecenyl acetate, the major storage precursor of the aldehyde pheromone of Choristoneurafumiferana, the eastern spruce budworm, has been found to be catalyzed by an acetyl-CoA: fatty alcohol acetyltransferase. In vitro, acetyltransferase activity was found almost exclusively in extracts from the pheromone producing gland, and could be demonstrated in vivo by topical application of ['4C]tetradecanol to the glands. Moreover, the activity was under developmental regulation, being low before and immediately after emergence of the moths from the pupal stage, and rising to a maximum in concert with the increase in glandular pheromone levels. Maximum activity with saturated alcohols was observed for acceptors of 12 to 15 carbons in chain length, with higher activities being found for the cis or trans monounsaturated analogs. The specificity of this enzyme with respect to substrate, morphological location and developmental regulation, indicates that it plays a key role in regulation of pheromone biosynthesis. Key Word Index: Insect pheromone, spruce budworm, acetate ester, acetyltransferase, aldehyde pheromone, Choristoneura fumiferana, tetradecenyl acetate, pheromone biosynthesis INTRODUCTION The identification and characterization of enzymes involved in pheromone metabolism are important steps leading to the understanding of the mechanism and regulation of pheromone biosynthesis in insects. Knowledge of the signal generating mechanisms for mating and their regulation is needed to understand how information is passed between individuals, and would be useful in the development of pest control methods designed to interrupt this process. However, very few studies have been initiated on the bio- synthesis of sex pheromones (Weaver, 1978; Blom- quist and Dillwith, 1983). The pheromone of the spruce budworm (Lepidop- tera:Tortricidae) is produced in a modified inter- segmental membrane located between the 8th and 9th abdominal segments (Roelofs and Feng, 1968; Percy and Weatherston, 1974). The pheromone gland can be recognized in the pupae of the female budworm several days before emergence of the adult moths, with its location and morphology being similar to glands from other lepidopteran species (Percy, 1974; Percy and Weatherston, 1974). The levels of the pheromone [(E):(Z)-ll-tetradecenal, 96:4] in the gland are low (~2 ng; Silk et al., 1980) compared to the amount (~40ng) of l l-tetradecenyl acetate *Present address: Harvard University, The Biological Lab- oratories, 16 Divinity Avenue, Cambridge, MA 02138, U.S.A. ?To whom correspondence should be addressed. (E:Z, 96:4) in the gland and the amount of pher- omone released by the insect each day (Silk et al., 1980; Morse et al., 1982). These observations have led to the proposal that the acetate ester was a precursor to the aldehyde, a conclusion supported by the identification of enzymes catalyzing this conversion (Morse and Meighen, 1984a) and by in vivo labelling studies showing that release of radiolabelled pher- omone coincided with a decrease in labelled glandu- lar acetate ester (More and Meighen, 1984b). Measurement of in vitro enzyme activities related to pheromone biosynthesis have only been recorded in a few instances (Clearwater, 1975; Hedin, 1977; Morse and Meighen, 1984a; Weatherston and Percy, 1976; Wolf and Roeiofs, 1983), and the enzymes have generally not been characterized. Such studies are hindered by the low amounts of material available for preparation of enzymes, the difficulty in establishing sensitive assay systems, and the often elaborate syn- theses required to prepare suitable radiolabelled sub- strates for in vivo analyses. Both in vitro and in vivo approaches are usually required, as the former is necessary for enzyme characterization while the latter is often necessary to position the enzyme correctly in the biosynthetic pathway. In this report we have combined the results of in vivo labelling studies with a radioactive assay devel- oped to directly measure acetyltransferase activity in vitro. Characterization of this enzyme activity showed that it was specifically located in the pheromone gland and increased concurrently with the measured aldehyde levels in the spruce budworm moth after 53