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 Pergamon Journals 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

54 DAVID MORSE and EDWARD MEIGHEN

emergence from the pupae (Grant et al., 1982). Since many other species of Lepidoptera use acetate esters directly as sex phermones (Inscoe, 1982), the mech- anism and regulation of the biosynthesis of the spruce budworm acetate ester may provide a general model for pheromone biosynthesis.

MATERIALS AND METHODS

Reagents

NADH, NADPH, acetyl-CoA, saturated alcohols and carboxylic ester hydrolase were supplied by Sigma. Un- saturated alcohols and aldehydes were obtained from Dr G. Grant of the Forest Pest Management Institute, Sault Ste Marie, Ontario, and were i>95% pure as judged by gas chromatography. [3H]Acetyl-CoA (200mCi/mmol), pre- pared by reaction of CoA with [3H]acetic anhydride (New England Nuclear), was purified by repeated ether extraction according to the method of Stadtman (l 957). Radiolabelled tetradecanols were prepared by reduction of either [9,10-3H]tetradecanoic acid (245mCi/mmol; custom syn- thesized by NEN) or [lJ4C]tetradecanoic acid (3t mCi/ mmol; Amersham, England) with LiAIH4 in dry ether, and were TLC purified prior to use. All solvents were supplied by Fischer, and were reagent grade. Phosphate buffers were prepared by mixing appropriate amounts of NaH2PO4 and K2HPO4.

Enzyme preparations

Spruce budworm moths, received as pupae from Dr G. Grant, Forest Pest Management Institute, Sault Ste Marie, Ontario, were allowed to emerge under natural window lighting. Crude gland homogenates containing 10excised glands/ml in 1.0 ml of 50 mM phosphate buffer, pH 7.0 (~200#g/ml of protein) were prepared at 4°C using a motor driven Teflon pestle (Morse and Meighen, 1984a). Clarified homogenates were obtained by centrifugation of the crude extract at 13,000g for 15 rain. The pellet of a further centrifugation at 105,000g for 90 min (designated the microsomal fraction) was resuspended in 50 mM phos- phate buffer, pH 7, by brief sonication.

Acetyltransferase assays The assay is based on the transfer of a radiolabelled

acetate moiety from an aqueous soluble substrate to the hexane soluble product. Typical assays were carried out by incubation of ~< 10/~g protein from the clarified homoge- hate (~< I/~g protein from the microsomal fraction), 10 nmol long chain alcohol and 31 nmol of [3H]acetyl-CoA in 1.0 ml phosphate buffer (0.05 M, pH 7.0) for 60 rain at room temperature (23 + I°C). Long chain alcohols could be added directly as a solution in hexane as long as the hexane was less than 0.1% v/v in the assay. The radioactive product was extracted from the aqueous phase with 1.0 ml aliquots of hexane after addition of either 30 #g (E)-1 l-tetradecenyl acetate or a mixture of 130/tg tetradecanyl acetate, 75 pg tetradecanol, 20/~g tetradecenal and 90/~g tetradecanoic acid. Extraction of the radioactivity was 60% with 1.0 ml hexane and 95% with 3 x 1.0 ml hexane. Several different 50 #1 aliquots of the hexane extract were dissolved in 10 ml Econofluor and counted with 45% efficiency in an Inter- technique scintillation counter. The units of enzyme activity were calculated as pmol acetate ester produced per rain from the specific radioactivity of the substrate (200 cpm/pmol) after subtraction of a background sample (without gland extract) carried through the same procedure. The back- ground increased linearly with the concentration of [3H]acetyl-CoA, so relatively low concentrations of this substrate were used in the assays. All activities were mea- sured in extracts from 2 to 6-day-old insects unless indicated otherwise.

Alcohol oxidase and acetate esterase assays

These activities were measured by the light emitted in the luminescence coupled assays for long chain aldehydes as described previously (Mors e and Meighen, 1984a).

Thin layer chromatography

Hexane extracts were generally chromatographed on Machery-Nagel silica gel TLC plates in a solvent of hexane~tiethyl etherqfcetic acid (90:10:2) as described (Morse and Meighen, t984b). AgNO3-silica gel plates were prepared by spraying the plates with a 7.5% solution of AgNO3 in 90% ethanol. Saponification of samples before analysis by TLC was carried out at 60'~C for 60 rain in a solution of 0.5 N KOH in 90% methanol. Radiolabelled lipids were visualized by autoradiography after a 3 7 day exposure to Kodak X-AR OMAT film at -70 'C.

In vivo labelling

The dimethyl sulfoxide treatment of Bjostad and Roelofs (1981) was used to introduce radiolabelled precursors into the gland. Lipids were extracted from the gland and run on TLC as described (Morse and Meighen, 1984b).

RESULTS

In vitro characterization o f acetyltransferase activity

Extracts from the pheromone gland of female spruce budworm moths were found to catalyze a reaction between [3H]acetyl-CoA and (E ) - l l - tetradecenol (E l I -14:OH) to form a radiolabelled product soluble in hexane. Format ion of this product was linear with time and required the presence of both the long chain alcohol and the gland extract (Fig. 1). Analysis of the reaction mixture by thin layer chromatography (Fig. 2) revealed that a single prod- uct was formed which comigrated with a standard of (E)- l l - te t radecenyl acetate (EII-14:Ac). Using a labelled alcohol ([3H]tetradecanol) and unlabelled acetyl-CoA, the analogous ester (tetradecanyl ace- tate) could also be synthesized by the gland extract. Identification of the product as an acetate ester was confirmed by saponification, which released the labelled fatty alcohol (Fig. 2). As neither malonyl- CoA nor acetate ( + ATP) could substitute for acetyl- CoA, this enzyme was designated as an acetyl- CoA:fa t ty alcohol-O-acetyltransferase. Over 80% of this activity could be centrifuged at 105,000g (90 min) indicating that it may be part of the micro- somal fraction. This step resulted in an increase in total activity of about 50% with an increase in specific activity (units enzyme activity/#g protein) of over 20-fold.

Kinetics studies on the acetyltransferase in the extract and on the partially purified enzyme (micro- somal fraction) give the same results for the de- pendence of activity (v) on substrate concentration (S). Hyperbolic plots for v versus S were obtained with acetyl-CoA and different alcohols (Fig. 3). Lineweaver-Burk plots were used to meaure appar- ent K~s of 8 p M for acetyl-CoA and 2-5 # M for the 14 carbon monounsaturated and saturated alcohols. A comparison of the specificity of the reaction for the chain length of the fatty alcohol (Fig. 4) showed that the enzyme had a clear preference for alcohols with chain lengths between 12 and 15 carbons. The en- zyme preferred monounsaturated fatty alcohols over the saturated isomers (Fig. 3) but could not dis-

55 Acetate ester biosynthesis

1400-

1200 .’

1000 .’

800.

600.

400.

0 IO 20 30 40

Time (minutes)

I

50 60

Fig. I. Formation of a hexane soluble product from [Wlacetyl-CoA and fatty alcohol by gland extracts. Reaction mixtures containing 10 PM El l-14:OH, 31 PM [‘Hlacetyl-CoA and 8 yg clarified gland homogenate in 1 .O ml 50 mM phosphate, pH 7.0, were extracted with 1 .O ml aliquots of hexane at the indicated times (+) and the radioactivity (in cpm) of 50 ~1 aliquots determined. No reaction was observed

in the absence of either gland homogenate (V) or tetradecanol (0).

tinguish between unsaturation at the A9 and All positions (data not shown).

Morphological distribution of acetyltransferase in the spruce budworm

The acetyltransferase activity was found to be located almost exclusively in the gland with the measured activity in extracts of other body parts of the adult female moth at background levels (< 10% of the gland extract). In an attempt to measure these lower activities, the background response was re- duced by purification of the hexane extract on TLC (as in Fig. 2) and the region on the TLC plate containing the acetate ester counted. The specific activity of the acetyltransferase in the gland extracts was found to be at least lOO-fold higher than in extracts from other parts of the insect (Table 1). Mixing of different extracts showed that the low levels of activity in parts of the insect other than the gland were not due to the presence of inhibitory factors present in those extracts (data not shown).

Dependence of acetyltransferase activity on insect development

The amount of acetyltransferase activity in the

Table 1. Morphological distribution of acctyltransferase activity*

pm01 ester formed/hr per

Body part pg protein

Gland 7.3 * 0.2 Head 0.05 + 0.03 Abdomen 0.02 f 0.02

kg 0.07 f 0.07 Th0riU 0.02 f 0.003

*The amount of ester formed after a 60min incubation in uirro with a clarified homogenate was calculated from the radioactivity comigrating with unlabelled (Q-1 1 -tetradccenyl acetate on silica gel TLC and the specific radioactivity of the [‘Hjaeetyl-CoA precursor. The data is the average of two experiments.

gland homogenates was found to vary with the age of the adult moth. As shown in Fig. 5, low levels of activity were found in extracts of glands from pupae as well as in extracts of adult moths just after emergence from the pupal cocoons. Activity in the gland homogenates reached a maximum by 2 days after emergence of the moths, and remained at a high

- 6’ .c (4 E 15 z P

;; 4.

Y

Q 3-

E2

1 *

0 12345 10

C AcCoA 1 (pM 1 C ALCOHOL 1 (pM )

Fig. 3. Kinetics of acetyltransferase. The standard reaction mixture contained 10 FM fatty alcohol, 31 PM [‘H]acetyl-CoA and 10 pg/ml clarilkd gland homogenate. The concentration of acetyl-CoA was varied in (A), while alcohol concentrations were varied in (B). Tetradecanol (a), El l-14:OH (0) and

21 I-14:OH (0) were the alcohol substrates employed.

56 DAVID MORSE and EDWARD MEIGHEN

level over the next 3-4 days. The enzyme activity in gland extracts of 2-6 day old moths was not inhibited by addition of gland extracts from pupae, showing that the higher activity of the acetyltransferase in extracts of adult moths did not arise from removal of an inhibitor. The development of this activity con- trasts sharply with the acetate esterase and alcohol oxidase activities in the gland which are independent of the age of the moth and have high levels in the glands from the pupae (Fig. 5). The acetyltransferase activity appears to closely parallel the levels of al- dehyde pheromone in the spruce budworm (Grant et aL, 1982). Before or immediately after (~<4hr) emergence from the pupae, there is less than 0.3 ng of aldehyde pheromone in the gland whereas the adult moth contains an average of 2 to 3 ng of pheromone.

In vivo labelling

To confirm the presence of acetyltransferase activ- ity in vivo, [14C]tetradecanol was applied to the pher- omone gland as a solution in dimethyl sulfoxide. The relative incorporation of the alcohol into the acetate ester was about four-fold higher than the incorpo- ration from tetradecanoic acid (Fig. 6A). Unlike the acetate ester produced from labelled tetradecanoic acid, the label from tetradecanol was found to be principally in the saturated isomer of the ester (Fig. 6B) with the amount of unsaturated ester formed being approximately the same when either the acid or the alcohol was used as a precursor (Fig. 6B). These results indicate that the saturated acetate ester was formed by direct incorporation of the long chain alcohol into ester and 15rovides strong evidence for the presence of acetyltratasferase activity in the gland of the living insect.

DISCUSSION

The specificity of the acetyltransferase enzyme for long chain fatty alcohols~ coupled with its presence only in mature glands of female moths capable of producing pheromone, provides strong evidence that this enzyme is directly on the pathway of pheromone

7,

6.

uJ 5 ,< nF

~->- 4

W

u 2

8 10 12 13 14 15 16 18

ALCOHOL CHAIN LENGTH

Fig. 4. Substrate specificity of acetyltransferase. Reaction mixtures contained 10#M saturated fatty alcohol of the indicated chain length, 195pM [3H]acetyl-CoA and 0.8 #g/ml microsomal protein. Acetyltransferase activities (pmol ester produced per min) are the a~erage ( _+ SD) of six

analyses.

biosynthesis. This result provides independent confirmation that the acetate ester is a precursor to the aldehyde pheromone (Silk et al., 1980; Morse and Meighen, 1984b). Enzymes capable of hydrolyzing the acetate ester to a fatty alcohol (acetate esterase) with oxidation to the aldehyde pheromone (alcohol oxidase) have already been demonstrated to be present in glandular extracts (Morse and Meighen, 1984a). Such a biosynthetic pathway may be advan- tageous for the insect since it produces a stable high energy precursor (acetate ester) that can be stored in the gland and then converted by simple hydrolysis

PUPA i A D U L T ~_ 12 ,60

o 1o ' ' 50 ~

>- F- < ~ 8 I " 4 0 H m l >~- LU LL ~, 6 ' ~ o b ~ z '~ ud n..-

0 • 0 -2 - I O 1 2 3 4 5 6 7

AGE AFTER ECLOSION (DAYS)

Fig. 5. Dependence of acetyltransferase activity on insect age. Activities, given as pmol product produced per min per gland, were measured in crude homogenates for acetyltransferase (O), alcohol oxidase (O) and acetate esterase (A) as described in Materials and Methods. Each point is the average of at least 12 assays, with the bars representing the standard deviation. The insect age before eclosion was estimated

relative to the time when the majority of the insects had emerged.

1 2 3 4 5

Tetrade A(

~L

Tetrade

Fig. 2. Characterization by TLC and autoradiography of the reaction product. Shown is the auto- radiogram of the silica gel TLC plate for hexane extracts from the different reaction mixtures. The labelled product from a reaction (60 min) between 1 #M E11-14:OH and 78 #M [3H]acetyl-CoA catalyzed by the gland homogenate (8 #g protein/ml) comigrates with a standard of (E)-I 1-tetradecenyl acetate (lane 1). Reaction between 0.8 gM [3H]tetradecanol and 78/~M acetyl-CoA catalyzed by the gland extract gives a product comigrating with tetradecanyl acetate (slightly above the unsaturated isomer) plus the labelled alcohol substrate (lane 3) whereas only the fatty alcohol is observed in the absence of gland extract (lane 2). Extraction of the ester from the TLC in lane 3 and rechromatography before (lane 4) and after

saponification (lane 5), shows the product contains an ester linkage.

57

A 1 2

Hydrocarbon Q

Wax Ester

TDA

Triglyceride

B

sat.TDA

E 11 .TDA

1 2

Z 11-T DA

Fatty Acid

Alcohol _~

Phospholipid Origi n ,

Fig. 6. In vivo labelling of glandular lipids with tetradecanoic acid and tetradecanol. (A) Autoradiogram of a silica gel TLC plate on which were run the glandular lipids incorporating radiolabel from [14C]tetradecanoic acid (lane 1) and [14C]tetradecanol (lane 2). Groups of 16 insects were labelled in vivo for 90 min with 105 cpm per gland. Unlabelled standards, run in parallel, were visualized with 12 vapour. In this system, hydrocarbons run with the solvent front while phospholipids remain at the origin. (B) Autoradiogram of an argentation TLC plate, on which were run samples of the acetate ester (TDA) (extracted from a silica gel TLC as in A) labelled in vivo with [~4C]tetradecanoic acid (lane 1) or [~4C]tetradecanol (lane 2). The acetate esters (TDA) can be resolved into the saturated (sat) as well as the

ciz (Z 1 l) and trans ( E l l ) isomers of tetradecenyl acetate.

58

Acetate ester

and oxidation to the less stable aldehyde pheromone without requiring additional energy.

Although both the acetyltransferase and the alco- hol oxidase utilize fatty alcohols as substrates at similar rates, these enzymes are presumably located in separate metabolic compartments in vivo. In this regard, the acetyltransferase is found in the micro- somal fraction whereas both the acetate esterase and alcohol oxidase enzymes remain in the soluble fraction on high speed centrifugation (Morse and Meighen, 1984a). The sedimentable fraction contain- ing the acetyltransferase would also contain any glandular smooth endoplasmic reticulum (SER), a subcellular fraction implicated in lipid metabolism in other systems. As electron microscopic studies have shown extensive SER in the gland cells (Percy, 1974) this is a possible intracellular location for the enzyme.

The high level of incorporation of radioactivity into the saturated ester on topical application of labelled tetradecanol to the gland of the female moth provides evidence that the acetyltransferase is re- sponsible for ester biosynthesis in vivo. Although label is also incorporated into the unsaturated ester both from tetradecanol and tetradecanoic acid, recent metabolic studies have shown that the incorporation of label into unsaturated ester from fatty acids arises by degradation and synthesis de novo of the long chain backbone (Morse and Meighen, 1984b). Incor- poration of radiolabel from tetradecanol into un- saturated ester (EZ11-14:Ac) could thus result from oxidation of the alcohol to acid followed by degra- dation and resynthesis. In contrast, incorporation of label into saturated ester (14:Ac) occurs only on topical application of radioactive tetradecanol and the total incorporation into the acetate ester is much larger (~4-fold) than with tetradecanoic acid. The increase in incorporation of radioactivity appears to arise exclusively from a specific increase in the bio- synthesis of 14:Ac by direct incorporation of labelled tetradecanol. This result also indicates that de- saturation of the long" chain backbone to form E Z 11-14: Ac does not occur at the level of the alcohol or ester but must occur at an earlier step in the biosynthetic pathway, presumably by desaturation of an activated acyl derivative (e.g. acyl-CoA) as has been described in other biological systems (James, 1977; Jeffcoat, 1979). In-this regard, the substrate specificity of the acetyltransferase in vitro does not account for the isomeric ratio ( Z / E ) of the un- saturated acetate ester (11-14:Ac). Although the de- saturase may be a logical candidate, additional con- trol of the isomeric ratio could be exerted on reduction of the fatty acid to form the alcohol.

The induction of acetyltransferase activity during the emergence of the insect from the pupae is clearly distinct from the daily rhythm of pheromone release which occurs during the early and middle scotophase (Morse et al., 1982). Consequently, the biosynthesis of the acetate ester can also occur in the photophase replacing any molecules converted to and released as the aldehyde pheromone at night, This acetyl- transferase activity, found only in the pheromone gland, has its maximum levels in adult moths and closely follows the levels of aldehyde pheromone in the glands with insect development (Grant et al., 1982). It is of interest that two neurohormones

biosynthesis 59

(juvenile hormone and fl-ecdysone) have been found to have increased titers in other insects during the late pupal and early adult stages (Riddiford and Truman, 1978) raising the possibility that these hormones might control the development of the pheromone biosynthetic machinery.

The existence of an acetyl-CoA:fatty alcohol acetyltransferase with a high specificity for fatty alcohols has not previously been noted in other biological systems perhaps reflecting the specialized evolution of the communication system in insects. As there are much larger numbers of insects producing ester pheromones compared to those using aldehydes (or alcohols) as pheromones (Inscoe, 1982), it may be that the latter compounds evolved from the original acetate ester pheromone resulting in reproductive isolation and the development of new insect species. If true, then the acetyltransferase activity may be a common biosynthetic enzyme in insects utilizing ace- tate esters, aldehydes and alcohols as pheromones and its presence could serve as a diagnostic test for insects producing these pheromones.

Acknowledgements--We thank Dr Gary Grant of the Forest Pest Management Institute, Sault Ste Marie, Ontario for his generous supply of spruce budworm pupae and his expert advice throughout the course of this work and Rosza Szittner for her assistance in many phases of the research. Supported by a Medical Research Grant (MT 4314) and a MRC Studentship (D.M.).

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