Journal of Chemical Ecology, Vol. 15, No. 10, 1989

IDENTIFICATION OF SEX PHEROMONE COMPONENT OF SPRUCE BUDMOTH Zeiraphera canadensis 1

P.J. SILK, 2 E.W. B U T T E R W O R T H , 2 L.P.S. K U E N E N , 2 C.J. N O R T H C O T T , 2 E. D U N K E L B L U M , 3 and E.G. K E T T E L A 4 ZPheromone Research Group, Research and Productivity Council P. O. Box 20000, College Hill Road Fredericton, New Brunswick Canada E3B 6C2 31nstitute of Plant Protection Volcani Institute Bet-Dagan, 50-250, Israel 4Forestry Canada--Maritimes P. O. Box 4000 Fredericton, New Brunswick Canada E3B 5P7

(Received November 9, 1987; accepted December 27, 1988) Abstract--The analyses of virgin female sex pheromone gland extracts by gas chromatography (GC), GC-electroantennographicdetection (GC-EAD) and GC-mass spectrometry (GC-MS) followed by field-trapping experiments, have identified(E)-9-tetradecenylacetate (E9-14 : Ac) as the primary sex pheromone component of the spruce budmoth, Zeiraphera canadensis. Dosages of 1.0-100.0/~g of E9-14 : Ac impregnatedin rubber septa provide effective trap baits. Key Words--Zeiraphera canadensis, spruce budmoth, (E)-9-tetradecenyl acetate, sex pheromone, Lepidoptem, Tortricidae, Eucosminae,trapping.

INTRODUCTION Two species in the budmoth group, the spruce budmoth, Zeiraphera canadensis Mutuura and Freeman, and the purplestriped shootworm, Z e i r a p h e r a unfortunana Powell [ = Z. destitutana (Walker)], are sympatric in New Brunswick and feed on white spruce [Picea glauca (Moench) Vossl. These species feed on 1Lepidoptera: Tortricidae: Eucosminae. 2435 0098-0331/89/1000-2435506.00/0 9 1989 Plenum Publishing Corporation

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SILK ET AL.

developing apical shoots (Martineau, 1984), which results in reduced tree height and bole deformation. The primary sex pheromone component of Z. unfortunana has recently been identified as (E)-9-dodecenyl acetate (E9-12 : Ac) (Silk et al., 1988). The pheromone for Z. canadensis has not been identified; however, (E)-9-tetradecenyl acetate (E9-14:Ac) has been found to be electrophysiologically active and effective in trapping experiments (Turgeon and Grant, 1988). We report here the identification of E9-14:Ac as the primary sex pheromone component of Z. canadensis. This was achieved by analyses of sex pheromone gland extracts using GC, GC-EAD, and GC-MS and field-trapping experiments.

METHODS AND MATERIALS

Insects. During 1986, white spruce foliage (50-cm branches), infested with Z. canadensis and Z. unfortunana larvae, was collected. The branches were suspended over trays of moist vermiculite in rooms at constant temperature (25~ and relative humidity (70-80%). Mature larvae subsequently dropped into the trays where pupation occurred. The two species were then initially separated on the basis of pupal size (Z. unfortunana is larger than Z. canadensis), sexed (Turgeon, 1985), and emerged adults were separated by differences in thoracic and wing maculations. Adults were maintained in small cages under the same environmental conditions as were pupae (Silk et al., 1988). Collection of Pheromone. Virgin female Z. canadensis and Z. unfortunana were held one to two days postemergence, and their sex pheromone glands were manually everted and excised during the first 3-4 hr of scotophase. Four hundred twenty Z. canadensis glands were placed in Spectrograde hexane, and 40 glands were placed in chloroform-methanol (2:1 v/v) for subsequent methanolysis (Bjostad and Roelofs, 1984). In addition, 21 Z. unfortunana glands were also placed in chloroform-methanol for methanolysis. Z. canadensis glands, in hexane, were sonicated for 30 rain, filtered through clean glass wool, and the extract was then used for subsequent GC, GC-EAD, and GC-MS analyses. All gland extracts were stored in solvent at - 10~ prior to analyses. GC-EAD Analysis. Portions of the pheromone gland extract in hexane were analyzed on a GC-EAD system (Struble and Am, 1984; Silk et al., 1988) using detector antennae of 2- to 3-day-old male Z. canadensis and Z. unfortunana moths; EAD responses and GC retention times were also recorded from synthetic standards. Fatty Acid Analyses. Forty virgin female glands of Z. canadensis in chloroform-methanol (2 : 1) were sonicated for 20 min, extracted as above, and the

PHEROMONE OF SPRUCE BUDMOTH

2437

extracts were subjected to alkaline methanolysis (Bjostad and Roelofs, 1984) followed by reaction with dimethyl disulfide (DMDS) (Dunkelblum et al., 1985). These DMDS adducts, in hexane, were analyzed by GC-MS as detailed below. Methanolized gland extracts of Z. unfortunana were derivatized and analyzed similarly. GC and GC-MS Analysis. GC analyses were performed as previously described (Silk et al., 1988). The following columns were used: Column A, 30 m x 0.32 mm DB-5 (Chromatographic Specialties Ltd.); Column B, 30 m x 0.32 mm SPB-5 (Supelco); Column C, 50 m x 0.30 mm Superox FA (Mandel Scientific Ltd.). GC-MS analyses on extracts, adducts, and synthetics were performed as previously described (Silk et al., 1988) using column A. Chemicals. Chemicals used in the above procedure were obtained from Chem. Samp. Co., Columbus, Ohio; Koken Fine and Aromatic Chemicals, Tokyo, Japan; or were synthesized in our laboratory using standard techniques; all chemicals were 98 % pure ( < 0.1% opposite isomers) as determined by capillary GC and used without further purification. Field Testing. Field-trapping studies were carried out in 1986 in the same plantations where larvae were collected. Plots ca. 50 m apart were chosen in homogeneous white spruce stands that were planted in 1976. Two trapping experiments were conducted; the first was conducted for 11 nights (July 19-30) in a 6 (treatments) x 10 (replicates) randomized block. The second experiment was conducted for 10 nights (July 20-30) in a 5 (treatments) x 5 (replicates)randomized block. In both trapping experiments, Pherocon-lC traps were baited with red rubber septa lures (Arthur H. Thomas Co.) and suspended from white spruce foliage ca. 1.5 m from the ground (mid-crown) with 25-m spacings between traps. All moths caught in the traps were identified (Z. canadensis and Z. unfortunana males) by examining their genitalia (Mutuura and Freeman, 1966). Log-transformed data were analyzed by two-way ANOVA and means compared using Tukey's HSD multiple-comparison test.

RESULTS

Chemical and GC-EAD Analyses. From the 420 Z. canadensis glands obtained for analysis, aliquots containing ca. 50 female-gland equivalents in hexane (2 14) were injected on columns A, B, and C for GC-EAD analysis using male Z. canadensis as detector antennae. Several gland components produced small EAD responses, but only one discernible GC peak produced a large EAD response (on all columns). The GC retention time (columns A and B) of this EAD-active component closely matched that of synthetic 9-tetradecenyl

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SILK ET AL.

acetate (9-14 : A c ) . T h e retention t i m e o f the c o m p o n e n t suggested the E configuration o f the d o u b l e b o n d w h e n G C - E A D analysis was carried out on colu m n C (E and Z i s o m e r s w e l l resolved). G C - E A D responses o f the gland c o m p o n e n t (largest E A D response) and synthetic c o m p o u n d s are s h o w n in T a b l e 1. F o r c o m p a r i s o n purposes, G C - E A D responses o f m a l e Z. unfortunana antennae to several synthetics are also presented (Silk et al., 1988) (Table 1). T h e 9 - 1 4 : A c s clearly g a v e m e d i u m to large E A D responses, w h i l e the saturated analogs, at 1-ng dosages, elicited no response in Z. canadensis antennae but a range o f responses in Z. unfortunana antennae (Silk et al., 1988) (Table 1). G C - M S analysis was carried out on the s a m e D B - 5 capillary c o l u m n with the r e m a i n i n g Z. canadensis gland extract (ca. 120 gland equivalents). O n e w e a k l y E A D - a c t i v e r e g i o n c o r r e s p o n d e d in retention t i m e and M S characteristics to E 9 - 1 2 " A c [m/e 61 (CH3COOH~-) 68, 82, 166 ( M + - 6 0 ) ] . T h e M S scan corresponding to the region o f highest E A D response corresponded to E 9 - 1 4 : A c

TABLE 1. MEAN EAD RESPONSEOF MALE Z. canadensis AND Z. unfortunana ANTENNAE TO SOME SYNTHETICSAND PHEROMONEGLAND EXTRACTS Stimulus a

EAD Response (mV)

Z. canadensis E11-14:Ac E9-14:Ac E7-14:Ac E7-12:Ac E9-12:Ac E9-14:OH Zll-14:Ac Z9-14:Ac Z7-14 : Ac Z7-12: Ac Z9-12:Ac 12:Ac 14 : Ac 12 : OH 14:OH Major gland component d

0.4 3.0 0.3 0.2 2.5 1.8 0.5 2.2

+ 0.5 (N = 3)b + 1.6(N = 10) 5:0.3 (N = 3) 5:0.4 (N = 3) + 1.3 (N = 5) + 1.1 (N = 2) + 0.09 (N = 2) + 0.18 (N = 2) 0.4 0.7 2.1 + 1.7 (N = 3) 0 0 0 0 2.7 (46 FE)

Z. unfortunana 0c 1.6 5:1.3 (N 0 0 4.2 5:1.5 (N 0 0 0 0 0 2.8 _ 0.7 (N 2.1 5:0.9 (N 0.6 0.4 0 2.3 (5 FE)

"1 ng stimulus, i.e., 2 ng injected on GC. bx 5:1 SD. CNot distinguishable from background response (ca. 0.05 mV). dForty-six female equivalents (FE); on SPB-5 capillary column, 50-210~ at 8~

= 6)

= 5)

= 3) = 3)

2439

PHEROMONE OF SPRUCE B U D M O T H

[m/e 61 ( C H 3 C O O H ~ - ) 67, 82 (base) 9 6 , 110, 124, a n d 194 ( M + - 6 0 ) ] . I n s u f ficient m a t e r i a l w a s a v a i l a b l e f o r d o u b l e - b o n d c o n f i r m a t i o n b y m i c r o c h e m i c a l means. Additional supportive evidence for double-bond assignment resulted from t h e fatty acid a n a l y s e s t h a t are s h o w n in T a b l e 2 ( n o t e t h e p r e s e n c e o f 1 1 14 : M e in Z. unfortunana). A s e x p e c t e d , 9 - 1 4 : M e ( m e t h y l 9 - t e t r a d e c e n o a t e ) w a s d e t e c t e d , a n d this fatty a c i d is t h e p r e s u m e d p r e c u r s o r to t h e 9 - 1 4 : A c ( R o e l o f s a n d B j o s t a d , 1984), t h e p r i m a r y E A D - a c t i v e g l a n d c o m p o n e n t . I n a d d i t i o n , 9 - 1 2 : M e ( m e t h y l 9 - d o d e c e n o a t e ) w a s d e t e c t e d as e x p e c t e d f r o m E A D a n d M S data. T h e s e d a t a s u p p o r t t h e d o u b l e - b o n d a s s i g n m e n t i n t h e g l a n d derived 9-dodecenyl and 9-tetradecenyl acetates. G C - E A D a n a l y s i s ( c o l u m n B) o f Z. canadensis s e x p h e r o m o n e g l a n d

TABLE 2. FATTY ACID ANALYSES OF Z. canadensis AND Z. unfortunana VIRGIN FEMALE SEX PHEROMONE GLAND EXTRACTS MSb characteristics (m/e) Fatty acida

M+

A+

B+

C+

Relative amount per glandc

306 334 334 362 376 390

89 159 117 145 159 173

217 175 217 217 217 217

185 143 185 185 185 185

VS VS VS L VS VL

334 334 334 362

89 159 117 89 145

217 175 217 245 217

185 143 185 213 185

VS VS VS VS L

390

173

217

185

VL

Z. canadensis A9-12 : Me 2x6-14 : Me 2x9-14 : Me 2x9-16 : Me 2x9-17 : Me A9-18 : Me

Z. unfortunana 2x9-12 : Me A6-14 : Me A9-14 : Me h i 1-14 : Me A9-16 : Me A9-17 : Me 2x9-18 : Me

aGenerated as methyl esters in the methanolysis step; z~9-12 :Me methyl 9-dodecenoate. bSee Dunkelblum et al. (1985)

cHsi'"

CH3(CH2)m-CH~CH- (CH2)n-COOCH3

1

M+

I SCH 3 A+

B+

C+(B+--32)

cVL, very large (ca. 1 /zg); L, large (ca. 100 ng); VS, very small ( 0.05). Our study with pure components and blends of E 9 - 1 4 : Ac and E9-12 : Ac indicate that both E 9 - 1 4 : Ac and E 9 - 1 2 : Ac at the 10-/~g dosage, were effective in capturing Z. canadensis and Z. unfortunana, respectively (Table 4). In the three blends of the two acetates tested, higher concentrations of a specific chemical constituent resulted in higher trap captures of the species specific to the given acetate (e.g., the higher the proportion of E9-12 : Ac, the higher the proportion of Z. unfortunana captured). Addition of E 9 - 1 2 : A c , identified as a

TABLE 3. TRAP CAPTUREOF MALE Zeiraphera canadensis IN PHEROCON-1CTRAPS Ac AND BLENDS OF E9-14 : Ac WITH Z 9 14 : Ac ANDE9-14 : OH

BAITED WITH DIFFERENT DOSAGES OF E9-14:

Compounds Eg-14:Ac (1) E9-14:Ac E9-14 : Ac (1) + 10% Z9-14:Ac (1) + 10% E9-14:OH Check (blank trap)

Dosage (/~g)

Trap capture (mean + 1 SD)~

1 10 100 1 1

37 +_ 13.0a 29 _+ 21.4a 38 • 15.9a 47 + 24.9a 36 + 39.2a 11 + 7.8b

aMeans followed by same letter are not significantly different; P > 0.05.

PHEROMONE OF SPRUCE BUDMOTH

2441

TABLE4. MEANNUMBEROFZeiraphera canadensis ANDZ. unfortunana CAUGHTIN TR~PS BA~TEDW~THPROPORTXONALBLENDSor E9-14 : Ac ANDE9-12 : Aca Blend (%) E9-14:Ac

E9-12:Ac

100 80 50 20 0

0 20 50 80 100

Z. canadensis

Z. unfortunana

(X + 1 SD)

(X _ 1 SD)

15 9 5 4 3

___ 10.3 + 6.9 + 3.3 + 3.1 +_ 1.9

0 3 16 38 52

+ 0 + 2.3 + 7.0 + 7.1 ___ 27.8

~A total of 10 ~g (see text for details).

gland component, to E9-14 :Ac, therefore, did not increase male Z. canadensis trap capture at the tested dosages. DISCUSSION

Field-trapping data in this work indicate that E9-14:Ac, impregnated in rubber septa, in the 1- to 100-/xg range, effectively traps Z. canadensis males. A dose-response, however, could not be demonstrated, and this could be explained by mid-crown placement of traps. Turgeon and Grant (1988), for example, clearly showed a dose-response with E9-14 :Ac when traps were placed at the tops of trees. Addition of either Z9-14 : Ac or E9-14 : OH (Table 3) to E9-14 : Ac does not affect trap capture. Traps with blends of E9-12 : Ac (a sex pheromone component ofZ. unfortunana; Silk et al., 1988) and E9-14 : Ac capture both species (Table 4), but the pure components appear to be reasonably specific in capturing one species only. The role of E 9 - 1 2 : A c in the sex pheromone glands of Z. canadensis is, therefore, unknown at this time. Several lepidopteran sex pheromone components have been shown to be biosynthesized by reaction of fatty acids with two key enzyme systems that are found only in sex pheromone glands (Roelofs and Bjostad, 1984). These are a microsomal/3-oxidation system, which yields limited chain shortening by two carbons, and a All-desaturase system. The characterized pheromones in the coniferophagous Choristoneura budworms, for example, can all be generated by these two enzyme systems starting from hexadecanoic acid (Wolf and Roelofs, 1987; Silk and Kuenen, 1988). The comparison of monounsaturated fatty acids in these two Zeiraphera species is interesting in this regard (Table 2): methyl 9-dodecenoate and methyl 9-tetradecenoate were identified in both spe-

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SILK ET AL,

cies, and corresponding acids are the presumed biosynthetic precursors to the identified unsaturated acetate pheromones. Other identified fatty acids may represent precursors to minor pheromone components. A plausible biosynthetic route to these acetate pheromones in both species is shown in Fig. 1. Both would be generated from hexadecanoate. For Z. canadensis, the A11-desaturase (E-specific) inserts the double bond followed by limited 13-oxidation to a (E)9-tetradecenoyl moiety. Reduction and acetylation would produce the required E9-14:Ac. With Z. unfortunana, /3-oxidation would occur first followed by ~ll-desaturation (E-specific); a second f3-oxidation step, then reduction and acetylation would yield E9-12 :Ac. The fatty acid analyses reported in Table 2 support this scheme. Second, recent trapping studies in white spruce stands in Nova Scotia have found E11-14:Ac to be potently active in capturing Zeiraphera fortunana (Butterworth and Silk, unpublished data). Z. fortunana is a relatively rare species found on white spruce in New Brunswick and Nova Scotia (Martineau, 1984). The male genitalis of Z. fortunana are morphologically

Acety~ CoA I

malonylCoA I NADPH ~

FattyAcid Synthase

Palmitoyl-• (16:Acyl) -All

I}

14:Acyl ~ -All

_EII-16:Acyl

E_ll-14:Acyl

E_9-14:Acyl Reductase

E9-12:Acyl ~ Reductase

E.E_ 9-14:0H

E_9-12:0H

transferase

transferase

E_g-14:Ac

E 9-12:Ac

Z. canadensis

Z. unfortunana

FIG. 1. Postulated divergent biosynthetic routes to the sex pheromones ofZ. canadensis and Z. unfortunana. After Roelofs and Brown (1982) and Roelofs and Bjostad (1984). -zXl 1 = A11-desaturase; t3 = limited 13-oxidationby two carbons.

PHEROMONE OF SPRUCE BUDMOTH

2443

very difficult to distinguish from Z. unfortunana and the two species are considered to be closely related (P. Dang, Biosystematics Institute, Agriculture Canada, personal communication). The fact that E11-14:Ac captures Z. fortunana is consistent with the postulated biosynthetic scheme outlined above and supports a close taxonomic relationship between Z. unfortunana and Z. forn~ana.

Roelofs and Brown (1982) suggest a similar relationship between the two forms of Z. diniana, which occur on different host species in Europe. Z. diniana (larch form) produces E l l - M : Ac (Roelofs et al., 1971; Guerin et al., 1984) while Z. diniana (cembran pine form) produces E9-12 :Ac (Baltensweiler et al., 1978, Guerin et al., 1984). Acknowledgments--We thank Rory Mclntosh, Azza Seif el Nasr, and Michael Chandra (RPC) for technical assistance; Sharon Knox and Charles Weaver (CFS) for insect maintenance; and J.D. Irving, Limited (Woodlands Division) for their assistance and for the use of their property. We thank J. Turgeon and G.G. Grant (FPMI, Forestry Canada) for their cooperation during this project. Financial support was provided through the Canada/New Brunswick Forestry Development Agreement.

REFERENCES BALTENSWEILER, W., PRIESNER, E., ARN, H., and DELUCCHI, V. 1978. Unterschiedliche Sexuallockstroffe bei Lfirchen und Arvenform des grauen L~irchenwicklers. Mitt. Schweiz. Entomol. Ges. 51:133-142. BJOSTAD, L.B., and ROELOFS, W.L. 1984. Sex pheromone biosynthetic precursors in Bombyx mori. Insect Biochem. 14:275-278. DUNKELBLUM,E., TAN, S.H. and SILK, P.J. 1985. Double-bond location in monounsaturated fatty acids by dimethyldisulfide derivatization and mass spectrometry: Application to analysis of fatty acids of four Lepidoptera. J. Chem. Ecol. 11:265-277. GUERIN, P.M., BALTENSWEILER,W., ARN, H., and BUSER, H.R. 1984. Host race pheromone polymorphism in the Larch Budmoth. Experientia 40:892-894. MARTINEAU,R. 1984. Insects harmful to forest trees. Canadian Forestry Service, Agriculture Canada, Multiscience Publications Ltd., Cat. No. Fo 64-32/1984E. MUTUURA, A., and FREEMAN, T.N. 1966. The North American species of the genus Zeiraphera. J. Res. Lepid. 5(3): 153-176. ROELOFS, W.L., and BJOSTAD,L.B. 1984. Biosynthesis of lepidopteran pheromones. Bioorg. Chem. 12:279-298. ROELOFS, W.L., and BROWN, R.L. 1982. Pheromones and evolutionary relationships of tortricidae. Annu. Rev. Ecol. Syst. 13:395-422. ROELOFS, W.L., CARDt~, R., BENZ, G., and VON SALIS, G. 1971. Sex attractant of the larch budmoth found by electroantennogram method. Experientia 27:1438-1439. SILK, P.J., and KUENEN, L.P.S. 1988. Sex pheromones and behavioral biology of the coniferophagous Choristoneura. Annu. Rev. Entomol. 33:83-101. SILK, P.J., BUTTERWORTH,E.W., KUENEN, L.P.S., NORTHCOTT, C.J., and KETTELA, E.G. 1988. A sex pheromone of the pulplestriped shootworm, Zeiraphera unfortunana. J. Chem. Ecol. 14:1417-1425.

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STRUBLE,D.L., and ARN, H. 1984. Combined gas chromatography and electroantennogram recording of insect olfactory responses, pp. 161-178, in H.E. Hummel and T.A. Miller (eds.). Techniques in Pheromone Research. Springer-Verlag, New York. TURGEON, J.J. 1985. Life cycle and behavior of the spruce budmoth, Zeiraphera canadensis (Lepidoptera: Olethreutidae) in New Brunswick. Can. Entomol. 117:1239-1247. TURGEON, J.J., and GRANT, G.G. 1988. Response ofZeiraphera canadensis (Lepidoptera: Olethreutinae) to candidate sex attractants and factors affecting trap catches. Environ. Entomol. 17:442-447. WOLF, W.A., and ROELOFS,W.L. 1987. Reinvestigation confirms action of 11-desaturases in spruce budworm moth sex pheromone biosynthesis. J. Chem. Ecol. 13:1019-1027.