Journal of Chemical Ecology, Vol. 17, No. 1, 1991

IDENTIFICATION OF SEX PHEROMONE OF TOMATO PINWORM, Keiferia lycopersicella (WALS.) 1

R.E.

C H A R L T O N , 2'* J . A . W Y M A N , 3'5 J . R . M c L A U G H L I N , J . - W . D U , 2'6 a n d W . L . R O E L O F S 2

4

2Department of Entomology, Cornell University New York State Agricultural Experiment Station Geneva, New York 14456 3Department of Entomology University of California Riverside, California 92521 41nsect Attractants, Behavior, and Basic Biology Research Laboratory Agricultural Research Service, USDA Gainesville, Florida 32604 (Received July 9, 1990; accepted September 6, 1990) Abstract--A sex pheromone produced by female Keiferia lycopersicella (Walsingham) was isolated and identified as (E)-4-tridecenyl acetate, based on chemical analyses, electroantennogram assays, and field trapping in California and Florida. Males were captured equally well in traps baited with (E)-4-tridecenyl acetate alone or a variety of (Z)- and (E)-4-tridecenyl acetate blends, although the Z isomer was not detected in extracts of female glands. Key Words--Sex pheromone, Keiferia lycopersicella, tomato pinworm, Lepidoptera, Gelechiidae, (E)-4-tridecenyl acetate.

INTRODUCTION T h e t o m a t o p i n w o r m ( T P W ) , Keiferia lycopersicella ( W a l s i n g h a m ) , is a m a j o r p e s t o f t o m a t o e s in N o r t h a n d C e n t r a l A m e r i c a . T P W l a r v a e are l e a f rollers a n d also b u r r o w into fruit, a n d as a r e s u l t t h e y are e f f e c t i v e l y s h e l t e r e d f r o m m o s t *To whom correspondence should be addressed. 1Lepidoptera: Gelechiidae. 5Present address: Department of Entomology, University of Wisconsin, Madison, Wisconsin 53706. 6Present address: Shanghai Institute of Entomology, Academia Sinica, Shanghai, P.R.C. 175 009843331/91/0100-0175506.50/09 1991PlenumPublishingCorporation

176

CHARLTON ET AL.

insecticides. There is thus considerable impetus to develop other control strategies, such as the use of the sex pheromone to directly suppress populations via mating disruption (Jim6nez et al., 1988; Jenkins et al., 1990) or as a monitoring tool in IPM programs. McLaughlin et al. (1979) were the first to demonstrate that female TPW emit a pheromone that can attract males in the laboratory and the field. We report here the isolation and identification of that pheromone as (E)-4-tridecenyl acetate and document its effectiveness as a trap lure for conspecific males in the field.

METHODS AND MATERIALS

Insects were shipped as pupae from Riverside, California, and Gainesville, Florida. The emerging adults were used directly for experiments or to establish a colony to provide additional adults. The insects were reared (Schuster and Burton, 1982) to fourth instar on tomato foliage in the greenhouse at 28 +_ 4~ and then were transferred to environmental chambers held at 27 ___ 1 ~ and on a 16L: 8D photoperiod. The pupae were segregated by sex, and adults were separated daily. Preparative glass GLC columns were packed with 3% OV-101 (methyl silicone) on 100-120 mesh Gas Chrom Q (2 m • 2 mm ID), and 10% XF1150 (50% cyanoethyl methyl silicone) on 100-120 mesh Chromosorb W-AWDMCS (3 m x 2 mm ID). The capillary columns used included a glass 7-m • 0.3-mm-ID Carbowax 20 M, and fused silica 30-m • 0.25-mm-ID Supelcowax 10 (Supelco) and 50-m • 0.25-mm-ID Silar 10C (Quadrex) columns. Extracts were obtained by extruding and excising the ovipositors, including the pheromone gland, of 2- to 4-day-old females during early scotophase; glands were extracted in redistilled Skelly B (mainly hexanes) or n-heptane. The crude extract was concentrated using a nitrogen stream and an aliquot was injected onto the XF-1150 or OV-101 column. Fractions (generally 1 min) were collected from the columns using chilled 30-cm glass capillary tubes. Active fractions were identified by recording electroantennograms (EAGs) from excised antennae of 1- to 4-day-old males as described in Roelofs (1984). Antennae also were screened with a series of synthetic positional isomers of 12-, 13-, and 14-carbon Z and E monounsaturated acetates and alcohols, using a 10-/~g loading on filter paper for each compound. The isolated compound was further characterized by microozonolysis and hydrolysis with ethanolic NaOH followed by acetylation with acetyl chloride (Bjostad et al., 1980), as well as by capillary GLC analysis, and GLC-mass spectrometry. Microozonolysis was carried out by dissolving samples in 50 txl of carbon disulfide in 3-ram • 3-cm glass tubes chilled in a Dry Ice/acetone

177

TOMATO PINWORM PHEROMONE

bath. A stream of ozone was bubbled through the solution for 20 sec. This solution then was analyzed immediately by capillary GLC, and the products compared with ozonized standards. A Hewlett-Packard 5880 gas chromatograph equipped with a splitless injector and a flame ionization detector was used for the GLC analyses. By chromatographing a series of synthetic tridecenyl acetate standards on polar and very polar capillary columns, we were able to separate and identify all of the geometric and positional isomers (Heath et al., 1980). Comparison of the retention time of the pheromone with those of the standards allowed confirmation of the position and geometry of its double bond. Chemical ionization mass spectra were obtained with a Hewlett-Packard 5985 mass spectrometer interfaced with a Supelcowax 10 capillary column with isobutane as the reagent gas. Electron impact mass spectra were obtained with a Hewlett-Packard 5970 series Mass Selective Detector coupled with a 30-m Supelcowax 10 capillary column using helium as the carrier. The (E)-4-tridecenyl acetate (E4-13 : OAc), (Z)-4-tridecenyl acetate (Z413 :OAc), (E)-8-tridecenyl acetate (E8-13 :OAc) and (E, E)-4,7-tridecadienyl acetate (E4, E7-13 :OAc) used in field tests were purchased from Farchan Chemical Co. GLC analyses of these compounds on X F - l l 5 0 and OV-101 revealed that they were > 99 % pure with no detectable amounts of other positional or geometric isomers. Trapping studies were conducted in tomato fields in California and Florida using Pherocon 1C (Zoecon Corp., Palo Alto, California) traps baited with rubber septa (rubber stoppers, sleeve type, 5 x 9 ram, A.H. Thomas Co.) loaded with test chemicals dissolved in dichloromethane or four caged virgin female moths. Traps were deployed in a randomized complete block design with treatments replicated three to five times and replicates separated by at least 50 m. Traps were hung on stakes at plant canopy height (0.3-1.8 m), and separated from one another by 3-15 m. Every 3-10 days, trapped males were counted and the traps were rerandomized within blocks. Data were transformed to (X + 0.5) 1/2 prior to analysis of variance and means were separated using the Ryan-Einot-Gabriel-Welsh multiple range test (SAS Institute, 1985) to control the experiment-wise type-I error rate (Day and Quinn, 1989).

RESULTS

Following fractionation of the crude extract on XF- 1150 (160 ~C), the largest peak and most of the EAG activity was found in the 6- to 8-min fraction; by comparison, saturated 13:OAc, and 14:OAc standards eluted at 6.7 rain and 9.4 rain, respectively. On OV-101 (150~ the highest activity was localized in the 5- to 6-rain fraction; under the same conditions, 12 : OAc elnted at 4.3 rain and 13:OAc at 6.1 rain. Thus, on polar and nonpolar columns, the

178

CHARLTON ET AL.

compound had retention times characteristic of an unsaturated 13-carbon acetate. Injection of this purified active fraction on the Carbowax 20 M capillary column revealed that only one major peak was present. Upon coinjection with a series of synthetic positional isomers of Z and E tridecenyl acetates on Supelcowax 10 and Silar 10C capillary columns, the retention time of the active peak was found to match that of synthetic E4-13 : OAc exactly. Saponification and reacetylation of the active fraction verified that the compound was an acetate. When the active fraction was hydrolyzed and the reaction products were injected onto the OV-101 and XF-1150 columns, all fractions were inactive by EAG. On both columns the retention time of the major peak now conformed to that of a monounsaturated alcohol. Following collection of the alcohol peak from OV-101, the fraction was acetylated and injected onto the OV-101 and XF-1150 columns, and activity was restored in the original 5- to 6-min and 6- to 8-min fractions, respectively. The 2~4 position of the double bond in the EAG-active compound was determined by microozonolysis. This procedure produced two peaks on the Supelcowax-10 capillary column whose retention times were identical to those of the 4-oxobutyl acetate and nonanal generated from ozonolysis of authentic E4-13 : OAc. Isobutane CI mass spectra of the active compound established that its molecular weight was 240 with diagnostic ions at 241 (M + 1) and 181 (M + 1 60, indicating the loss of acetic acid) again consistent with our interpretation that the compound was a monounsaturated 13-carbon acetate. The EI spectrum was identical in all respects to that of synthetic E4-13 : OAc; the parent ion (240) was not observed in the EI spectrum (Figure 1A). Standardized EAG responses from the synthetic compound series showed that the highest depolarizations were elicited by the monounsaturated 13-carbon acetates, followed by the 12-carbon and the 14-carbon acetates. The alcohols were mostly inactive. Within the 13-carbon acetate series, the best antennal responses were obtained for E4-13 : OAc; E8-13 :OAc gave only slightly less activity, with Z4, Z5, and E7 compounds also giving good responses. Despite the high EAG respones obtained from the latter four compounds, capillary GLC chromatograms of gland rinses showed only one major peak corresponding to E 4 - 1 3 : O A c with no evidence (detection limits > 0.1% of E4-13 :OAc) of Z 4 - 1 3 : O A c or other monounsaturated acetates (Figure 1B). The mean titer of E 4 - 1 3 : OAc in females whose glands were extracted at lightsoff on the day of emergence (age 2-24 hr) was 10.1 ng/female (based on two samples of 10 and 20 pooled glands). The first field test compared male captures in response to different loadings of E 4 - 1 3 : OAc, as well as to the highly EAG-active E8-13 : OAc alone or in combination with E4-13 :OAc. Also tested was another compound, E4, E 7 13 : OAc, which was discovered to be an attractant for TPW males during the

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FIG. 1. (A) Electron impact mass spectrum of (E)-4-tridecenyl acetate from female gland extract9 (B) Chromatogram of K. lycopersicella pheromone gland extract (pooled sample of 10 glands) with 10 ng 13:OAc added as internal standard. Arrow indicates retention time of tetradecanyl acetate. All monounsaturated acetates eluted between 13 : OAc and 14 : OAc under these conditions (30-m x 0.25-mmID Supelcowax 10; Temperature program: 80~ (2 min), then 4 ~ to 220~ (10 min).

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180

CHARLTON ET AL.

course of field screening tests for Phthorimaea operculella (Zeller) attractants (G. Kennedy, personal communication). The 100- and 300-/zg doses of E 4 13 : OAc captured the highest numbers of males but the 1-mg dose captured significantly fewer males (Table 1). These lures retained their potency over the entire 46 days they were deployed in the field. The 100-txg loading generally caught the highest number of males, but the relative effectiveness of the 300/zg and 1-mg dosages appeared to increase as the lures aged, probably due to declining emission rates. E8-13 :OAc and E4, E7-13 :OAc by themselves were ineffective as trap lures (Table 1). When varying amounts of E4, E7-13 :OAc were added to E 4 13 : OAc, it significantly depressed trap catch in a dose-dependent manner over the test as a whole and caused a reduction in captures during all but the last trapping interval (36-46 days); nevertheless, these traps still captured significantly more males than unbaited traps (Table 1). When E 8-13 :OAc was added to E 4 - t 3 : OAc, the results were more erratic: total trap catch was significantly lower for both loadings, but during several of the trapping intervals, the low

TABLE 1. TOMATO PINWORM MALES CAUGHT IN PHEROCON 1C TRAPS BAITED WITH ( E ) - 4 - T R I D E C E N Y L ACETATE A N D OTHER COMPOUNDS AT CHULA VISTA AND IRV1NE, CALIFORNIA, 1 9 7 6 ~

Moths/trap/night during trapping interval (lure age) 8/26-9/6

9/7-9/19

9/20-9/30

10/1-10/10

(0-12)

(12-25)

(25-36)

(36-46)

Mean (mean total capture/trap)

13.9 a 9.0 ab 3.9 c

18.1 a 14.6 ab 4.5 d

19.5 a 18.0 a 7.9 b

29.1 a 31.8 a 29.4"

19.7 a (907.6) 17.7 ~b (812.8) 10.6 c (486.8)

300/xg E 4 , E 7 - 1 3 : O A c

0.2 a

0.3 e

0.1 ~

0.4 c

0.3 e

(11.8)

300 # g E 8 - 1 3 : O A c

0.9 a

1.9 *

1.4 ~

9.1 b

3.1 a

(141.4)

300 tzg E 4 - 1 3 : O A c + 45 ~g E 8 - t 3 : O A c

9.8 a

9.9 bc

17.9 a

29.5 a

15.9 b

(732.2)

300 txg E 4 - 1 3 : O A c + 300/zg E8-13 : OAc

4.4 be

7.9 cd

11.5 b

24.5 a

11.5 ~

(527.4)

300 #g E 4 - 1 3 : O A c + 15/zg E 4 , E 7 - 1 3 : O A c

4 . 6 b~

7.2 cd

8.6 b

32.9 ~

12.4 ~

(571.6)

1.1 d 0.5 a

0.8 ~ 0.3 ~

1.2 ~ 0.2 d

18.0 b 0.6 c

2.9 d 0.4 ~

(135.0) (16.4)

Trap lure 100/*g E 4 - 1 3 : O A c 300 # g E 4 - 1 3 : O A c 1 mg E4-13:OAc

300/zg E 4 - 1 3 :OAc + 45/zgE4,E7-13:OAc

Unbaited

a Five replicates per treatment. Means in a column followed by the same letter are not significantly different according to Ryan-Einot-Gabriel-Welsh multiple range test (P > 0.05). All treatments dispensed from rubber septa except 1 mg loading of E 4 - 1 3 : OAc, which was released from poly-

ethylene caps.

TOMATO PINWORM PHEROMONE

181

dosage captured as many males as E 4 - 1 3 : O A c alone (Table 1). The lack of captures in the E 4 , E 7 - 1 3 : OAc-baited traps was inconsistent with the results from the P. operculella test; possibly, the baits used in the earlier study were contaminated with small amounts of E4-13 : OAc. The second set of trapping experiments compared different ratios of Z- and E 4 - 1 3 : O A c . In both California and Florida, TPW males responded equally well to a wide range of Z : E ratios with optimal catch occuring to the 3% Z blend (Table 2). Z4-13 :OAc alone captured significantly fewer males than any of the E: Z blends. Nonetheless, this compound had some intrinsic activity since it captured more males than did unbaited traps. In California, the best synthetic baits caught as many males as did virgin females (Table 2); females were not included in the results of the Florida test due to insecticide-induced mortality. DISCUSSION

The evidence presented here indicates that the sex pheromone of the tomato pinworm is (E)-4-tridecenyl acetate. This compound apparently is unique among identified lepidopteran sex pheromones (Am et al., 1986). Only a few moth species have been found to use 13-carbon acetates as pheromones (Baker et al., 1985) or attractants (Voerman and Heerebout, 1978; Willemse et al., 1987). Furthermore, pheromone components with unsaturation in the 4-position have TABLE 2. TOMATO PINWORM MALES CAPTURED /MEAN/TRAP (SE)] IN TRAPS BAITED WITH VARIOUS BLENDS OF (E)- AND (Z)-4-TRIDECENYL ACETATE IN FLORIDA AND CALIFORNIA TOMATO FIELDSa

Trap lure 100 t~g E4-13:OAc +3/xg Z4-13 :OAc + 10/zg Z4-13 :OAc + 30/zg Z4-13 : OAc + 100/xg Z4-13 :OAc 100/zg Z4-13 :OAc 30/xg E4-13 : OAc + 100/zg Z4-13:OAc Unbaited Virgin females

Homstead, Florida 6/3-6/21, 1977 69.0 a 85.0 a 62.3 a 67.0 a 75.3 ~ 19.0 b

Chula Vista, Irvine, California 11/2-11/12, 1976

(4.9) (9.9) (7.3) (4.2) (4.1) (5.8)

245.5 a (104.0) 293.5" (66.8) 185.3 ~ (50.7) 216.3 a (95.5) 205.0 a (80.4) 87.5 b (30.3)

49.7 a (11.8) 1.7 c (0.3)

228.5" (49.3) 3.3 c (1.3) 231.3" (75.9)

aThree and four replicates for Florida and California tests, respectively. Means in a column followed by the same letter are not significantly different according to Ryan-Einot-Gabriel-Welsh multiple range test (P _> 0.05).

182

CHARLTON ET AL.

been encountered from only a handful of species belonging to three lepidopteran families: Satumiidae (Bestmann, 1987), Gracillariidae (Beevor et al., 1986), and Gelechiidae. In the latter family, the only other identified A4 13-carbon acetate pheromone is the blend of di- and triunsaturated acetates, (E, Z)-4,7tridecadienyl acetate and (E, Z, Z)-4,7,10-tridecatrienyl acetate, which are used by P. operculella (Roelofs et al., 1975; Persoons et al., 1976). Willemse et al. (1987) found that (Z)-4-tridecenyl acetate was an attractant for males of another gelechiid, Sophronia semicostella (Hfibner), but it is not known whether this compound is actually produced by the females. The pheromones of almost all other gelechiids are even-carbon-numbered chain acetates ranging from 10 to 16 carbons in length with one or two double bonds at odd-numbered carbon positions. Our results show that 100/zg of pure E4-13 : OAc is an effective trap lure for monitoring K. lycopersicella populations. Curiously, even though Z 4 13 : OAc was not found in extracts of female glands, traps baited with a broad range of Z4/E4-13:OAc blends captured as many males as those baited with pure E4-13 : OAc. The Z4-13 :OAc is clearly not a behavioral antagonist, and therefore it should not be necessary to use geometrically pure E4-13 :OAc in field tests. In fact, a 96:4 E:Z mixture currently is generally used for monitoring traps and disruptant formulations (Jenkins et al., 1990). Optimal trap catch was obtained with pheromone dosages of 100 or 300/zg whereas 1 mg inhibited male attraction. These results are in agreement with those reported by Wyman (1979), who found that hollow-fiber dispensers emitting pheromone at lower rates captured significantly more males than did ones releasing high amounts. The TPW pheromone now is used widely to monitor infestations of this pest and to time insecticide treatment programs (Van Steenwyk et al., 1983). Moreover, it has been registered by the EPA, and commercial controlled-release formulations of the pheromone already provide effective control of TPW populations through mating disruption (Jim6nez et al., 1988; Jenkins et al., 1990). Acknowledgments--We thank Kathy Poole, Marlene Campbell, Frances Wadhams, and Robert Rutter for maintaining the insect cultures, and Henry Nakakahara and Anthony Antonio for assistance with field experiments. REFERENCES ARN, H., TOTH, M., and PRIESNER, E. 1986. List of Sex Pheromones of Lepidoptera and Related Attractants. International Organization for Biological Control, Paris. BAKER, R., HERBERT, R.H., NEEQUAYE,N.N., and RAO, K.N. 1985. Sex pheromone of tobacco stem borer, Scrobipalpa heliopa (Lower) (Lepidoptera: Gelechiidae). J. Chem. Ecol. t 1:989998. BEEVOR, P.S., CORK, A., HALL, D.R., NESBITT, B.F., DAY, R.K., and MUMFORD,J.D. 1986. Components of female sex pheromone of cocoa pod borer moth, Conopomorpha cramereIla. J. Chem. Ecol. 12:1-23.

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BESTMANN, H.J, ATTYGALE, A.B., BROSCHE, T., ERLER, J., PLATZ, H., SCHWARZ, J., VOSTROWSKY, O., WU, C.-H., KAISSLING, K.E., and CHEN, T.-M. 1987. Identification of three sex pheromone components of the female satumiid moth Antheraea pernyi (Lepidoptera: Satumiidae). Z. Naturforsch. 42C:631-636. BJOSTAD, L.B., TASCHENBERG,E.F., and ROELOFS, W.L. 1980. Sex pheromone of the chokecherry leafroller moth, Sparganothis directana. J. Chem. Ecol. 6:487-498. DAY, R.W., and QUINN, G.P. 1989. Comparison of treatments after an analysis of variance in ecology. Ecol. Monogr. 59:433-463. HEATH, R.R., BURNSED, G.E., TUMLINSON, J.H., and DOOLITTLE, R.E. 1980. Separation of a series of positional and geometrical isomers of olefinic aliphatic primary alcohols and acetates by capillary gas chromatography. J. Chromatogr. 189:199-208. JENKINS, J.W., DOANE, C.C., SCHUSTER, D.J., McLUGHLIN, J.R., and JIM~NEZ, M. 1990. Development and commercial application of sex pheromone for control of the tomato pinworm, pp. 269-280, in R.L. Ridgeway, R. M. Silverstein, and M.N. Inscoe (eds.). Behavior-Modifying Chemicals for Insect Management: Applications of Pheromones and Other Attractants. Marcel Dekker, New York. JIMI~NEZ, M.J., TOSCANO, N.C., FLAHERTY, D.L., ILIC, P., ZALOM, F.G., and KIDO, K. 1988. Controlling pinworm by mating disruption. Calif. Agric. 42(6): 10-12. MCLAUGHLIN, J.R., ANTONIO,A.Q., POE, S.L., and MINMCK, D.R. 1979. Sex pheromone biology of the adult tomato pinworm, Keiferia lycopersicella (Walsingham). Fla. Entomol. 62:34-41. PERSOON, C.J., VOERMAN, S., VERWIEL, P.E.J., RITTER, F.J., NOOYEN, W.J., and MINKS, A.K. 1976. Sex pheromone of the potato tuberworm moth, Phthorimaea operculella: Isolation, identification, and field evaluation. Entomol. Exp. Appl. 20:289-300. ROELOFS, W.L. 1984. Electroantennogram assays: rapid and convenient screening procedures for pheromones, pp. 131-159, in H.E. Hummel and T.A. Miller (eds.). Techniques in Pheromone Research. Springer-Verlag, New York. ROELOFS, W.L., KOCHANSKY, J.P., CARDE, R.T., KENNEDY, G.G., HENRICK, C.A., LABOVITZ, J.N., and CORBIN, V.L. 1975. Sex pheromone of the potato tuberworm moth, Phthorimaea operculella. Life Sci. 17:699-706. SAS INSTITUTE 1985. SAS Users Guide: Statistics. Version 5 (ed.). SAS Institute, Cary, North Carolina. SCHUSTER, D.J., and BURTON, R.L. 1982. Rearing the tomato pinworm (Lepidoptem: Gelichiidae) in the laboratory. J. Ecol. Entomol. 75:1164-1165. VAN STEENWYK,R.A., OATMAN,E.R., and WYMAN, J.A. 1983. Density treatment level for tomato pinworm (Lepidoptera: Gelechiidae) based on pheromone trap catches. J. Econ. Entomol. 76:440-445. VOERMAN, S., and HERREBOUT,W.M. 1978. A sex attractant for the leaf miner moth Lithocolletis corylifoliella and its influence on that ofL. blancardeIla (Lep., Gracillariidae). Entomol. Exp. Appl. 23:96-98. WILLEMSE, L.P.M., BOOIJ, C.J.H., and VOERMAN, S. 1987. New sex attractants for male Lepidoptera (Coleophoridae, Gelichiidae, Momphidae, Oecophoridae, and Yponomeutidae) found by field screeing in the Netherlands. J. Appl. Entomol. 103:508-515. WYMAN,J.A. 1979. Effect of trap design and sex attractant release rates on tomato pinworm catches. J. Econ. Entomol. 72:865-868.

Identification of sex pheromone of tomato pinworm,Keiferia lycopersicella (Wals.).

A sex pheromone produced by femaleKeiferia lycopersicella (Walsingham) was isolated and identified as (E)-4-tridecenyl acetate, based on chemical anal...
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