Journal of Chemical Ecology, Vol. 10, No. 2, 1984

ETHYL (Z)-9-HEXADECENOATE A SEX P H E R O M O N E OF Syndipnus rubiginosus, 1 A SAWFLY PARASITOID 2

F.J. E L L E R , 3 R.J. B A R T E L T , 4 R.L. J O N E S , and H.M. K U L M A N Department of Entomology, Fisheries, and Wildlife University of Minnesota St. Paul, Minnesota 55108

(Received December 17, 1983; revised May 9, 1983) Abstraet--A female-produced sex pheromone of Syndipnus rubiginosus Walley (Hymenoptera: Ichneumonidae), a parasitoid of the yellowheaded spruce sawfty, Pikonema alaskensis (Rohwer) (Hymenoptera: Tenthredinidae), was isolated and identifiedfrom hexane extracts of 250 virginfemales. Column chromatography (Florisil), gas chromatography, mass spectrometry, high performance liquid chromatography, and ozonolysis indicated the structure was ethyl (Z)-9-hexadecenoate. The optimum male response is at 300-1000 ng (3-10 FE). No cross-attraction between S. rubiginosus and the 8ympatric sawfly parasitoid S. gaspesianus (Provancher) could be demonstrated. Key Words--Sex pheromone, Hymenoptera, Ichneumonidae, Syndipnus rubiginosus, ethyl (Z)-9-hexadecenoate, parasitoid, Pikonema alaskensis, mass spectra, ozonolysis.

INTRODUCTION Since the first identification by B u t e n a n d t et al. (1959), sex p h e r o m o n e s have been d e m o n s t r a t e d in h u n d r e d s of species, most of which are destructive species in the orders L e p i d o p t e r a a n d Coleoptera. The sex p h e r o m o n e s that have been identified for H y m e n o p t e r a , for the most part, have also been in Hymenoptera: lchneumomdae. . . . . Paper No. 13,232 Scientific Journal Series, Minn. Agric. Exp. Stn., St. Paul, Minnesota 55108. Research supported by C.K. Blandin Foundation of Grand Rapids, Minnesota. 3Current address: Department of Entomology and Nematology, McCarty Hall, University of Florida, Gainesville, Florida 32611. 4Currentaddress: Department of Chemistry, Johnson Hall, Montana State University,Bozeman, Montana 59717. 2

291 0098 -0331 / 84,' 0200 -0291 $03.50,'0 9 1984 Plenum P ublishing Corporatior~

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ELLER ET AL.

destructive species (Ikan et al., 1969; Jewett et al., 1976; Bartelt et al., 1982b). The identification of the pheromone of the honeybee (Butler and Fairey, 1964), is an exception. Of the references to sex pheromones of parasitic Hymenoptera, most refer only to evidence of a sex pheromone. Such evidence has been demonstrated for the family Aphelinidae (Rao and Debach, 1969; Khasimuddin and Debach, 1975), Chalcididae (Leonard and Ringo, 1978; Simser and Coppel, 1980), Cynipidae (Read et al., 1970), Pteromalidae (King et al., 1969; Yoshida, 1978; Van den Assem et al., 1980), Scelionidae (Schwartz and Gerling, 1974), Braconidae (Genieys, 1925; Fink, 1926; Murr, 1930; Parker, 1931; Whitting, 1932; Hagen, 1953; Schlinger and Hall, 1960; Bousch and Baerwald, 1967; Cole, 1970; Read et al., 1970; Lewis et al., 1971; Weseloh, 1976; Tagawa, 1977; Askari and Coppel, 1978; Askari and Alishah, 1979), and Iehneumonidae (Champlain, 1921; Cole, 1970; Vinson, 1972; Robaeker et al., 1976; Coppel and Mertins, 1977). The sex pheromone of only one parasitic hymenopteran has been isolated and identified. Robacker and Hendry (1977), using laboratory bioassays, isolated neral and geranial from Itoplectis conquisitor (Say). No field results have been reported. Syndipnus rubiginosus Walley (Hymenoptera: Ichneumonidae) is a commonly reported parasitoid of the yellowheaded spruce sawfly, Pikonema alaskensis (Rohwer) (Hymenoptera: Tenthredinidae) (Forbes, 1949; Bradley, 1951; Raizenne, 1957; Rau, 1976; Houseweart and Kulman, 1976; Thompson and Kulman, 1980). Rau (1976) reported that S. rub iginosus attacks late (4th6th) instar yellowheaded spruce sawfly larvae. This paper describes the isolation and identification of a sex pheromone of S. rubiginosus. Field trials were conducted with synthetic pheromone, and cross-attraction between S. rubiginosus and the related species S. gaspesianus (Provancher) was studied also.

M E T H O D S AND MATERIALS

Insects. Yellowheaded spruce sawfly larvae were collected and handled as described by Bartelt et al. (1982a). Emerged S. rubiginosus were separated by sex. [Males have a yellow face and females have a black face (Walley, 1940).] Virgin females (2-4 days old) were killed by immersion in hexane and stored at -20 ~ C. Males were held at 15~ and provided with honey and water until used in bioassays. Greenhouse Bioassay. To monitor purification steps, a greenhouse bioassay cage containing 45 male S. rubiginosus was used (Barter et al., 1982a). Pheromone preparations were placed on 4-cm watch glasses, at the rate of ca. 1 female equivalent (FE) per plate. After the solvent had evaporated, the plates were positioned in the upwind end of the cage, in a row perpendicular to the air flow, and just above small (5 dm) spruce trees. Qualitatively, a positive response included: positive anemotaxis, down-

SAWFLY PARASITOID

293

wind hovering in front of plates, landing on plates, abdomen raising, wing fanning, and copulatory attempts with nearby males. Male response to various test materials was quantified by counting the number of males "hovering" within 2 cm directly in front of a watch glass every 15 sec over a 2-min period. The 2-min totals counts were analyzed as balanced incomplete block designs. The number of treatments, blocks, replications, and block size varied with the experiment. FieM Bioassays. Field studies were conducted in white spruce plantations near Grand Rapids, Minnesota, during June 1981-1982. Test materials were evaluated in the field by catches of males in Pherocon| II traps. Test materials were placed on 4-cm watch glasses and the solvent allowed to dry before placement in the traps. One hundred nanograms of the antioxidant, 2,5-di-tert-pentylhydroquinone, was also added to each watch glass. The traps were secured to branches of 3-5 m tall white spruce trees about 1.5 m from the ground and about 5 m apart. In 1981, field bioassays of Florisil fractions were run using ca. 1 FE per trap. These were tested in a randomized complete block design (RCB) containing five plots, each with one replication of the 10 treatments. Traps were checked after 2 days. The 1982 field test of various dosages (0, 10, 30, 100, 300, and 1000 ng) were tested in a RCB design, containing five plots, each with one replication of the six treatments. The experiment was repeated on five consecutive days. In the cross-attraction experiments between S. rubiginosus and S. gaspesianus, traps contained virgin females held in brass screen cages 4 cm long and 2 cm diameter. Control traps were empty. The experiment was a RCB design, with five plots, each with two replications of the three treatments. The experiment was repeated on four consecutive days. Chemical Analyses. Virgin females were either extracted by a hexane wash in an Erlenmeyer flask or with hexane in a Soxhlet extractor. The extract:was combined with the hexane used to kill the females, concentrated under vacuum, and stored at - 2 0 ~ until used for bioassay or chemical analysis. The concentrated crude extract was fractionated by column chromatography on Florisil (2.5% water by weight). The column (20 • 2.0 cm) (void volume ca. 40 ml) was eluted consecutively with 80-ml volumes of hexane, then 2.5, 5.0, 7.5, 10, 25, 50% ethyl ether in hexane (by volume), ethyl ether, acetone, and methanol. The effluent was collected in 10 fractions of 80 ml each and stored at - 2 0 ~ until used for bioassay or chemical analysis. Gas-liquid chromatography (GLC) was performed with a Hewlett Packard 5830A, equipped with a flame ionization detector, effluent splitter, and thermal gradient collector (similar to that described by Brownlee and Silverstein, 1968). The glass column (1.9 m • 1 mm ID) contained 5% Silar 5CP on 80/100 Gas Chrom Q and was eluted with N2 at 20 ml/min. For collections, the temperature program was 100~ for 1 min, then 5~ to 250 ~ For analysis of ozonolysis products and aldehyde standards, the tem-

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perature program was 50 ~ for 5 min, then 10~ to 250 ~ High-performance liquid chromatography ( H P L C ) was carried out with a Waters Associates M6000A p u m p and R401 differential refractometer detector. Samples (1-2 ~zg) were injected onto a 100 • 4 mm, 20% AgNO3 on silica column and eluted with toluene at 1.0 ml/min. Retention times were compared to standard esters. Mass spectra were obtained on a LKB-9000 gas c h r o m a t o g r a p h - m a s s spectrometer equipped with a 1.0-m • 2-mm ID 10% Alltech CS-5 column and electron ionization energy of 70 eV. Ozonolysis was conducted as described by Beroza and Bierl (1967). Samples (2/~g) were ozonized in 20 #1 of CS2 for 2 min at - 7 0 ~ . The ozonides were reduced with triphenylphosphine. Synthetic ethyl palmitoleate [ethyl (Z)-9-hexadecenoate, 99% pure] was obtained from Sigma Chemical C o m p a n y (St. Louis, Missouri 63178). RESULTS

The 1981 field evaluation of the Florisil fractions shows that a 5% ether in hexane Florisil fraction (prepared from an extract of 20 virgin females) caught an average (N = 5) of 9 males/trap in a 2-day test (Table 1). The other fractions had catches that did not differ significantly from zero. In greenhouse bioassays (winter 1981-1982), 5% and 7.5% ether in hexane fractions (prepared from an extract of 250 virgin females) were more attractive than the other fractions. The results are shown in Table 1. The 7.5% TABLE 1. FIELD AND GREENHOUSE BIOASSAY OF FLORISIL FRACTIONSa

Fraction Hexane Ether in hexane 2.5% 5.0% 7.5% 10.% 25.% 50.% Ether Acetone M ethanol

1981 Field mean 2-day catch b

Greenhouse mean response c

0.2a

0a

0a 9.0b 0.6a 0a 0a 0a 0.2a 0a 0a

0a 4.9b 6.0c 0a 0a 0a 0a 0a 0a

afield and greenhouse bioassays were done with two separate sets of Florisil fractions. bMean catch is given (N = 5); different letters denote significant differences at the 0.05 level by the least significant difference (LSD) method. CMean response is given (N = 9).

295

SAWFLY PARASITOID

fraction was significantly more attractive than the 5% fraction. These two were compared by G L C a n d contained peaks of similar retentions, but the 7.5% fraction had larger amounts. The 5% Florisil fraction used for the 1981 field bioassays also contained the same peaks as the other active Florisil fractions. The 7.5% Florisil fraction was collected in 13 fractions from the G L C (Figure 1). The collected fractions were tested in the greenhouse, and only one fraction, G L C fraction 4, showed any attractiveness. It had a mean response (N = 4) of 7.0, and all other fractions had responses of zero. The mass spectrum obtained for GLC fraction 4 (GLC inlet, 70 eV), is shown in Figure 2. The parent ion (M) was observed at rn / e 282. The M - 45, M - 46, M - 88 peaks observed were consistent with an ethyl ester of a 16carbon fatty acid with one double bond. To determine the double-bond location, G L C fraction 4 was ozonized, and the G L C retention times of the products were compared to those of standard aldehydes. The G L C c h r o m a t o g r a m of the ozonized G L C fraction 4 contained peaks at 5.89, equal to the retention time of heptanal, and at 17.99, equal to the retention time of ethyl-9-oxononanoate. These data indicated

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296

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that the double bond was in the 9 position and that the compound was an ethyl ester of 9-hexadecenoate. Ozonolysis of synthetic ethyl (Z)-9-hexadecenoate gave an identical GLC chromatogram. To determine the configuration about the double bond, the 7.5% Florisil fraction was subjected to H P L C on a AgNO3 silica column. Peaks appeared in the chromatogram (Figure 3) at 1.3 (hexane solvent), 1.8, and 3.2 ml after

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297

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CH3CH2CH2CH2CH2CH2C=CCH2CH2CH2CH2CH2CH2CH2COCH2CH3 ethyl (Z)-9-hexadecenoate (ethyl palmitoleate) FIG. 4. Structure of ethyl (Z)-9-hexadecenoate. injection. The peak at 1.8 ml was equal to the retention time of a saturated ester, and the peak at 3.2 ml was equal to the retention time of a cis monoene ester. The material was collected in four fractions (Figure 3) and bioassayed in the greenhouse. Only one fraction, H P L C fraction 4, had activity. It had a mean response (N = 6) of 7.7, and all other fractions had responses of zero. This fraction had the retention time of a cis ester, so the compound was tentatively identified as ethyl (Z)-9-hexadecenoate (or by its trivial name, ethyl palmitoleate) (Figure 4). Synthetic ethyl palmitoleate gave identical G L C retention time, mass spectrum, ozonolysis products, and H P L C retention time as the femalederived material. One FE (100 ng) of synthetic ethyl palmitoleate was tested in the greenhouse and elicited a significantly higher response (8.6) than a control (0.0). The synthetic ethyl palmitoleate elicited all of the characteristic male behaviors as elicited by female-derived materials. In the field, various dosages of synthetic ethyl palmitoleate were tested by catches of males in Pherocon II traps. Dosages of 0.1 FE and 0.3 FE did catch males but did not differ significantly f r o m controls, which caught none (Table 2). Attraction to 1 FE (2.96) was significantly higher than to controls, 0.1 FE, or 0.3 FE. Three FE and 10 FE caught significantly more males (5.32 TABLE 2. COMPARISON OF VARIOUS DOSES OF SYNTHETIC ETHYL PALMITOLEATE IN THE FIELD

Dose (female equivalents)

Mean l-day catch of S.r. malesa

Control 0.1 FE (0.01/~g) 0.3 FE (0.03/.~g) 1.0 FE (0.1 p.g) 3.0 FE (0.3/~g) 10. FE (1.0/~g)

0a 0.28a 0.56a 2.96b 5.32c 6.0c

~Mean catch is given (N = 25); different letters denote significant differences at the 0.05 level by the least significant difference (LSD) method.

298

ELLER ET AL. TABLE 3. CROSS-ATTRACTION BETWEEN S. rubiginosus AND S. gaspesianus

Bait Mean 4-day catch

S.r. 9

S.g. 9

Control

S. rubiginosus males S. gaspesianus males

3. lb 0. la

0a 19.5c

0a 0. Za

aMean response is given (N = 10); different letters denote significant differences at the 0.05 level by the least significant difference (LSD) method.

and 6.0) than 1 FE, but 3 FE and 10 FE did not differ significantly from each other. Although no direct comparison of virgin females to synthetic pheromone was made, traps baited with virgin females and synthetic pheromone (3 FE) in adjacent plots caught averages of 1.3 (N = 20) and 4.6 (N = 10) males per day, respectively. The cross-attraction study shows that females of each species attract only conspecific males, and that catches of the other species seem to be accidental, in that they do not differ significantly from controls (Table 3). DISCUSSION

We conclude that ethyl palmitoleate is a sex pheromone of S. rubiginosus, and, alone, it can elicit responses from males. The compound is active in relatively small amounts (100 ng can attract males in the field), and the optimum male response is to about 300 to 1000 ng. S. gaspesianus males are not attracted to ethyl palmitoleate and are not attracted to virgin female S. rubiginosus, but they are highly attracted to virgin female S. gaspesianus. This suggests the existence of a different sex pheromone in S. gaspesianus. Various means have been suggested for monitoring parasitoid populations to aid in management programs. Lewis et al. (1971) developed a femalebaided trap; Hrdy and Sedivy (1979) suggested using chemical attractants to detect both the pest and its parasitoids; and Robacker et al., (1976) suggested the use of parasitoid pheromones. With the identification of sex pheromones of both the yellowheaded spruce sawfly (Bartelt et al., 1982b) and its parasitoid S. rubiginosus, it is possible to monitor both by means of chemical attractants and use the information in management decisions. REFERENCES

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ASKARI,A., and ALISHAH,A. 1979. Courtship behavior and evidence for a sex pheromone in Diaeretiella rapae (Hymenoptera: Braconidae), the cabbage aphid primary parasitoid. Ann. Entomol. Soc. Am. 72:749-750. ASKARI,A., and COPPEL,H.C. 1978. Observations on courtship and mating behavior of Meteorus pulchricornus, a gypsy moth parasitoid. Ann. Entomol. Soc. Am. 71:364-366. BARTELT, R.J., JONES, R.L., and KULMAN,H.M. 1982a. Evidence for a multicomponent sex pheromone in the yetlowheaded spruce sawfly. J. Chem. Ecol. 8:83-94. BARTELT,R.J., JONES,R.L., and KULMAN,H.M. 1982b:,Hyd_rocarbon components of the yellowheaded spruce sawfly sex pheromone: A series of(Z,Z)-9,19 dienes. J. Chem. Ecol. 8:95-114. BEROZA, M., and BIERL, B.A. 1967. Rapid determination of olefin positions in organic compounds in microgram range by ozonolysis and gas chromatography. Anal. Chem. 39:113 I1135.

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