Journal of Chemical Ecology, Vol. 19, No. 11, 1993

EVIDENCE FOR A MULTICOMPONENT SEX PHEROMONE IN Eriborus terebrans (GRAVENHORST) (HYM.: ICHNEUMONIDAE), A LARVAL PARASITOID OF THE EUROPEAN CORN BORER

SHENGQ1ANG

S H U 1'3"* a n d R I C H A R D

L. J O N E S 2

IDepartment of Entomology 2College of Agriculture University of Minnesota St. Paul, Minnesota 55108 (Received December 8, 1992; accepted June 21, 1993)

Abstract--Sex pheromone activity of Eriborus terebrans (Gravenhorst) (Hymenoptera: Ichneumonidae) was recovered from acetone rinses of flasks that previously contained females. The acetone flask rinses elicited the following male responses: upwind anemotaxis, casting, hovering, landing, wingfanning, and mating attempts with other nearby males. Activity of the acetone flask rinse lasted up to four days on a glass substrate. Polar component and nonpolar components were demonstrated in the acetone flask rinse. The polar component elicited male behavioral responses similar to those by the acetone flask rinse, although retention of males at the pheromone source and the period of wing-fanning were of shorter duration. Chromatography data and chemical derivatizafion indicated that the polar component had the properties of a carboxylic acid with an additional oxygen-containing functional group. The nonpolar component acted as a synergist since it was inactive alone but increased male behavioral responses when added to the polar component. Florisil open column chromatography suggested that the nonpolar component was a hydrocarbon(s).

Key Words--Sex pheromone, multicomponent pheromone, parasitoid, bioassay, Hymenoptera, Ichneumonidae, Eriborus terebrans, Lepidoptera, Pyralidae, Ostrinia nubilalis. * TO whom correspondence should be addressed. 3Current address: Department of Entomology, Drawer EM, Mississippi State University, Mississippi State, Mississippi 39762. 2563 0098-0331/93/1100-2563507.00/0 9 1993PlenumPublishingCorporation

2564

SHU AND JONES INTRODUCTION

The use of natural enemies for biological control of insects is an integral part of a successful integrated pest management (IPM) program. An understanding of the chemical ecology of insect parasitoids can help to make their use more efficient. This includes the study and identification of sex pheromones of parasitoids, since such pheromones can be used to assess the activity of parasitoids, to monitor their density, and to predict rates of host parasitism. Morse and Kulman (1985) trapped the yellowheaded spruce sawfly, Pikonema alaskensis (Rohwer) and its parasitoid, Syndipnus rubiginosus Walley, using their respective synthetic sex pheromones. This information, along with tree height, was used to predict subsequent sawfly-caused defoliation of white spruce trees. Furthermore, sex pheromones can be useful as a chemotaxonomic character because the chemical composition of sex pheromones and/or the ratio of their components are often species-specific (Roelofs and Brown, 1982; Dor6 et al., 1986). Sex pheromones of three species of Hymenopterous parasitoids have been identified (Robacker and Hendry, 1977; Eller et al., 1984; Swedenborg and Jones, 1992; Swedenborg et al., 1993), but only two have been verified with synthesis and field studies. The occurrence of sex pheromones has been demonstrated in a few dozen species among at least nine families (Aphelinidae, Braconidae, Chalcididae, Cynipidae, Eulophidae, Ichneumonidae, Pteromalidae, Scelionidae, and Trichogrammatidae). Evidence for sex pheromones in Hymenopterous parasitoids and their courtship behavior have been reviewed in detail (Shu, 1992). Eriborus terebrans (Gravenhorst) (Hymenoptera: Ichneumonidae) is a larval parasitoid of the European corn borer, Ostrinia nubilalis (Hfibner). It was introduced to the United States from Europe and Asia in 1920s and 1930s and is now established in the corn-growing areas (Rolston et al., 1958; Hill et al., 1978; Andreadis, 1982; Lewis, 1982; Winnie and Chiang, 1982). Preliminary data showed that the presence of E. terebrans females elicited attraction and wing-fanning of conspecific males, indicating the existence of a female sex pheromone. This paper presents the evidence for a multicomponent sex pheromone in E. terebrans and describes the isolation and preliminary characterization of the pheromone. METHODS AND MATERIALS lnsects The colony of E. terebrans used in this study was field collected in Minnesota. E. terebrans and its host, O. nubilalis, were reared according to Ma et al. (1992) and Guthrie et al. (1971). Cocoons of E. terebrans were kept indi-

PhErOMONE OF Eriborus terebrans

2565

vidually to isolate sexes. Voucher specimens of the parasitoid have been deposited in the University of Minnesota Insect Collection (Shu, 1992).

Pheromone Collection (Flask Rinse) Active pheromone extracts were obtained by confining four virgin females (1-3 days old) in a 125-ml clear glass flask at ca. 25~ 85% relative humidity, and 16 : 8 hr light-dark for 24 hr. The females were removed and the flask was rinsed with 2 ml hexane once (hexane flask rinse, HFR) and 2 ml acetone twice (acetone flask rinse, AFR) by swirling the flask with the solvent for 30 sec each time. Both HFR and AFR were concentrated under a nitrogen stream and kept separately in a freezer at - 3 0 ~ until used. Some females were used repeatedly for pheromone collection. Females used more than once were fed with honey and water in a cotton sponge between collections. One female equivalent (FE) was equivalent to the total acetone rinse of a flask exposed to one female for 24 hr. Only AFR was used in this study. HFR was used for detailed studies of hydrocarbons (Shu and Jones, 1993; Shu et al., 1993).

Pheromone Isolation Isolation of AFR. Initially, AFR was separated into various fractions with Florisil open column chromatography consecutively eluted with aliquots of hexane, then 2.5, 5.0, 7.5, 10.0, 25.0, and 50.0% ether in hexane, ether, acetone, and methanol. Later, separation of AFR into two fractions was accomplished in the following way. AFR was dried under a stream of nitrogen and taken up in diethyl ether, which was then extracted three times in a separatory funnel with water saturated with NaHCO3. The ether layer (neutral phase) was saved, dried over MgSO4, and stored at - 3 0 ~ until used. The water extracts were combined, then acidified carefully with dilute HC1 and extracted with ether three times. The water layer was discarded. The ether layers (polar phase) were combined, dried over MgSO4 and either stored in a freezer at - 3 0 ~ or used immediately. Isolating Polar Component. High-performance liquid chromatography (HPLC) of the polar phase was conducted with a 25-cm • 0.46-cm Lichrosorb RP-8 column (5 tz, Alltech) employing a 1% aqueous phosphoric acid-methanol solvent running isocratically at a flow rate of 1 ml/min. The aqueous phosphoric acid-methanol ratio was 20:80. Collection of fractions began at 3 min after injection. A total of five 1-ml fractions was collected. Methanol in fractions was reduced under a stream of nitrogen, and the residue in water was extracted with ether. The ether extracts were then dried over MgSO4. Preparative thin-layer chromatography (TLC) of AFR was conducted with 5-cm • 20-cm Redi/Plate plates precoated with Silica Gel G (Analtech Inc., 250 #m thickness). The plates were activated overnight at 110 ~ then cooled

2566

SHu AND JONES

to room temperature before use. Plates were developed at room temperature with hexane-diethyl ether-acetic acid (70:30:2, v/v/v). Spots were visualized under UV light after spraying the plates with a 0.05 % ethanol solution of rhodamine B. Four fractions were collected. Fraction 1 was recovered by scraping off the band from an unsprayed plate at a location corresponding to a detected spot of 10-hydroxydecanoic acid (RF = 0.05) on a sprayed plate. Fraction 3 corresponded to a detected spot of octanoic acid (RF = 0.41). Fraction 2 was from a band between fraction 1 and fraction 3, and fraction 4 was from a band above fraction 3. The recovery procedure involved placement of the scraped gel into a glasswool-stopped pipet, which was then eluted with 2 ml acetone. Fractions 1 and 2 were combined, rechromatographed, and recovered in the same manner as described above. Isolation of Neutral Phase. The neutral phase was fractionated by open column chromatography on Florisil (2.5% water by weight). The column was eluted consecutively with aliquots of hexane, 5%, 7.5%, 10%, 25%, and 50% ether in hexane, ether, acetone, and methanol. Each fraction was bioassayed alone and in combination with the polar phase. Later, separations of the neutral phase were accomplished by collecting only two fractions from a Florisil column, one with hexane and one with ether. The volume of each eluant was twice the void volume of the column. Fractions were stored at - 3 0 ~ until used.

Derivatization Diazomethane in ether (CHzN2-ether) was obtained from N-methyl-N'nitro-N-nitrosoguanidine according to Black (1983). To esterify the extracts, freshly prepared diazomethane-ether with 10% methanol (volume ratio to CH2Nz-ether) was added to the crude extracts dried under a stream of nitrogen (Schlenk and Gellerman, 1960). The solution was kept at room temperature for at least 1 hr. It was then stored in - 3 0 ~ if not used. For a control, the crude extracts were dried under a stream of nitrogen and taken up in 10% methanol ether.

Female Body Rinse Female body rinse (FBR) was obtained by soaking virgin females in either hexane or acetone. After soaking for a day, the extracts were drawn off and the bodies were rinsed with acetone three times. The extracts and subsequent acetone rinses were combined and concentrated under nitrogen.

Gas Chromatography (GC) Hydrocarbons were analyzed by GC with a nonpolar column (DB-1) as described in Shu and Jones (1993).

PHEROMONEOF Eriborus terebrans

2567

Bioassay Bioassays were conducted in a rectangular wind tunnel (140 cm long x 90 cm wide x 94 cm high), which consisted of screens on each end, wood-framed glass walls (two side walls and top), and a plastic bottom. The wind-tunnel was set up in a ventilated laboratory room in which a 16 : 8 hr light-dark regime was maintained. A temperature of 22-26~ and 55-70% relative humidity were maintained in the wind tunnel. A 6-cm-square wood-board platform was erected at a height of 38 cm at 40 cm from the upwind end to hold a watchglass or a female-holding cage. A 22.5-in.-diam. fan was used at one end to draw air out of the wind tunnel at ca. 0.25 m/sec at the center of the tunnel. Air from the downwind end was exhausted from the room via a chemical hood. Two banks of four 40-W fluorescent lights above the wind tunnel ran parallel with the long axis of the tunnel. These lights were turned on when conducting bioassays. All the tests were performed between the first and third hour of the photo phase. A group of 50 unmated males, 3 days old or older, were used in every bioassay. Each group was used for no more than five consecutive days. No more than six bioassays were conducted each day. Diluted honey in cotton sponges and water in cotton wicks were provided in the wind tunnel. A virgin female (1-3 days old), when tested, was housed in a copper cylinder (4 x 6 cm) with one end screened and other end stopped with a watchglass and placed on the platform. Rinses or isolated fractions were applied on 6.5-cm watchglasses, and except for tests of effects of AFR age, all the chemical preparations were left to dry at the room temperature prior to bioassays for 24 hr. The number of male landings and the number of wing-fannings in a period of 5 rain were recorded as the response criteria for all the bioassays except where otherwise noted. Since males were not captured upon landing, the same male could be counted more than once. Only one wing-fanning per landing, if any, was recorded. All replications were conducted with new chemical preparations except for the test of effects of AFR age. A completely randomized block design was used for all the tests. All treatments in a test were bioassayed in random order on the same day, which was regarded as a block. Data were transformed with log (y + 1) to stabilize the variance. Multiple comparisons were made using Duncan's multiple-range tests at o~ < 0.05 (Duncan, 1955) after F test indicated significance. SAS computer program was used. RESULTS When virgin females or AFR were present in the wind tunnel, male behavior included upwind flight (anemotaxis), casting, hovering, landing, wing-fanning, and attempts to copulate with other males.

2568

SHu AND JONES

One FE AFR was significantly more active than one virgin female and than control in terms of both male landings and male wing-fannings (cr _< 0.05). One virgin female was significantly more active than control (c~ _< 0.05). The mean numbers (four replicates) of male landings and male wing-fannings on the AFR-treated watchglass were 126.25 (SE = 7.21) and 49.75 (SE = 3.87) respectively, while those on the female-holding cage were 89.25 (SE = 5.45) and 32.5 (SE = 1.39), respectively. The mean numbers of male landings and male wing-fannings on the control solvent-treated watchglass were 23.5 (SE = 2.02) and 0, respectively. The watchglass treated with A F R remained active up to four days (Figures 1 and 2). A F R on watchglasses between 0.5 FE and 2 FE was most active when it was 24 hr old. The 4 FE AFR maintained about the same activity from day 1 to day 4. The optimal dose for A F R when 1 day old was 2.5 FE. The pheromonal activity of AFR would not pass through a Florisil column. Neither a single Florisil fraction nor combination of all the Florisil fractions could restore the activity of AFR. Consequently the activity in AFR was partitioned between ether and water saturated with NaHCO3. The neutral phase (ether) was active, but additional activity was obtained by acidifying the water (saturated with NaHCO3) and reextracting with ether (polar phase). Both the

FIG. 1. Effect of AFR ages measured in male landings. The mean number of male landings is represented by y axis (mean based on six replications). FE = female equivalent; CONTR = control.

PHEROMONEOF Eriborus terebrans

2569

FIG. 2. Effect of AFR ages measured in male wing-fannings. The mean number of male wing-fannings is represented by y axis (mean based on six replications). FE = female equivalent; CONTR = control.

polar phase (1 FE) and the neutral phase (1 FE) were active, but not as active as AFR (1 FE) (Figure 3). Male behavior, in response to the polar phase, was similar to that obtained with AFR, but observations indicated that the retention of males on the watchglasses and the duration of their wing-fanning were briefer when the polar phase was present compared to AFR. Doubling the dose of the polar phase (2 FE) did not increase activity. Combination of 1 FE polar phase and 1 FE neutral phase increased the activity significantly, implying that more than one component was present. When the polar phase was further analyzed by HPLC, fraction 2 elicited the greatest behavioral activity and there was some activity in fraction 1 (Table 1). The retention times of 10-hydroxydecanoic acid and octanoic acid were 4.3 min and 6 rain, respectively. The retention time of 10-hydroxydecanoic acid corresponded to fraction 2, while the solvent (acetone) came off in fraction 1. Therefore, the active polar component was similar in polarity to 10-hydroxydecanoic acid under the chromatography conditions. When AFR was chromatographed by TLC, both TLC fractions 1 and 2 were active (Table 2), although TLC fraction 2 was more active than TLC fraction 1 in terms of male wing-fannings. TLC fraction 1 had an R F equivalent to that of 10-hydroxydecanoic acid. TLC fraction 3 had an R F equivalent to that

2570

S H u AND JONES

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Ft6. 3. Male responses (mean + SE) to isolated fractions of AFR. The figure is composed of two graphs. The x axis represents landings and wing-fannings. The number of male landings is represented by y axis on the left and the number of male wing-fannings is represented by y axis on the right (data based on four replications). The different letters above bars in the same category indicate significant difference at ~ < 0.05. IP = 1 FE (female equivalent) polar phase; 2P = 2 FE polar phase; 1N = 1 FE neutral phase; P + N = 1 FE polar phase plus l FE neutral phase; 1EXT = 1 FE AFR; CON = control.

TABLE 1. MEAN NUMBER OF MALE RESPONSES TO HPLC FRACTIONS (2 FE) OF POLAR PHASEa

Fraction 1 2 3 4 5 Control

Collection start to end (min) 3-4 4-5 5-6 6-7 7-8

Responses Male landings

Male wing-fannings

45.8a 64.0b 19.3c 18.3c 17.3c 20.5c

2.8a 11.8b 0 0 0 0

~The mean was obtained from four replications; HPLC was equipped with a Lichrosorb RP-8 column using a solvent system of 1% aqueous phosphoric acid-methanol (20 : 80, v/v) running isocratically. The means followed by different letters in the same column are significantly different at a < 0.05.

PHEROMONEOF Eriborus terebrans

2571

TABLE 2. MEANNUMBEROF MALE RESPONSESTO TLC Fme~CTION(5 FE) OF POLAR FRACTIONSa

Responses Fraction 1 2 3 4

Control

RF

value

0.05 0.18 0.41

Male landings

Male wing-farmings

55.0a 61.8a 22.5b 24.3b

19.0a 25.8a 0.5b 0

19.0b

0.3b

aThe means were obtained from four replications; TLC plate was precoated with Silica Gel G and

developed in a solvent system of: hexane-ether-acetic acid (70: 30 : 2, v/v/v). Polar fractions = TLC fractions 1 and 2 of AFR. The means followed by different letters in the same column are significantly different at ol _< 0.05. of octanoic acid. Therefore, the polar component of the sex pheromone had an RF smaller than that of a fatty acid and equal to or greater than that of a hydroxy acid. When A F R was esterified with diazomethane, it lost most activity. The mean numbers (six replications) of male landings and wing-fannings on watchglasses treated with the esterified A F R (1 FE) were 33.3 (SE = 5.401) and 7.7 (SE = 1.5), respectively, while those with AFR (1 FE) (taken up in the 10% methanol ether) were 98.5 (SE = 10.01) and 48.2 (SE = 5.48), respectively. The number of male landings and the number of male wing-fannings elicited by the esterified A F R were significantly different from those by unmodified A F R at a ___ 0.05. To further characterize the neutral phase of AFR, it was fractionated by open column chromatography on Florisil. Preliminary data suggested that only the hexane Florisit fraction was synergistically active. The neutral phase was consequently separated into two fractions--hexane Florisil fraction and ether Florisil fraction. Bioassay with a combination of either hexane Florisil fraction (2 FE) or ether Florisil fraction (2 FE) with the polar phase (1 FE) indicated that the hexane fraction from the neutral phase contained a component of the sex pheromone (Figure 4). The component was essentially inactive alone, but could increase activity to the polar phase, thus acting as a synergist. The hexane Florisil fraction elicited some male wing-fannings probably from older males. Observations indicated that when males became older and/or were more experienced with sex pheromone, they responded slightly to hydrocarbons (hexane Florisil fraction) alone and to other males with upwind anemotaxis, landing, and even wing-fanning. Extraction of virgin female bodies of E. terebrans with hexane or acetone

2572

SHu A N D

JONES

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FANNING [[221+HexFI EE~Eth ffX~+Eth

responses (mean -+ SE) to isolated fractions of the neutral phase by F l ori s i l column. The figure is composed of two graphs. The x axis represents landings and wingfannings. The number of male landings is represented by the y axis on the left and the number of male wing-fannings is represented by y axis on the right (data based on four replications). The different letters above bars in the same category indicate significant difference at ~ _< 0.05. C o n = control; 1P = 1 F E polar phase; HexF1 = 2 F E hexane Florisil fraction; + H e x F 1 = 1 F E polar phase + 2 F E h e x a n e F l ori s i l fraction; Eth = 2 F E ether F l o r i s i l fraction (eluted just after hexane); + E t h = 1 F E polar phase + 2 F E eth er Flo risil fraction. FIG. 4. M a l e

yielded extracts with very little activity (Table 3). However, FBR (by soaking females in hexane and rinsing the bodies with acetone after hexane extracts were drawn off) contained both the polar component (the polar phase) and the hydrocarbon component (hexane Florisil fraction of the neutral phase). These two components, isolated from FBR, were very active together, and the polar component from FBR was active alone. Based on GC analysis, soaking the females in hexane yielded about 15 times as much hydrocarbon as in AFR. It appeared that E. terebrans males responded to a range of ratios of hydrocarbons to polar component (Table 3, Figure 4). DISCUSSION

Active extracts were obtained by rinsing the female-holding container (glass flask) with acetone twice after rinsing with hexane once. HFR was slightly active, but much less active than AFR, indicating that most pheromone activity was still on the glass after rinsing with hexane. The sex pheromone was appar-

PHEROMONEOF Eriborus terebrans

2573

TABLE 3. MEAN NUMBEROF MALE RESPONSESTO FEMALEBODY RaNSES(FBR) AND ACETONE FLASKRaNSES(AFR) IN 2.5 MINUTES~ Mean number of male responses Treatments

N

Landings

Farmings

0.2 BE FBR 1 BE FBR 0.2 BE PC(FBR) 0.2 BE PC(FBR) + 0.2 BE HexFI(FBR) 1 FE PC(AFR) + 15 FE HexFI(AFR) 1 FE AFR Control

9 4 5 2 2 2 4

8.9 7.8 26.6 51.5 100 80 8.3

1.8 3 8 21.5 52.5 39.5 0

aTreatments were not blocked. Data were not statistically analyzed. BE (body equivalent) = total extracts obtained by soaking one virgin female in hexane; PC = polar component (polar phase), isolated as described in Shu and Jones (1993); HexF1 = hexane Florisil fraction.

enfly adsorbed strongly by glass, which indicated that the pheromone component(s) was very polar. This was supported by other evidence discussed below. Furthermore, GC analysis indicated that H F R contained the same profiles of hydrocarbons as those in A F R , but in greater quantities (4 • The activity o f A F R of E. terebrans on glass lingered for several days. Sex pheromones o f several other hymenopteran parasitoids have been reported to remain active for a long time. F o r example, a cube of plaster o f Paris, soaked in an aqueous suspension o f ground-up pupal shells o f Tortrix viridana (L.) from which females o f Phaeogenes invisor Thunberg had emerged, remained attractive to the males for at least a week (Cole, 1970). Dead female bodies o f Lariophagus distinguendus (F6rst) stayed attractive over periods o f months (Assem et al., 1980). The reason for this phenomenon is unknown. In the case o f E . terebrans, the fact that the pheromone was polar and thus strongly adsorbed to glass and/or the possible high molecular weight of the pheromone might account for the longevity. Near the optimal dose, A F R became most active when they were 24 hr old. The significance of this is unclear. Consequently, all the male responses to the pheromone in this study were assayed after the pheromone substance had been applied to watchglasses for 24 hr, except for the test o f effects of A F R age. The activity o f E. terebrans A F R could not be recovered from the Flofisil column probably because the major pheromone component(s) was too polar and adsorbed to the Flofisil too strongly to be eluted with an organic solvent. However, the pheromonal activity in A F R could be obtained with a liquid-liquid

2574

SHU AND JONES

extraction method. The separation seemed incomplete since the neutral phase was active as well as the polar phase. The active compound(s) in the polar phase had the properties of a carboxylic acid with an additional oxygen-containing function group. A carboxylic acid, being a "strong" acid relative to most organic compounds, would form a salt with a " w e a k " base (NaHCO3) that would be extracted with water and would be converted, after acidifying the solution, back to the carboxylic acid that would be extracted with ether. Comparison of retention times on HPLC indicated that the polar component was more polar than octanoic acid and about as polar as 10-hydroxydecanoic acid. The compound(s) in the polar component had the properties of either a carboxylic acid with less than eight carbons or a carboxylic acid with an additional polar function group. Fractionation by TLC was strictly by class and based on polarity (Holloway and Challen, 1966). Therefore, the RF of the polar component suggested that the compound(s) had the properties of a carboxylic acid with an additional oxygen-containing function group, assuming that the sex pheromone chemicals are composed of the three elements, carbon, hydrogen and oxygen. Diazomethane reacts with carboxylic acids to form methyl esters. AFR lost most of its activity, after esterification with diazomethane, suggesting that the polar component had the properties of a carboxylic acid. Base-hydrolysis of the esterified AFR failed to recover activity, which made purification of the polar compound(s) and subsequent identification by this method impossible. The second c6mponent of E. terebrans pheromone was isolated from the neutral phase of AFR. The neutral phase was eluted from a Florisil column with hexane followed only by ether. The ether Florisil fraction should contain all compounds that would come off the Florisil column when eluted with a gradient from 2.5% ether in hexane to 100% ether. Bioassays indicated that this ether fraction did not contain any pheromonal component. The synergistic component in the neutral phase was obtained from a Florisil column with hexane, indicating that it was a hydrocarbon(s). Extraction of virgin female bodies of E. terebrans with hexane or acetone (FBR) failed to yield an active extract. This could be due to several reasons. For example, an inhibitory agent or compounds, which reacted with or masked the sex pheromone, might have been extracted. Vinson (1972) reported that ethyl acetate extracts of females of Campoletis sonorensis (Cameron) elicited all the sexual behavioral elements in males. However, extracting females with ethyl acetate over 5 or 6 hr reduced the activity of the extracts. An unbalanced ratio of pheromone components also could reduce activity. Cuticular hydrocarbons have been reported to be sex pheromone components of two hymenopteran insects (Bartelt et al., 1982; Swedenborg and Jones, 1992). Because the cuticular hydrocarbons are easy to extract, soaking insects in an organic solvent might

PHEROMONE OF Eriborus terebrans

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o b t a i n the h y d r o c a r b o n c o m p o n e n t in e x c e s s o f t h e n a t u r a l ratio. Such extracts m a y n o t b e a c t i v e b e c a u s e o f t h e w r o n g ratio o f c o m p o n e n t s . In the case o f E. terebrans, the ratio o f h y d r o c a r b o n c o m p o n e n t to p o l a r c o m p o n e n t in sex p h e r o m o n e o f E. terebrans s e e m s to b e not critical, s i n c e the m a l e s a c t i v e l y r e s p o n d e d to a r a n g e o f ratios o f h y d r o c a r b o n s to p o l a r c o m p o n e n t ( T a b l e 3, F i g u r e 4). F u r t h e r m o r e , the p o l a r c o m p o n e n t a n d h y d r o c a r b o n s , isolated f r o m F B R , w e r e v e r y a c t i v e in c o n c e r t , w h i l e F B R itself w a s not active, s u g g e s t i n g F B R c o n t a i n e d a n e x t r a c o m p o n e n t ( s ) r e n d e r i n g F B R inactive. Acknowledgments--We wish to thank Paul D. Swedenborg for his help and advice; Dr. Timothy J. Kurtti, Dr. Christopher Bingham, and Dr. Essie Kariv-Miller at the University of Minnesota for their comments on draft of the manuscript; and Dr. Gary G. Grant at the Forest Pest Management Institute of Forestry Canada for his critical comments on the manuscript. Paper No. 20,455, Scientific Journal Series, Minnesota Agricultural Experiment Station, University of Minnesota, St. Paul, Minnesota 55108.

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