Journal of Chemical Ecology, Vol. 17, No. 2, 1991
MINT ROOT BORER, Fumibotys fumalis (Guenee) l: IDENTIFICATION AND FIELD TESTS OF FEMALE SEX PHEROMONE GLAND COMPONENTS
H . G . D A V I S , z L . M . M c D O N O U G H , 2'* C . L . S M I T H H I S L E R , 2 D . F . B R O W N , 2 B . A . L E O N H A R D T , 3 S. V O E R M A N , 4 and P.S. CHAPMAN 2
2Agricultural Research Service, USDA Yakima, Washington 98902 3Agicultural Research Service, USDA Bettsville, Maryland 20705 4Research Institute for Plant Protection P. O. Box 9060 6700 GW Wageningen, The Netherlands (Received July 12, 1990; accepted September 17, 1990) Abstract--Compounds identified in sex pheromone gland extracts of female Fumibotys fumalis (Guenee) consisted of (E, E )-10,12-tetradecadienyl acetate, (Z)-I 1-tetradecenyl acetate, (E)-11-tetradecenyl acetate, and (Z)-9tetradecenyl acetate in a ratio of 100:18 : 8:4, respectively. The compounds were identified by electroantennographic, gas chromatographic, mass spectrometric, and chemical derivatization procedures. In mint fields synthetic components in gray elastomeric septa at ratios found in the sex pheromone gland and at doses of 3 or 10 mg of the diene produced trap catch comparable to traps baited with three females. Key Words--Lepidoptem, Pyralidae, Pyraustinae, Pyraustini, Famibotys fumalis, sex pheromone, (E, E )-10,12-tetradecadienyl acetate, (Z)- 11-tetradecenyl acetate, (E)-11-tetradecenyl acetate, (Z)-9-tetradecenyl acetate.
INTRODUCTION T h e m i n t r o o t b o r e r , F u m i b o t y s f u m a l i s ( G u e n e e ) is w i d e l y d i s t r i b u t e d in N o r t h A m e r i c a ( M o n r o e , 1976). It w a s first r e p o r t e d to b e a p e s t o f a c u l t i v a t e d c r o p *To whom correspondence should be addressed. t Lepidoptera: Pyralidae. 249
250
DAVIS ET AL.
in 1971 when it was found damaging commercially grown peppermint, Mentha piperita L. in the Willamette Valley of Oregon (Berry, 1974). Subsequently, F. fumalis adapted to and damaged spearmint, M. spicata L., and became an established pest in the commercial mint fields of Washington, Idaho, and Oregon (Pike and Glazer, 1982). Damage occurs when the larvae bore into and feed on the mint rhizomes. Infested fields show de-~c'linin-'g c yields and shortened stand life (Pike et al., 1988). The taxonomy, morphology, and geographical distribution of F. fumalis were described by Monroe (1976). Studies of its biology and behavior (Forbes, 1923; Berry, 1974, 1977; Davis et al., 1984; Pike et al., 1988) and chemical and cultural control (Pike, 1979; Pike and Getzin, 1981; Pike and Glazer, 1982) have been reported. Because control procedures would be enhanced by a lure to monitor adult flight periods, we undertook a chemical and biological study in order to develop an effective sex pheromone attractant. An earlier biological study (Davis et al., 1984) demonstrated a female sex pheromone in F. fumalis, that maximum mating occurred between 12 PM and 1 AM, that females were maximally attractive from age 0.5-7 days, and that females were not attractive the night following mating. Here we report the identification of four components of the female sex pheromone gland and the development of an effective field lure.
METHODS AND MATERIALS
Insects. F. fumalis hibemaculae containing prepupae were collected from soil samples taken from infested peppermint fields near Harrah, Washington, in February and early March in 1987, 1988, and 1989. The hibernaculae were placed in clear plastic shoe boxes (31 x 17 x 9 cm) on a 5- to 6-cm layer of moistened peat moss, covered with clear plastic lids, and held in a rearing room at a temperature of 12.8 ~ relative humidity of 55-60 %, and a 12 : 12 hr lightdark cycle. As adults were needed to conduct laboratory or field studies, boxes were removed and placed in another facility at a temperature of 21.1~ and a 15 : 9 hr light-dark cycle. The onset of the dark cycle was set at 0800 hr for the flight-tunnel studies and at 2100 hr for the field studies. Emergent adults were collected daily at least 1 hr before the dark period and placed in individual vials. Collection of Pheromone. Female moths (3-4 days old) in their vials were collected 6 hr after the beginning of scotophase and placed in a refrigerator to inactivate them. They were removed individually and their abdominal tips containing the sex pheromone gland (SPG) were severed and steeped for 15-60 min in dichloromethane. Then the solution was removed with a syringe. To obtain pheromone that the females may have released while inside the vials, the vials were rinsed with dichloromethane and the rinses were combined.
SEX P H E R O M O N E OF M I N T R O O T BORER
251
Electroantennogram (EAG) Determinations. EAG determinations of model compounds were made with previously described apparatus (McDonough et al., 1980) except that 60-t~g charges were used. Duplicate determinations were made of each compound. The model compounds used were synthesized mainly by the procedure of Voerrnan (1988). A few were obtained from commercial sources. All compounds were at least 98% pure by capillary gas chromatographic analysis and contained 1% or less of their geometric isomers. The compounds used were: all of the saturated and monoene 12- and 14-carbon alcohols and corresponding acetate; Z and E isomers of 7-, 9-, and 11-16 : Ac; Z and E isomers of 7-, 8-, and 9-12 :A1; Z 7 - 1 4 :A1, Z9-14 :A1, Z11- and E11-14 : A1; Z5-16: A1, Z 7 - and E7-16 : A1, Z9-16 : A1, Z11- and E l 1 - 1 6 :A1, Z13-16 : A1. Gas Chromatography-Electroantennography (GC-EAG). Fractions of extracts from sex pheromone glands or vial washes containing female emissions were collected from a GC equipped with a 0.63-cm-OD x 180-cm-long glass column packed with 3 % methyl silicone (SE-30) liquid phase on 80-100 mesh Gas Chrom Q and connected to an effluent splitter. The initial column temperature was 120~ for 8 rain, then programmed at 4~ to 210~ and held at that temperature. The fractions were collected from time zero to the time corresponding to the complete elution of 10:Ac and then successively at times corresponding to the complete elution of each of the saturated acetates from 11 : Ac through 20 : Ac. Fractions were collected in 3-mm-OD, U-shaped, glass traps cooled by Dry Ice-acetone, and then dissolved in dichloromethane and deposited on the inside of glass tubes for EAG determinations. Fractions also were collected for EAG determination from a 0.63-cm-OD x 180-cm-long glass column packed with 5% Carbowax 20M liquid phase on 80-100 mesh Gas Chrom Q. Fraction 1 encompassed time zero to the beginning of the elution of 10:Ac; succeeding fractions each encompassed two carbon increments to the beginning of the elution of 20 : Ac. Gas Chromatography-Mass Spectrometry (GC-MS). A Hewlett-Packard (Avondale, Pennsylvania) gas chromatograph (model 5790) with a quadrupole mass spectrometer (model 5970) was equipped with either a DB-1 or DB-Wax capillary column, 60 m x 0.25 mm ID (J & W Scientific, Folsom, California). When the pheromone gland extracts were analyzed, the columns were held at 80~ for 2 rain, programmed at 2 0 ~ to 190~ for DB-1, and to 200~ for DB-Wax, and maintained isothermally thereafter. Total ion abundance was monitored. When the dimethyl disulfide derivatives of the monoenes were analyzed, the DB-1 column was used and was held at 80~ for 2 min, programmed at 2 0 ~ to 255~ and held at that temperature for 30 min. Determination of Positions and Configurations of Double Bonds of Monoenes. Monoenes from extract of sex pheromone glands were provisionally identified by comparing retention times on DB-1 (60 m x 0.25 mm; 2 rain at 80~ then 2 0 ~ to 190~ and DB-Wax (45 m x 0.20 mm ID at 145~
252
DAVISET AL.
GC capillary columns with synthetic monoenes. A Hewlett-Packard model 5880 GC was used. Rigorous determination of double-bond positions was accomplished by mass spectral determinations of fragmentation patterns of the dimethyl disulfide (DMDS) derivatives (Buser et al., 1983). To prepare the derivatives, monoenes (15 ng) isolated as a GC fraction from extract of SPG in 200/~1 of dichloromethane were transferred to a 1-ml Reacti-vial (Pierce Chemical Co., Rockford, Illinois). The solution was evaporated just to dryness in a stream of nitrogen, and the residue was dissolved in 50/xl heptane. Then 50/zl DMDS and 10/zl of a saturated solution of iodine in ether were added. The vial was sealed and heated overnight at 40~ in a convection oven. The solution was diluted with 200/zl of heptane and excess 12 was removed by shaking with 100 /A of 5 % of sodium bisulfite solution. The heptane solution was moved with a syringe and concentrated to 2 #1 for injection into the GC-MS.
Determination of Positions and Configurations of Double Bonds of Diene. The GC purified diene (144 ng) was ozonized in predistilled dichloromethane at - 7 0 ~ with an ultraviolet ozonizer (Orec Co. model 03V1, Phoenix, Arizona), and the ozonide was reduced with triply recrystallized triphenytphosphine. The ozonized sample was injected on a GC column (DB-1, 60 m x 0.25 mm), which was held at 80~ for 7 min, programmed at 6 ~ to 200~ and held at that temperature. When the configuration of the diene was determined, the same column was held at 80~ for 2 min and programmed at 20 ~ to 180 ~ and held at that temperature. Flight Tunnel. A flight tunnel constructed as described by Miller and Roelofs (1978) was used. The flight tunnel room was maintained at 22~ Males were released in groups of five. The number of males initiating and sustaining flight and contacting the lure were determined. All lures were formulated in gray elastomeric septa (formulation number 1888, size no. 1, West Co., Phoenixville, Pennsylvania) (Brown and McDonough, 1986) to prevent isomerization of the diene. Males were three to four days postemergence and were maintained on a reverse dark-light cycle ( 10 and 14 hr). Tests were conducted 3 hr after the onset of scotophase. Field Tests. Z9-, Z l l - , and E l l - 1 4 : A c used for the field tests were obtained from commercial sources and were pure by GC analysis and contained less than 1% of the geometric isomer. El0, E 1 2 - 1 4 : A c was synthesized by a modification of the method for Amorbia cuneana (McDonough et al., 1982; McDonough and Smithhisler, 1989). The synthesis was carried through until a mixture of El0, E l 2 - and El0, Z 1 2 - 1 4 : O H was obtained. Pure El0, E1214:OH was obtained by low-temperature recrystallization from pentane and then was converted to the acetate. Isomeric purity was 98.5% EE, 1% EZ and 0.5 % ZE for all of the experiments except the one in which the effect of purity was tested. Then the purity was 96.0% EE, 2.5% EZ, and 1.5 ZE. Candidate
253
SEX PHEROMONE OF MINT ROOT BORER
lures in 100 #1 of dichloromethane were impregnated into gray elastomeric septa; dichloromethane alone was added to control septa. Pherocon IC sticky traps (Trece, Corp., Salinas, California) were used. Traps were suspended from a movable metal arm (an 18 x 23-cm shelf bracket) attached by a metal hose clamp to a 120-cm-long wooden broom handle driven into the ground. This arrangement enabled the traps to be positioned just above the mint foliage, usually 45-60 cm above the ground, where most flight activity was believed to Occur.
The position of each trap in the experimental plot was drawn randomly and traps were deployed at approximately 10-m intervals. Females used in the field tests were placed in aluminum wire screen cages (9 x 9 • 2 cm, three females/ cage), which were suspended by a wire from the top of the trap equidistant between the top and bottom. There were four replicates of each lure tested and the trap data were transformed by (x + 0.5) 1/2 and compared by Duncan's (1955) multiple-range test ( P = 0.05).
RESULTS
AND
DISCUSSION
Exploratory Study of Structure of Pheromone Components. Among the model compounds tested, the strongest EAG responses were obtained from the 14-carbon acetates (Figure 1). The 10 and 12 positions ofunsaturation produced the strongest responses, and strong responses were also obtained from the 9 and 11 positions. After the 14-carbon acetates, the strongest responses were obtained from the 12-carbon acetates: E l 0 - and Z10-12 : Ac produced responses of about 50% of the standard (Z10-14:Ac) and the other positional isomers produced responses of about 25% of standard. Other tested compounds produced responses of 0-30% of the standard. lO0z ~
80-
_~
60-
txl a-
40-
ixl
~
w o_
EZ~E I Z
20 0
0
1
2
5
4
5
6
7
8
9
10 11 12 ! 3
DOUBLE BOND POSITION
Fic. 1. Electroantennographic responses of male mint root borers (Fumibotys fumalis) to 14-carbon acetates. Numbers indicate double-bond position; zero indicates saturated.
254
DAVIS ET AL.
The EAG response of GC fractions of SPG extract from the methyl silicone column was greatest for the fraction corresponding to the end of 14 : Ac through the end of 15 :Ac (1.9 mV vs. 0.1-0.8 mV for the other fractions), and from the Carbowax column for the fraction corresponding to the beginning of 16 : Ac to the beginning of 18 :Ac (0.7 mV vs. 0.2-0.4 mV for the other fractions). The EAG response to GC fractions (methyl silicone column) of the washes of the vials that held females was also greatest for the fraction corresponding to the end of 14:Ac through the end of 15:Ac (0.9 mV vs. 0.05-0.35 mV for the other fractions). GC-MS analysis (DB-1 column) of SPG extract (50 female equivalents) at the retention time period that produced the strong EAG response indicated the presence of a tetradecadienyl acetate [M +, 252; (M-60) § 192; CH3CO2H~-, 61] at a retention index (Ix) of 1838. Also, eluting prior to the tetradecadienyl acetate were three tetradecenyl acetates [(M-60) § 194; CH3CO2H~- , 61] at Ix values of 1776, 1781, and 1788. There was about 0.5 ng of the diene per female equivalent, and the ratio of the monoenes to the diene for each Ix value was 0.04 : 1 (1776), 0.08:1 (1781), and 0.18 : 1 (1788).
Positions and Configurations of Double Bonds of Pheromone Gland Components. Ozonolysis of the diene gave a single GC peak (methylsilicone column) with a retention time of 24.50 min near that of hexadecane (retention time = 24.77 min) and the same within experimental error ( + 0 . 0 4 min) as the ozonolysis product of 10,12-14:Ac (retention time = 24.52). The configuration of the diene was EE (retention time = 21.85 min) and the retention times of the four isomers of a synthetic standard (McDonough et al., 1982) were: ZE, 21.33; EE, 21.85; EZ, 22.10; ZZ, 22.29. A comparison of retention times of the pheromone tetradecenyl acetates with those of the synthetic tetradecenyl acetates on polar and nonpolar capillary GC columns indicated the pheromone tetradecenyl acetates were Z9-, Z11-, and E11-. Mass spectral analysis of the DMDS derivatives confirmed these assignments. The monoene derivatives and their diagnostic peaks (Buser et al., 1983) were: Z 9 - [M § 348 (33%); A, 117 (53%); B, 231 (100%)]; Z l l [M +, 348 (21%); A, 89 (34%); B, 259 (100%)]; E l l - [M +, 348 (19%); A, 89 (25%); B, 259 ( 100% )]. The retention times of the DMDS derivatives (in minutes) were: Z 9 - , 25.34; Z l l - , 27.12; E l l - , 27.54. The configurational assignments were confirmed by comparison of retention times with synthetic samples ( E 9 - DMDS derivative at 25.66 min). Male Response to Synthetic Pheromone Components. When lures containing 100/zg of E 1 0 , E 1 2 - 1 4 : A c plus 18, 8, and 4/zg of Z l l - , E l l - , and Z 9 tetradecenyl acetates were tested in a mint field (four replicated traps per test), total male captures (13) were not statistically different from blank traps but captures in female baited traps (98) were. In order to identify the reason for
SEX PHEROMONE OF MINT ROOT BORER
255
the lack of significant capture, limited tests in a flight tunnel were conducted (Table 1). When the pheromone components were tested individually (Table 1, tests 1-4), Z l l - 1 4 : A c was a weak attractant, but no response was obtained from the other components. When the four components were tested in the ratio found in the SPG and the dosage of EE was 100 #g (the same as the unsuccessful field test), 65 % of males exhibited upwind flight and 50 % touched the source (test 5). Lower doses of 33 and 10/xg of this ratio produced tess response (tests 6 and 7), while a higher dose produced the highest percent responding (test 8). Subtraction of E 1 1 - 1 4 : A c from the natural blend produced a response almost equivalent to the natural blend (test 9 vs. 5). Because of the limited numbers available for testing, more complete, systematic tests and statistical analysis were not undertaken. Nevertheless, collectively, these experiments established the attractiveness of the mixture of identified components and suggested that the initial field test may have produced low catches because the dose was too low. In further field tests (test 1, Table 2) when the ratio of EE, Z11-, and Z 9 TABLE 1. RESPONSE IN FLIGHT TUNNEL OF ADULT MALE f . LURES
Test 1 2 3 4 5
6
7
8
9
Lure (dose in/zg ) E 1 0 , E 1 2 - 1 4 : A c (100) Z11-14 : Ac (100) Z 9 - 1 4 : Ac (100) E l l - 1 4 : A c (100) E 1 0 , E 1 2 - 1 4 : A c (100) + Z I 1 14:Ac (18) + Z 9 - 1 4 : A c (4) + E l l - 1 4 : A c (8) E 1 0 , E 1 2 - 1 4 : A c (33) + Z l l 14:Ac (6) + Z 9 - 1 4 : A c (1.3) + E l l - 1 4 : A c (2.7) E 1 0 , E 1 2 - 1 4 : A c (10) + Z l l 14:Ac (1.8) + Z 9 - 1 4 : A c (0.4) + E l l - 1 4 : A c (0.8) E 1 0 , E 1 2 - 1 4 : A c (300) + Z l l 14:Ac (54) + Z 9 - 1 4 : A c (12) + E I I - 1 4 : A c (24) E 1 0 , E 1 2 - 1 4 : A c (100) + Z l l 1 4 : A c (18) + Z 9 - 1 4 : A c (4)
fumalis TO CANDIDATE
Number of males tested
Number flying upwind
Number contacting source
10 10 10 10 20
0 3 0 0 13
0 1 0 0 10
10
5
3
10
2
2
5
4
4
10
6
4
256
DAvis ET AL.
TABLE 2. CAPTURESOF NATIVE ADULT MALE F. fumalis IN PHEROCON 1C TRAPS BAITED WITH VARIOUS CANDIDATE LURES IN COMMERCIAL MINT FIELDS, HARRAH, WASHINGTON~ 1989 a
Test
Lure
i. July 27-28, 1989
Dose (#g/septum)
EIO,E12-14:Ac, Zll-14:Ac, Z9-14:Ac E10,E12-14:Ac, Z l l - t 4 : A c , Z9-14:Ac E10,E12-14:Ac, Zll-14:Ac, Z9-14:Ac E10,E12-14:Ac, Zll-14:Ac, Z9-14:Ac 3 females blank septa
2. August4-6, 1989
3. August 8, 1989
El0, El2-14 : Ac, E10,E12-14:Ac, El0, E12-14 : Ac, E10,E12-14:Ac, E11-14 : Ac 3 females blank septa
Z11-14 : Ac, Zll-14:Ac, Z11-14 : Ac, Zll-14:Ac,
El0, E12-14 : Ac, E10,E12-14:Ac, El0, E12-14 : Ac, E10,E12-14:Ac, 2 females blank septa
Z11-14 : Ac, Zll-14:Ac, Z11-14 : Ac, Zll-14:Ac,
Z9-14 : Ac Z9-14:Ac Z9-14 : Ac Z9-14:Ac,
100:18:4 300:54:12 1000:180:40 3000:540:120
3000:540:120 3000:540:120 b 10,000, 1800, 400 3000, 540, 120, 240
Cumulative captures 9 5 22 68 61 4
a a a b b a
105 128 278 119
a a b a
329 b 14 c Z9-14 : Ac Z9-14:Ac Z9-14 : Ac Z9-14:Ac
3000:1800:120 3000:5400:120 10,000:1800:400 10,000:5400:400
125 61 100 164 31 4
ab ac ab b c c
aGray elastomeric septa were .used as the controlled release substrate. There were four replicate traps per treatment. Letters after the numbers within a test number indicate significance according to Duncan's (1955) multiple range test (P = 0.05). bThe isomeric purity of El0, E12-14 :Ac in this test was 96 %; in all other tests the isomeric purity was 98.5 %.
f o u n d in the S P G was used and the dose o f EE was v a r i e d f r o m 100 to 3000 /~g, the 3000-/zg d o s e p r o d u c e d significant trap catch c o m p a r e d to controls and was e q u i v a l e n t to that p r o d u c e d by f e m a l e s . Captures p r o d u c e d by the l o w e r doses w e r e not statistically significant. In test 2, T a b l e 2, l o w e r i s o m e r i c purity of EL did not d e c r e a s e catch; the absence o f E 1 1 - 1 4 : A c did not d e c r e a s e catch; and the 10-mg d o s e p r o d u c e d h i g h e r catch than the 3 - m g dose. Catches f r o m b o t h doses w e r e statistically significant but only the 10 m g dose was e q u i v a l e n t to f e m a l e s . T e s t 3 was c o n d u c t e d to d e t e r m i n e if a h i g h e r proportion o f Z 1 1 14 : A c w o u l d increase catch. A t neither the 3- nor the 10-mg dose l e v e l did the increase in the p r o p o r t i o n o f Z l l - 1 4 : A c p r o d u c e statistically significant increases in trap catch. C o l l e c t i v e l y , t h e s e e x p e r i m e n t s d e m o n s t r a t e d that the three- o r f o u r - c o m p o n e n t lure in the ratio found in the sex p h e r o m o n e gland and at a dose o f 3 10 m g o f E 1 0 , E 1 2 - 1 4 : A c in gray e l a s t o m e r i c septa p r o d u c e d a v i a b l e attrac-
SEX P H E R O M O N E OF M I N T R O O T BORER
257
tant, competitive with female baited traps in field tests. Flight-tunnel studies i~ndicate at least two components are necessary to produce an effective attractant. E 1 1 - 1 4 : Ac may not be a component o f the pheromone emitted by females, since it is the expected biosynthetic precursor to E l 0 , E l 2 - 1 4 : A c (L6fstedt and Bengtsson, 1988). To fully define the importance o f each of the four components, field or flight-tunnel tests with subtraction experiments over a range of concentrations are needed. The relatively high septa doses required, coupled with the small amount o f pheromone found in the glands, were unexpected. Nevertheless, these facts do not necessarily indicate there are undetected pheromone components. The low amount o f pheromone in the glands could indicate close synchronization o f biosynthesis with emission rather than a low emission rate. Moreover, the emission rate of septa at the 3- to 10-mg doses is not excessive compared w i t h that required for some other Lepidoptera. Gray septa emit at rates about the same as red rubber septa (McDonough and Aller, unpublished). Also 16-carbon aldehydes and 14-carbon acetates emit at similar rates (McDonough, 1990). The most effective doses o f the sex pheromone of Heliothis zea ( 16-carbon aldehydes) in red rubber septa are 3 - 1 0 mg (Halfhill and McDonough, 1985), and the most effective doses of Heliothis virescens sex pheromone (mainly a 16carbon aldehyde) are even higher (Flint et al., 1979). Although this is the first report o f the chemical identification o f E l 0 , E 1 2 14 : Ac in the female sex pheromone gland o f a pyralid, in field screening tests Reed and Chisholm (1985) reported that Pyrausta fodinalis (L.) was captured in traps baited with E 1 0 , E 1 2 - 1 4 : A c . Taxonomically, P. fodinalis and F, fumalis are both grouped as Pyraustini. Acknowledgments--We thank Dr. Keith Pike, Washington State University Experiment Station, Prosser, for his suggestions; and L. Bert Dekker, Matthew Lommers, and on Rice for use of their mint fields.
REFERENCES BERRY, R.E. 1974. Biology of Pyrausta fumalis on peppermint in Oregon. Ann. EntomoL Soc. Am. 67:580-582. BERRY, R.E. 1977. Insects on mint. Pacific Northwest Coop. Ext. Pub. 182. 15 pp. Joint issue of the State Universities of Washington, Oregon, and Idaho. BROWN,D.F., and McDoNOUGH,L.M. 1986. Insect sex pheromones: Formulations to increase the stability of conjugated dienes. J. Econ. Entomol. 79:922-927. BUSER, H.R., ARN, H., GUERIN,P., and RAUSCHER,S. 1983. Determination of double bond position in mono-unsaturated acetates by mass spectrometry of dimethyl disulfide adducts. Anal. Chem. 55:818-822. DAVIS,H.G., MCDONOUGH,L.M., and PIKE,K.S. 1984. Attraction of male Fumibotysfumalis to females of the species. J. Entomol. Soc. B. C 81:25-28. DUNCAN,O.B. 1955. Multiple range and multiple F tests. Biometrics 11:1-41.
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FLINT, H.M., MCDONOOGH, L.M., SALTER, S.S., and WALTERS, S. 1979. Rubber septa: A long lasting substrate for (Z )-11-hexadecenal and (Z)-9-tetradecenal, the primary components of the sex pheromone of the tobacco budworm. J. Econ. Entomol. 72:798-800. FORBES, W.T.M. 1923. The Lepidoptera of New York and neighboring states. Memoir 68. Comell Univ. Agric. Exp. Sta. Ithica, New York. 729 pp. HALFHILL,J.E., and MCDONOUGH, L.M. 1985. Heliothis zea (Boddie): Formulation parameters for its sex pheromone in rubber septa. Southwest. Entomol. 10:176-180. LOFSTEDT, C., and BENGTSSON, M. 1988. Sex pheromone biosynthesis of (E,E)-8,10-dodecadienol in codling moth Cydia pomonella involves E9 desaturation. J. Chem. Ecol. 14:903915. McDoNOUGH, L.M. 1990. Controlled release of insect sex pheromones from a natural rubber substrate, in P. Hedin (ed.). Naturally Occurring Pest Bioregulators. ACS Symposium Series. American Chemical Society, Washington, D.C. In press. MCDONOUGH, L.M., and SMITHHISLER,C.L. 1989. Improved synthesis of the sex pheromone of the avocado leafroller, Amorbia cuneana (Walsingham). Southwest. Entomol. 14:153-157. MCDONOUCH, L.M., KAMM, J.A., and BIERL-LEONHARDT, B.A. 1980. Sex pheromone of the armyworm (Pseudoletia unipuncta (Haworth) (Lepidoptera: Noctuidae). J. Chem. Ecol. 6:565572. McDoNOUGH, L.M., HOFFMANN, M.P., BIERL-LEONHARDT, B.A., SMITHHISLER, C.L., BAILEY, J.B., and DAVIS, H.G. 1982. Sex pheromone of the avocado pest, Amorbia cuneana (Walsingham) (Lepidoptem: Tortricidae): Structure and synthesis. J. Chem. Ecol. 8:255-265. MILLER, J.R., and ROELOFS, W.L. 1978. Sustained flight tunnel for measuring insect responses to wind-borne sex pheromones. J. Chem. Ecol. 4:187-198. MONROE, E. 1976. Pyraloidea, pp. 26-28, in The Moths of America North of Mexico. Pyralidae (part) Fascicle 13.2A: E.W. Classey Ltd. and The Wedge Entomological Research Foundation, London. 304 + xx pp. PIKE, K.S. 1979. Peppermint, Fumibotysfumalis control, Washington. Insecticide Acaracide Tests 4:88. PIKE, K.S., and GETZIN, L.W. 1981. Persistence and movement of chlorpyrifos in sprinkler-irrigated soil. J. Econ. Entomol. 74:385-388. PIKE, K.S., and GLAZER,M. 1982. Strip rotary tillage: A management method for reducing Fumibotysfumalis (Lepidoptera: Pyralidae) in peppermint. J. Econ. Entomol. 75:1136-1139. PIKE, K.S., BAIRD, C.R., and BERRY, R.E. 1988. Mint root borer in the Pacific Northwest. Pacific Northwest Coop. Ext. Pub. 322. 8 pp. Joint issue of the State Universities of Washington, Oregon, and Idaho. REED, D.W., and CHISHOLM, M.D. 1985. Attraction of moth species of Tortricidae, Gelechiidae, Geometridae, Drepanidae, Pyralidae, and Gracillariidae families to field traps baited with conjugated dienes. J. Chem. Ecol. 11:1645-1657. VOERMAN,S. 1988. The pheromone bank: A collection of unsaturated compounds indispensable for discovery of sex attractants for Lepidoptera. Agric. Ecosyst. Environ. 21:31-41.
MINT ROOT BORER, Fumibotys fumalis (Guenee) l: IDENTIFICATION AND FIELD TESTS OF FEMALE SEX PHEROMONE GLAND COMPONENTS
H . G . D A V I S , z L . M . M c D O N O U G H , 2'* C . L . S M I T H H I S L E R , 2 D . F . B R O W N , 2 B . A . L E O N H A R D T , 3 S. V O E R M A N , 4 and P.S. CHAPMAN 2
2Agricultural Research Service, USDA Yakima, Washington 98902 3Agicultural Research Service, USDA Bettsville, Maryland 20705 4Research Institute for Plant Protection P. O. Box 9060 6700 GW Wageningen, The Netherlands (Received July 12, 1990; accepted September 17, 1990) Abstract--Compounds identified in sex pheromone gland extracts of female Fumibotys fumalis (Guenee) consisted of (E, E )-10,12-tetradecadienyl acetate, (Z)-I 1-tetradecenyl acetate, (E)-11-tetradecenyl acetate, and (Z)-9tetradecenyl acetate in a ratio of 100:18 : 8:4, respectively. The compounds were identified by electroantennographic, gas chromatographic, mass spectrometric, and chemical derivatization procedures. In mint fields synthetic components in gray elastomeric septa at ratios found in the sex pheromone gland and at doses of 3 or 10 mg of the diene produced trap catch comparable to traps baited with three females. Key Words--Lepidoptem, Pyralidae, Pyraustinae, Pyraustini, Famibotys fumalis, sex pheromone, (E, E )-10,12-tetradecadienyl acetate, (Z)- 11-tetradecenyl acetate, (E)-11-tetradecenyl acetate, (Z)-9-tetradecenyl acetate.
INTRODUCTION T h e m i n t r o o t b o r e r , F u m i b o t y s f u m a l i s ( G u e n e e ) is w i d e l y d i s t r i b u t e d in N o r t h A m e r i c a ( M o n r o e , 1976). It w a s first r e p o r t e d to b e a p e s t o f a c u l t i v a t e d c r o p *To whom correspondence should be addressed. t Lepidoptera: Pyralidae. 249
250
DAVIS ET AL.
in 1971 when it was found damaging commercially grown peppermint, Mentha piperita L. in the Willamette Valley of Oregon (Berry, 1974). Subsequently, F. fumalis adapted to and damaged spearmint, M. spicata L., and became an established pest in the commercial mint fields of Washington, Idaho, and Oregon (Pike and Glazer, 1982). Damage occurs when the larvae bore into and feed on the mint rhizomes. Infested fields show de-~c'linin-'g c yields and shortened stand life (Pike et al., 1988). The taxonomy, morphology, and geographical distribution of F. fumalis were described by Monroe (1976). Studies of its biology and behavior (Forbes, 1923; Berry, 1974, 1977; Davis et al., 1984; Pike et al., 1988) and chemical and cultural control (Pike, 1979; Pike and Getzin, 1981; Pike and Glazer, 1982) have been reported. Because control procedures would be enhanced by a lure to monitor adult flight periods, we undertook a chemical and biological study in order to develop an effective sex pheromone attractant. An earlier biological study (Davis et al., 1984) demonstrated a female sex pheromone in F. fumalis, that maximum mating occurred between 12 PM and 1 AM, that females were maximally attractive from age 0.5-7 days, and that females were not attractive the night following mating. Here we report the identification of four components of the female sex pheromone gland and the development of an effective field lure.
METHODS AND MATERIALS
Insects. F. fumalis hibemaculae containing prepupae were collected from soil samples taken from infested peppermint fields near Harrah, Washington, in February and early March in 1987, 1988, and 1989. The hibernaculae were placed in clear plastic shoe boxes (31 x 17 x 9 cm) on a 5- to 6-cm layer of moistened peat moss, covered with clear plastic lids, and held in a rearing room at a temperature of 12.8 ~ relative humidity of 55-60 %, and a 12 : 12 hr lightdark cycle. As adults were needed to conduct laboratory or field studies, boxes were removed and placed in another facility at a temperature of 21.1~ and a 15 : 9 hr light-dark cycle. The onset of the dark cycle was set at 0800 hr for the flight-tunnel studies and at 2100 hr for the field studies. Emergent adults were collected daily at least 1 hr before the dark period and placed in individual vials. Collection of Pheromone. Female moths (3-4 days old) in their vials were collected 6 hr after the beginning of scotophase and placed in a refrigerator to inactivate them. They were removed individually and their abdominal tips containing the sex pheromone gland (SPG) were severed and steeped for 15-60 min in dichloromethane. Then the solution was removed with a syringe. To obtain pheromone that the females may have released while inside the vials, the vials were rinsed with dichloromethane and the rinses were combined.
SEX P H E R O M O N E OF M I N T R O O T BORER
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Electroantennogram (EAG) Determinations. EAG determinations of model compounds were made with previously described apparatus (McDonough et al., 1980) except that 60-t~g charges were used. Duplicate determinations were made of each compound. The model compounds used were synthesized mainly by the procedure of Voerrnan (1988). A few were obtained from commercial sources. All compounds were at least 98% pure by capillary gas chromatographic analysis and contained 1% or less of their geometric isomers. The compounds used were: all of the saturated and monoene 12- and 14-carbon alcohols and corresponding acetate; Z and E isomers of 7-, 9-, and 11-16 : Ac; Z and E isomers of 7-, 8-, and 9-12 :A1; Z 7 - 1 4 :A1, Z9-14 :A1, Z11- and E11-14 : A1; Z5-16: A1, Z 7 - and E7-16 : A1, Z9-16 : A1, Z11- and E l 1 - 1 6 :A1, Z13-16 : A1. Gas Chromatography-Electroantennography (GC-EAG). Fractions of extracts from sex pheromone glands or vial washes containing female emissions were collected from a GC equipped with a 0.63-cm-OD x 180-cm-long glass column packed with 3 % methyl silicone (SE-30) liquid phase on 80-100 mesh Gas Chrom Q and connected to an effluent splitter. The initial column temperature was 120~ for 8 rain, then programmed at 4~ to 210~ and held at that temperature. The fractions were collected from time zero to the time corresponding to the complete elution of 10:Ac and then successively at times corresponding to the complete elution of each of the saturated acetates from 11 : Ac through 20 : Ac. Fractions were collected in 3-mm-OD, U-shaped, glass traps cooled by Dry Ice-acetone, and then dissolved in dichloromethane and deposited on the inside of glass tubes for EAG determinations. Fractions also were collected for EAG determination from a 0.63-cm-OD x 180-cm-long glass column packed with 5% Carbowax 20M liquid phase on 80-100 mesh Gas Chrom Q. Fraction 1 encompassed time zero to the beginning of the elution of 10:Ac; succeeding fractions each encompassed two carbon increments to the beginning of the elution of 20 : Ac. Gas Chromatography-Mass Spectrometry (GC-MS). A Hewlett-Packard (Avondale, Pennsylvania) gas chromatograph (model 5790) with a quadrupole mass spectrometer (model 5970) was equipped with either a DB-1 or DB-Wax capillary column, 60 m x 0.25 mm ID (J & W Scientific, Folsom, California). When the pheromone gland extracts were analyzed, the columns were held at 80~ for 2 rain, programmed at 2 0 ~ to 190~ for DB-1, and to 200~ for DB-Wax, and maintained isothermally thereafter. Total ion abundance was monitored. When the dimethyl disulfide derivatives of the monoenes were analyzed, the DB-1 column was used and was held at 80~ for 2 min, programmed at 2 0 ~ to 255~ and held at that temperature for 30 min. Determination of Positions and Configurations of Double Bonds of Monoenes. Monoenes from extract of sex pheromone glands were provisionally identified by comparing retention times on DB-1 (60 m x 0.25 mm; 2 rain at 80~ then 2 0 ~ to 190~ and DB-Wax (45 m x 0.20 mm ID at 145~
252
DAVISET AL.
GC capillary columns with synthetic monoenes. A Hewlett-Packard model 5880 GC was used. Rigorous determination of double-bond positions was accomplished by mass spectral determinations of fragmentation patterns of the dimethyl disulfide (DMDS) derivatives (Buser et al., 1983). To prepare the derivatives, monoenes (15 ng) isolated as a GC fraction from extract of SPG in 200/~1 of dichloromethane were transferred to a 1-ml Reacti-vial (Pierce Chemical Co., Rockford, Illinois). The solution was evaporated just to dryness in a stream of nitrogen, and the residue was dissolved in 50/xl heptane. Then 50/zl DMDS and 10/zl of a saturated solution of iodine in ether were added. The vial was sealed and heated overnight at 40~ in a convection oven. The solution was diluted with 200/zl of heptane and excess 12 was removed by shaking with 100 /A of 5 % of sodium bisulfite solution. The heptane solution was moved with a syringe and concentrated to 2 #1 for injection into the GC-MS.
Determination of Positions and Configurations of Double Bonds of Diene. The GC purified diene (144 ng) was ozonized in predistilled dichloromethane at - 7 0 ~ with an ultraviolet ozonizer (Orec Co. model 03V1, Phoenix, Arizona), and the ozonide was reduced with triply recrystallized triphenytphosphine. The ozonized sample was injected on a GC column (DB-1, 60 m x 0.25 mm), which was held at 80~ for 7 min, programmed at 6 ~ to 200~ and held at that temperature. When the configuration of the diene was determined, the same column was held at 80~ for 2 min and programmed at 20 ~ to 180 ~ and held at that temperature. Flight Tunnel. A flight tunnel constructed as described by Miller and Roelofs (1978) was used. The flight tunnel room was maintained at 22~ Males were released in groups of five. The number of males initiating and sustaining flight and contacting the lure were determined. All lures were formulated in gray elastomeric septa (formulation number 1888, size no. 1, West Co., Phoenixville, Pennsylvania) (Brown and McDonough, 1986) to prevent isomerization of the diene. Males were three to four days postemergence and were maintained on a reverse dark-light cycle ( 10 and 14 hr). Tests were conducted 3 hr after the onset of scotophase. Field Tests. Z9-, Z l l - , and E l l - 1 4 : A c used for the field tests were obtained from commercial sources and were pure by GC analysis and contained less than 1% of the geometric isomer. El0, E 1 2 - 1 4 : A c was synthesized by a modification of the method for Amorbia cuneana (McDonough et al., 1982; McDonough and Smithhisler, 1989). The synthesis was carried through until a mixture of El0, E l 2 - and El0, Z 1 2 - 1 4 : O H was obtained. Pure El0, E1214:OH was obtained by low-temperature recrystallization from pentane and then was converted to the acetate. Isomeric purity was 98.5% EE, 1% EZ and 0.5 % ZE for all of the experiments except the one in which the effect of purity was tested. Then the purity was 96.0% EE, 2.5% EZ, and 1.5 ZE. Candidate
253
SEX PHEROMONE OF MINT ROOT BORER
lures in 100 #1 of dichloromethane were impregnated into gray elastomeric septa; dichloromethane alone was added to control septa. Pherocon IC sticky traps (Trece, Corp., Salinas, California) were used. Traps were suspended from a movable metal arm (an 18 x 23-cm shelf bracket) attached by a metal hose clamp to a 120-cm-long wooden broom handle driven into the ground. This arrangement enabled the traps to be positioned just above the mint foliage, usually 45-60 cm above the ground, where most flight activity was believed to Occur.
The position of each trap in the experimental plot was drawn randomly and traps were deployed at approximately 10-m intervals. Females used in the field tests were placed in aluminum wire screen cages (9 x 9 • 2 cm, three females/ cage), which were suspended by a wire from the top of the trap equidistant between the top and bottom. There were four replicates of each lure tested and the trap data were transformed by (x + 0.5) 1/2 and compared by Duncan's (1955) multiple-range test ( P = 0.05).
RESULTS
AND
DISCUSSION
Exploratory Study of Structure of Pheromone Components. Among the model compounds tested, the strongest EAG responses were obtained from the 14-carbon acetates (Figure 1). The 10 and 12 positions ofunsaturation produced the strongest responses, and strong responses were also obtained from the 9 and 11 positions. After the 14-carbon acetates, the strongest responses were obtained from the 12-carbon acetates: E l 0 - and Z10-12 : Ac produced responses of about 50% of the standard (Z10-14:Ac) and the other positional isomers produced responses of about 25% of standard. Other tested compounds produced responses of 0-30% of the standard. lO0z ~
80-
_~
60-
txl a-
40-
ixl
~
w o_
EZ~E I Z
20 0
0
1
2
5
4
5
6
7
8
9
10 11 12 ! 3
DOUBLE BOND POSITION
Fic. 1. Electroantennographic responses of male mint root borers (Fumibotys fumalis) to 14-carbon acetates. Numbers indicate double-bond position; zero indicates saturated.
254
DAVIS ET AL.
The EAG response of GC fractions of SPG extract from the methyl silicone column was greatest for the fraction corresponding to the end of 14 : Ac through the end of 15 :Ac (1.9 mV vs. 0.1-0.8 mV for the other fractions), and from the Carbowax column for the fraction corresponding to the beginning of 16 : Ac to the beginning of 18 :Ac (0.7 mV vs. 0.2-0.4 mV for the other fractions). The EAG response to GC fractions (methyl silicone column) of the washes of the vials that held females was also greatest for the fraction corresponding to the end of 14:Ac through the end of 15:Ac (0.9 mV vs. 0.05-0.35 mV for the other fractions). GC-MS analysis (DB-1 column) of SPG extract (50 female equivalents) at the retention time period that produced the strong EAG response indicated the presence of a tetradecadienyl acetate [M +, 252; (M-60) § 192; CH3CO2H~-, 61] at a retention index (Ix) of 1838. Also, eluting prior to the tetradecadienyl acetate were three tetradecenyl acetates [(M-60) § 194; CH3CO2H~- , 61] at Ix values of 1776, 1781, and 1788. There was about 0.5 ng of the diene per female equivalent, and the ratio of the monoenes to the diene for each Ix value was 0.04 : 1 (1776), 0.08:1 (1781), and 0.18 : 1 (1788).
Positions and Configurations of Double Bonds of Pheromone Gland Components. Ozonolysis of the diene gave a single GC peak (methylsilicone column) with a retention time of 24.50 min near that of hexadecane (retention time = 24.77 min) and the same within experimental error ( + 0 . 0 4 min) as the ozonolysis product of 10,12-14:Ac (retention time = 24.52). The configuration of the diene was EE (retention time = 21.85 min) and the retention times of the four isomers of a synthetic standard (McDonough et al., 1982) were: ZE, 21.33; EE, 21.85; EZ, 22.10; ZZ, 22.29. A comparison of retention times of the pheromone tetradecenyl acetates with those of the synthetic tetradecenyl acetates on polar and nonpolar capillary GC columns indicated the pheromone tetradecenyl acetates were Z9-, Z11-, and E11-. Mass spectral analysis of the DMDS derivatives confirmed these assignments. The monoene derivatives and their diagnostic peaks (Buser et al., 1983) were: Z 9 - [M § 348 (33%); A, 117 (53%); B, 231 (100%)]; Z l l [M +, 348 (21%); A, 89 (34%); B, 259 (100%)]; E l l - [M +, 348 (19%); A, 89 (25%); B, 259 ( 100% )]. The retention times of the DMDS derivatives (in minutes) were: Z 9 - , 25.34; Z l l - , 27.12; E l l - , 27.54. The configurational assignments were confirmed by comparison of retention times with synthetic samples ( E 9 - DMDS derivative at 25.66 min). Male Response to Synthetic Pheromone Components. When lures containing 100/zg of E 1 0 , E 1 2 - 1 4 : A c plus 18, 8, and 4/zg of Z l l - , E l l - , and Z 9 tetradecenyl acetates were tested in a mint field (four replicated traps per test), total male captures (13) were not statistically different from blank traps but captures in female baited traps (98) were. In order to identify the reason for
SEX PHEROMONE OF MINT ROOT BORER
255
the lack of significant capture, limited tests in a flight tunnel were conducted (Table 1). When the pheromone components were tested individually (Table 1, tests 1-4), Z l l - 1 4 : A c was a weak attractant, but no response was obtained from the other components. When the four components were tested in the ratio found in the SPG and the dosage of EE was 100 #g (the same as the unsuccessful field test), 65 % of males exhibited upwind flight and 50 % touched the source (test 5). Lower doses of 33 and 10/xg of this ratio produced tess response (tests 6 and 7), while a higher dose produced the highest percent responding (test 8). Subtraction of E 1 1 - 1 4 : A c from the natural blend produced a response almost equivalent to the natural blend (test 9 vs. 5). Because of the limited numbers available for testing, more complete, systematic tests and statistical analysis were not undertaken. Nevertheless, collectively, these experiments established the attractiveness of the mixture of identified components and suggested that the initial field test may have produced low catches because the dose was too low. In further field tests (test 1, Table 2) when the ratio of EE, Z11-, and Z 9 TABLE 1. RESPONSE IN FLIGHT TUNNEL OF ADULT MALE f . LURES
Test 1 2 3 4 5
6
7
8
9
Lure (dose in/zg ) E 1 0 , E 1 2 - 1 4 : A c (100) Z11-14 : Ac (100) Z 9 - 1 4 : Ac (100) E l l - 1 4 : A c (100) E 1 0 , E 1 2 - 1 4 : A c (100) + Z I 1 14:Ac (18) + Z 9 - 1 4 : A c (4) + E l l - 1 4 : A c (8) E 1 0 , E 1 2 - 1 4 : A c (33) + Z l l 14:Ac (6) + Z 9 - 1 4 : A c (1.3) + E l l - 1 4 : A c (2.7) E 1 0 , E 1 2 - 1 4 : A c (10) + Z l l 14:Ac (1.8) + Z 9 - 1 4 : A c (0.4) + E l l - 1 4 : A c (0.8) E 1 0 , E 1 2 - 1 4 : A c (300) + Z l l 14:Ac (54) + Z 9 - 1 4 : A c (12) + E I I - 1 4 : A c (24) E 1 0 , E 1 2 - 1 4 : A c (100) + Z l l 1 4 : A c (18) + Z 9 - 1 4 : A c (4)
fumalis TO CANDIDATE
Number of males tested
Number flying upwind
Number contacting source
10 10 10 10 20
0 3 0 0 13
0 1 0 0 10
10
5
3
10
2
2
5
4
4
10
6
4
256
DAvis ET AL.
TABLE 2. CAPTURESOF NATIVE ADULT MALE F. fumalis IN PHEROCON 1C TRAPS BAITED WITH VARIOUS CANDIDATE LURES IN COMMERCIAL MINT FIELDS, HARRAH, WASHINGTON~ 1989 a
Test
Lure
i. July 27-28, 1989
Dose (#g/septum)
EIO,E12-14:Ac, Zll-14:Ac, Z9-14:Ac E10,E12-14:Ac, Z l l - t 4 : A c , Z9-14:Ac E10,E12-14:Ac, Zll-14:Ac, Z9-14:Ac E10,E12-14:Ac, Zll-14:Ac, Z9-14:Ac 3 females blank septa
2. August4-6, 1989
3. August 8, 1989
El0, El2-14 : Ac, E10,E12-14:Ac, El0, E12-14 : Ac, E10,E12-14:Ac, E11-14 : Ac 3 females blank septa
Z11-14 : Ac, Zll-14:Ac, Z11-14 : Ac, Zll-14:Ac,
El0, E12-14 : Ac, E10,E12-14:Ac, El0, E12-14 : Ac, E10,E12-14:Ac, 2 females blank septa
Z11-14 : Ac, Zll-14:Ac, Z11-14 : Ac, Zll-14:Ac,
Z9-14 : Ac Z9-14:Ac Z9-14 : Ac Z9-14:Ac,
100:18:4 300:54:12 1000:180:40 3000:540:120
3000:540:120 3000:540:120 b 10,000, 1800, 400 3000, 540, 120, 240
Cumulative captures 9 5 22 68 61 4
a a a b b a
105 128 278 119
a a b a
329 b 14 c Z9-14 : Ac Z9-14:Ac Z9-14 : Ac Z9-14:Ac
3000:1800:120 3000:5400:120 10,000:1800:400 10,000:5400:400
125 61 100 164 31 4
ab ac ab b c c
aGray elastomeric septa were .used as the controlled release substrate. There were four replicate traps per treatment. Letters after the numbers within a test number indicate significance according to Duncan's (1955) multiple range test (P = 0.05). bThe isomeric purity of El0, E12-14 :Ac in this test was 96 %; in all other tests the isomeric purity was 98.5 %.
f o u n d in the S P G was used and the dose o f EE was v a r i e d f r o m 100 to 3000 /~g, the 3000-/zg d o s e p r o d u c e d significant trap catch c o m p a r e d to controls and was e q u i v a l e n t to that p r o d u c e d by f e m a l e s . Captures p r o d u c e d by the l o w e r doses w e r e not statistically significant. In test 2, T a b l e 2, l o w e r i s o m e r i c purity of EL did not d e c r e a s e catch; the absence o f E 1 1 - 1 4 : A c did not d e c r e a s e catch; and the 10-mg d o s e p r o d u c e d h i g h e r catch than the 3 - m g dose. Catches f r o m b o t h doses w e r e statistically significant but only the 10 m g dose was e q u i v a l e n t to f e m a l e s . T e s t 3 was c o n d u c t e d to d e t e r m i n e if a h i g h e r proportion o f Z 1 1 14 : A c w o u l d increase catch. A t neither the 3- nor the 10-mg dose l e v e l did the increase in the p r o p o r t i o n o f Z l l - 1 4 : A c p r o d u c e statistically significant increases in trap catch. C o l l e c t i v e l y , t h e s e e x p e r i m e n t s d e m o n s t r a t e d that the three- o r f o u r - c o m p o n e n t lure in the ratio found in the sex p h e r o m o n e gland and at a dose o f 3 10 m g o f E 1 0 , E 1 2 - 1 4 : A c in gray e l a s t o m e r i c septa p r o d u c e d a v i a b l e attrac-
SEX P H E R O M O N E OF M I N T R O O T BORER
257
tant, competitive with female baited traps in field tests. Flight-tunnel studies i~ndicate at least two components are necessary to produce an effective attractant. E 1 1 - 1 4 : Ac may not be a component o f the pheromone emitted by females, since it is the expected biosynthetic precursor to E l 0 , E l 2 - 1 4 : A c (L6fstedt and Bengtsson, 1988). To fully define the importance o f each of the four components, field or flight-tunnel tests with subtraction experiments over a range of concentrations are needed. The relatively high septa doses required, coupled with the small amount o f pheromone found in the glands, were unexpected. Nevertheless, these facts do not necessarily indicate there are undetected pheromone components. The low amount o f pheromone in the glands could indicate close synchronization o f biosynthesis with emission rather than a low emission rate. Moreover, the emission rate of septa at the 3- to 10-mg doses is not excessive compared w i t h that required for some other Lepidoptera. Gray septa emit at rates about the same as red rubber septa (McDonough and Aller, unpublished). Also 16-carbon aldehydes and 14-carbon acetates emit at similar rates (McDonough, 1990). The most effective doses o f the sex pheromone of Heliothis zea ( 16-carbon aldehydes) in red rubber septa are 3 - 1 0 mg (Halfhill and McDonough, 1985), and the most effective doses of Heliothis virescens sex pheromone (mainly a 16carbon aldehyde) are even higher (Flint et al., 1979). Although this is the first report o f the chemical identification o f E l 0 , E 1 2 14 : Ac in the female sex pheromone gland o f a pyralid, in field screening tests Reed and Chisholm (1985) reported that Pyrausta fodinalis (L.) was captured in traps baited with E 1 0 , E 1 2 - 1 4 : A c . Taxonomically, P. fodinalis and F, fumalis are both grouped as Pyraustini. Acknowledgments--We thank Dr. Keith Pike, Washington State University Experiment Station, Prosser, for his suggestions; and L. Bert Dekker, Matthew Lommers, and on Rice for use of their mint fields.
REFERENCES BERRY, R.E. 1974. Biology of Pyrausta fumalis on peppermint in Oregon. Ann. EntomoL Soc. Am. 67:580-582. BERRY, R.E. 1977. Insects on mint. Pacific Northwest Coop. Ext. Pub. 182. 15 pp. Joint issue of the State Universities of Washington, Oregon, and Idaho. BROWN,D.F., and McDoNOUGH,L.M. 1986. Insect sex pheromones: Formulations to increase the stability of conjugated dienes. J. Econ. Entomol. 79:922-927. BUSER, H.R., ARN, H., GUERIN,P., and RAUSCHER,S. 1983. Determination of double bond position in mono-unsaturated acetates by mass spectrometry of dimethyl disulfide adducts. Anal. Chem. 55:818-822. DAVIS,H.G., MCDONOUGH,L.M., and PIKE,K.S. 1984. Attraction of male Fumibotysfumalis to females of the species. J. Entomol. Soc. B. C 81:25-28. DUNCAN,O.B. 1955. Multiple range and multiple F tests. Biometrics 11:1-41.
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