Journal of Chemical Ecology, VoL 15, No. 5, 1989
FEMALE SEX PHEROMONE OF IRIS BORER, Macronoctua onusta (LEPIDOPTERA: NOCTUIDAE)
J . A . K L U N , 1 J.W. N E A L , JR., 2 and B.A. L E O N H A R D T 1 ~Insect Chemical Ecology Laboratory 2Florist and Nursery Crops Laboratory U.S. Department of Agriculture, Agricultural Research Service Beltsville Agricultural Research Center Beltsville, Maryland 20705 (Received April 21, 1988; accepted July 25, 1988) Abstract--Chromatographic and mass spectrometry studies of heptane extracts of the ovipositors of the iris borer, Macronoctua onusta, showed that the females produce several compounds that are the same as those produced by females of the tobacco budworm, Heliothis virescens. In trapping experiments, a mixture of (Z)-1 t-hexadecenal, (Z)-11-hexadecen-1-ol, and (Z)-9tetradecenal (94 : 4 : 2) proved to be the minimum set of compounds required to cause effective capture of iris borer males in the field. Key Words--(Z)-ll-Hexadecenal, (Z)-ll-hexadecen-l-ol, (Z)-9-tetradecenal, iris, iris borer, Macronoctua onusta, tobacco budworm, Heliothis virescens, Lepidoptera, Noctuidae.
INTRODUCTION The iris borer ( M a c r o n o c t u a onusta, Grote) is the major lepidopterous pest insect in iris (Iris v e r s i c o l o r L.) throughout much of North America. Larvae, feeding in rhizomes, can destroy entire iris beds. Adults o f the species are nocturnal and they emerge, mate, and females lay eggs on iris in the fall (SeptemberOctober). Eggs hatch in spring, and larvae feed in the leaves and then bore into the rhizomes (Schread, 1970; Neiswander, 1961). W e became interested in determining the composition o f the female sex pheromone o f this insect because it was thought that a synthetic replica of the pheromone might prove useful to iris growers wishing to monitor flight activity o f the pest. 1559
1560
KLUN ET AL.
We report that the array of pheromonal compounds produced by the iris borer female resembles those previously found in the female tobacco budworm, Heliothis virescens (Fabricius) (Klun et al., 1980; Teal et al., 1986), and that a mixture of Z-11-hexadecenal, (Z)-ll-hexadecen-l-ol and (Z)-9-tetradecenal (94 : 4 : 2) is the minimum set of compounds required to effectively cause capture of iris borer males in the field.
METHODS AND MATERIALS
Insects. In August 1983 and 1984, rhizomes infested with late-instar larvae of the borer were collected from iris beds in the vicinity of Beltsville, Maryland, transplanted to potting soil, and held in a greenhouse. Pupae of the borer were recovered from the pots and transferred to screen-covered plastic containers containing ca. 5 cm moist soil to await emergence of adults. Adult females were held at ambient conditions of the greenhouse for two to three days after emergence. At that time, their ovipositors were excised at the onset of scotophase, soaked in ca. 25 /zl heptane/ovipositor for ca. 10 min, and then the solvent was drawn away from the ovipositors and stored at - 4 ~ in a screwcap vial. In one case, a set of six ovipositors was extracted twice: first by a brief soak (10 min) in 100/xl heptane followed by soaking in a second volume of solvent for 1 hr. The onset of scotophase was selected as the time for preparation of extracts because previous work (Schread, 1970) indicated that the adults mate nocturnally, and we surmised that pheromone titers in the females would likely be highest at that time. In all, we obtained extracts of four females in 1983 and 10 females in 1984 that permitted analyses by capillary gas chromatography (GC), using polar and nonpolar columns (Klun and Huettel, 1989), and also analysis by combined GC-mass spectrometry (MS), using instruments and operating conditions described by Klun et al. (1982). Attempts to obtain additional adult females by rearing early-instar fieldcollected larvae on artificial diets of the European corn borer (Reed et al., 1972) and corn earworm (Burton, 1970) were unsuccessful. Consequently, supplies of insects for chemical and field studies were severely restricted throughout this research and virgin females were available for inclusion in field trapping experiments in only the first year of the study. Chemicals and Male Trapping Tests. All compounds were previously synthesized at the Insect Chemical Ecology Lab and were purified by preparative liquid chromatography using two 25-cm x 10-mm (ID) stainless-steel tubes packed with 20% AgNO3-impregnated 10/xm Spherisorb and toluene as eluant. All compounds were geometrically pure and were greater than 95 % chemically pure according to GC analyses. In field trapping tests, conducted during October 1984-1987, mixtures of the compounds identified from female ovipositor
1561
PHEROMONE OF IRIS BORER
extracts were applied in microgram amounts to either cotton dental rolls (1984 season) or rubber septa (Thomas Scientific, catalog No. 1780-J07) that were positioned in Pherocon 1C, or Heliothis Scentry (1986 field tests only) insect traps (Great Lakes IPM, Vestaburg, Michigan 48891). The cotton rolls were used initially in the field tests because one of us (J.A.K.) had used them previously in a study of the tobacco budworm pheromone (Klun et al., 1980) and found that, when an appropriate mixture of compounds was evaporated from cotton rolls in field traps, they caused capture of males more effectively than traps baited with four virgin tobacco budworm females. Inasmuch as the compounds we were dealing with were the same as those of the tobacco budworm, we reasoned that cotton was a suitable substrate to use in the iris borer field tests. The cotton rolls were replaced nightly, while fresh septa were positioned in the traps weekly. The septa were chosen as evaporative substrates for the bulk of the field tests for convenience and because they have been used routinely to control the evaporation of compounds in studies of the pheromone systems of many moths (Heath et al., 1986). When Pherocon 1C traps were used, the lower interior surface of the traps was coated with Tack Trap to ensure trapcapture efficiency at the cool nighttime temperatures of October; Tack Trap is a polymer that is less viscous than the adhesive normally used by the commercial manufacturer. Virgin iris borer females were individually hung in traps within a small cylindrical cage. Traps were suspended ca. 1 m from the ground on stakes driven into the ground. The traps, containing specific mixtures of compounds, were positioned in urban areas known to have iris plantings. Traps were ca. 10 m apart, and all tests were replicated over time and were conducted using a randomized complete block design with 1-20 km between blocks (replicates). Duncan's multiple-range test was applied to test for significant differences between mean total male captures across replicates for each treatment.
RESULTS AND DISCUSSION
GC and GC-MS. Figure 1 shows the chromatogram obtained by analysis of an aliquot of sample (ca. 0.2 female equivalent) of the combined ovipositor extracts of four iris borer females in 1983 with a Carbowax 20 M capillary column. This analysis and subsequent analyses of samples taken from other extracts showed that they all contained the nine pheromone-like compounds. The identities of these compounds were first established by coincidence of their retention times with standard compounds on Carbowax 20 M and DB-1 capillary columns, and then all of them were verified by GC-MS; the mass spectra of compounds detected were identical to those of the standard compounds. The percentage composition of the pheromonal compounds in extracts of four females, the combined extracts of six females (rinse and soak), three females,
1562
KLuN ET AL, 6
HC 1
HC 2
FIG. 1. GC chromatogram (Carbowax 20M capillary) obtained by using a sample of the combined heptane extracts of four iris borer ovipositors. 1 = 14 : Aid, 2 = Z9-14 : Aid, 3 = 16:Ald, 4 = 14:OH, 5 = Z9-16:Ald, 6 = Z I I - 1 6 : A l d , 7 = Z9-14:OH, 8 = 16:OH, and 9 = Z l l - 1 6 : OH. HC1 = tricosane; HC2 = pentacosane. Other peaks in the chromatogram were contaminants in the solvent or did not appear regularly from one analysis to another.
and an individual female are shown in Table 1. It is noteworthy that the set o f compounds produced by females o f the iris borer are similar to those produced by the tobacco budworm, Heliothis virescens (Teal et al., 1986); all compounds found in the tobacco budworm, with exception o f Z 7 - 1 6 : Ald, occur in the iris borer. The major component in the extracts was ( Z ) - l l - h e x a d e c e n a l ( Z l l 16 : Aid), with exception o f a solution obtained by soaking the ovipositors in the solvent for an hour; it contained ( Z ) - l l - h e x a d e c e n - l - o l ( Z l l - 1 6 : OH) in greatest abundance (57%). Inasmuch as the 10-min rinse o f the same ovipositors contained proportionately less alcohol than that obtained by prolonged
1563
PHEROMONE OF IRIS BORER
TABLE 1.
PERCENTAGE COMPOSITIONS OF
IRIS BORER
OVIPOSITOR EXTRACTS a
Percentage composition 1983
1984 (DB-1)
4 females
6 females
Compound
DB-1
Carbowax 20 M
Rinse
Soak
3 females
1 female
14 :Ald Z9-14 :Ald 16 : Aid 14 :Oil Z9-16 : Aid Z11-16 : Ald Z9-14 : OH 16 : OH Z11-16 : OH
2.1 0.9 11.4 0.5 0.5 58.9 0.8 1.5 23.3
2.7 1.3 11.0 0.7 0.7 55.6 0.9 1.6 25.3
1.4 1.2 4.8 0.2 0.7 65.8 0.6 0.4 24.9
1.7 2.0 2.8 0.6 1.0 33.2 1.7 0.5 56.6
1.7 2.4 4.5 0.1 0.7 50.7 0.7 0.7 38.4
1.8 0.6 10.3 0.1 0.9 59.7 0.2 0.3 26.1
aDB-1 is data from a nonpolar column and Carbowax 20 M is data from a polar column.
extraction, it is probable that the large quantity o f alcohol found in the extracts is reflective o f a comparatively large alcohol metabolic pool that exists within the pheromone gland. The alcohol in the iris borer is quite likely a biosynthetic precursor o f Z 1 1 - 1 6 : A l d ; in Heliothis, Tumlinson and Teal (1987) have shown that an alcohol oxidase in the insect cuticle is responsible for generation o f pheromonal aldehydes, and it is reasonable to suspect that a similar enzyme occurs in the iris borer. The next most abundant compound in the iris borer extracts was hexadecanal (16 : A l d ) along with traces o f tetradecanal (14 :Ald), (Z)-9-tetradecenal ( Z 9 - 1 4 : A l d ) , t e t r a d e c a n - l - o l ( 1 4 : O H ) , (Z)-9-hexadecenal (Z9-16 : A l d ) , (Z)-9-tetradecen-l-ol (Z9-14 : OH), and hexadecan-l-ol (16: OH). In the first field-trapping experiment (1984), three mixtures o f compounds and virgin females were d e p l o y e d (Table 2). Treatment 1 contained all pheromonal compounds identified in the female extracts with exception of the trace amounts o f 14 : OH and 16 : OH; these were excluded to minimize the possible number o f permutations o f compounds to be tested. The ratio of Z l l - 1 6 : A l d to Z 1 1 - 1 6 : OH (73 : 27) in treatment 1 approximated the ratio observed in ovipositor extracts o f 1983. Treatment 2 lacked Z 1 1 - 1 6 : O H , and treatment 3 differed from treatment 1 in that the A I d - O H ratio was arbitrarily set at 9 6 : 4 . Proportionately less alcohol was selected for treatment three because we surmised that the ratio observed in the female extracts overestimated the amount o f alcohol that the females might actually release when calling for a mate. The
1564
KLUN ET AL.
TABLE 2. MIXTURES OF COMPOUNDS TESTED AGAINST IRIS BORER AND MALE CAPTURES IN FIELD TRAPSa
Treatment Compound (/xg)/dispenser Cotton dispenser 2.8 14: Ald 2.4 Z9-14 :Ald 9.8 16 :Ald 1.6 Z9-16:Ald 132.4 Z l l - 1 6 : A l d 1.2 Z9-14:OH 49.8 Z l l - 1 6 : O H 6.2 Z l l - 1 6 : O H
1
2
3
X X X X X X X
X X X X X X
X X X X X X
0
4
5
6
7
X
X
X
X
X X X
X X X X
8
9
Total male capture 1984 (6 nights, 4 replicates) Rubber septa 66.5 14 : Aid 58.7 Z9-14:AId 264.0 16 : Aid 39.1 Z9-16 : Aid 3,319.5 Z l l - 1 6 : A l d 31.3 Z9-14:OH 152.5 Z l l - 1 6 : O H
10ab
0b
35a
22ab X X X X X X X
X
X
X
X
X
Total male capture 1985 (14 nights, 5 replicates) 1986 (18 nights, 6 replicates) 1987 (14 nights, 3 replicates)
61a 18b 28a
79a 36a 31a
67a ---
67a ---
lb 0b 0b
-0b 0b
aTreatment 0 = virgin female iris borer. The Xs in each column indicate composition of each treatment. Male capture values in each row followed by the same letter are not significantly different from one another according to Duncan's multiple range test (alpha = 0.05).
t r a p p i n g test (table 2) i n d i c a t e d that this a s s u m p t i o n w a s s o u n d b e c a u s e the 9 6 : 4 ratio c a u s e d c a p t u r e o f m o r e m a l e s t h a n the 7 3 : 2 7 ratio o r the v i r g i n f e m a l e s . T h u s , t h e 9 6 : 4 A I d - O H ratio p r o b a b l y is a b e t t e r a p p r o x i m a t i o n o f t h e ratio p r o d u c e d b y the f e m a l e t h a n is t h e 73 : 27 ratio f o u n d in the o v i p o s i t o r e x t r a c t s . S i m i l a r results w e r e o b t a i n e d b y R a i n a et al. (1986) in a study o f the f e m a l e s e x p h e r o m o n e o f a n o t h e r n o c t u i d m o t h , H. phloxiphaga. T h e y f o u n d that a c o m b i n a t i o n o f Z 1 1 - 1 6 : A i d : Z 1 1 - 1 6 : O H w a s critical f o r elicitation o f o p t i m a l r e s p o n s e f r o m m a l e s a n d that p r o p o r t i o n a t e l y less a l c o h o l t h a n that
PHEROMONE OF IRIS BORER
1565
found in female extracts was the more effective male lure. Such a phenomenon has also shown to be the case for H. virescens (Ramaswamy et al., 1985; Shaver et al., 1987); with levels of alcohol at 0.25-1% of Z l l - 1 6 : A l d field trap captures were enhanced, but at 5.9 % Z 1 1 - 1 6 : O H (ca. the highest amount found in gland extracts according to Klun et al., 1980) captures were suppressed. In case of the iris borer, the question of what ratio is actually released by the female can only be answered definitively by analysis of the volatiles produced by the females. The 1984 trapping test definitively showed, however, that Z1116 : OH was an essential pheromonal component; no males were captured when it was deleted from the stimulus. Data collected in field tests from 1985 through 1987 (Table 2) revealed that the minimum set of compounds required to effectively cause capture of iris borer males was a mixture of Z l l - 1 6 : A l d , Z l l - 1 6 : O H , and Z9-14:Ald (94 : 4 : 2). This mixture can be used as an male attractant for monitoring flights of the pest. Acknowledgments--We thank Dr. Meyer Schwarz for synthesis of many of the compounds used in this study, E.D. DeVilbiss for gathering mass spectra, and E.C. Uebel for technical assistance.
REFERENCES BURTON, R.L. 1970. A low-cost artificial diet for the corn earworrn. J. Econ. Entomol. 63:19691970. HEATH, R.R., TEAL, P.E.A., TUMLINSON,J.H., and MENGELKOCH,L.J. 1986. Prediction of mlease ratios of multicomponent pheromones from rubber septa. J. Chem. Ecol. 12:2133-2143. KLUN, J.A., BIERL-LEONHARDT,B.A., PLIMMER,J.R., SPARKS,A.N., PRIM1ANI,M., CHAPMAN, O.L., LEPONE, G., and LEE, G.-H. 1980. Sex pheromone chemistry of the female tobacco budworm, Heliothis virescens. J. Chem. Ecol. 6:177-183. KLUN, J.A., and HUETTEL, M.D. 1989. Genetic regulationof sex pheromone production and response: Interaction of sympatric pheromonal types of the European corn borer, Ostrinia nubilalis (Lepidoptera: Pyralidae) J. Chem. Ecol. 14:2047-2061. KLUN, J.A., LEONHARDT,B.A., LOPEZ, J.D., JR., and LACHANCE,L.E. 1982. Female Heliothis subflexa (Lepidoptera: Noctuidae) sex pheromone: Chemistry and congeneric comparisons. Environ. Entomol. 11:1084-1090. NEISWANDER,C.R. 1961. The iris borer, Macronoctua onusta Grote, its behavior and methods of control. Research Bulletin 892. Ohio Agricultural Experiment Station, Wooster. RAMASWAMY,S.B., RANDLE, S.A., and MA, W.K. 1985. Field evaluation of the sex pheromone components of Heliothis virescens (Lepidoptera: Noctuidae) in cone traps. Environ. EntomoL 14:293-296. RAINA, A.K., KLUN, J.A., LOPEZ, J.D., and LEONHARDT, B.A. 1986. Female sex pheromone of Heliothis phloxiphaga (Lepidoptera: Noctuidae): Chemical identification, male behavioral response in the flight tunnel, and field tests. Environ. Entomol. 15:931-935. REED, G.L., SHOWERS,W.B., HUGGANS,J.L., and CARTER, S.W. 1972. Improved procedures for mass mating the European corn borer. J. Econ. Entomol. 65:1472-1476.
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SCHREAD,J.C. 1970. Iris borer and its control. Circular No. 235 of the Connecticut Agricultural Experiment Station, New Haven.
SHAVER,T.N., HENDRICKS,D.E., and LOPEZ, J.D. 1987. Enhancement of field performance of Heliothis virescens pheromone components by Z-11-hexadecen-l-ol in PVC dispenser. Belt-
wide Cotton Products Research Conference, pp. 307-309. TEAL, P.E.A., TUMLINSON,J.H., and HEATH, R.R. 1986. Chemical and behavioral analyses of volatile sex pheromone components released by calling Heliothis virescens (F.) females (Lepidoptera: Noctuidae). J. Chem. Ecol. 12:107-125. TUMLINSON, J.H., and TEAL, P.E.A. 1987. Relationship of structure and function to biochemistry in insect pheromone systems, pp. 3-26, in G.D. Prestwich, and G.J. Blomquist, (eds.). Pheromone Biochemistry, Academic Press, Orlando, Florida.
FEMALE SEX PHEROMONE OF IRIS BORER, Macronoctua onusta (LEPIDOPTERA: NOCTUIDAE)
J . A . K L U N , 1 J.W. N E A L , JR., 2 and B.A. L E O N H A R D T 1 ~Insect Chemical Ecology Laboratory 2Florist and Nursery Crops Laboratory U.S. Department of Agriculture, Agricultural Research Service Beltsville Agricultural Research Center Beltsville, Maryland 20705 (Received April 21, 1988; accepted July 25, 1988) Abstract--Chromatographic and mass spectrometry studies of heptane extracts of the ovipositors of the iris borer, Macronoctua onusta, showed that the females produce several compounds that are the same as those produced by females of the tobacco budworm, Heliothis virescens. In trapping experiments, a mixture of (Z)-1 t-hexadecenal, (Z)-11-hexadecen-1-ol, and (Z)-9tetradecenal (94 : 4 : 2) proved to be the minimum set of compounds required to cause effective capture of iris borer males in the field. Key Words--(Z)-ll-Hexadecenal, (Z)-ll-hexadecen-l-ol, (Z)-9-tetradecenal, iris, iris borer, Macronoctua onusta, tobacco budworm, Heliothis virescens, Lepidoptera, Noctuidae.
INTRODUCTION The iris borer ( M a c r o n o c t u a onusta, Grote) is the major lepidopterous pest insect in iris (Iris v e r s i c o l o r L.) throughout much of North America. Larvae, feeding in rhizomes, can destroy entire iris beds. Adults o f the species are nocturnal and they emerge, mate, and females lay eggs on iris in the fall (SeptemberOctober). Eggs hatch in spring, and larvae feed in the leaves and then bore into the rhizomes (Schread, 1970; Neiswander, 1961). W e became interested in determining the composition o f the female sex pheromone o f this insect because it was thought that a synthetic replica of the pheromone might prove useful to iris growers wishing to monitor flight activity o f the pest. 1559
1560
KLUN ET AL.
We report that the array of pheromonal compounds produced by the iris borer female resembles those previously found in the female tobacco budworm, Heliothis virescens (Fabricius) (Klun et al., 1980; Teal et al., 1986), and that a mixture of Z-11-hexadecenal, (Z)-ll-hexadecen-l-ol and (Z)-9-tetradecenal (94 : 4 : 2) is the minimum set of compounds required to effectively cause capture of iris borer males in the field.
METHODS AND MATERIALS
Insects. In August 1983 and 1984, rhizomes infested with late-instar larvae of the borer were collected from iris beds in the vicinity of Beltsville, Maryland, transplanted to potting soil, and held in a greenhouse. Pupae of the borer were recovered from the pots and transferred to screen-covered plastic containers containing ca. 5 cm moist soil to await emergence of adults. Adult females were held at ambient conditions of the greenhouse for two to three days after emergence. At that time, their ovipositors were excised at the onset of scotophase, soaked in ca. 25 /zl heptane/ovipositor for ca. 10 min, and then the solvent was drawn away from the ovipositors and stored at - 4 ~ in a screwcap vial. In one case, a set of six ovipositors was extracted twice: first by a brief soak (10 min) in 100/xl heptane followed by soaking in a second volume of solvent for 1 hr. The onset of scotophase was selected as the time for preparation of extracts because previous work (Schread, 1970) indicated that the adults mate nocturnally, and we surmised that pheromone titers in the females would likely be highest at that time. In all, we obtained extracts of four females in 1983 and 10 females in 1984 that permitted analyses by capillary gas chromatography (GC), using polar and nonpolar columns (Klun and Huettel, 1989), and also analysis by combined GC-mass spectrometry (MS), using instruments and operating conditions described by Klun et al. (1982). Attempts to obtain additional adult females by rearing early-instar fieldcollected larvae on artificial diets of the European corn borer (Reed et al., 1972) and corn earworm (Burton, 1970) were unsuccessful. Consequently, supplies of insects for chemical and field studies were severely restricted throughout this research and virgin females were available for inclusion in field trapping experiments in only the first year of the study. Chemicals and Male Trapping Tests. All compounds were previously synthesized at the Insect Chemical Ecology Lab and were purified by preparative liquid chromatography using two 25-cm x 10-mm (ID) stainless-steel tubes packed with 20% AgNO3-impregnated 10/xm Spherisorb and toluene as eluant. All compounds were geometrically pure and were greater than 95 % chemically pure according to GC analyses. In field trapping tests, conducted during October 1984-1987, mixtures of the compounds identified from female ovipositor
1561
PHEROMONE OF IRIS BORER
extracts were applied in microgram amounts to either cotton dental rolls (1984 season) or rubber septa (Thomas Scientific, catalog No. 1780-J07) that were positioned in Pherocon 1C, or Heliothis Scentry (1986 field tests only) insect traps (Great Lakes IPM, Vestaburg, Michigan 48891). The cotton rolls were used initially in the field tests because one of us (J.A.K.) had used them previously in a study of the tobacco budworm pheromone (Klun et al., 1980) and found that, when an appropriate mixture of compounds was evaporated from cotton rolls in field traps, they caused capture of males more effectively than traps baited with four virgin tobacco budworm females. Inasmuch as the compounds we were dealing with were the same as those of the tobacco budworm, we reasoned that cotton was a suitable substrate to use in the iris borer field tests. The cotton rolls were replaced nightly, while fresh septa were positioned in the traps weekly. The septa were chosen as evaporative substrates for the bulk of the field tests for convenience and because they have been used routinely to control the evaporation of compounds in studies of the pheromone systems of many moths (Heath et al., 1986). When Pherocon 1C traps were used, the lower interior surface of the traps was coated with Tack Trap to ensure trapcapture efficiency at the cool nighttime temperatures of October; Tack Trap is a polymer that is less viscous than the adhesive normally used by the commercial manufacturer. Virgin iris borer females were individually hung in traps within a small cylindrical cage. Traps were suspended ca. 1 m from the ground on stakes driven into the ground. The traps, containing specific mixtures of compounds, were positioned in urban areas known to have iris plantings. Traps were ca. 10 m apart, and all tests were replicated over time and were conducted using a randomized complete block design with 1-20 km between blocks (replicates). Duncan's multiple-range test was applied to test for significant differences between mean total male captures across replicates for each treatment.
RESULTS AND DISCUSSION
GC and GC-MS. Figure 1 shows the chromatogram obtained by analysis of an aliquot of sample (ca. 0.2 female equivalent) of the combined ovipositor extracts of four iris borer females in 1983 with a Carbowax 20 M capillary column. This analysis and subsequent analyses of samples taken from other extracts showed that they all contained the nine pheromone-like compounds. The identities of these compounds were first established by coincidence of their retention times with standard compounds on Carbowax 20 M and DB-1 capillary columns, and then all of them were verified by GC-MS; the mass spectra of compounds detected were identical to those of the standard compounds. The percentage composition of the pheromonal compounds in extracts of four females, the combined extracts of six females (rinse and soak), three females,
1562
KLuN ET AL, 6
HC 1
HC 2
FIG. 1. GC chromatogram (Carbowax 20M capillary) obtained by using a sample of the combined heptane extracts of four iris borer ovipositors. 1 = 14 : Aid, 2 = Z9-14 : Aid, 3 = 16:Ald, 4 = 14:OH, 5 = Z9-16:Ald, 6 = Z I I - 1 6 : A l d , 7 = Z9-14:OH, 8 = 16:OH, and 9 = Z l l - 1 6 : OH. HC1 = tricosane; HC2 = pentacosane. Other peaks in the chromatogram were contaminants in the solvent or did not appear regularly from one analysis to another.
and an individual female are shown in Table 1. It is noteworthy that the set o f compounds produced by females o f the iris borer are similar to those produced by the tobacco budworm, Heliothis virescens (Teal et al., 1986); all compounds found in the tobacco budworm, with exception o f Z 7 - 1 6 : Ald, occur in the iris borer. The major component in the extracts was ( Z ) - l l - h e x a d e c e n a l ( Z l l 16 : Aid), with exception o f a solution obtained by soaking the ovipositors in the solvent for an hour; it contained ( Z ) - l l - h e x a d e c e n - l - o l ( Z l l - 1 6 : OH) in greatest abundance (57%). Inasmuch as the 10-min rinse o f the same ovipositors contained proportionately less alcohol than that obtained by prolonged
1563
PHEROMONE OF IRIS BORER
TABLE 1.
PERCENTAGE COMPOSITIONS OF
IRIS BORER
OVIPOSITOR EXTRACTS a
Percentage composition 1983
1984 (DB-1)
4 females
6 females
Compound
DB-1
Carbowax 20 M
Rinse
Soak
3 females
1 female
14 :Ald Z9-14 :Ald 16 : Aid 14 :Oil Z9-16 : Aid Z11-16 : Ald Z9-14 : OH 16 : OH Z11-16 : OH
2.1 0.9 11.4 0.5 0.5 58.9 0.8 1.5 23.3
2.7 1.3 11.0 0.7 0.7 55.6 0.9 1.6 25.3
1.4 1.2 4.8 0.2 0.7 65.8 0.6 0.4 24.9
1.7 2.0 2.8 0.6 1.0 33.2 1.7 0.5 56.6
1.7 2.4 4.5 0.1 0.7 50.7 0.7 0.7 38.4
1.8 0.6 10.3 0.1 0.9 59.7 0.2 0.3 26.1
aDB-1 is data from a nonpolar column and Carbowax 20 M is data from a polar column.
extraction, it is probable that the large quantity o f alcohol found in the extracts is reflective o f a comparatively large alcohol metabolic pool that exists within the pheromone gland. The alcohol in the iris borer is quite likely a biosynthetic precursor o f Z 1 1 - 1 6 : A l d ; in Heliothis, Tumlinson and Teal (1987) have shown that an alcohol oxidase in the insect cuticle is responsible for generation o f pheromonal aldehydes, and it is reasonable to suspect that a similar enzyme occurs in the iris borer. The next most abundant compound in the iris borer extracts was hexadecanal (16 : A l d ) along with traces o f tetradecanal (14 :Ald), (Z)-9-tetradecenal ( Z 9 - 1 4 : A l d ) , t e t r a d e c a n - l - o l ( 1 4 : O H ) , (Z)-9-hexadecenal (Z9-16 : A l d ) , (Z)-9-tetradecen-l-ol (Z9-14 : OH), and hexadecan-l-ol (16: OH). In the first field-trapping experiment (1984), three mixtures o f compounds and virgin females were d e p l o y e d (Table 2). Treatment 1 contained all pheromonal compounds identified in the female extracts with exception of the trace amounts o f 14 : OH and 16 : OH; these were excluded to minimize the possible number o f permutations o f compounds to be tested. The ratio of Z l l - 1 6 : A l d to Z 1 1 - 1 6 : OH (73 : 27) in treatment 1 approximated the ratio observed in ovipositor extracts o f 1983. Treatment 2 lacked Z 1 1 - 1 6 : O H , and treatment 3 differed from treatment 1 in that the A I d - O H ratio was arbitrarily set at 9 6 : 4 . Proportionately less alcohol was selected for treatment three because we surmised that the ratio observed in the female extracts overestimated the amount o f alcohol that the females might actually release when calling for a mate. The
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TABLE 2. MIXTURES OF COMPOUNDS TESTED AGAINST IRIS BORER AND MALE CAPTURES IN FIELD TRAPSa
Treatment Compound (/xg)/dispenser Cotton dispenser 2.8 14: Ald 2.4 Z9-14 :Ald 9.8 16 :Ald 1.6 Z9-16:Ald 132.4 Z l l - 1 6 : A l d 1.2 Z9-14:OH 49.8 Z l l - 1 6 : O H 6.2 Z l l - 1 6 : O H
1
2
3
X X X X X X X
X X X X X X
X X X X X X
0
4
5
6
7
X
X
X
X
X X X
X X X X
8
9
Total male capture 1984 (6 nights, 4 replicates) Rubber septa 66.5 14 : Aid 58.7 Z9-14:AId 264.0 16 : Aid 39.1 Z9-16 : Aid 3,319.5 Z l l - 1 6 : A l d 31.3 Z9-14:OH 152.5 Z l l - 1 6 : O H
10ab
0b
35a
22ab X X X X X X X
X
X
X
X
X
Total male capture 1985 (14 nights, 5 replicates) 1986 (18 nights, 6 replicates) 1987 (14 nights, 3 replicates)
61a 18b 28a
79a 36a 31a
67a ---
67a ---
lb 0b 0b
-0b 0b
aTreatment 0 = virgin female iris borer. The Xs in each column indicate composition of each treatment. Male capture values in each row followed by the same letter are not significantly different from one another according to Duncan's multiple range test (alpha = 0.05).
t r a p p i n g test (table 2) i n d i c a t e d that this a s s u m p t i o n w a s s o u n d b e c a u s e the 9 6 : 4 ratio c a u s e d c a p t u r e o f m o r e m a l e s t h a n the 7 3 : 2 7 ratio o r the v i r g i n f e m a l e s . T h u s , t h e 9 6 : 4 A I d - O H ratio p r o b a b l y is a b e t t e r a p p r o x i m a t i o n o f t h e ratio p r o d u c e d b y the f e m a l e t h a n is t h e 73 : 27 ratio f o u n d in the o v i p o s i t o r e x t r a c t s . S i m i l a r results w e r e o b t a i n e d b y R a i n a et al. (1986) in a study o f the f e m a l e s e x p h e r o m o n e o f a n o t h e r n o c t u i d m o t h , H. phloxiphaga. T h e y f o u n d that a c o m b i n a t i o n o f Z 1 1 - 1 6 : A i d : Z 1 1 - 1 6 : O H w a s critical f o r elicitation o f o p t i m a l r e s p o n s e f r o m m a l e s a n d that p r o p o r t i o n a t e l y less a l c o h o l t h a n that
PHEROMONE OF IRIS BORER
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found in female extracts was the more effective male lure. Such a phenomenon has also shown to be the case for H. virescens (Ramaswamy et al., 1985; Shaver et al., 1987); with levels of alcohol at 0.25-1% of Z l l - 1 6 : A l d field trap captures were enhanced, but at 5.9 % Z 1 1 - 1 6 : O H (ca. the highest amount found in gland extracts according to Klun et al., 1980) captures were suppressed. In case of the iris borer, the question of what ratio is actually released by the female can only be answered definitively by analysis of the volatiles produced by the females. The 1984 trapping test definitively showed, however, that Z1116 : OH was an essential pheromonal component; no males were captured when it was deleted from the stimulus. Data collected in field tests from 1985 through 1987 (Table 2) revealed that the minimum set of compounds required to effectively cause capture of iris borer males was a mixture of Z l l - 1 6 : A l d , Z l l - 1 6 : O H , and Z9-14:Ald (94 : 4 : 2). This mixture can be used as an male attractant for monitoring flights of the pest. Acknowledgments--We thank Dr. Meyer Schwarz for synthesis of many of the compounds used in this study, E.D. DeVilbiss for gathering mass spectra, and E.C. Uebel for technical assistance.
REFERENCES BURTON, R.L. 1970. A low-cost artificial diet for the corn earworrn. J. Econ. Entomol. 63:19691970. HEATH, R.R., TEAL, P.E.A., TUMLINSON,J.H., and MENGELKOCH,L.J. 1986. Prediction of mlease ratios of multicomponent pheromones from rubber septa. J. Chem. Ecol. 12:2133-2143. KLUN, J.A., BIERL-LEONHARDT,B.A., PLIMMER,J.R., SPARKS,A.N., PRIM1ANI,M., CHAPMAN, O.L., LEPONE, G., and LEE, G.-H. 1980. Sex pheromone chemistry of the female tobacco budworm, Heliothis virescens. J. Chem. Ecol. 6:177-183. KLUN, J.A., and HUETTEL, M.D. 1989. Genetic regulationof sex pheromone production and response: Interaction of sympatric pheromonal types of the European corn borer, Ostrinia nubilalis (Lepidoptera: Pyralidae) J. Chem. Ecol. 14:2047-2061. KLUN, J.A., LEONHARDT,B.A., LOPEZ, J.D., JR., and LACHANCE,L.E. 1982. Female Heliothis subflexa (Lepidoptera: Noctuidae) sex pheromone: Chemistry and congeneric comparisons. Environ. Entomol. 11:1084-1090. NEISWANDER,C.R. 1961. The iris borer, Macronoctua onusta Grote, its behavior and methods of control. Research Bulletin 892. Ohio Agricultural Experiment Station, Wooster. RAMASWAMY,S.B., RANDLE, S.A., and MA, W.K. 1985. Field evaluation of the sex pheromone components of Heliothis virescens (Lepidoptera: Noctuidae) in cone traps. Environ. EntomoL 14:293-296. RAINA, A.K., KLUN, J.A., LOPEZ, J.D., and LEONHARDT, B.A. 1986. Female sex pheromone of Heliothis phloxiphaga (Lepidoptera: Noctuidae): Chemical identification, male behavioral response in the flight tunnel, and field tests. Environ. Entomol. 15:931-935. REED, G.L., SHOWERS,W.B., HUGGANS,J.L., and CARTER, S.W. 1972. Improved procedures for mass mating the European corn borer. J. Econ. Entomol. 65:1472-1476.
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SCHREAD,J.C. 1970. Iris borer and its control. Circular No. 235 of the Connecticut Agricultural Experiment Station, New Haven.
SHAVER,T.N., HENDRICKS,D.E., and LOPEZ, J.D. 1987. Enhancement of field performance of Heliothis virescens pheromone components by Z-11-hexadecen-l-ol in PVC dispenser. Belt-
wide Cotton Products Research Conference, pp. 307-309. TEAL, P.E.A., TUMLINSON,J.H., and HEATH, R.R. 1986. Chemical and behavioral analyses of volatile sex pheromone components released by calling Heliothis virescens (F.) females (Lepidoptera: Noctuidae). J. Chem. Ecol. 12:107-125. TUMLINSON, J.H., and TEAL, P.E.A. 1987. Relationship of structure and function to biochemistry in insect pheromone systems, pp. 3-26, in G.D. Prestwich, and G.J. Blomquist, (eds.). Pheromone Biochemistry, Academic Press, Orlando, Florida.
