Journal of Chemical Ecology, Vol. 15, No. 4, 1989
SEX PHEROMONE COMPONENTS OF CORN STALK BORER Sesamia nonagriodes (LEF.)t Isolation, Identification, and Field Tests
B.E.
MAZOMENOS
Institute of Biology N. R. C. ' "Democritos" P. O. Box 60228 GR 153 10 Aghia Paraskevi, Greece
(Received March 23, 1987; accepted June 6, 1988) Abstract--Z-11-Hexadecenyl acetate (Zll-16:OAc), dodecyl acetate (12 : OAc), Z- 11-hexadecenal (Z11-16 :Ald), and Z- 11-hexadecenol (Z1 I16:OH), were found in pheromone gland extracts of female Sesamia nonagriodes (Lef.) [Lepidoptera: Noctuidae]. These four components were also present in airborne volatiles collected from calling virgin females in a 65 : 18 : 8 : 9 ratio. Hexadecyl acetate (16 : OAc) was also detected but found to be inactive. The identification was based on multicolumn GC analysis, mass spectrometry, and field activity. Z11-16:OAc is the major sex pheromone component; the addition of the secondary components individually decreased male captures. The blend of the four synthetic components in 69 : 15 : 8 : 8 ratio was highly attractive to males; 200 #g per trap was the most effective concentration in field tests. Key Words--(Z)-I 1-Hexadecenyl acetate, dodecyl acetate, (Z)-I 1-hexadecenal, (Z)-I 1-hexadecenol, Sesamia nonagrioides, Lepidoptera, Noctuidae, sex pheromone.
INTRODUCTION T h e c o r n s t a l k b o r e r , S e s a m i a n o n a g r i o i d e s ( L e f . ) [ L e p i d o p t e r a : N o c t u i d a e ] , is a n i m p o r t a n t c o r n p e s t in m a n y M e d i t a r r a n e a n a n d c e n t r a l A f r i c a n c o u n t r i e s ( C . I . E . , 1979). W e r e p o r t e d p r e v i o u s l y ( M a z o m e n o s , 1984) t h a t v i r g i n f e m a l e a b d o m i n a l tip e x t r a c t s a t t r a c t e d m a l e s a n d t h a t t h e p h e r o m o n e p r o d u c e d is a m u l t i c o m p o n e n t p h e r o m o n e . T w o p h e r o m o n e c o m p o n e n t s w e r e i d e n t i f i e d as Lepidoptera: Noctuidae. 1241 0098-0331/89/0400-1241506.00/0
9 1989 Plenum Publishing Corporation
1242
MAZOMENOS
Z-11-hexadecenyl acetate (Z11-16 : OAc) and Z-11-hexadecenol (Z11-16 : OH) by Sreng et al. (1985). The same authors reported that hexadecyl acetate (16 :OAc) was present in the pheromone gland extracts, but its biological activity was not established. Here we report minor components of the sex pheromone of S. nonagriodes that improve male captures in field tests.
METHODS AND MATERIALS
Insects. The insects used in this study were collected as larvae or eggs from infested corn plants. The larvae were separated according to their instar, transferred to artificial diet (Tsitsipis et al., 1983), and allowed to continue their development under laboratory conditions. The temperature was kept at 27 + 2~ with a 16:8 hr light-dark regime and relative humidity of 60 + 5 %. The diet was replaced once a week. The eggs were transferred onto a filter paper impregnated with 0.3% propionic acid in Petri dishes for incubation at 27~ After the four- to five-day incubation period, the emerged larvae were placed on artificial diet. Larval development under the conditions described lasted ca. 30-40 days. The pupae were kept under the same conditions until moth emergence. Insects were sexed at the pupal stage, five to six days after pupation. The female adults were transferred in an incubator at 25~ with 16:8 hr light-dark regime, while males were placed in a large bioassay cage in a separate room, operated under the same conditions. Pheromone Collection. The pheromone was collected from the pheromone glands of 2- to 3-day-old virgin females exhibiting calling behavior; the peak of calling occurred in the fifth hour into the dark phase. The glands were removed with forceps and extracted in ether for 20 min. The ether extracts were filtered and concentrated through a 8-cm Vigreux column. Airborne volatiles from virgin females were collected using a Teckmar GLS-1 closed looping concentrator. Females 2 to 4 days old were placed in the l-liter sampler during the dark period, and the volatiles emitted from the females were swept by the recirculating headspace gas and absorbed on 1.5 mg of activated charcoal. The trap was then removed and a collection vial was connected to the trap on the end close to the charcoal. Aliquots of 5 /~1 hexane were added twice to the trap. The bottom of the trap-vial assembly was cooled with a Dry Ice cube. This caused the air in the vial to contract, creating a partial vacuum that pulled the solvent through the charcoal. After pulling the solvent down, it was pushed back through the charcoal three times by warming the bottom of the trap with hand contact. The extracts collected were subjected to GC analysis.
CORN STALK BORER PHEROMONE
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Gas Chromatography-Mass Spectrometry. Concentrated crude extracts were injected on a 2-m x 1.8-mm (ID) column packed with 5% Carbowax 20 M on Chromosorb G/HP 80-100 mesh; the column temperature was held at 90~ for 10 min and then was programmed to 180~ at 6~ Nitrogen was used as carrier with a flow of 20 cc/min. Consecutive 3-min fractions were collected in liquid-nitrogen-cooled glass capillaries, and each was tested for biological activity in laboratory bioassays. The active fractions recovered from the Carbowax 20 M column were further purified on a 2-m x 1.8-mm (ID) column packed with 3% OV-101 on Chromosorb Q 80-100 mesh operated isothermally at 180~ and collected in 1-min fractions. The active peaks were analyzed on 3% OV-101 (2 m x 1.8 m m ID) on Chromosorb Q 80-100 mesh at 180~ 5% DEGS (2 m x 1.8 mm ID) on Chromosorb G H/P 80-100 mesh at 160~ and 15% OV-225 (2 m x 1.8 mm ID) on Chromosorb Q 80-100 mesh at 150~ The mass-spectral analyses of the active components were performed using a GC-MS Finnigan MAT 112s spectrometer in the electron-impact mode. The GC glass column was 3.05 m x 0.32 cm (ID), packed with 3.5% OV-101 on Chromosorb Q 100-200 mesh. Helium was the carder gas at a flow rate of 25 cc/min. Chemicals. Z11-16 : OAc, Z11-16 : Aid, and Z11-16 : OH were obtained from Sigma Chemical Co. (St. Louis., Missouri); and 12 : OAc was synthesized in our laboratory. The components were found to be 96-98% pure when analyzed by GC on OV-101 and OV-225 columns. Bioassays. Bioassays were conducted in a screen cage described by Mazomenos (1984). Usually 25-40, 1- to 3-day-old males were present in the cage during the bioassay. The bioassays were conducted 5-6 hr into the dark phase under a uniform low level of light (ca. 5 lux). Samples of the crude extract and the fractions obtained during the purification were pipetted onto a piece of Whatman No. 1 filter paper that was suspended about 3 cm below the top of the cage. The response of the males to the sample was observed for 10 min. During each testing period samples were tested individually. The same volume of solvent on filter paper was used as control. Field Tests. For the field tests conducted during August 13-September 15, 1985, July 3-30, and September 3-October 2, 1986, funnel-type moth traps (Phytophyl, Averof 16, Athens, Greece) were suspended across a corn field in Kopais, central Greece, 1.5 m above the ground, approx. 50 m apart. In 1985, the traps were baited with rubber septa (J12 7 x 11 mm, A.H. Thomas Co., Philadelphia) loaded with 250 /zg of each pheromone component and blends containing the Z11-16: OAc and one, two, or three secondary components. In 1986, the traps were baited with different concentrations of the four-component blend. Three replicates for each treatment were used in a complete randomized design. A slow-release formulation of DVP (Vapona) was used as killing agent.
1244
MAZOMENOS
Traps were serviced once a week. Weekly trap catches were subjected to log(x + 1) transformation prior to statistical analysis. Mean comparisons were made with Duncan's multiple-range test (Steel and Torrie, 1960).
RESULTS AND DISCUSSION
Gas Chromatographic Analysis. Female pheromone gland extracts were injected on a Carbowax 20 M column and collected in 3-min fractions. The fractions were tested for biological activity in laboratory bioassays. Activity was found in the fractions eluted 9-12 (fraction 4) and 21-24 (fraction 8) min after injection. Further purificatio'n of the active fractions on OV-101 column resulted in the isolation of two active components with retention times of 4.3 and 18.7 min for fractions 4 and 8, respectively. The retention times of the two components corresponded to those of synthetic 12 : OAc and Z11-16 : OAc. GC-MS analyses of the two components supported the chromatographic data. The mass spectrum for component with a retention time of 4.3 min showed diagnostic peaks of m/z 228 for P and 168 for P-60. This spectrum was identical with that obtained for the synthetic 12 : OAc. The mass spectrum for the component with a retention time of 18.7 min established that it was a C~6 monounsaturated acetate (m/z 282 for P and 222 for P-60). The double-bond geometry and position are not revealed by the spectrum, but the spectrum was identical to that of synthetic Z11-16: OAc. These two synthetic components tested separately or in combination in laboratory bioassays produced an activity level less than that elicited from the crude extract. Since Z 1 1 - 1 6 : O H and 16:OAc have been reported as sex pheromone components (Sreng et al., 1985), we reexamined the pheromone gland extracts and the airborne volatiles collected from virgin females. Three other components were found that had retention times matching those of monounsaturated 16 : OH and 16 : Aid and saturated 16 : OAc. Since the 16 : OAc and 1 6 : O H were reported (Sreng et al., 1985) to be of Z-11 configuration, it was assumed that the 16 : Aid detected was also of the Z-11 configuration; no other aldehydes were considered. Hexadecyl acetate had no effect on male behavior and was not included in field tests. The ratio of the other four components Z1116 : OAc/12 : OAc/Z11-16 : Ald/Z11-16t: OH was found to be 65 : 18 : 8 : 9, respectively. In laboratory tests, the components Z11-16 : OH and Z11-16 : Aid, when tested individually, were not attractive to males; however, when the two components were added to the two acetates (Z11-16 : OAC and 12 : OAC), male attractiveness was increased. Field Tests. Results of field experiments indicated that Z l l - 1 6 : O A c attracted quite a high number of males. The blend of the four synthetic corn-
CORN STALK BORER PHEROMONE
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ponents in a 69 : 15 : 8 : 8 ratio (similar to that found in the female volatiles) attracted more males than Z l l - 1 6 : O A c alone (Table 1). The secondary components, 12 : OAc, Z11-16 : Aid, and Z11-16 : OH, when added individually to the Z l l - 1 6 : O A c in 20, 10, and 10% amounts, respectively, decreased male captures. On the other hand, male capture were increased when two of the three secondary components were added to Z11-16 : OAc. The effect of the pheromone concentration on male captures was studied in 1986, during July with low insect population and in September when the insect population density was high. Concentrations ranging from 50 to 400 ~g/ trap were tested, and all attracted males. A trend of higher male attraction was observed in traps baited with 200/zg of the pheromone blend (Table 2). The results confirmed that Z11-16 : OAc is the major sex pheromone component. Many moths of the Noctuidae family are known to use Z11-16 : OAc as their major sex pheromone component; Sesamia inferents (Nesbitt et al., 1976), Pseudaletia unipuncta (Hill and Roelofs, 1980; McDonough et al., 1980; Farine et al., 1981), and Mamestra brassicae (Descoins et al., 1978). Some of these species require the addition of the corresponding alcohol or aldehyde for optimum attraction. The three components were isolated from the species Mamestra suasa (Toth et al., 1986) and are essential for male attraction of the cutworm moth, Crymodes devastator (Steck et al., 1980). Dodecyl acetate has also been isolated from other lepidopterous species (Inscoe, 1982), and in most cases this compound acts as a synergist. In our field tests, traps baited with Z11-16 : OAc also attracted a high numTABLE 1. MALES. nonagrioides CAPTUREDIN TRAPSBAITEDWITHDIFFERENT COMBINATIONSOF SYNTHETICSEX PHEROMONECOMPONENTS,KOPAIS, GREECE, 1985 Components (/~g) Z11-16 : OAc
12 : OAc
Z11-16 :Ald
Z1 I - 16 : OH
Males caught/ trap/nighta
225 225 225 250 225 200 225 225
50 50 0 0 50 50 0 0
25 25 25 0 0 0 25 0
25 0 25 0 25 0 0 25
44.1 a 27.1b 10.6c 9.6c 2.8d 3.6d 2.2d 4.3d
~Means followed by the same letter are not significant different (Duncan's multiple-range test P = 0.05).
1246
MAZOMENOS TABLE 2. MALE S. nonagrioides CAPTURED IN TRAPS BAITED WITH VARIOUS CONCENTRATION OF SYNTHETIC SEX PHEROMONE BLEND, a AT A
RATIOOF 69 : 15 : 8 : 8, KOPAIS, GREECE, 1986 Males caught~trap~nightb
Concentration (/zg)
Test 1 (July 3-30, 1986)
50 100 150 200 250 300 350 400
0.5b 0.8ab 1.lab 1.5a 0.8ab 0.9ab 0.8ab 0.4b
Test 2 (Sept. 5-Oct. 2, 1986)
12.5a 16.1a 18.5a 13.7a
"Z11-16 : Oac/12 : OAc/Z11-16: AId/Z11-16 : OH. bMeans followed by the same letter in the same column are not significant different (Duncan's multiple-range test, P = 0.05).
ber of P. unipuncta males (14.7 males/trap/night). Male P. unipuncta catches were significantly reduced with the addition of the three secondary components (1 male/trap/night). The synthetic blend of Z11-16 : OAc, 12 : OAc, Z11-16 : Aid, and Z1116 : OH (69 : 15 : 8 : 8) at a concentration of 200/~g provides a specific attractant for monitoring S. nonagrioides population. Acknowledgments--The author wish to thank Mr. J.G. Pomonis for mass spectral determination and Mrs. T. Pantazi-Mazomenou for technical assistance.
REFERENCES C.I.E. (COMMONWEALTHINSTITUTEOF ENTOMOLOGY). 1979. Distribution Maps of Pests. Series A (Agricultural), Map No. 399 Pest: Sesamia nonagriodes (Lef.) Commonwealth Agricultural Bureau, London. DESCOINS, C., PRIESNER, E., GALLOIS, M., ARN, H., and MARTIN, G. 1978. Sur la secretion pheromonale des femalles vlerges de Mamestra brassicae L. et Mamestra oleracea L. (Lepidoptera Noctuidae, Hadenidae). C.R. Acad. Sci. Ser. D 286:77-80. FARINE, J.P., FREROT, B., and ISORT, J. 1981. Chemical isolation factors in pheromonal secretions of two noctuids (Hadeninae): Mamestra brassicae (L.) and Pseudaletia unipuncta (Haw.) C.R. Acad. Sci. III:IOI-IlM. HILL, A.S., and ROELOFS, W.L. 1980. A female produced sex pheromone component and attractant for males in the armyworm moth, Pseudaletia unipuncta. Environ. Entomol. 9:408-411.
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INSCOE, M.N. 1982. Insect attmctants, attmctant pheromones and related compounds, pp. 201298, in A.F. Kydonieus and M. Beroza (eds.). Insect Suppression with Controlled Release Pheromone Systems, Vol. 2. CRC Press, Boca Raton, Florida. MAZOMENOS, B.E. 1984. A sex attractant of the corn stalk borer, Sesamia nonagrioides (Lef.). Partial chemical purification and its biological activity under laboratory conditions. Med. Fac. Landbouww. Rikjsuniv. Gent, 49(3a):675-681. MCDONOUGH, L.M., KAMM,J.A., and BIERL-LEONARDT. 1980. Sex pheromone of the armyworm, Pseudaletia unipuncta (Haworth), (Lepidoptera: Noctuidae). J. Chem. Ecol. 6:565-572. NESBITT, B.F. BEEVOR, P.S., HALL, D.R., LESTER, R., and DYSK, V.A. 1976. Identification of the female sex pheromone of the purple stem borer moth, Sesamia inferents. Insect Biochem. 6:105-107. SRENG, I., MAUNE, B., and FREROT, B. 1985. Analyse de la sdcr6tion phdromonale produite par les femelles vierges de Sesamia nonagrioides (Lef.). C.R. Acad. Sci. III 301:439-442. STECK, W.F., UNDERHILL, E.W., BAILEY, B.K., and CHISHOLM, M.D. 1980. Improved attractant blend for adult males of the grassy cutworm, Grymodes devastator (Lepidoptem: Noctuidae). Can. Entomol. 112:751-752. STEEL, R.C.D., and TORRIE, J.H. 1960. Principles and Procedures of Statistics, with Special Reference to the Biological Sciences. McGraw-Hill, New York. TOTH, M., Szocs, G., LOFSTEDT, C., HANSSON, B.S., and SUBCHEV, M. 1986. Sex pheromone components of Mamestra suasa: Chemical analysis, electrophysiotogical activity, wind tunnel activity and field tests in two European countries. Entomol. Exp. Appl. 22:291-299. TS[TSlPIS, J.A., MAZOMENOS, B.E., CHRISTOULAS,C., MOULOUDIS, S., STEFANAKIS, M., PAPAGEORGIOU,G., GLIATIS, A., and SINIS, G. 1983. Report on the Lepidopterous insect attacking corn in Greece with emphasis on the corn stalk borer, Sesamia nonagriodes. 9th Interbalkanic Plant Protection Conference, Athens, Greece, November 7-11, 1983.
SEX PHEROMONE COMPONENTS OF CORN STALK BORER Sesamia nonagriodes (LEF.)t Isolation, Identification, and Field Tests
B.E.
MAZOMENOS
Institute of Biology N. R. C. ' "Democritos" P. O. Box 60228 GR 153 10 Aghia Paraskevi, Greece
(Received March 23, 1987; accepted June 6, 1988) Abstract--Z-11-Hexadecenyl acetate (Zll-16:OAc), dodecyl acetate (12 : OAc), Z- 11-hexadecenal (Z11-16 :Ald), and Z- 11-hexadecenol (Z1 I16:OH), were found in pheromone gland extracts of female Sesamia nonagriodes (Lef.) [Lepidoptera: Noctuidae]. These four components were also present in airborne volatiles collected from calling virgin females in a 65 : 18 : 8 : 9 ratio. Hexadecyl acetate (16 : OAc) was also detected but found to be inactive. The identification was based on multicolumn GC analysis, mass spectrometry, and field activity. Z11-16:OAc is the major sex pheromone component; the addition of the secondary components individually decreased male captures. The blend of the four synthetic components in 69 : 15 : 8 : 8 ratio was highly attractive to males; 200 #g per trap was the most effective concentration in field tests. Key Words--(Z)-I 1-Hexadecenyl acetate, dodecyl acetate, (Z)-I 1-hexadecenal, (Z)-I 1-hexadecenol, Sesamia nonagrioides, Lepidoptera, Noctuidae, sex pheromone.
INTRODUCTION T h e c o r n s t a l k b o r e r , S e s a m i a n o n a g r i o i d e s ( L e f . ) [ L e p i d o p t e r a : N o c t u i d a e ] , is a n i m p o r t a n t c o r n p e s t in m a n y M e d i t a r r a n e a n a n d c e n t r a l A f r i c a n c o u n t r i e s ( C . I . E . , 1979). W e r e p o r t e d p r e v i o u s l y ( M a z o m e n o s , 1984) t h a t v i r g i n f e m a l e a b d o m i n a l tip e x t r a c t s a t t r a c t e d m a l e s a n d t h a t t h e p h e r o m o n e p r o d u c e d is a m u l t i c o m p o n e n t p h e r o m o n e . T w o p h e r o m o n e c o m p o n e n t s w e r e i d e n t i f i e d as Lepidoptera: Noctuidae. 1241 0098-0331/89/0400-1241506.00/0
9 1989 Plenum Publishing Corporation
1242
MAZOMENOS
Z-11-hexadecenyl acetate (Z11-16 : OAc) and Z-11-hexadecenol (Z11-16 : OH) by Sreng et al. (1985). The same authors reported that hexadecyl acetate (16 :OAc) was present in the pheromone gland extracts, but its biological activity was not established. Here we report minor components of the sex pheromone of S. nonagriodes that improve male captures in field tests.
METHODS AND MATERIALS
Insects. The insects used in this study were collected as larvae or eggs from infested corn plants. The larvae were separated according to their instar, transferred to artificial diet (Tsitsipis et al., 1983), and allowed to continue their development under laboratory conditions. The temperature was kept at 27 + 2~ with a 16:8 hr light-dark regime and relative humidity of 60 + 5 %. The diet was replaced once a week. The eggs were transferred onto a filter paper impregnated with 0.3% propionic acid in Petri dishes for incubation at 27~ After the four- to five-day incubation period, the emerged larvae were placed on artificial diet. Larval development under the conditions described lasted ca. 30-40 days. The pupae were kept under the same conditions until moth emergence. Insects were sexed at the pupal stage, five to six days after pupation. The female adults were transferred in an incubator at 25~ with 16:8 hr light-dark regime, while males were placed in a large bioassay cage in a separate room, operated under the same conditions. Pheromone Collection. The pheromone was collected from the pheromone glands of 2- to 3-day-old virgin females exhibiting calling behavior; the peak of calling occurred in the fifth hour into the dark phase. The glands were removed with forceps and extracted in ether for 20 min. The ether extracts were filtered and concentrated through a 8-cm Vigreux column. Airborne volatiles from virgin females were collected using a Teckmar GLS-1 closed looping concentrator. Females 2 to 4 days old were placed in the l-liter sampler during the dark period, and the volatiles emitted from the females were swept by the recirculating headspace gas and absorbed on 1.5 mg of activated charcoal. The trap was then removed and a collection vial was connected to the trap on the end close to the charcoal. Aliquots of 5 /~1 hexane were added twice to the trap. The bottom of the trap-vial assembly was cooled with a Dry Ice cube. This caused the air in the vial to contract, creating a partial vacuum that pulled the solvent through the charcoal. After pulling the solvent down, it was pushed back through the charcoal three times by warming the bottom of the trap with hand contact. The extracts collected were subjected to GC analysis.
CORN STALK BORER PHEROMONE
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Gas Chromatography-Mass Spectrometry. Concentrated crude extracts were injected on a 2-m x 1.8-mm (ID) column packed with 5% Carbowax 20 M on Chromosorb G/HP 80-100 mesh; the column temperature was held at 90~ for 10 min and then was programmed to 180~ at 6~ Nitrogen was used as carrier with a flow of 20 cc/min. Consecutive 3-min fractions were collected in liquid-nitrogen-cooled glass capillaries, and each was tested for biological activity in laboratory bioassays. The active fractions recovered from the Carbowax 20 M column were further purified on a 2-m x 1.8-mm (ID) column packed with 3% OV-101 on Chromosorb Q 80-100 mesh operated isothermally at 180~ and collected in 1-min fractions. The active peaks were analyzed on 3% OV-101 (2 m x 1.8 m m ID) on Chromosorb Q 80-100 mesh at 180~ 5% DEGS (2 m x 1.8 mm ID) on Chromosorb G H/P 80-100 mesh at 160~ and 15% OV-225 (2 m x 1.8 mm ID) on Chromosorb Q 80-100 mesh at 150~ The mass-spectral analyses of the active components were performed using a GC-MS Finnigan MAT 112s spectrometer in the electron-impact mode. The GC glass column was 3.05 m x 0.32 cm (ID), packed with 3.5% OV-101 on Chromosorb Q 100-200 mesh. Helium was the carder gas at a flow rate of 25 cc/min. Chemicals. Z11-16 : OAc, Z11-16 : Aid, and Z11-16 : OH were obtained from Sigma Chemical Co. (St. Louis., Missouri); and 12 : OAc was synthesized in our laboratory. The components were found to be 96-98% pure when analyzed by GC on OV-101 and OV-225 columns. Bioassays. Bioassays were conducted in a screen cage described by Mazomenos (1984). Usually 25-40, 1- to 3-day-old males were present in the cage during the bioassay. The bioassays were conducted 5-6 hr into the dark phase under a uniform low level of light (ca. 5 lux). Samples of the crude extract and the fractions obtained during the purification were pipetted onto a piece of Whatman No. 1 filter paper that was suspended about 3 cm below the top of the cage. The response of the males to the sample was observed for 10 min. During each testing period samples were tested individually. The same volume of solvent on filter paper was used as control. Field Tests. For the field tests conducted during August 13-September 15, 1985, July 3-30, and September 3-October 2, 1986, funnel-type moth traps (Phytophyl, Averof 16, Athens, Greece) were suspended across a corn field in Kopais, central Greece, 1.5 m above the ground, approx. 50 m apart. In 1985, the traps were baited with rubber septa (J12 7 x 11 mm, A.H. Thomas Co., Philadelphia) loaded with 250 /zg of each pheromone component and blends containing the Z11-16: OAc and one, two, or three secondary components. In 1986, the traps were baited with different concentrations of the four-component blend. Three replicates for each treatment were used in a complete randomized design. A slow-release formulation of DVP (Vapona) was used as killing agent.
1244
MAZOMENOS
Traps were serviced once a week. Weekly trap catches were subjected to log(x + 1) transformation prior to statistical analysis. Mean comparisons were made with Duncan's multiple-range test (Steel and Torrie, 1960).
RESULTS AND DISCUSSION
Gas Chromatographic Analysis. Female pheromone gland extracts were injected on a Carbowax 20 M column and collected in 3-min fractions. The fractions were tested for biological activity in laboratory bioassays. Activity was found in the fractions eluted 9-12 (fraction 4) and 21-24 (fraction 8) min after injection. Further purificatio'n of the active fractions on OV-101 column resulted in the isolation of two active components with retention times of 4.3 and 18.7 min for fractions 4 and 8, respectively. The retention times of the two components corresponded to those of synthetic 12 : OAc and Z11-16 : OAc. GC-MS analyses of the two components supported the chromatographic data. The mass spectrum for component with a retention time of 4.3 min showed diagnostic peaks of m/z 228 for P and 168 for P-60. This spectrum was identical with that obtained for the synthetic 12 : OAc. The mass spectrum for the component with a retention time of 18.7 min established that it was a C~6 monounsaturated acetate (m/z 282 for P and 222 for P-60). The double-bond geometry and position are not revealed by the spectrum, but the spectrum was identical to that of synthetic Z11-16: OAc. These two synthetic components tested separately or in combination in laboratory bioassays produced an activity level less than that elicited from the crude extract. Since Z 1 1 - 1 6 : O H and 16:OAc have been reported as sex pheromone components (Sreng et al., 1985), we reexamined the pheromone gland extracts and the airborne volatiles collected from virgin females. Three other components were found that had retention times matching those of monounsaturated 16 : OH and 16 : Aid and saturated 16 : OAc. Since the 16 : OAc and 1 6 : O H were reported (Sreng et al., 1985) to be of Z-11 configuration, it was assumed that the 16 : Aid detected was also of the Z-11 configuration; no other aldehydes were considered. Hexadecyl acetate had no effect on male behavior and was not included in field tests. The ratio of the other four components Z1116 : OAc/12 : OAc/Z11-16 : Ald/Z11-16t: OH was found to be 65 : 18 : 8 : 9, respectively. In laboratory tests, the components Z11-16 : OH and Z11-16 : Aid, when tested individually, were not attractive to males; however, when the two components were added to the two acetates (Z11-16 : OAC and 12 : OAC), male attractiveness was increased. Field Tests. Results of field experiments indicated that Z l l - 1 6 : O A c attracted quite a high number of males. The blend of the four synthetic corn-
CORN STALK BORER PHEROMONE
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ponents in a 69 : 15 : 8 : 8 ratio (similar to that found in the female volatiles) attracted more males than Z l l - 1 6 : O A c alone (Table 1). The secondary components, 12 : OAc, Z11-16 : Aid, and Z11-16 : OH, when added individually to the Z l l - 1 6 : O A c in 20, 10, and 10% amounts, respectively, decreased male captures. On the other hand, male capture were increased when two of the three secondary components were added to Z11-16 : OAc. The effect of the pheromone concentration on male captures was studied in 1986, during July with low insect population and in September when the insect population density was high. Concentrations ranging from 50 to 400 ~g/ trap were tested, and all attracted males. A trend of higher male attraction was observed in traps baited with 200/zg of the pheromone blend (Table 2). The results confirmed that Z11-16 : OAc is the major sex pheromone component. Many moths of the Noctuidae family are known to use Z11-16 : OAc as their major sex pheromone component; Sesamia inferents (Nesbitt et al., 1976), Pseudaletia unipuncta (Hill and Roelofs, 1980; McDonough et al., 1980; Farine et al., 1981), and Mamestra brassicae (Descoins et al., 1978). Some of these species require the addition of the corresponding alcohol or aldehyde for optimum attraction. The three components were isolated from the species Mamestra suasa (Toth et al., 1986) and are essential for male attraction of the cutworm moth, Crymodes devastator (Steck et al., 1980). Dodecyl acetate has also been isolated from other lepidopterous species (Inscoe, 1982), and in most cases this compound acts as a synergist. In our field tests, traps baited with Z11-16 : OAc also attracted a high numTABLE 1. MALES. nonagrioides CAPTUREDIN TRAPSBAITEDWITHDIFFERENT COMBINATIONSOF SYNTHETICSEX PHEROMONECOMPONENTS,KOPAIS, GREECE, 1985 Components (/~g) Z11-16 : OAc
12 : OAc
Z11-16 :Ald
Z1 I - 16 : OH
Males caught/ trap/nighta
225 225 225 250 225 200 225 225
50 50 0 0 50 50 0 0
25 25 25 0 0 0 25 0
25 0 25 0 25 0 0 25
44.1 a 27.1b 10.6c 9.6c 2.8d 3.6d 2.2d 4.3d
~Means followed by the same letter are not significant different (Duncan's multiple-range test P = 0.05).
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MAZOMENOS TABLE 2. MALE S. nonagrioides CAPTURED IN TRAPS BAITED WITH VARIOUS CONCENTRATION OF SYNTHETIC SEX PHEROMONE BLEND, a AT A
RATIOOF 69 : 15 : 8 : 8, KOPAIS, GREECE, 1986 Males caught~trap~nightb
Concentration (/zg)
Test 1 (July 3-30, 1986)
50 100 150 200 250 300 350 400
0.5b 0.8ab 1.lab 1.5a 0.8ab 0.9ab 0.8ab 0.4b
Test 2 (Sept. 5-Oct. 2, 1986)
12.5a 16.1a 18.5a 13.7a
"Z11-16 : Oac/12 : OAc/Z11-16: AId/Z11-16 : OH. bMeans followed by the same letter in the same column are not significant different (Duncan's multiple-range test, P = 0.05).
ber of P. unipuncta males (14.7 males/trap/night). Male P. unipuncta catches were significantly reduced with the addition of the three secondary components (1 male/trap/night). The synthetic blend of Z11-16 : OAc, 12 : OAc, Z11-16 : Aid, and Z1116 : OH (69 : 15 : 8 : 8) at a concentration of 200/~g provides a specific attractant for monitoring S. nonagrioides population. Acknowledgments--The author wish to thank Mr. J.G. Pomonis for mass spectral determination and Mrs. T. Pantazi-Mazomenou for technical assistance.
REFERENCES C.I.E. (COMMONWEALTHINSTITUTEOF ENTOMOLOGY). 1979. Distribution Maps of Pests. Series A (Agricultural), Map No. 399 Pest: Sesamia nonagriodes (Lef.) Commonwealth Agricultural Bureau, London. DESCOINS, C., PRIESNER, E., GALLOIS, M., ARN, H., and MARTIN, G. 1978. Sur la secretion pheromonale des femalles vlerges de Mamestra brassicae L. et Mamestra oleracea L. (Lepidoptera Noctuidae, Hadenidae). C.R. Acad. Sci. Ser. D 286:77-80. FARINE, J.P., FREROT, B., and ISORT, J. 1981. Chemical isolation factors in pheromonal secretions of two noctuids (Hadeninae): Mamestra brassicae (L.) and Pseudaletia unipuncta (Haw.) C.R. Acad. Sci. III:IOI-IlM. HILL, A.S., and ROELOFS, W.L. 1980. A female produced sex pheromone component and attractant for males in the armyworm moth, Pseudaletia unipuncta. Environ. Entomol. 9:408-411.
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INSCOE, M.N. 1982. Insect attmctants, attmctant pheromones and related compounds, pp. 201298, in A.F. Kydonieus and M. Beroza (eds.). Insect Suppression with Controlled Release Pheromone Systems, Vol. 2. CRC Press, Boca Raton, Florida. MAZOMENOS, B.E. 1984. A sex attractant of the corn stalk borer, Sesamia nonagrioides (Lef.). Partial chemical purification and its biological activity under laboratory conditions. Med. Fac. Landbouww. Rikjsuniv. Gent, 49(3a):675-681. MCDONOUGH, L.M., KAMM,J.A., and BIERL-LEONARDT. 1980. Sex pheromone of the armyworm, Pseudaletia unipuncta (Haworth), (Lepidoptera: Noctuidae). J. Chem. Ecol. 6:565-572. NESBITT, B.F. BEEVOR, P.S., HALL, D.R., LESTER, R., and DYSK, V.A. 1976. Identification of the female sex pheromone of the purple stem borer moth, Sesamia inferents. Insect Biochem. 6:105-107. SRENG, I., MAUNE, B., and FREROT, B. 1985. Analyse de la sdcr6tion phdromonale produite par les femelles vierges de Sesamia nonagrioides (Lef.). C.R. Acad. Sci. III 301:439-442. STECK, W.F., UNDERHILL, E.W., BAILEY, B.K., and CHISHOLM, M.D. 1980. Improved attractant blend for adult males of the grassy cutworm, Grymodes devastator (Lepidoptem: Noctuidae). Can. Entomol. 112:751-752. STEEL, R.C.D., and TORRIE, J.H. 1960. Principles and Procedures of Statistics, with Special Reference to the Biological Sciences. McGraw-Hill, New York. TOTH, M., Szocs, G., LOFSTEDT, C., HANSSON, B.S., and SUBCHEV, M. 1986. Sex pheromone components of Mamestra suasa: Chemical analysis, electrophysiotogical activity, wind tunnel activity and field tests in two European countries. Entomol. Exp. Appl. 22:291-299. TS[TSlPIS, J.A., MAZOMENOS, B.E., CHRISTOULAS,C., MOULOUDIS, S., STEFANAKIS, M., PAPAGEORGIOU,G., GLIATIS, A., and SINIS, G. 1983. Report on the Lepidopterous insect attacking corn in Greece with emphasis on the corn stalk borer, Sesamia nonagriodes. 9th Interbalkanic Plant Protection Conference, Athens, Greece, November 7-11, 1983.
