Journal of Chemical Ecology, Vol. 12, No. 6, 1986
THE FEMALE SEX PHEROMONE OF SUGARCANE STALK BORER, Chilo auricilius Identification of Four Components and Field Tests
BRENDA
F. N E S B I T T , 1 P. S. B E E V O R , 1 A. C O R K , j D. R. H A L L , 1 H. D A V I D , 2 and V. N A N D A G O P A L 2 Tropical Development and Research Institute, 56/62 Gray's Inn Road, London WC1X 8LU, England, 2Sugarcane Breeding Institute, Coimbatore 641007, India (Received June 27, 1985; accepted September 17, 1985)
Abstract--Four pheromonal components have been detected in ovipositor washings and volatiles from female sugarcane stalk borers, Chilo auricilius Dudgeon (Lepidoptera: Pyralidae), using combined gas chromatographyelectroantennography. The components have been identified as (I) (Z)-7-dodecenyl acetate, (If) (Z)-8-tridecenyl acetate, (III) (Z)-9-tetradecenyl acetate, and (IV) (Z)-10-pentadecenyl acetate by comparison of their gas chromatographic behavior with that of synthetic standards. In field tests carried out in northern India during 1982-1984, a combination of II, III, and IV in their naturally occurring ratio (8:4:1) was shown to provide a highly attractive synthetic source for trap use. (Z)-7-Dodecenyl acetate was found to reduce catches of male C. auricilius, both when dispensed with the other three components and when released from dispensers surrounding a trap baited with the other three components. Key Words--Sugarcane stalk borer, Chilo auricilius, Lepidoptera, Pyralidae, sex pheromone, sex attractant; (Z)-7-dodecenyl acetate, (Z)-8-tridecenyl acetate, (Z)-9-tetradecenyl acetate, (Z)-10-pentadecenyl acetate.
INTRODUCTION Chilo auricilius D u d g e o n ( L e p i d o p t e r a : Pyralidae) is an important pest o f sugarcane found in India, Southeastern A s i a , I n d o n e s i a , and northern Australia. T h e larvae d a m a g e the c a n e by attacking shoots and by internode boring, w h i c h impairs g r o w t h and results in losses o f w e i g h t o f c a n e and sugar r e c o v e r y ; yield 1377 0098 0331/86/0600 1377505.00/0 9 1986 Plenum Publishing Corporation
1378
NEsBnn" ET AL.
losses of up to 30% have been recorded in India. Sorghum and rice are also attacked by this species. Its life history and factors affecting its incidence are described by Gupta et al. (1981), who also reported that no suitable and effective control measures were available. The presence of a female sex pheromone was demonstrated in northern India by trapping of male C. auricilius in virgin female baited traps (Kalra and David, 1971). Identification and synthesis of the female sex pheromone was undertaken with the aim of providing a control agent compatible with existing cultural practices in India.
METHODS AND MATERIALS
Insect Material. Pupae were field collected in northern India and sent by air freight to London. They were sexed and the sexes segregated and maintained in an environmental cabinet until adult emergence (conditions: reversed lightdark cycle, 12 hr: 12 hr; temperature 26~ photophase, 24~ scotophase; relative humidity 85 %). Adult moths were maintained under the same conditions with a supply of 10% sucrose solution. Pheromone Collection. Ovipositor washings in carbon disulfide or heptane were usually prepared from virgin female moths 6-8 hr into the first dark period after emergence. Some extracts were prepared after 189 3, 4, 5, and 1 ~ hr and from 2- to 3-day-old females in attempts t o obtain higher pheromone yields. Volatiles were collected from individual virgin female moths as described previously (Nesbitt et al., 1979). Electroantennography (EAG). Recording of male moth antennal responses to GC column effluent (GC-EAG) was carried out essentially as described previously (Moorhouse et al., 1969). " P u f f " tests were carried out as described by Nesbitt and et al. (1977), with a 7-min recovery period between stimulations. Gas Chromatography (GC). For analyses on packed columns, with and without simultaneous recording of EAG responses to part of the column effluent, columns and conditions were as follows: glass columns (1.8 m x 2 mm ID) packed with (A) 2.5% SE30 and 0.5% Carbowax 20 M on Chromosorb G AW DMCS, programmed from 120 to 225~ at 4~ and (B) 1.5% Carbowax 20 M on Chromosorb G AW DMCS programmed from 120 to 200~ at 4~ Carrier gas for A and B was 25 ml/min nitrogen. For analyses on fused silica capillary columns, columns were as follows (except where otherwise stated): 50 m x 0.32 mm ID Chrompack columns coated with (C) CP Sil 5CB (chemically bonded methyl silicone), and (D) CP wax 57CB (chemically bonded, cross-linked polyethylene glycol). Cartier gas for C and D was helium 0.8 kg/cm 2. A Grob splifless injector was used. Columns were maintained at 70~ for 2 min at the start of each analysis (except for analyses of pentadecenyl isomers); subsequent temperature profiles were as follows: For dodecenyl isomers: C and D, 70-120~ at 20~ then isothermal; for tridecenyl iso-
PHEROMONE OF THE S U G A R C A N E STALK BORER
1379
mers: C, 70-135~ at 20~ then isothermal; D, 70-100~ at 20~ then isothermal (25 m x 0.24 mm ID column). For tetradecenyl isomers: C, 70-145~ at 20~ then isothermal; D, 7 0 - t 4 0 ~ at 20~ then isothermal. Conditions for pentadecenyl isomers were as follows: C, 80~ for 1 min, 80-120~ at 40~ 120-200~ at l~ D 70~ for 2 min, 70-110~ at 20~ isothermal for 34 min, 110-200~ at 2~ Synthetic Chemicals. (Z)- and (E)-6-tridecenyl acetate, (Z)- and (E)-7tridecenyl acetate, and (Z)- and (E)-10-tridecenyl acetate were purchased from the Institute for Pesticide Research, Wageningen, The Netherlands. The other monounsaturated acetates were synthesized by standard Wittig or acetylenic routes. The pheromone components used in field tests were purified by liquid chromatography on silica gel impregnated with 20 % silver nitrate so that they contained less than 0.1 ~ of the opposite geometric isomer. FieM Tests. These were carried out at two centers in Uttar Pradesh, N. India, during 1982-1984. The traps used were large water traps (60 cm x 60 cm) on 75 cm legs, similar to those described by Campion et al. (1974) but without lids; pheromone dispensers were suspended centrally just above the water from a cross rod. Dispensers for the synthetic compounds were polythene vials (35 mm x 8 mm • 1.5 mm thick); an equal weight of 2,6-di-tert-butyl4-methylphenol (BHT) was added to the synthetic materials as antioxidant. Synthetic pheromone sources were not renewed during experiments. Virgin female moths used to bait traps were caged singly in small mesh containers positioned in the same way as the polythene vials. The female moths were renewed every three days. For attractancy experiments involving a small number of treatments (Tables 1 and 2), traps were set out randomly in a row, crosswind, approximately 30 m apart. For the experiment detailed in Table 3, treatments were placed randomly in an 8 x 5 grid with approximately 30 m between traps. For the experiment detailed in Table 4, a 6 x 6 lattice design was used with trap spacing within blocks 30 m and blocks at least 100 m apart. For communication disruption experiments, six vials containing the test chemical were placed evenly in a circle around a trap baited with synthetic attractant at 1 m distance and at the same height above the ground as the attractant vial. In the experiment detailed in Table 5, the "surrounded" traps and control traps were set out randomly in two parallel rows, with approximately 30 m between traps in each row and 60 m between rows. In the two-trap experiments (Tables 6 and 7) traps were approximately 30 m apart and replicates were at least 100 m apart. In the experiments summarized in Tables 1, 2, 3, and 5, treatments were randomized at the beginning of the experiment only. Statistical analyses were based on a factorial random block design with treatments and nights as the two factors. Catches were converted to (x + 0.5) and subjected to analysis of variance. Differences between treatment means were tested for significance by least significant differences (LSD) tests. The
1380
NESBITT ET AL.
experiment described in Table 4 was a Latin square design and treatments were rerandomized each night. In the disruption experiments (Tables 6 and 7), the positions of treatment and control traps were exchanged each night. RESULTS AND DISCUSSION
Structure Determination GC-EAG analyses of ovipositor washings and female volatiles on nonpolar and polar packed columns (A and B) showed four EAG responses. Equivalent chain lengths of the EAG-active compounds based on the retention times of n-alkyl acetates (ECLs) suggested that these compounds were C~2, C13, C~4, and C15 monounsaturated acetates o r C14 , C15 , C16 , and C17 lnonounsaturated aldehydes (column A ECLs 11.9, 12.9, 13.9, 14.9; column B ECLs 12.4, 13.3., 14.3, 15.4). The EAG-active compounds, assumed to be pheromonal components, were designated I-IV in order of elution; the largest EAG response was to component II. In ovipositor washings, the average ratio of the GC peak heights for components I, II, HI, and IV was 4 : 8 : 4 : 1. Entrained female volatiles were enhanced in the more volatile components I and II, and the ratio of the four components was very variable. The best yield of pheromone was 6 ng/ female from ovipositor washings; usually much lower yields were obtained. When male moth EAG responses to tridecyl acetate and pentadecanal were compared (GC-EAG link, 10 ng injected), a 1.0-mV response was recorded to the acetate and no response to the aldehyde, suggesting that component II at least, was an acetate rather than an aldehyde. When GC retention times of components I-IV were compared with those of the positional and geometric isomers of dodecenyl, tridecenyl, tetradecenyl, and pentadecenyl acetates, respectively, on fused silica capillary columns C and D, the results described below were obtained: Component L All dodecenyl isomers were available for comparison, and the only one with retention times consistent with those of component I on both columns was (Z)-7-dodecenyl acetate, (Z7-12 : Ac) (Figure 1). Component II. Z- and E6, 7, 8, 9, 10-tridecenyl acetates were available for comparison. Component II had retention times consistent only with the Z8 isomer, (Z8-13 : Ac) (Figure 2). By analogy with the retention times of the full series of dodecenyl and tetradecenyl acetates, which both follow a similar pattern, the (Z)-8-tridecenyl acetate retention times are thought to be unambiguous. Component IlL All tetradecenyl acetate isomers were available for comparison. The retention times for component III fitted onty with those of (Z)-9tetradecenyl acetate (Z9-14 : Ac) on both columns (Figure 3). Component IV. The structural assignment for this component is more tentative than for I-III; it was the least abundant component in ovipositor washings and yields in entrained female volatiles were further reduced by its relative
1381
PHEROMONE OF THE S U G A R C A N E STALK BORER
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THE FEMALE SEX PHEROMONE OF SUGARCANE STALK BORER, Chilo auricilius Identification of Four Components and Field Tests
BRENDA
F. N E S B I T T , 1 P. S. B E E V O R , 1 A. C O R K , j D. R. H A L L , 1 H. D A V I D , 2 and V. N A N D A G O P A L 2 Tropical Development and Research Institute, 56/62 Gray's Inn Road, London WC1X 8LU, England, 2Sugarcane Breeding Institute, Coimbatore 641007, India (Received June 27, 1985; accepted September 17, 1985)
Abstract--Four pheromonal components have been detected in ovipositor washings and volatiles from female sugarcane stalk borers, Chilo auricilius Dudgeon (Lepidoptera: Pyralidae), using combined gas chromatographyelectroantennography. The components have been identified as (I) (Z)-7-dodecenyl acetate, (If) (Z)-8-tridecenyl acetate, (III) (Z)-9-tetradecenyl acetate, and (IV) (Z)-10-pentadecenyl acetate by comparison of their gas chromatographic behavior with that of synthetic standards. In field tests carried out in northern India during 1982-1984, a combination of II, III, and IV in their naturally occurring ratio (8:4:1) was shown to provide a highly attractive synthetic source for trap use. (Z)-7-Dodecenyl acetate was found to reduce catches of male C. auricilius, both when dispensed with the other three components and when released from dispensers surrounding a trap baited with the other three components. Key Words--Sugarcane stalk borer, Chilo auricilius, Lepidoptera, Pyralidae, sex pheromone, sex attractant; (Z)-7-dodecenyl acetate, (Z)-8-tridecenyl acetate, (Z)-9-tetradecenyl acetate, (Z)-10-pentadecenyl acetate.
INTRODUCTION Chilo auricilius D u d g e o n ( L e p i d o p t e r a : Pyralidae) is an important pest o f sugarcane found in India, Southeastern A s i a , I n d o n e s i a , and northern Australia. T h e larvae d a m a g e the c a n e by attacking shoots and by internode boring, w h i c h impairs g r o w t h and results in losses o f w e i g h t o f c a n e and sugar r e c o v e r y ; yield 1377 0098 0331/86/0600 1377505.00/0 9 1986 Plenum Publishing Corporation
1378
NEsBnn" ET AL.
losses of up to 30% have been recorded in India. Sorghum and rice are also attacked by this species. Its life history and factors affecting its incidence are described by Gupta et al. (1981), who also reported that no suitable and effective control measures were available. The presence of a female sex pheromone was demonstrated in northern India by trapping of male C. auricilius in virgin female baited traps (Kalra and David, 1971). Identification and synthesis of the female sex pheromone was undertaken with the aim of providing a control agent compatible with existing cultural practices in India.
METHODS AND MATERIALS
Insect Material. Pupae were field collected in northern India and sent by air freight to London. They were sexed and the sexes segregated and maintained in an environmental cabinet until adult emergence (conditions: reversed lightdark cycle, 12 hr: 12 hr; temperature 26~ photophase, 24~ scotophase; relative humidity 85 %). Adult moths were maintained under the same conditions with a supply of 10% sucrose solution. Pheromone Collection. Ovipositor washings in carbon disulfide or heptane were usually prepared from virgin female moths 6-8 hr into the first dark period after emergence. Some extracts were prepared after 189 3, 4, 5, and 1 ~ hr and from 2- to 3-day-old females in attempts t o obtain higher pheromone yields. Volatiles were collected from individual virgin female moths as described previously (Nesbitt et al., 1979). Electroantennography (EAG). Recording of male moth antennal responses to GC column effluent (GC-EAG) was carried out essentially as described previously (Moorhouse et al., 1969). " P u f f " tests were carried out as described by Nesbitt and et al. (1977), with a 7-min recovery period between stimulations. Gas Chromatography (GC). For analyses on packed columns, with and without simultaneous recording of EAG responses to part of the column effluent, columns and conditions were as follows: glass columns (1.8 m x 2 mm ID) packed with (A) 2.5% SE30 and 0.5% Carbowax 20 M on Chromosorb G AW DMCS, programmed from 120 to 225~ at 4~ and (B) 1.5% Carbowax 20 M on Chromosorb G AW DMCS programmed from 120 to 200~ at 4~ Carrier gas for A and B was 25 ml/min nitrogen. For analyses on fused silica capillary columns, columns were as follows (except where otherwise stated): 50 m x 0.32 mm ID Chrompack columns coated with (C) CP Sil 5CB (chemically bonded methyl silicone), and (D) CP wax 57CB (chemically bonded, cross-linked polyethylene glycol). Cartier gas for C and D was helium 0.8 kg/cm 2. A Grob splifless injector was used. Columns were maintained at 70~ for 2 min at the start of each analysis (except for analyses of pentadecenyl isomers); subsequent temperature profiles were as follows: For dodecenyl isomers: C and D, 70-120~ at 20~ then isothermal; for tridecenyl iso-
PHEROMONE OF THE S U G A R C A N E STALK BORER
1379
mers: C, 70-135~ at 20~ then isothermal; D, 70-100~ at 20~ then isothermal (25 m x 0.24 mm ID column). For tetradecenyl isomers: C, 70-145~ at 20~ then isothermal; D, 7 0 - t 4 0 ~ at 20~ then isothermal. Conditions for pentadecenyl isomers were as follows: C, 80~ for 1 min, 80-120~ at 40~ 120-200~ at l~ D 70~ for 2 min, 70-110~ at 20~ isothermal for 34 min, 110-200~ at 2~ Synthetic Chemicals. (Z)- and (E)-6-tridecenyl acetate, (Z)- and (E)-7tridecenyl acetate, and (Z)- and (E)-10-tridecenyl acetate were purchased from the Institute for Pesticide Research, Wageningen, The Netherlands. The other monounsaturated acetates were synthesized by standard Wittig or acetylenic routes. The pheromone components used in field tests were purified by liquid chromatography on silica gel impregnated with 20 % silver nitrate so that they contained less than 0.1 ~ of the opposite geometric isomer. FieM Tests. These were carried out at two centers in Uttar Pradesh, N. India, during 1982-1984. The traps used were large water traps (60 cm x 60 cm) on 75 cm legs, similar to those described by Campion et al. (1974) but without lids; pheromone dispensers were suspended centrally just above the water from a cross rod. Dispensers for the synthetic compounds were polythene vials (35 mm x 8 mm • 1.5 mm thick); an equal weight of 2,6-di-tert-butyl4-methylphenol (BHT) was added to the synthetic materials as antioxidant. Synthetic pheromone sources were not renewed during experiments. Virgin female moths used to bait traps were caged singly in small mesh containers positioned in the same way as the polythene vials. The female moths were renewed every three days. For attractancy experiments involving a small number of treatments (Tables 1 and 2), traps were set out randomly in a row, crosswind, approximately 30 m apart. For the experiment detailed in Table 3, treatments were placed randomly in an 8 x 5 grid with approximately 30 m between traps. For the experiment detailed in Table 4, a 6 x 6 lattice design was used with trap spacing within blocks 30 m and blocks at least 100 m apart. For communication disruption experiments, six vials containing the test chemical were placed evenly in a circle around a trap baited with synthetic attractant at 1 m distance and at the same height above the ground as the attractant vial. In the experiment detailed in Table 5, the "surrounded" traps and control traps were set out randomly in two parallel rows, with approximately 30 m between traps in each row and 60 m between rows. In the two-trap experiments (Tables 6 and 7) traps were approximately 30 m apart and replicates were at least 100 m apart. In the experiments summarized in Tables 1, 2, 3, and 5, treatments were randomized at the beginning of the experiment only. Statistical analyses were based on a factorial random block design with treatments and nights as the two factors. Catches were converted to (x + 0.5) and subjected to analysis of variance. Differences between treatment means were tested for significance by least significant differences (LSD) tests. The
1380
NESBITT ET AL.
experiment described in Table 4 was a Latin square design and treatments were rerandomized each night. In the disruption experiments (Tables 6 and 7), the positions of treatment and control traps were exchanged each night. RESULTS AND DISCUSSION
Structure Determination GC-EAG analyses of ovipositor washings and female volatiles on nonpolar and polar packed columns (A and B) showed four EAG responses. Equivalent chain lengths of the EAG-active compounds based on the retention times of n-alkyl acetates (ECLs) suggested that these compounds were C~2, C13, C~4, and C15 monounsaturated acetates o r C14 , C15 , C16 , and C17 lnonounsaturated aldehydes (column A ECLs 11.9, 12.9, 13.9, 14.9; column B ECLs 12.4, 13.3., 14.3, 15.4). The EAG-active compounds, assumed to be pheromonal components, were designated I-IV in order of elution; the largest EAG response was to component II. In ovipositor washings, the average ratio of the GC peak heights for components I, II, HI, and IV was 4 : 8 : 4 : 1. Entrained female volatiles were enhanced in the more volatile components I and II, and the ratio of the four components was very variable. The best yield of pheromone was 6 ng/ female from ovipositor washings; usually much lower yields were obtained. When male moth EAG responses to tridecyl acetate and pentadecanal were compared (GC-EAG link, 10 ng injected), a 1.0-mV response was recorded to the acetate and no response to the aldehyde, suggesting that component II at least, was an acetate rather than an aldehyde. When GC retention times of components I-IV were compared with those of the positional and geometric isomers of dodecenyl, tridecenyl, tetradecenyl, and pentadecenyl acetates, respectively, on fused silica capillary columns C and D, the results described below were obtained: Component L All dodecenyl isomers were available for comparison, and the only one with retention times consistent with those of component I on both columns was (Z)-7-dodecenyl acetate, (Z7-12 : Ac) (Figure 1). Component II. Z- and E6, 7, 8, 9, 10-tridecenyl acetates were available for comparison. Component II had retention times consistent only with the Z8 isomer, (Z8-13 : Ac) (Figure 2). By analogy with the retention times of the full series of dodecenyl and tetradecenyl acetates, which both follow a similar pattern, the (Z)-8-tridecenyl acetate retention times are thought to be unambiguous. Component IlL All tetradecenyl acetate isomers were available for comparison. The retention times for component III fitted onty with those of (Z)-9tetradecenyl acetate (Z9-14 : Ac) on both columns (Figure 3). Component IV. The structural assignment for this component is more tentative than for I-III; it was the least abundant component in ovipositor washings and yields in entrained female volatiles were further reduced by its relative
1381
PHEROMONE OF THE S U G A R C A N E STALK BORER
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