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Effective pheromone lures for monitoring the rice leaffolder moth, Cnaphalocrocis medinalis (Lepidoptera: Crambidae)
ARTICLE IN PRESS
Crop Protection 23 (2004) 589–593
Effective pheromone lures for monitoring the rice leaffolder moth, Cnaphalocrocis medinalis (Lepidoptera: Crambidae) Kei Kawazua,*, Takeshi Kamimurob, Hidemi Kamiwadac, Kenji Nagatad, Tadashi Matsunagad, Hajime Sugiee, Takehiko Fukumotof, Taro# Adatig, Sadahiro Tatsukia a
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan b Kagoshima Prefectural Agricultural Experiment Station, Kagoshima 891-0116, Japan c Kagoshima Agricultural Improvement and Advisory Center, Kagoshima 892-0817, Japan d Sankei Chemical Company, Tokyo 110-0015, Japan e National Institute of Agro-Environmental Sciences, Ibaraki 305-8604, Japan f Shin-Etsu Chemical Company, Tokyo 100-0004, Japan g International Institute of Tropical Agriculture, Kano Station, PMB 3112, Kano Nigeria Received 28 July 2003; received in revised form 29 September 2003; accepted 14 November 2003
Abstract Several factors affecting the attractiveness of traps baited with synthetic sex pheromone components of Cnaphalocrocis medinalis were examined in the laboratory and field. In the laboratory, among the four pheromone components Z11-18:Ald, Z13-18:Ald, Z1118:OH and Z13-18:OH, the major component, Z13-18:Ald, was shown to be essential for attraction and Z11-18:Ald was a potent synergist for the major component. The alcohols also showed an apparent synergistic effect in the laboratory bioassay when their mixture was combined with an aldehyde mixture. The addition of the geometric isomers of the four components at levels greater than 5% of the purified 4-component blend showed significant inhibitory effects on the pheromonal activity in the laboratory. In contrast, no synergistic effect of the alcohols on the aldehydes was shown in the field. A range of loadings of the pheromone rubber septa (0.01–3.42 mg) was tested and all remained attractive for at least 1 month with optimum attractiveness of 0.86 mg/septum. No significant effects were obtained when the ratio of Z11-18:Ald to Z13-18:Ald was changed. Rubber septa containing 0.86 mg of the four components (E-isomers o5%) at the natural ratio are being effectively used for monitoring C. medinalis. r 2003 Elsevier Ltd. All rights reserved. Keywords: Rice leaffolder; Cnaphalocrocis medinalis; Synthetic pheromone
1. Introduction The rice leaffolder, Cnaphalocrocis medinalis, is a migratory rice pest in Japan. Moth occurrence in Japan is highly variable by year and by place. Thus, an effective monitoring system is especially important to investigate migration of C. medinalis. Although light traps have often been used for monitoring insect pests, they are of little use for C. medinalis which is not particularly sensitive to light. Therefore, an effective monitoring system is required and the application of synthetic sex pheromones may provide this.
*Corresponding author. E-mail address: [email protected] (K. Kawazu). 0261-2194/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2003.11.009
In a previous study, we identified the female sex pheromone of C. medinalis in Japan as a mixture of (Z)-11-octadecenal (Z11-18:Ald), (Z)-13-octadecenal (Z13-18:Ald), (Z)-11-octadecen-1-ol (Z11-18:OH) and (Z)-13-octadecen-1-ol (Z13-18:OH) in a ratio of 11:100:24:36 (Kawazu et al., 2000). In field trials using purified synthetic compounds (>99.9%) it was shown that the two aldehydes, Z11-18:Ald and Z13-18:Ald, were essential for male attraction and the two alcohols, Z11-18:OH and Z13-18:OH, showed a weak synergistic effect on the aldehydes (Kawazu et al., 2000). For developing monitoring tools using synthetic sex pheromone, an understanding of the effects of the geometric isomers, composition and blend ratios in the synthetic pheromone used and of the loading of the pheromone on the attractiveness to male moths is important.
ARTICLE IN PRESS 590
K. Kawazu et al. / Crop Protection 23 (2004) 589–593
The unpurified synthetic pheromone compounds contain small proportions (usually o10%) of their (E)isomers. If the presence of the geometric isomers in the pheromone blend at the above levels show no inhibitory effect on the attractiveness, purification of the synthetic compounds would be unnecessary. The effect of the geometric isomers on the attractiveness of purified synthetic compounds was therefore examined in the laboratory. Furthermore, the effect of pheromone composition and blend ratios was examined. In addition, the synergistic effect of the two alcohols on the aldehydes was re-examined, since it was unclear in the previous field test (Kawazu et al., 2000). To investigate the optimum dosage range for attraction, the effect of the loadings of synthetic pheromone components was examined in the field.
2. Materials and methods 2.1. Chemicals The synthetic pheromone components, Z11-18:Ald, Z13-18:Ald, Z11-18:OH and Z13-18:OH, were supplied by Shin-Etsu Chemical Company (Tokyo, Japan). Analyses by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) of the synthetic Z11-18:Ald, Z13-18:Ald, Z11-18:OH and Z13-18:OH showed that these compounds contained 1–2% of their (E)-isomers. They were thus purified by high-performance liquid chromatography (HPLC) with an HP-1050 system using a silver ion-coated Nucleosil 100-SA column (250 4 mm ID, 5 mm particle size, GL Science, Tokyo, Japan). The (E)-isomers used for the bioassay were obtained from the above separation of the synthetic pheromone compounds. The aldehydes components were further purified by HPLC on an Inertsil ODS-2 column (250 4 mm ID, 5 mm particle size, GL Science, Tokyo, Japan) to remove trace impurities (o1%). The solvent used for the both HPLC purifications was methanol at a flow rate of 0.45 ml/min. Purity of all compounds was determined to be greater than 99.9% by GC and GC–MS using a DB-23 column (Kawazu et al., 2000). 2.2. Laboratory bioassay The C. medinalis used for laboratory bioassay were from a stock culture originally collected in Osaka prefecture (Japan) in 1985. They were supplied by Sumitomo Chemical Company (Tokyo, Japan) and have been successively reared on an artificial diet (Silkmate 2(S), Nihon Nosan Kogyo, Yokohama, Japan) for over 100 generations at 2571 C under a long-day cycle (15L-9D). After emergence, female and male adults were kept separately under the same
conditions as above and provided with 5% sucrose solution as food. The moths after emergence were designated as age 0 during the scotophase in which they emerged. The subsequent ages were started at the time of each lights on. Laboratory bioassays were conducted with a screen cage (32 22 30 cm). Virgin males of 3–7 days after emergence were bioassayed at 4–8 h after lights off under the usual 15L:9D, 25 C. Twenty naive males were released in the cage. A filter paper strip (2 4 cm) impregnated with 2 ml hexane solution containing the test blend of synthetic pheromone was suspended in the cage from the center of the top screen 5 cm under the top. The number of contacts of the males with claspers extruded to the filter paper strip was recorded for 5 min, not counting accidental contacts. The males attracted by the pheromone showed zigzag flight when approaching within 10 cm of the pheromone source. A dimmed red lamp for visual observation was used during the dark period. Each bioassay was replicated 4–10 times. For statistical analyses, the differences among the mean numbers of contacts of the males were tested for significance by Tukey-Kramer’s test.
2.3. Field bioassays Baits were prepared using gray halo-butyl isoprene blend elastomer septa (West Co., Singapore). The required amounts and ratios of the synthetic compounds each dissolved in 200 ml hexane were applied onto the inner surface of rubber septa. After evaporation of the solvent, the baits were placed in sealed aluminumlaminated polyethylene bags, and stored at 20 C until use. The pheromone lures were renewed everymonth in the field. All bioassays were conducted in paddy fields in Kagoshima City, Kagoshima prefecture during the emergence season of the moth. Sticky traps (SE-Traps, Sankei Chemical Company, Kagoshima, Japan), with a triangle section, dimensions of 29 32 8 cm height, and a 24 30 cm sticky plate, were used in 1997. Cone traps slightly modified from those reported by Kawasaki and Sugie (1990) were used in 1998, 1999 and 2000. Modifications were made to the length (height) of the cone traps which were 2 cm longer than those reported by Kawasaki and Sugie (1990). Pheromone traps were set at the boundary of the paddy fields and at the height of the top of rice plants. The distance between traps was greater than 10 m. After the numbers of trapped male C. medinalis were checked every morning, the traps were moved round one position to reduce any bias due to trap location. For statistical analyses, the number of trapped pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi males at each trap was transformed to ðX þ 0:5Þ and the differences among the means of caught moths were tested for significance by Tukey’s test after analysis of variance.
ARTICLE IN PRESS K. Kawazu et al. / Crop Protection 23 (2004) 589–593
3. Results
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3.1. Laboratory bioassay 3.1.1. Effect of the addition of E-isomers to the purified pheromone To examine the antagonistic effects of the E-isomers of the pheromone components, we compared the activities of synthetic blends containing 1, 3, 5, 10 or 50% of each E-isomer with those of the purified pheromone blends (3.4 ng). The antagonistic effect of geometric isomers was shown in a blend ratio-dependent manner (Fig. 1). The addition of the geometric isomers at greater than 5% of the purified pheromone blends caused an antagonistic effect on the attractiveness of the pheromone. However, when 1, 3 or 5% geometric isomers were added, the attractiveness to male moths was not affected. 3.1.2. Effect of composition of synthetic pheromone components The major component, Z13-18:Ald, alone showed significant attractiveness, while the other aldehyde component, Z11-18:Ald, alone was not attractive (Fig. 2). The addition of Z11-18:Ald to Z13-18:Ald in the natural ratio significantly increased the activity of the major component. The addition of a mixture of the two alcohols to the two aldehydes in the natural ratio significantly increased the activity. The mixture of the two alcohols at their natural ratio was not attractive. 3.2. Field test 3.2.1. Effect of loaded amount of synthetic pheromone components The effect of loading of the pheromone components in the rubber septa was evaluated with varying doses of the synthetic 4-component blend in the naturally occurring ratio. In 1997, there were no significant 50
Total number of contacts
a
a
a ab
40
30
b
20
c
10
Total number of contacts
a 40
b 30
20
c 10
d
d
d 0
Solvent control
Z13-18:Ald
Z11-18:Ald
Z13-18:Ald + Z11-18:Ald
Z13-18:Ald + Z11-18:Ald + Z13-18:OH + Z11-18:OH
Z13-18:OH + Z11-18:OH
Fig. 2. Effect of composition of synthetic pheromone components on male responses in the laboratory. The number of contacts of 20 males with claspers extruded to the treated filter paper was recorded for 5 min. Each bioassay was replicated 4–10 times. Means with the same letter at each figure are not significantly different at Po0:05 by Tukey– Kramer’s test.
differences in attractiveness among doses ranging from 0.43 to 3.42 mg/septum (Table 1). Therefore, we conducted further field tests with lower, in addition to wider, range of doses from 0.01 to 1.71 mg. In 1998, we again found no significant differences in attractiveness among the above dosages. In 1999, however, 0.21 mg lures caught the greatest number of males. The attractiveness of 0.21 mg lures was not significantly different from that of the 0.86 mg lure, while it was significantly greater than those of the lower (0.01 and 0.06 mg) and the higher (1.71 mg) doses. 3.2.2. Effects of composition and blend ratio of synthetic pheromone components In 2000, a field test was conducted to examine the following two effects on male attraction; (1) the addition of the two alcohols to the two aldehydes (2-component blend vs. 4-component blend) and (2) ratio of the Z1118:Ald to Z13-18:Ald (2-component blends). No synergistic effect of the alcohols on trap catches was shown when they were added to the aldehydes in the naturallyoccurring ratio (Table 2). No significant effects were detected even when the amount of Z11-18:Ald was changed from one-third to three fold that in the natural ratio.
c 0
4-component blend
+ 1% E
+ 3% E
+ 5% E
+ 10% E
+ 50% E
Solvent control
Fig. 1. Effect of the addition of E-isomers to the purified pheromone on male responses in the laboratory. The number of contacts of 20 males with claspers extruded to the treated filter paper was recorded for 5 min. Each bioassay was replicated 4–10 times. Means with the same letter at each figure are not significantly different at Po0:05 by Tukey–Kramer’s test.
4. Discussion In contrast to the results in the previous field test (Kawazu et al., 2000), in the laboratory the major component, Z13-18: Ald, alone showed weak, but significant attractiveness (Fig. 2). Since neither
ARTICLE IN PRESS K. Kawazu et al. / Crop Protection 23 (2004) 589–593
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Table 1 Effect of loaded amount of synthetic pheromone components on trap catches of male C. medinalis in the field Test period
Total amounts loadeda (mg/septum)
No. males caught/ trap/nightb,c
Aug.14–Sep.12, 1997
0.43 0.86 1.71 3.42
0.3a 1.0a 0.4a 0.5a
Jul.16–Sep.13, 1998
0 0.01 0.06 0.21 0.86 1.71
0.1a 1.1b 1.3b 0.8b 1.1b 1.3b
Jul.19–Sep.15, 1999
0 0.01 0.06 0.21 0.86 1.71
0.3a 4.0b 3.7b 7.8c 5.3b,c 4.6b
a Z11-18:Ald, Z13-18:Ald, Z11-18:OH and Z13-18:OH (11:100:24:36). b Values followed by the same letters in each test are p not significantly ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi different at the 5% level by Tukey’s test using ðX þ 0:5Þ for transformed data. c Values obtained from two sticky traps in each treatment in the 1997 test, from three cone traps in each treatment in the 1998 and 1999 test.
Table 2 Effects of blend ratio and composition of synthetic pheromone components on trap catches of male C. medinalis in the field Component (mg/septum) Z11-18:Ald 0.02 0.06c 0.17 0.06c
Z13-18:Ald 0.5 0.5 0.5 0.5
No. males caught/ trap/nighta,b Z11-18:OH — — — 0.12
Z13-18:OH — — — 0.18
1.6a 2.4a 1.3a 2.1a
a
Values followed by the same letters are not significantly different at pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi the 5% level by Tukey’s test using ðX þ 0:5Þ for transformed data. b Values obtained from three cone traps in each treatment during August 7–September 28, 2000. c Blends at the natural ratio.
Z11-18:Ald alone nor the alcohols showed attractive activity, the major component, Z13-18:Ald, is an essential component for attraction. The addition of Z11-18:Ald to Z13-18:Ald at the natural ratio increased the activity of the major component. Therefore, Z1118:Ald was shown to be a synergist of Z13-18:Ald under the laboratory conditions. In the previous field test (2000), we could not observe a significant attractiveness for Z13-18:Ald alone.
In a previous study, the alcohols showed a weak synergistic effect in the field when added to the mixture of the aldehydes, although the effect was not significant (Kawazu et al., 2000). In the present study, no synergistic effect of the alcohols could be found in the field (Table 2). On the other hand, in the laboratory bioassay, the alcohols showed an apparent synergistic effect on the aldehydes (Fig. 2). These results suggest that the addition of the alcohols to the aldehydes has a synergistic effect under certain environmental conditions. There is a possibility that slight differences in pheromone blends may occur in bioassays performed under controlled laboratory conditions, but may not result in significant differences in trap catches in the field. If the closely related moth species that share the same habitat and the same flight season use common sex pheromone compositions, the alcohols may play an important role in reproductively isolating the sexual communication channel, as in the case of Mamestra configurata (Underhill et al., 1977; Struble et al., 1984). From these considerations, although the synergistic effect of the alcohols in the field is still ambiguous, the four components may be recommended for practical use in monitoring C. medinalis. In the laboratory bioassay, the addition of the geometric isomers to the pheromone blend showed a dose-dependent antagonistic effect on the sex pheromone activity (Fig. 1). In many moths, e.g. Maruca vitrata (Adati and Tatsuki, 1999), Eupoecilia ambiguella (Arn et al., 1986), Eucosma notanthes (Hung et al., 2001) and Endopiza viteana (Witzgall et al., 2000), addition of the geometric isomers above critical dosages causes a significant inhibitory effect on the attractiveness of the synthetic pheromones. When the synthetic pheromone of C. medinalis is used as an effective monitoring tool, compounds with >95% geometric purities are required. Among doses used ranging from 0.43 to 3.42 mg/ septum, there were no significant differences in attractiveness in 1997 (Table 1). In the 1998 test with a lower dosage series, again no significant differences in attractiveness were found among doses used ranging 0.01–1.71 mg (Table 1). However, in the 1999 test with the same dosage series as in 1998, significantly more male catches were obtained by the loading of 0.21 and 0.86 mg. Significant differences in the 1998 test may have been due to the low population density. Although the pheromone lures of a wide range of loadings showed attractiveness in the field, the result of the 1999 test suggested the presence of an optimum range of loading between 0.06 and 0.86 mg/septum. Rubber septa containing 0.86 mg of the four components (E-isomers o5%) at the natural ratio are now being used effectively for monitoring C. medinalis.
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Acknowledgements This research was partly supported by the Research Project on the Development of Integrated Pest Management Systems to Reduce Environmental Load, the Ministry of Agriculture, Forestry and Fisheries, Japan. We express our thanks to Shin-Etsu Chemical Company (Tokyo, Japan) for the gift of the synthetic pheromones and also to Sankei Chemical Company (Tokyo, Japan) for supplying the pheromone traps and rubber septa. This study was partly supported by Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 07306003, No. 13460021).
References Adati, T., Tatsuki, S., 1999. Identification of female sex pheromone of the legume pod borer, Maruca vitrata and antagonistic effects of geometrical isomers. J. Chem. Ecol. 25, 105–115. Arn, H., Rauscher, S., Buser, H., Guerin, P.M., 1986. Sex pheromone of Eupoecilia ambiguella female: analysis and male response to ternary blend. J.Chem. Ecol. 12, 1417–1429.
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Hung, C.C., Hwang, J.S., Hung, M.D., Yen, Y.P., Hou, R.F., 2001. Isolation, identification and field tests of the sex pheromone of the carambola fruit borer, Eucosma notanthes. J. Chem. Ecol. 27, 1855–1866. Kawasaki, K., Sugie, H., 1990. A simple sex pheromone cone trap. Jpn. J. Appl. Entomol. Zool. 34, 317–319 (in Japanese). Kawazu, K., Hasegawa, J., Honda, H., Ishikawa, Y., Wakamura, S., Sugie, H., Kamiwada, H., Kamimuro, T., Yoshiyasu, Y., Tatsuki, S., 2000. Geographical variation in female sex pheromones of the rice leaffolder moth, Cnaphalocrocis medinalis: identification of pheromone components in Japan. Entomol. Exp. Appl. 96, 103–109. Struble, D.L., Ayre, G.L., Byers, J.R., 1984. Minor sex-pheromone components of Mamestra configurata (Lepidoptera: Noctuidae) and improved blends for attraction of male moths. Can. Entomologist 116, 103–105. Underhill, E.W., Steck, W.F., Chisholm, M.D., 1977. A sex pheromone mixture for the bertha armyworm moth, Mamestra configurata: (Z)-9-tetradecen-1-ol acetate and (Z)-11-hexadecen-1ol acetate. Can. Entomol. 109, 1335–1340. Witzgall, P., Bengtsson, M., Trimble, R.M., 2000. Sex pheromone of grape berry moth (Lepidoptera: Tortricidae). Envron. Entomol 29, 433–436.
Crop Protection 23 (2004) 589–593
Effective pheromone lures for monitoring the rice leaffolder moth, Cnaphalocrocis medinalis (Lepidoptera: Crambidae) Kei Kawazua,*, Takeshi Kamimurob, Hidemi Kamiwadac, Kenji Nagatad, Tadashi Matsunagad, Hajime Sugiee, Takehiko Fukumotof, Taro# Adatig, Sadahiro Tatsukia a
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan b Kagoshima Prefectural Agricultural Experiment Station, Kagoshima 891-0116, Japan c Kagoshima Agricultural Improvement and Advisory Center, Kagoshima 892-0817, Japan d Sankei Chemical Company, Tokyo 110-0015, Japan e National Institute of Agro-Environmental Sciences, Ibaraki 305-8604, Japan f Shin-Etsu Chemical Company, Tokyo 100-0004, Japan g International Institute of Tropical Agriculture, Kano Station, PMB 3112, Kano Nigeria Received 28 July 2003; received in revised form 29 September 2003; accepted 14 November 2003
Abstract Several factors affecting the attractiveness of traps baited with synthetic sex pheromone components of Cnaphalocrocis medinalis were examined in the laboratory and field. In the laboratory, among the four pheromone components Z11-18:Ald, Z13-18:Ald, Z1118:OH and Z13-18:OH, the major component, Z13-18:Ald, was shown to be essential for attraction and Z11-18:Ald was a potent synergist for the major component. The alcohols also showed an apparent synergistic effect in the laboratory bioassay when their mixture was combined with an aldehyde mixture. The addition of the geometric isomers of the four components at levels greater than 5% of the purified 4-component blend showed significant inhibitory effects on the pheromonal activity in the laboratory. In contrast, no synergistic effect of the alcohols on the aldehydes was shown in the field. A range of loadings of the pheromone rubber septa (0.01–3.42 mg) was tested and all remained attractive for at least 1 month with optimum attractiveness of 0.86 mg/septum. No significant effects were obtained when the ratio of Z11-18:Ald to Z13-18:Ald was changed. Rubber septa containing 0.86 mg of the four components (E-isomers o5%) at the natural ratio are being effectively used for monitoring C. medinalis. r 2003 Elsevier Ltd. All rights reserved. Keywords: Rice leaffolder; Cnaphalocrocis medinalis; Synthetic pheromone
1. Introduction The rice leaffolder, Cnaphalocrocis medinalis, is a migratory rice pest in Japan. Moth occurrence in Japan is highly variable by year and by place. Thus, an effective monitoring system is especially important to investigate migration of C. medinalis. Although light traps have often been used for monitoring insect pests, they are of little use for C. medinalis which is not particularly sensitive to light. Therefore, an effective monitoring system is required and the application of synthetic sex pheromones may provide this.
*Corresponding author. E-mail address: [email protected] (K. Kawazu). 0261-2194/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2003.11.009
In a previous study, we identified the female sex pheromone of C. medinalis in Japan as a mixture of (Z)-11-octadecenal (Z11-18:Ald), (Z)-13-octadecenal (Z13-18:Ald), (Z)-11-octadecen-1-ol (Z11-18:OH) and (Z)-13-octadecen-1-ol (Z13-18:OH) in a ratio of 11:100:24:36 (Kawazu et al., 2000). In field trials using purified synthetic compounds (>99.9%) it was shown that the two aldehydes, Z11-18:Ald and Z13-18:Ald, were essential for male attraction and the two alcohols, Z11-18:OH and Z13-18:OH, showed a weak synergistic effect on the aldehydes (Kawazu et al., 2000). For developing monitoring tools using synthetic sex pheromone, an understanding of the effects of the geometric isomers, composition and blend ratios in the synthetic pheromone used and of the loading of the pheromone on the attractiveness to male moths is important.
ARTICLE IN PRESS 590
K. Kawazu et al. / Crop Protection 23 (2004) 589–593
The unpurified synthetic pheromone compounds contain small proportions (usually o10%) of their (E)isomers. If the presence of the geometric isomers in the pheromone blend at the above levels show no inhibitory effect on the attractiveness, purification of the synthetic compounds would be unnecessary. The effect of the geometric isomers on the attractiveness of purified synthetic compounds was therefore examined in the laboratory. Furthermore, the effect of pheromone composition and blend ratios was examined. In addition, the synergistic effect of the two alcohols on the aldehydes was re-examined, since it was unclear in the previous field test (Kawazu et al., 2000). To investigate the optimum dosage range for attraction, the effect of the loadings of synthetic pheromone components was examined in the field.
2. Materials and methods 2.1. Chemicals The synthetic pheromone components, Z11-18:Ald, Z13-18:Ald, Z11-18:OH and Z13-18:OH, were supplied by Shin-Etsu Chemical Company (Tokyo, Japan). Analyses by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) of the synthetic Z11-18:Ald, Z13-18:Ald, Z11-18:OH and Z13-18:OH showed that these compounds contained 1–2% of their (E)-isomers. They were thus purified by high-performance liquid chromatography (HPLC) with an HP-1050 system using a silver ion-coated Nucleosil 100-SA column (250 4 mm ID, 5 mm particle size, GL Science, Tokyo, Japan). The (E)-isomers used for the bioassay were obtained from the above separation of the synthetic pheromone compounds. The aldehydes components were further purified by HPLC on an Inertsil ODS-2 column (250 4 mm ID, 5 mm particle size, GL Science, Tokyo, Japan) to remove trace impurities (o1%). The solvent used for the both HPLC purifications was methanol at a flow rate of 0.45 ml/min. Purity of all compounds was determined to be greater than 99.9% by GC and GC–MS using a DB-23 column (Kawazu et al., 2000). 2.2. Laboratory bioassay The C. medinalis used for laboratory bioassay were from a stock culture originally collected in Osaka prefecture (Japan) in 1985. They were supplied by Sumitomo Chemical Company (Tokyo, Japan) and have been successively reared on an artificial diet (Silkmate 2(S), Nihon Nosan Kogyo, Yokohama, Japan) for over 100 generations at 2571 C under a long-day cycle (15L-9D). After emergence, female and male adults were kept separately under the same
conditions as above and provided with 5% sucrose solution as food. The moths after emergence were designated as age 0 during the scotophase in which they emerged. The subsequent ages were started at the time of each lights on. Laboratory bioassays were conducted with a screen cage (32 22 30 cm). Virgin males of 3–7 days after emergence were bioassayed at 4–8 h after lights off under the usual 15L:9D, 25 C. Twenty naive males were released in the cage. A filter paper strip (2 4 cm) impregnated with 2 ml hexane solution containing the test blend of synthetic pheromone was suspended in the cage from the center of the top screen 5 cm under the top. The number of contacts of the males with claspers extruded to the filter paper strip was recorded for 5 min, not counting accidental contacts. The males attracted by the pheromone showed zigzag flight when approaching within 10 cm of the pheromone source. A dimmed red lamp for visual observation was used during the dark period. Each bioassay was replicated 4–10 times. For statistical analyses, the differences among the mean numbers of contacts of the males were tested for significance by Tukey-Kramer’s test.
2.3. Field bioassays Baits were prepared using gray halo-butyl isoprene blend elastomer septa (West Co., Singapore). The required amounts and ratios of the synthetic compounds each dissolved in 200 ml hexane were applied onto the inner surface of rubber septa. After evaporation of the solvent, the baits were placed in sealed aluminumlaminated polyethylene bags, and stored at 20 C until use. The pheromone lures were renewed everymonth in the field. All bioassays were conducted in paddy fields in Kagoshima City, Kagoshima prefecture during the emergence season of the moth. Sticky traps (SE-Traps, Sankei Chemical Company, Kagoshima, Japan), with a triangle section, dimensions of 29 32 8 cm height, and a 24 30 cm sticky plate, were used in 1997. Cone traps slightly modified from those reported by Kawasaki and Sugie (1990) were used in 1998, 1999 and 2000. Modifications were made to the length (height) of the cone traps which were 2 cm longer than those reported by Kawasaki and Sugie (1990). Pheromone traps were set at the boundary of the paddy fields and at the height of the top of rice plants. The distance between traps was greater than 10 m. After the numbers of trapped male C. medinalis were checked every morning, the traps were moved round one position to reduce any bias due to trap location. For statistical analyses, the number of trapped pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi males at each trap was transformed to ðX þ 0:5Þ and the differences among the means of caught moths were tested for significance by Tukey’s test after analysis of variance.
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3. Results
591
50
3.1. Laboratory bioassay 3.1.1. Effect of the addition of E-isomers to the purified pheromone To examine the antagonistic effects of the E-isomers of the pheromone components, we compared the activities of synthetic blends containing 1, 3, 5, 10 or 50% of each E-isomer with those of the purified pheromone blends (3.4 ng). The antagonistic effect of geometric isomers was shown in a blend ratio-dependent manner (Fig. 1). The addition of the geometric isomers at greater than 5% of the purified pheromone blends caused an antagonistic effect on the attractiveness of the pheromone. However, when 1, 3 or 5% geometric isomers were added, the attractiveness to male moths was not affected. 3.1.2. Effect of composition of synthetic pheromone components The major component, Z13-18:Ald, alone showed significant attractiveness, while the other aldehyde component, Z11-18:Ald, alone was not attractive (Fig. 2). The addition of Z11-18:Ald to Z13-18:Ald in the natural ratio significantly increased the activity of the major component. The addition of a mixture of the two alcohols to the two aldehydes in the natural ratio significantly increased the activity. The mixture of the two alcohols at their natural ratio was not attractive. 3.2. Field test 3.2.1. Effect of loaded amount of synthetic pheromone components The effect of loading of the pheromone components in the rubber septa was evaluated with varying doses of the synthetic 4-component blend in the naturally occurring ratio. In 1997, there were no significant 50
Total number of contacts
a
a
a ab
40
30
b
20
c
10
Total number of contacts
a 40
b 30
20
c 10
d
d
d 0
Solvent control
Z13-18:Ald
Z11-18:Ald
Z13-18:Ald + Z11-18:Ald
Z13-18:Ald + Z11-18:Ald + Z13-18:OH + Z11-18:OH
Z13-18:OH + Z11-18:OH
Fig. 2. Effect of composition of synthetic pheromone components on male responses in the laboratory. The number of contacts of 20 males with claspers extruded to the treated filter paper was recorded for 5 min. Each bioassay was replicated 4–10 times. Means with the same letter at each figure are not significantly different at Po0:05 by Tukey– Kramer’s test.
differences in attractiveness among doses ranging from 0.43 to 3.42 mg/septum (Table 1). Therefore, we conducted further field tests with lower, in addition to wider, range of doses from 0.01 to 1.71 mg. In 1998, we again found no significant differences in attractiveness among the above dosages. In 1999, however, 0.21 mg lures caught the greatest number of males. The attractiveness of 0.21 mg lures was not significantly different from that of the 0.86 mg lure, while it was significantly greater than those of the lower (0.01 and 0.06 mg) and the higher (1.71 mg) doses. 3.2.2. Effects of composition and blend ratio of synthetic pheromone components In 2000, a field test was conducted to examine the following two effects on male attraction; (1) the addition of the two alcohols to the two aldehydes (2-component blend vs. 4-component blend) and (2) ratio of the Z1118:Ald to Z13-18:Ald (2-component blends). No synergistic effect of the alcohols on trap catches was shown when they were added to the aldehydes in the naturallyoccurring ratio (Table 2). No significant effects were detected even when the amount of Z11-18:Ald was changed from one-third to three fold that in the natural ratio.
c 0
4-component blend
+ 1% E
+ 3% E
+ 5% E
+ 10% E
+ 50% E
Solvent control
Fig. 1. Effect of the addition of E-isomers to the purified pheromone on male responses in the laboratory. The number of contacts of 20 males with claspers extruded to the treated filter paper was recorded for 5 min. Each bioassay was replicated 4–10 times. Means with the same letter at each figure are not significantly different at Po0:05 by Tukey–Kramer’s test.
4. Discussion In contrast to the results in the previous field test (Kawazu et al., 2000), in the laboratory the major component, Z13-18: Ald, alone showed weak, but significant attractiveness (Fig. 2). Since neither
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Table 1 Effect of loaded amount of synthetic pheromone components on trap catches of male C. medinalis in the field Test period
Total amounts loadeda (mg/septum)
No. males caught/ trap/nightb,c
Aug.14–Sep.12, 1997
0.43 0.86 1.71 3.42
0.3a 1.0a 0.4a 0.5a
Jul.16–Sep.13, 1998
0 0.01 0.06 0.21 0.86 1.71
0.1a 1.1b 1.3b 0.8b 1.1b 1.3b
Jul.19–Sep.15, 1999
0 0.01 0.06 0.21 0.86 1.71
0.3a 4.0b 3.7b 7.8c 5.3b,c 4.6b
a Z11-18:Ald, Z13-18:Ald, Z11-18:OH and Z13-18:OH (11:100:24:36). b Values followed by the same letters in each test are p not significantly ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi different at the 5% level by Tukey’s test using ðX þ 0:5Þ for transformed data. c Values obtained from two sticky traps in each treatment in the 1997 test, from three cone traps in each treatment in the 1998 and 1999 test.
Table 2 Effects of blend ratio and composition of synthetic pheromone components on trap catches of male C. medinalis in the field Component (mg/septum) Z11-18:Ald 0.02 0.06c 0.17 0.06c
Z13-18:Ald 0.5 0.5 0.5 0.5
No. males caught/ trap/nighta,b Z11-18:OH — — — 0.12
Z13-18:OH — — — 0.18
1.6a 2.4a 1.3a 2.1a
a
Values followed by the same letters are not significantly different at pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi the 5% level by Tukey’s test using ðX þ 0:5Þ for transformed data. b Values obtained from three cone traps in each treatment during August 7–September 28, 2000. c Blends at the natural ratio.
Z11-18:Ald alone nor the alcohols showed attractive activity, the major component, Z13-18:Ald, is an essential component for attraction. The addition of Z11-18:Ald to Z13-18:Ald at the natural ratio increased the activity of the major component. Therefore, Z1118:Ald was shown to be a synergist of Z13-18:Ald under the laboratory conditions. In the previous field test (2000), we could not observe a significant attractiveness for Z13-18:Ald alone.
In a previous study, the alcohols showed a weak synergistic effect in the field when added to the mixture of the aldehydes, although the effect was not significant (Kawazu et al., 2000). In the present study, no synergistic effect of the alcohols could be found in the field (Table 2). On the other hand, in the laboratory bioassay, the alcohols showed an apparent synergistic effect on the aldehydes (Fig. 2). These results suggest that the addition of the alcohols to the aldehydes has a synergistic effect under certain environmental conditions. There is a possibility that slight differences in pheromone blends may occur in bioassays performed under controlled laboratory conditions, but may not result in significant differences in trap catches in the field. If the closely related moth species that share the same habitat and the same flight season use common sex pheromone compositions, the alcohols may play an important role in reproductively isolating the sexual communication channel, as in the case of Mamestra configurata (Underhill et al., 1977; Struble et al., 1984). From these considerations, although the synergistic effect of the alcohols in the field is still ambiguous, the four components may be recommended for practical use in monitoring C. medinalis. In the laboratory bioassay, the addition of the geometric isomers to the pheromone blend showed a dose-dependent antagonistic effect on the sex pheromone activity (Fig. 1). In many moths, e.g. Maruca vitrata (Adati and Tatsuki, 1999), Eupoecilia ambiguella (Arn et al., 1986), Eucosma notanthes (Hung et al., 2001) and Endopiza viteana (Witzgall et al., 2000), addition of the geometric isomers above critical dosages causes a significant inhibitory effect on the attractiveness of the synthetic pheromones. When the synthetic pheromone of C. medinalis is used as an effective monitoring tool, compounds with >95% geometric purities are required. Among doses used ranging from 0.43 to 3.42 mg/ septum, there were no significant differences in attractiveness in 1997 (Table 1). In the 1998 test with a lower dosage series, again no significant differences in attractiveness were found among doses used ranging 0.01–1.71 mg (Table 1). However, in the 1999 test with the same dosage series as in 1998, significantly more male catches were obtained by the loading of 0.21 and 0.86 mg. Significant differences in the 1998 test may have been due to the low population density. Although the pheromone lures of a wide range of loadings showed attractiveness in the field, the result of the 1999 test suggested the presence of an optimum range of loading between 0.06 and 0.86 mg/septum. Rubber septa containing 0.86 mg of the four components (E-isomers o5%) at the natural ratio are now being used effectively for monitoring C. medinalis.
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Acknowledgements This research was partly supported by the Research Project on the Development of Integrated Pest Management Systems to Reduce Environmental Load, the Ministry of Agriculture, Forestry and Fisheries, Japan. We express our thanks to Shin-Etsu Chemical Company (Tokyo, Japan) for the gift of the synthetic pheromones and also to Sankei Chemical Company (Tokyo, Japan) for supplying the pheromone traps and rubber septa. This study was partly supported by Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 07306003, No. 13460021).
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