Journal of Chemical Ecology, Vol. 13, No. 2, 1987
PLANT SECONDARY COMPOUNDS AS OVIPOSITION DETERRENTS FOR CABBAGE BUTTERFLY, Pieris rapae (LEPIDOPTERA: PIERIDAE)
BRUCE E. T A B A S H N I K Department of Entomology, University of Hawaii Honolulu, Hawaii 96822
(Received November 12, 1985; accepted February 26, 1986) Abstract--Oviposition by Pieris rapae butterflies was deterred by spraying the plant secondary compounds coumarin and turin on cabbage plants in greenhouse choice tests. In no-choice tests ranging from 5 min to 24 hr, acceptance of rutin-treated plants for oviposition increased with trial duration. Both coumarin and rutin deterred oviposition primarily by affecting prealighting rather than postalighting behavior, indicating that deterrence was mediated by noncontact cues. Key Words--Oviposition deterrence, oviposition behavior, Pieris rapae, cabbage butterfly, Lepidoptera, Pieridae, coumarin, rutin, plant secondary compounds, insect-plant interactions, cabbage, Cruciferae. INTRODUCTION For insects such as Lepidoptera, host selection by mobile adults is crucial in determining the fitness of their less mobile offspring (Scriber, 1984; Tabashnik and Slansky, 1987). Egg-laying females identify suitable plants by chemical and physical stimuli that characterize such plants (Miller and Strickler, 1984). Plant secondary compounds (allelochemicals) typical of a given plant may be oviposition stimulants for insects which feed on that plant (e.g., Gupta and Thorsteinson, 1960; Rodman and Chew, 1980; Renwick and Radke, 1983). In contrast, oviposition by a particular insect may be deterred by compounds that occur at high levels in unsuitable plants, but not in suitable plants (Tingle and Mitchell, 1984; Mitchell and Heath, 1985). Plants suitable for a particular insect may also contain compounds that deter oviposition by that insect (Renwick and Radke 1981, 1985). Oviposition deterrence by plant compounds could have significant consequences for management of crop pests. 309 0o98-0331/87/0260-0309505.0o/09 1987PlenumPublishingCorporation
310
TABASHNIK
Previous work showed that chemical extracts from noncruficerous plants deter oviposition by the crucifer-feeding cabbage butterfly, Pieris rapae L. (Lundgren, 1975; Renwick and Radke, 1985). However, relatively little is known about the effects of individual compounds as oviposition deterrents. The plant secondary compounds coumarin and rutin are potential oviposition deterrents for P. rapae because they do not occur in crucifers at substantial levels, but they are present at high concentrations in many noncruciferous herbs. Coumarin occurs in members of several plant families (e.g., Compositae, Lauraceae, Leguminosae, Umbelliferae), in some cases as the major aromatic constituent (Leung, 1980). Rutin is a nonvolatile flavonoid pigment found in numerous plant families, with concentrations of up to 24% of leaf dry weight recorded for some species (Leung, 1980). Both coumarin and rutin deterred oviposition by the crucifer pest, Plutella xylostella L. (Tabashnik, 1985). The study reported here examined how P. rapae oviposition is affected by application of coumarin and rutin to cabbage plants. The effects of the compounds on oviposition, prealighting behavior, and postalighting behavior were investigated using choice and no-choice behavioral assays. METHODS AND MATERIALS Insects. Adults were obtained from a laboratory colony 5-8 generations removed from field populations at the Pearl City Instructional Facility community garden, Oahu, Hawaii. Crucifers available for oviposition at the Pearl City field site include broccoli, cabbage, and watercress; larvae were fed broccoli in the laboratory colony (Haji-Mamat, 1984). Plants. Cabbage plants were grown from seed ("C-G Cross," Takii and Co., Ltd.) in the greenhouse in plastic pots (9.5 • 10 x 10 cm) filled with vermiculite. Seedlings were fertilized with dilute Ortho-Gro | Liquid Plant Food 12-6-6 (1 ml : 200 ml H20) within one week after planting. Plants used in behavioral assays were treated with either a control solution of 0.5 % Tween 80 (polyoxyethylene sorbitan monooleate, Sigma) in water or a solution of 0.5% Tween 80 in water plus the test material. Solutions tested were coumarin (Kodak) and rutin (Sigma). Solutions were thoroughly shaken in a 235-ml polyethylene jar, then sprayed on upper and lower plant surfaces for about 3 sec using aerosol propellant (Sigma spray kit). The amount of solution applied to leaves was estimated by weighing 10 leaves immediately before and after spraying. The estimated mean volume of solution sprayed on leaves was 0.294 ml (+0.022 SE)/g leaf fresh weight, indicating that leaves sprayed with coumarin solutions of 0.1 M and 0.01 M had approximately 4.3 mg and 0.43 mg coumarin/g leaf fresh weight, respectively. Estimated values for applied rutin concentration were 19.6 and 1.96 mg/g leaf fresh weight, for 0.1 M and 0.01 M solutions, respectively.
DETERRENCE OF CABBAGE BUTTERFLY OVIPOSITION
311
Behavioral Assays. All behavioral assays were conducted between February and June 1984 in the Entomology Department greenhouse at the University of Hawaii Manoa Campus. Daily maximum temperatures were 29 _+ 2~ and daily minimums were 22 _+ 2~ the natural photoperiod (ca. 12 hr light) was used. Groups of females (3-10) with approximately equal numbers of males were tested in screen cages (50 cm on each side) in all experiments. Cotton dental wicks immersed in dilute honey-water (1 m l : 5 ml) solutions in open vials were suspended from the cage tops to provide food for adults. Within the constraints imposed by confinement, behavior of females in the assays was similar to their field behavior (Chew 1977); females laid their eggs singly and usually flew off the plant after oviposition. Choice Tests. One plant treated with control solution and another treated with either a coumarin or rutin solution were placed in diagonally opposite comers of each cage. Trials lasted 2-3 days. Eggs were counted after each trial, and plant positions in the cage were altemated in successive trials. No-Choice Tests. Females were offered either two control plants or two treated plants (either coumarin or rutin at 0.1 M). Plants were placed in diagonally opposite comers of the cage. In the 5-min no-choice tests, each group of females was tested for 10 trials, consisting of an alternating sequence of control and treated plants offered for 5-min periods, with less than 1 min between trials. For no-choice tests lasting 15 min, 60 rain, and 24 hr, groups of females were paired so that one group was offered two control plants while, at the same time, a matched group of equal numbers and age was offered treated plants. In the next trial, the plants were switched so that the first group received treated plants while the second group received controls. Pre- and Postalighting Behavior. Females were observed during behavioral assays to determine how coumarin and rutin influenced their pre- and postalighting behavior. "Contacts" were recorded when females touched a plant with one or more legs. Analysis. G tests for goodness of fit, adjusted for sample size as necessary (Sokal and Rohlf, 1969), were used throughout. The statistical significance of deterrence was determined by contrasting observed frequencies of behaviors (ovipositions or contacts) on control vs. test plants with the 1 : 1 frequency of behaviors expected if butterflies did not differentiate between treatments. Ratios of behaviors (eggs laid/contact) on control vs. test plants were contrasted by testing for independence in 2 • 2 contingency tables (Sokal and Rohlf, 1969). The 2 • 2 contingency analysis was also used to compare frequencies of eggs on control vs. plants treated with mtin (0.1 M) in no-choice tests of various duration. Means (_+ SE) for percentages of eggs on treated plants were calculated to provide a rough index of variation among replicates. Statistical inferences from ANOVA on the percentages were generally consistent with the G tests.
312
TABASHNIK RESULTS
Choice Tests. Both coumarin and rutin deterred oviposition by P. rapae (Table 1). At both concentrations tested, coumarin was a weak deterrent. Rutin at 0.1 M caused a fivefold reduction in oviposition, but 0.01 M rutin did not deter oviposition. No-Choice Tests. Responses to cabbage plants treated with 0.1 M rutin in no-choice tests depended on the trial duration, with observed deterrence inversely related to trial duration (Table 2). Deterrence was strongest in the 5rain no-choice test, intermediate in 15- and 60-min no-choice tests, and weakest in the 24-hr no-choice test. Pre- and Postalighting Behavior. Coumarin and rutin deterred oviposition primarily by affecting prealighting rather than postalighting behavior. Direct behavioral observations showed that both compounds significantly reduced the number of contacts with plants (Table 3). Analysis of the ratio of eggs laid/ contact showed that after contacting a plant, females were somewhat less likely TABLE l. EFFECTS OF COUMARIN AND RUTIN ON OVIPOSITION BY Pieris rapae ~N CHOICE TESTS
Total eggs laid Treatment Coumarin Coumarin Rutin Rutin
0.1 0.01 0.1 0.01
M M M M
Trials
Control
Treated
7 5 6 6
1155 918 1928 949
605" 668" 375" 886
% of eggs on treated (-~ +_ SE) 40.9 42.5 18.5 44.8
+ 5.2 _+ 2.5 + 4.1 +_ 6. l
ap < 0.001 by G tests on pooled data.
TABLE 2. EFFECT OF RUTIN (0.1 M) ON OVlPOSlTION BY Pieris rapae IN N o - C H o I c E TESTS OF VARIOUS DURATIONS
Total eggs laid '* Trial duration
Trials
Control
Treated
5 min 15 min 60 min 24 hr
10 4 4 16
27 46 116 924
0a 19 b 48 b 752 c
% of eggs on treated (X + SE) 0.0 28.4 29.0 38.7
+ 0.0 _+ 2.1 _+ 3.6 + 7.7
aRatios of eggs (control:treated) followed by different letters are significantly different from each other by analysis of 2 • 2 contingency tables (P < 0.05).
DETERRENCE OF CABBAGEBUTTERFLYOVIPOSITION
313
TABLE 3. EFFECTSOF COUMARINAND RUTIN (0.1 M) ON PRE- AND POSTALIOr~TING BEHAVIOROF Pieris rapae Contacts
Eggs/contact
Assay
Treatment
Trials
Control
Treated
Control
Treated
Choice Choice No-choicea No-choice~ No-choiceb
Coumafin Rutin Coumafin Rutin Rutin
10 .10 10 10 4
75 25 42 76 142
35c 4c 14c 14c 61c
0.21 0.76 0.31 0.36 0.41
0.14 0.51 0.00 a 0.00 d 0.39
a5 rain. %0 min. cp < 0.001 by G tests on pooled data. d p < 0.05 by analysis of 2 x 2 contingency tables. tO oviposit on treated plants than on control plants (Table 3). However, the effects o f coumarin and mtin on postalighting behavior were small compared to the prealighting effects.
DISCUSSION The results o f this study support the view that host acceptance by insect herbivores is mediated by positive and negative stimuli from plants and by internal levels o f satiation (Dethier, 1982). Both coumarin and mtin deterred oviposition by P. rapae, yet females laid some eggs on treated plants in nearly all experiments. These results suggest that the cabbage plants provided strong positive stimuli, whereas the applied compounds were negative stimuli that reduced the acceptability o f treated cabbage plants significantly, but not completely. In no-choice tests, acceptance o f rutin-treated plants relative to control plants increased with trial duration. It is unlikely that the reduced deterrence o f rutin seen in prolonged no-choice tests was due to rutin concentration declining through time. Rutin's deterrence remained effective in choice tests lasting 2 - 3 days (Table 1) and plants treated with rutin 24 hr before testing did not show reduced deterrence compared to plants treated with rutin immediately before testing. Another possibility is that the control plants became less acceptable as the number o f eggs per control plant increased in no-choice tests o f increasing duration. Egg-load assessment is reported to ocur for some P. rapae populations (Rothschild and Schoonhoven, 1977) but not others (Ives, 1978; Traynier, 1979; Shapiro, 1981). Results from a no-choice test in the present study show that the rate o f oviposition on a clean control plant (9.3 eggs laid/female/hr) was not greater than the rate o f oviposition on a control plant with 56 eggs (10.0 eggs
314
TABASHNIK
laid/female/hr), suggesting that acceptance of control plants in no-choice tests did not decline due to egg-load assessment. It seems that increased acceptance of rutin-treated plants occurred because females were deprived of the opportunity to oviposit on untreated plants. This type of behavior is analogous to increased acceptance of nonpreferred host-plant species when preferred species are unavailable (e.g., Singer, 1982, 1983; Tabashnik, 1983a, b; Tabashnik et al., 1985). Time available for oviposition may be a limiting factor for butterflies like P. rapae, which lay their eggs singly (Tabashnik 1983a). Thus, single egglayers may accept nonpreferred plants for oviposition more readily than cluster egg-layers. The results show that during 5-min trials, P. rapae consistently rejected treated plants, while consistently accepting untreated plants (Table 2)~ During trials of 15 min or longer, however, females accepted treated plants, indicating that the "discrimination phase" (Singer, 1982) for P. rapae in these experiments was between 5 and 15 min. These results are consistent with earlier findings (Tabashnik, 1983a) supporting the hypothesis that single egg-laying butterflies have relatively short discrimination phases. Analysis of pre- and postalighting behavior revealed that the compounds reduced oviposition primarily by lowering the rate at which females contacted plants, indicating that deterrence was mediated by noncontact cues. These observations conflict with the notion that host-plant chemistry is most important in determining postalighting rather than prealighting behavior. However, secondary compounds were applied topically in this study, and thus their impact on behavior may not mimic the effects of compounds incorporated in leaves. The volatile compound, coumarin, probably deterred oviposition by alteration of olfactory cues. The noncontact effects of the nonvolatile pigment, rutin, were probably mediated by vision. The results suggest that plants producing a secondary compound typical of nonhosts might gain some protection from specialist herbivores by reducing their acceptance to ovipositing females. If the observed responses to coumarin and rutin by P. rapae and Plutella xylostella (Tabashnik, 1985) are indicative of general trends, then this protection would only be partial--even when a compound was produced in relatively high quantities. If the compound reduced the plant's suitability, selection would be expected to increase discrimination against it. Alternatively, if the compound had a neutral or positive effect on suitability, selection would act to reduce the deterrent effect of the compound. The results imply that nonhost-plant secondary compounds might confer partial crop protection by deterring oviposition. Due to increasing acceptance of treated plants when untreated plants are absent, treatments would be most effective when some plants were left untreated as a "trap crop,,' Application of plant secondary compounds to protect crops poses many of the same problems and hazards as do conventional pesticides, yet this technique offers potential as a tool to be integrated with other pest control measures (Schoonhoven,
DETERRENCE OF CABBAGEBUTTERFLY OVIPOSITION
315
1982). Attempts to deter oviposition by mobile adults may be more fruitful than tactics aimed at reducing consumption by immatures. Acknowledgments--I am especially grateful to J.A.A. Renwick for stimulating my interest in deterrents and for sharing his insights. I thank R.F. Chapman, F. Gould, M.O. Harris, M.W. Johnson, W.J. Mattson, J.R. Miller, L.M. Schoonhoven, M.C. Singer, and C. Wiklund for their comments and encouragement. H.B. Haji-Mamat shared knowledge and butterflies, F. Chang gave excellent technical advice, and S. Arnold provided expert laboratory assistance. Special thanks to R.H. Krakauer for unfaltering moral support. Financial support was provided by a grant from the Research and Training Fund, University of Hawaii and USDA-HAW00947H. This is paper no. 2997 of the Hawaii Inst. of Trop. Ag. and Human Res. Journal Series.
REFERENCES CHEW, F.S. 1977. Coevolution of Pierid butterflies and their cmciferous food plants. II. The distribution of eggs on potential food plants. Evolution 31:568-579. DETHIER, V.G. 1982. Mechanisms of host-plant recognition. Entomol. Exp. AppL 31:49-56. GUPTA,P.D., and THORSTEINSON, A.G. 1960. Food plant relationships of the diamond-back moth Plutella maculipennis (Curt.) II. Sensory regulation of oviposition by the adult female. Entomol. Exp. Appl. 3:305-314. HAII-MA~AT, H.B. 1984. Some pathological manifestations in adult Artogeia rapae (L.) (Lepidoptera: Pieridae) developing from larvae infected with Nosema sp. MS thesis. University of Hawaii. IrEs, P.M. 1978. How discriminating are cabbage butterflies? Aust. J. Ecol. 3:261-276. LEUNG, A.Y. 1980. Encyclopedia of Common Natural Ingredients Used in Food, Drugs, and Cosmetics. John Wiley & Sons, New York. LUNDGREN,L. 1975. Natural plant chemicals acting as oviposition deterrents on cabbage butterflies [Pieris brassicae (L.), P. rapae (L.), P. napi (L.)]. Zool. Ser. 4: 253-258. M1LLER, J.R., and STR~C~:LER,K.L. 1984. Finding and accepting host plants, pp. 127-157, in W.J. Bell and R.T. Card6 (eds.). Chemical Ecology of Insects. Sinauer Associates, Sunderland, Massachusetts. M~TCHELL, E.R., and HEATH, R.R. 1985. Influence of Amaranthus hybridus L. allelochemics on oviposition behavior of Spodoptera exigua and S. eridania (Lepidoptera: Noctuidae). J. Chem. Ecol. 11 :609-617. RENW~CK,J.A.A., and RADKE,C.D. 1981. Host plant constituents as oviposition deterrents for the cabbage looper, Trichoplusia ni (Lepidoptera: Noctuidae). Entomol. Exp. Appl. 30:201-204. RENWICK, J.A.A., and RADKE, C.D. 1983. Chemical recognition of host plants for oviposition by the cabbage butterfly, Pieris rapae (Lepidoptera: Pieridae). Environ. Entomol. 12:446-450. RENWICK, J.A.A., and RADKE, C.D. 1985. Constituents of host and nonhost plants deterring oviposition by the cabbage butterfly, Pieris rapae. Entomol. Exp. Appl. 39:21-26. RODMAN, J.E., and CHEW, F.S. 1980. Phytochemical correlates of herbivory in a community of native and naturalized Cruciferae. Biochem. Syst. Ecol. 8:43-50. ROTHSCHILD, M., and SCHOONHOVEN,L.M. 1977. Assessment of egg load by Pieris brassicae (Lepidoptera: Pieridae). Nature 266:352-355. SCHOONHOVEN, L.M. 1982. Biological aspects of antifeedants. Entomol. Exp. Appl. 31:57-69. SCRmER, J.M. 1984. Host-plant suitability, pp. 159-202, in W.J. Bell and R.T. Card6 (eds.). Chemical Ecology of Insects. Sinauer Associates, Sunderland, Massachusetts. SHAPIRO, A.M. 1981. The pierid red-egg syndrome. Am. Nat. 117:276-294. SINGER, M.C. 1982. Quantification of host specificity by manipulation of oviposition behavior in the butterfly Euphydryas editha. Oecologia (Berlin) 52:224-229.
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TABASHNIK
SINGER, M.C. 1983. Determinants of multiple host use by a phytophagous insect population. Evolution 25:383-389. SOKAL, R.R., and ROHLE, F.I. 1969. Biometry. Freeman, San Francisco. TABASHNIK, B.E. 1983a. Oviposition specificity in single vs. cluster egg-laying butterflies: A discrimination phase in Colias eurytheme ? Oecologia (Berlin) 58:278-279. TABASHNIK, B.E. 1983b. Host range evolution: The shift from native legume hosts to alfalfa by the butterfly, Colias philodice eriphyle. Evolution 37:150-162. TABASHNIK,B.E. 1985. Deterrence of diamondback moth (Lepidoptera: Plutellidae) oviposition by plant compounds. Environ. Entomol. 14:575-578. TABASHNIK,B.E., and SLANSKY,F., JR. 1987. Nutritional ecology of forb fbliage-chewing insects, in F. Slansky, Jr., and J.G. Rodriguez (eds.). Nutritional Ecology of Insects, Mites, and Spiders. John Wiley & Sons, New York. In press. TABASHr~IK,B.E., MATTSON, W.J., and MILLER, J.R. 1985. Host acceptance behavior of the red pine cone beetle ( Conophthorus resinosae). Entomol. Exp. Appl. 37:3-7. TINGLE, F.C., and MITCHELL, E.R. 1984. Aqueous extracts from indigenous plants as oviposition deterrents for Heliothis vireseens (F.) J. Chem. Ecol. 10:101-113. TRAYNIER, R.M.M. 1979. Long-term changes in the oviposition behavior of the cabbage butterfly, Pieris rapae, induced by contact with plants. Physiol. Entomol. 4:87-96.
PLANT SECONDARY COMPOUNDS AS OVIPOSITION DETERRENTS FOR CABBAGE BUTTERFLY, Pieris rapae (LEPIDOPTERA: PIERIDAE)
BRUCE E. T A B A S H N I K Department of Entomology, University of Hawaii Honolulu, Hawaii 96822
(Received November 12, 1985; accepted February 26, 1986) Abstract--Oviposition by Pieris rapae butterflies was deterred by spraying the plant secondary compounds coumarin and turin on cabbage plants in greenhouse choice tests. In no-choice tests ranging from 5 min to 24 hr, acceptance of rutin-treated plants for oviposition increased with trial duration. Both coumarin and rutin deterred oviposition primarily by affecting prealighting rather than postalighting behavior, indicating that deterrence was mediated by noncontact cues. Key Words--Oviposition deterrence, oviposition behavior, Pieris rapae, cabbage butterfly, Lepidoptera, Pieridae, coumarin, rutin, plant secondary compounds, insect-plant interactions, cabbage, Cruciferae. INTRODUCTION For insects such as Lepidoptera, host selection by mobile adults is crucial in determining the fitness of their less mobile offspring (Scriber, 1984; Tabashnik and Slansky, 1987). Egg-laying females identify suitable plants by chemical and physical stimuli that characterize such plants (Miller and Strickler, 1984). Plant secondary compounds (allelochemicals) typical of a given plant may be oviposition stimulants for insects which feed on that plant (e.g., Gupta and Thorsteinson, 1960; Rodman and Chew, 1980; Renwick and Radke, 1983). In contrast, oviposition by a particular insect may be deterred by compounds that occur at high levels in unsuitable plants, but not in suitable plants (Tingle and Mitchell, 1984; Mitchell and Heath, 1985). Plants suitable for a particular insect may also contain compounds that deter oviposition by that insect (Renwick and Radke 1981, 1985). Oviposition deterrence by plant compounds could have significant consequences for management of crop pests. 309 0o98-0331/87/0260-0309505.0o/09 1987PlenumPublishingCorporation
310
TABASHNIK
Previous work showed that chemical extracts from noncruficerous plants deter oviposition by the crucifer-feeding cabbage butterfly, Pieris rapae L. (Lundgren, 1975; Renwick and Radke, 1985). However, relatively little is known about the effects of individual compounds as oviposition deterrents. The plant secondary compounds coumarin and rutin are potential oviposition deterrents for P. rapae because they do not occur in crucifers at substantial levels, but they are present at high concentrations in many noncruciferous herbs. Coumarin occurs in members of several plant families (e.g., Compositae, Lauraceae, Leguminosae, Umbelliferae), in some cases as the major aromatic constituent (Leung, 1980). Rutin is a nonvolatile flavonoid pigment found in numerous plant families, with concentrations of up to 24% of leaf dry weight recorded for some species (Leung, 1980). Both coumarin and rutin deterred oviposition by the crucifer pest, Plutella xylostella L. (Tabashnik, 1985). The study reported here examined how P. rapae oviposition is affected by application of coumarin and rutin to cabbage plants. The effects of the compounds on oviposition, prealighting behavior, and postalighting behavior were investigated using choice and no-choice behavioral assays. METHODS AND MATERIALS Insects. Adults were obtained from a laboratory colony 5-8 generations removed from field populations at the Pearl City Instructional Facility community garden, Oahu, Hawaii. Crucifers available for oviposition at the Pearl City field site include broccoli, cabbage, and watercress; larvae were fed broccoli in the laboratory colony (Haji-Mamat, 1984). Plants. Cabbage plants were grown from seed ("C-G Cross," Takii and Co., Ltd.) in the greenhouse in plastic pots (9.5 • 10 x 10 cm) filled with vermiculite. Seedlings were fertilized with dilute Ortho-Gro | Liquid Plant Food 12-6-6 (1 ml : 200 ml H20) within one week after planting. Plants used in behavioral assays were treated with either a control solution of 0.5 % Tween 80 (polyoxyethylene sorbitan monooleate, Sigma) in water or a solution of 0.5% Tween 80 in water plus the test material. Solutions tested were coumarin (Kodak) and rutin (Sigma). Solutions were thoroughly shaken in a 235-ml polyethylene jar, then sprayed on upper and lower plant surfaces for about 3 sec using aerosol propellant (Sigma spray kit). The amount of solution applied to leaves was estimated by weighing 10 leaves immediately before and after spraying. The estimated mean volume of solution sprayed on leaves was 0.294 ml (+0.022 SE)/g leaf fresh weight, indicating that leaves sprayed with coumarin solutions of 0.1 M and 0.01 M had approximately 4.3 mg and 0.43 mg coumarin/g leaf fresh weight, respectively. Estimated values for applied rutin concentration were 19.6 and 1.96 mg/g leaf fresh weight, for 0.1 M and 0.01 M solutions, respectively.
DETERRENCE OF CABBAGE BUTTERFLY OVIPOSITION
311
Behavioral Assays. All behavioral assays were conducted between February and June 1984 in the Entomology Department greenhouse at the University of Hawaii Manoa Campus. Daily maximum temperatures were 29 _+ 2~ and daily minimums were 22 _+ 2~ the natural photoperiod (ca. 12 hr light) was used. Groups of females (3-10) with approximately equal numbers of males were tested in screen cages (50 cm on each side) in all experiments. Cotton dental wicks immersed in dilute honey-water (1 m l : 5 ml) solutions in open vials were suspended from the cage tops to provide food for adults. Within the constraints imposed by confinement, behavior of females in the assays was similar to their field behavior (Chew 1977); females laid their eggs singly and usually flew off the plant after oviposition. Choice Tests. One plant treated with control solution and another treated with either a coumarin or rutin solution were placed in diagonally opposite comers of each cage. Trials lasted 2-3 days. Eggs were counted after each trial, and plant positions in the cage were altemated in successive trials. No-Choice Tests. Females were offered either two control plants or two treated plants (either coumarin or rutin at 0.1 M). Plants were placed in diagonally opposite comers of the cage. In the 5-min no-choice tests, each group of females was tested for 10 trials, consisting of an alternating sequence of control and treated plants offered for 5-min periods, with less than 1 min between trials. For no-choice tests lasting 15 min, 60 rain, and 24 hr, groups of females were paired so that one group was offered two control plants while, at the same time, a matched group of equal numbers and age was offered treated plants. In the next trial, the plants were switched so that the first group received treated plants while the second group received controls. Pre- and Postalighting Behavior. Females were observed during behavioral assays to determine how coumarin and rutin influenced their pre- and postalighting behavior. "Contacts" were recorded when females touched a plant with one or more legs. Analysis. G tests for goodness of fit, adjusted for sample size as necessary (Sokal and Rohlf, 1969), were used throughout. The statistical significance of deterrence was determined by contrasting observed frequencies of behaviors (ovipositions or contacts) on control vs. test plants with the 1 : 1 frequency of behaviors expected if butterflies did not differentiate between treatments. Ratios of behaviors (eggs laid/contact) on control vs. test plants were contrasted by testing for independence in 2 • 2 contingency tables (Sokal and Rohlf, 1969). The 2 • 2 contingency analysis was also used to compare frequencies of eggs on control vs. plants treated with mtin (0.1 M) in no-choice tests of various duration. Means (_+ SE) for percentages of eggs on treated plants were calculated to provide a rough index of variation among replicates. Statistical inferences from ANOVA on the percentages were generally consistent with the G tests.
312
TABASHNIK RESULTS
Choice Tests. Both coumarin and rutin deterred oviposition by P. rapae (Table 1). At both concentrations tested, coumarin was a weak deterrent. Rutin at 0.1 M caused a fivefold reduction in oviposition, but 0.01 M rutin did not deter oviposition. No-Choice Tests. Responses to cabbage plants treated with 0.1 M rutin in no-choice tests depended on the trial duration, with observed deterrence inversely related to trial duration (Table 2). Deterrence was strongest in the 5rain no-choice test, intermediate in 15- and 60-min no-choice tests, and weakest in the 24-hr no-choice test. Pre- and Postalighting Behavior. Coumarin and rutin deterred oviposition primarily by affecting prealighting rather than postalighting behavior. Direct behavioral observations showed that both compounds significantly reduced the number of contacts with plants (Table 3). Analysis of the ratio of eggs laid/ contact showed that after contacting a plant, females were somewhat less likely TABLE l. EFFECTS OF COUMARIN AND RUTIN ON OVIPOSITION BY Pieris rapae ~N CHOICE TESTS
Total eggs laid Treatment Coumarin Coumarin Rutin Rutin
0.1 0.01 0.1 0.01
M M M M
Trials
Control
Treated
7 5 6 6
1155 918 1928 949
605" 668" 375" 886
% of eggs on treated (-~ +_ SE) 40.9 42.5 18.5 44.8
+ 5.2 _+ 2.5 + 4.1 +_ 6. l
ap < 0.001 by G tests on pooled data.
TABLE 2. EFFECT OF RUTIN (0.1 M) ON OVlPOSlTION BY Pieris rapae IN N o - C H o I c E TESTS OF VARIOUS DURATIONS
Total eggs laid '* Trial duration
Trials
Control
Treated
5 min 15 min 60 min 24 hr
10 4 4 16
27 46 116 924
0a 19 b 48 b 752 c
% of eggs on treated (X + SE) 0.0 28.4 29.0 38.7
+ 0.0 _+ 2.1 _+ 3.6 + 7.7
aRatios of eggs (control:treated) followed by different letters are significantly different from each other by analysis of 2 • 2 contingency tables (P < 0.05).
DETERRENCE OF CABBAGEBUTTERFLYOVIPOSITION
313
TABLE 3. EFFECTSOF COUMARINAND RUTIN (0.1 M) ON PRE- AND POSTALIOr~TING BEHAVIOROF Pieris rapae Contacts
Eggs/contact
Assay
Treatment
Trials
Control
Treated
Control
Treated
Choice Choice No-choicea No-choice~ No-choiceb
Coumafin Rutin Coumafin Rutin Rutin
10 .10 10 10 4
75 25 42 76 142
35c 4c 14c 14c 61c
0.21 0.76 0.31 0.36 0.41
0.14 0.51 0.00 a 0.00 d 0.39
a5 rain. %0 min. cp < 0.001 by G tests on pooled data. d p < 0.05 by analysis of 2 x 2 contingency tables. tO oviposit on treated plants than on control plants (Table 3). However, the effects o f coumarin and mtin on postalighting behavior were small compared to the prealighting effects.
DISCUSSION The results o f this study support the view that host acceptance by insect herbivores is mediated by positive and negative stimuli from plants and by internal levels o f satiation (Dethier, 1982). Both coumarin and mtin deterred oviposition by P. rapae, yet females laid some eggs on treated plants in nearly all experiments. These results suggest that the cabbage plants provided strong positive stimuli, whereas the applied compounds were negative stimuli that reduced the acceptability o f treated cabbage plants significantly, but not completely. In no-choice tests, acceptance o f rutin-treated plants relative to control plants increased with trial duration. It is unlikely that the reduced deterrence o f rutin seen in prolonged no-choice tests was due to rutin concentration declining through time. Rutin's deterrence remained effective in choice tests lasting 2 - 3 days (Table 1) and plants treated with rutin 24 hr before testing did not show reduced deterrence compared to plants treated with rutin immediately before testing. Another possibility is that the control plants became less acceptable as the number o f eggs per control plant increased in no-choice tests o f increasing duration. Egg-load assessment is reported to ocur for some P. rapae populations (Rothschild and Schoonhoven, 1977) but not others (Ives, 1978; Traynier, 1979; Shapiro, 1981). Results from a no-choice test in the present study show that the rate o f oviposition on a clean control plant (9.3 eggs laid/female/hr) was not greater than the rate o f oviposition on a control plant with 56 eggs (10.0 eggs
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laid/female/hr), suggesting that acceptance of control plants in no-choice tests did not decline due to egg-load assessment. It seems that increased acceptance of rutin-treated plants occurred because females were deprived of the opportunity to oviposit on untreated plants. This type of behavior is analogous to increased acceptance of nonpreferred host-plant species when preferred species are unavailable (e.g., Singer, 1982, 1983; Tabashnik, 1983a, b; Tabashnik et al., 1985). Time available for oviposition may be a limiting factor for butterflies like P. rapae, which lay their eggs singly (Tabashnik 1983a). Thus, single egglayers may accept nonpreferred plants for oviposition more readily than cluster egg-layers. The results show that during 5-min trials, P. rapae consistently rejected treated plants, while consistently accepting untreated plants (Table 2)~ During trials of 15 min or longer, however, females accepted treated plants, indicating that the "discrimination phase" (Singer, 1982) for P. rapae in these experiments was between 5 and 15 min. These results are consistent with earlier findings (Tabashnik, 1983a) supporting the hypothesis that single egg-laying butterflies have relatively short discrimination phases. Analysis of pre- and postalighting behavior revealed that the compounds reduced oviposition primarily by lowering the rate at which females contacted plants, indicating that deterrence was mediated by noncontact cues. These observations conflict with the notion that host-plant chemistry is most important in determining postalighting rather than prealighting behavior. However, secondary compounds were applied topically in this study, and thus their impact on behavior may not mimic the effects of compounds incorporated in leaves. The volatile compound, coumarin, probably deterred oviposition by alteration of olfactory cues. The noncontact effects of the nonvolatile pigment, rutin, were probably mediated by vision. The results suggest that plants producing a secondary compound typical of nonhosts might gain some protection from specialist herbivores by reducing their acceptance to ovipositing females. If the observed responses to coumarin and rutin by P. rapae and Plutella xylostella (Tabashnik, 1985) are indicative of general trends, then this protection would only be partial--even when a compound was produced in relatively high quantities. If the compound reduced the plant's suitability, selection would be expected to increase discrimination against it. Alternatively, if the compound had a neutral or positive effect on suitability, selection would act to reduce the deterrent effect of the compound. The results imply that nonhost-plant secondary compounds might confer partial crop protection by deterring oviposition. Due to increasing acceptance of treated plants when untreated plants are absent, treatments would be most effective when some plants were left untreated as a "trap crop,,' Application of plant secondary compounds to protect crops poses many of the same problems and hazards as do conventional pesticides, yet this technique offers potential as a tool to be integrated with other pest control measures (Schoonhoven,
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1982). Attempts to deter oviposition by mobile adults may be more fruitful than tactics aimed at reducing consumption by immatures. Acknowledgments--I am especially grateful to J.A.A. Renwick for stimulating my interest in deterrents and for sharing his insights. I thank R.F. Chapman, F. Gould, M.O. Harris, M.W. Johnson, W.J. Mattson, J.R. Miller, L.M. Schoonhoven, M.C. Singer, and C. Wiklund for their comments and encouragement. H.B. Haji-Mamat shared knowledge and butterflies, F. Chang gave excellent technical advice, and S. Arnold provided expert laboratory assistance. Special thanks to R.H. Krakauer for unfaltering moral support. Financial support was provided by a grant from the Research and Training Fund, University of Hawaii and USDA-HAW00947H. This is paper no. 2997 of the Hawaii Inst. of Trop. Ag. and Human Res. Journal Series.
REFERENCES CHEW, F.S. 1977. Coevolution of Pierid butterflies and their cmciferous food plants. II. The distribution of eggs on potential food plants. Evolution 31:568-579. DETHIER, V.G. 1982. Mechanisms of host-plant recognition. Entomol. Exp. AppL 31:49-56. GUPTA,P.D., and THORSTEINSON, A.G. 1960. Food plant relationships of the diamond-back moth Plutella maculipennis (Curt.) II. Sensory regulation of oviposition by the adult female. Entomol. Exp. Appl. 3:305-314. HAII-MA~AT, H.B. 1984. Some pathological manifestations in adult Artogeia rapae (L.) (Lepidoptera: Pieridae) developing from larvae infected with Nosema sp. MS thesis. University of Hawaii. IrEs, P.M. 1978. How discriminating are cabbage butterflies? Aust. J. Ecol. 3:261-276. LEUNG, A.Y. 1980. Encyclopedia of Common Natural Ingredients Used in Food, Drugs, and Cosmetics. John Wiley & Sons, New York. LUNDGREN,L. 1975. Natural plant chemicals acting as oviposition deterrents on cabbage butterflies [Pieris brassicae (L.), P. rapae (L.), P. napi (L.)]. Zool. Ser. 4: 253-258. M1LLER, J.R., and STR~C~:LER,K.L. 1984. Finding and accepting host plants, pp. 127-157, in W.J. Bell and R.T. Card6 (eds.). Chemical Ecology of Insects. Sinauer Associates, Sunderland, Massachusetts. M~TCHELL, E.R., and HEATH, R.R. 1985. Influence of Amaranthus hybridus L. allelochemics on oviposition behavior of Spodoptera exigua and S. eridania (Lepidoptera: Noctuidae). J. Chem. Ecol. 11 :609-617. RENW~CK,J.A.A., and RADKE,C.D. 1981. Host plant constituents as oviposition deterrents for the cabbage looper, Trichoplusia ni (Lepidoptera: Noctuidae). Entomol. Exp. Appl. 30:201-204. RENWICK, J.A.A., and RADKE, C.D. 1983. Chemical recognition of host plants for oviposition by the cabbage butterfly, Pieris rapae (Lepidoptera: Pieridae). Environ. Entomol. 12:446-450. RENWICK, J.A.A., and RADKE, C.D. 1985. Constituents of host and nonhost plants deterring oviposition by the cabbage butterfly, Pieris rapae. Entomol. Exp. Appl. 39:21-26. RODMAN, J.E., and CHEW, F.S. 1980. Phytochemical correlates of herbivory in a community of native and naturalized Cruciferae. Biochem. Syst. Ecol. 8:43-50. ROTHSCHILD, M., and SCHOONHOVEN,L.M. 1977. Assessment of egg load by Pieris brassicae (Lepidoptera: Pieridae). Nature 266:352-355. SCHOONHOVEN, L.M. 1982. Biological aspects of antifeedants. Entomol. Exp. Appl. 31:57-69. SCRmER, J.M. 1984. Host-plant suitability, pp. 159-202, in W.J. Bell and R.T. Card6 (eds.). Chemical Ecology of Insects. Sinauer Associates, Sunderland, Massachusetts. SHAPIRO, A.M. 1981. The pierid red-egg syndrome. Am. Nat. 117:276-294. SINGER, M.C. 1982. Quantification of host specificity by manipulation of oviposition behavior in the butterfly Euphydryas editha. Oecologia (Berlin) 52:224-229.
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SINGER, M.C. 1983. Determinants of multiple host use by a phytophagous insect population. Evolution 25:383-389. SOKAL, R.R., and ROHLE, F.I. 1969. Biometry. Freeman, San Francisco. TABASHNIK, B.E. 1983a. Oviposition specificity in single vs. cluster egg-laying butterflies: A discrimination phase in Colias eurytheme ? Oecologia (Berlin) 58:278-279. TABASHNIK, B.E. 1983b. Host range evolution: The shift from native legume hosts to alfalfa by the butterfly, Colias philodice eriphyle. Evolution 37:150-162. TABASHNIK,B.E. 1985. Deterrence of diamondback moth (Lepidoptera: Plutellidae) oviposition by plant compounds. Environ. Entomol. 14:575-578. TABASHNIK,B.E., and SLANSKY,F., JR. 1987. Nutritional ecology of forb fbliage-chewing insects, in F. Slansky, Jr., and J.G. Rodriguez (eds.). Nutritional Ecology of Insects, Mites, and Spiders. John Wiley & Sons, New York. In press. TABASHr~IK,B.E., MATTSON, W.J., and MILLER, J.R. 1985. Host acceptance behavior of the red pine cone beetle ( Conophthorus resinosae). Entomol. Exp. Appl. 37:3-7. TINGLE, F.C., and MITCHELL, E.R. 1984. Aqueous extracts from indigenous plants as oviposition deterrents for Heliothis vireseens (F.) J. Chem. Ecol. 10:101-113. TRAYNIER, R.M.M. 1979. Long-term changes in the oviposition behavior of the cabbage butterfly, Pieris rapae, induced by contact with plants. Physiol. Entomol. 4:87-96.
