Effects of Foliage Color on the Landing Response of Pieris rapae (Lepidoptera: Pieridae) Author(s): Jun Tsuji and Lauren Coe Source: Environmental Entomology, 43(4):989-994. Published By: Entomological Society of America URL: http://www.bioone.org/doi/full/10.1603/EN14084
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BEHAVIOR
Effects of Foliage Color on the Landing Response of Pieris rapae (Lepidoptera: Pieridae) JUN TSUJI1
AND
LAUREN COE
Biology Department, Siena Heights University, 1247 E. Siena Heights Drive, Adrian, MI 49221
Environ. Entomol. 43(4): 989Ð994 (2014); DOI: http://dx.doi.org/10.1603/EN14084
ABSTRACT The effects of foliage color on the selection of host plants by Pieris rapae (L.) were investigated using choice tests between Brassica rapa (L.) varieties with green, variegated, and yellowÐ green leaves. Gravid-naive females displayed a Þrst landing preference for the green and variegated Brassica varieties when the plants were freely accessible. Comparable results were observed when the plants were enclosed in glass jars, demonstrating that visual cues were sufÞcient to induce the landing response. The Þrst landing choice was positively correlated with oviposition preference and larval survival. These results suggest that leaf color is an important visual cue used by P. rapae for intraspeciÞc host selection. KEY WORDS intraspeciÞc host plant selection, imported cabbageworm, Brassicaceae, visual cue
Adult females of the imported cabbageworm Pieris rapae (L.) (Lepidoptera: Pieridae) are specialist that land and lay single eggs primarily on members of the Brassicaceae family (Root and Kareiva 1984). The selection of suitable host plants by ovipositing females is critical to the survival of this insect species because the newly hatched larvae have limited mobility and can only successfully grow on certain plant species (Ives 1978, Hern et al. 1996). Many studies of host plant selection have examined the response of these female butterßies to different plant species (Richards 1940, Chew 1977, Jones 1977). For instance, when P. rapae females were simultaneously presented with cabbage and lettuce plants in choice tests, the butterßies exhibited a landing preference for cabbage even when both plants were covered with perforated black plastic (Ikeura et al. 2010). These observations suggest that the female butterßies can use olfactory cues to distinguish members of the cabbage family from other plants. In addition to interspeciÞc host selection, P. rapae females can also discriminate between individuals of the same plant species (Ives 1978). When a female butterßy enters a cabbage Þeld, for example, she lands and oviposits on certain individual plants, but not on others (Jones 1977, Ives 1978, Langan et al. 2001). Visual rather than olfactory cues are believed to be important in intraspeciÞc host selection because healthy individuals of the same plant species release the same volatile chemicals (Pierre et al. 2011), P. rapae searches and lays eggs near mid-day when the air is most turbulent (Root and Kareira 1984, Ohsaki and Sato 1994), and volatiles from healthy leaves do not 1
Corresponding author, e-mail: [email protected].
enhance oviposition (Renwick and Radke 1983). The most important visual stimulus that elicits butterßy landings on host plants appears to be leaf color (Renwick and Radke 1988). Past studies have shown that P. rapae females can differentiate between different colors during their landing response. For instance, the butterßies exhibited a landing preference for green and yellow paper cards, instead of red, orange, blue, and violet, after Þrst landing on green cabbage (Traynier 1979). Similarly, P. rapae can learn to distinguish and land on green or blue paper after conditioning them with glucosinolates (Traynier 1984). Because gravid females displayed a landing preference for green over red cabbage varieties (Snell-Rood and Papaj 2009) and green rather than a greenÐwhite variegated Arabidopsis plant (Zheng et al. 2010), P. rapae can also discriminate between different colored plants of the same species. The landing response of P. rapae to different colored foliage may be related to its oviposition preference. With cabbage, for example, P. rapae females not only preferred to land, but also laid more eggs on green varieties rather than on red (Radcliffe and Chapman 1966, Jankowska 2006, Snell-Rood and Papaj 2009). Similarly, Pieris females exhibited more Þrst landings and oviposited more on green Arabidopsis than on a virus-induced, greenÐwhite variegated plant (Zheng et al. 2010). These positive correlations suggest that foliage color may serve as a visual cue for landing and oviposition decisions (Meyers 1985). The relationship between the landing preference and larval performance of P. rapae has received little attention. With Arabidopsis, female butterßies preferred to land on plants that also promoted larval
0046-225X/14/0989Ð0994$04.00/0 䉷 2014 Entomological Society of America
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growth, as larvae reared on green Arabidopsis plants gained greater weight than those that fed on greenÐ white variegated plants (Zheng et al. 2010). To determine whether the observations with P. rapae and the model plant Arabidopsis describe responses that can be generalized to an economically important genus, we conducted similar experiments using rapidcycling green, variegated, and yellowÐ green varieties of Brassica rapa (L.) (Williams and Hill 1986). The goal of this investigation was to study the landing response of P. rapae to different colored varieties of B. rapa and to examine its relationship to oviposition preference and larval performance. Materials and Methods Insects and Plants. A laboratory population of P. rapae was established using eggs purchased from Carolina Biological Supply Co. (Burlington, NC). Larvae were reared on heads of caulißower (Brassica oleracea L. variety botrytis) in cages (60 by 30 by 35 cm) constructed using 1.27-cm-diameter polyvinyl chloride pipe frames and covered with shear polyester netting. The larvae were maintained at room temperature, and the larvae were allowed to pupate in the cage. Afterwards, the pupae were transferred to a separate cage without any plants, where the adults emerged. The butterßies were provided with an artiÞcial nectar solution consisting of 10% sucrose and were allowed to mate before use (Poelman et al. 2008). Because larval food plants do not affect the oviposition behavior of adults (Chew 1977) and the butterßies were not exposed to any plants before the experiments, the adult females were considered to be naive (Smallegange et al. 2006). B. rapa seeds of the green (stock no. 1Ð33), variegated (1Ð28), and yellowÐ green (1Ð 46) varieties were obtained from the Rapid Cycling Brassica Collection (University of Wisconsin). Plants were grown in potting soil with a photoperiod of 14:10 (L:D) h under ßuorescent lights (10,000 lux) at room temperature. Three- to four-week-old plants grown in 12.7-cm-diameter, dark green, plastic pots were used in the experiments. Plants of similar size and shape were tested to minimize the effect of these visual cues in the experiment. Flowers and buds, if present, were removed before use. First Landing Choice. The Þrst landing response of naive gravid female butterßies was studied using choice tests between the three varieties of Brassica (Zheng et al. 2010). Two pots of each of the three varieties (two plants per pot) were placed on a tan ßoor at one end of a 321 (length) by 160 (width) by 165 cm (height) test cage constructed of nylon netting supported by a polyvinyl chloride pipe (1.27-cm in diameter) frame. The pots were randomly arranged in two rows separated by 45 cm between the centers of each pot. Female butterßies were released from the opposite end of the cage and were observed for their Þrst landing on the leaves of either the green, variegated, or yellowÐ green plant. After the landing choice was noted, the butterßy was removed and the pots
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were rearranged to minimize positional effects. The process was then repeated with other butterßies over a 2- to 3-h period. The experiment was conducted between 1200 and 1700 hours (GMT-5) on Þve separate days using fresh plants on each date. The Þrst landing choices of a total of 127 female butterßies were recorded using 20 plants of each variety. The choice tests were conducted indoors at room temperature with overhead ßuorescent illumination. The Þrst landing response was also observed using plants enclosed in air-tight, clear, glass jars to exclude olfactory cues (Reeves 2011). Individual green, variegated, and yellowÐ green Brassica plants were transplanted into 266-ml clear plastic cups. Each cup was then place on a clear, glass, 250-ml beaker to elevate the plant so that the leaves were pressed against the inner surface of an inverted, 1.89-liter clear glass jar. The opening of the jar, located beneath the beaker, was sealed using a screw-cap metal lid with a plastic gasket. Two plants, in separate jars, of each of the three Brassica varieties were used in choice experiments using the same procedure as described with the freely accessible plants. The Þrst landing preference of individual females on the glass jar surrounding either the green, variegated, or yellowÐ green plant was recorded. Landing experiments were performed between 1200 and 1700 hours (GMT-5) on seven separate days using fresh plants on each date. The Þrst landing choices of 127 female butterßies were observed using 14 plants of each variety. Oviposition Preference. The egg laying preferences of naõ¨ve Pieris females were tested using 24 pots (one to two plants per pot) containing 14 plants of each of the three Brassica varieties. The pots were placed at one end of the 321 (length) by 160 (width) by 165 cm (height) test cage and arranged in four rows of six pots, with two pots of each variety randomly placed in each row, separated by 20 cm between the middle of each pot. At 1130 hours (GMT-5), 18 gravid females were simultaneously released at the opposite end of the cage. After 1.5 h, the pots were randomly rearranged to minimize positional effects. After an additional 1.5 h, the butterßies were collected, and the number of eggs deposited on each plant was counted. Oviposition preference was expressed as the median number of eggs deposited per plant for each Brassica variety (Zheng et al. 2010). Larval Performance. As in the oviposition preference test, 15 gravid females laid eggs on 16 plants of each of the three Brassica varieties for 3 h. Afterwards, the butterßies were collected, and the number of eggs deposited on each plant was counted (mean ⫾ SE; 11.2 ⫾ 1.7 eggs per plant on green, 11.1 ⫾ 1.5 eggs per plant on variegated, 6.8 ⫾ 0.8 eggs per plant on yellowÐ green). Because larval mortality is independent of egg density (Root and Kareiva 1984, Jones et al. 1987), the number of eggs on each plant was unaltered. The plants were then transferred under ßuorescent illumination. Ten days after oviposition, but before the plants had become depleted, the larvae (third instars) were counted and individually weighed to determine their fresh body mass. Because the larval mortality of
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Fig. 1. First landing response of P. rapae to freely accessible and enclosed green, variegated, and yellowÐgreen B. rapa. N ⫽ 127 female butterßies for the freely accessible Brassica varieties, and N ⫽ 127 females for the enclosed plants. Percentages with the same letters above the columns are not signiÞcantly different (2 test, z-test, P ⬎ 0.05).
Fig. 2. Median (⫾ Þrst and third quartile) number of eggs laid by P. rapae on green, variegated, and yellowÐgreen B. rapa. N ⫽ 14 plants per variety. Medians with the same letters above the columns are not signiÞcantly different (Wilcoxon signed rank test, P ⬎ 0.05).
laboratory populations is greatest during the Þrst two instars (Jones and Ives 1979), and late-instar larvae can be difÞcult to locate because they migrate (Mauricio and Bowers 1990) and leave the plant to pupate (Jones et al. 1987), the larva to egg ratio on each Brassica plant at the third instar was calculated and used as a measure of larval survival (Radcliffe and Chapman 1966). Data Analyses. The distribution of butterßy landings on the three varieties of freely accessible plants as well as the distribution of landings on the enclosed plants was analyzed using chi-square tests followed by post hoc z-tests (Ikeura et al. 2010). A chi-square test was also used to compare the number of Þrst landings on the freely accessible plants to the enclosed B. rapa (Zheng et al. 2010). A Wilcoxon signed rank test was used to evaluate the median number of eggs deposited on each green, variegated, and yellowÐ green plant (Poelman et al. 2008, Zheng et al. 2010). The mean larva to egg ratio for each of the three varieties were compared using student t-tests. A one-way analysis of variance (ANOVA) was used to compare the weights of larvae reared on the different Brassica varieties (McKenzie and Goldman 2005).
females oviposited on the glass immediately adjacent to the enclosed plant. The Þrst landing response to the enclosed plants was comparable to the response to the freely accessible plants for the green (41.0 vs. 39.4%), variegated (41.7 vs. 40.1%), and yellowÐ green (17.3 vs. 20.5%) varieties (2 ⫽ 0.77; df ⫽ 2; P ⫽ 0.679; Fig. 1). Oviposition Preference. In the oviposition choice test, female butterßies laid more eggs per plant on the green and variegated Brassica plants than on the yellowÐ green variety (Fig. 2). SigniÞcantly more eggs were deposited on the green variety than on the yellowÐ green plants (median; 18 eggs per plant on green vs. 3 eggs per plant on yellowÐ green, n ⫽ 14; P ⫽ 0.002). Likewise, more eggs were laid on the variegated plants than on the yellowÐ green variety (median; 13 eggs per plant on variegated vs. 3 eggs per plant on yellowÐ green, n ⫽ 14; P ⫽ 0.004). The median number of eggs oviposited on the green Brassica variety did not differ signiÞcantly from that on the variegated plants (P ⫽ 0.363). The choice test was conducted three times with comparable results. Larval Performance. Larval survival to the third instar was lower on the yellowÐ green variety than on the green plants (mean larva per egg ⫾ SE; 0.212 ⫾ 0.048 on yellowÐ green vs. 0.394 ⫾ 0.046 on green; t ⫽ 2.74; df ⫽ 30; P ⫽ 0.010; Fig. 3). Larvae reared on the
Results First Landings. In the Þrst landing choice experiment between the three varieties of the freely accessible plants, more female P. rapae butterßies landed Þrst on the green (39.4%) and variegated (40.1%) B. rapa than on the yellowÐ green (20.5%) variety (2 ⫽ 9.47; df ⫽ 2; P ⫽ 0.009; Fig. 1). The percentage of Þrst landings on the green and variegated plants were statistically equivalent (z ⫽ 0.128; P ⫽ 0.890). When the Þrst landing choice experiment was repeated using plants enclosed in glass jars to exclude olfactory cues, female butterßies landed on the jars surrounding the green (41.0%) and variegated (41.7%) Brassica plants more often than on the glass covering the yellowÐ green (17.3%) variety (2 ⫽ 14.67; df ⫽ 2; P ⬍ 0.001; Fig. 1). Nearly identical percentages of females landed on the enclosed green plants as on the enclosed variegated plants (z ⫽ 0.127; P ⫽ 0.890). When given the opportunity, some of the
Fig. 3. Larval survival of P. rapae reared on green, variegated, and yellowÐgreen B. rapa. Data are expressed as the mean (⫾SE) survivorships of the larvae on 16 plants per variety. Means with same letters above the bars are not signiÞcantly different (t-test; P ⬎ 0.05).
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Fig. 4. Mean (⫾SE) fresh weights of P. rapae larvae reared on green, variegated, and yellowÐgreen B. rapa. N ⫽ 30 larvae per variety. Means with same letters above the bars are not signiÞcantly different (ANOVA, P ⬎ 0.05).
yellowÐ green plants also had a lower survivorship than larvae that fed on the variegated Brassicas (mean larva per egg ⫾ SE; 0.212 ⫾ 0.048 on yellowÐ green vs. 0.407 ⫾ 0.072 on variegated; t ⫽ 2.25; df ⫽ 30; P ⫽ 0.032). The survival of larvae that fed on the green and on the variegated plants were comparable (t ⫽ ⫺0.15; df ⫽ 30; P ⫽ 0.885). Larvae raised on either the green, variegated, or yellowÐ green plants were equivalent in weight (mean ⫾ SE; 18.9 ⫾ 2.4 mg on green vs. 19.9 ⫾ 1.8 mg on variegated vs. 20.8 ⫾ 1.6 mg on yellowÐ green; F ⫽ 0.30; df ⫽ 2; P ⫽ 0.741; Fig. 4). Similar larval survival and mass results were obtained in three independent trials. Discussion Foliage color had a signiÞcant effect on the landing response of P. rapae, as more Þrst landings occurred on freely accessible green and variegated plants than on the yellowish variety. For many herbivorous insects, yellow can act as a superoptimal stimulus that elicits alightment on living plants (Prokopy and Owens 1983). Although B. rapa ßower petals are yellow and P. rapae females prefer to land on yellow artiÞcial ßowers (Kandori et al. 2009), we saw no evidence that the female butterßies were especially attracted to the yellowÐ green leaves or mistook the leaves for yellow ßowers and nectaring. Our landing results are similar to those reported in previous studies that have shown an innate landing preference for green foliage (SnellRood and Papaj 2009, Zheng et al. 2010). The landing response of female P. rapae butterßies to the variegated Brassica plant differed from that reported with the variegated Arabidopsis. The variegated phenotype used in our study is a maternally inherited, chloroplast DNA trait, and the Brassica plants displayed mostly green foliage with yellow and white sectors (Williams 1985). None of the leaves were entirely white. The variegated Arabidopsis, however, is a virus-induced phytoene desaturase-silenced plant with many white photobleached leaves (Zheng et al. 2010). The different landing responses seen with the Brassica and Arabidopsis plants suggest that female P. rapae butterßies have the ability to distinguish between different levels of variegation. Mostly green
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variegated leaves appear to be more acceptable to the female butterßies than mostly white plants. The Þrst landing response to the enclosed Brassica plants was comparable with the response to the freely accessible plants. Because the enclosed plants were in air-tight jars, olfactory cues were unavailable to the butterßies. Consequently, visual cues were sufÞcient to orient the butterßies and to induce a landing response. Interestingly, when given the opportunity, some of the gravid females oviposited on the glass jars surrounding the enclosed plants. This egg-laying response may be related to the motivational status of the females (Chew and Robbins 1984), as they were reared in the absence of host plants before the choice tests. However, motivation-based models of host plant selection do not explain the oviposition behavior of P. rapae well (Hopkins and van Loon 2001). Alternatively, these observations suggest that physical contact with the leaf surface is not needed to elicit egg laying. When the gravid females were given the opportunity to land multiple times and lay eggs for 3 h, they oviposited more on the green and variegated Brassica plants than on the yellowÐ green variety. These results are similar to those of previous studies that have shown that P. rapae has an innate egg-laying preference for green artiÞcial substrates (Horvanitz and Chang 1964) and dark green fertilized plants (Meyers 1985, Letourneau and Fox 1989, Chen et al. 2004). We also observed that the oviposition preference was positively correlated with the landing choice on B. rapa, which is also consistent with the results reported from studies conducted with cabbage (Radcliffe and Chapman 1966, Jankowska 2006, Snell-Rood and Papaj 2009) and Arabidopsis (Zheng et al. 2010). Unlike plant age (Jones 1977) and leaf shape (Renwick and Radke 1988), foliage color appears to be an important visual cue used to elicit landings that lead to the acceptance of plants as oviposition sites. Larval survival to the third instar was greater on the green and variegated Brassica plants than on the yellowÐ green variety. These results are in accordance with the oviposition preferenceÐ offspring performance hypothesis (Jaenike 1978), which predicted that reproductive success would result from appropriate host selection. Because cruciferous plants can vary in their suitability as larval food, larval survival and development rates can vary depending on their host (Chew 1975, Ohsaki and Sato 1994). In particular, green foliage can support a greater proportion of surviving larvae than yellowÐ green (Doak et al. 2006), where nutritional limitations could lead to food-related mortality as well as egg cannibalism (Watanabe and Yamaguchi 1993). Such nutritional variances, along with differences in predator and parasitoid avoidance (Loader and Damman 1991, Ohsaki and Sato 1994), can contribute to the selection for female P. rapae butterßies that can discriminate one host plant from another (Darwin 1909). While larval survival was lower on the yellowÐ green variety, the surviving larvae had similar weights as those reared on the green and variegated plants. These results are similar to those reported by Slansky
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and Feeny (1977) and Chen et al. (2004), who studied larval performance on dark green fertilized plants and light green unfertilized plants. Although P. rapae preferred to lay eggs on green leaves of fertilized plants, which supported faster larval development (Meyers 1985, Chen et al. 2004), larvae raised on light green leaves of unfertilized plants compensated by spending more of their time feeding (Loader and Damman 1991), consuming food faster, and using nitrogen more efÞciently (Slansky and Feeny 1977). Consequently, larvae reared on light green unfertilized plants had growth rates and Þnal weights as high as those grown on dark green fertilized plants (Slansky and Feeny 1977, Chen et al. 2004). In our study, the similar weights of the larvae that fed on the different B. rapa varieties suggest that compensatory feeding may have occurred on the yellowÐ green variety. The growth of the larvae reared on the variegated Brassica observed in our study differed from the larval performance reported with the variegated Arabidopsis (Zheng et al. 2010). Larvae raised on the variegated Brassica were equivalent in weight to those reared on the green variety, whereas larvae that fed on the variegated Arabidopsis plants weighed signiÞcantly less than those that fed on the green controls (Zheng et al. 2010). Larval growth is especially limited by the availability of nitrogen in their food (Slansky and Feeny 1977). Because the greenness of leaves is an indication of their nitrogen content (Loader and Damman 1991), our results suggest that the mostly green leaves of the variegated Brassica had sufÞcient nitrogen to support normal larval growth, while the mostly white leaves of the variegated Arabidopsis did not. This study is the Þrst to examine the effects of green, variegated, and yellowÐ green Brassica foliage on the intraspeciÞc host selection of P. rapae. Female butterßies preferred to land on green and variegated plants, and visual rather than olfactory cues were sufÞcient to induce a landing response. The landing choice was positively correlated with oviposition preference and larval survival. Different results were also noted between the variegated Brassica described in this study and the variegated Arabidopsis examined by Zheng et al. (2010). Taken together, our results suggest that leaf color is an important visual cue used by female P. rapae butterßies to select hosts that are suitable larval food plants. Acknowledgments We thank Tim Husband for assistance with the statistical analyses.
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Ohsaki, N., and Y. Sato. 1994. Food plant choice of Pieris butterßies as a trade-off between parasitoid avoidance and quality of plants. Ecology 75: 59 Ð 68. Pierre, P. S., J. J. Jansen, C. A. Hordijk, N. M. van Dam, A. M. Cortesero, and S. Dugravot. 2011. Differences in volatile proÞles of turnip plants subjected to single and dual herbivory above-and belowground. J. Chem. Ecol. 37: 368 Ð377. Poelman, E. H., C. Broekgaarden, J.J.A. van Loon, and M. Dicke. 2008. Early season herbivore differentially affects plant defence responses to subsequently colonizing herbivores and their abundance in the Þeld. Mol. Ecol. 17: 3352Ð3365. Prokopy, R. J., and E. D. Owens. 1983. Visual detection of plants by herbivorous insects. Ann. Rev. Entomol. 28: 337Ð364. Radcliffe, E. B., and R. K. Chapman. 1966. Varietal resistance to insect attack in various cruciferous crops. J. Econ. Entomol. 59: 120 Ð125. Reeves, J. L. 2011. Vision should not be overlooked as an important sensory modality for Þnding host plants. Environ. Entomol. 40: 855Ð 863. Renwick, J.A.A., and C. D. Radke. 1983. Chemical recognition of host plants for oviposition by the cabbage butterßy, Pieris rapae (Lepidoptera: Pieridae). Environ. Entomol. 12: 446 Ð 450. Renwick, J.A.A., and C. D. Radke. 1988. Sensory cues in host selection for oviposition by the cabbage butterßy, Pieris rapae. J. Insect Physiol. 34: 251Ð257. Richards, O.W. 1940. The biology of the small white butterßy (Pieris rapae), with special reference to the factors controlling its abundance. J. Anim. Ecol. 9: 243Ð288. Root, R. B., and P. M. Kareiva. 1984. The search for resources by cabbage butterßies (Pieris rapae): ecological consequences and adaptive signiÞcance of markovian
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movements in a patchy environment. Ecology 65: 147Ð 165. Slansky, F., and P. Feeny. 1977. Stabilization of the rate of nitrogen accumulation by larvae of the cabbage butterßy on wild and cultivated food plants. Ecol. Monogr. 47: 209 Ð228. Smallegange, R. C., T. C. Everaarts, and J.J.A. van Loon. 2006. Associative learning of visual and gustatory cues in the large cabbage white butterßy, Pieris brassicae. Anim. Biol. 56: 157Ð172. Snell-Rood, E. C., and D. R. Papaj. 2009. Patterns of plasticity in common and rate environments: a study of host use and color learning in the cabbage white butterßy Pieris rapae. Am. Nat. 173: 615Ð 631. Traynier, R.M.M. 1979. Long-term changes in the oviposition behavior of the cabbage butterßy, Pieris rapae, induced by contact with plants. Physiol. Entomol. 4: 87Ð96. Traynier, R.M.M. 1984. Associative learning in the ovipositional behavior of the cabbage butterßy, Pieris rapae. Physiol. Entomol. 9: 465Ð 472. Watanabe, M., and H. Yamaguchi. 1993. Egg cannibalism and egg distribution of two Pieris butterflies, Pieris rapae and P. melete (Lepidoptera, Pieridae) on a host plant, Rorippa indica (Cruciferae). Jpn. J. Ecol. 43: 181Ð188. Williams, P. H. 1985. The crucifer genetics cooperative. Plant Mol. Biol. Report. 3: 129 Ð144. Williams, P. H., and C. B. Hill. 1986. RapidÐ cycling populations of Brassica. Science 232: 1385Ð1389. Zheng, S.-J., T.A.L. Snoeren, S. W. Hogewoning, J.J.A. van Loon, and M. Dicke. 2010. Disruption of plant carotenoid biosynthesis through virus-induced gene silencing affects oviposition behavior of the butterßy Pieris rapae. New Phytol. 186: 733Ð745. Received 31 March 2014; accepted 9 June 2014.
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BEHAVIOR
Effects of Foliage Color on the Landing Response of Pieris rapae (Lepidoptera: Pieridae) JUN TSUJI1
AND
LAUREN COE
Biology Department, Siena Heights University, 1247 E. Siena Heights Drive, Adrian, MI 49221
Environ. Entomol. 43(4): 989Ð994 (2014); DOI: http://dx.doi.org/10.1603/EN14084
ABSTRACT The effects of foliage color on the selection of host plants by Pieris rapae (L.) were investigated using choice tests between Brassica rapa (L.) varieties with green, variegated, and yellowÐ green leaves. Gravid-naive females displayed a Þrst landing preference for the green and variegated Brassica varieties when the plants were freely accessible. Comparable results were observed when the plants were enclosed in glass jars, demonstrating that visual cues were sufÞcient to induce the landing response. The Þrst landing choice was positively correlated with oviposition preference and larval survival. These results suggest that leaf color is an important visual cue used by P. rapae for intraspeciÞc host selection. KEY WORDS intraspeciÞc host plant selection, imported cabbageworm, Brassicaceae, visual cue
Adult females of the imported cabbageworm Pieris rapae (L.) (Lepidoptera: Pieridae) are specialist that land and lay single eggs primarily on members of the Brassicaceae family (Root and Kareiva 1984). The selection of suitable host plants by ovipositing females is critical to the survival of this insect species because the newly hatched larvae have limited mobility and can only successfully grow on certain plant species (Ives 1978, Hern et al. 1996). Many studies of host plant selection have examined the response of these female butterßies to different plant species (Richards 1940, Chew 1977, Jones 1977). For instance, when P. rapae females were simultaneously presented with cabbage and lettuce plants in choice tests, the butterßies exhibited a landing preference for cabbage even when both plants were covered with perforated black plastic (Ikeura et al. 2010). These observations suggest that the female butterßies can use olfactory cues to distinguish members of the cabbage family from other plants. In addition to interspeciÞc host selection, P. rapae females can also discriminate between individuals of the same plant species (Ives 1978). When a female butterßy enters a cabbage Þeld, for example, she lands and oviposits on certain individual plants, but not on others (Jones 1977, Ives 1978, Langan et al. 2001). Visual rather than olfactory cues are believed to be important in intraspeciÞc host selection because healthy individuals of the same plant species release the same volatile chemicals (Pierre et al. 2011), P. rapae searches and lays eggs near mid-day when the air is most turbulent (Root and Kareira 1984, Ohsaki and Sato 1994), and volatiles from healthy leaves do not 1
Corresponding author, e-mail: [email protected].
enhance oviposition (Renwick and Radke 1983). The most important visual stimulus that elicits butterßy landings on host plants appears to be leaf color (Renwick and Radke 1988). Past studies have shown that P. rapae females can differentiate between different colors during their landing response. For instance, the butterßies exhibited a landing preference for green and yellow paper cards, instead of red, orange, blue, and violet, after Þrst landing on green cabbage (Traynier 1979). Similarly, P. rapae can learn to distinguish and land on green or blue paper after conditioning them with glucosinolates (Traynier 1984). Because gravid females displayed a landing preference for green over red cabbage varieties (Snell-Rood and Papaj 2009) and green rather than a greenÐwhite variegated Arabidopsis plant (Zheng et al. 2010), P. rapae can also discriminate between different colored plants of the same species. The landing response of P. rapae to different colored foliage may be related to its oviposition preference. With cabbage, for example, P. rapae females not only preferred to land, but also laid more eggs on green varieties rather than on red (Radcliffe and Chapman 1966, Jankowska 2006, Snell-Rood and Papaj 2009). Similarly, Pieris females exhibited more Þrst landings and oviposited more on green Arabidopsis than on a virus-induced, greenÐwhite variegated plant (Zheng et al. 2010). These positive correlations suggest that foliage color may serve as a visual cue for landing and oviposition decisions (Meyers 1985). The relationship between the landing preference and larval performance of P. rapae has received little attention. With Arabidopsis, female butterßies preferred to land on plants that also promoted larval
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growth, as larvae reared on green Arabidopsis plants gained greater weight than those that fed on greenÐ white variegated plants (Zheng et al. 2010). To determine whether the observations with P. rapae and the model plant Arabidopsis describe responses that can be generalized to an economically important genus, we conducted similar experiments using rapidcycling green, variegated, and yellowÐ green varieties of Brassica rapa (L.) (Williams and Hill 1986). The goal of this investigation was to study the landing response of P. rapae to different colored varieties of B. rapa and to examine its relationship to oviposition preference and larval performance. Materials and Methods Insects and Plants. A laboratory population of P. rapae was established using eggs purchased from Carolina Biological Supply Co. (Burlington, NC). Larvae were reared on heads of caulißower (Brassica oleracea L. variety botrytis) in cages (60 by 30 by 35 cm) constructed using 1.27-cm-diameter polyvinyl chloride pipe frames and covered with shear polyester netting. The larvae were maintained at room temperature, and the larvae were allowed to pupate in the cage. Afterwards, the pupae were transferred to a separate cage without any plants, where the adults emerged. The butterßies were provided with an artiÞcial nectar solution consisting of 10% sucrose and were allowed to mate before use (Poelman et al. 2008). Because larval food plants do not affect the oviposition behavior of adults (Chew 1977) and the butterßies were not exposed to any plants before the experiments, the adult females were considered to be naive (Smallegange et al. 2006). B. rapa seeds of the green (stock no. 1Ð33), variegated (1Ð28), and yellowÐ green (1Ð 46) varieties were obtained from the Rapid Cycling Brassica Collection (University of Wisconsin). Plants were grown in potting soil with a photoperiod of 14:10 (L:D) h under ßuorescent lights (10,000 lux) at room temperature. Three- to four-week-old plants grown in 12.7-cm-diameter, dark green, plastic pots were used in the experiments. Plants of similar size and shape were tested to minimize the effect of these visual cues in the experiment. Flowers and buds, if present, were removed before use. First Landing Choice. The Þrst landing response of naive gravid female butterßies was studied using choice tests between the three varieties of Brassica (Zheng et al. 2010). Two pots of each of the three varieties (two plants per pot) were placed on a tan ßoor at one end of a 321 (length) by 160 (width) by 165 cm (height) test cage constructed of nylon netting supported by a polyvinyl chloride pipe (1.27-cm in diameter) frame. The pots were randomly arranged in two rows separated by 45 cm between the centers of each pot. Female butterßies were released from the opposite end of the cage and were observed for their Þrst landing on the leaves of either the green, variegated, or yellowÐ green plant. After the landing choice was noted, the butterßy was removed and the pots
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were rearranged to minimize positional effects. The process was then repeated with other butterßies over a 2- to 3-h period. The experiment was conducted between 1200 and 1700 hours (GMT-5) on Þve separate days using fresh plants on each date. The Þrst landing choices of a total of 127 female butterßies were recorded using 20 plants of each variety. The choice tests were conducted indoors at room temperature with overhead ßuorescent illumination. The Þrst landing response was also observed using plants enclosed in air-tight, clear, glass jars to exclude olfactory cues (Reeves 2011). Individual green, variegated, and yellowÐ green Brassica plants were transplanted into 266-ml clear plastic cups. Each cup was then place on a clear, glass, 250-ml beaker to elevate the plant so that the leaves were pressed against the inner surface of an inverted, 1.89-liter clear glass jar. The opening of the jar, located beneath the beaker, was sealed using a screw-cap metal lid with a plastic gasket. Two plants, in separate jars, of each of the three Brassica varieties were used in choice experiments using the same procedure as described with the freely accessible plants. The Þrst landing preference of individual females on the glass jar surrounding either the green, variegated, or yellowÐ green plant was recorded. Landing experiments were performed between 1200 and 1700 hours (GMT-5) on seven separate days using fresh plants on each date. The Þrst landing choices of 127 female butterßies were observed using 14 plants of each variety. Oviposition Preference. The egg laying preferences of naõ¨ve Pieris females were tested using 24 pots (one to two plants per pot) containing 14 plants of each of the three Brassica varieties. The pots were placed at one end of the 321 (length) by 160 (width) by 165 cm (height) test cage and arranged in four rows of six pots, with two pots of each variety randomly placed in each row, separated by 20 cm between the middle of each pot. At 1130 hours (GMT-5), 18 gravid females were simultaneously released at the opposite end of the cage. After 1.5 h, the pots were randomly rearranged to minimize positional effects. After an additional 1.5 h, the butterßies were collected, and the number of eggs deposited on each plant was counted. Oviposition preference was expressed as the median number of eggs deposited per plant for each Brassica variety (Zheng et al. 2010). Larval Performance. As in the oviposition preference test, 15 gravid females laid eggs on 16 plants of each of the three Brassica varieties for 3 h. Afterwards, the butterßies were collected, and the number of eggs deposited on each plant was counted (mean ⫾ SE; 11.2 ⫾ 1.7 eggs per plant on green, 11.1 ⫾ 1.5 eggs per plant on variegated, 6.8 ⫾ 0.8 eggs per plant on yellowÐ green). Because larval mortality is independent of egg density (Root and Kareiva 1984, Jones et al. 1987), the number of eggs on each plant was unaltered. The plants were then transferred under ßuorescent illumination. Ten days after oviposition, but before the plants had become depleted, the larvae (third instars) were counted and individually weighed to determine their fresh body mass. Because the larval mortality of
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Fig. 1. First landing response of P. rapae to freely accessible and enclosed green, variegated, and yellowÐgreen B. rapa. N ⫽ 127 female butterßies for the freely accessible Brassica varieties, and N ⫽ 127 females for the enclosed plants. Percentages with the same letters above the columns are not signiÞcantly different (2 test, z-test, P ⬎ 0.05).
Fig. 2. Median (⫾ Þrst and third quartile) number of eggs laid by P. rapae on green, variegated, and yellowÐgreen B. rapa. N ⫽ 14 plants per variety. Medians with the same letters above the columns are not signiÞcantly different (Wilcoxon signed rank test, P ⬎ 0.05).
laboratory populations is greatest during the Þrst two instars (Jones and Ives 1979), and late-instar larvae can be difÞcult to locate because they migrate (Mauricio and Bowers 1990) and leave the plant to pupate (Jones et al. 1987), the larva to egg ratio on each Brassica plant at the third instar was calculated and used as a measure of larval survival (Radcliffe and Chapman 1966). Data Analyses. The distribution of butterßy landings on the three varieties of freely accessible plants as well as the distribution of landings on the enclosed plants was analyzed using chi-square tests followed by post hoc z-tests (Ikeura et al. 2010). A chi-square test was also used to compare the number of Þrst landings on the freely accessible plants to the enclosed B. rapa (Zheng et al. 2010). A Wilcoxon signed rank test was used to evaluate the median number of eggs deposited on each green, variegated, and yellowÐ green plant (Poelman et al. 2008, Zheng et al. 2010). The mean larva to egg ratio for each of the three varieties were compared using student t-tests. A one-way analysis of variance (ANOVA) was used to compare the weights of larvae reared on the different Brassica varieties (McKenzie and Goldman 2005).
females oviposited on the glass immediately adjacent to the enclosed plant. The Þrst landing response to the enclosed plants was comparable to the response to the freely accessible plants for the green (41.0 vs. 39.4%), variegated (41.7 vs. 40.1%), and yellowÐ green (17.3 vs. 20.5%) varieties (2 ⫽ 0.77; df ⫽ 2; P ⫽ 0.679; Fig. 1). Oviposition Preference. In the oviposition choice test, female butterßies laid more eggs per plant on the green and variegated Brassica plants than on the yellowÐ green variety (Fig. 2). SigniÞcantly more eggs were deposited on the green variety than on the yellowÐ green plants (median; 18 eggs per plant on green vs. 3 eggs per plant on yellowÐ green, n ⫽ 14; P ⫽ 0.002). Likewise, more eggs were laid on the variegated plants than on the yellowÐ green variety (median; 13 eggs per plant on variegated vs. 3 eggs per plant on yellowÐ green, n ⫽ 14; P ⫽ 0.004). The median number of eggs oviposited on the green Brassica variety did not differ signiÞcantly from that on the variegated plants (P ⫽ 0.363). The choice test was conducted three times with comparable results. Larval Performance. Larval survival to the third instar was lower on the yellowÐ green variety than on the green plants (mean larva per egg ⫾ SE; 0.212 ⫾ 0.048 on yellowÐ green vs. 0.394 ⫾ 0.046 on green; t ⫽ 2.74; df ⫽ 30; P ⫽ 0.010; Fig. 3). Larvae reared on the
Results First Landings. In the Þrst landing choice experiment between the three varieties of the freely accessible plants, more female P. rapae butterßies landed Þrst on the green (39.4%) and variegated (40.1%) B. rapa than on the yellowÐ green (20.5%) variety (2 ⫽ 9.47; df ⫽ 2; P ⫽ 0.009; Fig. 1). The percentage of Þrst landings on the green and variegated plants were statistically equivalent (z ⫽ 0.128; P ⫽ 0.890). When the Þrst landing choice experiment was repeated using plants enclosed in glass jars to exclude olfactory cues, female butterßies landed on the jars surrounding the green (41.0%) and variegated (41.7%) Brassica plants more often than on the glass covering the yellowÐ green (17.3%) variety (2 ⫽ 14.67; df ⫽ 2; P ⬍ 0.001; Fig. 1). Nearly identical percentages of females landed on the enclosed green plants as on the enclosed variegated plants (z ⫽ 0.127; P ⫽ 0.890). When given the opportunity, some of the
Fig. 3. Larval survival of P. rapae reared on green, variegated, and yellowÐgreen B. rapa. Data are expressed as the mean (⫾SE) survivorships of the larvae on 16 plants per variety. Means with same letters above the bars are not signiÞcantly different (t-test; P ⬎ 0.05).
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Fig. 4. Mean (⫾SE) fresh weights of P. rapae larvae reared on green, variegated, and yellowÐgreen B. rapa. N ⫽ 30 larvae per variety. Means with same letters above the bars are not signiÞcantly different (ANOVA, P ⬎ 0.05).
yellowÐ green plants also had a lower survivorship than larvae that fed on the variegated Brassicas (mean larva per egg ⫾ SE; 0.212 ⫾ 0.048 on yellowÐ green vs. 0.407 ⫾ 0.072 on variegated; t ⫽ 2.25; df ⫽ 30; P ⫽ 0.032). The survival of larvae that fed on the green and on the variegated plants were comparable (t ⫽ ⫺0.15; df ⫽ 30; P ⫽ 0.885). Larvae raised on either the green, variegated, or yellowÐ green plants were equivalent in weight (mean ⫾ SE; 18.9 ⫾ 2.4 mg on green vs. 19.9 ⫾ 1.8 mg on variegated vs. 20.8 ⫾ 1.6 mg on yellowÐ green; F ⫽ 0.30; df ⫽ 2; P ⫽ 0.741; Fig. 4). Similar larval survival and mass results were obtained in three independent trials. Discussion Foliage color had a signiÞcant effect on the landing response of P. rapae, as more Þrst landings occurred on freely accessible green and variegated plants than on the yellowish variety. For many herbivorous insects, yellow can act as a superoptimal stimulus that elicits alightment on living plants (Prokopy and Owens 1983). Although B. rapa ßower petals are yellow and P. rapae females prefer to land on yellow artiÞcial ßowers (Kandori et al. 2009), we saw no evidence that the female butterßies were especially attracted to the yellowÐ green leaves or mistook the leaves for yellow ßowers and nectaring. Our landing results are similar to those reported in previous studies that have shown an innate landing preference for green foliage (SnellRood and Papaj 2009, Zheng et al. 2010). The landing response of female P. rapae butterßies to the variegated Brassica plant differed from that reported with the variegated Arabidopsis. The variegated phenotype used in our study is a maternally inherited, chloroplast DNA trait, and the Brassica plants displayed mostly green foliage with yellow and white sectors (Williams 1985). None of the leaves were entirely white. The variegated Arabidopsis, however, is a virus-induced phytoene desaturase-silenced plant with many white photobleached leaves (Zheng et al. 2010). The different landing responses seen with the Brassica and Arabidopsis plants suggest that female P. rapae butterßies have the ability to distinguish between different levels of variegation. Mostly green
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variegated leaves appear to be more acceptable to the female butterßies than mostly white plants. The Þrst landing response to the enclosed Brassica plants was comparable with the response to the freely accessible plants. Because the enclosed plants were in air-tight jars, olfactory cues were unavailable to the butterßies. Consequently, visual cues were sufÞcient to orient the butterßies and to induce a landing response. Interestingly, when given the opportunity, some of the gravid females oviposited on the glass jars surrounding the enclosed plants. This egg-laying response may be related to the motivational status of the females (Chew and Robbins 1984), as they were reared in the absence of host plants before the choice tests. However, motivation-based models of host plant selection do not explain the oviposition behavior of P. rapae well (Hopkins and van Loon 2001). Alternatively, these observations suggest that physical contact with the leaf surface is not needed to elicit egg laying. When the gravid females were given the opportunity to land multiple times and lay eggs for 3 h, they oviposited more on the green and variegated Brassica plants than on the yellowÐ green variety. These results are similar to those of previous studies that have shown that P. rapae has an innate egg-laying preference for green artiÞcial substrates (Horvanitz and Chang 1964) and dark green fertilized plants (Meyers 1985, Letourneau and Fox 1989, Chen et al. 2004). We also observed that the oviposition preference was positively correlated with the landing choice on B. rapa, which is also consistent with the results reported from studies conducted with cabbage (Radcliffe and Chapman 1966, Jankowska 2006, Snell-Rood and Papaj 2009) and Arabidopsis (Zheng et al. 2010). Unlike plant age (Jones 1977) and leaf shape (Renwick and Radke 1988), foliage color appears to be an important visual cue used to elicit landings that lead to the acceptance of plants as oviposition sites. Larval survival to the third instar was greater on the green and variegated Brassica plants than on the yellowÐ green variety. These results are in accordance with the oviposition preferenceÐ offspring performance hypothesis (Jaenike 1978), which predicted that reproductive success would result from appropriate host selection. Because cruciferous plants can vary in their suitability as larval food, larval survival and development rates can vary depending on their host (Chew 1975, Ohsaki and Sato 1994). In particular, green foliage can support a greater proportion of surviving larvae than yellowÐ green (Doak et al. 2006), where nutritional limitations could lead to food-related mortality as well as egg cannibalism (Watanabe and Yamaguchi 1993). Such nutritional variances, along with differences in predator and parasitoid avoidance (Loader and Damman 1991, Ohsaki and Sato 1994), can contribute to the selection for female P. rapae butterßies that can discriminate one host plant from another (Darwin 1909). While larval survival was lower on the yellowÐ green variety, the surviving larvae had similar weights as those reared on the green and variegated plants. These results are similar to those reported by Slansky
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and Feeny (1977) and Chen et al. (2004), who studied larval performance on dark green fertilized plants and light green unfertilized plants. Although P. rapae preferred to lay eggs on green leaves of fertilized plants, which supported faster larval development (Meyers 1985, Chen et al. 2004), larvae raised on light green leaves of unfertilized plants compensated by spending more of their time feeding (Loader and Damman 1991), consuming food faster, and using nitrogen more efÞciently (Slansky and Feeny 1977). Consequently, larvae reared on light green unfertilized plants had growth rates and Þnal weights as high as those grown on dark green fertilized plants (Slansky and Feeny 1977, Chen et al. 2004). In our study, the similar weights of the larvae that fed on the different B. rapa varieties suggest that compensatory feeding may have occurred on the yellowÐ green variety. The growth of the larvae reared on the variegated Brassica observed in our study differed from the larval performance reported with the variegated Arabidopsis (Zheng et al. 2010). Larvae raised on the variegated Brassica were equivalent in weight to those reared on the green variety, whereas larvae that fed on the variegated Arabidopsis plants weighed signiÞcantly less than those that fed on the green controls (Zheng et al. 2010). Larval growth is especially limited by the availability of nitrogen in their food (Slansky and Feeny 1977). Because the greenness of leaves is an indication of their nitrogen content (Loader and Damman 1991), our results suggest that the mostly green leaves of the variegated Brassica had sufÞcient nitrogen to support normal larval growth, while the mostly white leaves of the variegated Arabidopsis did not. This study is the Þrst to examine the effects of green, variegated, and yellowÐ green Brassica foliage on the intraspeciÞc host selection of P. rapae. Female butterßies preferred to land on green and variegated plants, and visual rather than olfactory cues were sufÞcient to induce a landing response. The landing choice was positively correlated with oviposition preference and larval survival. Different results were also noted between the variegated Brassica described in this study and the variegated Arabidopsis examined by Zheng et al. (2010). Taken together, our results suggest that leaf color is an important visual cue used by female P. rapae butterßies to select hosts that are suitable larval food plants. Acknowledgments We thank Tim Husband for assistance with the statistical analyses.
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Chew, F. S. 1977. Coevolution of pierid butterßies and their cruciferous foodplants. II. The distribution of eggs on potential foodplants. Evolution 31: 568 Ð579. Chew, F. S., and R. K. Robbins. 1984. Egg-laying in butterßies, pp. 65Ð79. In R. I. Vane-Wright and P. R. Ackery (eds.), The Biology of Butterßies. Academic, London, Great Britian. Darwin, C. 1909. Essay of 1844. In F. Darwin (ed.), The Foundations of the Origin of Species, Cambridge University Press, Cambridge, London, United Kingdom. Doak, P., P. Kareiva, and J. Kingsolver. 2006. Fitness consequences of choosy oviposition for a time-limited butterßy. Ecology 87: 395Ð 408. Hern, A., G. Edwards-Jones, and R. G. McKinlay. 1996. A review of the pre-oviposition behavior of the small cabbage white butterßy, Pieris rapae (Lepidoptera: Pieridae). Ann. Appl. Biol. 128: 349 Ð371. Hopkins, R., and J.J.A. van Loon. 2001. The effect of host acceptability on oviposition and egg accumulation by the small white butterßy, Pieris rapae. Physiol. Entomol. 26: 149 Ð157. Horvanitz, W., and V.C.S. Chang. 1964. Adult oviposition responses in Pieris rapae. J. Res. Lepid. 3: 159 Ð172. Ikeura, H., F. Kobayashi, and Y. Hawata. 2010. How do Pieris rapae search for Brassicaceae host plants? Biochem. Syst. Ecol. 38: 1199 Ð1203. Ives, P. M. 1978. How discriminating are cabbage butterßies? Aus. J. Ecol. 3: 261Ð276. Jaenike, J. 1978. On optimal oviposition behavior in phytophagous insects. Theor. Pop. Biol. 14: 350 Ð356. Jankowska, B. 2006. The occurrence of some Lepidoptera pests on different cabbage vegetables. J. Plant Protect. Res. 46: 181Ð190. Jones, R. E. 1977. Movement patterns and egg distribution in cabbage butterßies. J. Anim. Ecol. 46: 195Ð212. Jones, R. E., and P. M. Ives. 1979. The adaptiveness of searching and host selection behavior in Pieris rapae (L.). Aus. J. Ecol. 4: 75Ð 86. Jones, R. E., V. G. Nealis, P. M. Ives, and E. Scheermeyer. 1987. Seasonal and spatial variation in juvenile survival of the cabbage butterßy Pieris rapae: evidence for patchy density-dependence. J. Anim. Ecol. 56: 723Ð737. Kandori, I., T. Yamaki, S.-I. Okuyama, N. Sakamoto, and T. Yokoi. 2009. InterspeciÞc and intersexual learning rate differences in four butterßy species. J. Exp. Biol. 212: 3810 Ð3816. Langan, A. M., C. P. Wheater, and P. J. Dunleavy. 2001. Does the small white butterßy (Pieris rapae L.) aggregate eggs on plants with greater gas exchange activity? J. Insect Behav. 14: 459 Ð 468. Letourneau, D. K., and L. R. Fox. 1989. Effects of experimental design and nitrogen on cabbage butterßy oviposition. Oecologia 80: 211Ð214. Loader, C., and H. Damman. 1991. Nitrogen content of food plants and vulnerability of Pieris rapae to natural enemies. Ecology 72: 1586 Ð1590. Mauricio, R., and M. D. Bowers. 1990. Do caterpillars disperse their damage?: larval foraging behavior of two specialist herbivores, Euphydryas phaeton (Nymphalidae) and Pieris rapae (Pieridae). Ecol. Entomol. 15: 153Ð161. McKenzie, J. D., and R. Goldman. 2005. The student guide to MINITAB release 14. Pearson Addison-Wesley, Boston, MA. Meyers, J. H. 1985. Effect of physiological condition of the host plant on the ovipositional choice of the cabbage white butterßy, Pieris rapae. J. Anim. Ecol. 54: 193Ð204.
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