Sensory cues in host selection for oviposition by the cabbage butterfly, Pieris rapae

Odl22-19lO/88 53.00+O.OO

J,hecr Physid.Vol. 34,No. 3.pp.251-257.1988

Pergamon Press plc

Printed in Great Britain

SENSORY CUES IN HOST SELECTION FOR OVIPOSITION BY THE CABBAGE BUTTERFLY, PIERIS RAPAE J. A. A. RENWICK and CELIA D. RADKE Boyce Thompson Institute, Cornell University, Ithaca, NY 14853, U.S.A. Abstract-The behavioural events leading up to oviposition by Pieris rapae and the sensory modalities involved in host selection by these butterflies are reviewed. The process of host finding and acceptance is divided into three phases: searching, landing and contact evaluation. Little is known about sensory aspect of searching, but visual stimuli play some role. Landing appears to be mediated primarily by visual cues, of which colour is the most important. Choice experiments using artificial leaves of identical spectral and chemical properties revealed little or no preference for shape or size of oviposition substrate. Contact evaluation depends on positive or negative chemical stimuli at the leaf surface. Oviposition stimulant was extracted from cabbage leaves and partially isolated by solvent extraction and a series of chromatographic separations. Chemical constituents other than glucosinolates appear to be responsible for the discriminatory behaviour of ovipositing butterflies. These stimulatory compounds are water-soluble but many are less polar than the glucosinolates, and may be active alone or in combinations. Identification of these compounds would greatly improve our understanding of the mechanism of host acceptance by cabbage butterflies. Key Word Index: Pieris rupue, cabbage butterfly, oviposition, chemoreception,

INTRODUCTION of host selection by insects feeding on plants of the family Cruciferae has been the subject of numerous studies during the last few decades. The reason for this interest lies partly in the economic importance of cruciferous crops and in the relatively well known, distinctive chemistry of the plants. A close link between plant chemistry and herbivory by crucifer specialists was first pointed out by Verschaffelt (191 l), who noted that the host range of the cabbage butterflies, Pieris brussicae L. and P. rapae L., is limited to plants containing mustard oil glucosides (glucosinolates). This observation has triggered extensive studies on chemical aspects of crucifer-herbivore interactions. The glucosinolates are believed to represent a major line of chemical defence against invading organisms (Feeny, 1977), but a wide array of insects has apparently adapted to these compounds. In fact, most of the pests of brassica crops are specialists (Root, 1973) and many of these insects actually use the glucosinolates or their hydrolysis products (mustard oils) as positive signals for recognition of suitable host plants (reviewed by Schoonhoven, 1972, Feeny et al., 1983, and Chew and Robbins, 1984). Despite the clear correlation between the presence of glucosinolates and the host range of many insects attacking crucifers, these glycosides alone are not always responsible for host finding and acceptance by specialist insects (Chew, 1987; Neilsen, 1979; Nielsen et al., 1979; Renwick, 1983; Renwick and Radke, 1983). Nevertheless, emphasis on the role of glucosinolates and the mustard oils in host finding and acceptance by crucifer specialists has led to many assumptions and efforts to demonstrate such a role. As a result, other aspects of host selection by cruciferThe process

host selection, vision

feeding insects have received less attention, and there are several gaps in our understanding of the sensory modalities involved. The behaviour of each insect species needs to be studied separately, without any preconceptions about the involvement of specific groups of plant chemicals. The small white cabbage butterfly, P. rapae, has been widely studied in different parts of the world. Many of these studies have shown close similarities in host preferences and behaviour of this species to the large white cabbage buttefly, P. brassicae. However, since P. brassicae lays its eggs in clusters, while P. rapae lays single eggs, these species are likely to have different strategies for locating suitable sites for oviposition (Davies and Gilbert, 1985), and careful analysis of the similarities and differences is necessary to obtain a clear picture of host finding and acceptance by each species. In this paper we review our present knowledge of the sensory cues leading to oviposition by P. rapae and describe experiments to provide additional information about the process of host selection by these butterflies. The behavioural events leading up to oviposition by P. rapae include a searching flight, landing, and contact evaluation of potential host plants.

SEARCHING The sensory modalities involved in the initial searching phase of host finding by P. rapae are not

well understood, but certain patterns of flight have been observed. The first task of the butterflies is to locate suitable habitats, and then to identify patches of vegetation that contain potential host plants. Cabbage butterflies appear to restrict their search to open areas (Klots, 1951; Voss and Wagner, 1956). 251

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and avoid cool, shaded woodlands even when host plants are available in these areas (Cromartie, 1975a). These observations are consistent with the fact that gravid butterflies do not oviposit during overcast weather (Gossard and Jones, 1977) and high light intensity is required for oviposition in the laboratory (Renwick and Radke, unpublished results). The flight paths of ovipositing P. rapae tend to be linear and independent of wind direction or position of the sun (Root and Kareiva, 1984). Since a gravid female usually lays only a single egg on each plant and the females pass over many suitable hosts, Root and Kareiva (1984) have suggested that this behaviour represents an “egg-spreading syndrome”. However, differences in flight patterns between P. rapae populations in Canada and Australia have been noted (Jones, 1977; Jones and Ives, 1979), so searching strategies may vary slightly between geographically separated populations. Other factors, such as the attractiveness of host plants that appear to be in better physiological condition, may also affect searching behaviour. Myers (1985) observed deviation from the usual linear flight paths of butterflies when particularly attractive plants were encountered. In such cases butterflies would tend to remain in the area and lay several eggs on these plants.

LANDING

The sensory cues that elicit landing of butterflies on potential host plants play a critical role in the process of host selection, since the final signal for oviposition or rejection usually depends on contact chemoreception of stimulants or deterrents (Dethier, 1964). The two sensory systems that are most likely to influence the “decision” to land on a plant are vision and olfaction. Visual stimuli

The role of vision in the orientation of flying insects to potential host plants has been reviewed by Prokopy and Owens (1983) and the most important stimulus for most insects, including P. rapae, appears to be colour. Gravid females of the cabbage butterfly have been shown to respond to green and blue/green colours for oviposition (Hovanitz and Chang, 1964). Detailed studies on P. brassicae have elegantly demonstrated the wavelength-specific requirements of this species for drumming behaviour and egg-laying (Kolb and Scherer, 1982). The precise spectral characteristics necessary for landing of P. rapae on artificial leaves have recently been investigated, and a preference was shown for surfaces with a maximal reflectance at 550 nm (unpublished results). Furthermore, a degree of associative learning in these butterflies has been demonstrated (Traynier, 1984, 1986). Butterflies preferred colours that were previously associated with the presence of a chemical stimulant (sinigrin). Other visual stimuli that affect landing for oviposition by butterflies include leaf shape and size (Rausher, 1978; Stanton, 1982). The response of P. rapae to different plant or leaf size has been studied by Ives (1978) and Jones and Ives (1979) who found that landing is more likely to occur on larger plants.

and

CELIA D. RADKE

However, landings on smaller, younger plants are more likely to result in oviposition, so more eggs are eventually laid on plants of intermediate size. In studies with different cabbage varieties, two groups of investigators concluded that larger plants were preferred for oviposition (Latheef and Irwin, 1979; Radcliffe and Chapman, 1965). One problem in interpretation of these results is that plants or leaves of different age differ in their colour and chemistry. Also, the effect of leaf shape on oviposition by cabbage butterflies has not been studied. In this paper we present results of tests to examine the effects of leaf shape and size on oviposition preferences using artificial leaves that have identical spectral and chemical qualities. Olfactory stimuli

The involvement of olfaction in the location of host plants by P. rapae adults has yet to be demonstrated. The remarkable ability of butterflies to find host plants in uncultivated plots containing diverse vegetation (Cromartie, 1975b; Root and Kareiva, 1984) would suggest that stimuli other than vision must be involved. However, Hovanitz and Chang (1964) found that the mustard oil, allylisothiocyanate, was not attractive to ovipositing butterflies and may be slightly repellent. We have similarly shown that the volatiles from homogenized cabbage leaves are not attractive and may in fact have a slight inhibitory effect (Renwick and Radke, 1983). A role of non-host-plant volatiles in the distribution of eggs by P. rapae has been suggested. A mixture of herbs interplanted with collard plants appeared to cause clumping of eggs on the collards (Latheef and Ortiz, 1983) and a similar concentration of eggs on collards surrounded by tomato plants occurred (Maguire, 1984). These results indicate that butterflies are repelled by some non-host plants. Hence the involvement of olfaction in landing appears to be restricted to an avoidance response to non-hosts and the absence of negative signals from potentially acceptable plants. CONTACT EVALUATION

Once a butterfly has landed on a plant, tactile and contact chemical stimuli are likely to be the major factors affecting acceptance or rejection of the site for egg deposition. Tactile stimuli

Information on the involvement of tactile stimuli in oviposition behaviour of P. rapae is limited. In one of the few studies of different leaf textures, varietal comparisons indicated little or no effect of dimpling (Latheef and Irwin, 1979). Laboratory tests of different substrates treated with cabbage extracts have suggested a preference for smooth hard surfaces such as index cards over rougher and softer textures like blotting paper or felt (Renwick and Radke, unpublished). Many butterfly species are known to undergo a “drumming reaction”. or rapid movement of the forelegs across the surface of a leaf after alighting (Use, 1937). The behaviour which is also observed in P. rapae (Stadler and Renwick, un.oublished observations) is believed to provide both

Oviposition by Pieris rapae physical and chemical information about the suitability of the plant. Since leaves of most brassica plants are well COVered by epicuticular wax (Martin and Juniper, 1970) drumming may serve to dislodge wax crystals so that the polar stimulants for oviposition can be detected (Stldler, 1986). Chemical stimuli

The chemistry of host recognition by P. rapae after landing remains to be elucidated at this time. Traynier (1979) has shown that the butterfly’s ovipositor is not involved in assessing the chemical suitability of a site, but that tarsal chemoreception provides the critical information for acceptance of a plant for oviposition. Chemical stimulants can be extracted from leaves of host plants using polar solvents, and an inert green substrate treated with plant fractions or individual compounds has been used to evaluate stimulatory activity (Renwick and Radke, 1983). The general idea that glucosinolates play a significant role in the specificity of crucifer-herbivore interactions has led to some confusion as to the extent of their involvement (Chew, 1987). Sinigrin alone does not release the highly selective oviposition behaviour in P. rapae that is obtained with cabbage extracts (Renwick and Radke, 1983), although some ovipositional response of the butterflies to sinigrin was obtained when this compound was used in associative learning experiments (Traynier, 1984, 1986). Also, oviposition preferences are not related to glucosinolate levels in plants grown under different nutritional conditions (Wolfson, 1980). One problem that contributes to apparently conflicting results is the variation in responses of individual insects. Most P. rapae females in our laboratory ignore green cards treated with sinigrin solution, and withhold their eggs if nothing more suitable is presented to them. However, oviposition is triggered in a few individuals, and these butterflies lose their ability to discriminate between control cards and those treated with the normally stimulating cabbage extract (Renwick, 1987). In general, butterflies in our greenhouse cages never lay eggs on substrates that are not coated with stimulant. But in Australian experiments, (Traynier, 1986) butterflies often laid eggs on substrates treated with water alone. So differences in discriminatory ability of geographically separate populations may occur. Results from bioassays using solvent extracts of a wide variety of plant species have revealed the presence of oviposition deterrents to P. rapae in host as well as non-host plants (Renwick and Radke, 1985). Non-polar extracts of hosts including cabbage were deterrent, but polar extracts had no effect. However, both polar and non-polar extracts of unacceptable plants were deterrent. Two of these plants, Erysimum cheiranthoides L. and Capsella bursa-pastoris (L.), are crucifers, which one might expect to be hosts. Subsequent experiments have shown that E. cheiranthoides contains oviposition stimulants as well as deterrents (Renwick and Radke, 1987). These results support the idea that a balance of positive and negative stimuli determines whether a plant is accepted for oviposition (Dethier, 1982; Miller and Strickler, 1984).

253

The chemistry of plants that serve as hosts for cabbage buttertlies is quite variable. Despite the common link provided by the presence of glucosinolates in all the host plants, these glycosides have extremely diverse aglycones. It is therefore unlikely that any one compound can explain the stimulatory activity resulting in oviposition. In this paper we describe experiments to isolate oviposition stimulants for cabbage and the development of a general separation scheme which might lead to identification of the chemical constituents involved. MATERIALS AND METHODS

Insects

The butterflies used in all bioassays were from a colony maintained on cabbage plants in the laboratory at about 22°C under fluorescent lights providing a photoperiodic regime of 16 h light-8 h dark. The colony was renewed annually with fieldcollected butterflies so that experimental insects had completed no more than 16 generations in the laboratory at any time. Pupae were separated by sex (Richards, 1940) and after eclosion, buttedies were transferred to greenhouse cages for mating and oviposition on cabbage plants. Butterflies in colony and bioassay cages were provided with vials of 10% sucrose solution containing yellow food colouring and a cotton wick to facilitate feeding. Supplementary lighting in the greenhouse was supplied by 400 W multivapour, high-intensity discharge lamps with a 16 h photophase. Plants

Cabbage plants (var. “Golden Acre”) for rearing insects and preparation of extracts were grown in artificial soil mix (Cornell mix A, with osmocote; Boodley and Sheldrake, 1977) in a greenhouse with supplementary lighting. The plants were generally 68 weeks old when used for extracts. Bioussays

The isolation of oviposition stimulant from extracts of cabbage was monitored using a standard assay in screened cages (48 x 48 x 48 cm) in the greenhouse (Renwick and Radke, 1983). Test extracts and fractions were painted on green index cards, 12.7 x 7.6 cm (Esselte Pendaflex Corp., Garden City, New York) supported on vertical sticks. Five pairs of butterflies were offered a choice of treated cards or control cards, which were painted with solvent alone. The eggs on each card were counted after a 24 h period. For experiments on the effects of shape and size on oviposition, the same cards were painted with standard water extract of cabbage (Renwick and Radke, 1983) at a concentration of 5 g fresh weight leaf equivalent/ml. The treated cards were cut into squares (6.3 x 6.3 cm), circles (7.1 cm diam.), triangles (10.4 x 9.1 x 9.1 cm), and rectangles (12.7 x 3.2 cm) of approximately equal area (40 cm*). The effect of size of surrogate plants on oviposition preference was investigated using treated cards cut into small (2.5 x 2.5 cm), medium (5.0 x 5.0 cm), and large (7.5 x 7.5 cm) squares. Substrate size and shape effects were tested separately in binary choice tests between all possible pairs of cards, using two of each

254

J. A.

A.

RENWICK

and

CELIA

D.

RADKE

in diagonally opposite corners of cages to minimize position effects. All tests were replicated with different batches of 5 pairs of butterflies.

EoAing ethanol Evaporation Hexane wash water Wml

Chemical isolation

Extraction of stimulants from cabbage foliage was performed as described previously (Renwick and Radke, 1983) using boiling ethanol, followed by lipid removal with hexane and taking up the active material in water. Partitioning of the water extracts with n-butanol resulted in removal of inactive material, and the water fraction was then subjected to cation exchange chromatography (Bio-Rad AG SOW-X8, ammonium form). After desorption of cations with 0.5 N NH,OH, all the stimulatory activity was found in the unabsorbed anions and neutral fraction. The aqueous extract was further separated by size exclusion chromatography on a column of Bio-Gel P-2, 200-400 mesh (Bio-Rad Laboratories, Richmond, Calif.) using water for elution. All activity was obtained in the late fractions (low molecular weight). The active material was finally separated according to polarity by high-performance liquid chromatography on a semi-preparative reversed phase column (Varian MCH-10, 30 cm x 8 mm). The solvent programme from water to acetonitrile was completed in 40 min at a flow rate of 3 ml/min. The eluate was collected as three major fractions, A, B and C, according to major areas of ultraviolet absorption at 254 nm.

Eflect of shape

Choice tests to determine the effects of artificial leaf shape on oviposition showed no preference (Table 1). I. Oviposition

by P. rapae on stimulant-treated of different shapes*

Number of eggs replicates -______

green cards

1

2

3

Total No. of eggs

Square Triangle

48 51

I5 42

83 46

I46 139

Square Rectangle

48 79

32 51

84 69

164 199

Circle Square

43 44

51 30

62 59

156 133

Circle Triangle

45 41

I6 21

65 55

126 129

Rectangle Triangle

46 48

43 61

81 81

176 190

Rectangle Circle

40 45

20 33

48 52

108 130

Shape

*No significant

differences

Table 2. Oviposition by P. Butterflies (5 pair/replication)

N -8utanol extraction

r *Hz0

L BUOH Cation exchanger

A

A

B t

C l

1. Separation scheme for isolation of oviposition stimulant for P. rapae from host plants. *Indicates fractions with the highest stimulatory activity.

Fig.

Total area, 2 cards (cm2 )

EfSect of size

When provided with a choice of oviposition substrates that were identical except for size, butterflies tended to lay more eggs on the larger of the two sizes of cards, but the differences were not significant (Table 2). When compared on the basis of area presented, the density of eggs on the smaller of each pair was significantly higher (Table 2). Partial isolation of oviposition stimulant from cabbage leaves was accomplished according to the separation scheme in Fig. 1. Fresh leaves were dropped into boiling ethanol, which was allowed to cool after 5 min and the tissue was homogenized in a Waring blender. The homogenate was filtered through glass wool under vacuum and the filtrate evaporated to dryness. The resulting residue was extracted first with hexane and then with water, and each extract was filtered. All the stimulatory activity was in the water fraction. Partitioning of the water

rqm on stimulant-treated pairs of cards of different sizes. were offered a choice of two cards of each size in each test No. of replications

Mean No. of eggs on 2 cards &SE

Mean No. of eggs/c& & SE

Large Medium

112.6 50.0

5

54.0 i 21.5 42.4 k 14.5”’

0.5 f 0.2 0.9 f 0.3’

Medium Small

50.0 12.3

7

54.6 + 13.0 43.6 i I I .9”’

I.1 kO.3 3.5 f 0.9**

112.6 12.3

7

54.3 f 8.8”” 34.9 + 9.6

0.5 * 0.1 2.8 + 0.8**

Large Small

soluble

Isolation of stimulant

at c[ = 0.05 (1’ test).

Size

*Water

Squares, circles, triangles and rectangles were equally acceptable in all possible pairwise comparisons.

RESULTS

Table

i L Hexone soluble

**P < 0.01; *P G 0.05; “‘P

> 0.05; Wilcoxon

signed rank test.

255

Oviposition by Pieris rapae

Table 3. Oviposition by P. rqme on cards treated with crude cabbage extract and HPLC fractions* Total eggs

J

20% CH3CN 100%CH3CN

HP0 10%CH3CN

Fig. 2. HPLC chromatogram of oviposition-stimulating isolate of cabbage extract (detection by ultraviolet at 254 nm). with n-butanol resulted in removal of considerable colour and other inactive material into the butanol. Cations were removed by the ion-exchange chromatography, and the active unadsorbed material was further fractionated by size exclusion chromatography. The activity was found in the low molecular weight, late fractions. At each step, the activity was clearly present in one fraction only, and the oviposition response of the butterflies resulted in almost perfect discrimination between test and control cards. The final step in the isolation scheme was high pressure liquid chromatography (HPLC), which gave three major areas of ultraviolet absorption with monitoring at 254 nm (Fig. 2). Bioassays of the three fractions indicated a degree of stimulatory activity in each of the fractions A, B and C (Table 3). However, butterffies exposed to fraction A did not discriminate well between test and control cards. Twenty-four per cent of the eggs were laid on the controls, which lacked stimulant. The stimulatory effect of B and C was more similar to that of the original crude extract, which elicits more precise discrimination (Table 3). Collection and bioassay of multiple small fractions from the HPLC column showed that the main areas of activity were clearly separated by inactive regions. Thus, different compounds are responsible for the stimulatory activity of each fraction. DISCUSSION

These results suggest that the visual stimuli affecting landing for oviposition by P. rapae are relatively simple. Butterflies showed no discrimination between surrogate leaves of different shapes, and leaf size appeared to have little effect on choice of oviposition site. Although some more eggs were laid on large squares than on small ones in a choice test (Table 2), no clear preference was shown. As a result, a much higher density of eggs was obtained on the smaller surface (Table 2). One might expect the visual image presented by a large leaf or plant to be more attractive to butterflies as a more plentiful source of food for their progeny. Also, the more conspicuous, larger surface is more readily available for landing. However, the dominant visual stimulus for cabbage butterflies appears to be colour, so landing on small plants is almost as likely as on large plants of the same colour. Field observations of more frequent landing on larger plants (Ives, 1978; Jones and Ives, 1979; Latheef and Irvin, 1979; Radcliffe and

Fraction

N

T

C

Crude extract A B C

6 I 9 6

818 699 900 592

220 50 26

I

Mean OPlt f SE

100.0 * 0.02” 42.6 f 2.4b 94.2 + 1.9” 92.4 + 0.6’

*Means followed by the same letter not significantly different in multiple pairwise comparisons by unpaired t-test at c( = 0.009 (experimentwise Q = 0.05); data transformed to arcsin ,/P for analysis. tOGposition Preference Index [(T - C)/(T + C)] x IM).

Chapman, 1965) may be explained by changes in colour with plant age. The intense blue/green of older leaves of many brassica plants is probably more attractive than the pale yellow/green of young leaves (Hovanitz and Chang, 1964). The chemical mechanism of host recognition by P. rapae appears to be much more complex than the visual mechanism. The isolation of fractions containing oviposition stimulants from cabbage and subsequent separation by HPLC indicates that several different compounds are involved. The activity of fractions B and C resembles that of original water extracts of cabbage, but the less discriminatory behaviour triggered by fraction A is more similar to the response to sinigrin (Renwick and Radke, 1983). When the glucosinolates, sinigrin and glucotropaeolin, were separated on the HPLC system using the same solvent programme, these eluted early in fraction A. The activity of the cabbage fraction A may therefore be explained by the presence of glucosinolates. However, the discriminatory response to fractions B and C suggests that less polar compounds are involved in the precise process of host recognition. Additive effects of different host constituents are likely, and the possibility of synergistic action cannot be discounted. Preliminary studies on extracts of other host plants indicated that oviposition can be stimulated by different compounds or groups of compounds from the different sources, and the fraction that would contain glucosinolates is not always involved (Renwick and Radke, unpublished). The individual chemical constituents responsible for the stimulation of oviposition on a host plant remain to be identified. CONCLUSIONS

The sensory cues that mediate host selection for oviposition by cabbage butterflies include visual, tactile and chemical information. The most crucial stimuli seem to be colour (to induce landing) and chemistry of the leaf surface for final evaluation. Although many other factors may play a role in host finding and acceptance, leaf shape and size are not important, and butterllies do not appear to be attracted by plant volatiles (Renwick and Radke, 1983). In contrast to some other crucifer specialists, P. rapae adults do not respond to the volatile mustard oils, and the involvement of glucosinolates in acceptance is questionable. Tarsal receptors appear to be involved in the assessment of leaf surface chemistry,

J. A. A.

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and oviposition deterrents can play a major role in the rejection of unsuitable plants. Stimulation of oviposition on host plants depends on the presence of one or more polar compounds that may differ from one host plant to another. These chemical stimulants may be structurally related, but remain to be identified. Based on an evaluation of the literature and our own results, the behavioural events leading to oviposition by P. rapae and the sensory modalities involved may be summarized as follows: Searching and landing. This phase of the hostfinding process is probably guided primarily by avoidance of shady areas, and the use of colour cues to locate suitable patches of vegetation. No evidence for orientation to olfactory stimuli has been found. The predominant stimulus inducing butterflies to land is the spectral quality of potential host plants. Olfaction does not play a role in positive orientation to hosts, but may be involved in avoidance of nonhost plants. Contact evaluation. Little is known about the tactile cues that might be involved in assessing the suitability of a leaf for oviposition. Drumming behaviour may provide some tactile information, but is more likely to result in better access to chemical cues. Acceptance of a site for oviposition depends on tarsal contact with polar stimulants, but the presence of deterrents can counteract this effect, and oviposition may be regulated by the balance of these positive and negative factors. Despite the extensive volume of studies on P. rapae, several questions remain to be answered. Can butterflies recognize host plants before landing? How do volatiles from non-host plants affect the behaviour of butterflies? Are butterflies receiving the same sensory information from different stimulants in different plants? Are deterrents and stimulants detected by different receptors? The chemistry of the leaf surface is obviously very important, and identification of the compounds involved could provide valuable information to address some of these questions. Acknowledgements-We thank Dr Michael B. Dimock and Dr P. R. Hughes for comments on the manuscript. This material is based upon work supported by the U.S. Department of A&culture under Agreement No. 86-CRCR-l-2007 and by a grant from the Cornell University Biotechnology Program. REFERENCES

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