GLUCOSINOLATES and DIFFERENTIAL HERBIVORY in WILD POPULATIONS of Brassica Oleracea

Journal of Chemical Ecology, Vol. 26, No. 11, 2000

GLUCOSINOLATES AND DIFFERENTIAL HERBIVORY IN WILD POPULATIONS OF Brassica oleracea

CATHERINE L. MOYES,1,* HAMISH A. COLLIN,2 GEORGE BRITTON,2 and ALAN F. RAYBOULD1

1 Institute of Terrestrial Ecology Furzebrook Research Station Wareham Dorset, BH20 5AS, UK 2 Department of Biological Sciences University of Liverpool P.O. Box 147, Liverpool, L39 3BX, UK

(Received February 25, 2000; accepted July 13, 2000)

Abstract—Glucosinolates are known to elicit responses from Brassica herbivores in laboratory studies. To study their importance in interactions with herbivores in the field, glucosinolate profiles and levels of herbivory were ascertained for wild cabbage plants growing in four neighboring populations in the UK. Glucosinolate profiles differed between plant populations, but not between different habitats within populations. Within habitats, there was no link between individual plant glucosinolate profiles and herbivory by Pieris spp., slugs and snails, flea beetles or aphids. Plants attacked by the micromoth, Selania leplastriana, contained higher levels of 2-hydroxy-3-butenylglucosinolate and 3-indolylmethylglucosinolate than plants within the same population that were not attacked. It is concluded that the differences in glucosinolate profiles between the plant populations are unlikely to be due to differential selection pressures from herbivores feeding on the mature plants over the two years studies.

Key Words—Defense compounds, cabbage, invertebrate herbivory, host choice, Brassica, glucosinolates.

INTRODUCTION

To understand the importance of plant secondary metabolites in interactions with herbivores more completely, we need information on these compounds and their

*To whom correspondence should be addressed at: Brassica and Oilseeds Research Department, John Innes Centre, Colney Lane, Norwich, NR47UH, UK; e-mail: [email protected]

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0098-0331/ 00/ 1100-2625$18.00/ 0  2000 Plenum Publishing Corporation 2626 MOYES, COLLIN, BRITTON, AND RAYBOULD interactions in natural plant communities. Many of the vast number of plant secondary metabolites are thought to play a role in defense against herbivory (Ben- net and Wallsgrove, 1994). However, much of the data supporting their role in herbivore choices comes from laboratory trials. Effects that are statistically significant in a controlled environment are not necessarily ecologically significant (Gurevitch and Collins, 1994). Glucosinolates are sulfur- and nitrogen-containing compounds found in the order Capparales. They are subdivided into three classes, aliphatic, indolyl, and aromatic. Glucosinolates have frequently been quoted as both a defense against generalist herbivores and an agent for host choice by specialist herbivores (Louda and Mole, 1991; Herms and Mattson, 1992; Renwick, 1988). Differences in glucosinolate profiles between species within the Cruciferae have been linked to the host preferences of specialist herbivores (Rodman and Chew, 1980), although other differences between these species may confound these results (Stadler¨ et al., 1995). Despite the wealth of studies on herbivore responses to these compounds in controlled environments (reviewed in Chew, 1988), there is little evidence for the importance of their role in determining levels of herbivory on plants in natural populations. The only study to address this issue was limited by the methods for glucosinolate analysis, which did not allow the quantification of each individual glucosinolate (Louda and Rodman, 1983). As many herbivores of the Cruciferae have been shown, in the laboratory, to respond differentially to each glucosinolate this is a serious drawback. More recently Mithen et al. (1995) analyzed the individual glucosinolate profiles of Brassica oleracea subsp. oleracea in natural populations. They found differences in allele frequencies at the loci controlling aliphatic glucosinolate side-chain structure and differences in quantities of glucosinolates produced between populations at Kimmeridge and St Aldhelm’s Head on the Dorset coast, England. Mithen et al. (1995) proposed that these find- ings could reflect differential selection pressures from generalist and specialist herbivores at these sites. The first aim of this study was to look in more detail at the differences in glucosinolate profiles between and within the above populations and two flank- ing populations. If herbivores do indeed exert a selection pressure on the alle- les controlling glucosinolate production, they must first show a preference for individual plants based on their glucosinolate profiles. In this study, patterns of herbivory were compared to glucosinolate profiles of individual plants, within each population, to look for evidence of host choice.

METHODS AND MATERIALS

Site Description and Transect Setup. Four discrete cabbage populations were used in this study and their locations are shown in Figure 1. The cab- GLUCOSINOLATES AND HERBIVORY IN Brassica 2627

FIG. 1. Map of part of the Dorset coast, England showing the positions of each plant population and transect. bage populations cover a variable habitat within each site, with plants growing at the base of cliffs in shingle, on cliff faces, and on cliff tops in sparse to dense vegetation. To include variability in habitats within plant populations, two transects were set up at each of the Durdle Door, Kimmeridge, and St Aldhelm’s Head sites. Approximate positions of each transect are given in Figure 1. Trans- ect 1 at Durdle Door (DD1) was situated along the base of a cliff with moderate vegetation cover adjacent to a pebble beach. Transect 2 at Durdle Door (DD2) was situated on a shallow cliff with moderate vegetation cover, running down to the sea. The transect at Handfast Point (HP) was situated at the top of a sheer cliff face at the junction between dense vegetation on the cliff top and sparse vegetation on the cliff face. Transect 1 at Kimmeridge (K1) was situated on a vertical shale cliff face with sparse vegetation, at approximately 1.5 m above the stone beach. Transect 2 at Kimmeridge (K2) was situated in dense vegetation on the cliff top approximately 5 m from the cliff edge. Transect 1 at St Aldhelm’s Head (SA1) was situated in dense vegetation on the cliff top approximately 50 m back from the cliff edge. Transect 2 at St Aldhelm’s Head (SA2) was situated on a rocky scree with moderate vegetation cover approximately 10 m back from the cliff edge. In October 1994, 49-m lines were marked with canes at DD1, DD2, K2, SA1, and SA2 in the middle of a patch of cabbage plants. At 1-m intervals, starting from 0 m, the closest plant perpendicular to the line was marked. This gave 50 marked plants. At HP and K1 this was not possible due to the inacces- sibility of the plants. At HP, the 50 plants were marked at irregular intervals at points where the cliff face populations reached the cliff top. At K1, 36 plants 2628 MOYES, COLLIN, BRITTON, AND RAYBOULD were marked at irregular intervals where the cliff face populations came down to head height. High mortalities were sustained in the first year (largely through cliff erosion), and so in October 1995 replacement plants were marked. Plants marked were those closest to the individuals lost. Herbivores. All herbivores found on the foliage of marked plants were identified (Kerney et al., 1987; Kirk and Gray, 1992; W. E. Rispin, Institute of Terrestrial Ecology, UK, personal communication, P. Stirling, Dorset County Council, UK, Personal communication) and recorded together with associated damage, every three weeks from October 1994 to September 1996. The total leaf area was estimated from the area of five leaves, calculated by using a grid printed onto an acetate sheet, multiplied by the total number of leaves. The proportion of leaf area damaged was estimated by counting the number of leaves with 0/ 5, 1/ 5, 2/ 5, 3/ 5, 4/ 5, and 5/ 5 of leaf tissue lost, and then the mean of these values was used to calculate the proportion damaged per plant. The proportion of leaf tissue lost was combined with the total leaf area to give an estimate of leaf area eaten. This method of estimating proportions and areas of leaf tissue consumed is not the most accurate, as the leaves are not all the same size. Therefore, under- and overestimates may be obtained if the leaves damaged are bigger or smaller, respectively, than the mean leaf size (Williams and Abbot, 1991). However, B. oleracea subsp. oleracea is a slow growing plant with little variation in leaf size within individual plants, and this method enabled damage to be recorded for a large number of plants at frequent intervals. Aphid infestation of flowering racemes was scored on every raceme as either 0 (no aphids), 1 (occasional aphid), 2 (part of raceme covered), or 3 (entire raceme covered). A mean score was then calculated for each plant. Leaf Extraction and Conversion to Desulfoglucosinolates. Leaves were collected from every marked plant in the week commencing October 9, 1995. A range of mature, but not senescent, leaves was taken from each plant with a minimum number collected of three. The leaves were immediately frozen at −708C, freeze dried, and stored at 48C over silica gel. The sample of freeze dried material (0.4 g or 0.3 g where material was limited) was coarsely ground and placed in 10 ml methanol at 708. The standard (300 ml) of 8 mM 2-propenylglucosinolate (Sigma; sinigrin monohydrate from horseradish) was added (each sample was extracted with and without the standard), and the extract was incubated at 708C for 20 min, vortexing twice. The extract was then centrifuged at 1000g for 3 min, and 3 ml of the supernatant was passed through a DEAE Sephadex A-25 column. This was washed with 2 × 0.5 ml water and 2 × 0.5 ml sodium acetate (pH 5). Bottles were placed under the columns and 75 ml of purified sulfatase was layered on top of the resin and left overnight. The resulting desulfoglucosinolates were eluted with 2 × 0.5 ml and 1 × 0.25 ml of water. GLUCOSINOLATES AND HERBIVORY IN Brassica 2629

Separation and Quantification of Glucosinolates. The high-performance liquid chromatography (HPLC) system used consisted of a Perkin-Elmer Series 3 liquid chromatograph attached to an Adsorbosphere HS C18 column (250 × 4.6 mm, 5 mm). The desulfoglucosinolate-containing solution (5 ml) was injected into the system (Rheodyne injection system). The mobile phase had a flow rate of 1 ml/ min and the solvents used were: (A) 25% (v/ v) water (twice distilled), 25% acetonitrile (HPLC grade, BDH) and 50% methanol (HPLC grade, BDH) and (B) water (twice distilled). The program consisted of 15% A for 5 min, a linear gradient over 20 min to 99.9% A, held constant for 5 min and 10 min to return to equilibrium. The eluted desulfoglucosinolates were monitored with a Perkin-Elmer LC-75 spectrophotometric detector (6-mm pathlength flow cell, 8-ml cell volume) at 229 nm and 2 aufs. Chromatograms were recorded by means of a Hewlett-Packard model 3396A integrator. Leaf samples were injected in a completely randomized order and 30 samples were repeated. Identification of Individual Glucosinolates. The peaks separated by HPLC were identified by mass spectrometry. The HPLC column was linked to a VG- Quattro Quadrupole mass spectrometer to obtain atmospheric pressure chemical ionization (APCI) spectra. The conditions used for the HPLC separation were the same as above and for the mass spectrometry—corona 4.13 kV, cone 73 kV, skimmer offset 5 kV, 1208C source temperature, and the probe was heated to 2008C. The spectra were acquired by scanning from m/ z 80 to m/ z 700 every 3 secs. The use of APCI enabled us to obtain protonated molecular ions, (M+H)+, which could be used together with the protonated fragment ions to identify the HPLC peaks. 2-Propenylglucosinolate was identified by the presence of ions at m/ z 118. We identified 3-butenylglucosinolate by the presence of ions at m/ z 294 and 132, and 2-hydroxy-3-butenylglucosinolate was identified by the presence of ions at m/ z 310, 116, 131, and 148. 3-Indolylmethylglucosinolate was identified by the presence of ions at m/ z 369, 130, and 207. 1-Methoxy-3-indolylmethylglucosinolate was identified by the presence of ions at m/ z 399, 160, and 237. The structures of these ions are given in Hogge et al. (1988).

RESULTS

Herbivores. The herbivores found were Phyllotreta Foudras spp. (Coleop- tera: Chrysomelidae), Pieris brassicae (Lepidoptera: Pieridae), P. rapae (Lep- idoptera: Pieridae), Selania leplastriana (Tortricoides: Olethreutinae), Brevico- ryne brassicae (Homoptera: Aphididae), and slugs and snails (Tables 1–4). Pieris napi (Lepidoptera: Pieridae) and Plutella xylostella (Lepidoptera: Plutellidae) were recorded on rare occasions. Slugs and snails are generalists, whereas the other groups are specialist herbivores of crucifers. Glucosinolates. Five principal glucosinolates were found in this study, 2- 2630 MOYES, COLLIN, BRITTON, AND RAYBOULD

TABLE 1.LEAF AREA DAMAGED BY FLEA BEETLES AND MOLLUSCS PER PLANT OVER 12 MONTHS, FOR EACH TRANSECT

Area damaged (%, mean ± SE)

Flea beetles Mollusks

Transecta 1994/ 51995/ 61994/ 5 1995/ 6

DD10.19 ± 0.04 0.47 ± 0.09 1.88 ± 0.21 0.85 ± 0.11 DD21.99 ± 0.23 0.50 ± 0.13 2.14 ± 0.16 1.50 ± 0.33 HP 1.90 ± 0.30 0.63 ± 0.13 2.40 ± 0.28 0.76 ± 0.10 K10.83 ± 0.20 1.78 ± 0.25 K22.54 ± 0.25 1.20 ± 0.13 2.43 ± 0.21 1.59 ± 0.28 SA10.65 ± 0.08 0.34 ± 0.08 1.80 ± 0.11 0.53 ± 0.09 SA20.27 ± 0.05 0.06 ± 0.02 1.00 ± 0.08 0.32 ± 0.05 a Each transect consisted of 50 plants, except K1which had 36. propenyl-, 3-butenyl-, 2-hydroxy-3-butenyl-, 3-indolylmethyl-, and 1-methoxy- 3-indolylmethylglucosinolate. All ﬁve glucosinolates were found at all of the study sites. However, there were signiﬁcant differences in their quantities and abundance between and within sites (Figures 2 and 3). The mean quantities at each site are affected by the number of plants at each site producing the glucosinolate in question and the quantity of glucosinolate that each of these plants produces. These two factors have different causes and implications, so they are considered separately. Both of the indolyl glucosinolates were found in all plants, but there were between-site differences in quantities (Table 5).

TABLE 2.PROPORTION OF PLANTS DAMAGED AND PEAK PERCENT OF LEAF AREA DAMAGED PER PLANT BY PIERID CATERPILLARSa

P. brassicae P. rapae

1995 1996 1995 1996

Transect Prop Dam Prop Dam Prop Dam Prop Dam

DD10.35 3.0 ± 1.20.27 5.65 ± 1.90.48 1.70 ± 0.50.18 0.41 ± 0.2 DD20 0 0 0 0.21 0.72 ± 0.30.44 0.44 ± 0.3 HP 000.03 0.90 ± 0.90.42 1.34 ± 0.40.21 1.46 ± 0.8 K10 0 0.30 0.64 ± 0.3 K20 0 00.81 ± 0.80.41 1.14 ± 0.30.28 0.78 ± 0.3 SA10 0 0 0 0.13 0.21 ± 0.10.39 2.04 ± 0.6 SA20 0 0 0 0.34 1.05 ± 0.30.18 0.53 ± 0.3 a Prop c proportion of plants attacked; Dam c mean ± SE percent of leaf area damaged per plant. GLUCOSINOLATES AND HERBIVORY IN Brassica 2631

TABLE 3.PROPORTION OF PLANTS DAMAGED BY S. leplastriana CATERPILLARS AND MEAN ± SE PEAK PERCENT OF LEAF AREA DAMAGED PER PLANT IN 1996

Transect Proportion Damage (%)

DD10.34 1.46 ± 0.45 DD20.18 0.67 ± 0.33 HP 0.36 1.91 ± 0.67 K1 K20.42 3.41 ± 0.95 SA10.48 4.50 ± 1.07 SA20.30 0.63 ± 0.29

The aliphatic glucosinolates were not found in all plants within each site and their abundance varied among sites (Table 6). There were also between-site differences in the quantities of aliphatic glucosinolates produced (Table 7). There were no differences in indolyl or aliphatic glucosinolate quantities between transects within sites, except for 2-propenylglucosinolate at Durdle Door (Bonfer- roni, a c 0.05). Herbivore Choice of Glucosinolate Profiles. Where possible, every marked plant was used in the following analyses. However, leaves for glucosinolate analysis were only taken in 1995 and a number of plants had died before harvest- ing, principally due to physical factors such as cliff erosion. In 1995–1996 only plants that survived long enough to have five recordings were used to calculate mean mollusk damage. Only those plants with 10 recordings were used for mean flea beetle damage, as this was more variable. Caterpillar damage and aphid infestation occurred late in the fieldwork year, so lower numbers of plants

TABLE 4. SEVERITY OF CABBAGE APHID INFESTATION PER PLANT AT EACH TRANSECT

1995 1996

Transect N Scorea N Scorea

DD1150.50 ± 0.22 22 0.005 ± 0.002 DD2130.45 ± 0.21 29 0 HP 23 0.87 ± 0.23 39 0 K191.07 ± 0.29 22 0.04 ± 0.02 K2260.47 ± 0.13 38 0 SA1300.20 ± 0.07 45 0 SA2170.26 ± 0.09 28 0 a Infestation was scored on each raceme from 0 (no aphids) to 3 (entire raceme covered) and a mean ± SE value calculated for each plant. 2632 MOYES, COLLIN, BRITTON, AND RAYBOULD

FIG. 2. Frequency histograms of the aliphatic glucosinolate quantities in plants at each transect. Glucosinolates were extracted from mature leaves of plants in the ﬁeld which had been freeze dried. the bar on the left of each graph shows the number of plants that do not contain that particular glucosinolate. had caterpillar damage recorded. The numbers of plants used in the analyses are summarized in Table 8. Plants at transect K1 were excluded altogether due to their high mortality from cliff erosion. A large number of statistical tests were performed, so the probability of making a type I error was reduced by making a Bonferroni adjustment (a c 0.05) to each set of tests. To consider the effect of the presence and quantity of each glucosinolate on herbivore damage (leaf damage or aphid severity), general linear models (GLM) were used. The presence of each individual aliphatic glucosinolate and transect was used as a factor, and size (in terms of leaf area or raceme number GLUCOSINOLATES AND HERBIVORY IN Brassica 2633

FIG. 3. Frequency histograms of the indolyl and total glucosinolate quantities in plants at each transect. Glucosinolates were extracted from mature leaves of plants in the field which had been freeze dried. for aphid severity) and glucosinolate quantities were used as covariates. Inter- actions between transects and glucosinolates also were included. Herbivore data from each year were considered separately. Neither presence of the individual aliphatic glucosinolates nor glucosinolates quantities had any significant effect on any measure of herbivore damage. To test for a link between individual glucosinolate presence and caterpillar presence, chi-square tests were performed for each year, transect, and caterpillar species. This gave 76 comparisons. No significant links were found. To test for a link between glucosinolate quantity and caterpillar species presence, GLMs were used with presence of each caterpillar species, transect, and 2634 MOYES, COLLIN, BRITTON, AND RAYBOULD

TABLE 5. INDOLYL GLUCOSINOLATES AT EACH SITEa

Glucosinolates (mmol/ g dry wt)

Site Ind Mind

Durdle Door 30.0 ± 3.62 a 2.60 ± 0.36 ab Handfast Point 11.7 ± 1.63 b 0.97 ± 0.16 a Kimmeridge 13.4 ± 1.73 b 4.69 ± 0.98 b St Aldhelm’s Head 5.45 ± 0.96 b 1.76 ± 0.43 a a Values are mean ± SE; values followed by the same letter, within each glucosinolate type, are not signiﬁcantly different (Bonferroni LSD, a c 0.05). Ind c 3-indolylmethylglucosinolate; Mind c 1-methoxy-3-indolylmethylglucosinolate.

TABLE 6. PROPORTION OF PLANTS AT EACH SITE PRODUCING ALIPHATIC GLUCOSINOLATESa

Glucosinolates (mmol/ g dry wt)

Site Pro But Ohc

Durdle Door 0.75 a 0.91 a 0.99 a Handfast Point 0.88 a 0.90 a 0.59 b Kimmeridge 0.84 a 0.41 b 0.82 a St Aldhelm’s Head 0.83 a 0.92 a 0.41 b a Values within each column followed by the same letter are not signiﬁcantly different (chi-square, P c 0.05). Pro c 2-propenylglucosinolate; But c 3-butenylglucosinolate; Oh c 2-hydroxy-3- butenylglucosinolate.

TABLE 7. ALIPHATIC GLUCOSINOLATE IN PLANTS PRODUCING EACH GLUCOSINOLATEa

Site Pro But Oh

Durdle Door 8.60 ± 1.17 a 3.80 ± 0.58 a 17.0 ± 2.08 a Handfast Point 7.20 ± 0.93 ab 4.96 ± 1.16 a 3.01 ± 0.80 b Kimmeridge 5.89 ± 0.79 b 1.71 ± 0.56 a 1.25 ± 0.22 b St Aldhelm’s Head 6.93 ± 0.79 ab 12.3 ± 2.06 b 1.49 ± 0.34 b a Values are mean ± SE; Values followed by the same letter, within each glucosinolate type, are not different (Bonferroni LSD, ac 0.05). Pro c 2-propenylglucosinolate; But c 3-butenylglucosinolate; Oh c 2-hydroxy-3-butenylglucosinolate. interaction between the two as factors, leaf area as a covariate, and individual glucosinolate quantity as the response. There were no differences in glucosinolate quantities in plants whether or not they were infested by P. brassicae or P. rapae. Levels of 2-hydroxy-3-butenylglucosinolate and 3-indolylmethylglucosi- GLUCOSINOLATES AND HERBIVORY IN Brassica 2635

TABLE 8.PLANTS (N) INCLUDED IN ANALYSES OF LINKS BETWEEN HERBIVORY AND GLUCOSINOLATE CONTENT

Transect

Damage DD1 DD2 HP K2 SA1 SA2

1994/ 5 Mollusk 28 38 40 37 33 36 Flea beetle 28 38 37 36 33 35 Caterpillar 25 38 37 36 33 35 Aphid 12 11 23 24 20 14 1995/ 6 Mollusk 48 50 49 49 49 50 Flea beetle 48 10 48 48 48 50 Caterpillar 39 0 36 32 26 35 Aphid 15 8 33 27 35 21 nolate were higher in plants supporting S. leplastriana at Durdle Door in 1996 (Figure 4) but no differences were found for the other glucosinolates (Bonfer- roni, a c 0.05, comparisons N c 20). This herbivore was not found feeding on leaves in 1995.

DISCUSSION

Five glucosinolates were found in these populations of wild cabbage. These were the aliphatic glucosinolates 2-propenyl-, 3-butenyl-, and 2-hydroxy- 3-butenylglucosinolate and the indolyl glucosinolates 3-indolylmethyl- and 1-methoxy-3-indolylmethylglucosinolate. Glucosinolate profiles varied among plant populations in terms of presence/ absence of the aliphatic glucosinolates and quantities of both the aliphatic and the indolyl glucosinolates, in agreement with the previous study (Mithen et al., 1995). The new populations included in this study were different, particularly Durdle Door. The majority of the differences in glucosinolate profiles occurred at the population level, with few differences in glucosinolate profiles between different habitats within plant populations. Most of these differences are likely to have a strong genetic basis. The presence of individual aliphatic glucosinolates is known to be controlled by a small number of loci, and their ratios remain constant in variable environments (Giamoustaris and Mithen, 1996a). Aliphatic glucosinolate concentration is under strong genetic control from four to five loci, whereas indolyl glucosinolate concentration is under weaker genetic control from two to three loci and is more influenced by environmental factors (Rucker¨ and Robbelson,¨ 1994; Toroser et al., 1995). 2636 MOYES, COLLIN, BRITTON, AND RAYBOULD

FIG. 4. Mean (± SE) 2-hydroxy-3-butenyl- and 3-indolylmethylglucosinolate quantities in plants infested (shaded) or not (unshaded) by the caterpillar S. leplastriana within each plant population. Glucosinolates were measured in mature leaf samples which had been freeze-dried.

On a smaller scale, there were differences in the glucosinolate profile of neighboring individuals. These differences included presence/ absence of the individual aliphatic glucosinolates and quantities of both the aliphatic and indolyl glucosinolates. Thus, the potential for host choice existed. One herbivore species did respond to the range of glucosinolate profiles found in the wild cabbage plants. The micromoth S. leplastriana is highly specialized and has been recorded on wild cabbage almost exclusively (Bradley et al., 1979). Although caterpillars fed more frequently on plants containing higher quantities of 2-hydroxy-3-butenylglucosinolate and 3-indolylmethylglucosinolate, plants not containing 2-hydroxy-3-butenylglucosinolate were equally likely to be attacked, so this compound is not a prerequisite for herbivory by S. leplastriana. All plants contained 3-indolylmethylglucosinolate, so it is not possible to say whether its presence is required. These preferences were only evident in plant populations containing individuals with high levels of these glucosinolates. The fact that this species was as common and fed as readily within plant populations containing no plants with high levels shows that these amounts are not necessary, simply preferred. There was no evidence for host choice by flea beetles, P. brassicae, P. rapae, B. brevicoryne, or slugs and snails on the basis of glucosinolate profiles. GLUCOSINOLATES AND HERBIVORY IN Brassica 2637

In this study, glucosinolate profiles were measured only once: after the first year of herbivory was recorded and before the second. Previously, Mithen et al. (1995) measured the glucosinolates in two of these populations at different times of the year and found no significant differences in the aliphatic glucosinolates but significant differences in the indolyl glucosinolates. Population differences in aliphatic glucosinolates remained constant when plants were grown under glasshouse conditions, although absolute quantities were lower (Mithen et al., 1995; Moyes 1997). We have found that although between-site differences in quantities of indolyl glucosinolates were reduced in the glasshouse, they were still detectable (Moyes, 1997). Studies have shown that indolyl glucosinolate quantities increase after various forms of damage (Koritsas et al., 1989; Bart- let et al., 1999). However, in this study no plant was ever undamaged. We were interested in herbivore choices between individual plants, so it is the relative differences in glucosinolate profiles that are of most interest. However, the absolute amounts may influence whether the range presented to the herbivores falls within the range that they can detect or respond to. Many studies indicate that glucosinolates may be important in host choice. Slugs reduce the number and severity of attacks on oilseed rape seedlings containing higher concentrations of glucosinolates (Glen et al., 1990; Moens et al., 1992). The aphid B. brassicae can detect and respond to 3-butenyl- and 2-propenylglucosinolate breakdown products (Nottingham et al., 1991; Visser et al., 1996). Flea beetles are attracted to sticky traps baited with the breakdown products of 2-propenylglucosinolate (Vincent and Stewart, 1984; Pivnick et al., 1992), and P. brassicae and P. rapae are stimulated to oviposit by 3-indolylmethylglucosinolate and 2-propenylglucosinolate (Traynier and Truscott, 1991; Renwick et al., 1992; van Loon et al., 1992; Stadler¨ et al., 1995). However, none of these studies of specialist herbivores used in vivo glucosinolates, and the studies of slugs used crop plants in the laboratory. Other studies have also found a lack of interaction between herbivores and glucosinolates in the field. Responses to oilseed rape glucosinolate profiles described for flea beetles and pollen beetles (Giamoustaris and Mithen, 1995, 1996b) were not found in other studies with different ranges of glucosinolate profiles (Milford et al., 1989; Williams, 1989). There are a number of reasons that may explain why results found in the laboratory are not translated into an effect in our wild plant populations. All of the plants in this study contained 3-indolylmethylglucosinolate, so the herbivores were only presented with a choice of quantity, not presence/ absence for this glucosinolate. Glucosinolate concentrations have been found to have an effect in studies using isolated chemicals (Huang and Renwick, 1994), although this study did not look at varying glucosinolate concentrations in vivo. The point of detection of the glucosinolates may be more important than the glucosinolate concentration in the whole leaf. Thus, compounds in the waxy layer may be important to ovipositing Lepidoptera (van Loon et al., 1992), 2638 MOYES, COLLIN, BRITTON, AND RAYBOULD whereas the volatile breakdown products of glucosinolates released into the atmosphere may be more important for attraction of flea beetles and aphids (see above). If attraction to volatiles is important, this may not be specific to individual plants, and invertebrates may land on neighbors releasing volatile attractants (Read et al., 1970). These factors may be further complicated by the physiol- ogy of glucosinolate production and breakdown (Louda and Mole, 1991) and the presence of other secondary metabolites (Usher and Feeny, 1983; Broadway and Colvin, 1992; Stadler¨ et al., 1995). Behavior of the herbivore species may reduce the importance of glucosinolates in host choice. For example, P. rapae butterflies fly in a straight line land- ing at regular intervals regardless of the nubmer of suitable plants they pass over (Jones, 1977; Courtney, 1982), and flea beetles are highly vagile, continuously switching plants (Kareiva, 1982). Furthermore, learning behavior can be important as slugs and snails may prefer plants encountered when they are juveniles (Teyke, 1995; Cook et al., 1997). P. rapae can learn to associate glucosinolate presence with color, leading to a loss of preference on the basis of glucosinolates in the laboratory (Traynier, 1986; Traynier and Truscott, 1991). All of these factors mean that the situation in the wild is likely to be much more complicated than laboratory studies predict, and this study highlights the lack of a detectable relationship between glucosinolates and host choice by flea beetles, P. brassicae, P. rapae, B. brassicae, and slugs and snails. The selection of individual plants on the basis of their glucosinolate profiles is essential if herbivores are to act as a selection pressure on glucosinolates. Mauricio and Rausher (1997) found evidence for selection on total glucosinolate concentration in Arabidopsis thaliana, by a combination of herbivores and plant pathogens. Clearly some of these organisms must have distinguished between glucosinolate profiles. The species behind this effect were not identified, so we do not know if they include those studied here. A further difference between the two studies is the plant species used. A. thaliana is an annual plant of dis- turbed habitats, whereas B. oleracea subsp. oleracea is a perennial plant surviv- ing for up to 20 years. Wild cabbage is also much larger than A. thaliana and grows above most of the surrounding vegetation. Chew and Courtney (1991), studying pierid butterflies feeding on members of the order Capparales, found that more apparent plants growing in more predicatable populations accumulate greater herbivore loads and have more specialized herbivores. They suggested that such plants either evolve novel chemistry to escape herbivores or maintain stable host–herbivore associations. Unapparent or unpredictable plants have the option of escaping herbivores through ecological disappearance. We do not know whether our plants contain novel defense compounds, but it is not surprising that they are exposed to different selection pressures than A. thaliana. In order to act as a selection pressure, herbivores must alter plant fitness. The herbivores studied in our work have not been found to affect seed pro- GLUCOSINOLATES AND HERBIVORY IN Brassica 2639 duction or plant survival, but herbivores of the seed and seedling stages have (Moyes, 1997). The selection of glucosinolate profiles by these herbivores will be discussed in future papers. In conclusion, a range of glucosinolate profiles exists in natural populations of wild cabbage. This range was sufficient to stimulate host choice by the specialist herbivore S. leplastriana. There was no evidence for host selection on the basis of glucosinolates by the other herbivores studied.

Acknowledgments—This work was funded by a BBSRC CASE studentship to C. L. Moyes, with the support of the UK Department of the Environment’s genetically modiﬁed organisms research programme.

REFERENCES

BARTLET, E., KIDDLE, G., WILLIAMS, I., and WALLSGROVE, R. 1999. Wound-induced increases in the glucosinolate content of oilseed rape and their effect on subsequent herbivory by a crucifer specialist. Entomol. Exp. Appl. 91:164–167. BENNET, R. N., and WALLSGROVE, R. M. 1994. Tansley review no. 72. Secondary metabolites in plant defense mechanisms. New Phytol. 127:617–633. BRADLEY, J. D., TREMEWAN, W. G., and SMITH, A. 1979. British Tortricoid Moths: Tortricidae: Olethreutinae. The Ray Society, London. BROADWAY, R. M., and COLVIN, A. A. 1992. Influence of cabbage proteinase inhibitors in situ on the growth of larval Trichoplusia ni and Pieris rapae. J. Chem. Ecol. 18:1009–1024. CHEW, F. S. 1988. Biological effects of glucosinolates, pp. 155–181, in H. Cutler (ed.). Biologically Active Natural Products. American Chemical Society, Washington, DC. CHEW, F. S., and COURTNEY, S. P. 1991. Plant apparency and evolutionary escape from insect herbivory. Am. Nat. 138:729–750. COOK, R. T., BAILEY, S. E. R., and MCCROHAN, C. R. 1997. The potential for common weeds to reduce slug damage to winter wheat: Laboratory and field studies. J. Appl. Ecol. 34:79–87. COURTNEY, S. P. 1982. Coevolution of pierid butterflies and their cruciferous foodplants IV. Crucifer apparency and Anthocharis cardamines (L.) oviposition. Oecologia 52:258–265. GIAMOUSTARIS, A., and MITHEN, R. 1995. The effect of modifying the glucosinolate content of oilseed rape (Brassica napus ssp. olerifera) on its interaction with specialist and generalist pests. Ann. Appl. Biol. 126:347–363. GIAMOUSTARIS, A., and MITHEN, R. 1996a. Genetics of aliphatic glucosinolates. IV. Side-chain mod- ification in Brassica oleracea. Theor. Appl. Genet. 93:1006–1010. GIAMOUSTARIS, A., and MITHEN, R. 1996b. The effect of flower colour and glucosinolates on the interaction between oilseed rape and pollen beetles. Entomol. Exp. Appl. 80:206–208. GLEN, D. M., JONES, H., and FIELDSEND, J. K. 1990. Damage to oilseed rape seedlings by the field slug Deroceras reticulatum in relation to glucosinolate concentration. Ann. Appl. Biol. 117:197–207. GUREVITCH, J., and COLLINS, S. L. 1994. Experimental manipulation of natural plant communities. Trends Ecol. Evol. 9:94–97. HERMS, D. A., and MATTSON, W. J. 1992. The dilemma of plants: To grow or defend. Q. Rev. Biol. 67:283–335. HOGGE, L. R., REED, D. W., and UNDERHILL,E.W.1988. The identification of desulphoglu- cosinolates using thermospray liquid chromatography/ mass spectrometry. J. Chromatogr. Sci. 26:348–351. 2640 MOYES, COLLIN, BRITTON, AND RAYBOULD

HUANG, X., and RENWICK, J. A. A. 1994. Relative activities of glucosinolates as oviposition stim- ulants for Pieris rapae and P. napi oleracea. J. Chem. Ecol. 20:1025–1037. JONES, R. E. 1977. Movement patterns and egg distribution in cabbage butterflies. J. Anim. Ecol. 46:195–212. KAREIVA, P. 1982. Experimental and mathematical analyses of herbivore movement: Quantifying the influence of plant spacing and quality on foraging discrimination. Ecol. Monogr. 53:261–282. KERNEY, M. P., CAMERON, R. A. D., and RILEY, G. 1987. Land Snails of Britain and North-West Europe. HarperCollins, Hong Kong. KIRK, W. D. J., and GRAY, M. 1992. Insect on Cabbages and Oilseed Rape. The Richmond Publishing Co. Ltd., Slough, UK. KORITSAS, V. M., LEWIS, J. A., and FENWICK, G. R. 1989. Accumulation of indole glucosinolates in Psylliodes chrysocephala L. -infested, or -damaged tissues of oilseed rape (Brassica napus L.). Experientia 45:493–495. LOUDA, S., and MOLE, S. 1991. Glucosinolates: Chemistry and ecology, pp. 123–164, in G. A. Rosen- thal and M. R. Berenbaum (eds.). Herbivores: Their Interactions with Secondary Plant Metabo- lites. Academic Press, New York. LOUDA, S. M., and RODMAN, J. E. 1983. Ecological patterns in the glucosinolate content of a native mustard, Cardamine cordifolia, in the Rocky mountains. J. Chem. Ecol. 9:397–423. MAURICIO, R., and RAUSHER, M. D. 1997. Experimental manipulation of putative selective agents provides evidence for the role of natural enemies in the evolution of plant defense. Evolution 51:1435–1444. MILFORD, G. F. J., PORTER, A. J. R., FIELDSEND, J. K., MILLER, C. A., LEACH, J. E., and WILLIAMS, I. H. 1989. Glucosinolates in oilseed rape (Brassica napus) and the incidence of pollen beetles (Meligethes aeneus). Ann. Appl. Biol. 115::375–380. MITHEN, R., RAYBOULD, A. F., and GIAMOUSTARIS,A.1995. Divergent selection for secondary metabolites between wild populations of Brassica oleracea and its implication for plant–herbivore interactions. Heredity 75:472–484. MOENS, R., COUVREUR, R., and CORS, F. 1992. Influence de la teneur en glucosinolates des variet´ es´ de colza d’hiver sur les deg´ atsˆ de limaces. Bull. Rech. Agron. Gemblaux 27:289–307. MOYES, C. L. 1997. The role of glucosinolate production in populations of the wild cabbage. PhD dissertation. University of Liverpool, Liverpool, UK. NOTTINGHAM, S. F., HARDIE, J., DAWSON, G. W., HICK, A. J., PICEKTT, J. A., WADHAMS, L. J., and WOODCOCK, C. M. 1991. Behavioral and electrophysiological responses of aphids to host and nonhost plant volatiles. J. Chem. Ecol. 17:1231–1242. PIVNICK, K. A., LAMB, R. J., and REED, D. 1992. Response of flea beetles, Phyllotreta spp., to mustard oils and nitriles in field trapping experiments. J. Chem. Ecol. 18:863–873. READ, D. P., FEENY, P. P., and ROOT, R. B. 1970. Habitat selection by the aphid parasite Diaere- tialla rapae (Hymenoptera: Braconidae) and hyperparasite Charips brassicae (Hymenoptera: Cynipidae). Can. Entomol. 102:1567–1578. RENWICK, J. A. A. 1988. Comparative mechanisms of host selection by insects attacking pine trees and crucifers, pp. 303–316, in K. C. Spencer (ed.). Chemical Mediation on Coevolution. Har- court Brace Jovanovitch, San Diego. RENWICK, J. A. A., RADKE, C. D., SACHDEV-GUPTA, K., and STADLER¨ , E. 1992. Leaf surface chemicals stimulating oviposition by Pieris rapae (Lepidoptea: pieridae) on cabbage. Chemoecology 3:33–38. RODMAN, J. E., nad CHEW, F. S. 1980. Phytochemical correlates of herbivory in a community of native and naturalized Cruciferae. Biochem. Syst. Ecol. 8:43–50. RUCKER¨ , B., and ROBBELSON¨ , G. 1994. Inheritance of total and individual glucosinolate contents in seeds of winter oilseed rape (Brassica napus L.). Plant Breed. 113:206–216. STADLER¨ , E., RENWICK, J. A. A., RADKE, C. D., and SACHDEV-GUPTA, K. 1995. Tarsal contact GLUCOSINOLATES AND HERBIVORY IN Brassica 2641

chemoreceptor response to glucosinolates and cardenolides mediating oviposition in Pieris rapae. Physiol. Entomol. 20:175–187. TEYKE,T.1995. Food attraction conditioning in the snail, Helix pomatia. J. Comp. Physiol. 177:409–414. TOROSER, D., THORMANN, C. E., OSBORN, T. C., and MITHEN, R. 1995. RFLP mapping of quantitative trait loci controlling seed aliphatic glucosinolate content in oilseed rape (Brassica napus L.). Theor. Appl. Genet. 91:802–808. TRAYNIER, R. M. M. 1986. Visual learning in assays of sinigrin solution as an oviposition releaser for the cabbage butterfly, Pieris rapae. Entomol. Exp. Appl. 40:25–33. TRAYNIER, R. M. M., and TRUSCOTT, R. G. W. 1991. Potent natural egg-laying stimulant for the cabbage butterfly, Pieris rapae. J. Chem. Ecol. 17:1371–1380. USHER, B. F., and FEENY, P. 1983. Atypical secondary compounds in the family Cruciferae: Tests for toxicity to Pieris rapae, an adapted crucifer-feeding insect. Entomol. Exp. Appl. 34:257–262. VAN LOON, J. J. A., BLAAKMEER, A., GRIEPINK, F. C., VAN BEEK, T. A., SCHOOVEN, L. M., and DE GROOT, A. 1992. Leaf surface compound from Brassica oleracea (Cruciferae) induces oviposition by Pieris brassicae (Lepidoptera: Pieridae). Chemoecology 3:39–44. VINCENT, C., and STEWART, R. K. 1984. Effect of allyl isothiocyanate on field behaviour of crucifer- feeding flea beetles (Coleoptera: Chrysomelidae). J. Chem. Ecol. 10:33–39. VISSER, J. H., PIRON, P. G. M., and HARDIE, J. 1996. The aphids’ peripheral perception of plant volatiles. Entomol. Exp. Appl. 80:35–38. WILLIAMS, I. H. 1989. Pest incidence on single low and double low oilseed rape cultivars. Aspects Appl. Biol. 23:277. WILLIAMS, M. R., and ABBOTT, I. 1991. Quantifying average defoliation using leaf level measure- ments. Ecology 72:1510–1511.