the effect of glucosinolates on responses of young phyllotreta nemorum larvae to non-host plants

Entomol. exp. appl. 51: 249-259, 1989. �9 1989 Kluwer Academic Publishers. Printed in Belgium. 249

The effect of glucosinolates on responses of young Phyllotreta nemorum larvae to non-host plants

Jens Kvist Nielsen Department of Chemistry, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark

Accepted: March 6, 1989

Key words: Host plant selection, crucifer specialist, leaf miner, Chrysomelidae, Alticinae, flea beetle, Brassicaceae, Cruciferae, Capparales, sinigrin, allelochemicals

Abstract

All recorded host plants of Phyllotreta nemorum L. (Coleoptera: Chrysomelidae) contain glucosinolates and belong to the plant families Brassicaceae (Cruciferae), Resedaceae and Capparaceae. The acceptability of 56 plant species from 28 other plant families (non-hosts) for young larvae has been studied in the laboratory. None of these species were fully acceptable for initiations of leaf mines when intact untreated leaves were presented, and only one species, Malva silvestris L. (Malvaceae), was partially acceptable. The acceptability of some species increased when leaf discs were presented instead of intact leaves; but the highest percentages of mine initiations occurred in leaf discs treated with the glucosinolate, sinigrin. A stimulatory effect of sinigrin could be demonstrated in experiments with 7 plant species: Papaver dubium L., Papaver rhoeas L., Fumaria officinalis L., Malva silvestris L., Pisum sativum L., Campanula latifolia L. and Laetuca sativa L. The majority of species remained unacceptable even after treatment with glucosinolates.

The main causes for these differences between plant species are supposed to be differences in contents of deterrents and/or other stimulants for mine initiation. These possibilities are discussed in relation to the content of allelochemicals in acceptable plants and the position of these plants in botanical classifications.

Introduction

Most Phyllotreta species (Coleoptera: Chryso- melidae) are associated with Cruciferae and related glucosinolate containing families (Nielsen, 1988). The available information is based mainly on observations and experiments with adult beetles, while few studies have been made with larvae. Food plants and feeding habits of species with soil dwelling larvae are largely unknown (Bracken & Bucher, 1986; Newton, 1928), while

more information is available on the leaf mining species, e.g.P, nemorum L. (Buhr, 1954, 1956; Nielsen, 1977, 1988, 1989). According to Buhr (1954, 1956) larvae ofP. nemorum can be found in most if not all genera of Cruciferae and Resedaceae, but they are never found in plants which do not contain glucosinolates. In a field experiment, the host plant range was shown to be more restricted as only a few of the available glucosinolate containing species were attacked (Nielsen, 1977). Transfer experiments with In'st

250

instar larvae in the field and in the laboratory suggested that the larvae had a broader host plant range than adults, but no mines were found in plants without glucosinolates (Nielsen, 1977, 1989). Buhr (1956) obtained similar results with older larvae.

Glucosinolates are known to stimulate feeding in adults of several Phyllotreta species (Hicks, 1974; Nielsen, 1978a,b; Nielsen etaL, 1979a). Moreover, glucosinolates are known to stimulate feeding in larvae of two other crucifer feeding leaf beetles Entomoscelis americana Brown and Phaedon cochleariae (F.) (Mitchell, 1978; Tanton, 1965). No information is available on the effect of glucosinolates on larvae of Phyllotreta species.

Larvae of P. nemorum emerge from the eggs in the soil and have to search for their host plants. They initiate a mine, usually in a lower leaf on the plant. Later they may change several times to new mines in other leaves and even in other plants before they finally pupate in the soil. The present paper describes how newly emerged first instar larvae respond to plants without a natural content of glucosinolates and how these responses are modified by treatments with glucosinolates.

Methods and materials

Rearing conditions for P. nemorum and the origin and handling of the larvae have been described elsewhere (Nielsen, 1989). Larvae were less than 24 h old and had no access to plant material prior to the start of the experiment. With few excep- tions, plants were grown in field plots at the agricultural experimental station, Hojbakkegaard, or collected at natural growth sites. Experiments were started not later than 24 h after cutting of the leaves in the field.

Intact leaves of test plants were presented in plastic vials together with a piece of moist filter paper. The size of the vials (12.5, 25 and 160 ml) varied with the size of the leaves. Five larvae were transferred to each vial. All experiments with intact leaves were non-choice tests. Intact leaves were never treated with glucosinolates or other compounds.

T 2 . 7 cm

1 1 f

I 2.7 cm 4

Fig. 1. Device used for testing the acceptability of leaf discs to first instar larvae of Phyllotreta nemorum. 1: leaf disc; f: moist filter paper rolled into a cylinder.

Leaf discs were cut with a cork borer (d = 14 mm). From narrow and divided leaves, leaf pieces were cut with a pair of scissors. Leaf discs (or leaf pieces) were presented to the larvae in small plastic vials (V = 12.5 ml). A small piece of moist filter paper was rolled into a cylinder. The length of this cylinder was a little longer than the diameter of the vial, so that it could be firmly fixed at the bottom of the vial. Two leaf discs were then fastened between the cylinder and the walls of the vial (Fig. 1). It seemed to be important that the leaf discs touched the walls probably because the larvae were crawling there until they found a site for initiating a mine. Most experiments with leaf discs were non-choice tests with only one treatment per vial, but in a few cases choice tests were performed where the two leaf discs had received different treatments. In these cases, either the test or the control discs were then given a mark with a pin.

For treatments of leaf discs with glucosinolates, the vacuum infiltration technique described by Harris & Mohyuddin (1965) was used initially. This technique was abandoned when it turned out that infiltration with pure water might increase the numbers of larvae initiating a mine (Table 2). In later experiments, leaf discs were dipped in solutions of sinigrin in water, which at the same time contained 0.05 ~ sodium dodecylsulphate (detergent). When the solvent had evaporated, the discs were fastened in the vials and one larva was intro- duced to each vial. The numbers of larvae initiat-

ing a mine were counted after 24 h. Later, the numbers of larvae which were still alive after 3 days were counted. Further details are given by Nielsen (1989).

As a rule, the Fisher exact probability Test (one tailed) was used for the statistical analysis when N < 30. This test is the most suitable test for small sample sizes and for small cell frequencies (Siegel, 1956). The Chi-square test was used when N > 30 and when the expected cell frequencies were higher (> 5). Plant names and the arrangement in the tables follow a recent treatise of the Danish flora (Hansen, 1981), except for the use of the common name for dandelion, Taraxacum vulgare L.

Results

Most plant species without a natural content of glucosinolates were unsatisfactory for mine initiation by first instar larvae of P. nemorum. Fifty- six species belonging to 28 families were presented to the larvae both as intact leaves and as leaf discs treated with the glucosinolate, sinigrin. Sugar beet (Beta vulgaris L. var. altissima Drll) was arbi- trarily chosen as a reference species because it was completely unacceptable for mine initiation even after treatment with sinigrin (Table 1).

Intact leaves of all investigated species were unacceptable for mine initiation (Table 1). The most acceptable of the investigated non-host plants seemed to be Malva silvestris, but even this species did not receive significantly more mines than sugar beet (p > 0.05; Fisher exact probability Test). Forty-seven species (84~o)remained unacceptable after treatment with sinigrin, but 9 species received significantly more mines than sugar beet when presented as leaf discs treated with sinigrin. These species were: Papaver dubium, P. rhoeas, Fumaria officinalis, Malva silvestris, Melilotus alba, Vicia faba, Pisum sativum, Campanula latifolia and Lactuca sativa.

Further experiments were performed with these 9 species in order to investigate the role of glucosinolates in mine initiation and survival. Larger numbers of larvae initiated mines in

251

sinigfin-treated than in solvent-treated leaf discs of 7 species (Table 2). The same tendency was found in Melilotus alba, but the difference was not significant. In Viciafaba, there was no difference between untreated and sinigrin-treated leaf discs and no experiments were performed with solvent- treated discs. The stimulatory effect of sinigrin and two other glucosinolates could also be demonstrated in choice tests with pea leaf discs (Table 3).

Although these experiments showed the importance of sinigrin as a stimulant for mine initiation, it was equally evident that other factors were important, too. Plant species which were most acceptable after sinigrin-treatment (e.g. Malva silvestris, Papaver dubium and Pisum sativum ) were also partially acceptable even without sinigrin treatment. Pea was by far the most acceptable of the investigated non-glucosinolate containing plants. About 50 ~o of the larvae initiated mines in untreated leaf discs of this plant compared to 5 ~/o in intact leaves. Most of the larvae (92 ~o) entered solvent-treated and untreated leaf discs of pea at the edge, where there was a direct access to the mesophyll, while only few penetrated the epidermis. These observations suggest that the mesophyll of pea contains some compounds which stimulate mine initiation. The effect of wound healing on the availability of the stimulatory compounds in the mesophyll was investigated by cutting leaf discs of pea 48 h prior to the treatments and start of the experiments. A smaller percentage of larvae entered sinigrin-free leaf discs (untreated or solvent treated) presented after wound healing compared to discs which were presented immediately after cutting (Chi 2 = 5.40; df -- 1 ; p < 0.05) (Table 2).

Vacuum infiltration with pure water increased the acceptability of Pisum sativum and Campanula latifolia for mine initiation (Table 2). Therefore, this method was abandoned and instead leaf discs were dipped in an aqueous solution containing 0.05~o sodium dodecylsulphate as a detergent. Occasionally, treatments with this solvent also increased the numbers of mine initiations (Table 2), but the effect was less pronounced than when the vacuum infiltration technique was used.

252

Table 1. Acceptability of non-host plants for mine initiation by first instar larvae of Phyllotreta nemorum in laboratory assays. Plants were presented either as intact leaves or as leaf discs treated with sinigrin. N: number of larvae tested; I: number of larvae initiating a mine. Significance levels (p) are given for comparisons of sinigrin-treated leaf discs of test plants with sinigrin-treated leaf discs of a completely unacceptable plant (sugar beet). None of the test plants were significantly more acceptable than sugar beet when presented as intact leaves

Plant species Intact Leaf discs treated with leaves sinigrin a

N I N I I(%) p

DICOTYLEDONES

Ranunculaceae Caltha palustris L. 10 0 10 0 0

Papaveraceae Papaver dubium L. 30 0 25 21 84 Papaver rhoeas L. 20 0 55 22 40

Fumariaceae Fumaria officinalis L. 15 0 25 6 24

Caryophyllaceae Stellaria media (L.) Viii. 15 0 15 0 0

Chenopodiaceae Beta vulgaris L.** 15 0 40 0 0 Chenopodium album L. 15 0 15 1 7

Polygonaceae Rumex obtusifolius L.** 20 0 15 0 0 Polygonum convolvulus L. 15 0 15 0 0

Malvaceae Malva silvestris L.* 40 5 15 13 87

Violaceae Viola arvensis Murray 15 0 15 0 0 Viola riviniana Reichenb. 10 0 15 0 0

Rosaceae Geum rivale L. 15 0 15 0 0

Fabaceae (Papilionaceae) Melilotus alba Medicus* 10 0 30 9 30 Astragalus glycyphyllus L. 15 0 15 0 0 Vicia cracca L. 15 0 15 0 0 Viciafaba L.* 15 2 15 6 40 Vicia sepium L. 15 0 15 1 7 Pisum sativum L. cv. Alaska 20 1 30 24 80 P. sativum cv. Kelvedon Wonder 30 1 30 25 83

Onagraceae (Oenotheraceae) Chamaenerion angustifolium (L.) Scop. 15 0 15 0 0 Epitobium hirsutum L. 15 0 15 0 0

Lythraceae L ythrum salicaria L. 15 0 15 0 0

Euphorbiaceae Mercurialis perennis L. 20 0 15 0 0 Euphorbia helioscopia L.** 15 0 15 0 0

Geraniaceae Geranium molle L. 15 0 15 0 0

Balsaminaceae Impatiens parviflora L.** 15 0 15 0 0

NS

< 0.001 < 0.001

<0.01

NS

NS

NS NS

< 0.001

NS NS

NS

<0.001 NS NS <0.001 NS <0.001 < 0.001

NS NS

NS

NS NS

NS

NS

Table 1. (continued)

253

Plant species Intact leaves

Leaf discs treated with sinigrin a

N I N I I(%) p

Apiaceae (Umbelliferae) Anthriscus sylvestris (L.) Hoffm. 15 0 15 1 7 NS Aegopodium podagraria L. 15 0 15 0 0 NS Pastinaca sativa L. 15 0 15 0 0 N S

Asclepiadaceae Vincetoxicum hirundinaria Medicus 15 0 15 0 0 NS

Solanaceae Solanum dulcamara L. 15 0 15 0 0 NS

Convolvulaceae Convolvulus arvensis L.** 15 0 20 0 0 NS

Boraginaceae Anchusa officinalis L. 20 0 15 0 0 NS Pulmonaria obscura Dumort. 15 0 15 0 0 NS

Lamiaceae (Labiatae) Galeopsis tetrahit L.** 15 0 20 0 0 NS Lamium album L. 15 0 15 0 0 NS Lycopus europaeus L. 25 0 15 0 0 NS

Plantaginaceae Plantago lanceolata L.** 15 0 15 0 0 NS Plantago major L. ssp. major 15 0 20 0 0 NS

Scrophulariaceae Scrophularia nodosa L. 15 0 15 0 0 NS Veronica agrestis L.** 15 0 15 0 0 NS Veronica beccabunga L. 15 0 15 0 0 NS

Campanulaceae Campanula latifolia L. 40 0 25 13 52 <0.001 Campanula rapunculoides L. 15 0 10 1 0 NS

Dipsacaceae Knautia arvensis (L.) Coulter 10 0 10 0 0 NS

Asteraceae (Compositae) Galinsoga parviflora Cav.** 15 0 15 2 13 N S Cirsium arvense (L.) Scop.** 15 0 20 2 10 NS Cirsium oleraceum (L.) Scop. 15 0 15 0 0 NS Lactuca sativa L.* 15 0 25 11 44 < 0.001 Lactuca muralis (L.) Gaertner 15 1 15 2 13 NS Sonchus oleraceus L.* 30 0 15 1 7 NS Taraxacum vulgare L.** 15 0 20 0 0 NS

MONOCOTYLEDONES

Poaceae (Graminae) Hordeum vulgare L. 15 0 15 0 0 NS Avena sativa L. 15 0 15 0 0 NS

Lilliaceae Allium moly 15 0 15 2 13 NS Polygonatum multiflorum (L.) All. 15 0 15 0 0 NS

a In most cases, the leaf discs were dipped in the sinigrin solution, but in a few cases (*) the vacuum infiltration technique was used and in others (**) both techniques were used with similar results.

254

Table 2. The effect ofsinigrin on mine initiation by first instar larvae ofPhyllotreta nemorum in selected non-hos t plants. S: number of larvae surviving for three days; other symbols are explained in Table 1. Significance levels (pl and ps ) show whether solvent t rea ted leaf discs from a particular plant are different from untrea ted discs of the same plant, and whether sinigrin t rea ted leaf discs are different from solvent t rea ted leaf discs; pl compare da ta for mine initiations and ps da ta for larval survival

Plant species Trea tment Conc. N I I% pI S S/I.100 ps (mM)

Vacuum infiltration with sinigrin dissolved in water

Malva silvestris Unt rea ted - 20 4 20 4 (100) Solvent - 15 5 33 NS 4 (80) - Sinigrin 4 15 13 87 <0.01 11 85 -

Melilotus alba Unt rea ted - 20 0 0 0 - Solvent - 20 2 10 NS 1 - Sinigrin 4 30 9 30 NS 4 44 -

Vicia faba Unt rea ted - 20 5 25 0 0 Sinigrin 4 15 6 40 NS 0 0 -

Pisum sativum a Untrea ted - 203 94 46 48 51 cv. Kelvedon Wonder Solvent - 127 78 61 <0.01 56 72 0.01

Sinigrin 0.01 116 92 79 < 0.01 70 76 NS Sinigrin 0.1 121 96 79 <0.01 78 81 NS

Campanula latifolia Unt rea ted - 15 2 13 2 - Solvent - 15 9 60 < 0.05 6 67 - Sinigrin 4 15 14 93 <0.05 12 86 NS

Lactuca sativa a Unt rea ted - 20 1 5 0 - Solvent - 30 1 3 NS 0 - - Sinigrin 4 25 11 44 < 0.001 5 45 -

Dipping in sinigrin dissolved in water containing 0.05 % sodium

Papaver dubium

Papaver rhoeas

Fumaria officinalis

Pisum sativum cv. Alaska

Pisum sativum cv. Kelvedon Wonder

Pisum sativum ev. Kelvedon Wonder 48 h after cutting

the discs Campanula latifolia

Unt rea ted Solvent Sinigrln Unt rea ted Solvent Sinigrln Unt rea ted Solvent Sinigrm Unt rea ted Solvent Sinigrm Unt rea ted Solvent Sinigrln Unt rea ted Solvent Sinigrm

dodecylsulphate (detergent)

-- 2 5 4 1 6 2 ( 5 0 )

-- 2 5 9 36 NS b 4 44 8 25 21 84 <0.001 b 11 52

- 4 5 0 0

- 45 6 13 <0.02 6 100 8 55 22 40 <0.01 16 73 - 15 0 0

- 15 0 0 N S 0 -

8 25 6 24 < 0.05 0 0 - 30 15 50 7 47 - 30 15 50 NS b 10 67 8 30 24 80 <0.02 b 18 75 - 30 15 50 4 27 - 30 16 53 NS b 7 44 8 30 25 83 <0 .02 b 14 56

- 3 5 1 0 2 9 - -

- 35 12 34 NS - -

8 35 25 71 <0.01 - -

Un t rea ted - 25 1 4 0 - Solvent - 25 3 12 NS b 3 (100) Sinigrin 8 25 13 52 <0.01 b 10 77

NS NS

NS NS

NS NS

NS NS

a Plants grown in the greenhouse; all o ther plants are collected in the field or at natural growth sites. b Chi 2 test used although N < 30 (see text).

255

Table 3. Site of first mine initiation in choice tests where first instar larvae of Phyllotreta nemorum had access to one pea leaf disc treated with a glucosinolate and another pea leaf disc treated with the solvent

Compound First choice

N glucosinolate solvent No mine p a

treated treated initiation discs discs

Sinigrin b 40 26 6 8 < 0.001 Sinigrin ~ 47 30 10 7 < 0.01 Glucoiberin ~ 51 29 12 10 < 0.01 Glucocheirolin c 42 27 9 6 < 0.01

a C h i 2 test of the hypothesis that equal numbers entered glucosinolate and solvent treated leaf discs. b Pea plants grown in the field, and glucosinolate treatment performed by dipping in 8 mM solution. c Pea plants grown in the greenhouse and glucosinolate treatment performed by vacuum infiltration with 4 mM solutions.

Larvae which initiated a mine in Fumaria

officinalis or Vicia faba died within 3 days; but larvae were able to survive and develop in several other plant species for at least 3 days (Table 2). Survival in these plant species was not increased by the treatments with sinigrin. However, vacuum infiltration with water had a small, but significant effect on survival in pea leaf discs compared to untreated discs (Table 2).

Discussion

The present experiments demonstrate that plants without glucosinolates are rarely attacked by first instar larvae of Phyllotreta nemorum. However, a small proportion of these plants become acceptable after treatment with the glucosinolate, sinigrin. A stimulatory effect of glucosinolates on feeding behaviour has also been demonstrated in larvae of other crucifer feeding Coleoptera (Mitchell, 1978; Tanton, 1965), Lepidoptera (David & Gardiner, 1966; Gupta & Thorsteinson, 1960; Ma, 1972; Nayar & Thorsteinson, 1963; Verschaffelt, 1910) and Hymenoptera (Bogawat & Srivastava, 1968). The glucosinolates are also important for adults of crucifer specialist insects. They are feeding stimulants for several Chrysornelidae, and o,4i- position stimulants for certain Lepidoptera and Diptera (Chew, 1988; Mitchell, 1988; Nielsen, 1988; St~tdler, 1986). The lowest concentration

tested (10-5 M) was still stimulatory, and therefore, P. nemorum larvae might be as sensitive to glucosinolates as larvae ofPieris brassicae. In this species, the threshold for detection of glucosinolates was found to be around 1 0 - 6 M in behavioural assays (David & Gardiner, 1966; Ma, 1972), and even lower in electrophysiological recordings from the glucosinolate sensitive cells on the maxillae (Schoonhoven, 1967). For the present experiments, it is important to notice that the concentration of sinigrin used for treatments of various plant species (Table 1)is far above the detection threshold.

A large proportion of plants (84~o) remained unacceptable to P. nemorum larvae even after treatment with glucosinolates (Table 1). Similar results were obtained with Plutella xylostella (L.) (Gupta & Thorsteinson, 1960). Plants which remain unacceptable after treatment with sinigrin may contain deterrent compounds or lack some compounds which are stimulatory to mine initiation. The importance of feeding deterrents from plants outside Capparales has been demonstrated in other crucifer feeding larvae (Jermy, 1966; Ma, 1972; Mitchell & Sutcliffe, 1984). Deterrents may be important for larvae of P. nemorum, too, but a satisfactory assay for measuring deterrency in this leaf mining species has not yet been developed (Nielsen, unpubl.). There are few reports on the presence of deterrents for crucifer feeding larvae in unacceptable crucifers (Usher & Feeny, 1983).

256

P. nemorum larvae do not initiate mines in some glucosinolate containing plants, but it is not known whether these plants contain deterrents (Nielsen, 1989). There is a strong influence of feeding and oviposition deterrents on host plant acceptance in adults of crucifer specialists (Nielsen, 1978a, b; Renwick & Radke, 1987; Rothschild et al., 1988).

The role of primary compounds like sugars, certain amino acids and ascorbic acid on feeding behaviour ofcrucifer feeding larvae has been demonstrated several times (Ma, 1972; Mitchell, 1978; Nayar & Thorsteinson, 1963). On artificial diets, the stimulatory effect of sinigrin could only be demonstrated if glucose or sucrose were present, too (Ma, 1972; Mitchell, 1978; Nayar & Thorsteinson, 1963). However, there is no evi- dence that non-host plants become more acceptable after treatment with these sugars. Primary compounds may also be important for mine initiation in P. nemorum larvae. Some larvae initiate mines in leaf discs of pea and a few other plants even in the absence of glucosinolates. The compounds involved have not been identified. They may be primary compounds or other compounds with a wide distribution within the plant kingdom, because the plants which are acceptable in the absence ofglucosinolates belong to different botanical taxa with no obvious similarities in secondary chemistry (Dahlgren et al., 1981; Gershenzon & Mabry, 1983).

Initiations of leaf mines occurred more readily in leaf discs compared to intact leaves and in vacuum infiltrated leaf discs compared to untreated leaf discs (Table 2). These treatments may break some plant cells and make the stimulatory compounds more available to the larvae. Another explanation would be that the leaf surface contained some deterrent compounds or some mechanical barriers which were removed by the treatments. Mechanical barriers could inhibit mine initiation, but also the transport of active compounds from the mesophyll to the leaf surface (Stadler, 1986).

If larvae succeeded in initiating a mine, they were usually able to feed and survive for at least three days even in discs which did not contain

glucosinolates. These results might indicate that glucosinolates are less important for continuous feeding within the mines. However, it has not been possible to rear P. nemorum during all three larval instars on any non-glucosinolate containing plant with or without sinigrin treatment. One reason for this failure is that larvae do not readily accept intact leaves, and that leaf discs start to deteriorate before larval development has finished. Further studies on these aspects and on the influence of glucosinolates on continuous feeding within the mines are in progress.

Plants which become acceptable after sinigrin treatment belong to the following plant orders: Papaverales (Papaveraceae and Fumariaceae), Malvales (Malvaceae), Fabales (Fabaceae), Campanulales (Campanulaceae) and Asterales (Asteraceae). As only one or a few members of many plant families have been tested, it is still uncertain whether the orders mentioned above contain a higher proportion of plants which become acceptable after sinigrin treatment compared to other plant orders. In most botanical classifications (Cronqvist, 1968; Dahlgren et al., 1981), the orders mentioned above are widely separated from each other and from the Capparales, which contain the natural host plants of P. nemorum. Moreover, there are no obvious similarities in contents of allelochemicals in these plant orders. The present Papaverales were once placed together with the present Capparales in the order Rhoeodales. However, there is now general agreement between botanists that Papaverales should be placed together with Ranunculales and other families containing benzylisoquinoline alkaloids (Gershenzon & Mabry, 1983). These compounds are present in Papaverales, but not in Capparales. On the other hand, the Capparaies contain glucosinolates which are never found in the Papaverales (Dahlgren et al., 1981 ; Gershen- zon & Mabry, 1983).

Members of Papaverales have not often been investigated in experimental studies on the host plant range of crucifer feeding insects. Papaver somniferum L. was unacceptable to older larvae of P. nemorum, but supported development of two other crucifer feeding leaf miners, Ceutorhynchus

contractus (Marsh.) (Coleoptera: Curculionidae) and Scaptomyza flava (Fall.) (Diptera: Droso- philidae) (Buhr, 1937, 1956). S. f lava has been reported to feed on members of the Papaverales (Hering, 1951), but this information needs confir- mation because of uncertainties in the taxonomy of this genus (Stein, 1963).

The most acceptable plant in the present experiments was Pisum sativum (Fabaceae). Even without sinigrin treatment, more than 50 ~o of the larvae initiated mines in leaf discs of this plant (Table 2). The reason for this outstanding position is unknown. Pea is known to contain kaempferol glycosides similar to those found in many Brassica species (Durkee & Harborne, 1973; Weissenb0ck et al., 1986). Some kaempferol glycosides are feeding stimulants for adult Phyllotreta species (Nielsen, 1978b; Nielsen etal., 1979b), but similar effects have not yet been demonstrated in larvae. Pea did not contain feeding deterrents for larvae of Pieris brassicae and Athalia rosae (Jermy, 1966) and together with other members of Fabaceae it was among the most acceptable plants for Plutella xylostella after sinigrin treatment (Gupta & Thorsteinson, 1960).

Malva silvestris, Campanula latifolia, and Lactuca sativa became more or less acceptable for young P. nemorum larvae after sinigrin treatment. M. silvestris did not contain feeding deterrents for Pieris brassicae and Athalia rosae (Jermy, 1966), and Lactuca sativa was acceptable for Plutella xylostella after sinigrin treatment (Gupta & Thorsteinson, 1960).

A few plants included in the present study are host plants for chrysomelid species which are related to some crucifer specialists (Mohr, 1966): Hordeum vulgare and A vena sativa (host plants for Phyllotreta vittula Redt.), Solanum dulcamara (host plant for several Psylliodes species) and Veronica beccabunga (host plant for Phaedon armoraciae (L.)). None of these plant species became acceptable for P. nemorum larvae after treatment with sinigrin.

The present results demonstrate that sinigrin and other glucosinolates stimulate mine initiations by P. nemorum larvae, but that the effect of sinigrin is different in different chemical and/or

257

physical environments. Both the absence of inhibitory compounds and the presence of other stimulatory compounds may be important for creating suitable chemical environments for the glucosinolates (Nielsen, 1978a, b; Nielsen et al., 1979b; Renwick & Radke, 1987; Rothschild etal., 1988; SchOni etal., 1987). Since the pioneering work of Verschaffelt (1910) much emphasis has been placed on the effects of glucosinolates and their degradation products in host plant selection of crucifer feeding insects. Future work will probably pay more attention to the identification of other compounds which have similar effects either alone or in combinations with the glucosinolates.

Zusammenfassung

Die Wirkung von Glukosinolaten auf die Reak- tionen der Junglarven von Phyllotreta nemorum an Nichtwirts-Pflanzen.

Phyllotreta nemorum L. ist ein oligophager Erdfloh, der an Cruciferen und anderen Gluko- sinolat-haltigen Pflanzenarten gebunden ist. Die Imagines fressen LOcher in die Blatter und die Weibchen legen ihre Eier in den Boden. Die Larven sind Blattminierer. Nach dem SchlOpfen im Boden klettern sie an die Pflanzen hoch, und die Einbohrung und der Anfang der Minierung erfolgt in eines der unteren Blatter der Wirtspflanze.

Die Wirkung von Glukosinolaten auf die Einbohrung von Junglarven in Pflanzenarten, die keine natt~rliche Inhalt von Glukosinolaten haben, ist in Laborexperimenten untersucht worden. 56 Pflanzenarten aus 28 Familien wurden prasentiert teils als unbehandelte Bl~ttter und teils als Glukosinolatbehandelte Blatt- scheiben. Unbehandelte Bl~itter yon nicht-Gluko- sinolathaltigen Arten waren immer unbefriedi- gend for die Larven. Nur in eine Art, Malva silvestris L. war die Frequenz der Einbohrung ein bisschen hOher als 10~o. Eine signifikante ErhOherung der Anzahl eingebohrten Larven nach der Sinigrin-behandlung erfolgte in 7

258

Pflanzenarten: Papaver dubium L., P. rhoeas L., Fumaria officinalis L., Malva silvestris L., Pisum sativum L., Campanula latifolia L. und Lactuca sativa L. Doch blieben die meisten Pflanzenarten (84~o) auch nach der Sinigrin-Behandlung unbesiedelt.

Pflanzenarten, die nach der Sinigrin-Behand- lung nicht besiedelt werden enthalten vielleicht frasshemmende Stoffe, oder ihnen fehlen noch weitere Frass-stimulierende Stoffe. Diese MOg- lichkeiten werden diskutiert in Zusammenhang mit den Inhalt von Allelochemikalien in besiedel- ten Pflanzenarten und mit ihrer taxonomischer Position.

Acknowledgements

I am grateful to Kirsten Larsen, Marianne K. Petersen and Ame H. Kirkeby-Thomsen for tech- nical assistance, to Lis Park for drawing the figure and to Tove Narager for typing the manuscript. Support from the Danish Agricultural and Veteri- nary Research Council and the Carlsberg Foundation is gratefully acknowledged.

References

Bogawat, J.K. & B.K. Srivastava, 1968. Discovery of sinigrin as a phagostimulant by Athalia proxima Klug (Hymenoptera: Tenthredinidae). Indian J. Entomol. 30: 89.

Bracken, G. K. & G. E. Bucher, 1986. Yield losses in eanola caused by adult and larval flea beetles, Phyllotreta cruciferae (Coleoptera: Chrysomelidae). Can. Entomol. 118: 319-324.

Buhr, H., 1937. Parasitenbefall und Pflanzenverwandtschaft. Botan. Jahrb. 68: 142-198.

Buhr, H., 1954. Zur Kenntnis der Biologie und der Ver- breitung minierender K~er. Arch. Freunde Nat. Gesch. Mecklenb. 1: 289-381.

Buhr, H., 1956. Zur Kenntnis der Biologie und der Ver- breitung minierender K~ffer, Fortsetzung. Ibid. 2: 35-108.

Chew, F. S., 1988. Searching for defensive chemistry in the Cruciferae, or, Do glueosinolates always control inter- actions of Cruciferae with their potential herbivores and symbionts? No! In: K. C. Spencer (ed.), Chemical Media- tion of Coevolution. Plenum Press, New York: pp. 8 i-112.

Cronquist, A., 1968. The Evolution and Classification of

Flowering Plants. Thomas Nelson and Sons, Ltd., London: 396 pp.

Dahlgren, R. M. T., S. R. Jensen & B.J. Nielsen, 1981. A revised classification of the Angiosperms with comments on correlation between chemical and other characters. In: D. A. Young & D. S. Seigler (eds.), Phytochemistry and Angiosperm Phylogeny. Praeger Publ., New York: pp. 149-204.

David, W. A. L. & B.O.C. Gardiner, 1966. Mustard oil glucosides as feeding stimulants for Pieris brassicae larvae in a semi-synthetic diet. Entomol. exp. appl. 9: 247-255.

Durkee, A. B. & J. B. Harborne, 1973. Flavonol glycosides in Brassica and Sinapis. Phytochemistry 12: 1085-1089.

Gershenzon, J. & T. J. Mabry, 1983. Secondary metabolites and the higher classification of angiosperms. Nord. J. Bot. 3: 5-34.

Gupta, P. D. & A. J. Thorsteinson, 1960. Food plant relationships of the diamond-back moth (Plutella maculipennis (Curt.)). I. Gustation and olfaction in relation to botanical specificity of the larva. Entomol. exp. appl. 3:241-250.

Hansen, K. (ed.), 1981. Dansk Feltflora. Gyldendal, Copen- hagen: 777 pp.

Harris, P. & A. I. Mohyuddin, 1965. The bioassay of insect feeding tokens. Can. Entomol. 97: 830-833.

Hering, E. M., 1951. Biology of the leaf miners. Dr. W. Junk, 's-Gravenhage: 420 pp.

Hicks, K.L., 1974. Mustard oil glucosides: Feeding stimulants for adult cabbage flea beetles, Phytlotreta cruciferae (Coleoptera: Chrysomelidae). Ann. ent. Soc. Am. 67: 261-264.

Jermy, T., 1966. Feeding inhibitors and food preference in chewing phytophagous insects. Entomol. exp. appl. 9: 1-12.

Ma, W.C., 1972. Dynamics of feeding responses in Pieris brassicae Linn. as a function of chemosensory input: A behavioural, ultrastructural and electrophysiological study. Meded. Landbouwhogesehool Wageningen 72-11; 162 pp.

Mitchell, B. K., 1978. Some aspects ofgustation in the larval red turnip beetle, Entomoscelis americana, related to feeding and host plant selection. Entomol. exp. appI. 24: 540-549.

Mitchell, B. K., 1988. Adult leaf beetles as models for explor- ing the chemical basis of host-plant recognition. J. insect Physiol. 34: 213-225.

Mittchell, B. K. & J. F. Sutcliffe, 1984. Sensory inhibition as a mechanism of feeding deterrence: effect of three alkaloids on leaf beetle feeding. Physiol. Entomol. 9: 57-64.

Mohr, K.-H., 1966. Chrysomelidae. In: H. Freude, K.W. Harde & G.A. Lohse (eds.), Die K~fer Mitteleuropas, Band 9. Goecke & Evers Verlag, Krefeld: pp. 95-299.

Nayar, J. K. & A.J. Thorsteinson, 1963. Further investi- gations into the chemical basis of insect-host plant relationships in an oligophagous insect, Plutella maculipennis (Curtis) (Lepidoptera: Plutellidae). Can. J. Zool. 41: 923-929.

Newton, H. C. F., 1928. The biology of the flea beetles (Phyllotreta) attacking cultivated Cruciferae. J. s.-east Agric. Coll. Wye. 25: 90-115.

Nielsen, J. K., 1977. Host plant relationships of Phyllotreta nemorum L. (Coleoptera: Chrysomelidae). I. Field studies. Z. angew. Entomol. 84: 396-407.

Nielsen, J.K., 1978a. Host plant discrimination within Cruciferae: Feeding responses of four leaf beetles (Coleop- tera: Chrysomelidae) to glucosinolates, cueurbitacins and cardenolides. Entomol. exp. appl. 24: 41-54.

Nielsen, J. K., 1978b. Host plant selection of monophagous and oligophagous flea beetles feeding on crucifers. Entomol. exp. appl. 24: 562-569.

Nielsen, J. K., 1988. Crucifer-feeding Chrysomelidae: Mecha- nisms of host plant finding and acceptance. In: P. Jolivet, E. Petitpierre & T. H. Hsiao (Eds.), Biology of Chryso- melidae. Kluwer Acad. Publ., Dordrecht, Boston, Lancaster: pp. 25-40.

Nielsen, J.K., 1989. Host plant relations of Phyllotreta nemorum L. (Coleoptera: Chrysomelidae). II. Various defensive mechanisms in plants and their role in defining the host plant range. J. appl. Entomol. 107: 193-202.

Nielsen, J. K., L. Dalgaard, L. M. Larsen & H. Sorensen, 1979a. Host plant selection of the horseradish flea beetle Phyllotreta armoraciae (Coleoptera: Chrysomelidae): Feeding responses to glucosinolates from several crucifers. Entomol. exp. appl. 25: 227-239.

Nielsen, J. K., L.M. Larsen & H. Sorensen, 1979b. Host plant selection of the horseradish flea beetle Phyllotreta armoraciae (Coleoptera: Chrysomelidae): Identification of two flavonol glycosides stimulating feeding in combination with glucosinolates. Entomol. exp. appl. 26: 40-48.

Renwick, J. A. A. & C. D. Radke, 1987. Chemical stimulants and deterrents regulating acceptance or rejection of crucifers by cabbage butterflies. J. Chem. Ecol. 13: 1771-1776.

Rothschild, M., H. Alborn, G. Stenhagen & L.M. Schoonhoven, 1988. A Strophanthidin glycoside in

259

Siberian wallflower: A contact deterrent for the large white butterfly. Phytochemistry 27: 101-108.

SchOni, R., E. St/idler, J. A. A. Renwick & C.D. Radke, 1987. Host and non-host plant chemicals influencing the oviposition behaviour of several herbivorous insects. In: V. Labeyrie, G. Fabres & D. Lachalse (eds.), Insects-Plants, Proc. 6 th Int. Symp. Insect-Host Plant Relationships (Pau, 1986). Dr. W. Junk Publ., Dordrecht, Boston, Lancaster: pp. 31-36.

Schoonhoven, L. M., 1967. Chemoreception of mustard oil glucosides in larvae of Pieris brassicae L. Med. Kon. Ned. Akad. Wet. Amsterdam, Ser. C. 5: 556-568.

Siegel, S., 1956. Nonparametric statistics for the behavioral sciences. Mc. Graw-Hill, New York: 312 pp.

St/idler, E., 1986. Oviposition and feeding stimuli in leaf surface waxes. In: B. Juniper & R. Southwood (eds.), Insects and the plant surface. Edward Arnold Publ., London: pp. 105-121.

Stein, E., 1963. Zur Biologie von Drosophila tiara Fall. (Diptera, Drosophilidae), einer Minierfliege an Kultur- cruciferen. Z. angew. Entomol. 52: 39-56.

Tanton, M. T., 1965. Agar and chemostimulant concentra- tions and their effect on intake of synthetic food by larvae of the mustard beetle, Phaedon cochleariae Fab. Entomol. exp. appl. 8: 74-82.

Usher, B.F. & P. Feeny, 1983. Atypical secondary compounds in the family Cruciferae: tests for toxicity to Pieris rapae, an adapted crucifer-feeding insect. Entomol. exp. appl. 34: 257-265.

Verschaffelt, E., 1910. The cause determining the selection of food in some herbivorous insects. Proc. K. Akad. Wet. Amsterdam, Sect. Sci. 13: 536-542.

Weissenb6ck, G., R. Hedrich & G. Sachs, 1986. Secondary phenolic products in isolated guard cells, epidermal cell and mesophyll cell protoplasts from pea (Pisum sativum L.) leaves: Distribution and determination. Protoplasma 134: 141-148.

the effect of glucosinolates on responses of young phyllotreta nemorum larvae to non-host plants

Documents