J. Appl. Ent. 107 (1989), 193-202 0 1989 Verlag Paul Parey, Hamburg und Berlin ISSN 0044-2240

Royal Veterina y and Agricultural University, Department of Chemistry, Frederiksberg, Denmark

Host plant relations of Phyllotreta nemorum L. (Coleoptera, Chrysomelidae)

11. Various defensive mechanisms in plants and their role in defining the host plant range

By J. K. NIELSEN

Abstract

Responses of adults and larvae of Phyllotretu nemorum to 28 plants which contain glucosinolates and 4 plants without these compounds were studied in the laboratory. Results were compared with the results from a previous field experiment using the same plants. Various defensive mechanisms against attack by P. nemorum were discovered in plants: 1. Content of feeding inhibitors against adult insects, 2. low acceptability for initiation of leaf mines by larvae, 3. presence of barriers to leaf mine initiation on the leaf surface and 4. low survival rates of larvae. Host plant utilization in the field experiment could be explained by presence vs. absence of defensive mechanisms in the plants.

1 Introduction

Phyllotretu nemorum L. is an oligophagous flea beetle feeding mainly on a few plant species belonging to the family Cruciferae (Brassicaceae). All members of this and a few other families e.g. Capparaceae, Resedaceae and Tropaeolaceae share a characteristic group of sulfur containing compounds: the glucosinolates (DAHLGREN et al. 1981; FENWICK et al. 1983). These compounds are feeding stimulants for adult P. nemorum and for several other crucifer feeding insects (LERIN 1980; NIELSEN 1978a, b, 1988).

In a field experiment it was shown that a natural population of P. nemorum utilized only a few of the available glucosinolate containing species (NIELSEN 1977). Radish (Ruphunus sutiwus L.) and turnip (Brussicu rupu L. var. rupu) were by far the most infested plant species; black and white mustard (Brussicu nigm [L.]) Koch and Sinupis ulbu [L.]), a few other Brussicu species, Cheirunthus cheiri L. and Eryrimum hieraciifolium L. were also infested by the natural population, but 15 glucosinolate containing and 4 non-glucosinolate containing species were not infested. The glucosinolate contents of most crucifers were satisfactory as feeding stimulants for adult P. nemorum (NIELSEN 1978b). Plants which are unacceptable may then contain some defensive mechanisms in addition to the glucosinolates (FEENY 1977).

P. nemorum hibernate in the adult stage. In spring, the beetles colonize and feed on various crucifers (HEIKERTINGER 192~; NEWTON 1928). Oviposition starts after a few days of feeding. Oviposition behaviour has not been studied, but eggs are laid in the soil probably close to the food plants of the adult females (NIELSEN 1977). After hatching, the young larvae climb the plant and initiate a mine, usually in one of the lower leaves. There are three larval instars (NEWTON 1928). Larval development lasted about 20 days in the field (NIELSEN 1977). Larvae may change to new mines several times during the whole larval period. Pupation takes place in the soil.

Several critical steps seems to be involved in successful utilization of a plant by P. nemorum: 1. the plant should be acceptable for adult feeding and oviposition, 2. the plant

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194 /. K. Nielsen

should be acceptable for mine ;nitiation by newly emerged first instar larvae and 3. the plant should support larval development. Plant defenses might be directed against the insects at any of these steps. In order to investigate these possibilities, laboratory experiments were performed with the same plant species which were included in the field experiment (NIELSEN 1977). The purpose of these experiments was dual: 1 . to investigate the defensive mechanisms utilized by plants against P. nemoyum and 2. to discover whether it was possible to explain the utilization of plants in the field by presence vs. absence of defensive mechanisms in the plants.

2 Materials and methods

2.1 Insects

P. nemorum was reared in the laboratory on radish. The culture was supplemented by field collected s ecimen every year. Adult beetles were kept at 24 +- 1 "C and a photoperiod of 18 h light and 6 h Lrkness in 400 ml plastic vials with a gypsum charcoal bottom layer (NIELSEN 1978a). Beetles used in the experiments were at least 3 days old and had been feeding on radish from the day of emergence.

Eggs were laid in the bottom layer. Egg develo ment lasted 5-6 days at 24 "C, and every 5 days the beetles were removed and transferred to new viak After hatching and hardening of the cuticle, the larvae were found crawling on the walls and lids of the vials. These larvae were used in the experiments. They were less than 24 h old and had no access to plant material prior to the experiments. The health of the larvae was judged from their behaviour in the vials. Only healthy larvae were used in the experiments.

2.2 Plants

The experiments included the same 28 glucosinolate containing plants (species and/or varieties) and 4 non-glucosinolate containing species as in the field experiment. A detailed list of species and cultivars is given in NIELSEN (1977) together with information on the origin of seeds. The experiments included the most important cultivated species and varieties of Cruciferae as well as glucosinolate containing species from various taxa: Four families (Cruciferae, Capparaceae, Resedaceae, and Tropaeolaceae) and seven subfamilies of Cruciferae (SCHULZ 1960). Latin names are used exce t for some of the cultivated crucifers. Two cultivars of radish, turnip and oilseed rape were includei R. s. var. radicula cv. Copenhagen Market (Radish r), R. s. var. niger cv. Chinese Rose-red (Radish n), B. c. var. rapa cv. Snowball (Turnip s; a vegetable), B. c. var. rupu cv. Fynsk Bortfelter (Turnip f; a fodder crop), B. nupus L. var. oleifera cv. Bronowski (Rape b; with low glucosinolate levels in the seeds) and B. n. var. oleifera cv. Gyllen (Rape g; with high glucosinolate levels in the seeds). Another cultivar of oilseed rape with low seed glucosinolate levels, Line (Rape I) was used in laboratory experiments with larvae instead of cv. Bronowski.

Plants were grown in the field at the experimental station Hajbakkegaard, Taastrup. Plants were in the same stages as used in the field experiment, e.g. biennials like Hesperis mutronalis L. and Lunaria annua L. (= L. biennis Mnch.) were in the vegetative stages. Experiments were started not later than 24 h after cutting of the leaf material in the field.

2.3 Exper imenta l conditions

Leaf disc tests were used in experiments with adult beetles. Leaf discs (d = 14 mm) were cut with a cork borer. Four leaf discs of the test plant (test discs) were presented together with four leaf discs from Radish r (reference discs) in 400 ml vials similar to those used for rearing. Beetles had access to the leaf discs for 24 hours. Temperature and light conditions were as described above. The numbers of mmz eaten from test and reference discs were counted under a preparation microscope. A preference index Q = T/(T+R) was calculated; T = number of mm2 eaten from the four test discs and R = the number of mm2 eaten from the four reference discs. Thus, the Q-values ranged from 0 (no feeding on test discs) to 1 (no feeding on reference discs). Further details on the bioassay are given by NIELSEN (1978a).

Experiments with larvae were non-choice tests with only one plant species in each test. Intact leaves were presented in plastic vials together with a small piece of moist filter paper. The size of the vials (12.5, 25 or 160 ml) varied with the size of leaves. Experiments with leaf discs (d = 14 mm) were performed in the smallest vials (12.5 ml). A small piece of moist filter paper was rolled into a cylinder which was a little longer than the diameter of the vial. The paper cylinder was firmly fixed close to the

Host plant relations of Phyllotreta nemorum L. 195

bottom of the vial, and the leaf discs were placed so that they touched both the cylinder and the walls of the vial.

Larvae were transferred to the ex erimental vials by means of a small piece of soft and wet paper held by a pair of forceps. This proce&re proved to be the least dama ing to the larvae. In ex eriments with intact leaves, 5 larvae were usually transferred to each vial. In a i experiments with IeaPdiscs and in some experiments with intact leaves, only one larva was present in each vial. Experiments were run in darkness at 22 + 2 "C. After 24 h, the number of mines was counted and after 3 days the number of surviving larvae was counted. Larvae could not survive for a period of 3 days without feeding.

2.4 Statistics

Sugar beet (Beta vulgaris L.) was chosen as an unacceptable reference plant. This plant is unacceptable to all stages of P. nemorum even after treatment with glucosinolates (NIELSEN, in prep.). Plants were divided into three categories according to their acceptability compared to radish and sugar beet: 1 . highly acceptable plants which are not significantly different from Radish r, but significantly different from sugar beet (++), 2. plants of intermediate acceptability which are significantly less acceptable than Radish r, but significantly more acceptable than sugar beet (+), and 3. unacceptable plants which are significantly different from Radish r, but not significantly more acceptable than sugar beet (0).

In experiments with adults, the means of Q-values as well as the 95 % confidence intervals were calculated. Test plants are significantly different from Radish r, if these confidence intervals do not include 0.5, the theoretical Q-value for Radish r which was present as a reference in all ex eriments. The Mann-Whitney U test was used in the comparisons with sugar beet. In tests with Ervae, the Fisher exact probability test was used if N 5 30 and the xz test if N > 30 (SIEGEL 1956).

3 Results

The 28 glucosinolate containing plants (species and/or cultivars) differred very much in acceptability for feeding by adult P. nemorum. Only 6 plants (21 YO) were fully acceptable: 2 cultivars of radish (including the reference cultivar Radish r), 2 cultivars of turnip and black and white mustard (table 1). Another 12 (43 %) were partially acceptable, while 10 (36%) were nearly or totally unacceptable. The results with some species, e.g. Cleome aurea Celak and Tropaeolum majus L. were variable. A small amount of feeding occurred in a few cases, but not in others, and there were no significant differences between these plants and sugar beet. Sugar beet and 3 other plants without glucosinolates were unaccept- able for feeding by adult P. nemorum (table 1).

Larvae seemed to have a broader host plant range than adults. 13 glucosinolate containing plants (46 Yo) were fully acceptable for mine initiation by first instar larvae (table 2). O u t of these 13 plants, 6 were fully acceptable for adult feeding, 5 were partially acceptable and 2 were unacceptable (table 1). Another 10 glucosinolate containing plants (36 %) were of intermediate acceptability, while 3, Hespevis rnatronalis and 2 Tropaeolum species, were unacceptable. In Zberis species, older plants were less satisfactory for mine initiation than younger plants (table 2). The Brassica species with glaucous leaves and a blueish wax bloom (oilseed rape, swede, cabbage and cauliflower) were relatively unac- ceptable for mine initiation when intact leaves were presented. However, they were fully acceptable when presented as leaf discs (table 3). Results with other plant species did not depend to the same extent on the way of presentation (table 3). There were no differences in responses of larvae or adults to cultivars of oilseed rape with high vs. low seed glucosinolate levels (tables 1-3).

A few larvae entered mines in Malwa silvestris L. although this plant does not contain glucosinolates. No larvae initiated mines in 3 other species which d o not contain these compounds (table 2 ) .

The fate of larvae after mine initiation was also different in different plant species. In most plants, there was a high rate of survival, but in Zberis umbellata L., Bunzas erucago L., Matthiolaparwiflora (Schousb.) R.BR., and Reseda alba L. all larvae died within 3 days (table 2). The larvae usually left their initial mines in these plants after a few hours, but in a

196 J . K. Nielsen

Table 1. Acceptability of plants to adult Phyllotreta nemorum in leaf disc tests in the laboratory

Cruciferae Radish n Turnip f Turnip s Rape b Rape g Swede Cabbage Cauliflower Crambe hispanica Black mustard White mustard Capsella bursa-pastoris Iberis amara I . umbellata Bunias erucago Lunaria annua Matthiola pamiflora Cheivanthus cheiri Erysimum hieracii olium Hesperis matrona if is

Resedaceae Reseda alba R. luteola R. odorata

Cap araceae d o m e aurea C. violacea

Tropaeolaceae Tropaeolum majus T. peregrinum

Pisum sativum (pea) Lactuca sativa (lettuce) Malva silvestris Beta vulgarzs (sugar beet)

Plants without glucosinolates

+ 95 % conf. intervals

0.52 f 0.088 0.50 f 0.083 0.53 f 0.111 0.11 f 0.120 0.26 f 0.128 0.27 f 0.081 0.05 f 0.042 0.08 f 0.042 0.03 f 0.066 0.43 f 0.133 0.47 f 0.063 0.01 f 0.025 0.00 f 0.004 0.00 f 0.005 0.05 f 0.080 0.01 f 0.009 0.13 f 0.087 0.04 f 0.029 0.21 f 0.170

0

0.29 f 0.117 0.03 f 0.021

U

0.04 f 0.059 0.10 f 0.113

0.10 f 0.181 0

0 ?. U

0 0

N Category

6 ++ 5 ++ 8 ++ 6 + 6 + 6 + 6 + 6 + 5 0 6 +f 6 ++ 6 0 5 0 5 0 6 + 6 0 6 + 5 + 5 + 6 0

6 + 5 + 5 0

6 0 6 +

6 0 6 0

5 0 5 0 5 0 5 -

++: complete acceptability; not significantly different from Radish r; +: intermediate acceptabil- ity; significantly less acceptable than Radish r, but more acceptable than sugar beet (see text); 0: unacceptable; not significantly more acceptable than sugar beet; -: reference plant, or category cannot be determind.

few cases they died within the mines. Some larvae made 3 4 mines in these plants before they actually died. In these cases, the percentage of larvae which initiated a mine could not be measured if several larvae were present in each vial, and additional experiments with 1 larva per vial were performed (table 2).

Data from the laboratory experiments are summarized and compared with results from the field experiment in table 4. The laboratory experiments explain to a large extent the observations from the field. The most acceptable plants in the field, radish and turnip, were fully acceptable to both larvae and adults in the laboratory. The low infestation of the 2 mustard species in the field is unexpected from the present laboratory data, but may be explained by their growth form (see discussion). Cheivanthus cheivi and Erysimum hieruciifolium were fully acceptable for the larvae, but they were only partially acceptable

Host plant relations of Phyllotreta nemorum L. 197

Table 2. Acceptability of intact leaves of various plants for initial steps in host plant selection of PhyIlotreta nemorum larvae

Plants Numbers of Numbers of larvae Numbers of larvae Categoriesb larvae initiating a minea surviving for 3 started days

N I I ( % ) s S/I.lOO CI cs

Cruciferae Radish r Radish n Turnip f Turnip s Rape 1

Swede Cabbage Cauliflower Crambe hispanica Black mustard White mustard Capsella bursa-pastoris Iberis amara, vegetative Iberis amara, bud stage Iberis amara, flowering Iberis umbellata, vegetative Iberis umbellata, bud stage Iberis umbellata, flowering Bunias erucago Bunias erucago (I larvahial) Lunaria annua Matthioloa pamiflora M. parutflora (1 larvahial) Cheiranthus cheiri Erysimum hieraciifolium Hesperis matronalis

Cap araceae CEome aurea C. viofacea

Resedaceae Reseda alba (1 larvahial) R. luteola R . odorata

Tropaeolaceae Tropaeolum majus T. preegrinum

Pisum sativum (pea) Lactuca sativa (lettuce) Malva sifvestris Beta vulgarzs (sugar beet)

Rape g

Plants without glucosinolates

25 15 15 55 30 35 25 70 40 30 20 15 25

105 65 65 30 20 35 15 40 30 15 10 50 20 40

15 25

30 25 15

40 15

20 30 40 25

25 100 15 100 15 100 52 (1) 95 15 50 15 43 5 20

19 27 17 43 18 (3) 60 18 (1) 90 14 93 23 92 63 60 48 74 13 20 14 47 5 (2) 25 1(1) 3

11 (2) 37

4 (11) - 34 85

8 (7) i0 9

40 80 20 100

6 15

12 80 21 84

12 40

5 33 11 (3) 44

2 (3) ; 1

0 0 0 0 5 13 0 0

25 15 15 50 10 13 3

19 17 17 17 13 23 24

2 1 0 0 0 0 0 4 0 0

33 20 1

12 20

0 11 5

0 0

0 0 5 0

100 100 100 96 66 87 60

100 100 94 94 93

100 38 4 8 0 0 0 0 0

36 0 0

83 100 16

100 95

0 100 100

0 0

0 0

100 0

- ++ ++ ++ + + 0 + + + ++ ++ ++ + + + 0

0 0 - ++ + ++ i f ++

-

0

++ ++

+ + +

0 0

0 0 0 -

- ++ ++ ++ + + + + + ++ ++ ++ +

0

0 0 0 0 0 0 0 0 0 0 + ++ 0

++ ++

0 + +

0 0

0 0 0 -

a Numbers of empty mines (in brackets) should perhaps be added to I (see text). - CI: category concerning mine initiation; C,: category concerning larval survival; see Table 1 for explanation of symbols.

for the adults and only slightly infested in the field. The low infestation of the glaucous Brussica species in the field agreed with the reduced acceptability of these plants for feeding by adults and mine initiation by larvae, although there is a high survival rate for larvae which manage to initiate mines.

198 J . K. Nielsen

Table 3. Acceptability of leaf discs of various plants for initial steps in host plant selection of Pbyllotreta nemorum larvae

Plants Numbers Numbers of larvae of larvae initiating a mine started

N I I (Yo)

Numbers of larvae surviving for 3 days

S S/I.lOO pa

Cruciferae Radish r Rape 1 Rape g Swede Cauliflower Cabbage Iberis amara I . umbellata Bunias erucago Matthiola pamiflora Cheiranthus cheiri Hesperis matronalis

Reseda alba Resedaceae

25 25 20 25 30 25 10 10 15 15 15 30

15

25 100 25 100 19 95 25 100 28 93 22 88

1 10 1 10

11 73 14 93 10 67 10 33

12 80

25 100 14 56 15 79 20 80 26 93 13 59 0 0 0 0 0 0 0 0 9 90 0 0

NS p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 NS NS NS NS NS NS

0 0 p<0.05

a Significance levels for com arisons of responses to leaf discs with the responses to intact leaves of the same plants given in Tahe 2; xz test.

Table 4. Comparison of laboratory data with results obtained in a previous field experiment Symbols are used as described in table 1

Acceptability to adult feeding

Acceptability to mine

initiation

Acceptability for larval

development

Infestation by field population

Examples

++ ++ + + 0 (+) 0 + + 0 0

++ ++ ++ + ++ + ++ + + 0

++ ++ ++ ++ ++ (++I 0 0 0 -

++ + + (+) 0 0 0 0 0 0

Radish, turnip Black and white mustard Cheiranthus, Erysimum Glaucous Bvassica spp. Capsella, Cleome spp. Cvambe, Reseda odorata Matthiola, Bunias Reseda alba young Ibevis spp., Lunavia mature Iberis, Hesperis, and Tropaeolum spp.

All other plant species were not infested in the field, and in all cases a serious hindrance to attack has been documented in the laboratory experiments. Capsellu bursa-pustovis (L.) and the two Cleome species were only slightly acceptable for adult feeding although they were acceptable for mine initiation and development of larvae. In B u n k erucago, Matthiola parv i fora and Reseda alba there was also some reduction in adult feeding, but the most serious hindrance to attack in these species seemed to be the failure to support larval development. The remaining 9 glucosinolate containing species seemed to be protected against both larvae and adults.

Host plant relations of Pbyllotreta nemorum L.

4 Discussion

199

The present results demonstrate that both larvae and adults of P. nemouum are closely associated with glucosinolate containing plants. Glucosinolates stimulate feeding in adults (NIELSEN 1978a, b) and mine initiation in larvae (NIELSEN, in prep.). Glucosinolates from both host and non-host crucifers were stimulatory to adult feeding (NIELSEN 1978b). All glucosinolate containing species are therefore potentially susceptible to attack by P. nemoyum if they do not have other defensive mechanisms which counteract the effect of the glucosinolates. The present results demonstrate that such defensive mechanisms are present in many glucosinolate containing plants, and that they may be directed against larvae as well as against adult beetles.

Content of feeding deterrents against adult beetles seems to be a common and effective defensive mechanism in glucosinolate containing plants. In a number of cases, the acceptability of a particular plant species to adults were found to be inversely correlated with the content of feeding deterrents (NIELSEN 1978b). The low acceptability of many crucifers to adults in the present experiments (table 1) is caused by the presence of feeding deterrents ( NIELSEN 1978a, b). The most active feeding deterrents in Iberis species have been identified as the cucurbitacins (NIELSEN 1978a; NIELSEN et al. 1977). Further feeding deterrents have not yet been identified from glucosinolate containing plants.

In contrast to other Chrysomelidae, P. nemouum is relatively insensitive to cardenolides (NIELSEN 1978a), and the cardenolide containing species, Cheiuunthus cheiri and Evy- simum hieuaciifolium are relatively acceptable both in the laboratory and in the field (table 4). Cheiranthus cheivi proved to be less acceptable to adult feeding in the present experiments compared to earlier experiments with plants grown in the greenhouse (NIELSEN 1978a). A possible reason might be that leaves of this plant became very hard when grown in the field. Hardness of leaves were also suggested to be the reason why Crambe hispanica was unacceptable to adult feeding even though it did not contain extractable feeding deterrents (NIELSEN 1978b). Hairiness may be another factor affecting responses to crucifers in both larvae and adults (NIELSEN, unpubl.).

Defensive measures seems to be more often directed against adults than against larvae. Only 6 % of the investigated glucosinolate containing plants were fully acceptable for adult feeding while 46 % were fully acceptable for mine initiation in larvae. This difference may have been exaggerated because choice tests were performed with the adults and non-choice tests with the larvae. This difference in experimentzl design was considered to be most appropriate for simulating the conditions met by the two stages in the field. Under natural conditions larvae do not often meet a choice situation, because they are unlikely to find several crucifers or other acceptable plants within their limited search area. Adults are much more mobile and may much easier investigate a number of possible host plants before they finally settle for continuous feeding and oviposition. Several other cases are known where adults are more discriminatory than larvae, but the opposite may occur as well (THOMPSON 1988 and refs. therein).

Different types of defensive mechanisms against larvae were demonstrated. Hesperis matuonalis, Iberis species (especially older plants) and Tropaeolum species were un- acceptable for mine initiation, and reduced rates of mine initiation were observed in many other glucosinolate containing plants, too. These plants might contain deterrents to mine initiation. A different type of defensive mechanism was demonstrated in Bunius euucago, Matthiola pauviflora and Reseda alba. A high percentage of larvae initiated mines in these species, but no larvae survived for three days. In most cases larvae left the mines again rapidly, but in a few cases dead larvae were found within the mines. Therefore, these plants seemed to be toxic to P. nemorum larvae, although at present it cannot be ruled out that these plants may contain deterrents or lack some stimulants which influence continuous feeding in the mines, but not mine initiation.

200 J. K. Nielsen

The Brassica species with glaucous leaves were relatively unacceptable when presented as intact leaves. However, the results were variable and probably depending on the development of the wax layer. These plants became much more acceptable when presented as leaf discs. Mine initiations usually occurred in edges of leaf discs of these plants where there was a direct access to the mesophyll. In glossy leaves like radish, the larvae usually penetrated the epidermis somewhere on the leaf surface (NIELSEN, unpubl.). These observations suggest, that there is a barrier to attack by young P. nemorum larvae on the surface of glaucous leaves of these Brassica species. This barrier may be the wax layer itself. A negative effect of the wax layer might be the interference with attachment to the leaf surface as demonstrated in the mustard beetle, Phaedon cochleariae (STORK 1980). The failure to detect any differences in responses to oilseed rape cultivars with different seed glucosinolate levels is in agrement with similar findings in other insects and suggest that glucosinolate levels in green arts may not be correlated with the levels in the seeds (LAMB

glaucous plants is in progress. In early experimental work on leaf mining beetle larvae BUHR (1954, 1956) used a

technique which is different from the one used here. Older larvae in their original mines in various crucifers were transplanted to the mesophyll of the experimental plants. It was then observed whether feeding occurred in the new plant and whether development proceeded until pupation and adult emergence. These transplantations were mainly made to plants where no natural infestation was observed. BUHR reported some natural infestation in most glucosinolate containing species at least in the Botanical Garden of Rostock, and few transplantation experiments were performed with species included in the present experi- ments. No feeding occurred on plants without glucosinolates, but Reseda luteola, Reseda odorata, and Tropaeolum peregrinum were acceptable in BUHR’S experiments. These results demonstrate less discrimination between glucosinolate containing plants than observed in the present experiments, probably because older larvae were used and they are able to utilize a larger range of food sources than younger larvae (NIELSEN, unpubl.).

The discrimination shown by larvae and adults in the present laboratory experiments seems to agree with experiments from the field (table 4). In all cases, a zero in the column showing field infestation is followed by one or more zeroes in the columns showing results from the laboratory experiments. The high rates of infestation in radish and turnip and the intermediate rates in Cheiranthus cheiri and Erysimum hieraciifolium in the field correlate with differences in adult feeding rates, while all these species are fully acceptable for the larvae. The low rates of field infestation of mustard species by the natural population in the field cannot be explained for the moment because these plants are as acceptable as radish and turnip to both larvae and adults in the laboratory. It might be caused by differences in growth form. Mustard species stretch early and do not have a permanent rosette of leaves while radish and turnip remained in the rosette stage throughout the period of the field experiment. Therefore, the larvae have to climb a longer distance in order to find a site for mine initiation in mustard species than in radish and turnip. This factor has been shown to be important in field experiments with artificial infestation (NIELSEN 1977). Plant height and growth form might also influence adult feeding and oviposition behaviour, and there is some indirect evidence from the field experiments that fewer eggs are laid close to the mustard species (NIELSEN 1977).

A critical point in the present laboratory investigations is whether it is possible to judge the suitability of a plant for larval development after only three days. This period cannot be prolonged in experiments with excised leaves which start to deteriorate after a period of this length. The first three days are probably very critical for the larvae, because older larvae are able to attack plants which are unacceptable to first instar larvae (NIELSEN, unpubl.). The validity of the method is supported by the results of a field experiment with artificial infestation. In this experiment, some larvae developed until pupation in all

1988; LARSEN et al. 1985; 8: HMAN 1982). A more detailed study of larval responses to

Host plant relations of Phyllotreta nemorum L. 201

glucosinolate containing plants which supported larval development for three days in the laboratory (NIELSEN 1977).

The present s tudy suggest that simple laboratory experiments can give a fairly good judgement on whether a plant will be infested by a free living population of P. nemorum. The methods may be used in screening for resistance in crop plants, and in the evaluation of wild plants (weeds) as possible natural host plants for populations of P. nemorum. Several defensive mechanisms were discovered which deserve further investigations, e.g. the nature of the barrier to mine initiation in Brussicu species with glaucous leaves, and the apparently toxic properties of certain wild plants. Furthermore, there is a severe lack on information on oviposition behaviour in flea beetles and in crucifer feeding Chrysomelidae in general (NIELSEN 1988). It would be very interesting to evaluate how oviposition behaviour in P. nemorum relates to the present data on feeding behaviour in adults and larvae.

Acknowledgements

I am very rateful to Dr. E. STADLER, Wadenswil for critical comments. The study was supported by The Danisk agricultural and veterinary research Council and The Carlsberg Foundation.

Zusammenfassung

Uber die Beziehungen von Phyllotreta nemorum (Col., Chrysomelidae) Z U T Wirtspfanze. II. Verteidigungsmechanismen in Pjlanzen und ihre Rolle fur die Abgrenzung

des Wirtspflanzenspektrums Phyllotretu nemorum ist ein oligophager Erdfloh, der an Cruciferen und andere glukosinolathaltigen Arten gebunden ist. Die Imagines fressen Locher in die Blatter, und die Larven sind Blattminierer.

Das Verhalten der Larven und Imagines gegen 28 glukosinolathaltige und 4 nicht-glukosinolathal- tige Pflanzen wurde im Labor untersucht. Die Resultate der Laboruntersuchungen wurden mit fruheren Freilanduntersuchungen verglichen. Verschiedene Verteidigungsmechanismen der Pflanzen gegen die Besiedlung durch P. nemorum waren ersichtlich: 1. Vorhandensein von fraghemmenden Stoffen (Deterrenten) gegen Imagines; 2. niedrige Akzeptanz fur die Einbohrung und den Anfang der Minierung der Larven; 3. Barrieren gegen die Einbohrung der Larven an der Blattoberflache; 4. geringe Fahigkeit, die Larvenentwicklung zu fordern. Pflanzenarten, die im Freilandversuch besiedelt waren, zeigten weniger Verteidigungsmechanismen. Pflanzen mit sehr wirksamen Verteidigungsme- chanismen wurden im Freiland nicht besiedelt.

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DAHLGREN, R. M. T.; JENSEN, S. R.; NIELSEN, B. J., 1981: A revised classification of the Angiosperms with comments on correlation between chemical and other characters. In: Phytochemistry and Angiosperm Phylogeny. Ed. by D. A. YOUNG and D. S. SEIGLER. New York: Praeger Sci. Publ. pp. 149-204.

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Author’s address: JENS KVIST NIELSEN, Royal Veterinary and Agricultural University, Department of Chemistry, Thorvaldsensvej 40, DK-1871 Frederiksberg c, Denmark