Effects of snowdrop lectin (GNA) delivered via artificial diet and transgenic plants on the development of tomato moth (Lacanobia oleracea) larvae in laboratory and glasshouse trials

Pergamon PII: S0022-1910(97)00042-5

J. Insect Physiol. Vol. 43, No. 8, pp. 727–739, 1997  1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0022-1910/97 $17.00 + 0.00

Effects of Snowdrop Lectin (GNA) Delivered Via Artificial Diet and Transgenic Plants on the Development of Tomato Moth (Lacanobia oleracea) Larvae in Laboratory and Glasshouse Trials ELAINE FITCHES*, ANGHARAD M. R. GATEHOUSE,*† JOHN A. GATEHOUSE* Received 24 January 1996; revised 17 March 1997

The effects of snowdrop lectin (Galanthus nivalis agglutinin, GNA) on Lacanobia oleracea larval growth, development, consumption, and survival, were examined by 3 distinct bioassay methods. Larvae were reared on artificial diet containing GNA at 2% (w/w) dietary protein; on excised leaves of transgenic potato expressing GNA at approx. 0.07% of total soluble proteins; and on transgenic potato plants expressing GNA at approx. 0.6% of total soluble proteins in glasshouse trials. Significant effects on larval growth were observed with all three treatments. At 21 days after hatch mean larval biomass was reduced by 32 and 23%, in the artificial diet and excised leaf bioassays respectively. In glasshouse trials a 48% reduction in insect biomass per plant was observed after 35 days. The artificial diet and excised leaf assays also showed that GNA significantly slowed larval development as assessed by instar duration. GNA caused a 59% overall reduction in mean daily consumption in the artificial diet assay, and a significant reduction in leaf damage in glasshouse trials. However, prolonged compensatory feeding by larvae in the excised leaf assay resulted in their consuming 15% more total leaf material than the control group. Adaptation to low levels of GNA, in terms of biomass recovery and compensatory feeding, was observed within one larval generation in the detached leaf assay. No significant effects of GNA on larval survival were observed in the artificial diet and detached leaf bioassays, whereas survival was decreased by approx. 40% in the glasshouse bioassay. The assays show that the insecticidal effects of GNA can be observed both in vitro when fed in artificial diet and in planta, and can be demonstrated in the glasshouse as well as under growth cabinet conditions.  1997 Elsevier Science Ltd Snowdrop lectin Insect resistance Lepidopteran larvae

INTRODUCTION

Problems associated with widespread pesticide usage, together with the development of insect resistance to Bt toxins in genetically engineered crops, has resulted in greater interest in the potential for exploiting plant defensive proteins, such as lectins, to help combat insectrelated crop damage (for review see Gatehouse and Hilder, 1994). Lectins have been suggested to have toxic

*Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, U.K. †To whom all correspondence should be addressed.

Transgenic plants Lacanobia oleracea

effects towards lepidopteran larvae, although such effects are variable (Shukle and Murdock, 1983; Boulter et al., 1990; Czapla and Lang, 1990). GNA (Galanthus nivalis agglutinin; snowdrop lectin), a lectin exhibiting a strict specificity for alpha-d-mannose, belongs to a group of lectins isolated from bulbs of species in the plant family Amaryllidaceae (Van Damme et al., 1987). GNA is toxic towards a number of important insect pests; these include Homoptera such as aphids (Rahbe´ et al., 1995; Sauvion et al., 1996) and the rice brown plant hopper (Powell et al., 1993), and Coleoptera, such as bruchid beetles (Gatehouse et al., 1995). However, the effects of GNA ingestion vary from

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species to species of insect. GNA incorporated in artificial diet at 0.1% w/v concentration had a significant effect upon both development and survival of rice brown planthopper (Powell et al., 1993), with 90% corrected mortality observed over 5 days. However, at the same concentration in artificial diet, GNA had only marginal effects upon survival of glasshouse potato aphids (Aulacorthum solani), although it significantly decreased both the development and fecundity (Down et al., 1996). Variability in the insecticidal effects of GNA between insect species may be accounted for by differences in the mechanisms involved in lectin action, which remain to be clarified. As part of a wider investigation into the mechanisms of lectin toxicity towards insects, the present paper examines the effects of GNA upon larvae of the tomato moth (Lacanobia oleracea), a member of the economically important noctuid group of Lepidopteran pests. This polyphagous species is widely distributed throughout the greater part of Europe and Asia Minor, and is an occasional pest of glasshouse tomatoes in the U.K. (Lloyd, 1920; Burgess and Jarrett, 1976). Recently, transgenic potato plants expressing GNA have been shown to have significantly enhanced resistance to attack by L. oleracea larvae when tested under growth cabinet conditions (Gatehouse et al., 1997). The present paper reports detailed bioassays of the effects of GNA upon L. oleracea larvae when incorporated into an artificial diet, and when expressed in leaves from transgenic potato plants, and shows that similar effects are observed in glasshouse trials of whole plants. MATERIALS AND METHODS

Insect culture A culture of Lacanobia oleracea was originally obtained from Central Science Laboratory, Slough, Berkshire. The insects were maintained at 25°C with a 16:8 L:D regime. Plant material Virus-free, sterile plantlets of Solanum tuberosum L. cv. Desiree´ were obtained from the Scottish Office, Agriculture and Fisheries Department, Edinburgh, U.K. Shoot cultures were maintained in test tubes containing 10 ml potato medium (PM) as described in Newell et al. (1991). The cultures were grown at 22°C with a 16 h photoperiod. Shoots were subcultured monthly by excising approx. 1 cm of the shoot tip and transferring to fresh PM. When the shoots were approx. 12 cm in length, the internode sections were used in transformation experiments. Insect bioassays on artificial diets GNA was supplied by Drs W. Peumans and E. van Damme, Catholic University of Leuven, Belgium. The purity of the GNA was estimated by spectrophotometry and SDS–PAGE to be ⬎ 90%, and the protein was

shown to be fully functionally active by haemagglutination assays (titre equivalent to published values for GNA). All other dietary components were obtained from BDH (Poole, U.K.), or Sigma (Dorset, U.K.). A diet based on freeze-dried potato leaf powder was used; the composition is given in Table 1. Ratios of water to dry wt of components were adjusted to provide an optimal diet consistency. Leaf material was prepared by immediate freeze drying following removal from the plant, to prevent any accumulation of compounds induced by wounding; freeze-dried leaves were then ground to a fine powder in a mortar and pestle. Fresh diet was prepared every 4–5 days and stored at 4°C in airtight containers. GNA was incorporated into this diet at a single concentration of 2% of dietary protein dry wt (7.3 mg GNA per g dry wt of diet). The control diet was supplemented with an equivalent weight of casein to account for the extra protein (i.e. GNA) added to the experimental diet. For each treatment 30 first instar larvae aged ⬍ 24 h were placed individually into 250 ml clear plastic airtight pots. To prevent artificial diet dessication a small piece of wetted filter paper was placed alongside the diet. Fresh diet (0.1–0.5 g wet wt, depending on the size of the larva) was given daily, and filter paper replaced regularly to prevent bacterial contamination. Small airholes were made in the lids 6 days after transfer. Survival and larval instar were recorded daily, for each replicate pot. Once larvae were large enough to handle without causing damage (7 days after hatch) individual wet wts ( ± 0.1 mg) were recorded. Larvae were starved for approx. one hour prior to weighing, which was carried out as much as possible at the same time each day. Measurements of food consumption and faecal production were made on a dry, rather than wet wt basis, to eliminate errors due to variable water content. Remaining diet and faeces were freeze-dried in order to record dry wts; diet supplied was weighed as ‘wet’, and the equivalent dry wt was determined from a calibration curve for diet wet wt vs dry wt determined separately (see below).

TABLE 1. Artificial diet composition. Components (A) were mixed thoroughly and added to boiled agar and water (C) Weight/volume 0.2525 g 0.101 g 2 ml 0.0101 g 0.005 g 0.0088 g 0.00363 g 0.125 g 3 ml

Ingredient

Category

Potato leaf powder Casein Distilled water Vitamin C Methyl-p-hydroxybenzoate Aureomycin GNA or casein Agar Distilled water

A A A B B B B C C

Vitamins, antibiotics and GNA (B) were added once this mixture had cooled to below 45°C.

EFFECTS OF GNA ON LACANOBIA OLERACEA

Production of transgenic plants Details of plasmid construction are given elsewhere (Gatehouse et al., 1997). In brief, the GNA gene was obtained as clone LECGNA2 (Van Damme et al., 1991) and the coding region fragment was cloned between the cauliflower mosaic virus (CaMV) 35S promoter sequence and the nopaline synthase (nos) transcriptional terminator sequence to produce an expression cassette. This was introduced into a binary vector system, containing the uidA gene encoding ␤-glucuronidase (GUS) as a screenable marker, and the nos-neo gene encoding neomycin phosphotransferase (giving resistance to the antibiotic kanamycin) and mobilized into Agrobacterium tumefaciens (strain LBA 4404). Transformation of stem internode explants was performed according to the method of Newell et al. (1991), with the following exceptions. The stem sections were incubated in 10 ml of Agrobacterium suspension in a Petri dish, 100 sections per dish, for 30 min; following inoculation they were transferred to co-culture plates containing a feeder layer of 1.5 ml of a Nicotiana benthamiana cell suspension (see Gatehouse et al., 1997 for details) and covered by a sterile filter paper. Shoots which developed were initially screened for GUS activity with the indigogenic substrate, 5-bromo-4-chloro-3-indolyl-␤-d-glucuronide (X-gluc), according to the method of Jefferson (1987). Sections of stem were immersed in substrate and incubated at 37°C overnight before being transferred to 70% ethanol. Once confirmed, transgenic plantlets were transferred to rooting medium, with antibiotic selection (100 mg l−1 kanamycin) being maintained. Plant propagation Plants derived from two original transformants, designated PWG6#85 and PGNA2#28, were used in this work, and are referred to as lines PWG6#85 and PGNA2#28, respectively. All plants used in insect bioassays, including non transformed control plants, were propagated in tissue culture by means of shoot cuttings. Antibiotic selection was not maintained for these propagation steps. Once a good root system had developed, plantlets were potted out in John Innes no. 3 compost and a polythene bag was placed over each plant. When the plant had reached a height of 10 cm, the top of the bag was opened to enable ventilation; after a week the bag was removed. Plants were grown at a temperature of 20°C under a 16:8 L:D lighting regime. Determination of transgene expression Confirmation of transgene expression was obtained by Western blotting, and expression of GNA in transgenic plants used for insect bioassays was estimated quantitatively using an immunodot-blot detection system. In both cases the primary antibody was raised in rabbits against GNA. For Western blotting and the immunodot blot assays carried out to determine expression levels for the detached leaf bioassay, 125I-labelled donkey anti-rabbit

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IgG (Amersham) was used as the secondary antibody. For immunodot blot assays carried out on transgenics used in the glasshouse trials the protein was visualized using the enhanced chemiluminescence (ECL) method (Amersham) with affinity purified goat anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad) as the secondary antibody. Assays were carried out on extracts prepared from leaves sampled from control and transgenic plants. For determination of expression levels it is important to ensure that leaves from the different plants are of comparable age and position. Full details of these two methods are given elsewhere (Gatehouse et al., 1997). The dot-blots were calibrated using known amounts of GNA ‘spiked’ into control potato leaf extract, and quantified using densitometry of the final images on X-ray film (Fuji XR) after autoradiography or exposure to chemiluminescence. GNA levels were quantified as a percentage of total extractable protein, as estimated by the Coomassie Blue G-250 dye-binding method (BioRad; Bradford, 1976). Tissue blots from plants in the glasshouse were taken just prior to the plants being infested with the neonate larvae, to confirm GNA expression. Leaves from both transgenic and control plants were cut in half and the cut edges touched down on to nitrocellulose; since potato contains peroxidase activity, prior to processing the blots when using the ECL Method, the blots were soaked in 2% periodic acid for 10 min in order to inactivate the endogenous peroxidase activity. Following this treatment, blots were washed with water and then visualized as described above. In all immunoassay methods for GNA expression in transgenic plants, the lower limit of detection for GNA was approx. 0.01% of total protein; no expression of GNA was detected in control plants. Bioassays on detached leaves For each of the two treatments, 150 first instar larvae aged ⬍ 24 h were placed into 250 ml clear plastic airtight pots (10 larvae per pot). Small airholes were made in the lids 6 days after transfer. Larvae were fed daily on freshly excised control or transgenic (line PGNA2#28) potato leaves. After two weeks larvae were transferred into 500 ml rectangular plastic boxes covered with clear pierced lids. Containers were cleaned daily. A shallow layer of vermiculite was added when larvae reached the pre-pupal phase, to facilitate burrowing and to aid subsequent pupation. Survival and larval instar were recorded for each replicate pot daily, from 0 to 24 h post hatch until the onset of pupation. Larvae of each instar were identified by head capsule widths, and the formation of new head capsules prior to ecdysis. For control and experimental treatments 40 newly eclosed third instar larvae (4 larvae from each of 5 randomly chosen replicate pots per treatment) were selected to compare growth, consumption, and faecal production between treatments. Individual larval wet wts ( ± 0.1 mg) were recorded at the same time each day. Larvae were

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starved for approx. 1 h prior to weighing to minimize any potentially significant error resulting from the production of faecal material during the course of weighing. Measurements of food consumption and faecal production were made on a dry, rather than wet wt basis to eliminate errors due to variable water content. Remaining leaf and faeces were freeze-dried in order to record dry wts; leaf material supplied was weighed as ‘wet’, and the equivalent dry wt was determined from a calibration curve for leaf wet wt vs dry wt determined separately (see below). Glasshouse trials A large scale glasshouse trial of mature transgenic potato plants expressing GNA was set up. Prior to carrying out the trial, environmental variability within the glasshouse was determined in order to aid experimental design. For this, 40 plots arranged on a grid were set up, each planted with 12 radish seeds (var. French Breakfast). Percent germination, plant height and weight, and root length were recorded. Based on the experimental variation observed amongst these plots, a lattice design for the experimental beds was employed. The glasshouse was arranged with four beds each of 10 × 2.5 ft, each subdivided into plots of 2.5 × 2.5 ft, thus giving a total of 16 plots; these were separated from one another by fine aphid-proof meshing attached to a frame. The beds had a soil depth of approx. 9 in., and were filled with a loam-based compost over a 2 in. gravel base. Each plot was planted out with either control or transgenic plants, and plot type (control or transgenic) alternated in the lattice grid. Larvae were free to migrate between plants within a plot, but not between plots. Two full glasshouse bioassays were carried out. Mature plants were infested with neonate larvae of L. oleracea, and the trials were allowed to run for 5 weeks. At the conclusion of each trial, plant damage was visually scored, on a scale of 0 (undamaged, or almost undamaged) to 5 (very heavily damaged), and surviving insect numbers and insect biomass per plot were determined. The trials differed in the potato transformants used; the first trial used plants of line PWG6#85, and the second used plants of line PGNA2#28. Seven plants per plot were used for the first trial, and six for the second; the plants were each infested with eight (PWG6#85) or four (PGNA2#28) neonate larvae. Data analysis All data analysis was carried out with the Statview (v4.5; Abacus Concepts, Berkely, Ca, U.S.A.) software package on Apple Macintosh computers. The acceptance level of statistical significance was P ⬍ 0.05 in all instances. Data from glasshouse trials were subjected to ANOVA (analysis of variance) to separate effects of location of sub-beds within the glasshouse from the effects of the treatment, i.e. transgene expression. In the glasshouse trials leaf damage results were subjected to analysis by a Mann–Whitney U-test for differences. For

the detached leaf, and artificial diet bioassays the time taken (days) for each larva to reach each successive instar was tabulated to facilitate statistical analysis of instar duration under the different feeding regimes. A Mann– Whitney U-test was employed to detect significant differences in the median time taken to reach successive instars between the control and experimental samples. To detect significant differences in daily mean larval wet wts between treatments analysis of variance (ANOVA) and a subsequent Fishers PLSD (probability least significant difference) test was employed. Dry/wet leaf weight, and dry/wet artificial diet weight calibrations were carried out by recording the initial wet wt of 10 variably sized leaves/diet pieces and respective dry wts following freeze-drying of the samples. Linear regressions with r2 values of 0.973 and 0.995, for leaf and artificial diet calibrations respectively, were obtained. As larvae in the artificial diet bioassay were kept as individuals, analysis of consumption could be made on an individual basis. An ANOVA as above was used to detect any significant differences in mean daily consumption between control and experimental treatments. RESULTS

Artificial diet bioassays Artificial diet bioassays were carried out starting from neonate larvae. The diet was based on potato leaf powder, and was formulated to eliminate components that contain additional lectins or sugars (e.g. haricot bean) that could potentially mask the effects of GNA. The diet supported normal growth and development of larvae up to the fifth or sixth instar, but was not optimized for the full life cycle of the insect. All experimental diets were made up with a constant level of GNA, adjusted to be 2% of estimated total dietary protein; this represents a concentration of approx. 1.4 × 10 − 5 M in the diet. Insect survival was not affected by feeding larvae artificial diet containing GNA [Fig. 1(B)]. Both control and experimentally reared larvae exhibited a gradual decline in survival during the first five instars, with 20% mortality recorded for both treatments from day 0 to day 25. A subsequent steeper decline in survival was observed with 33% of control, and 36% of experimental larvae lost from days 27 to 30, and days 28–31 respectively. Although the suboptimal nature of the diet prevented larvae from attaining pupation, 66% of control, and 56% of experimentally reared larvae reached sixth instar. In contrast to the lack of effect on survival, the presence of GNA in artificial diet significantly inhibited larval development. Larvae reared on artificial diet containing GNA consistently lagged behind control larvae in development, and for all stages after first instar this translated into a delay of approx. 2 days in reaching instars [Fig. 2(A)]. The delay is largely attributable to the particularly slow development of around 10% of the experimental group. When subjected to a Mann–Whitney U-

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FIGURE 1. (A) Mean larval weight (wet) for L. oleracea larvae feeding on artificial diet, and artificial diet containing GNA (snowdrop lectin) at a level of 2% of total protein. (B) Survival of L. oleracea larvae in the diet bioassay shown in (A).

test the median time taken to reach all six instars was significantly greater for GNA-fed larvae relative to the control group. The presence of GNA in artificial diet also exerts a negative influence upon larval growth, as shown in Fig. 1(A). The experimental group of larvae, fed on GNAcontaining diet, have a consistently lower mean biomass (by approx. 20–30%) compared to the control group. Following a one way analysis of variance, the difference in mean larval biomass was seen to be significant from the onset of measurement (day 7 post hatch) until day 27, with no obvious trend in the ratio of control mean biom-

ass:experimental mean biomass with time. The sharp increase in mortality after day 27, combined with a gradual increase in variance, largely accounted for the nonsignificance of the differences between control and experimental groups in the statistical analysis during the later stages of development. A peak in mean biomass was observed for both treatments at 30 days post hatch at which point 80% of the surviving control group but only 46% of the surviving experimental group had reached their final instar. The maximum mean biomass of larvae fed control artificial diet was seen to be significantly greater (1.36 × ), than that recorded for larvae

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FIGURE 2. (A) Mean times to instars for L. oleracea larvae in artificial diet bioassay shown in Fig. 1(A). Error bars show means ± SE; differences between control and GNA-fed groups are signifciant at *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001. (B) Mean times to instars for L. oleracea larvae in detached leaf bioassay shown in Fig. 4(A). Error bars show means ± SE; differences between control and GNA-fed groups are significant at *P ⬍ 0.05,**P ⬍ 0.01, ***P ⬍ 0.001.

reared on GNA-containing diet following a one way analysis of variance. In addition, reduced fitness of the experimental group was indicated by the ability of control larvae to withstand a significantly greater mean loss in weight prior to death (ANOVA) compared to GNAfed larvae (data not presented). The consumption of diet by larvae reared on artificial diet with and without the incorporation of GNA showed a significant decrease caused by the lectin. As shown in Fig. 3(A), on a daily mean basis, larvae fed diet containing GNA consumed less than the control group throughout the bioassay. This difference was seen to be significant, following a one way analysis of variance, for 13 out of the 37 days for which consumption was assessed. Total daily consumption by the experimental group was

also significantly less (P = 0.0063, ANOVA) than that recorded for the control group. Overall, larvae raised on GNA-containing diet consumed 40% less diet than that consumed by larvae fed control diet [see Fig. 5(A)]. Detailed analysis of diet consumption and faecal production on a per instar basis from third to sixth instars was also carried out. Although moulting was not simultaneous, values were collated (e.g. values for day 14–19 correspond to fourth instar) to enable comparative analysis. Larvae reared on GNA-containing diet consumed less and produced less frass than the control group during each of the instars for which parameters were assessed (data not presented). Values were highest for the fifth instar during which control and experimentally fed larvae consumed 32.5 and 36.6% of total consumption, and

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FIGURE 3. (A) Mean daily diet consumption (dry wt) for L. oleracea larvae in the artificial diet bioassay shown in Fig. 1. Error bars show means ± SE; differences between control and GNA-fed groups are significant at *P ⬍ 0.05, **P ⬍ 0.01. (B) Mean daily diet consumption as a fraction of mean larval body wt, plotted against time for the artificial diet bioassay with L. oleracea larvae shown in Fig. 1.

excreted 28.8 and 32.7% of total frass production respectively. This correlates with results given in Fig. 1(A) and Fig. 2(A), which show that the greatest gain in mean biomass was achieved by both groups during this instar (day 19–24). The greatest difference in consumption and faecal production between the two treatments occurred in the final instar, during which control larvae consumed 2 × , and produced 1.7 × that of the experimental group. If the consumption of diet is assessed as a ratio of

body wt, both control and experimental groups of insects show a steady decline in this ratio throughout development [Fig. 3(B)]. The data can be fitted to linear regressions of (consumption/weight) against time (r ⬎ 0.85) for both control and experimental groups; there are no significant differences between control and experimental parameters (data not presented). The groups do not differ significantly when a non-parametric paired sign test is used. These results suggest that although GNA depresses growth, and thus decreases diet consumption,

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it does not cause any long-term inhibition of feeding, since consumption as a fraction of body wt does not differ between control and experimental groups. Production and selection of transgenic plants Potato plants expressing GNA under the control of the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter were produced by Agrobacterium tumefaciensmediated gene transfer technology (see section 2). Transformants were selected by antibiotic resistance and assay for expression of the screenable marker gene; further selection of primary transformants was made on the basis of assay for GNA expression, using immunological techniques (see section 2). These assays showed that GNA was expressed in the transgenic plants as a polypeptide of approx. 12,000 Mr. Selected transformants were propagated vegetatively in tissue culture to give lines of clonal replicate plants. GNA was estimated to be present in a number of these lines at levels of 0.5–1.5% of total protein; subsequent purification of GNA from potato line PGNA2#28 showed that the protein was fully functional by haemagglutination assay, and had the correct N-terminal sequence (Gatehouse et al., 1997). This line was selected for both the detached leaf bioassays and for the large scale glasshouse trials on the basis of previous trials carried out under growth room conditions (reported in Gatehouse et al., 1997). A preliminary glasshouse bioassay was also carried out using plants of line PWG6#85. Potato lines were maintained by repeated vegetative propagation in tissue culture. For in planta bioassays, transgenic and control plants were multiplied by clonal propagation in tissue culture before being planted out, so that the assays were carried out on genetically uniform plants. Transgenic plant bioassays with detached leaves Plants of line PGNA2#28 used for the detached leaf bioassay were maintained in tissue culture for approx. 18 months prior to planting out clonal replicates in soil. Dot-blot immunoassays of GNA in leaf samples taken from these mature transgenic plants showed that expression levels had decreased from those originally observed. GNA levels in different leaf samples ranged from ⬍ 0.01–0.19% of total leaf soluble protein, with a mean of 0.068% (SE = ± 0.018; n = 12). It was considered worthwhile to determine whether these relatively low levels of expression of the foreign protein would still have measurable effects on L. oleracea larval development. As in the artificial diet bioassay, insect survival was not affected by the presence of GNA in leaves from transgenic potatoes [Fig. 4(B)]. Larvae fed control and transgenic leaves exhibited an initial (0–15 days) gradual decline in survival to approx. 80%. Survival stabilized during the third instar (15–22 days), and subsequently declined more steeply during fourth, fifth, and sixth instars. 45% of both the control and transgenic potato fed larvae attained pupation.

The development of larvae reared on transgenic potato leaves is inhibited by the presence of dietary GNA [Fig. 2(B)]. When subject to a Mann–Whitney U-test the median time taken to reach second, third, fourth, and fifth instars was significantly greater for larvae reared on transgenic leaves compared to larvae reared on control potato leaves. The difference was greatest for time taken to reach fourth instar; on average, control insects reached fourth instar 1.3 days earlier than did insects reared on transgenic leaves. This initial delay in development was, however, counterbalanced by shorter mean durations observed for fourth, fifth and sixth instars in larvae reared on transgenic leaves (4.7, 4.9, 8.4 days respectively) compared to the control group (5.3, 5.0, 8.8 days respectively). Hence the total mean development time observed for larvae fed control potato leaves and GNA expressing potato leaves was similiar, at 33.5 and 33.7 days respectively. The effect of control and transgenic leaves upon larval growth is illustrated by Fig. 4(A). Expression of GNA in the transgenic leaves exerts a negative influence upon growth, as indicated by the lower mean biomass observed from days 13 to 31 in larvae fed transgenic plant material, relative to the control group. The difference between treatments was maximal on days 21, 22, and 24, where a significant difference between means (P ⬍ 0.001; P = 0.001; P = 0.007 respectively) was observed following a one-way analysis of variance. By day 21, 75% of control and 85% of transgenic fed larvae were in fifth instar, and by day 24, 45% of control and 20% of transgenic fed larvae had reached sixth instar. A peak in mean biomass was recorded on day 29 when 63% of control and 41% of transgenic fed larvae were in their sixth instar. The subsequent reduction in mean biomass largely reflects the onset of a sedentary non-feeding prepupal phase. During this phase the mean biomass of larvae fed transgenic leaves became greater than that of the control group. This difference was maximal on day 37, by which time 100% of the control group, but only 81% of the experimental group had pupated, and is indicative of compensatory feeding prior to the onset of pupation. Subsequently a similiar mean ( ± SE) pupal biomass of 0.288 ± 0.009 g and 0.296 ± 0.008 g was recorded for control and transgenic fed larvae respectively. Although daily consumption figures were measured for larvae in this bioassay, the variability in the data precluded analysis of daily mean consumptions. Data were therefore plotted as cumulative consumption for the whole group, and are presented in Fig. 5(B); data for the artificial diet bioassay have been plotted in this format for comparison [Fig. 5(A)]. Estimated cumulative consumption was very similiar for both treatments in the detached leaf bioassay. However, 100% of surviving larvae fed control potato leaves had pupated by day 34, at which point 25% of surviving larvae fed transgenic leaves continued feeding and did not pupate until day 40. Consequently the experimental group actually consumed 0.880 g (dry wt) more than the control group even though

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FIGURE 4. (A) Mean larval weight (wet) for Lacanobia oleracea larvae feeding on detached leaves from control potato plants, and transgenic potato plants of line PGNA2#28, expressing GNA at approx. 0.07% of total protein. (B) Survival of L. oleracea larvae in the detached leaf bioassay shown in (A).

10% less of the larvae fed transgenic leaves actually survived to the sixth instar. As for the artificial diet bioassay, consumption was also estimated on a per instar basis. Fourth and fifth instar larvae fed transgenic potato leaves consumed less than the control group; differences in frass production were small, as was the difference for consumption in the fourth instar, but consumption in the fifth instar was decreased by 8.0% (data not presented). This correlated with the lower mean biomass relative to control larvae, observed during this time [see Fig. 2(B), Fig. 4(A)].

Glasshouse bioassays with transgenic potato plants An initial glasshouse trial was carried out with transgenic plants of line PWG6#85. Plants of this line were estimated to be expressing GNA at 1.5% of total protein when assayed in tissue culture; expression of GNA was confirmed by tissue blots of all transgenic plants at maturity in the glasshouse, immediately prior to insect infestation. Results in this trial were compromised by some plants suffering a fungal infection, but nevertheless, differences between control and transgenic plants were apparent. The

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FIGURE 5. (A) Cumulative consumption of diet (total dry wt) by L. oleracea larvae in the artificial diet bioassay shown in Fig. 1. Days on which consumption by control insects was significantly different from that of GNA-fed insects are denoted by *P ⬍ 0.05. (B) Cumulative consumption of leaf tissue (total dry wt) by L. oleracea larvae in the detached leaf bioassay shown in Fig. 4. (C) Leaf damage by L. oleracea larvae after 21 days in the glasshouse bioassay of transgenic potato plants of line PGNA2#28 (expressing GNA at approx. 0.6% of total protein) and controls, shown in Fig. 6. Leaf damage is the result of cumulative consumption by larvae, and thus the data are equivalent to a single timepoint in (A) and (B). Damage was estimated by a visual scoring system; difference between control and GNA-expressing plants was estimated as significant by non-parametric statistical analysis at *P ⬍ 0.05.

control plants had a mean leaf damage score of 4.0, whilst the transgenic plants had a reduced mean score of 2.6 (significantly different at P ⬍ 0.002 by Mann–Whitney U-test). Total insect biomass per control plant was 640 ± 75 mg as opposed to only 368 ± 42 mg per transgenic plant (significantly different at P ⬍ 0.01 by unpaired t-test). Insect survival was also better on control plants ( ⬎ 90% of initial inoculum) than on transgenic plants (approx. 60% of control; significantly different to control at P ⬍ 0.001 by Mann–Whitney U-test). The glasshouse trial was repeated with transgenic plants of line PGNA2#28. These plants were estimated to be expressing GNA at 0.6% of total protein when assayed in tissue culture; as before, GNA expression was confirmed in mature plants prior to exposure to insects by tissue blots. Results from this trial are shown in Fig.

6(A) and (B). Data were subjected to ANOVA to separate effects of location of sub-beds within the glasshouse (which, in one case did show a significant effect) from effects of the treatment, i.e. transgene expression. As shown in Fig. 6(A), although the parameters measured showed lower larval survival and biomass, and leaf damage, in most plots containing transgenic plants than any plot containing control plants, one or two transgenic plots showed worse performance than the best of the control plots. However, the overall analysis showed that the PGNA2#28 plants were significantly more resistant to attack by tomato moth than the controls in all parameters measured [Fig. 6(B), Fig. 5(C)]. Larval biomass per plot was decreased by approx. 50% in transgenic plants (significantly different at P ⬍ 0.01), and survival was decreased by approx. 35% (significantly different at P ⬍

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FIGURE 6. (A) Representative data by plot from the glasshouse bioassay of transgenic potato plants of line PGNA2#28 (expressing GNA at approx. 0.6% of total protein) and controls. (B) ANOVA of plot data from glasshouse bioassay of transgenic potato plants of line PGNA2#28 and controls. Differences in larval biomass/plot between controls and GNAexpressing plants were significant at **P ⬍ 0.01; differences in larval survival/plot were significant at *P ⬍ 0.05.

0.02); control survival was again ⬎ 90% of the inoculum. Plant damage was also decreased, by nearly 2 units on the visual scoring system described above [significantly different at P ⬍ 0.02 by Mann–Whitney U-test; Fig. 5(C)]. Plants in this trial suffered no infections or other compromises on growth. In both glasshouse trials insects developed from neonate larvae to as far as the final instar, although the stage of development varied considerably from individual to individual. No clear pattern of differences in distributions of insects among larval instars was apparent when control and transgenic plants were compared. DISCUSSION

The present study provides a detailed analysis of the effects of GNA upon L. oleracea survival, development, and consumption throughout the larval instars, and provides novel data on the effects of this lectin on a lepidopteran larva; no data on its effects on lepidoptera in artificial diet have been published previously, and the published data with transgenic plants only consider parameters measured at an arbitrary point near the end of

larval development. In agreement with earlier data, this work has shown that GNA exerts a significantly detrimental effect upon larval development, growth, and consumption, but it has less effect on survival. This is similar to effects of GNA on glasshouse potato aphids (Aulacorthum solani), where GNA was seen to significantly affect development and fecundity (Down et al., 1996), but contrasts with its toxic effects on rice brown planthopper (Nilaparvata lugens; Powell et al., 1993). This may reflect the feeding habits of the different species; L. oleracea and A. solani are polyphagous, whereas N. lugens is effectively monophagous. Detoxification mechanisms, or resistance to antimetabolic effects, are likely to be more developed in polyphagous species than in a monophagous insect which has adapted to its specific host. A greater capacity to overcome the effects of toxins or antimetabolites will lead to less sensitivity to the effects of GNA. The feeding experiments in artificial diet provide the strongest evidence for deleterious effects of GNA. However, comparison of larval growth with experiments where larvae are feeding directly on plant material (Figs 1 and 4) suggest that the diet is nutritionally suboptimal

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for larger larvae, and thus results from the later stages (after approx. 22 days) must be viewed with caution. A further caveat to the conclusions drawn stems from the composition of the artificial diet, which might be better termed ‘semi-artificial’, since the potato leaf powder used in its formulation contains a number of potential antimetabolites, including small amounts of a chitin-binding lectin. However, the clear difference between larval performance on control and experimental ( + GNA) diets can only result from the added protein, and the diet is at least comparable in its composition to plant tissue. Larvae fed excised leaves from potato plants developed well, and nearly half of the initial number reached pupation. The effects of GNA expression in these leaves were relatively small, but significant, and similar to the effects seen in artificial diet, supporting the validity of the diet assay. The low levels of GNA expression observed in these potato plants was unexpected, and contrasts with the relatively high levels of expression observed in plants used for the greenhouse trial. The plants used in the greenhouse trial came from stocks that had been maintained in tissue culture for a relatively short time ( ⬍ 6 months), whereas stocks used to produce plants for the detached leaf bioassays had been maintained in tissue culture for up to 18 months, without antibiotic selection. Although the original material was clonal, it is apparent that expression from the foreign gene construct had decreased with repeated vegetative propagation. Further experiments are currently underway to attempt to clarify this observation. The relatively small deleterious effects of GNA observed in the detached leaf bioassay suggest that the effects of the lectin do depend on the dosage, as would be expected. Previous results have not supported a direct relationship between the accumulation levels of GNA in different transgenic potato lines and insecticidal effect (Gatehouse et al., 1997). Other factors, such as potential ‘knockouts’ of endogenous genes by insertion of foreign DNA, could play a part in the effects observed in transgenic plants; alternatively, there may be a complex relationship between lectin dosage and effect, as has been observed with aphids in this laboratory (R. Down, personal communication). Previous growth room trials examining the effects of GNA on L. oleracea showed a 64% reduction in larval feeding as assessed by leaf damage analysis (Gatehouse et al., 1997). A similar reduction (59%) in overall mean daily consumption was observed in the artificial diet bioassay (data not presented) and significant reduction in leaf damage was observed in the glasshouse after a 35 day trial period [Fig. 5(C); cf. Fig. 5(A)]. However, the data from artificial diet show that reduced feeding is a consequence of smaller larval size, and thus the present data provide no evidence for GNA exerting a feeding deterrent effect, as was observed for rice brown planthopper (Powell et al., 1995). The reduction in larval biomass, particularly in the excised leaf assay where consumption was almost identical for both control and GNA-

fed larvae [Fig. 4(A) and Fig. 5(B)], suggests that GNA acts by inhibiting nutrient uptake in L. oleracea. The mechanism for this remains to be established, but by analogy to the situation in mammals (Pusztai, 1991), insecticidal effects of lectins may be primarily determined by binding to suitably glycosylated targets in the insect gut, causing inhibition of nutrient absorption and/or midgut cell disruption (Gatehouse et al., 1984; Eisemann et al., 1994). Indeed, proteins which bind GNA in vitro have been identified in L. oleracea gut tissue preparations (data not presented), and characterization studies are presently underway. Alternatively, lectins may bind to and block the peritrophic membrane protecting the mid-gut surface (Eisemann et al., 1994). The relevance of the presented results to crop protection in the field has yet to be tested. However, this investigation indicates that GNA, when expressed at relatively high levels (i.e. 0.6–2% total soluble proteins) in transgenic potatoes, will afford some degree of protection against L. oleracea. REFERENCES Boulter D., Edwards G. A., Gatehouse A. M. R., Gatehouse J. A. and Hilder V. A. (1990) Additive protective effects of different plantderived insect resistance genes in transgenic tobacco plants. Crop Protection 9, 351–354. Bradford M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Burgess H. D. and Jarrett P. (1976) Adult behaviour and oviposition of five noctuid and tortricid moth pests and their control in glasshouses. Bulletin of Entomological Research 66, 501–510. Czapla T. H. and Lang B. A. (1990) Effect of plant lectins on the larval development of European corn borer (Lepidoptera: Pyralidae) and Southern corn rootworm (Coleoptera: Chrysomelidae). Journal of Economic Entomology 83, 2480–2485. Down R. E., Gatehouse A. M. R., Hamilton W. D. O. and Gatehouse J. A. (1996) Snowdrop lectin inhibits development and decreases fecundity of the glasshouse potato aphid (Aulacorthum solani) when administered in vitro and via transgenic plants both in laboratory and glasshouse trials. Journal of Insect Physiology 42, 1035–1045. Eisemann C. H., Donaldson R. A., Pearson R. D., Cadogan L. C., Vuocolo T. and Tellam R. L. (1994) Larvacidal activity of lectins on Lucilia cuprina: mechanism of action. Entomologia Experimentalis et Appplicata 72, 1–10. Gatehouse A. M. R., Dewey F. M., Dove J., Fenton K. A. and Pusztai A. (1984) Effect of seed lectin from Phaseolus vulgaris on the development of larvae of Callosobruchus maculatus: mechanism of toxicity. Journal of the Science of Food and Agriculture 35, 373–380. Gatehouse, A. M. R. and Hilder, V. A. (1994) Genetic manipulation of crops for insect resistance. In Molecular Perspectives in Crop Protection, eds G. Marshall and R. D. Walkers, pp. 177–201. Chapman and Hall, London. Gatehouse, A. M. R., Powell, K. S., Peumans, W. J., Van Damme, E. J. M. and Gatehouse, J. A. (1995) Insecticidal properties of lectins; their potential in plant protection. In Lectins: Biomedical perspectives, eds A. J. Pusztai and S. Bardocz, pp. 35–58. Taylor and Francis, Hants, U.K. Gatehouse A. M. R., Davidson G. M., Newell C. A., Merryweather A., Hamilton W. D. O., Burgess E. P. J., Gilbert R. J. C. and Gatehouse J. A. (1997) Transgenic potato plants with enhanced resistance to the tomato moth Lacanobia oleracea: growth room trials. Molecular Breeding 3, 49–63.

EFFECTS OF GNA ON LACANOBIA OLERACEA Jefferson R. A. (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Molecular Biology Reporter 5, 387–405. Lloyd L. (1920) The habits of the glasshouse tomato moth, Hadena (Polio) oleracea and its control. Annals Applied Biology 7, 66–102. Newell C. A., Rozman R., Hinchee M. A., Lawson E. C., Haley L., Sanders P., Kaniewski W., Tumer N. E., Horsch R. B. and Fraley R. F. (1991) Agrobacterium-mediated transformation of Solanum tuberosum L. cv. Russet Burbank. Plant Cell Reports 10, 30–34. Powell K. S., Gatehouse A. M. R., Hilder V. A. and Gatehouse J. A. (1993) Antimetabolic effects of plant lectins and plant and fungal enzymes on the nymphal stages of two important rice pests, Nilaparvata lugens and Nephotettix cinciteps. Entomologia Experimentalis et Appplicata 75, 61–65. Powell K. S., Gatehouse A. M. R., Hilder V. A. and Gatehouse J. A. (1995) Antifeedant effects of plant lectins and an enzyme on the adult stage of the rice brown planthopper, Nilaparvata lugens. Entomologia Experimentalis et Appplicata 75, 51–59. Pusztai, A. (1991) Plant Lectins. Chemistry and Pharmacology of Natural Products Series, pp. 263. Cambridge Univ. Press, Cambridge. Rahbe´ Y., Sauvion N., Febvay G., Peumans W. J. and Gatehouse A. M. R. (1995) Toxicity of lectins and processing of ingested proteins in the pea aphid Acyrhosiphon pisum. Entomologia Experimentalis et Appplicata 76, 143–155. Sauvion N., Rahbe´ Y., Peumans W. J., Van Damme E. J. M., Gatehouse J. A. and Gatehouse A. M. R. (1996) Effects of GNA and other mannose binding lectins on development and fecundity of the peach potato aphid Myzus persicae. Entomologia Experimentalis et Appplicata 79, 285–293.

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Shukle R. H. and Murdock L. L. (1983) Lipoxygenase, trypsin inhibitor, and lectin from soybeans: effects on larval growth of Manduca sexta (Lepidoptera: Sphingidae). Environmental Entomology 12, 787–791. Van Damme E. J. M., Allen A. K. and Peumans W. J. (1987) Isolation and characterisation of a lectin with exclusive specificity towards mannose from snowdrop (Galanthus nivalis) bulbs. FEBS Letters 215, 140–144. Van Damme E. J. M., De Clerq N., Claessens F., Hemschoote K., Peeters B. and Peumans W. J. (1991) Molecular cloning and characterisation of multiple isoforms of the snowdrop (Galanthus nivalis) lectin. Planta 186, 35–43.

Acknowledgements—The authors would like to thank Drs W.D.O. Hamilton, C. Newell and A. Merryweather of Axis Genetics Ltd for producing and providing the initial transgenic plant material, Miss G.M. Davison for tissue culture and maintenance of plants and Drs L.N. Gatehouse and R.R.D. Croy for producing anti-GNA antibodies. Prof. J. Edwards and Dr. G. Marris (MAFF CSL) are thanked for advice on the culture of L. oleracea, and for supplying insects; Dr V. Hilder is thanked for advice on assessing the uniformity of glasshouse growing conditions. The authors would also like to acknowledge with gratitude funding from MAFF (CASE Studentship; EF), the Scottish Office (SOAEFD Project No. UDH/818/95) and the Department of Trade and Industry in a LINK Programme with Axis Genetics. Work involving transgenic potato plants was carried out under MAFF licence number PHF 346B/1459/35.