Direct and indirect sublethal effects of Galanthus nivalis agglutinin (GNA) on the development of a potato-aphid parasitoid, Aphelinus abdominalis (Hymenoptera: Aphelinidae)

Snowdrop lectin (Galanthus nivalis agglutinin, GNA), has been shown to confer partial resistance to two potato aphids Myzus persicae and Aulacorthum solani, when incorporated in artificial diet and/or expressed in transgenic potato. First-tier laboratory-scale experiments were conducted to assess the potential effect of GNA on the aphid parasitoid Aphelinus abdominalis. GNA (0.1% w/v) was successfully delivered to Macrosiphum euphorbiae via artificial diet and induced a reduced growth rate and increased mortality compared to aphids fed a control diet. As aphid parasitoid larvae are endophagous, they may be exposed to GNA during their larval development and potential "chronic toxicity" on A. abdominalis was investigated. The amounts of GNA present in aphid and parasitoid tissues were estimated by western blotting. Results suggest that parasitoids excrete most of the GNA ingested. Sublethal effects of GNA on several parasitoid fitness parameters (parasitism success, parasitoid development and size, emergence success, progeny survival and sex ratio) were studied. No direct detrimental effect of GNA on A. abdominalis was observed. However, GNA had an indirect host-size-mediated effect on the sex ratio and the size of parasitoids developing in GNA-fed aphids. This work highlights the need to determine the exact "causes and effects" when assessing the ecological impact of transgenic plants on non-target beneficial insects. Such bioassays form the basis of a tiered risk assessment moving from laboratory studies assessing individuals towards field-scale experiments assessing populations.     

Evaluation of lectin-expressing transgenic sugarcane against stalkborers (Lepidoptera: Pyralidae): effects on life history parameters

The impact of snowdrop lectin (Galanthus nivalis agglutinin, GNA) expressed in transgenic sugarcane on life history parameters of Mexican rice borer [Eoreuma loftini (Dyar)] and sugarcane borer [Diatraea saccharalis (F.)] (both Lepidoptera: Pyralidae) was evaluated. In the laboratory, lyophilized sugarcane leaf sheath tissue was incorporated in a meridic diet resulting in a GNA concentration of 0.47% of total protein, and used for insect bioassays over two successive generations. Deleterious effects of GNA were not observed on survival, weight, and developmental periods of larvae and pupae, nor on adult fecundity and egg viability of D. saccharalis. Moreover, in the first generation, addition of transgenic sugarcane tissue to the diet enhanced larval growth in D. saccharalis resulting in higher larval and pupal weight compared with diet with nontransgenic sugarcane, but this effect was not observed in the second generation. In contrast, larval survival, percent adult emergence, and female fecundity of E. loftini were significantly reduced when fed transgenic sugarcane diet compared with nontransgenic sugarcane diet. In addition, a substantial reduction of female pupal weight of E. loftini was observed in the second generation. For both species, the only consistent effect of GNA in both generations was a reduction in adult female longevity. Life table parameters showed that GNA at the level found in the transgenic diet negatively affected development and reproduction of E. loftini, whereas it had a nil to positive effect on development and reproduction of D. saccharalis.     

Effects of artificial diet containing GNA and GNA-expressing potatoes on the development of the aphid parasitoid Aphidius ervi Haliday (Hymenoptera: Aphidiidae)

Aphid parasitoids are important biological control agents. The possibility arises that whilst foraging on insect-resistant transgenic plants, they are themselves at risk from direct and indirect effects of the expression of a transgene used to control the pest species. A liquid artificial diet was successfully used to deliver the snowdrop lectin (Galanthus nivalis agglutinin; GNA) to the peach-potato aphid, Myzus persicae. Bioassays utilising artificial diet incorporating GNA, and excised leaves of the GNA-expressing transgenic potato line, GNA2#28, were performed to assess the potential effects of GNA on the development of the aphid parasitoid Aphidius ervi. The results indicate that GNA delivered via artificial diet to the aphids can be transferred through the trophic levels and has a dose-dependent effect on parasitoid development. Parasitoid larvae excreted most of the ingested GNA in the meconium but some of it was detected in the pupae. Although A. ervi development was not affected when developing within hosts feeding on transgenic potato leaves, this probably reflected sub-optimal expression of the toxin in the transgenic potato line used     

Effects of Galanthus nivalis agglutinin (GNA) expressed in tomato leaves on larvae of the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae) and the effect of GNA on the development of the endoparasitoid Meteorus gyrator (Hymenoptera: Braconidae)

The effect of ingestion of transgenic tomato leaves expressing the plant lectin Galanthus nivalis agglutinin (GNA) on development of larvae of Lacanobia oleracea (Linnaeus) was studied under laboratory conditions. When L. oleracea larvae were fed on tomato line 14.1H, expressing approximately 2.0% GNA, significant increases in the mean larval weight and in the amount of food consumed were found. This resulted in an overall reduction in the mean development time to the pupal stage of approximately 7 days. A significant increase in the percentage survival to the adult moth was also recorded when newly hatched larvae were reared on transgenic tomato leaves (72%) compared to larvae reared on untransformed leaves (40%). The effects of ingestion of GNA by L. oleracea larvae, via artificial diet or the leaves of transgenic tomato or potato plants, on the subsequent development of its solitary endoparasitoid Meteorus gyrator (Thunberg) was also studied. No significant effects on the life cycle parameters of M. gyrator developing in L. oleracea fed on GNA-containing diets were observed. Experiments with transgenic potato plants indicated that the stadium of the host larvae at parasitism had a greater influence on M. gyrator development than the presence of GNA. Potential GNA-binding glycoproteins were detected in the gut and body tissues of larval M. gyrator. Despite detection in host tissues, GNA could not be detected in adult M. gyrator and therefore it is likely that at the time of pupation M. gyrator are able to void the GNA in the meconial pellet.     

Effects of snowdrop lectin on Mexican rice borer (Lepidoptera: Pyralidae) life history parameters

The effects of the snowdrop lectin, Galanthus nivalis agglutinin (GNA), delivered through an artificial diet, on growth, development, and life history parameters of the Mexican rice borer, Eoreuma loftini (Dyar), were evaluated in the laboratory. Incorporation of GNA at three treatment levels, 0.5, 1.0, and 2.0% of total dietary protein, in the larval diet significantly decreased larval survivorship and percentage of adults emerging relative to a control diet lacking GNA, whereas differences were not observed among the three treatment levels. Both larvae and pupae in the control were 8-25% larger than those in the GNA treatments, but differences were not observed between larvae in the GNA treatments. Furthermore, presence of GNA did not affect larval and pupal developmental periods, longevities, and fecundities compared with the control. Mexican rice borer life history parameters, such as net reproductive rate and intrinsic rate of increase, were substantially reduced by the presence of GNA in the diet, but differences were not evident among the three GNA treatment levels.     

Screening for fire blight resistance in apple (Malus pumila) using excised leaf assays from in vitro and in vivo grown material

The apple cultivars Greensleeves and Novole and somaclones derived from Greensleeves were tested for resistance to fire blight (Erwinia amylovora) using excised leaf bioassays. Cv. Greensleeves was consistently susceptible to fire blight in in vitro, and in glasshouse and excised leaf bioassays whilst cv. Novole was resistant. Greensleeves somaclones, identified as having intermediate levels of fire blight resistance in glasshouse and in vitro screening tests, also showed intermediate levels of resistance in excised leaf bioassays. The length of leaves and the inoculum concentration employed affected the severity of symptoms observed. Excised leaf bioassays were unsuitable as tests for fire blight resistance using field grown material.     

Use of an artificial diet system to study the toxicity of gut-active insecticidal compounds on larvae of the green lacewing Chrysoperla sinica

A semi-liquid artificial diet was established and found to be a suitable food source for Chrysoperla sinica larvae, comparable to aphid prey. Using the artificial diet, we established and validated a dietary exposure assay by using the insecticidal potassium arsenate (PA) as positive control. Dose-dependent responses were documented for all observed life-table parameters of C. sinica larvae such as survival rate, pupation rate, larval weight, and larval development time. Thus, the dietary assay can detect the effects of insecticidal compounds on the survival and development of C. sinica larvae. Using the established dietary assay, we subsequently tested the toxicity of Cry1Ab, Cry1Ac, and Cry2Aa proteins (which are produced by transgenic maize, cotton or rice plants) to C. sinica larvae. Artificial diets containing Galanthus nivalis agglutinin (GNA) or PA were included as positive controls. Survival and development of C. sinica larvae were not affected when the artificial diet contained purified Cry1Ab, Cry1Ac, or Cry2Aa at 200 μg/g diet. In contrast, C. sinica larvae were adversely affected when the diet contained PA and GNA. The stability and bioactivity of the Cry proteins in the diet and Cry protein uptake by the lacewing larvae were confirmed by bioassay with a Cry-sensitive insect species and by ELISA. The current study describes a suitable experimental system for assessing the potential effects of gut-active insecticidal compounds on green lacewing larvae. The experiments with the Cry proteins demonstrate that C. sinica larvae are not sensitive to Cry1Ab, Cry1Ac, and Cry2Aa.     

Digestive proteolytic activity in larvae of tomato moth,Lacanobia oleracea; effects of plant protease inhibitors in vitro and in vivo

Three distinct digestive protease activities, with strongly alkaline pH optima, were identified in the gut of tomato moth (Lacanobia oleracea) larvae, and characterised using specific synthetic substrates and inhibitors. These were; a trypsin-like activity, a chymotrypsin-like activity specific for substrates and inhibitors containing more than one amino acid residue, and an elastase-like activity, accounting for 40%, 30% and 20% of overall proteolysis respectively. The protease activities differed in their sensitivities to inhibition by different plant protein protease inhibitors (PIs), as estimated by I(50) values. Soya bean Kunitz trypsin inhibitor (SKTI) was the only plant PI tested to inhibit all three digestive protease activities at concentrations <40 &mgr;g/ml (approx. 5x10(-6)M). Incorporation of SKTI into a potato leaf-based artificial diet at 2% of total protein, decreased larval survival and growth (by approx. 33% and 40% respectively after 21 days) and retarded development (by approx. 2 days). However, when SKTI was expressed in transgenic potato plants at approx. 0.5% of total protein, only marginal effects on L. oleracea larvae were observed, which decreased with time. Whilst the presence of SKTI in artificial diet increased endogenous larval trypsin-like activity by up to four-fold, no effects on this activity were observed in larvae feeding on transgenic plants.     

Transgenic GNA Expressing Potato Plants Augment the Beneficial Biocontrol of Lacanobia Oleracea (Lepidoptera: Noctuidae) by the parasitoid Eulophus Pennicornis (Hymenoptera; Eulophidae)

The effect of expressing the gene encoding snowdrop lectin (Galanthus nivalis agglutinin, GNA) in transgenic potato plants, on parasitism of the phytophagous insect pest Lacanobia oleracea by the gregarious ectoparasitoid Eulophus pennicornis, was investigated in glasshouse trials. Expression of GNA (approx. 1.0% total soluble protein) by transgenic plants significantly reduced the level of pest damage, thus confirming previous studies. Furthermore, the presence of the parasitoid significantly reduced the levels of damage incurred either by the transgenic or control plants when compared to those plants grown in the absence of the parasitoid. For the GNA expressing plants the presence of the parasitoid resulted in further reductions (ca. 21%) in the level of damage caused by the pest species. The ability of the wasp to parasitise and subsequently develop on the pest larvae was not altered by the presence of GNA in the diet of the host. E. pennicornis progeny that developed on L. oleracea reared on GNA expressing plants showed no significant alteration in fecundity when compared with wasps that had developed on hosts fed on control potato plants, although mean size and longevity of female parasitoids was significantly reduced. The number of F ₂ progeny produced by parasitoids derived from hosts fed on GNA-expressing plants was not significantly different to those produced by parasitoids from hosts fed control plants. Results from the present study demonstrate that the use of transgenic plants expressing insecticidal proteins can be compatible with the deployment of beneficial insects and that the two factors may interact in a positive manner.     

Seasonal capacity of attached and detached vineyard roots to support grape phylloxera (Homoptera: Phylloxeridae)

Field experiments were conducted to evaluate population densities and survival, developmental rate, and fecundity of grape phylloxera, Daktulosphaira vitifoliae (Fitch), as influenced by root attachment or detachment from mature, field-grown, Vitis vinifera L. grapevines through the growing season. Experiments were performed using artificial infestations of California biotype A grape phylloxera. Thirty-day bioassays on attached- and detached-roots were repeated monthly from May to September in 1997 (cultivar 'Carignane') and April to September in 1998 (cultivar 'Thompson Seedless'). The bioassays showed that attached roots had lower population densities than detached roots in all months of both years. Densities varied by month, tending to be higher in spring than in summer. Of the population parameters studied, survival was most influenced by attachment condition, being higher on detached than on attached roots by up to 25-fold. These results imply the importance of vine-related mortality factors to grape phylloxera population density. Influence of root attachment condition on developmental rate and fecundity was not uniform across bioassay months for either year; however, in the four out of 21 assays where there was a significant difference it favored detached roots by twofold. Fruit harvest resulted in higher survival in the July assay but not for assays in August and September; however, neither developmental rate nor fecundity was affected by harvest in any ofthe assays. We conclude that mortality rather than nutritional factors are most limiting for field populations on susceptible vines. This work suggests that detachment of roots as occurs with root girdling by root pathogens may increase grape phylloxera populations on infested, susceptible vines. These results imply that excised root bioassays over-estimate grape phylloxera virulence and underestimate rootstock resistance.     

Concanavalin A inhibits development of tomato moth (Lacanobia oleracea) and peach-potato aphid (Myzus persicae) when expressed in transgenic potato plants

The effects of concanavalin A (ConA), a glucose/mannose-specific lectin from jackbean (Canavalia ensiformis), on insect crop pests from two different orders, Lepidoptera and Homoptera, were investigated. When fed to larvae of tomato moth (Lacanobia oleracea) at a range of concentrations (0.02–2.0% of total protein) in artificial diet, ConA decreased survival, with up to 90% mortality observed at the highest dose level, and retarded development, but had only a small effect on larval weight. When fed to peach-potato aphids (Myzus persicae) at a range of concentrations (1–9μM) in liquid artificial diet, ConA reduced aphid size by up to 30%, retarded development to maturity, and reduced fecundity (production of offspring) by >35%, but had little effect on survival. With both insects, there was a poor correlation between lectin dose and the quantitative effect. Constitutive expression of ConA in transgenic potatoes driven by the CaMV 35S promoter resulted in the protein accumulating to levels lower than predicted, possibly due to potato not being able to adequately reproduce the post-translational processing of this lectin which occurs in jackbean. However, the expressed lectin was functionally active as a haemagglutinin. Bioassay of L. oleracea larvae on ConA-expressing potato plants showed that the lectin retarded larval development, and decreased larval weights by >45%, but had no significant effect on survival. It also decreased consumption of plant tissue by the larvae. In agreement with the diet bioassay results, ConA-expressing potatoes decreased the fecundity of M. persicae by up to 45%. ConA thus has potential as a protective agent against insect pests in transgenic crops. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of snowdrop lectin (GNA) delivered via artificial diet and transgenic plants on Eulophus pennicornis (Hymenoptera: Eulophidae), a parasitoid of the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae)

Snowdrop lectin (Galanthus nivalis agglutinin, GNA) has previously been shown to confer significant levels of protection against the lepidopteran pest Lacanobia oleracea when expressed in transgenic potato. The effect of GNA on the parasitism of L. oleracea by the gregarious ectoparasitoid Eulophus pennicornis was investigated. Maize-based, and potato leaf-based diets containing GNA, and excised transgenic potato leaves expressing GNA, were fed to L. oleracea larvae from the beginning of either the third or fourth larval instar. Lacanobia oleracea larvae were individually exposed to single mated adult female E. pennicornis parasitoids from the fifth instar onwards.The success of the wasp was not reduced by the presence of GNA in any of the diets, or by the length of feeding of the host prior to parasitism. However, the mean number of wasps that developed on L. oleracea reared from the third instar on the GNA-containing maize diet was significantly higher than on the controls (20.6 and 9.3 adults/host respectively). In all other cases differences were not significant. Eulophus pennicornis progeny that developed on L. oleracea reared on GNA-containing diets showed little or no alteration in size, longevity, egg load and fecundity when compared with wasps that had developed on hosts fed the respective control diets.The results suggest that expression of GNA in transgenic crops to confer resistance to lepidopteran pests will not adversely affect the ability of the ectoparasitoid E. pennicornis to utilise the pest species as a host.     

Snowdrop lectin (GNA) has no acute toxic effects on a beneficial insect predator,the 2-spot ladybird (Adalia bipunctata L.)

Two-spot ladybird (Adalia bipunctata L.) larvae were fed on aphids (Myzus persicae (Sulz.)) which had been loaded with snowdrop lectin (Galanthus nivalis agglutinin; GNA) by feeding on artificial diet containing the protein. Treatment with GNA significantly decreased the growth of aphids. No acute toxicity of GNA-containing aphids towards the ladybird larvae was observed, although there were small effects on development. When fed a fixed number of aphids, larvae exposed to GNA spent longer in the 4th instar, taking 6 extra days to reach pupation; however, retardation of development was not observed in ladybird larvae fed equal weights of aphids. Ladybird larvae fed GNA-containing aphids were found to be 8-15% smaller than controls, but ate a significantly greater number of aphids (approx. 40% to pupation). GNA was shown to be present on the microvilli of the midgut brush border membrane and within gut epithelial cells in ladybird larvae fed on GNA-dosed aphids, although disruption of the brush border was not observed. It is hypothesised that GNA does not have significant direct toxic or adverse effects on developing ladybird larvae, but that the effects observed may be due to the fact that the aphids fed on GNA are compromised and are thus a suboptimal food.     

In vitro and in vivo binding of snowdrop (Galanthus nivalis agglutinin; GNA) and jackbean (Canavalia ensiformis; Con A) lectins within tomato moth (Lacanobia oleracea) larvae; mechanisms of insecticidal action

When fed in semi-artificial diet the lectins from snowdrop (Galanthus nivalis: GNA: mannose-specific) and jackbean (Canavalia ensiformis: Con A: specific for glucose and mannose) were shown to accumulate in vivo in the guts, malpighian tubules and haemolymph of Lacanobia oleracea (tomato moth) larvae. Con A, but not GNA, also accumulated in the fat bodies of lectin-fed larvae. The presence of glycoproteins which bind to both lectins in vitro was confirmed using labelled lectins to probe blots of polypeptides extracted from larval tissues. Immunolocalisation studies revealed a similar pattern of GNA and Con A binding along the digestive tract with binding concentrated in midgut sections. Binding of lectins to microvilli appeared to lead to transport of the proteins into cells of the gut and malpighian tubules. These results suggested that both lectins are able to exert systemic effects via transport from the gut contents to the haemolymph across the gut epithelium. The delivery of GNA and Con A to the haemolymph was shown to be dependent on their functional integrity by feeding larvae diets containing denatured lectins. Con A, but not GNA, was shown to persist in gut and fat body tissue of lectin-fed larvae chased with control diet for three days. Con A also shows more extensive binding to larval tissues in vitro than GNA, and these two factors are suggested to contribute to the higher levels of toxicity shown by Con A, relative to GNA, in previous long term bioassays.     

Are fecundity and longevity of female Aphelinus abdominalis affected by development in GNA‐dosed Macrosiphum euphorbiae?

Abstract. Snowdrop lectin ( Galanthus nivalis agglutinin, GNA) confers partial resistance to several aphid species when incorporated into an artificial diet and/or expressed in transgenic potato. First-tier laboratory-scale experiments were conducted to assess the potential effect of GNA on the longevity and fecundity of female parasitoid Aphelinus abdominalis (Dalman) that had developed in Macrosiphum euphorbiae (Thomas) fed artificial diet containing 0.1% GNA (w/v). In a previous study it was shown that GNA ingested by A. abdominalis larvae is not acutely toxic. It was also shown that GNA has a host-size mediated effect on parasitoid sex ratio and larval development, but no apparent direct effect.         In this study, we report that A. abdominalis larvae that developed in GNA-dosed aphids that were smaller than control aphids of the same age, produced smaller adults with a reduced longevity and fecundity. Aphelinus abdominalis larvae that developed in GNA-dosed aphids older than the control but of the same size, produced adults of similar size that lived as long as the control but had a reduced fecundity.         Our results suggest that GNA fed to aphids in artificial diet has both a host-mediated effect (via aphid-size) and a direct effect on adult parasitoid fecundity. It is not known how GNA affects parasitoid larval development and subsequently adult fecundity, but it is hypothesized that GNA acted as an antifeedant to parasitoid larvae, thus disturbing nutrient assimilation and conversion necessary for egg maturation.     

Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids

The range of sap-sucking insect pests to which GNA, (the mannose specific lectin from snowdrops (Galanthus nivalis) has been shown to be insecticidal in artificial diets has been extended to include the peach potato aphid (Myzus persicae). A gene construct for constitutive expression of GNA from the CaMV35S gene promoter has been introduced into tobacco plants. A transgenic tobacco line which expresses high levels of GNA has been shown to have enhanced resistance toM. persicae in leaf disc and whole plant bioassays,demonstrating the potential for extending transgenic plant technology to the control of sap-sucking insect pests.     

Potential effects of oilseed rape expressing oryzacystatin-1 (OC-1) and of purified insecticidal proteins on larvae of the solitary bee Osmia bicornis

Despite their importance as pollinators in crops and wild plants, solitary bees have not previously been included in non-target testing of insect-resistant transgenic crop plants. Larvae of many solitary bees feed almost exclusively on pollen and thus could be highly exposed to transgene products expressed in the pollen. The potential effects of pollen from oilseed rape expressing the cysteine protease inhibitor oryzacystatin-1 (OC-1) were investigated on larvae of the solitary bee Osmia bicornis (= O. rufa). Furthermore, recombinant OC-1 (rOC-1), the Bt toxin Cry1Ab and the snowdrop lectin Galanthus nivalis agglutinin (GNA) were evaluated for effects on the life history parameters of this important pollinator. Pollen provisions from transgenic OC-1 oilseed rape did not affect overall development. Similarly, high doses of rOC-1 and Cry1Ab as well as a low dose of GNA failed to cause any significant effects. However, a high dose of GNA (0.1%) in the larval diet resulted in significantly increased development time and reduced efficiency in conversion of pollen food into larval body weight. Our results suggest that OC-1 and Cry1Ab expressing transgenic crops would pose a negligible risk for O. bicornis larvae, whereas GNA expressing plants could cause detrimental effects, but only if bees were exposed to high levels of the protein. The described bioassay with bee brood is not only suitable for early tier non-target tests of transgenic plants, but also has broader applicability to other crop protection products.     

Toxicity and repellency of Tephrosia candida to larval and adult Diaprepes root weevil (Coleoptera: Curculionidae)

Leaves of the tropical legume Tephrosia candida DC deterred feeding by adults of the Diaprepes root weevil, Diaprepes abbreviatus (L.), compared with leaves of Citrus macrophylla Wester, a common citrus rootstock, or T. vogelii Hook. f. When larvae were placed in pots containing plants of the three species for 28 d in a growth chamber, larval survival and weight gain were significantly reduced in pots containing plants of T. candida compared with larvae in pots with C. macrophylla or T. vogelii. Diet incorporation of lyophilized roots of T. candida into an artificial diet increasingly inhibited the growth of larvae and increased larval mortality with increased concentration of roots, whereas roots from C. macrophylla or T. vogelii had no effect compared with the diet-only control. T. candida, but not T. vogelii, contains at least one constituent that acts as an antifeedant toward adult D. abbreviatus and as a toxicant toward larvae. No antifeedant effect of roots of T. candida toward larvae was observed in no-choice pot tests or in a diet incorporation bioassay. In pots, larval feeding damage to roots of T. candida was evident. In the diet incorporation assay, 97% of larvae survived 29 d on a diet of cellulose powder (a nutritionally inert filler) despite losing weight. We conclude that decreased survival and weight gain of larvae-fed fresh or lyophilized roots of T. candida were the result of ingestion of a toxicant and not deterrence from feeding.     

Tritrophic Interactions Between Transgenic Potato Expressing Snowdrop Lectin (GNA), an Aphid Pest (Peach–Potato Aphid; Myzus Persicae (Sulz.) and a Beneficial Predator (2-Spot Ladybird; Adalia Bipunctata L.)

Tritrophic interactions between transgenic potato expressing the insecticidal lectin from snowdrop (Galanthus nivalis agglutinin; GNA), an aphid pest, Myzus persicae (Sulz.), and a beneficial predator, the 2-spot ladybird (Adalia bipunctata L.) were investigated. Clonal plants expressing GNA at 0.1–0.2% total soluble protein in leaves were used. No significant effects on development and survival of ladybird larvae fed on aphids from these transgenic plants were observed, with larval survival in the experimental group being 90% compared to 89% for controls. There were also no effects on subsequent female or male longevity. Female fecundity was also investigated. Although no significant differences (p > 0.05) were observed in egg production between control and experimental groups, a 10%, reduction (p < 0.01) in egg viability (determined by % hatch) occurred in ladybirds fed aphids reared on transgenic plants. Additional studies were carried out using aphids fed on artificial diet containing GNA, to deliver quantified levels of the protein to ladybird adults. GNA had no deleterious effects upon adult longevity, but resulted in a consistent trend for improved fecundity. Egg production was increased by up to 70% and egg viability also increased significantly. The results suggest that GNA is not deleterious to ladybirds. Results from these studies highlight the need to discriminate between direct and indirect effects when studying tritrophic interactions between plants/pests/natural enemies. Furthermore, it emphasises the importance of demonstrating cause and effect.     

Transgenic potato plants with enhanced resistance to the tomato moth,Lacanobia oleracea: growth room trials

Insecticidal effects of three plant-derived genes, those encoding snowdrop lectin (GNA), bean (Phaseolus vulgaris) chitinase (BCH) and wheat -amylase (WAI), were investigated and compared with effects of the cowpea trypsin inhibitor gene (CpTI). Transgenic potato plants containing each of the three genes singly, and in pairwise combinations were produced. All the introduced genes were driven by the CaMV 35S promoter; expression was readily detectable at the RNA level in transformants, but not detectable accumulation of WAI could be detected in transgenic potatoes containing its encoding gene. GNA and BCH were accumulated at levels up to 2.0% of total soluble protein; both proteins were expressed in a functional form, and GNA was shown to undergo 'correct' N-terminal processing. Accumulation levels of individual proteins were higher in plants containing a single foreign gene than in plants containing two foreign genes.Resistance of the transgenic plants to insect attack was assayed by exposing the plants to larvae of the tomato moth, Lacanobia oleracea. All the plants tested which were expressing GNA showed an enhanced level of resistance. Leaf damage was reduced by more than 50% compared to controls; total insect biomass per plant was reduced by 45-65%, but larval survival was only slightly reduced (20%). These results support the hypothesis that GNA has a significant antifeedant effect on insects. Expression of BCH had no protective effect against this insect. Expression of CpTI in transgenic potatoes had similar effects to expression of GNA on total insect biomass and survival, but did not afford protection against insect damage to the plant.