Non-target impacts of forest defoliator management options: Decision for no spraying may have worse impacts on non-target Lepidoptera than Bacillus thuringiensis insecticides

Management programs for major forest defoliators such as gypsy moths or forest tent caterpillars, and crop pests such as the European corn borer have shifted from broad-spectrum insecticides to more environmentally benign microbial pesticides such as Bacillus thuringiensis (foliage sprays and transgenic toxin expression in plant tissues). Phytochemically resistant host plants and natural enemies have been used as alternative pest management strategies (including generalist tachinid flies such as Compsilura, viruses, microsporidians, and fungi), but all of these have some non-target impacts, as described from literature review. A sequence of lab and field studies were conducted to determine non-target impacts on native Lepidoptera in North America. The conclusions reached are that a decision not to spray Bt pesticides (i.e. to allow defoliation and natural pest outbreaks to run their course) could be as bad or worse for non-target Lepidoptera as the microbial insecticides would be. The important concept that must be maintained is that all pest management programs have some risk of negative non-target impacts, but it is the magnitude and relative importance that will remain the most critical issue for environmental impacts and pest management.     

The detection and decay of Cry1Ab Bt-endotoxins within non-target slugs,Deroceras reticulatum (Mollusca: Pulmonata), following consumption of transgenic corn

With greater acreages being planted to transgenic crops, the exposure of non-target species to bioengineered material is increasing. Although the slug, Deroceras reticulatum (Müller), is a major agricultural pest throughout the world, Bacillus thuringiensis crops were not intended to target these species. Molluscs are readily consumed by many generalist predators; if these Cry1Ab-endotoxins are taken up by slugs during feeding on transgenic plants, predators would therefore be exposed to elevated endotoxin concentrations. Using a biochemical assay, we tested the hypothesis that slugs fed transgenic corn would accumulate detectable quantities of Cry1Ab-endotoxins for prolonged periods of time. Characterization indicated that at low dilution rates, Cry1Ab-endotoxins were detectable in slugs fed Bt-corn but no reactivity was elicited by specimens fed non-transgenic food. It was possible to detect Cry1Ab-endotoxins in slugs for 95.9 h after consumption of Bt-corn. Although quantities were small, these long detection periods indicated potential exposure of generalist predators to low concentrations of transgenic insecticidal toxins in the field.     

Effects of Bt maize on the herbivore <Emphasis Type="Italic">Spodoptera littoralis</Emphasis> (Lepidoptera: Noctuidae) and the parasitoid <Emphasis Type="Italic">Cotesia marginiventris</Emphasis> (Hymenoptera: Braconidae).

Recent studies have shown that transgenic insect resistant plants can have negative effects on non-target herbivores as well as on beneficial insects. The study of tritrophic interactions gives insight into the complex mechanisms of food webs in the field and can easily be incorporated into a tiered risk assessment framework. We investigated the effects of transgenic maize (Zea mays) expressing insecticidal proteins derived from Bacillus thuringiensis (Bt maize) on Spodoptera littoralis, a non-target herbivore, and on the hymenopteran parasitoid Cotesia marginiventris. In a laboratory study, S. littoralis larvae were reared for their whole lifespan on a mixture of leaves and stems from 2–4-week old Bt maize plants. S. littoralis survival, developmental times and larval weights were significantly affected by Bt maize diet. However, adult moths, which survived development on Bt maize, were the same size as the adults from the control group.C. marginiventris survival, developmental times and cocoon weights were significantly negatively affected if their S. littoralis host larva had been fed Bt maize. ELISA tests confirmed that S. littoralis larvae ingest high amounts of Cry1A(b) toxin while feeding on Bt maize. In S. littoralis pupae and in C. marginiventris cocoon silk, only traces of the toxin could be detected. No toxin was found in S. littoralis and C. marginiventris adults. Thus the toxin is not accumulating in the trophic levels and in fact appears to be excreted. Our results suggest that the effects on C. marginiventris when developing in susceptible S. littoralis larvae are indirect (host mediated). The biological relevance of those results and the significance of this study in risk assessment are discussed.     

Biological Activity of Cry1Ab Toxin Expressed by Bt Maize Following Ingestion by Herbivorous Arthropods and Exposure of the Predator Chrysoperla carnea

A major concern regarding the deployment of insect resistant transgenic plants is their potential impact on non-target organisms, in particular on beneficial arthropods such as predators. To assess the risks that transgenic plants pose to predators, various experimental testing systems can be used. When using tritrophic studies, it is important to verify the actual exposure of the predator, i.e., the presence of biologically active toxin in the herbivorous arthropod (prey). We therefore investigated the uptake of Cry1Ab toxin by larvae of the green lacewing (Chrysoperla carnea (Stephens); Neuroptera: Chrysopidae) after consuming two Bt maize-fed herbivores (Tetranychus urticae Koch; Acarina: Tetranychidae and Spodoptera littoralis (Boisduval); Lepidoptera: Noctuidae) by means of an immunological test (ELISA) and the activity of the Cry1Ab toxin following ingestion by the herbivores. Moreover, we compared the activity of Cry1Ab toxin produced by Bt maize to that of purified toxin obtained from transformed Escherichia coli, which is recommended to be used in toxicity studies. The activity of the toxin was assessed by performing feeding bioassays with larvae of the European corn borer (Ostrinia nubilalis (Hübner); Lepidoptera: Crambidae), the target pest of Cry1Ab expressing maize. ELISA confirmed the ingestion of Bt toxin by C. carnea larvae when fed with either of the two prey species and feeding bioassays using the target pest showed that the biological activity of the Cry1Ab toxin is maintained after ingestion by both herbivore species. These findings are discussed in the context of previous risk assessment studies with C. carnea. The purified Cry1Ab protein was more toxic to O. nubilalis compared to the plant-derived Cry1Ab toxin when applied at equal concentrations according to ELISA measurements. Possible reasons for these findings are discussed.     

Assessing the utilization of a carbohydrate food source and the impact of insecticidal proteins on larvae of the green lacewing, Chrysoperla carnea

A concern with the widespread use of insecticidal transgenic crops is their potential to adversely affect non-target organisms, including biological control agents such as larvae of the green lacewing, Chrysoperla carnea (Neuroptera: Chrysopidae). Since the insecticidal proteins expressed by the current transgenic plants are active only after ingestion, dietary bioassays are required to test direct effects on non-target organisms. After showing that C. carnea larvae utilize carbohydrate foods, we exposed them to insecticidal proteins dissolved in a sucrose solution. Feeding on snowdrop lectin (Galanthus nivalis agglutinin, GNA) as a model compound, the larvae were negatively affected in a number of life-table parameters. Interestingly, GNA caused a prolongation in first instar development, but had no effect on subsequent utilization of prey resulting in an increased weight of second instars. Comparable studies with avidin, a biotin-binding protein, revealed strong effects on C. carnea survival at the concentration tested. Despite the fact that the proteolytic digestion of C. carnea larvae is reported to be dominated by serine proteases, ingestion of soybean trypsin inhibitor (SBTI) did not cause any detrimental effects. Similarly, two Cry proteins derived from Bacillus thuringiensis (Cry1Ac and Cry1Ab) did not cause negative effects on C. carnea, what is consistent with earlier studies. The here presented bioassay provides a valuable tool to assess direct impacts of insecticidal proteins to C. carnea larvae and other predators that are known to feed on carbohydrate solutions.     

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.     

The effect of stem diameter on European corn borer behavior and survival: potential consequences for IRM in Bt-corn

The ability of non-crop plants to support complete development of insect pests is an important factor for determining the impact of those plants on resistance management programs for transgenic crops. We assessed the effect of one physical factor, plant stem diameter, on the ability of plants to support full development of the European corn borer (ECB), Ostrinia nubilalis Hübner (Lepidoptera: Crambidae), the target pest of transgenic Bt-corn. In the field, European corn borer larvae were significantly more likely to tunnel and survive in plants with larger stem diameters. Larvae were 40× more likely to survive on corn, the largest plant tested, compared to many of the smaller plants. In the laboratory, larvae were more likely to survive in and less likely to abandon the largest diet-filled artificial stems that varied only in stem diameter. In conditions simulating those that an ECB larvae would encounter upon abandoning a host, larvae survived up to three weeks and were able to locate corn as a new host with a significantly higher frequency than would be expected if they were foraging randomly. These results indicate that the probability of ECB larval survival to maturity on a plant other than corn is relatively low and thus these smaller stemmed non-corn plants may not make a substantial contribution to the pool of susceptible adults. Conversely, since more mature larvae are not as susceptible as neonates, any larvae that partially develop on non-corn plants and subsequently colonize Bt-corn may not be exposed to a lethal dose of the toxin. Since some proportion of the individuals that survive could be partially resistant heterozygotes the presence of non-corn host plants could facilitate the development of resistant ECB populations.     

Evaluation of dietary effects of transgenic corn pollen expressing Cry3Bb1 protein on a non-target ladybird beetle, Coleomegilla maculata

A transgenic corn event (MON 863) has been recently developed by Monsanto Company for control of corn rootworms, Diabrotica spp. (Coleoptera: Chrysomelidae). This transgenic corn event expresses the cry3Bb1 gene derived from Bacillus thuringiensis (Berliner), which encodes the insecticidal Cry3Bb1 protein for corn rootworm control. A continuous feeding study was conducted in the laboratory to evaluate the dietary effect of MON 863 pollen expressing the Cry3Bb1 protein on the survival, larval development, and reproductive capacity of the non-target species, Coleomegilla maculata DeGeer (Coleoptera: Coccinellidae). First instar C. maculata (less than 24 h old) and newly emerging adults (less than 72 h old) were fed individually on a diet mixture containing 50% of MON 863 pollen, non-transgenic (control) corn pollen, bee pollen (a component of normal rearing diet), or potassium arsenate-treated control corn pollen. In the larval tests, 96.7%, 90.0%, and 93.3% of C. maculata larvae successfully pupated and then emerged as adults when fed on MON 863 pollen, non-transgenic corn pollen, and bee pollen (normal rearing) diets, respectively. Among the larvae completing their development, there were no significant differences in the developmental time to pupation and adult emergence among the transgenic corn pollen, non-transgenic corn pollen, and bee pollen diet treatments. All larvae fed on arsenate treated corn pollen diet died as larvae. For tests with adults, 83.3%, 80.0%, and 100% of adult C. maculata survived for the 30 days of the test period when reared on diets containing 50% of MON 863 pollen, non-transgenic corn pollen, and bee pollen respectively. While the adult survival rate on MON 863 pollen diet was significantly less than that on the bee pollen diet, there was no significant difference between the MON 863 and non-transgenic corn pollen treatments. During the period of adult testing, an average of 77, 80, and 89 eggs per female were laid by females fed on the MON 863 pollen, control corn pollen, and bee pollen, respectively; no significant differences were detected in the number of eggs laid among these treatments. These results demonstrate that when offered at 50% by weight of the dietary component, transgenic corn (MON 863) pollen expressing Cry3Bb1 protein had no measurable negative effect on the survival and development of C. maculata larvae to pupation and adulthood nor any adverse effect on adult survival and reproductive capacity. Relevance of these findings to ecological impacts of transgenic Bt crops on non-target beneficial insects is discussed.     

Potential use of a serpin from Arabidopsis for pest control

Although genetically modified (GM) plants expressing toxins from Bacillus thuringiensis (Bt) protect agricultural crops against lepidopteran and coleopteran pests, field-evolved resistance to Bt toxins has been reported for populations of several lepidopteran species. Moreover, some important agricultural pests, like phloem-feeding insects, are not susceptible to Bt crops. Complementary pest control strategies are therefore necessary to assure that the benefits provided by those insect-resistant transgenic plants are not compromised and to target those pests that are not susceptible. Experimental GM plants producing plant protease inhibitors have been shown to confer resistance against a wide range of agricultural pests. In this study we assessed the potential of AtSerpin1, a serpin from Arabidopsis thaliana (L). Heynh., for pest control. In vitro assays were conducted with a wide range of pests that rely mainly on either serine or cysteine proteases for digestion and also with three non-target organisms occurring in agricultural crops. AtSerpin1 inhibited proteases from all pest and non-target species assayed. Subsequently, the cotton leafworm Spodoptera littoralis Boisduval and the pea aphid Acyrthosiphon pisum (Harris) were fed on artificial diets containing AtSerpin1, and S. littoralis was also fed on transgenic Arabidopsis plants overproducing AtSerpin1. AtSerpin1 supplied in the artificial diet or by transgenic plants reduced the growth of S. littoralis larvae by 65% and 38%, respectively, relative to controls. Nymphs of A. pisum exposed to diets containing AtSerpin1 suffered high mortality levels (LC₅₀ = 637 µg ml⁻¹). The results indicate that AtSerpin1 is a good candidate for exploitation in pest control.     

Cry1Ab protein levels in phytophagous insects feeding on transgenic corn: implications for secondary exposure risk assessment

Enzyme-linked immunosorbent assays (ELISA) and bioassays were used to estimate levels of Cry1Ab protein in four species of phytophagous insects after feeding on transgenic Bt-corn plants expressing Cry1Ab protein or artificial diets containing Cry1Ab protein. The level of Cry1Ab in insects feeding on sources containing the Cry1Ab protein was uniformly low but varied with insect species as well as food source. For the corn leaf aphid, Rhopalosiphum maidis (Fitch), feeding on diet solutions containing Cry1Ab protein, the level of the protein in the aphid was 250–500 times less than the original levels in the diet, whereas no Cry1Ab was detected by ELISA in aphids feeding on transgenic Bt-Corn plants. For the lepidopteran insects, Ostrinia nubilalis (Hübner), Helicoverpa zea (Boddie), and Agrotis ipsilon (Hufnagel), levels of Cry1Ab in larvae varied significantly with feeding treatment. When feeding for 24 h on artificial diets containing 20 and 100 ppm of Cry1Ab, the level of Cry1Ab in the larvae was about 57 and 142 times lower, respectively, than the original protein level in the diet for O. nubilalis, 20 and 34 times lower for H. zea, and 10 to 14 times lower for A. ipsilon. Diet incorporation bioassays with a susceptible insect (first instar O. nubilalis) showed significant Cry1Ab bioactivity present within whole body tissues of R. maidis and O. nubilalis that had fed on diet containing a minimum of 20 ppm or higher concentrations (100 or 200 ppm) of Cry1Ab, but no significant bioactivity within the tissues of these insects after feeding on transgenic Bt-corn plants. The relevance of these findings to secondary exposure risk assessment for transgenic Bt crops is discussed.     

Landscape refuges delay resistance of the European corn borer to Bt-maize: a demo-genetic dynamic model

We constructed a reaction-diffusion model of the development of resistance to transgenic insecticidal Bt crops in pest populations. Kostitzin’s demo-genetic model describes local interactions between three competing pest genotypes with alleles conferring resistance or susceptibility to transgenic plants, the spatial spread of insects being modelled by diffusion. This new approach makes it possible to combine a spatial demographic model of population dynamics with classical genetic theory. We used this model to examine the effects of pest dispersal and of the size and shape of the refuge on the efficiency of the “high-dose/refuge” strategy, which was designed to prevent the development of resistance in populations of insect pests, such as the European corn borer, Ostrinia nubilalis Hübner (Lepidoptera, Crambidae). We found that, with realistic combinations of refuge size and pest dispersal, the development of resistance could be considerably delayed. With a small to medium-sized farming area, contiguous refuge plots are more efficient than a larger number of smaller refuge patches. We also show that the formal coupling of classical Fisher–Haldane–Wright population genetics equations with diffusion terms inaccurately describes the development of resistance in a spatially heterogeneous pest population, notably overestimating the speed with which Bt resistance is selected in populations of pests targeted by Bt crops.     

Effects of soybean proteinase inhibitors on development of the soil mite Scheloribates praeincisus (Acari: Oribatida)

Proteinase inhibitors (PI) are present in plant tissues, especially in seeds, and act as a defense mechanism against herbivores and pathogens. Serine PI from soybean such as Bowman–Birk (BBPI) and Kunitz have been used to enhance resistance of sugarcane varieties to the sugarcane borer Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae), the major pest of this crop. The use of these genetically-modified plants (GM) expressing PI requires knowledge of its sustainability and environmental safety, determining the stability of the introduced characteristic and its effects on non-target organisms. The objective of this study was to evaluate direct effects of ingestion of semi-purified and purified soybean PI and GM sugarcane plants on the soil-dwelling mite Scheloribates praeincisus (Berlese) (Acari: Oribatida). This mite is abundant in agricultural soils and participates in the process of organic matter decomposition; for this reason it will be exposed to PI by feeding on GM plant debris. Eggs of S. praeincisus were isolated and after larvae emerged, immatures were fed milled sugarcane leaves added to semi-purified or purified PI (Kunitz and BBPI) or immatures were fed GM sugarcane varieties expressing Kunitz and BBPI type PI or the untransformed near isogenic parental line variety as a control. Developmental time (larva-adult) and survival of S. praeincisus was evaluated. Neither Kunitz nor BBPI affected S. praeincisus survival. On the other hand, ingestion of semi-purified and purified Kunitz inhibitor diminished duration of S. praeincisus immature stages. Ingestion of GM senescent leaves did not have an effect on S. praeincisus immature developmental time and survival, compared to ingestion of leaves from the isogenic parental plants. These results indicate that cultivation of these transgenic sugarcane plants is safe for the non-target species S. praeincisus.     

Development of the predator Chrysoperla carnea (Stephens) as affected by feeding on Helicoverpa armigera (Hübner) reared on transgenic Cry 2Ab tomato plants

Investigations were conducted using Helicoverpa armigera fed on transgenic tomato plants containing a synthetic Cry2ab gene aiming to evaluate its impact on the development of the predator Chrysoperla carnea. Prey consumption and development parameters of C. carnea were assessed. No obvious differences were observed in the development of C. carnea that preyed on H. armigera fed on transgenic Bt tomato plants as compared to the control. This shows obvious that transgenic Bt tomato plants can be safely used as an efficient tool for the biocontrol of H. armigera with no effect on its predator, C. carnea.     

Prey-mediated effects of transgenic canola on a beneficial, non-target, carabid beetle

Transgenic plants producing insecticidal proteins from Bacillus thuringiensis (Bt) can control some major insect pests and reduce reliance on sprayed insecticides. However, large scale adoption of this technology has raised concerns about potential negative effects, including evolution of pest resistance to Bt toxins, transgene flow from Bt crops to other plants, and harm to non-target beneficial organisms. Furthermore, concern has also been expressed over the effects this technology may have on biodiversity in general. Ecologically relevant risk assessment is therefore required (Risk = Hazard × Exposure). Transgenic plants that produce Bt toxins to kill insect pests could harm beneficial predators. This might occur directly by transmission of toxin via prey, or indirectly by toxin-induced reduction in prey quality (Hazard). To test these hypotheses, we determined the effects of Bt-producing canola on a predatory ground beetle (Pterostichus madidus) fed larvae of diamondback moth (Plutella xylostella) that were either susceptible or resistant to the Bt toxin. Survival, weight gain, and adult reproductive fitness did not differ between beetles fed prey reared on Bt-producing plants and those fed prey from control plants. Furthermore, while Bt-resistant prey was shown to deliver high levels of toxin to the beetle when they were consumed, no significant impact upon the beetle was observed. Subsequent investigation showed that in choice tests (Exposure), starved and partially satiated female beetles avoided Bt-fed susceptible prey, but not Bt-fed resistant prey. However, in the rare cases when starved females initially selected Bt-fed susceptible prey, they rapidly rejected them after beginning to feed. This prey type was shown to provide sufficient nutrition to support reproduction in the bioassay suggesting that Bt-fed susceptible prey is acceptable in the absence of alternative prey, however adults possess a discrimination ability based on prey quality. These results suggest that the direct effects of Bt-producing canola on predator life history was minimal, and that predators’ behavioural preferences may mitigate negative indirect effects of reduced quality of prey caused by consumption of Bt-producing plants. The results presented here therefore suggest that cultivation of Bt canola may lead to conservation of non-target predatory and scavenging organisms beneficial in pest control, such as carabids, and may therefore provide more sustainable agricultural systems than current practices. In addition, minimal impacts on beneficial carabids in agro-ecosystems suggest that Bt canola crops are likely to be compatible with integrated pest management (IPM) systems.     

Field trials to evaluate effects of Bt-transgenic silage corn expressing the Cry1Ab insecticidal toxin on non-target soil arthropods in northern New England, USA

Traditionally, control of European corn borer (Ostrinia nubilalis) Hübner has been achieved through the use of chemical insecticides. With increasing emphasis on reducing pesticide inputs in agricultural production, alternative management technologies are now being used including transgenic silage corn modified to express Cry1Ab protein toxins derived from Bacillus thuringiensis (Bt) Berliner. The Cry1Ab toxin is expressed by all plant cells and throughout the growing season. Furthermore, the toxins are exuded from corn plant roots into the rhizosphere, raising concerns over possible side-effects on non-target beneficial organisms in the same habitat. In addition, detrivores are exposed to crop residues containing the toxin when incorporated into the soil. The current 2-year study (2003, 2004) evaluated effects of two silage-corn varieties: Pioneer var. 38A25 (Bt-corn expressing the Cry1Ab toxin) and Pioneer var. 38A24 (parent isoline) on species diversity and evenness of carabid beetles and Collembola. Pitfall traps were used to collect surface-dwelling species on a bimonthly schedule from April to October. Soil cores were taken once a month from April to October to sample subterranean species, which were extracted using Berlese funnels. All individuals were recorded and identified where possible to species level for analysis in the Simpson’s D and Shannon–Wiener H’ diversity indices. Evenness was measured using Simpson’s E’, after which dominant species were analyzed in a multivariate ordination analysis. Results showed Bt-corn had no negative effects on any of the organisms analyzed. There was a significant year effect on the abundance of surface-dwelling Collembola and on species diversity of soil-dwelling Collembola. Our findings suggest that crop management practices and/or environmental conditions (e.g., heavy rainfall during the 2004 growing season) had the greatest impact on species diversity and evenness, rather than the crop itself (Bt or isoline).     

Influence of transgenic cottons with <Emphasis Type="Italic">Bacillus thuringiensis cry1Ac</Emphasis> gene on the natural enemies of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Transgenic cotton has been released for cultivation in several parts of the world to increase crop productivity. However, concerns have been raised regarding the possible undesirable effects of genetically modified crops on non-target organisms in the eco-system. Therefore, we studied the effects of transgenic cottons with cry1Ac gene from Bacillus thuringiensis Berliner (Bt) on the natural enemies of cotton bollworm/legume pod borer, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) under field and laboratory conditions. There was no apparent effect of transgenic cotton on the relative abundance of predatory spiders (Clubiona sp. and Neoscona sp.), coccinellid (Cheilomenes sexmaculatus Fab.), and the chrysopid (Chrysoperla carnea Stephens). However, the abundance of spiders, coccinellids, and chrysopids was quite low in insecticide protected plots towards end of the cropping season. There was a significant reduction in cocoon formation and adult emergence of the ichneumonid parasitoid, Campoletis chlorideae Uchnida reared on H. armigera larvae fed on the leaves of transgenic cottons before and after parasitization. However, no Bt toxins were detected in H. armigera larvae and the parasitoid cocoons with enzyme linked immunosorbent assay. Reduction in cocoon formation was because of early mortality of the H. armigera larvae due to Bt toxins in the leaves of transgenic cotton. There was a slight reduction in adult weight and fecundity, and prolongation of the larval period when the parasitoid was raised on H. armigera larvae fed on the leaves of transgenic cotton before and after parasitization. Survival and development of C. chlorideae was also poor when H. armigera larvae were fed on the leaves of cotton hybrid Mech 184. The adverse effects of transgenic cotton on survival and development of C. chlorideae were largely due to early mortality, and possibly poor nutritional quality of H. armigera larvae due to toxic effects of the transgene.     

Expression of Sambucus nigra agglutinin (SNA-I′) from elderberry bark in transgenic tobacco plants results in enhanced resistance to different insect species

Tobacco plants (Nicotiana tabacum cv Samsun NN) have been transformed with the gene encoding the type-2 ribosome-inactivating protein (RIP) SNA-I′ from elderberry (Sambucus nigra) under the control of the Cauliflower Mosaic Virus 35S promoter. Previous research confirmed that these plants synthesize, correctly process and assemble a fully active RIP. Variability in protein expression was observed within the transgenic lines. The effects of the type-2 RIP SNA-I′ delivered through a leaf feeding assay were evaluated in the laboratory on two economically important pest insects belonging to the orders of Hemiptera, the tobacco aphid (Myzus nicotianae) and Lepidoptera, the beet armyworm (Spodoptera exigua). In the experiment with aphids, significant effects were observed on the life parameters, such as survival, intrinsic rate of increase, net reproductive rate, mean generation time and mean daily offspring, whereas with caterpillars significant reduction in fresh weight as well as retardation in development were observed. In addition, significant increases in mortality were noted for insects fed on the transgenic lines as compared to wild type plants. This information provides further support for RIPs having a role in plant resistance to insect pest species.     

Insecticidal activity of a cryia(c) transgene in callus derived from regeneration-recalcitrant cotton (Gossypium hirsutum L.)

Summary The insecticidal effectiveness of a δ-endotoxin Cry protein from Bacillus thuringiensis in non-regenerable callus of a commercial Gossypium hirsutum L. variety was investigated. Two transgenic callus types were generated. The first callus type harbored the cry1A(c) gene and the hygromycin B phosphotransferase hpt selectable marker gene. The second callus type, the transgenic control, carried the marker genes β-glucuronidase (GUS) and hpt. Growth and survival rates of three major cotton moth species, Pectinophora gossypiella, Helicoverpa armigera, and Spodoptera littoralis, were examined with aseptic neonates reared on callus. Normal larval development occurred in all species supplied with non-transgenic callus, but insects died, or their growth was severely restricted, when reared on transgenic callus harvested from hygromycin B-supplemented medium. Development of larvae on transgenic control and on non-transgenic callus became very much alike after the transgenic control tissue had been subcultured on a hygromyein B-free medium for about 100 d prior to the insect-callus bioassay. Accordingly, for detection of Bt toxin activity without the interference of the influence of hygromycin B on insects, cry1A(c) callus was infested with insects after it had been propagated for more than 100 d on a medium free of the antibiotic. Under these experimental conditions all P. gossypiella and H. armigera, and most S. littoralis neonates died, and the growth (e.g., weight increment) of S. littoralis survivors was markedly impeded by cry1A(c) callus. Three new findings emerge from this study: first, P. gossypiella, a pest feeding in the field on bolls only, can be grown in vitro on cotton callus; second, in a host which is recalcitrant in terms of plant regeneration, the biological potency of an insectdetrimental transgene can nevertheless be evaluated by generating a transgenic host callus and conducting in vitro transgenic callus-insect assays; and third, our results suggest that hygromycin B is toxic to lepidopteran larvae.     

GFP基因在新疆小拟南芥和拟南芥中的表达分析

以质粒pMCB30为模板,扩增GFP基因,连接到载体pCMBIA2300-35S-OCS上,构建过量表达载体p35S:GFP,将其转入农杆菌GV3101.通过农杆菌介导法将p35S:GFP载体分别转入新疆特色植物小拟南芥和拟南芥中.T0代经含有卡那霉素的1/2MS培养基筛选,获得了T1代转基因小拟南芥2株,T1代转基因拟南芥9株.通过激光共聚焦显微镜观察,在转基因小拟南芥和拟南芥的根尖细胞中均可检测到GFP绿色荧光蛋白;对转基因植株进行PCR扩增,均可检测到GFP基因,表明GFP基因已成功转入小拟南芥和拟南芥中.该研究建立了小拟南芥的遗传转化体系,为进一步利用GFP基因和进一步研究小拟南芥的功能基因奠定基础.     

Transgenic rice plants with a synthetic cry1Ab gene from Bacillus thuringiensis were highly resistant to eight lepidopteran rice pest species

To fully explore the resistance potential of transgenic rice produced by Agrobacterium-mediated transformation, an elite line KMD1 was assessed for its resistance to eight lepidopteran rice pest species. KMD1 contained a synthetic cry1Ab gene from Bacillus thuringiensis under the control of a maize ubiquitin promoter. It was derived from a commercial japonica Chinese rice variety Xiushui 11, and bred true for both agronomic traits and a cry1Ab gene when the bioassays were done in 1998 in the R₅ generation. The eight lepidopteran pest species were: four Pyralidae species: Chilo suppressalis (striped stem borer, SSB), Scirpophaga incertulas (yellow stem borer, YSB), Cnaphalocrocis medinalis (leaf folder), Herpitogramma licarisalis; two Noctuidae: Sesamia inferens (pink stem borer, PSB) and Naranga anescens; one Stayridae: Mycalesis gotama; and one Hesperiidae, Parnara guttata. In laboratory bioassays, 100% mortality was observed in all insect species when their newly hatched or third-instar larvae were fed KMD1 leaf tissues, whereas only 9.65% of the neonates and none of the third-instar larvae died when fed the leaf tissues of non-transgenic control. Moreover, the leaf area of control tissues consumed in four days by stem borers was 20 to 40 times higher than that of KMD1 tissues, and the area of control tissues eaten by leaf-feeding species was 120 to 180 times greater than that of the transgenic tissues. Under natural infestation, no KMD1 plant was visibly damaged by the SSB, YSB and leaf folder in field evaluation. On the other hand, 80, 9.3 and 88.7% of control plants were injured by SSB, YSB, and leaf folder, respectively. These data disclosed that the transgenic line was highly resistant to a broad spectrum of lepidopteran insect species and could be useful in insect resistance breeding programs.