Uptake of Bt-toxin by herbivores feeding on transgenic maize and consequences for the predator Chrysoperla carnea

Abstract  1. Chrysoperla carnea is an important predatory insect in maize. To assess the ecological effects of Bt-maize, expressing the Cry1Ab protein, on larvae of this predator, the following factors were examined: (1) the performance of three prey herbivores ( Rhopalosiphum padi , Tetranychus urticae , and Spodoptera littoralis ) on transgenic Bt and non‐transgenic maize plants; (2) the intake of the Cry1Ab toxin by the three herbivores; and (3) the effects on C. carnea when fed each of the prey species.
2. The intrinsic rate of natural increase (r_m) was used as a measure of performance for R. padi and T. urticae . No difference in this parameter was observed between herbivores reared on Bt or non‐transgenic plants. In contrast, a higher mortality rate and a delay in development were observed in S. littoralis larvae when fed Bt-maize compared with those fed the control maize plants.
3. The ingestion of Cry1Ab toxin by the different herbivores was measured using an immunological assay (ELISA). Highest amounts of Cry1Ab toxin were detected in T. urticae , followed by S. littoralis , and only trace amounts detected in R. padi .
4. Feeding C. carnea with T. urticae , which were shown to contain the Cry1Ab toxin, or with R. padi , which do not ingest the toxin, did not affect survival, development, or weight of C. carnea . In contrast, a significant increase in mortality and a delay in development were observed when predators were fed S. littoralis larvae reared on Bt-maize.
5. A combined interaction of poor prey quality and Cry1Ab toxin may account for the negative effects observed on C. carnea when fed S. littoralis . The relevance of these findings to the ecological risks of Bt-maize on C. carnea is 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.     

Effects of Bt maize expressing Cry1Ab and Bt spray on Spodoptera littoralis

Various studies have been conducted to assess the damage caused by secondary lepidopteran pests to transgenic Bt maize expressing Cry1Ab. However, to date little is known on the effects of transgenic maize on Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), a polyphagous herbivore which is considered a pest in Mediterranean maize growing areas. Here we present results on the effects of Bt maize (Bt‐11) and Bt spray (Dipel) on the various life stage parameters of this herbivore. We further assess the expression of Cry1Ab in different leaves and leaf parts in maize at a given plant growth stage, and determine whether the feeding damage of 3rd instar S. littoralis is influenced by Bt toxin expression. Contrary to previous literature reporting that S. littoralis is not sensitive to Bt Cry1Ab toxin, our results show that insects fed on either transgenic or Bt sprayed plants were negatively affected. Young S. littoralis larvae (1st and 2nd instars) were found to be the most sensitive to the Bt toxin. This was represented by a higher mortality and a slower developmental time of larvae maintained on transgenic or sprayed plants when compared to insects maintained on control plants. Moreover, Bt maize had a stronger and prolonged detrimental effect on insects when compared to Bt spray in maize. This was revealed by the fact that insects maintained on transgenic plants from 3rd instar to pupation took longer to reach adult emergence compared to insects that were maintained on sprayed plants. This was likely due to the continuous exposure of insects to the toxin when kept on transgenic maize. ELISA results showed a variation in the amount of Bt toxin among different leaf sections in transgenic maize at a given plant growth stage. These differences in Bt toxin were primarily found in the youngest leaf of growing plants. Although the lowest amounts of Bt toxin were detected in the growing leaf section of young leaves, this difference did not appear to influence the feeding behavior of 3rd instar S. littoralis.     

Effects of Bt maize on Frankliniella tenuicornis and exposure of thrips predators to prey-mediated Bt toxin

As a part of a risk assessment procedure, the impact of Bt maize expressing Cry1Ab toxin on the thrips Frankliniella tenuicornis (Uzel) (Thysanoptera: Thripidae) was investigated, and the potential risks for predators feeding on thrips on Bt maize were evaluated. The effects of Bt maize on F. tenuicornis were assessed by measuring life‐table parameters when reared on Bt and non‐Bt maize. The content of Cry1Ab toxin in different stages of F. tenuicornis reared on Bt maize and the persistence of the toxin in adults where determined in order to evaluate the possible exposure of predators when feeding on thrips. In addition, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) was used as a model predator to assess how the behaviour of prey and predator may influence the exposure of a natural enemy to the Bt toxin. Life‐table parameter results showed that F. tenuicornis was not affected when it was reared on Bt maize. This indicates that the potential for prey quality‐mediated effects on predators is low. Bt content was highest in thrips larvae and adults, and negligible in the non‐feeding prepupal and pupal stages. The persistence of the Cry1Ab toxin in adult F. tenuicornis was short, resulting in a decrease of 97% within the first 24 h. Predation success by young C. carnea larvae varied among the thrips stages, indicating that exposure of predators to Bt toxin can additionally depend on the prey stage. When combining the current knowledge of the susceptibility of major thrips predators with our findings showing no potential for prey quality‐mediated effects, relatively low toxin content in thrips as well as short persistence, it can be concluded that the risks for predators when feeding on thrips in or next to Bt maize fields are negligible.     

Effects of Bt-toxin Cry1Ac on Propylaea japonica Thunberg (Col., Coccinellidae) by feeding on Bt-treated Bt-resistant Helicoverpa armigera (Hübner) (Lep., Noctuidae) larvae

Abstract:  Propylaea japonica is an important predatory insect of common cotton pests. To assess the ecological effects of transgenic Bt cotton, expressing Cry1Ac toxin, on this predator, we examined the life history parameters of P. japonica for two generations by feeding them with Bt-resistant Helicoverpa armigera . After ingesting Bt-treated Bt-resistant H. armigera larvae in the third and fourth instar, the body mass and body length of adult P. japonica decreased, a combined effect of poor prey quality and Cry1Ac Bt-toxin may account for these effects. However, larval survivorship and development in these two instars, pupal mortality, fecundity and adult longevity of P. japonica were not affected in both the generations. These results suggest that ingesting Bt-toxin Cry1Ac-treated pests in advanced larval stage might have no significant effect on the fitness of predator P. japonica .     

Prey-mediated effects of Cry1Ab toxin and protoxin and Cry2A protoxin on the predator Chrysoperla carnea

Laboratory feeding experiments were carried out to study prey-mediated effects of artificial diet containing Bacillus thuringiensis proteins on immature Chrysoperla carnea. Activated Cry1Ab toxin and the protoxins of Cry1Ab and Cry2A were mixed into standard meridic diet for Spodoptera littoralis (Boisduval) larvae at the following concentrations; for Cry1Ab toxin, 25, 50, 100 g g^–1 diet were used; for Cry1Ab protoxin, the concentration was doubled (50 g g^–1 diet, 100 g g^–1 diet and 200 g g^–1 diet) to give relative comparable levels of toxin concentration. Cry2A protoxin was incorporated into the meridic diet at one concentration only (100 g g^–1 diet). For the untreated control, the equivalent amount of double distilled water was added to the meridic diet. Individual C. carnea larvae were raised on S. littoralis larvae fed with one of the respective treated meridic diets described above. The objectives were to quantify and compare the resulting effects on mortality and development time of C. carnea with those observed in two previous studies investigating prey-mediated effects of transgenic Cry1Ab toxin-producing corn plants and the other studying effects of Cry1Ab toxin fed directly to C. carnea larvae. Mean total immature mortality for chrysopid larvae reared on B. thuringiensis-fed prey was always significantly higher than in the control (26%). Total immature mortality of C. carnea reared on Cry1Ab toxin 100 g g^–1 diet-fed prey was highest (78%) and declined with decreasing toxin concentration. Cry1Ab protoxin-exposed C. carnea larvae did not exhibit a dose response. Prey-mediated total mortality of Cry1Ab protoxin-exposed chrysopid larvae was intermediate (46–62%) to Cry1Ab toxin exposed (55–78%) and Cry2A protoxin (47%) exposed C. carnea. In agreement with the previous studies, total development time of C. carnea was not consistently, significantly affected by the Bt-treatments except at the highest Cry1Ab toxin concentration. However, both highest mortality and delayed development of immature C. carnea raised on Cry1Ab toxin 100 g g^–1 diet – fed prey may have been confounded with an increased intoxication of S. littoralis larvae that was observed at that concentration. At all other B. thuringiensis protein concentrations S. littoralis was not lethally affected. Comparative analysis of the results of this study with those of the two previous studies revealed that in addition to prey/herbivore by B. thuringiensis interactions, also prey/herbivore by plant interactions exist that contribute to the observed toxicity of B. thuringiensis – fed S. littoralis larvae for C. carnea. These findings demonstrate that tritrophic level studies are necessary to assess the long-term compatibility of insecticidal plants with important natural enemies.     

Prey mediated effects of Bt maize on fitness and digestive physiology of the red spider mite predator Stethorus punctillum Weise (Coleoptera: Coccinellidae)

The present study investigated prey-mediated effects of two maize varieties expressing a truncated Cry1Ab, Compa CB (event Bt176) and DKC7565 (event MON810), on the biology of the ladybird Stethorus punctillum. Although immuno-assays demonstrated the presence of Cry1Ab in both prey and predator collected from commercial maize-growing fields, neither transgenic variety had any negative effects on survival of the predator, nor on the developmental time through to adulthood. Furthermore, no subsequent effects on ladybird fecundity were observed. As a prerequisite to studying the interaction of ladybird proteases with Cry1Ab, proteases were characterised using a range of natural and synthetic substrates with diagnostic inhibitors. These results demonstrated that this predator utilises both serine and cysteine proteases for digestion. In vitro studies demonstrated that T. urticae were not able to process or hydrolyze Cry1Ab, suggesting that the toxin passes through the prey to the third trophic level undegraded, thus presumably retaining its insecticidal properties. In contrast, S. punctillum was able to activate the 130 kDa protoxin into the 65 kDa fragment; a fragment of similar size was also obtained with bovine trypsin, which is known to cleave the protoxin to the active form. Thus, despite a potential hazard to the ladybird of Bt-expressing maize (since the predator was both exposed to, and able to proteolytically cleave the toxin, at least in vitro), no deleterious effects were observed.     

Consumption of Bt maize pollen expressing Cry1Ab or Cry3Bb1 does not harm adult green Lacewings, Chrysoperla carnea (Neuroptera: Chrysopidae)

Adults of the common green lacewing, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae), are prevalent pollen-consumers in maize fields. They are therefore exposed to insecticidal proteins expressed in the pollen of insect-resistant, genetically engineered maize varieties expressing Cry proteins derived from Bacillus thuringiensis (Bt). Laboratory experiments were conducted to evaluate the impact of Cry3Bb1 or Cry1Ab-expressing transgenic maize (MON 88017, Event Bt176) pollen on fitness parameters of adult C. carnea. Adults were fed pollen from Bt maize varieties or their corresponding near isolines together with sucrose solution for 28 days. Survival, pre-oviposition period, fecundity, fertility and dry weight were not different between Bt or non-Bt maize pollen treatments. In order to ensure that adults of C. carnea are not sensitive to the tested toxins independent from the plant background and to add certainty to the hazard assessment, adult C. carnea were fed with artificial diet containing purified Cry3Bb1 or Cry1Ab at about a 10 times higher concentration than in maize pollen. Artificial diet containing Galanthus nivalis agglutinin (GNA) was included as a positive control. No differences were found in any life-table parameter between Cry protein containing diet treatments and control diet. However, the pre-oviposition period, daily and total fecundity and dry weight of C. carnea were significantly negatively affected by GNA-feeding. In both feeding assays, the stability and bioactivity of Cry proteins in the food sources as well as the uptake by C. carnea was confirmed. These results show that adults of C. carnea are not affected by Bt maize pollen and are not sensitive to Cry1Ab and Cry3Bb1 at concentrations exceeding the levels in pollen. Consequently, Bt maize pollen consumption will pose a negligible risk to adult C. carnea.     

Harmonia axyridis (Coleoptera: Coccinellidae) Exhibits No Preference between Bt and Non-Bt Maize Fed Spodoptera frugiperda (Lepidoptera: Noctuidae)

A recent shift in managing insect resistance to genetically engineered (GE) maize consists of mixing non-GE seed with GE seed known as “refuge in a bag”, which increases the likelihood of predators encountering both prey fed Bt and prey fed non-Bt maize. We therefore conducted laboratory choice-test feeding studies to determine if a predator, Harmonia axyridis, shows any preference between prey fed Bt and non-Bt maize leaves. The prey species was Spodoptera frugiperda, which were fed Bt maize (MON-810), expressing the single Cry1Ab protein, or non-Bt maize. The predators were third instar larvae and female adults of H. axyridis. Individual predators were offered Bt and non-Bt fed prey larvae that had fed for 24, 48 or 72 h. Ten and 15 larvae of each prey type were offered to third instar and adult predators, respectively. Observations of arenas were conducted at 1, 2, 3, 6, 15 and 24 h after the start of the experiment to determine the number and type of prey eaten by each individual predator. Prey larvae that fed on non-Bt leaves were significantly larger than larvae fed Bt leaves. Both predator stages had eaten nearly all the prey by the end of the experiment. However, in all combinations of predator stage and prey age, the number of each prey type consumed did not differ significantly. ELISA measurements confirmed the presence of Cry1Ab in leaf tissue (23–33 µg/g dry weight) and S. frugiperda (2.1–2.2 µg/g), while mean concentrations in H. axyridis were very low (0.01–0.2 µg/g). These results confirm the predatory status of H. axyridis on S. frugiperda and that both H. axyridis adults and larvae show no preference between prey types. The lack of preference between Bt-fed and non-Bt-fed prey should act in favor of insect resistance management strategies using mixtures of GE and non-GE maize seed.     

Lack of detrimental effects of Bacillus thuringiensis Cry toxins on the insect predator Chrysoperla carnea: a toxicological, histopathological, and biochemical analysis

The effect of Cry proteins of Bacillus thuringiensis on the green lacewing (Chrysoperla carnea) was studied by using a holistic approach which consisted of independent, complementary experimental strategies. Tritrophic experiments were performed, in which lacewing larvae were fed Helicoverpa armigera larvae reared on Cry1Ac, Cry1Ab, or Cry2Ab toxins. In complementary experiments, a predetermined amount of purified Cry1Ac was directly fed to lacewing larvae. In both experiments no effects on prey utilization or fitness parameters were found. Since binding to the midgut is an indispensable step for toxicity of Cry proteins to known target insects, we hypothesized that specific binding of the Cry1A proteins should be found if the proteins were toxic to the green lacewing. In control experiments, Cry1Ac was detected bound to the midgut epithelium of intoxicated H. armigera larvae, and cell damage was observed. However, no binding or histopathological effects of the toxin were found in tissue sections of lacewing larvae. Similarly, Cry1Ab or Cry1Ac bound in a specific manner to brush border membrane vesicles from Spodoptera exigua but not to similar fractions from green lacewing larvae. The in vivo and in vitro binding results strongly suggest that the lacewing larval midgut lacks specific receptors for Cry1Ab or Cry1Ac. These results agree with those obtained in bioassays, and we concluded that the Cry toxins tested, even at concentrations higher than those expected in real-life situations, do not have a detrimental effect on the green lacewing when they are ingested either directly or through the prey.     

A Sensitive Bioassay for the Detection of Cry1A Toxin Expression in Transgenic Cotton

In the process of development of insect resistant transgenic plants and also to evaluate the consistency in expression of the toxin under greenhouse and field conditions, immunological and bioassays are commonly used. The assay being described in this report, is based on the high levels of sensitivity of a cotton leaf feeding insect, the semilooper, Anomis flava (Fabricius) to Cry toxins (Cry1Aa, Cry1Ab and Cry1Ac). The assay is sensitive, quick and reproducible. Cry1Ac was the most toxic followed by Cry1Ab and Cry1Aa. LC 50 s of the three toxins on first instar larvae ranged from 0.79-6.08 ng cm -2 of leaf. LC 50 s of Cry1Ac for the fourth instar larvae ranged from 12.91-21.14 ng cm -2 while LC 50 s for Cry1Aa and Cry1Ab were in the range 53.0-138 ng cm -2 . The fiducial limits (at 95% probability) of the probit assay data indicated that there was no difference in response between the three different populations to each of the three toxins. The data from all assays were pooled for each of the three toxins separately and subjected to regression analysis to obtain a cumulative log dose response for first and fourth instar larvae. These can be used as standard curves to quantify toxin expression in plants based on mortality response of either first or fourth instar A. flava larvae. Apart from being used to detect expression in putative Bt cotton transgenic plants, the assay can also be used to follow the activity of Cry toxins in transgenic cotton plants in the field during the growing season.     

Toxicity and characterization of cotton expressing Bacillus thuringiensis Cry1Ac and Cry2Ab2 proteins for control of lepidopteran pests

Cry1Ac protoxin (the active insecticidal toxin in both Bollgard and Bollgard II cotton [Gossypium hirsutum L.]), and Cry2Ab2 toxin (the second insecticidal toxin in Bollgard II cotton) were bioassayed against five of the primary lepidopteran pests of cotton by using diet incorporation. Cry1Ac was the most toxic to Heliothis virescens (F.) and Pectinophora gossypiella (Saunders), demonstrated good activity against Helicoverpa zea (Boddie), and had negligible toxicity against Spodoptera exigua (Hübner) and Spodoptera frugiperda (J. E. Smith). Cry2Ab2 was the most toxic to P. gossypiella and least toxic to S. frugiperda. Cry2Ab2 was more toxic to S. exigua and S. frugiperda than Cry1Ac. Of the three insect species most sensitive to both Bacillus thuringiensis (Bt) proteins (including H. zea), P. gossypiella was only three-fold less sensitive to Cry2Ab2 than Cry1Ac, whereas H. virescens was 40-fold less sensitive to Cry2Ab2 compared with CrylAc. Cotton plants expressing Cry1Ac only and both Cry1Ac and Cry2Ab2 proteins were characterized for toxicity against H. zea and S.frugiperda larvae in the laboratory and H. zea larvae in an environmental chamber. In no-choice assays on excised squares from plants of different ages, second instar H. zea larvae were controlled by Cry1Ac/Cry2Ab2 cotton with mortality levels of 90% and greater at 5 d compared with 30-80% mortality for Cry1Ac-only cotton, depending on plant age. Similarly, feeding on leaf discs from Cry1Ac/Cry2Ab2 cotton resulted in mortality of second instars of S.frugiperda ranging from 69 to 93%, whereas exposure to Cry1Ac-only cotton yielded 20-69% mortality, depending on plant age. When cotton blooms were infested in situ in an environmental chamber with neonate H. zea larvae previously fed on synthetic diet for 0, 24, or 48 h, 7-d flower abortion levels for Cry1Ac-only cotton were 15, 41, and 63%, respectively, whereas for Cry1Ac/Cry2Ab2 cotton, flower abortion levels were 0, 0, and 5%, respectively. Cry1Ac and Cry2Ab2 concentrations were measured within various cotton tissues of Cry1Ac-only and Cry1Ac/Cry2Ab2 plants, respectively, by using enzyme-linked immunosorbent assay. Terminal leaves significantly expressed the highest, and large leaves, calyx, and bracts expressed significantly the lowest concentrations of Cry1Ac, respectively. Ovules expressed significantly the highest, and terminal leaves, large leaves, bracts, and calyx expressed significantly (P < 0.05) the lowest concentrations of Cry2Ab2. These results help explain the observed differences between Bollgard and Bollgard II mortality against the primary lepidopteran cotton pests, and they may lead to improved scouting and resistance management practices, and to more effective control of these pests with Bt transgenic crops in the future.     

Using field-evolved resistance to Cry1F maize in a lepidopteran pest to demonstrate no adverse effects of Cry1F on one of its major predators

Spodoptera frugiperda (JE Smith) represents the first documented case of field-evolved resistance to a genetically engineered crop expressing an insecticidal protein from Bacillus thuringiensis (Bt). In this case it was Cry1F-expressing maize (Mycogen 2A517). The ladybird beetle, Coleomegilla maculata, is a common and abundant predator that suppresses pest populations in maize and many other cropping systems. Its larvae and adults are polyphagous, feeding on aphids, thrips, lepidopteran eggs and larvae, as well as plant tissues. Thus, C. maculata may be exposed to Bt proteins expressed in genetically engineered crops by several pathways. Using Cry1F-resistant S. frugiperda larvae as prey, we evaluated the potential impact of Cry1F-expressing maize on several fitness parameters of C. maculata over two generations. Using Cry1F resistant prey removed any potential prey-mediated effects. Duration of larval and pupal stages, adult weight and female fecundity of C. maculata were not different when they were fed resistant S. frugiperda larvae reared on either Bt or control maize leaves during both generations. ELISA and insect-sensitive bioassays showed C. maculata were exposed to bioactive Cry1F protein. The insecticidal protein had no effect on C. maculata larvae, even though larvae contained 20?C32?ng of Cry1F/g by fresh weight. Over all, our results demonstrated that the Cry1F protein did not affect important fitness parameters of one of S. frugiperda??s major predators and that Cry1F protein did not accumulate but was strongly diluted when transferred during trophic interactions.     

Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) larval feeding behavior on transgenic maize (MON 863) and its isoline

Diabrotica species (Coleoptera: Chrysomelidae) larval behavior studies have posed a challenge to researchers because of the subterranean life cycle of this pest. To fully understand how the western corn rootworm, Diabrotica virgifera virgifera LeConte, injures the maize, Zea mays L., root system, its behavior must be studied. For example, larvae that can detect an area of the root that has a lower amount of toxin, whether from an insecticide or a transgenic maize plant, have an increased chance of survival. This study assessed D. v. virgifera larval feeding behavior on rootworm-susceptible maize and maize containing a biotechnology-derived trait (MON 863) with resistance to D. v. virgifera first instar feeding. Maize plants were grown in a medium that allowed for direct observation and measurements during feeding of larval stadia. Neonates were placed on maize seedlings, and data were taken at 3, 6, 9, and 12 d postinfestation on resistant and susceptible maize. On rootworm-susceptible maize, neonate larvae aggregated at the root tips and began actively feeding, and then they moved to older root tissue. Conversely, some larvae that ingested Cry 3Bb1 from the resistant maize exhibited no movement. Other larvae on the resistant maize moved continuously, sampling root hairs or root tissue but not actively feeding. The continuously moving larvae had visibly empty guts, suggesting possible nonpreference for the resistant root. This study contributes to our understanding of D. v. virgifera larval behavior and provides insight into questions surrounding the potential evolution of behavioral and biochemical resistance to Cry3Bb1.     

Effect of Bt maize on the reproduction and development of saprophagous Diptera over multiple generations

A laboratory experiment was used to quantify the effects of Bt maize on Drosophila melanogaster and Megaselia scalaris, representatives of two saprophagous dipteran families (Drosophilidae, Phoridae). Freshly hatched larvae were reared on a diet containing decaying maize leaves. Two transgenic maize varieties, expressing Cry3Bb1 or Cry1Ab, and their corresponding isolines were tested. In an additional treatment, a solution of pure Cry1Ab was added to the maize diet. According to quantitative ELISA analyses, all Bt diets and all larvae feeding on Bt maize contained low concentrations of Cry proteins but Cry proteins were not detected in adults, thus, predators of the larvae are exposed to Cry proteins whereas predators of adult flies are not. Highest concentrations were in larvae feeding on a maize diet supplemented with a Cry1Ab protein solution. The developmental time and fertility (offspring/female) were measured over four generations for D. melanogaster and over three generations for M. scalaris. Only a few significant differences were found between transgenic and non-transgenic treatments but the differences were not consistent and did not indicate any negative effects of Bt proteins. We conclude that D. melanogaster and M. scalaris larvae are not affected in the long term when feeding and developing on decaying Cry1Ab and Cry3Bb1 maize leaves.     

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.     

Effect of Bt broccoli and resistant genotype of Plutella xylostella (Lepidoptera: Plutellidae) on life history and prey acceptance of the predator Coleomegilla maculata (Coleoptera: Coccinellidae)

The ecological implications on biological control of insecticidal transgenic plants, which produce crystal (Cry) proteins derived from the soil bacterium Bacillus thuringiensis (Bt), remains a contentious issue and affects risk assessment decisions. In this study, we used a unique system of resistant insects, Bt plants and a predator to critically evaluate this issue. The effects of broccoli type (normal or expressing Cry1Ac protein) and insect genotype (susceptible or Cry1Ac-resistant) of Plutella xylostella L. (Lepidoptera: Plutellidae) were examined for their effects on the life history of the predator, Coleomegilla maculata DeGeer (Coleoptera: Coccinellidae) over two generations. Additional behavioral studies were conducted on prey choice. C. maculata could not discriminate between Bt-resistant and susceptible genotypes of P. xylostella, nor between Bt and normal broccoli plants with resistant genotypes of P. xylostella feeding on them. The larval and pupal period, adult weight and fecundity of each female were not significantly different when C. maculata larvae fed on different genotypes (Bt-resistant or susceptible) of insect prey larvae reared on Bt or non-Bt broccoli plants. The life-history parameters of the subsequent generation of C. maculata fed on Bt broccoli-reared resistant P. xylostella were also not significantly different from those on non-Bt broccoli. These results indicated that Cry1Ac did not harm the life history or prey acceptance of an important predator after two generations of exposure. Plants expressing Cry1Ac are unlikely to affect this important predator in the field.     

Resistance to Bt maize in Mythimna unipuncta (Lepidoptera: Noctuidae) is mediated by alteration in Cry1Ab protein activation

Bt maize cultivars based on the event MON810 (expressing Cry1Ab) have shown high efficacy for controlling corn borers. However, their efficiency for controlling some secondary lepidopteran pests such as Mythimna unipuncta has been questioned, raising concerns about potential outbreaks and its economic consequences. We have selected a resistant strain (MR) of M. unipuncta, which is capable of completing its life cycle on Bt maize and displays a similar performance when feeding on both Bt and non-Bt maize. The proteolytic activation of the protoxin and the binding of active toxin to brush border membrane vesicles were investigated in the resistant and a control strain. A reduction in the activity of proteolytic enzymes, which correlates with impaired capacity of midgut extracts to activate the Cry1Ab protoxin has been observed in the resistant strain. Moreover, resistance in larvae of the MR strain was reverted when treated with Cry1Ab toxin activated with midgut juice from the control strain. All these data indicate that resistance in the MR strain is mediated by alteration of toxin activation rather than to an increase in the proteolytic degradation of the protein. By contrast, binding assays performed with biotin labelled Cry1Ab suggest that binding to midgut receptors does not play a major role in the resistance to Bt maize. Our results emphasize the risk of development of resistance in field populations of M. unipuncta and the need to consider this secondary pest in ongoing resistance management programs to avoid the likely negative agronomic and environmental consequences.     

Laboratory toxicity studies demonstrate no adverse effects of Cry1Ab and Cry3Bb1 to larvae of <Emphasis Type="Italic">Adalia bipunctata</Emphasis> (Coleoptera: Coccinellidae): the importance of study design

Scientific studies are frequently used to support policy decisions related to transgenic crops. Schmidt et al., Arch Environ Contam Toxicol 56:221–228 (2009) recently reported that Cry1Ab and Cry3Bb were toxic to larvae of Adalia bipunctata in direct feeding studies. This study was quoted, among others, to justify the ban of Bt maize (MON 810) in Germany. The study has subsequently been criticized because of methodological shortcomings that make it questionable whether the observed effects were due to direct toxicity of the two Cry proteins. We therefore conducted tritrophic studies assessing whether an effect of the two proteins on A. bipunctata could be detected under more realistic routes of exposure. Spider mites that had fed on Bt maize (events MON810 and MON88017) were used as carriers to expose young A. bipunctata larvae to high doses of biologically active Cry1Ab and Cry3Bb1. Ingestion of the two Cry proteins by A. bipunctata did not affect larval mortality, weight, or development time. These results were confirmed in a subsequent experiment in which A. bipunctata were directly fed with a sucrose solution containing dissolved purified proteins at concentrations approximately 10 times higher than measured in Bt maize-fed spider mites. Hence, our study does not provide any evidence that larvae of A. bipunctata are sensitive to Cry1Ab and Cry3Bb1 or that Bt maize expressing these proteins would adversely affect this predator. The results suggest that the apparent harmful effects of Cry1Ab and Cry3Bb1 reported by Schmidt et al., Arch Environ Contam Toxicol 56:221–228 (2009) were artifacts of poor study design and procedures. It is thus important that decision-makers evaluate the quality of individual scientific studies and do not view all as equally rigorous and relevant.     

Immunological analysis of phloem sap of Bacillus thuringiensis corn and of the nontarget herbivore Rhopalosiphum padi (Homoptera: Aphididae) for the presence of Cry1Ab

Phloem sap of transgenic Bacillus thuringiensis (Bt) corn expressing a truncated form of the B. thuringiensis delta-endotoxin Cry1Ab, sap sucking aphids feeding on Bt corn and their honeydew were analysed for presence of Cry1Ab using ELISA. Phloem sap of Bt and non-Bt corn was collected using a newly developed technique with a microcapillary being directly inserted into the phloem tubes. Using this technique, no Cry1Ab was detected in the phloem sap. In contrast, measurable concentrations of Cry1Ab in the range of 1 ppb were detected when phloem sap of pooled leaf samples was extracted using EDTA buffer. This was probably because of Cry1Ab toxin released from damaged cells. When analysing apterous adults of Rhopalosiphum padi L. and their honeydew, no Cry1Ab could be detected. In contrast, Cry1Ab was clearly detected in both larvae of the leaf chewing herbivore Spodoptera littoralis (Boisduval) and their faeces, showing that Cry1Ab is detectable after ingestion and excretion by herbivores. These results suggest that R. padi ingests or contains no or only very low concentrations of Cry1Ab in the range of the detection limit. In consequence it is hypothesized that R. padi as an important prey for beneficial insects in corn is unlikely to cause any harm to its antagonists due to mediating Bt toxin.