Michael Meissle

Transgenic crops expressing Bacillus thuringiensis toxins and biological control

The area devoted to growing transgenic plants expressing insecticidal Cry proteins derived from Bacillus thuringiensis (Bt) is increasing worldwide. A major concern with the adoption of Bt crops is their potential impact on nontarget organisms including biological control organisms. Regulatory frameworks should advocate a step-wise (tiered) approach to assess possible nontarget effects of Bt crops. Laboratory and glasshouse studies have revealed effects on natural enemies only when Bt-susceptible, sublethally damaged herbivores were used as prey or host, with no indication of direct toxic effects. Field studies have confirmed that the abundance and activity of parasitoids and predators are similar in Bt and non-Bt crops. In contrast, applications of conventional insecticides have usually resulted in negative impacts on biological control organisms. Because Bt-transgenic varieties can lead to substantial reductions in insecticide use in some crops, they can contribute to integrated pest management systems with a strong biological control component.

Effects of Bt maize on the herbivore Spodoptera littoralis (Lepidoptera: Noctuidae) and the parasitoid Cotesia marginiventris (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.

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.

Effects of Bt maize-fed prey on the generalist predator Poecilus cupreus L. (Coleoptera: Carabidae).

We investigated the effects of transgenic maize (Zea mays) expressing Bacillus thuringienses toxin (Bt maize) on larval and adult Poecilus cupreus carabid beetles in laboratory studies. In no-choice trials, neonate P. cupreus larvae were fed exclusively with Spodoptera littoralis caterpillars, which had been raised on Bt maize. S. littoralis raised on conventional maize or “high quality” Calliphora sp. pupae were fed to the beetle larvae in two control treatments. Bt-maize-fed caterpillar prey increased mortality to 100 within 40 days. The experiment was repeated with 10-day-old beetle larvae. Bt treatment resulted in fewer pupae than in both controls, and in a higher mortality than in the Calliphora control. S. littoralis was suitable as exclusive prey in no-choice tests, at least for 40 days, although prey quality seemed to be low compared to Calliphora pupae. The observed effects are most likely indirect effects due to further reduced nutritional prey quality. However, direct effects cannot be excluded. In the second part of the study, exposure of P. cupreus to Bt intoxicated prey was examined in paired-choice tests. Adult beetles were offered a choice between different prey conditions (frozen and thawed, freshly killed or living), prey types (S. littoralis caterpillars, Calliphorasp. pupae, cereal aphids) and prey treatments (raised on Bt or conventional maize). Living prey was preferred to frozen and dead prey. Caterpillars were only preferred to fly pupae and aphids when living. Prey treatment seemed to be least important for prey selection. The tests showed that P. cupreus ingested caterpillars readily and there was no evidence of them avoiding Bt containing prey, which means exposure in the field could occur. The presented protocols are a first step towards ecological risk assessment for carabid beetles.

Bt maize and integrated pest management - a European perspective

The European corn borer (Ostrinia nubilalis), the Mediterranean corn borer (Sesamia nonagrioides) and the western corn rootworm (Diabrotica virgifera virgifera) are the main arthropod pests in European maize production. Practised pest control includes chemical control, biological control and cultural control such as ploughing and crop rotation. A pest control option that is available since 1996 is maize varieties that are genetically engineered (GE) to produce insecticidal compounds. GE maize varieties available today express one or several genes from Bacillus thuringiensis (Bt) that target corn borers or corn rootworms. Incentives to growing Bt maize are simplified farm operations, high pest control efficiency, improved grain quality and ecological benefits. Limitations include the risk of resistance evolution in target pest populations, risk of secondary pest outbreaks and increased administration to comply with licence agreements. Growers willing to plant Bt maize in the European Union (EU) often face the problem that authorisation is denied. Only one Bt maize transformation event (MON810) is currently authorised for commercial cultivation, and some national authorities have banned cultivation. Spain is the only EU member state where Bt maize adoption levels are currently delivering farm income gains near full potential levels. In an integrated pest management (IPM) context, Bt maize can be regarded as a preventive (host plant resistance) or a responsive pest control measure. In any case, Bt maize is a highly specific tool that efficiently controls the main pests and allows combination with other preventive or responsive measures to solve other agricultural problems including those with secondary pests.

Insecticidal activity of Cry3Bb1 expressed in Bt maize on larvae of the Colorado potato beetle, Leptinotarsa decemlineata

Environmental risk assessment for genetically modified crops producing insecticidal Cry proteins derived from Bacillus thuringiensis (Bt) includes the evaluation of adverse effects on non‐target organisms. Although ELISA concentration measurements indicate the presence of Cry proteins, sensitive insect bioassays determine whether there is biological activity. The insecticidal activity of the coleopteran‐active Cry3Bb1 expressed in different tissues of Bt maize, contained in maize‐fed herbivores, and in spiked soil was measured in sensitive insect bioassays using larvae of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). Biological activity was confirmed of Cry3Bb1 contained in pulverized Bt maize pollen, roots, leaves, silk, and Bt maize‐fed spider mites and western corn rootworm adults. When test substances were incorporated into artificial diet at the same concentrations of Cry3Bb1 (measured by ELISA), maize pollen and leaf litter exhibited lower toxicity than fresh plant material and maize‐fed arthropods. This suggests that nutritional quality of food and degradation of Cry proteins may influence toxicity to insects. When soil was spiked with Cry3Bb1, the Bt protein was highly adsorbed and retained its full biological activity. Because toxicity of Cry proteins contained in different matrices cannot always be determined from ELISA values alone, sensitive insect bioassays can improve hazard and exposure assessments in environmental risk assessment of Bt crops.

Impact of Insect-resistant Transgenic Crops on Above-ground Non-target Arthropods

Genetically modified (GM) maize and cotton varieties that express insecticidal proteins derived from Bacillus thuringiensis (Bt) have become an important component in integrated pest management programmes worldwide. A number of other crops producing Bt toxins, or more broad-spectrum insecticidal proteins, are likely to enter commercial production in the near future. Because insecticidal GM crops target insect pests, an important part of the environmental risk assessment is their potential impact on nontarget arthropods. Those include protected species and organisms providing important ecological services such as biological control of herbivores. Non-target arthropods can be exposed to the plant-produced insecticidal proteins through various routes, but mainly by feeding on GM plant material or herbivores that have consumed GM plant material. The Bt proteins produced in todays GM plants appear to have no direct effects on natural enemies due to their narrow spectrum of activity. Furthermore, it has become clear that in crop systems where the deployment of Bt varieties has led to a decline in insecticide use, biological control organisms have benefited significantly. Future GM plants that produce broader-spectrum insecticidal proteins will need to be assessed for their potential non-target effects case by case and compared to the impact of the conventional pest control methods that they replace.

Use of maize pollen by adult Chrysoperla carnea (Neuroptera: Chrysopidae) and fate of Cry proteins in Bt-transgenic varieties.

We investigated the use of maize pollen as food by adult Chrysoperla carnea under laboratory and field conditions. Exposure of the insects to insecticidal Cry proteins from Bacillus thuringiensis (Bt) contained in pollen of transgenic maize was also assessed. Female C. carnea were most abundant in a maize field when the majority of plants were flowering and fresh pollen was abundant. Field-collected females contained an average of approximately 5000 maize pollen grains in their gut at the peak of pollen shedding. Comparable numbers were found in females fed ad libitum maize pollen in the laboratory. Maize pollen is readily used by C. carnea adults. When provided with a carbohydrate source, it allowed the insects to reach their full reproductive potential. Maize pollen was digested mainly in the insects mid- and hindgut. When Bt maize pollen passed though the gut of C. carnea, 61% of Cry1Ab (event Bt176) and 79% of Cry3Bb1 (event MON 88017) was digested. The results demonstrate that maize pollen is a suitable food source for C. carnea. Even though the pollen grains are not fully digested, the insects are exposed to transgenic insecticidal proteins that are contained in the pollen.

Potential use of an arthropod database to support the non-target risk assessment and monitoring of transgenic plants

Abstract Worldwide, plants obtained through genetic modification are subject to a risk analysis and regulatory approval before they can enter the market. An area of concern addressed in environmental risk assessments is the potential of genetically modified (GM) plants to adversely affect non-target arthropods and the valued ecosystem services they provide. Environmental risk assessments are conducted case-by-case for each GM plant taking into account the plant species, its trait(s), the receiving environments into which the GM plant is to be released and its intended uses, and the combination of these characteristics. To facilitate the non-target risk assessment of GM plants, information on arthropods found in relevant agro-ecosystems in Europe has been compiled in a publicly available database of bio-ecological information during a project commissioned by the European Food Safety Authority (EFSA). Using different hypothetical GM maize case studies, we demonstrate how the information contained in the database can assist in identifying valued species that may be at risk and in selecting suitable species for laboratory testing, higher-tier studies, as well as post-market environmental monitoring.

Impact of Cry3Bb1-expressing Bt maize on adults of the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)

BACKGROUND Genetically engineered maize producing insecticidal Cry3Bb1 protein from Bacillus thuringiensis (Bt) is protected from root damage by corn rootworm larvae. An examination was made to establish whether western corn rootworm (Diabrotica virgifera virgifera) adults are affected by Cry3Bb1-expressing maize (MON88017) when feeding on above-ground tissue. RESULTS In laboratory bioassays, adult D. v. virgifera were fed for 7 weeks with silk, leaves or pollen from Bt maize or the corresponding near-isoline. Male, but not female, survival was reduced in the Bt-leaf treatment compared with the control. Female weight was lower when fed Bt maize, and egg production was reduced in the Bt-silk treatment. ELISA measurements demonstrated that beetles feeding on silk were exposed to higher Cry3Bb1 concentrations than beetles collected from Bt-maize fields in the United States. In contrast to silk and pollen, feeding on leaves resulted in high mortality and low fecundity. Females feeding on pollen produced more eggs than on silk. C:N ratios indicated that silk does not provide enough nitrogen for optimal egg production. CONCLUSIONS Direct effects of Cry3Bb1 on adult beetles could explain the observed effects, but varietal differences between Bt and control maize are also possible. The impact of Bt maize on adult populations, however, is likely to be limited.