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Effects of Bt maize containing three Bt proteins on arthropods

(2008 – 2011) RWTH Aachen University, Institute of Biology III (Plant Physiology), Worringer Weg 1, 52074 Aachen

Topic

The aim of this project was to investigate the potential effects of the genetically modified Bt maize cultivar MON89034xMON88017 on herbivorous insects – mirids, cicadas and thrips – which come into contact with Bt proteins as a result of their diet.

Herbivorous insects are themselves a food source for predatory insects, which could in turn ingest Bt proteins through their prey. They could also be harmed indirectly due to reduced prey density and/or quality.

This project investigated the following questions:

  • Is the occurrence and density of mirids, cicadas, thrips and their predators in trial plots of genetically modified maize lower than in plots of conventional maize?
  • Is there a difference between the conventional varieties in this respect and if so, how great is it?
  • How much Bt protein is absorbed by herbivorous and predatory insects in fields of genetically modified maize?
  • Once ingested, are the Bt proteins bioavailable and bioactive?

Summary

There was no indication during the three years of the trial that the genetically modified Bt maize had any impact on arthropods in the vegetation layer or their predators. However, some varietal differences were observed.

In the full life cycle test in the laboratory with the mirid bug Trigonotylus caelestialium differences were found for the various development parameters, e.g. hatching rate, length of time to pupation and mortality, between the different maize varieties. However, no clear Bt maize effect was found. There were no differences in the feeding behaviour of the bugs between the different maize variants.

No accumulation of Bt proteins was observed in the bugs. The Bt proteins that the bugs ingested through their food were still active in the biotest with European corn borer larvae

Experiment description

The genetically modified variety, its isogenic parent variety and two further conventional control varieties were grown in a field trial. This method enables researchers to distinguish between potential Bt effects and varietal effects. In addition, the isogenic variety in one part of the plot was treated with an insecticide so that the effect of conventional pest-control methods can also be included in the study.

Insects were shaken out of the male inflorescences when the plants were in flower.

Sticky trap

Sticky traps were set up over a period of six weeks.

A net was used to catch insects in the herbaceous layer.

Determining the density and diversity of herbivorous and predatory insects

Flying insects and insects living in the maize foliage in specific sections of the field were caught with nets on three occasions during each growing season. Sticky boards were set up over a period of six to eight weeks and left on the field for a week at a time. Thrips were collected by shaking the male inflorescences when the maize plants were in full bloom. All the insects caught were identified in the laboratory to species level where possible. Samples were collected over three successive growing seasons to take account of natural population fluctuations and medium-term effects

Determining the exposure of the insects to the Bt proteins

The intake of Bt proteins, the path they take and how they are broken down were investigated using the bug T.caelestialium as a model. This bug is suitable for use as a model organism because it is common and feeds directly on the plant, so it is exposed to the Bt protein.

  • Individual adult insects were caught in Bt maize plots on the trial field and the amount of detectable Bt protein was measured using the ELISA detection method.
  • To check whether Bt proteins accumulate in the bugs or are excreted, the bugs were first kept on Bt maize and then on conventional maize.
  • A breeding programme for mirid bugs was set up using insects from the trial field. Feeding experiments were conducted that covered the entire life cycle of the insects and were repeated for all the maize varieties. Various different parameters were documented, including the hatching rate, length of time to pupation and mortality.
  • In another feeding experiment, the feeding behaviour of the bugs was investigated in relation to the maize variety by calculating the area eaten by a bug each day.

Determining the toxicity and bioavailability of the Bt proteins

Extracts from insects in which Bt protein had been detected were added to a synthetic nutrient medium, which was then fed to European corn borer larvae. Visible impairment of caterpillar development provides an indication of the bioactivity and availability of the Bt proteins.

Results

Determining the density and diversity of herbivorous and predatory insects

The composition of the species community on the trial field was comparable with that on the release site of the previous project. However, the proportions of the various species in the total community differed.

Statistical analyses were carried out for all three years to see how the numbers of individual species were distributed across the different maize varieties and whether there were any significant differences. Varietal differences were found, but there was no indication of any significant impact of the genetically modified Bt maize on arthropods in the vegetation layer or their predators.

  • Thrips: A statistically significant difference was found in the flower-visiting thrips in 2008 between the early-flowering and late-flowering maize varieties in samples taken when the plants were in full bloom. No differences between the maize varieties were found for samples taken at the same time.

  • Beetles: The main beetles found were ladybirds. The main species were the harlequin ladybird (Harmonia axyridis) and the 14-spotted ladybird (Propylea quatuordecimpunctata). The numbers at species level were very low, so it was only possible to carry out a statistical evaluation on a few occasions and only for some species. Taking all the ladybirds together, it was possible to observe a statistically significant difference between the Benicia variety and the other maize varieties at certain times.

  • Leafhoppers: The predominant species at the start of the growing season was Javesella pellucida. Over the course of the growing season, there was a shift towards Zyginidia scutellaris, which was the dominant species overall. When the leafhoppers were observed as a whole, no varietal effects were found. At various sampling times, statistically significant differences were found, particularly between the Benicia variety on the one hand and the Bt variety, the near-isogenic variety and the variety treated with insecticide on the other.

  • Bugs: The rice leaf bug T. caelestialium was the dominant bug species. The numbers of these bugs found in the plots with the Benicia variety stood out. Whereas the adult bugs were usually found in small numbers on plots with this maize variety, the nymphs were consistently more numerous on these plots than on all the plots with other maize varieties.

Determining the exposure of the insects to the Bt proteins

The Bt protein levels measured in the insects from the trial field were comparable with the levels in the insects raised on Bt maize in the breeding programme. An average of 7.91 micrograms per gram was measured.

Comparison of mortality rates during juvenile development in the F1 generation
MON = Bt maize MON89034xMON88017
ISO = near-isogenic line BEN = conventional Benicia variety
DKC = conventional DKC 4250 variety

Comparison of mortality rates during juvenile development in the F2 generation

When bugs were transferred to the near-isogenic variety after spending 24 hours on Bt maize, the Bt proteins could no longer be detected in the insects after six hours. There is evidently no accumulation in the bugs that could then have impacts in the food web.

In the full life cycle test with rice leaf bugs, no statistically significant differences were found in egg laying or the length of time to emergence between the different maize varieties. The hatching rate, length of time to pupation and mortality showed differences between the various maize varieties, but no clear Bt maize effect.

No differences were found in the feeding behaviour studies between the different maize variants.

Determining the toxicity and bioavailability of the Bt proteins

A total of 812 corn borer larvae were tested. Extracts from mirid bugs in which Bt proteins were detected were combined into two collective samples. In the test with the concentrate from the two collective samples, 17 per cent of the larvae did not survive, which indicates that the Bt proteins were still bioactive.