Jul 19, 2010

Research Projects

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Effects of Bt maize containing three Bt proteins on ground beetles and spiders

(2008 – 2011) Bavarian State Research Center for Agriculture, Institute for Plant Protection (IPS 2d), Lange Point 10, 85354 Freising

Topic

The aim of this project is to investigate the potential effects of the genetically modified Bt maize cultivar MON89034xMON88017 on ground beetles and spiders found in maize fields. This maize produces three Bt proteins, making it resistant to both the European corn borer and the Western corn rootworm.

Ground beetles and spiders are key organisms in the maize field ecosystem. They are exposed to Bt proteins both directly through ingesting maize litter and maize pollen, and indirectly through eating prey which feeds on the maize plants (e.g. the European corn borer).

This project aims to examine the following questions:

• Does the cultivation of MON89034xMON88017 maize cause significant changes in the incidence and density of ground beetles and spiders?
• Is it possible to determine statistically a typical species pattern for ground beetles and spiders in the maize field, which will then serve as the basis of assessment for the long-term monitoring of genetically modified maize varieties?
• Are Bt proteins transported to other locations, and therefore to other food chains outside the field?
• To what extent do Bt proteins remain biologically active and potentially toxic in the insects’ gastro-intestinal tract?

Experiment description

Field and laboratory experiments will be used to investigate the questions listed above. Not only will the study compare the genetically modified variety with its isogenic parent variety, it will also include two further conventional maize varieties so that a distinction can be made between maize varietal effects and Bt effects. In addition, the isogenic variety will be treated with a soil insecticide.

Field investigations

Soil traps: Soil traps will be set up in the various trial field plots to record the species pattern and the density of individual species. These will be emptied on a weekly basis from sowing until harvesting. The trapped insects will be identified to species level. Other maize fields which differ from the trial field e.g. in terms of soil quality or crop sequence will also be included in the investigation.

Radius of action of ground beetles: Some of the ground beetles caught in the trial field will be individually marked and then released. The recapture of individual ground beetles in soil traps even outside the trial field will enable researchers to estimate the routes they take and their range. This should explain how far ground beetles exposed to Bt protein absorbed through their food can spread beyond the field.

Laboratory investigations

The contents of the digestive tract of different ground beetle species will be examined in the laboratory at various periods during the growing season. The amount of Bt protein will be measured using marked antibodies and the Bt proteins will also be examined to find out how intact they have remained. To determine the potential residual toxicity of the digested Bt proteins, bioassays will be conducted which will involve feeding these digested proteins to European corn borers and Colorado potato beetles.

The ground beetles may be harmed not only directly through absorbing Bt proteins, but also indirectly through their prey. To investigate this, ground beetles will be offered European corn borer larvae that have been fed on the genetically modified or isogenic variety. In further bioassays, the rate of reproduction of ground beetles will be measured following exposure to Bt proteins.

Results

Field investigations

Soil traps: A total of 80 pit traps (two in each maize plot) were emptied weekly between June and September in 2008 and 2009.

In 2008, a total of 27,365 ground beetles and 3,405 spiders were counted on the 12 sampling dates. The abundance of ground beetles and spiders did not differ significantly between the Bt maize plots and the conventional maize plots. By contrast, on a few sampling dates there were clear differences between the Bt maize variant and the plots with soil insecticide and between individual conventional varieties.

The most common ground beetle (30%) was Pterostichus melanarius, followed by Calathus melanocephalus (27%), Trechus quadristriatus (20%) and Calathus fuscipes (9%).

Laboratory investigations

ELISA-analyses: Ground beetles were caught alive on the trial field on two dates – before and after the start of the maize-flowering period – with the help of pit traps without trap fluid, and were frozen at -50 ºC to be preserved for protein analysis at a later date. 150 beetles caught before the start of the maize-flowering period were examined. The Bt protein Cry3Bb1 was found in 29 (45.3 %) of the 64 beetles tested from the Bt maize plots. The average protein concentration was 51.7 nanograms per gram fresh weight (ng/g). Cry3Bb1 was found in 15 (17.4 %) of the 86 beetles from the conventional maize plots. Here, the average protein concentration was 26.3 ng/g.

Significantly more beetles were found containing the Bt protein Cry3Bb1 among the 154 ground beetles caught after the start of the maize-flowering period: 72.3 per cent of the beetles from the Bt maize plots and 43.3 per cent of the beetles from the plots with conventional maize. However, the concentrations were similar: 53.4 ng/g and 34.10 ng/g respectively. These concentrations are much lower than the amount of protein needed to harm the target organism – the Western corn rootworm in the case of Cry3Bb1.

Further tests with the Bt proteins Cry1A.105 and Cry2Ab2 are not yet complete/have not yet been conducted.

Feeding trials along the food chain maize > European corn borer > ground beetle have been carried out and are currently being evaluated.

Feeding trials with Bt proteins: A breeding programme for ground beetles was set up successfully using Poecilus cupreus. Over 600 beetle larvae were tested in six types of trial, each with at least ten repetitions. The concentrations of the Bt proteins were based on the maximum Bt protein level in the Bt maize plants. A preliminary evaluation showed no negative impacts on the pupation rate, hatching rate, development time or hatching weight of the beetles. Fertility levels are currently being tested in order to cover the full life cycle (full life cycle test).