Dec 29, 2008

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Impacts of the cultivation of Diabrotica-resistant Bt maize on the maize ecosystem

(2005 – 2008) RWTH Aachen University, Institute of Environmental Research (Biology V) Chair of Ecology, Ecotoxicology and Ecochemistry

Topic

The aim of the project was to compare the impact of Bt maize, which is resistant to the European corn borer, and conventional maize on various organisms in the maize ecosystem.

Key research points were the effects of the Bt protein (Cry3Bb1) on organisms living in the soil which are involved in breaking down the maize plant and are therefore exposed to the Bt toxin.

Summary

In the field trial a comparison of the feeding activity on the plots with the different maize variants found no differences in any of the three years of the trial. A comparison of the feeding activity on different types of bait found variety-specific differences in some years.

Some differences were observed in the incidence of springtails, predatory mites and wood mites when environmental parameters were taken into account – including between the Bt and isogenic variants. The difference was not noticeable when the environmental data was ignored.

No significant differences in feeding activity between the variants was observed in the mesocosms. In terms of the species spectrum, there were no significant differences in the numbers of wood mites or springtails before the maize litter was added in any of the years. After the addition of maize litter, there were some differences in the numbers of springtails and wood mites in some years. However, these differences were not attributable to the Bt maize.

Impacts on epigeous (above-ground) predatory arthropods: No significant differences between the different maize varieties were observed either in terms of relative numbers of the different ground beetle species or in terms of the composition of the ground beetle communities. Over the three years of observation the species community of ground beetles changed and a composition typical of maize fields became established. The Bt protein was detected in the ground beetles. The level of Bt protein was significantly higher after the maize-flowering period. The level of Bt protein decreased by at least a factor of 20 along the food chain (maize - European corn borer - ground beetle).

Bioassay: The bioassays did not find any acute Bt effect on ground beetles.

Experiment description

A total of 32 plots were arranged on the trial field. Eight plots were planted with Bt maize (MON88017), eight with an isogenic line and eight each with two other conventional varieties. The plots with the different varieties are distributed at random, but in compliance with two conditions: no two adjoining plots contain the same variety and there must be the same number of plots of each variety at the edge of the field and in the middle. Bt maize was grown on the same plots in all years so that any accumulation effects in the soil could be observed.

Effects of Bt maize cultivation on soil-dwelling organisms

Maize litter is decomposed directly by wood mites and earthworms among other organisms. These primary decomposers are in turn eaten by other organisms like springtails and mites.

The decomposition process is studied both in the field and in mesocosms.

Field

Feeding activity: To determine the feeding activity of the soil fauna, bait strip tests are carried out after the harvest. A bait substance is inserted into the bait strips and placed in the soil. Two variants are used:

• Feeding activity on one bait type: 16 bait strips (each with 16 holes) are placed in each plot of each maize variety. The strips are baited with isogenic maize and cellulose.
• Feeding activity on different bait types: 16 bait strips containing the different maize variants (transgenic, isogenic and two other conventional varieties, each with cellulose) are placed in each of the isogenic maize plots.

Species spectrum: The species spectrum of the soil fauna in the field is determined by extracting ‘soil cores’ (diameter 5 cm, depth 5 cm). Five soil cores are taken from each plot of the trial field (=160 samples).

Mesocosms

In line with the field experiments, in 2005 and 2006 a comparative study was made of the feeding activity of the soil fauna in mesocosms. Each year in August 32 soil samples (diameter 30 cm, height 40 cm) were taken from a field close to the trial field. In the experiment, 76 grams of chopped maize litter of each of the different varieties were mixed into the top ten centimetres. Eight bait strips (bait substance: isogenic maize litter + cellulose) were placed in each mesocosm. Soil samples were examined to assess the species spectrum and abundance six times between September and May of the following year.

Impacts on epigeous (above-ground) predatory arthropods

Ground beetles (Carabidae) are important predators in the species community of a maize field. To measure their the activity density in Bt maize compared with the conventional varieties, a pit trap was placed in each plot. The traps were emptied once a week (11 to 21 times). The samples were sorted into animal groups and identified down to species level.

ELISA: At the same time, live traps were set in order to study the ground beetles for Cry3Bb1 using the ELISA method. The ground beetles could have ingested the Bt protein via their prey (e.g. springtails, mites, aphids), or from plant food sources such as maize litter.

On three dates in each summer ground beetles that had been caught alive were frozen at -50 degrees Celsius. These beetles were later tested for their Cry3Bb1 content.

Biotest: In complementary biotests in the laboratory, ground beetles were fed larvae from the European corn borer. The ground beetles were given the choice between corn borers that had previously been fed on Bt maize and corn borers that had been fed on isogenic maize (choice trials). In additional feeding experiments, the four most common species (Calathus fuscipes, Pseudophonus rufipes, Calathus ambiguus and Pterostichus melanarius) were fed with the Bt protein, either directly or indirectly.

Results

Impacts of Bt maize cultivation on soil-dwelling organisms

Field trial

Feeding activity: A comparison of the feeding activity on one type of bait on the plots with the different maize variants found no differences in any of the three years of the trial (see fig. 1).

Whereas there were several differences in feeding activity on the different bait types in 2005 and 2006, the bait strip experiment conducted during the 2007 maize-flowering period found no significant differences (see fig. 1).

Species spectrum: The feeding activity during the trial correlated with the incidence of wood mites (Oribatida). However, it was not possible to establish any correlation between feeding activity and the incidence of springtails (Collembola). No significant differences were found at first between the different maize varieties for the wood mites or the springtails. Significant differences were found for one variety for the predatory mites.

Taking environmental (soil) parameters into account, however, significant differences were found in some years between Bt maize and the isogenic variety, as well as other varietal differences in the incidence of wood mites, springtails and predatory mites.

Mesocosms

Feeding activity: No significant differences in feeding activity were found in either year between the different maize varieties.

Species spectrum: In 2005 the incidence of wood mites and springtails did not differ significantly between the varieties before the maize litter was added to the mesocosms. After the maize litter was added, the incidence of springtails in the isogenic variety was significantly different from all the other varieties in the trial, including the Bt maize. By contrast, the incidence of wood mites was significantly different in the Bt maize from all the conventional varieties.

Impacts on epigeous (above-ground) predatory arthropods

The dominant species among the ground beetles was the Calathus fuscipes, but the Pseudophonus rufipes and Pterostichus melanarius were also common. In 2005 a total of 3660 ground beetles were found from 33 different species. More than twice as many ground beetles (8575) were found and identified in 2006. It is assumed that a ground beetle community typical of a maize field became established in the trial field over this time. A comparison of the average numbers of ground beetles per variety and year revealed no significant differences between the different maize varieties in 2005 and 2006.

ELISA: Averaged over the three years, Bt protein was found in 33 per cent of ground beetles collected on the Bt plots before flowering, and in 69 per cent of the ground beetles found on these plots after flowering.

In the conventional maize plots, the Cry3Bb1 protein was found in 15 per cent of the beetles captured before flowering, and in 39 per cent of the beetles caught after flowering. The findings of Bt protein in ground beetles from the conventional maize plots can be attributed to movement between the Bt plots and the conventional plots.

In the ground beetles from the conventional plots that tested positive for Cry3Bb1, an average of 12 ng of Cry protein per gram of beetle was found before harvesting. After flowering, the average level found was 66 ng/g. In the ground beetles from the Bt plots, the average level before flowering was 26 ng/g. After flowering, the average level of Cry3Bb1 was 300 ng/g.

The food chain analysis found an average of 7 micrograms of Bt protein per gram in the fresh Bt maize leaves, 2 micrograms per gram in the European corn borer larvae, and 0.2 micrograms per gram in the ground beetles.

Bioassay: The bioassays found no acute effect of Bt protein on ground beetles.