Oct 8, 2010

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Genetically modified Bt maize and non-target organisms

Mirid bugs not keen on all types of maize

The stacks of Petri dishes appear at first to contain nothing but pieces of maize leaves. It is only on closer inspection that the tiny creatures living inside the plastic dishes become visible. They are mirids in all stages of development. Eva Schultheis of RWTH Aachen University provides the insects with fresh leaf material. Throughout their entire lifecycle the bugs are fed exclusively on leaves from one of four maize varieties, including genetically modified Bt maize. The feeding experiment is designed to show whether Bt maize harms the bugs.

For the feeding experiment, Eva Schultheis caught live mirid bugs of the species Trigonotylus caelestialium (rice leaf bugs) on the maize trial field and brought them to Aachen. “Since we do not know how old the captured insects are and their development has not been recorded, we started the feeding experiment with the first batches of eggs produced by these bugs.” Eva Schultheis carefully opens one of the containers, inserts new maize material and sprays the underside of the lid with water. A sufficient moisture supply is important to ensure that the maize does not dry out.

Four maize varieties are being used in this trial. They are the same ones that are growing on the maize trial field: genetically modified Bt maize, the isogenic variety used to produce the GM maize, and two other conventional varieties. It is expected that various conventional varieties may also differ in the nutritional value they offer insects.

Twenty insects per maize variety are being observed closely and documented in a lifecycle test. When do the insects hatch and how high is the hatching rate? How long do they take to mature into adults? How many eggs do they lay and how many of the insects die? “We observed a particularly high mortality rate during the last sloughing,” says Eva Schultheis, carefully removing a dead insect from the dish with a brush. “We have to check very carefully to assess what the insects died off. There could be mould in the dish, it could be that we have injured an insect by accident - it all has to be precisely documented.”

Dominant species on the field

Trigonotylus caelestialium, the rice leaf bug, was chosen as the model organism in a previous research project. This bug was found to be the dominant species in the bug community on the maize trial field and was very common. “It is also easy to work with. The individual insects can be examined and even weighed,” Eva Schultheis adds. The bug also makes a good model organism because it is ‘exposed’, in other words, it takes in a lot of the Bt protein; also Bt toxin produced in the Bt maize. It feeds on the mesophyll, the internal leaf tissue, of the maize leaves. Laboratory tests on the insects detected as much as a sixth of the Bt protein levels measured in the leaf.

The field trial will compare the incidence and species composition of individual insects in the different maize variants over a total of 40 plots. Nets are used to catch insects like T. caelestialium that live in the herbaceous layer of the maize plants at three different points in time within defined field segments. In addition, for a period of six to eight weeks, one sticky trap is set up on each plot and left on the field for a week at a time. 2010 is the third and final year of the trial.

T. caelestialium came to the scientists’ attention in a previous project after all the insect catches had been evaluated. At certain times of year, seven to ten times as many of these insects were found in one of the conventional varieties than in the other. And the same held true in all three years. However, there was no difference between the Bt maize and the isogenic variety, indicating that some maize varieties do not agree with the bugs as much as others.

The rice leaf bug is very common on the current maize trial field as well. Although evaluation of the current trial is not yet complete, the same clear trend is visible here too. So far, varietal differences have been found, but no influence attributable to the genetically modified Bt maize.

Successful breeding programme

As well as conducting the feeding trial, Eva Schultheis is running a bug-breeding programme. In captivity, the insects are fed only on isogenic maize material. At a constant temperature of 25 °C, 70 % humidity and a day-night rhythm of 16:8 hours, it could be possible to produce generation after generation all year round with no winter rest period. The insects are required for special tests. For instance, the project is currently investigating how the insects feed on the leaf and what traces they leave behind. This could provide insights into what influence the surface structure of a maize leaf has on whether the bugs like it or prefer it to the leaves of other varieties or whether they tend to avoid it. To test how much Bt protein the bugs ingest, they are kept on Bt maize and the level of Bt protein in the insects is measured.

After hatching, the mirid bugs shed their skin several times until they eventually mature into adult insects without pupating. Once they reach sexual maturity they are given wheat seedlings for the females to lay their eggs in, as well as their maize food. Eva Schultheis carefully opens up a wheat seedling with tweezers and finds what she is looking for: three eggs of a somewhat darker green than the seedling lie close together, making them difficult to spot with the naked eye. A batch of eggs can contain up to 25 eggs.

The feeding experiment has been running for two generations, long enough to stake stock of the results so far. As in the field trial, the bugs in this experiment are less keen on one of the conventional varieties than on the other maize lines. Genetically modified Bt maize does not appear to have any influence.