Dec 8, 2010

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Genetically modified Bt maize and non-target organisms

Nematodes: An indicator of soil quality

There are micrograph enlargements of nematodes, or roundworms, on nearly all the walls of the Institute for Animal Ecology at the University of Bielefeld. The pictures show elegant worms with clearly recognisable mouthparts and sexual and digestive organs. The staff here seem to have a special affinity for these tiny soil-dwellers. Their fascination with the creatures is almost tangible. Sebastian Höss has been studying nematodes for years and knows how beneficial they are for soil fertility. This is why they make such good bioindicators for testing the effect of toxic substances. At the moment, Sebastian Höss is conducting research on behalf of the Institute for Biodiversity in Regensburg, on whether nematodes are sensitive to genetically modified Bt maize.

Nematodes are found almost everywhere in the soil, in rivers and lakes, and even in the sea. They are the most common of the multicellular animals and have the greatest number of species. Not all of them are free living and beneficial. There is also a significant number of parasitic species, found both in plants and in animals, including humans.

Sebastian Höss is studying nematodes that live in agricultural soil. “In the top 20 cm of one square metre of ground you can find up to 40 million nematodes. To illustrate, this means that in a handful of earth there are around 1000 nematodes and a dozen species,” he says. They live in a liquid film between the soil particles and feed on bacteria, fungi and plant remains, or prey on other tiny creatures. “They have different mouthpieces depending on what they feed on – some have a spike,” says Sebastian Höss, “and some, like this one, even have teeth” he adds, pointing to a giant picture of a worm with a kind of mouth at one end.

Caenorhabditis elegans – a model organism

Sebastian Höss gives a whole list of reasons why he believes it makes sense to take a closer look at nematodes in conjunction with genetically modified Bt maize. One of the main reasons is that nematodes occupy an important position in the soil food web. “The ones that eat fungi and bacteria in particular stimulate microbial degradation in the soil,” he explains. Secondly, nematodes in the soil are exposed to the Bt proteins produced by Bt maize and are therefore potentially at risk. Bt proteins enter the soil primarily through rotting plant remains. The Bt maize being studied in this project (MON89034 x MON88017) contains three different Bt proteins.

Nematodes have also been well researched. They have long been used as bioindicators for toxic chemicals. Studying nematodes gives researchers an idea of soil quality. The bacteria-eating nematode Caenorhabditis elegans is a particular favourite for this type of research. There are already standard methods for determining the toxicity of chemicals in the soil using this model organism (ISO 10872).

Sebastian Höss carried out one of these tests for all three proteins, both individually and in combination. Caenorhabditis elegans was exposed to the pure Bt proteins in an aqueous solution. The nematodes responded to all three proteins with a significantly reduced breeding rate, although only at concentrations far above those found in the soil of a Bt maize field of this type by another team of researchers working in the same project group. When the three proteins were combined, the toxic effect was not as high as expected.

Sebastian Höss would like to find out why it is that nematodes are sensitive to these Bt proteins even though they are not the target organism. The insecticidal Bt proteins produced by Bt maize target certain maize pests very specifically. Sebastian Höss goes into a bit more detail: “It is known that there are some Bt or Cry proteins that are specifically effective against nematodes. These are nematicidal Cry proteins. They harm the nematodes via a similar mechanism to that used for example by the Cry proteins in Bt maize to harm the European corn borer.” Sebastian Höss therefore wants to mark the proteins with a fluorescent dye so that he can follow them in the worms’ intestines. Bt proteins should work only if there are special receptors on the intestinal wall for them to bind to. If they do this in the nematode intestines, it will become apparent in an increase in fluorescence along the intestinal wall.

Extracting nematodes

Today, at the institute at the University of Bielefeld, nematodes are being extracted from soil samples taken from the maize trial field. Over the past three years, several conventional varieties and the genetically modified Bt maize line MON89034 x MON88017 have been grown here on a total of 40 plots. “The field trials are about investigating whether the nematode communities differ between the different plots, both in terms of numbers and in terms of species composition.” A member of the project team demonstrates on a patch of grass on the institute’s premises how to lift a small strip of earth using a sampler measuring just 2 cm in diameter. One of these was used on three different dates – shortly after sowing, during the maize-flowering period and after harvest – to take soil samples from the trial field. Only the top 20 cm of soil were removed, from six different sites on each plot. The individual samples from a plot were combined into composite samples and mixed carefully. A quantity of between 15 and 20 cubic centimetres from each composite sample was analysed in the laboratory.

The samples are first fixed with formalin, which kills the animals, but preserves their tissue. To make it easier to count them under the binocular microscope later on, rose bengal dye is added. The nematodes form part of the organic substance in the soil. So, in the next step, density separation is used to separate the mineral soil constituents from the organic ones. Then the arduous counting process under the binocular microscope can begin. Some soil samples contain more than 2000 nematodes. In 2008, a figure of between 5 and 27 million nematodes per square metre was extrapolated for the maize trial field – a huge number. In 2009 the figure was even higher, at between 8 and 28 million.

Colonizers and persisters

50 nematodes are fished out of every sample. A special tool was invented for this purpose with an eyelash on one end. The 50 nematodes are then examined more closely under the microscope at 1000 times magnification. Where possible, they are identified down to genus level and categorised according to food type and reproductive strategy.

There are some species of nematode that reproduce quickly and in large numbers. These are called ‘colonizers’ and are regarded as being tolerant of disturbance. The ‘persisters’ are very different. They have a longer generation time and are better adapted to stable sites, but do not cope as well with disturbance. The nematode species are assigned a value on a colonizer-persister scale of one to five, so that their maturity index (MI), a special stress index, can be calculated. A toxic effect caused by Bt proteins in the soil could represent a disturbance that would be detected in a lower MI value.

The evaluations of the soil samples for 2008 and 2009 show that the main types of nematode in all plots on the maize trial field were bacteria- and plant-eating nematodes. Over the course of the growing season, the proportion of bacteria eaters fell and the proportion of predatory nematodes increased. In the spring, after the soil had been tilled, the colonizers were dominant. Later in the year, the persisters became more numerous, which led to an increase in the MI value. It was clear, therefore, that the nematode communities alter their composition over the course of the growing season.

Unlike in a previous project with Bt maize MON88017, the different maize varieties did not have any impact on the nematode communities on any of the three sampling dates. They were not affected by the Bt proteins or by the insecticide treatment. There were, however, differences in the communities that can be attributed to differences in sand content in the soil on the plots.