Jul 26, 2010

Research Projects

Send

Effects of Bt maize containing three Bt proteins on nematodes

(2008 – 2011) Institute for Biodiversity Network (ibn), Drei-Kronen-Gasse 2, 93047 Regensburg

Topic

The aim of this project is to investigate the effects of the genetically modified Bt maize cultivar MON89034xMON88017, which produces three different Bt proteins, on nematodes (roundworms).

Nematodes are the richest group of organisms present in the soil both in terms of number of species and individuals and are found in virtually all habitats. By specialising in a range of different feeding habits, they occupy key sites in food webs and play a vital role in the soil nutrient cycle. This makes nematodes particularly good bio-indicators for assessing soil quality.

Bt proteins enter the soil via root exudates, plant residues and excretions from herbivorous insects. An accumulation of Bt proteins in the soil could be harmful to soil-dwelling organisms. Certain classes of Bt proteins are known to have a toxic effect on nematodes; as with insects, these proteins damage the nematodes’ intestinal wall.

This project will examine the following questions:

• Does the cultivation of Bt maize change the structure of nematode communities in the soil?
• Do nematodes with different feeding habits differ in terms of their absorption of Bt proteins?
• Does the absorption of Bt proteins from the Bt maize under investigation reduce the rate of reproduction in the roundworm Caenorhabditis elegans?

Experiment description

The genetically modified variety, its isogenic parent variety and two further conventional control varieties will be 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 will be treated with an insecticide so that the effect of conventional pest control methods can also be included in the study.

Structure of nematode communities in the soil

The effect of Bt proteins on the structure of nematode communities will be investigated in the field and in the laboratory. During the first and second crop years, several root soil samples will be taken from each plot on the trial field at three different times.

In addition, during the second crop year microcosms (artificial ecosystems) will be set up in the laboratory. Small containers will be filled with soil from the trial field and with the associated native soil fauna which has been identified during the first year. Pure Bt proteins or varying quantities of plant material from Bt maize will be added to the microcosms. By way of comparison, some microcosms will be treated with an insecticide in various concentrations. Microcosms with plant material from the isogenic maize variety and untreated microcosms will serve as a control. Soil samples will be taken on four occasions.

Nematodes isolated from the field and laboratory samples will be identified to genus level. The genera will be subdivided into feeding types based on their mouthparts and classified according to their reproductive strategies.

Absorption of Bt proteins in nematodes with different feeding habits

Nematodes with different feeding habits will be kept for short periods in cultures containing fluorescently marked Bt proteins. They will then be cleaned, place on a specimen slide and observed under a fluorescent microscope. These investigations will provide insights into whether and to what extent Bt proteins are absorbed in the worms’ intestines and whether they bind to the intestinal wall. Different controls will be carried out, for example using cultures containing pure fluorescent dye.

Feeding trials with Caenorhabditis elegans

Nematodes belonging to the species Caenorhabditis elegans will be exposed to various materials containing Bt protein (pure Bt proteins, plant material from Bt maize, soil samples from the Bt maize plots and transgenic bacteria that produce Bt proteins). As usual, the same medium containing no toxic material or containing soil and plant material from the trial plots with the isogenic maize variety will be used as a control.

After 96 hours the worms’ growth and reproduction will be determined. After just 24 hours some of the worms will be removed, chemically treated and frozen. These samples will be used to investigate the expression of specific genes which are known to react to certain cry proteins that are toxic to nematodes.

Results

Incidence of nematodes in the field: Six samples were taken from the top 20 cm of the rhizosphere from each of the 40 plots on the trial field shortly after sowing, during the maize growth period and after harvesting, and combined into mixed samples.

A very large number of nematodes were found: between five and 27 million per square metre in 2008 and between eight and 28 million per square metre in 2009. The incidence of nematodes fluctuated slightly on all plots over the course of the study. On most of the sampling dates there were no significant differences between the maize varieties. A significant difference was found between the number of nematodes on the Bt maize plots and on one of the conventional plots only on the last sampling date (October 2009).

Composition of nematode communities in the field: Once the number of nematodes had been assessed, 50 were taken from each sample and dissected so that they could be identified. The nematodes were classified according to food type (e.g. those that feed on plants and those that feed on bacteria) and according to reproductive strategy.

The first project year has been evaluated (40 plots, 3 sampling dates = 120 plot samples). Although a change could be observed in the composition of the different food types in all plots over the course of the growing season, with one exception, there were no significant differences between the different maize varieties. In terms of reproductive strategy too, with one exception, no differences were observed between the different maize varieties.

However, a correlation was found with the location of the plots within the trial field. There were differences between the nematode communities on the five plot rows on all three sampling dates, which indicates a soil condition effect. The soil analysis conducted by one of the project group partners shows that the proportion of sand declines from row A to row E, while the proportion of silt and clay increases. Correlation analyses showed a significant correlation between the number and composition of species in the nematode communities and the particle size distribution in the soil.

Toxicity of the Bt proteins (Cry1A.105, Cry2Ab2, Cry3Bb1)

Solutions of the neat Bt proteins were tested for their toxic effect on C.elegans nematodes individually in aqueous solution and in equimolar (1:1) mixtures. Clear dose-effect curves were calculated for all three proteins and protein mixtures. Cry3Bb1 displayed the highest toxicity, followed by Cry1A.105 and Cry2Ab2. The mixture was less toxic than expected.