Feb 1, 2008

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Testing a nematode biotest

(2005 – 2008) Institute for Biodiversity - Experts Network, Regensburg

Topic

In this project, a biotest using the nematode Caenorhabditis elegans was investigated for detecting Bt-toxins. The test was developed using the transgenic maize strain MON88017, which is resistant to the Western corn rootworm (Diabrotica virgifera virgifera).

Analysis of environmental samples using the nematode biotest (comparing transgenic and isogenic maize varieties) should facilitate assessing the risk potential of a release of new Bt-maize lines.

In the final year of the trial, the composition of the nematode community in the soil in different areas of the test fields was examined.

Summary

Nematode biotest

No Bt effect was measured in the soil samples. This is attributed to the fact that the concentration in the soil never exceeded 1 ng/g dry weight of soil. In the laboratory, an effect was first measured concentrations above 36 mg/L; i.e., the concentration in the soil was far below the concentration that can produce an observable effect on the nematodes.

In addition, no differences in nematode growth were found in tests with different leaf samples.

Mode of action

There is evidence to show that the Bt-toxin Cry3Bb1 does not affect the nematodes in the same way as the effective nematode-specific Cry-toxins (nematicide). However, Cry3Bb1 and Cry1Ab appear to bind to the Cry protein-specific receptors.

Nematode communities

A large number of nematodes were found in the soil samples – two to four million per square metre. No significant differences were seen in the composition of the communities in terms of feeding types or diversity between the different maize varieties.

Experiment description

Nematode biotest

For the nematode biotest, five young nematodes (C. elegans) in the first juvenile stage (J1) were placed in an aqueous suspension or solution of the test material (Cry toxins, powdered plants, sieved soil). The test was terminated after 4 days – representing a complete generation cycle at 20°C.

The parameters observed were the growth and reproduction (progeny per test organism) of the test organisms.

The test was used to examine the following types of material:

Leaf material. Old, dried maize leaves and freeze-dried maize leaves were tested. In each case, leaf material from Bt-maize (MON88017) and an isogenic variety (DKX 5134) were available. For the dried leaves, the impact on egg production was also recorded. The dried leaves were ground with sand using a pestle and mortar; 10, 50 and 100 mg of leaf/sand mixture were used for the tests. The freeze-dried leaves were shredded very finely and then sieved.

Cry3Bb toxin. Dose-effect experiments were carried out using neat toxin on two nematode strains – one with low and one with high sensitivity to nematode-specific Bt-toxins (nematocides). The toxin was supplied by one of the project partners.

Soil from test field (soil attached to roots): Von jeder Parzelle der verschiedenen Maissorten (transgener Mais, isogene Sorte, sowie zwei weitere konventionelle Maissorten) wurde Wurzelanhangserde von zwei Maisentwicklungsstadien (zur Blüte, nach der Ernte) beprobt und mit dem Nematodentest untersucht.

Soil with plant litter (mesocosms): Soil samples from the field trial were placed in trial containers (mesocosms) with plant litter from the four different maize varieties. These mesocosms were observed and sampled in a greenhouse (AG Schuphan, RWTH Aachen). Soil samples from the mesocosms were investigated using the nematode biotest.

Mode of action

The toxicity of Cry3Bb1 and Cry1Ab was compared between the two nematode strains to determine whether the mechanism of action of Cry3Bb1 and Cry1Ab was comparable with that of nematocidal Bt toxins. In the trials, both nematode strains were offered three different E-coli strains as feed (= feed bacteria): two genetically modified E. coli strains (one of which carried the Cry3Bb1 gene and one the Cry1Ab gene), and one without any genetic modification. The different bacterial strains were offered to the nematodes as feed in various concentrations and ratios as well as for different lengths of time (16, 24, 48 hours).

Subsequently, two nematode genes (ttm-1 and ttm-2) expressing enzymes that are involved in the defence against Cry proteins were examined using PCR (cooperation with Ralph Menzel, HU Berlin).

Investigation of the nematode community

To isolate nematodes, in 2007, samples were taken from the top 20 cm of soil just after sowing, during flowering and after harvesting from all 32 areas of the test field. After counting the nematodes, 50 individuals at a time were examined as far as possible to determine their variety. The nematodes were classified according to their feeding strategy into different types (herbivore, fungivore, bacterivore, predator, algae eater, omnivore). In addition, a stress index especially developed for nematodes, the maturity index, was calculated.

Results

Nematode biotest

Before this project was started, the nematode biotest had been performed on Bt-maize (MON810), which is resistant to the European corn borer. The test proved to be sensitive for the transgenic varieties.

Old, dried leaves: The development of the nematodes worsened as the quantities of plant material increased. This was the case with both Bt and isogenic (Iso) plant material. No difference in growth was observed between samples of Bt and isogenic maize.

Freeze-dried leaves: The nematodes showed no development on the freeze-dried leaf material. The fine powder was evidently not a suitable substrate for the nematode biotest.

Cry3Bb toxin: Dissolved Cry3Bb1 showed a clear toxic effect on C. elegans, with an EC50 (concentration required to produce 50% inhibition of the toxicity parameters) for growth and reproduction of ~30 mg/L (Fig. 1). No difference was found between the two nematode strains in terms of their sensitivity to Cry3Bb1 (Fig. 1).

Soil from trial field (soil attached to roots): No significant differences in growth were found either within a particular maize type or between the maize varieties (Fig. 2). The fertility was 100% in all the samples tested. Reproduction varied more within the treatment groups than between the maize varieties (Fig. 2). On average, the highest numbers of progeny were found in the soil of one of the two conventional maize varieties. Therefore, no Bt effect was found.

Significance of the toxicity data: The concentrations in the soil never exceeded 1 ng/g soil dry weight. In a liquid medium the concentration below which no effect could be observed (LOEC; no observed effect concentration) was 36 mg/g. This means that the concentrations measured in the soil were way below the concentrations required for an effect to be observed on the nematodes (see Fig. 4). This explains why no direct effects of the Cry3Bb1 toxin were observed on C. elegans in the soil samples in project years 2005 and 2006.

Soil with plant litter (mesocosms): With the Bt soil samples growth was significantly lower than with the soil samples from the isogenic line. However, this was also observed for the two conventional varieties, so that no clear Bt effect was apparent.

In all the samples tested, the fertility of C. elegans was higher than 90%. Thus, again, no Bt effect was found for reproduction. The explanation for this is most likely due to the very low toxin concentrations in the soil, as seen with the soil samples from the test field.

Mode of action

Feeding trials with transgenic E. coli, which produce the toxin, also indicated no increased sensitivity of a Bt-sensitive strain to Cry3Bb1. This suggests that Cry3Bb1 does not exert it effect through the same mechanism as the nematocidal Cry toxins.

From previous tests with Cry1Ab-producing E. coli, it was suspected that Cry1Ab acts through the same mechanism as nematocidal Cry toxins (Cry5A; Cry14A; Cry21A). The toxicity of Cry1Ab and Cry3Bb1 on C. elegans is, however, more than two orders of magnitude greater than that of the nematocidal Cry proteins.

In the presence of Cry1Ab, the ttm genes were more strongly expressed for defence. In the presence of Cry3Bb1, this was only the case for ttm-2. This could indicate that Cry1Ab and Cry3Bb1 can bind to receptors that are specific for Cry proteins.

Investigation of the nematode community

A large number of nematodes were found in the soil samples –two to four million per square metre (Fig. 3). Over the observation period, the number decreased slightly. No significant differences were seen between the different maize varieties. The communities were dominated in all plots and at both time points by the herbivores and bacterivores. The composition of the community with respect to feeding type and diversity did not differ either between the different time points or between the maize varieties (Fig. 4). The communities did, however, differ between the plots, and are therefore dependent on the location. No Bt effect could therefore be established.