Testing a nematode biotest

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

Topic

The aim is to test a biotest with the nematode Caenorhabditis elegans used to detect Bt toxins . The test is being developed with the transgenic maize variety Mon88017, which is resistant to the Western corn rootworm (Diabrotica virgifera virgifera).

Using the nematode test to examine environmental samples (comparison of transgenic and isogenic maize varieties) is intended to make it easier to assess the risk potential of a release of new Bt maize varieties.

In addition, in the final year of the trial, the plan is to investigate the composition of the nematode community in the soil of the different plots on the trial field to take into account any Bt effects at a higher ecological level.

Experiment description

Culture of C. elegans on agar: various stages of development (photo taken with stereo microscope, approx. 40x magnification)
Photo: Sebastian Höss

For the nematode test, five young nematodes (C. elegans) in the first juvenile stage (J1) are placed in an aqueous suspension of the test material (toxin solution, powdered plants, sieved soil).

After four days – a complete generation cycle at 20 °C – the test is completed. Each test is repeated five times. The test parameters are the growth and reproduction (progeny per test organism) of the test organisms.

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

Leaf material: Old, dried maize leaves and freeze-dried maize leaves are tested. In each case, leaf material is provided from Bt maize, an isogenic variety and, in some cases, from two other conventional varieties. Growth and reproduction (number of progeny) are recorded and, in the case of the dried leaves, the impact on egg production. The dried leaves are ground with sand using a mortar. 10, 50 and 100 mg of leaf/sand mixture were used for the tests. The freeze-dried leaves are mashed very finely and then sieved.

Cry3Bb toxin: Dose-effect experiments are to be conducted with the neat toxin on two nematode strains – one with low sensitivity and one with high sensitivity to Bt toxins that are specifically effective against nematodes (nematocides). The toxin is being supplied by a project group partner.in addition, the toxicity of Cry3Bb1 and Cry1Ab is being investigated and compared between the two nematode strains. This is designed to test whether the mechanism of action of Cry3Bb1 and Cry1Ab is comparable with that of nematocidal Bt toxins. In the trials, both nematode strains are offered three different E coli strains as food (= food bacteria): two genetically modified E-coli strains, one of which carries the Cry3Bb1 gene and one of which carries the Cry1Ab gene, and one without any genetic modification. The various bacterial strains are offered to the nematodes as food in various concentrations and in various mix ratios.

Soil from trial field (soil attached to roots): Samples of root soil were taken from each plot of the different maize varieties (transgenic maize, the isogenic variety and two other conventional maize varieties) at two maize development stages (BBCH60, after harvest) and investigated using the nematode test.

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 are being observed in the greenhouse and samples are taken (AG Schuphan, RWTH Aachen). Soil samples from the mesocosms have been investigated using the nematode test.

Results

Before the project began, the nematode test had already been carried out on Bt maize (Mon810), which is resistant to the European corn borer. The test proved sensitive to the transgenic varieties.

In this project the test has already been carried out on the materials listed below.

Fig.1: Growth (µm) and reproduction (progeny per test organism) of two C. elegans strains (N2: wild type; sek-1: Bt-sensitive strain) after 96 hours’ exposure in an aqueous solution of Cry3Bb1; C = buffer control.

Fig. 2: Growth (top) and progeny per test organism (bottom) of C. elegans in soil attached to roots from a total of 32 plots on the trial field (8 for each of the four maize varieties); average (bars) and standard deviation (n=5).

Fig.3: Growth (top) and progeny per test organism (bottom) of C. elegans in soil attached to roots from the various maize varieties; average (bars) and standard deviation from average values of the individual plots (n=8). Different letters mean a significant difference

Fig.4: Growth (top) and progeny per test organism (bottom) of C. elegans after 96 hours’ exposure in soil samples from mesocosms with maize litter for the different maize varieties; average (bars) and standard deviation from average values of the individual mesocosms (n=8). Different letters mean a significant difference.

Fig.5: Concentration range of Cry3Bb1 in rhizosphere soil in comparison with the effect range of Cry3Bb1 in the liquid medium

Leaf material

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

In the test with the 50 mg leaf/sand mixture a significantly higher number of eggs was observed in the body, indicating delayed egg laying.

Reproduction took place only in the tests with the 10 and 50 mg leaf/sand mixture. With 100 mg sample material per test, the nematodes hardly developed at all. With 50 mg sample material per test reproduction was significantly lower with Bt leaf material than with isogenic leaf material.

Freeze-dried leaves: The nematodes did not develop with the freeze-dried leaf material. The fine powder was evidently not a suitable substrate for the nematode test.

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 at around 30 mg l-1 (Fig. 1). No difference was found between the two nematode strains in terms of their sensitivity to Cry3Bb1 (Fig. 1). Feeding trials with transgenic E. coli which produce the toxin did not show increased sensitivity of a Bt-sensitive strain to Cry3Bb1 either. This suggests that Cry3Bb1 does not have the same mechanism of action as the nematocidal cry toxins. From previous tests with Cry1Ab-producing E.coli, it is suspected that Cry1Ab does have the same mechanism of action 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.

Soil from trial field (soil attached to roots): For the years 2005 and 2006, soil samples from all plots were investigated at two points in time (BBCH60 and after harvest) using the nematode test.

No significant differences in growth were found either within the maize varieties or between the maize varieties (Fig.2 BBCH60).

In all the samples tested, fertility was one hundred per cent. Reproduction varied within the treatment variants more 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. In other words, no Bt effect was found.

Interpreting the toxicity data: The concentrations in the soil are never greater than one nanogram per gram of soil (dry weight). In the liquid medium the concentration at which no effect was observed (LOEC; no observed effect concentration) was 36 milligrams per gram. This means that the concentrations measured in the soil are way below the concentrations required for an effect to be observed on the nematodes (s. 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 (Fig. 3). However, this can also be observed for the two conventional varieties, so does not indicate a clear Bt effect.

In all the samples that were tested the fertility of C. elegans was greater than 90%. Neither was any Bt effect found for reproduction. The most likely explanation for this is that, as with the soil samples from the trial field, the soil held very low toxin concentrations.