Jun 8, 2007

Research Projects

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Monitoring and resistance management for the sustainable use of Bt toxins

(2002 – 2006) RWTH Aachen University, Institute of Environmental Research (Biology V) Chair of Ecology, Ecotoxicology and Ecochemistry (coordinator)

Topic

Genetically modified Bt maize produces a Bt toxin which makes it resistant to certain insect pests. This project is concerned with Bt maize that is effective in controlling the European and Mediterranean corn borer (ECB, Ostrinia nubilalis; MCB, Sesamia nonagrioides).

If Bt maize is grown on a large scale, the pest itself could potentially develop resistance to the Bt toxin.

A dataset on corn borer populations has been compiled to form the basis of a resistance management plan. This could accompany the introduction of Bt maize in Europe to ensure that it is used in a safe and sustainable way.

Twelve partners from eight countries (ES, FR, I, SK, GR, DE, AU and USA) were involved in the joint EU project, which concluded in April 2006. In Germany these included the State Education and Research Institute for Agriculture (SLFA) in Neustadt, the Max Planck Institute (MPI) in Jena and the Frauenhofer Institute for Molecular Biology and Applied Ecology (IME) in Schmallenberg, in addition to the project coordinator, RWTH Aachen University.

Summary

The susceptibility of corn borers to Bt toxins was recorded throughout the EU. An ECB and MCB data and specimen bank is available for comparative studies in the event of the potential development of resistance. The following conclusions were drawn up to form the basis of a resistance management plan to accompany the introduction of transgenic Bt maize in the European Union:

• Pest susceptibility is comparable throughout the EU.
• Gene flow is high enough for “high-dose” refuge management (HDR). For monitoring purposes, only a few populations per country need to be studied as representative populations.
• Recessive Bt resistance genes are rare in European populations, below 10^-3. In the early stages of resistance development, the database could be used to adapt the resistance management strategy.
• Knowledge of potential resistance development mechanisms can be used for resistance management. For example, a combination of two specific Bt toxins may be recommended.

Experiment description

Studying corn borer populations

Specimens of corn borer populations were collected throughout the EU. ECB were collected in Italy, France, Germany, Spain, Slovakia and Greece, and MCB in Spain and Greece.

Larval susceptibility/resistance: To study larval susceptibility, the larvae were raised in the laboratory and exposed to an artificial diet containing different concentrations of Cry1Ab protein. To find out which larvae were resistant, the laboratory cultures were split, with one group being selected using Bt toxin treatments and one not.

Gene flow: Gene flow both within countries (Germany and Spain) and within Europe (Germany, Italy, France, Slovakia, Romania and Greece) was calculated and compared using various techniques (AFLP, RAPD etc.).

To study gene exchange between corn borers in different maize fields, corn borers in Germany, France and Slovakia were marked, released and then recaptured.

Monitoring potential resistance genes: Potential resistance genes are difficult to detect before they are known. A very labour-intensive monitoring method (F2 screening) was used, which can detect rare recessive resistance genes.

Characterising resistance mechanisms

Special enzymes known as proteases, which are found in the intestinal fluid of the corn borer larvae, split the Bt proteins, thereby activating them. The proteins then bind to specific receptors in the intestinal wall. Here they become integrated in the membrane and form pores. The perforated intestinal wall causes the death of the insect. Insect resistance to Bt toxins can occur at any of these stages.

Protease-mediated resistance: Trypsins are thought to play a role in protease-mediated resistance. Therefore the occurrence of trypsins and the activity of genes for trypsin-like enzymes in the mid-guts of MCB larvae were studied.

Binding studies with Bt toxins: The binding behaviour of different marked Bt toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca and Cry1Fa) in the “brush border membrane vesicles” (BBMV) was studied. These are fragments of intestinal wall isolated from the larvae, to which the toxins bind. Resistant strains (from laboratory cultures in the USA) were compared with susceptible strains. The pore-forming activity of active Cry1 Bt toxins in BBMV was also studied (measurement of potential in the membrane).

Results

Recording corn borer populations

The specimens of different ECB and MCB populations gathered throughout the EU are stored in a specimen bank at the Frauenhofer IME, where they can be used as reference material.

Larval susceptibility/resistance: Susceptibility of the pests (LC50) was comparable everywhere, with the exception of Spain. Minor differences in susceptibility were attributed to natural variation and to potential loss of toxin activity, which may have occurred during shipping when they were sent from one researcher to another.

Surviving larvae in Bt maize were collected from Slovakia and the Czech Republic and reared in the laboratory. However, these larvae collected in the field proved to be susceptible to the Bt toxin in the laboratory. In other words, the larvae were not resistant, but had survived due to the fact that a small percentage of plants in Bt maize fields fail to produce the Bt toxin.

In experiments to produce resistance artificially in the laboratory, the susceptibility of ECB larvae decreased after several generations, depending on the toxin dose (four to eight generations for the high-dose strategy and 23 generations for the low dose). As far as the MCB larvae were concerned, only the Spanish strain showed a reduction in susceptibility, whilst no changes in susceptibility were observed with the Greek strain. This was attributed to toxin variations.

Infecting the larvae with microsporidia changed neither the susceptibility of the larvae (ECB) nor the maize damage.

Gene flow: Only minor differences were found between the individual corn borer populations. This suggests that gene flow is high enough for “high-dose” refuge management (HDR). This means that only a few populations per country or geographically similar region are needed as representative populations for susceptibility screening and monitoring.

Monitoring potential resistance genes: No resistance genes to Bt maize were found. This indicates that recessive resistance genes are rare in European populations (less than 10^-3). The data and specimen bank can be used if outbreaks of resistance occur. If this happens, an early warning system and the resistance management strategy can be adapted to safeguard the future use of Bt maize.

Characterising resistance mechanisms

Protease-mediated resistance: Four different trypsin-like proteases and their genes were found in the midgut of MCB larvae. The susceptibility of trypsin activity to specific protease inhibitors changes at different larval stages, as does the relative proportion of enzymes. These changes can play a role in protease-mediated resistance to Bt maize.

Binding studies with Bt toxins: Resistance found in at least one laboratory strain (USA) was linked to modified receptor binding, although the precise mechanism is not known.

Cry1Aa, Cry1Ab and Cry1Ac share a binding site, whereas Cry1Ca and Cry1Fa bind to different sites. Therefore a combination of Cry1Ab and Cry1Fa is recommended to delay any development of resistance.

It was shown that Cry1Ab and Cry1Fa in vivo increase the membrane permeability to potassium. This was not observed with Cry1Da. The results also indicated that the active toxins affect amino acid transportation.