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Investigations for the early detection of resistance development in the European corn borer and for understanding the resistance mechanisms

(2001 – 2004) Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Biological Control; Darmstadt

Topic

European corn borer larva in the stem of a maize plant. A typical corn borer feeding pathway.

European corn borer larva in the stem of a maize plant

For the early detection of resistance development in the European corn borer, corn borer larvae were collected from Bt maize fields, where they had survived despite the fact that the maize plants expressed the Bt toxin, which is toxic to chewing insects.

The aim of the project was to investigate:

  • whether survival could be attributed to a higher tolerance to the toxin or
  • whether modified grazing behaviour of the European corn borer larvae enabled the larvae to survive in the Bt maize fields.

Another part of the project looked into the mechanisms involved in a possible resistance, in other words why the Bt toxin is not converted to a toxic form or is not effective in resistant insects.

  • Are different resistance mechanisms found?
  • Can other variants of the Bt toxin be used to break this resistance?

Summary

Over a three-year period European corn borer larvae were gathered from Bt maize fields and tested for Bt sensitivity/resistance. A surviving larva does not necessarily have to be resistant. According to company information, up to two per cent of the plants in a Bt maize field in fact contain no toxin.

It must be noted in conclusion that no resistant insects were found, despite the fact that toxin sensitivity tests were carried out not only on the second filial generation – as originally anticipated – but also on subsequent generations.

Experiment description

The project focussed primarily on the following tasks:

  • Collecting the surviving European corn borer larvae in the Bt maize fields. Testing the offspring for increased ability to detect the Bt toxin in the Bt maize and for reduced sensitivity to the activated Bt toxin Cry 1Ab.
  • Experimental resistance challenge in the laboratory.
Prepared midgut of a European corn borer. In order to investigate resistance mechanisms, researchers studied the conversion of Bt toxin and possible binding sites in the intestine.

Prepared midgut of the European corn borer

Photo: Dr. R. Kaiser-Alexnat, BBA Darmstadt

To explain possible resistance mechanisms, the natural conversion of the Bt toxin in the intestine of the larvae was tracked in the laboratory by studying the relevant enzymes and the conversion products.

In addition, special investigative techniques were used to determine whether the Bt toxin is stored at special binding sites in the intestine of the corn borer larva.

Results

Rapid on-site Bt test.Bt maize plants in which living corn borer larvae are found do not usually contain Bt toxin. According to company data, up to two per cent of plants in a Bt maize field will not produce toxin.

Rapid on-site Bt test

Eggs. A female has laid her eggs of the lid of the glass container

Breeding in the laboratory: A female has laid eggs on the lid of the glass container after mating.

Emergence of larvae (L1)

Emergence of the small F1 larvae

Photos top and bottom: Dr. R. Kaiser-Alexnat, BBA Darmstadt

Selecting for Bt toxin resistance

In autumn 2001, 2002 and 2003, European corn borer larvae were collected from approximately 1.9 million plants in Bt maize fields.

On average one corn borer larva was found per 1000 plants. According to preliminary tests in the field, however, not all plants under attack by the larvae actually contained the Bt toxin. According to information from the company, up to two percent of plants in a Bt maize field fail to express the toxin.

After the diapause (winter rest period) of the larvae, the offspring of the collected corn borers were subjected to F2-screening tests, which involved testing the second filial generation for toxin sensitivity. The second, or next-but-one generation of the collected insects was studied because monogenically recessively inherited resistance genes do not take effect until this generation.

In these trials the amounts of toxin involved enabled less sensitive larvae to survive. Consequently, it was possible that reduced sensitivity could evolve in subsequent generations. Ultimately it was hoped that resistant insects would be found, which could further our understanding of the ways in which resistance develops, but this was not the case. Even with the use of selection pressure in the laboratory, no resistant corn borers could be found.

Resistance mechanisms

It is only when it enters the intestine of the chewing insects that the Bt protein is converted into a toxic variant, which then destroys the intestine of the insect.

One possible cause of Bt toxin resistance is a change in the complement of important digestive enzymes. Therefore enzymes present in the intestine were identified and their role in activating the toxin was discovered.

It was confirmed that the enzymes trypsin, chymotrypsin, elastase and aminopeptidase are also active in German European corn borers. All detected proteases except aminopeptidase modified the toxin.