Sep 29, 2005
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
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?
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.
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 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.
Rapid on-site Bt test
Breeding in the laboratory: A female has laid eggs on the lid of the glass container after mating.
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.
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.
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Bundesministerium für Bildung und Forschung
Untersuchungen zur frühzeitigen Entdeckung einer Resistenzentwicklung des Maiszünslers und zur Aufklärung der Resistenzmechanismen
Dr. Renate Kaiser-Alexnat
Biologische Bundesanstalt für Land und Forstwirtschaft (BBA)
(seit 2008 Julius Kühn-Institut (JKI))
Institut für biologischen Pflanzenschutz
Kaiser-Alexnat, R., Wagner W., Langenbruch G.-A., Kleespies R.G., Keller B., Meise T., Hommel B. (2005) Selection of resistant European Corn Borer (Ostrinia nubilalis) to B.t.-corn and preliminary studies for the biochemical characterization IOBC wprs bulletin 28 (3), 115-118
Kaiser-Alexnat, R., Meise T., Langenbruch G.-A., Hommel B., Huber J. (2005) Untersuchungen zur frühzeitigen Entdeckung einer Resistenzentwicklung des Maiszünslers (Ostrinia nubilalis) gegenüber dem B.t.-Mais-Toxin Cry1Ab. Nachrichtenblatt des Deutschen Pflanzenschutzdienstes 57 (7), 144-151
Kaiser-Alexnat, R., Wagner W., Langenbruch G.-A., Kleespies R.G. , Keller B., Hommel B. (2004) European corn borer (Ostrinia nubilalis): Studies on proteinase activity and proteolytical processing of the B.t.-toxin Cry1Ab in transgenic corn. IOBC wprs bulletin 27 (3), 97-102
Project: Bt maize (2001-2004)
- Production of a Bt toxin standard, DLR Rheinpfalz
- Effects of Bt maize on flower-visiting insects and predatory spiders, LBP Freising
- Effects on aphids and their antagonists, University of Göttingen
- Effects on various arthropods, TH Aachen
- Impacts on sciarid fly larvae, BBA Braunschweig
- Effects on butterflies and their antagonists, MPI Jena
- Toxicity for parasitic wap, BBA Darmstadt
- Effects on the honeybee, University of Jena
- Resistance development in the European corn borer, BBA Darmstadt
- Gene transfer to digestive flora in cattle, TU München / BLT Grub
- Breakdown of Bt maize in soils and impacts on micro-organisms, FAL Braunschweig
- Is Bt toxin bound in the soil?, University of Trier