Research into the activation of Bt proteins in the Western corn rootworm

(2005 - 2008) Federal Biological Research Centre for Agriculture and Forestry (BBA) (since 2008 Julius Kühn Institute (JKI)), Institute for Biological Control, Darmstadt

Topic

When Bt maize is cultivated there is a risk that maize pests will develop resistance to the active Bt toxin in question.

In this project, test systems are being developed that can be used in the event of resistance developing in the Western corn rootworm to investigate possible resistance mechanisms.

In order to understand the research approach, an understanding of the effect mechanism of Bt toxins is necessary: Bt toxins are proteins. These proteins are ingested by insects along with their food and dissolved in the digestive tract. They are degraded by special enzymes in the intestinal juice, so-called proteases. This activates them and they are then bound to specific receptors in the intestinal wall. Eventually, they are integrated into the membrane and pores are formed. This perforates the intestinal wall, leading to the death of the insect.

Insect resistance to Bt toxins can begin at any of these stages. The types of resistance to Bt toxins described so far are mostly protease- or receptor-induced. For this reason, this project is investigating which proteases are active in the intestinal juice of the Western corn rootworm and which of the identified proteases are involved in activating the Bt toxin. The project is also studying the binding of the active Bt toxin to the receptors in the intestinal wall. If Bt-resistant pests are found in the course of the project, they are to be investigated to shed light on the resistance mechanism.

Experiment description

Since the effect mechanism of Bt toxins takes place in the intestinal tract of the insects, midguts of larvae are needed for the research. The larvae are bred and prepared by a project partner (BTL Bio-Test Labor, Sagerheide). At the BBA intestinal juice or intestinal wall material is then obtained from the midguts.

First the pH value of the intestinal juice is measured, since the activity of the enzymes that break down the toxin (proteases) is dependant on the pH value. Since the pH value can vary from one intestinal area to another, the pH was also measured for different intestinal sections. This involved dividing 170 midguts into front, middle and rear sections and measuring the pH value of the intestinal juice for each section.

Detecting protease activity

Photometric tests with specific substrates and specific protease inhibitors are used to identify the active proteases in the intestinal juice of the Western corn rootworm.

Diluting the intestinal juice: If resistant insects emerge, it is desirable to be able to work with as low a quantity of intestinal juice as possible, since larger quantities of midguts can be made available only after a fairly long reproduction phase. The team has therefore carried out additional dilution experiments. They tested at which maximum dilution it is still possible to detect the proteases in question with certainty.

Degradation and binding of the toxin

To understand the degradation route of the toxin, intestinal juice or the detected enzymes are mixed with the toxin and the degradation products are characterised using electrophoretic methods.

To investigate the binding of the Bt toxin to the intestinal wall, marked Bt toxin is added to isolated intestinal wall sections. The aim is to characterise the receptors involved in the binding process. The Bt toxin was supplied by Monsanto.

Results

The pH values of the intestinal juice samples were strikingly homogeneous. They were in the slightly acid range (pH 5.75 on average). Research into the different midgut areas indicated a slight increase in pH from the front to the back section (pH 5.75 to 6.03). However, the standard deviations were in some cases higher than the differences between the gut sections.

Detection of protease activity

Aminopeptidases were detected in the intestinal juice of L3 Western corn rootworm larvae in addition to the serine proteases trypsin, chymotrypsin and elastase. The cysteine proteases cathepsin L (with papain), cathepsin B and cathepsin H were also found to be highly active. These proteases are responsible for protein breakdown in many beetles, including Chrysomelidae, the family to which the Western corn rootworm belongs.

Protease activity: A comparison of all proteases identified so far shows that cysteine proteases are by far the most active in the intestinal juice of the Western corn rootworm.

In the Western corn rootworm a different inhibitor works on trypsin than in the European corn borer. This could indicate that different trypsin types are active in these insects.

Diluting the intestinal juice: It was still possible to detect individual proteases at certain levels of dilution.

Degradation of the toxin

The Bt toxin was broken down after intestinal juice was added. In the control experiment with heated intestinal juice, the protein was not processed because the proteases were inactivated by the heat.

The results were obtained with a ten-minute incubation period for the protein and protease. But even after an incubation period of 24 hours the same results were obtained. Proteases that did not break down the protein after ten minutes, did not break it down after 24 hours either, and vice versa.