Breeding and characterising the Western corn rootworm (Diabrotica v. virgifera)

(2005 - 2008) BTL Bio-Test Labor GmbH Sagerheide

Topic

The Western corn rootworm (Diabrotica v.virgifera) is a leaf beetle from the USA that has become firmly established in South-Eastern Europe and is spreading further. First evidence of the pest’s arrival in southern Germany was found in the summer of 2007. Because of the economic damage it causes to maize crops, however, the beetle is still classed as a quarantine pest in EU member states. Because of this, continuous laboratory breeding programmes have to be set up if biological research is to be conducted on Diabrotica in Germany.

This project is pursuing two main objectives:

Variations in wing sheaths in a Hungarian field strain of Diabrotica v. virgifera. Top: ‘typical’ wing sheath markings. The larger insect in each case is the female.

Setting up a breeding programme for the mass production of the Western corn rootworm: In order to make Diabrotica available for other working groups within the group project, permanent breeding programmes for various Diabrotica strains are being established in compliance with the safety conditions.
Comparing the biological performance of different Diabrotica strains: Since both the introduction of the beetle in Europe and years of laboratory breeding programmes can cause changes to the biology of the beetles, researchers checked whether results from the USA and from laboratory strains can be applied to other strains. A comparative biotest on the toxicity of the Bt protein Cry3Bb1 compared the biological performance of field and laboratory strains of the Western corn rootworm.

Summary

By adapting existing breeding methods, mass breeding programmes were established for different Diabrotica strains. 10,000 Diabrotica larvae were raised and prepared for a project being conducted by a team at the Julius Kühn Institute in Darmstadt.

The results of this project show,

that transgenic MON 88017 maize has no impact on the mortality or fertility of the adult Western corn rootworm
and that there are significant differences between the laboratory and Hungarian field strains used in the experiment. Results obtained from the laboratory strain therefore need to be checked for transferability.

Experiment description

Experiments with the quarantine species Diabrotica are conducted exclusively in special safety laboratories and greenhouses.

Breeding cages and hatching box

Fresh fodder

Adult beetles eating leaves

Preparation of mid-guts and gut epithelia

Setting up a mass breeding programme and preparation of larvae

Breeding methods have been developed that ensure a constant supply of the test organisms for the project group’s experiments.

A total of 10,000 mid-guts and gut epithelia have been prepared from laboratory-bred Diabrotica larvae to test for possible resistance mechanisms against Bt toxins (part-project at JKI Darmstadt)

Diet comparison

To test the suitability and acceptability of artificial diets for adult beetles, selection tests are being conducted on one field and one laboratory strain. The beetles are offered both fresh fodder and artificial diets in the same space. In order to carry out biotests on Diabrotica larvae as well, an artificial diet is also being developed for Diabrotica larvae.

Comparing the biological performance of different Diabrotica strains/biotest

Continuous breeding programmes for corn rootworms from different sources – laboratory strain (USA) and field (SE Europe) – were established in the laboratory.

The biological performance (mortality, weight, fertility, etc.) was assessed in the laboratory strain in a biotest using four different diets (transgenic MON88017 maize, isogenic DKC 5143 maize, control variety (DK 315) and an artificial diet). At the same time, beetles from the Hungarian field strain were tested with the artificial diet and the control variety (DK 315). In each variant, four females were held with six males over eight weeks, with twelve repetitions. Mortality/lifespan and the number of eggs laid were recorded. The Cry3Bb1 content of the feed was checked using the ELISA detection method.

Results

Diabrotica larvae in the first and second larval stages (F1 and F2) on the artificial diet

Third larval stage (L3) of the Western corn rootworm

Biotest with adult Western corn rootworm

Average number of eggs laid per female after feeding on the different maize varieties
Survival rate of the females after feeding on the different maize varieties

Diet comparison

Testing adult beetles with artificial and fresh diets showed that the laboratory strain copes with the artificial diet better than field beetles. Since the different diets varied in their ease of handling, a mix of fresh and artificial diet was chosen for the breeding programmes.

In the diet comparison test, beetles from the laboratory strain that were fed the artificial diet laid twice as many eggs on average as those that were given fresh food. The survival rates of the beetles that received the artificial diet were no higher than for the other variants.

The development of an artificial diet for Diabrotica larvae proved very difficult because of contamination with fungi and bacteria. This was caused by the (unsterile) larvae and led to higher mortality rates. Tested antibiotics and preservatives were either not sufficiently effective or themselves increased the mortality rates of the larvae. By refining the methods (disinfecting the eggs), contamination of the food was reduced to below ten per cent. The diet for larvae was used only in the biotests and not in the breeding programme.

Comparison of different Diabrotica strains

Knowledge about the extent of these differences can be used to check the transferability of data obtained in America and/or using laboratory strains. This avoids unnecessary repetition of research, and also prevents misinterpretation.

The tests on beetles bred from an American laboratory strain displayed differences compared with insects of European origin in terms of biology, habit and tissue texture (evident during preparation of the guts).

The females of the laboratory strain laid almost eight times as many eggs when fed on the artificial diet as the beetles from Hungary. When fed on maize, they laid four times as many. The mortality rates were the same for all variants.

Biotest

In the biotests to examine the effects of Cry3Bb1 on adult Western corn rootworms, no differences were found between the transgenic (MON 88017), isogenic and control varieties for the parameters investigated. There were no differences in the number of eggs laid or in the mortality rate of the insects.