Aug 26, 2011

Research Projects

Send

Effects of Bt maize containing three Bt proteins on butterflies and moths

(2008 – 2011) RWTH Aachen University, Institute of Biology III (Plant Physiology), Worringer Weg 1, 52074 Aachen

Topic

The aim of this project is to investigate the potential effects of the genetically modified Bt maize cultivar MON89034xMON88017 on butterflies and moths. This maize produces three Bt proteins, two of which are designed to control the European corn borer, which is a moth. Other butterfly and moth species in the area surrounding a Bt maize field may also be at risk as a result of Bt pollen deposits on their food plants, for example.

This project will examine the following questions:

• How much pollen from genetically modified maize plants is deposited on the food plants of moths and butterflies in the area surrounding the maize field?
• How much Bt protein do caterpillars ingest via these food plants and does this have any observable effect on their development?
• How great is the risk potential for a butterfly population subject to the structure of the local agricultural landscape?

Experiment description

To be included in the investigation, butterfly and moth species must be commonly found in the agricultural landscape; their larvae must develop when the maize plants are in flower; their caterpillars must be easy to spot and they must feed on only one host plant. The small tortoiseshell and the peacock butterfly, the commonest butterfly species in most agricultural landscapes, meet these selection criteria. Their caterpillars feed on nettle leaves.

Maize pollen on butterfly and moth host plants

Pollen traps will be set up on each side of the maize trial field during the flowering period to record maize pollen dispersal and density. The traps will be placed directly at the field edge and at intervals of 5, 10, 15, 20 and 30 metres, with additional traps 50 metres from the edge in the direction of the prevailing wind. Each pollen trap will be set up for two 8-hour periods, day and night.

Nettle plants in pots will be placed alongside the pollen traps and regularly sampled to record how much maize pollen adheres to the nettle plant leaves. Maize pollen on the petri dishes and on the nettle leaves will be counted under the binocular microscope in the laboratory.

Absorption and toxicity of the Bt proteins

The absorption of Bt proteins by butterfly larvae and their effect will be examined initially in the laboratory. The results will then be verified by field trials.

Caterpillars will be bred in climatic exposure test cabinets in the laboratory and fed on nettle leaves coated with specific quantities of Bt protein in varying Bt protein combinations, with Bt maize pollen or, by way of control, with pollen from a conventional maize variety. Caterpillar feeding and development will be recorded by measuring their weight and length, pupation rate and time, and mortality. The ELISA detection method will be used to measure Bt protein concentrations in maize pollen.

Choice experiments let caterpillars choose between stinging nettle leaves that have been dusted with maize pollen and untreated leaves. In another choice experiment, the caterpillars can choose between leaves containing Bt maize pollen and leaves with pollen from conventional maize plants. The researchers keep a record of which leaves the caterpillars choose.

Estimating the risk potential

The risk to butterfly larvae posed by Bt proteins from genetically modified maize will be assessed on the basis of the recorded pollen distribution and the laboratory results for pollen toxicity.

Landscape ecology parameters will also be included in the risk assessment, in particular the actual spatial distribution of butterfly populations and host plants in the area surrounding maize fields. Other parameters will also be considered, such as field size, the geographical location of maize fields and the percentage of agricultural land under maize cultivation. These parameters will be recorded for several maize-growing areas in Germany and evaluated using digital field maps.

Results

Maize pollen on butterfly food plants

A total of 76 pollen traps and stinging nettle pots were set up at the start of the maize-flowering period in 2008, 2009 and 2010 at various distances from the field edge. The Petri dishes were exchanged every eight hours in 2008 and leaves were taken from the stinging nettles once a day. In 2009 and 2010 the Petri dishes were exchanged once a day and a leaf was taken from each of the stinging nettle plants each day.

The distribution of pollen depended mainly on wind direction, distance from the field edge and time of day. In the direction of the prevailing wind (north-east), the pollen quantities were several times higher than on the more sheltered sides of the trial field. Pollen quantities also declined rapidly with increasing distance from the field. The greatest pollen volumes were found in the morning, while hardly any pollen was released at night. It was noticeable that the stinging nettle leaves held several times less pollen per cm² than the pollen traps placed at the same sampling points.

In all three years the maximum values (833 pollen grains per cm² on the pollen traps and 212 pollen grains per cm² on the stinging nettles) were measured right next to the eastern edge of the field. The average number of pollen grains per cm² found next to the field edge was 150 on the pollen traps and 34 on the stinging nettle leaves.

Pollen intake and toxicity of the Bt proteins

The project was able to set up its own breeding programme for small tortoiseshell and peacock butterflies.

Feeding experiments were conducted on caterpillars of both species from the breeding programme. Individuals in the third larval stage were each fed one piece of stinging nettle leaf (1 cm2) to which a drop of a defined pollen suspension had been applied. The test was conducted on the small tortoiseshell caterpillars with 200, 250, 300, 400, 500, 1000 and 2000 pollen grains per cm², and on caterpillars of the peacock butterfly with 10, 200 and 400 pollen grains per cm².

The insects were kept in a climate chamber at a constant temperature of 25°C and a day:night rhythm of 16:8 hours and subsequently given untreated food. When given 1000 pollen grains per cm², the mortality rate of the larvae given Bt maize pollen was significantly higher than that of the control groups. A dose of 300 pollen grains produced no difference between the different maize variants.

In a second experiment, caterpillars were each fed one piece of leaf containing pollen solution in the morning and evening. This continued until they had reached the final larval stage (on average 7 days). The insects were given between ten and 200 pollen grains per cm². The effects were not found to be any greater than with the single pollen dose.

In the choice experiments, the caterpillars did not appear to show any preference for or actively avoid any particular type of leaf offering.

Estimating the risk potential using real agricultural landscapes

The two study regions, Wesecke and Schwarzenau, were mapped in their entirety during the maize-flowering period in 2008, 2009 and 2010. Field crops and landscape structures and populations of stinging nettles were recorded. All sites with stinging nettles were inspected for nests of the small tortoiseshell and peacock butterfly. The mapping was carried out using a geographic information system (GIS).

No butterfly nests were found in either of the two study regions in 2008. In 2009 and 2010, nests of both the small tortoiseshell and peacock butterfly were found.

In the region with high levels of maize cultivation, more than 60 per cent of the nests were in the vicinity of maize fields. In the region with low levels of maize cultivation, over 50 per cent of all butterfly nests were more than 50 metres away from the nearest maize field.