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


The aim of this project was 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 were 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 examined 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?


The pollen traps contained far more pollen than the nettles. The stinging nettles next to the field margin contained on average 34 pollen grains per square centimetre. The maximum level found on one plant was 212 pollen grains per square centimetre.

The feeding experiments produced first Bt maize pollen effects at doses of between 200 and 300 pollen grains per square centimetre of leaf. At these doses the larvae ate less and developed more slowly than the control insects. A much higher rate of mortality was measured at 1000 pollen grains per square centimetre.

In the agricultural landscapes investigated, caterpillars of both species of butterfly were found during the maize-flowering period, both in the vicinity of the maize fields and at distances of more than 50 metres from the fields.

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.

Petri dish serving as a pollen trap: Maize pollen adheres to the agarose film.

Pollen traps were set up at various distances from the edge of the maize trial field.

The pollen traps are suspended one metre above the ground on vertical wooden posts. A nettle plant in a pot can be seen beside the post.

Feeding experiment: Small caterpillars are offered defined amounts of pollen on pieces of stinging nettle leaf

In the lab, small tortoiseshell butterfly larvae are fed with specific amounts of Bt maize pollen.

Maize pollen on butterfly and moth host plants

Pollen traps were 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.

Pollen intake and toxicity of the Bt proteins

Intake of Bt proteins by butterfly larvae and their effects were investigated in the laboratory. Caterpillars were kept in climate chambers at a constant temperature of 25°C and a day:night rhythm of 16:8 hours. As soon as they attained the third larval stage, they were given a piece of stinging nettle leaf coated in a defined quantity of Bt maize pollen or, as a control, with pollen from a conventional maize variety. Afterwards the larvae were given untreated food. The caterpillars’ feeding habits and development were recorded by measuring feeding activity, weight, development time and mortality. The ELISA detection method was used to measure Bt protein concentrations in maize pollen.

In a second experiment, caterpillars of the small tortoiseshell butterfly were given a piece of leaf to which a pollen solution had been applied, morning and evening until the larvae reached the final larval stage (on average 7 days).

Choice experiments were conducted in which the caterpillars could choose between stinging nettle leaves that had been dusted with maize pollen and untreated leaves. In another choice experiment, the caterpillars could choose between leaves containing Bt maize pollen and leaves with pollen from conventional maize plants. A record was kept of which leaves the caterpillars chose.

Estimating the risk potential

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

Landscape ecology parameters were also to be included in the risk assessment. Here the main focus was on the actual geographical distribution of butterfly populations and host plants in the agricultural landscape – particularly in terms of their proximity to maize fields. Other parameters were also of interest, including field size, the geographical location of maize fields and the percentage of agricultural land under maize cultivation. These parameters were recorded for two maize-growing regions in Germany during the maize-flowering period. 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).


Maize pollen on butterfly food plants

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. 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 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.

The first effects became visible in the range of 200 to 300 pollen grains per cm². The larvae ate less and developed more slowly than the control insects. 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.

In the experiment with several treated leaf pieces, 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 active avoidance of, any particular type of pollen offering.

Estimating the risk potential using real agricultural landscapes

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 both regions.

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.

The risk posed to the butterfly species studied here by the Bt maize was judged to be negligible because the quantities of pollen that led to effects in the laboratory experiment were found in only isolated instances in the field. Moreover, only a part of the butterfly populations was found to develop in the vicinity of maize fields.