Effects of Bt maize pollen on the honeybee

Topic

This project aimed to answer to the following question:

Does large-scale cultivation of Bt maize have an impact on the honeybee? The honeybee is of paramount importance as a pollinator in the natural environment and agriculture. Honeybees pollinate 70 percent of all flowering plants in our part of the world.

As reductions in efficiency of bee colonies have immediate ecological consequences for flora, pesticides have to be tested for incompatibility with honeybees using a standardized process. (OECD Guidelines, 1996). To date there are no regulations or recognized methods for testing the compatibility of genetically modified plants with bees.

The aim of this research project was to develop a simple, reproducible test procedure for the effects of transgenic plants on the honeybee. The resulting procedure could be incorporated into the approval procedure as well as being used during post-market monitoring .

Summary

Overall it was not possible to prove the existence of any chronic toxic effects of Bt176 and Mon810 Bt maize varieties on healthy honeybee colonies.

In view of the extreme conditions under which the trial was carried out (six-week duration, high Bt toxin content), the wide-ranging investigations carried out show that toxic effects on healthy bees under natural conditions can be excluded with a high degree of certainty.

This result is further supported by the fact that honeybees only collect small quantities of maize pollen, even in areas cultivated with large maize plots, when other plants are available as sources of pollen (less than three percent).

Experiment description

Testing the effect of Bt maize pollen in the laboratory

Maize pollen collected from the trial field (Bt176) and a toxin which was genetically produced from E.coli for the joint maize project were used for the laboratory tests.

The impact of the Bt diet was tested in terms of the following criteria:

• Death rate of pupae (mortality)
• Weight on emergence and viability of newly emerged bees
• Fluctuations in the rearing rates for larvae in their different stages

Definition of rearing rates serves as a measure of "normal" brood care behavior.

Toxicity for adult honeybees. In cage trials, bees were fed Bt maize pollen or Bt maize pollen and Bt toxin over a period of four days. A maximum of 100 times the quantity of the concentration occurring in the pollen was added.

Chronic toxicity for honeybee larvae. Larvae were reared in vitro in accordance with a particular method (Wittmann et al.,1981). The semi-synthetic larval food was mixed with the Bt toxin. The larvae were subjected to the toxin in this way for the entire feeding phase.

Laboratory test for testing chronic toxicity on mini colonies. The aim was to develop a standard laboratory procedure for testing chronic toxicity and to test a series of influencing factors such as presence of the queen bee, number of bees, temperature, water supply, honey intake and size of honeycomb for the rearing of 200 larvae.

Testing the effect of Bt maize pollen in the field

In four consecutive years, eight bee colonies were placed in flight tents in a meadow scattered with fruit trees. The pollen was fed to the bee colonies over a longer period than the normal flowering period of the maize plant (six weeks = two successive brood cycles). Under natural conditions, maize pollen is often only available to bee colonies as a source of protein for a few days, or two weeks at most.

The colonies were observed for foraging and brood care activity and for the number of bees.

Testing for chronic toxicity. In the first two years the bee colonies were fed Bt maize pollen to which a 10-fold concentration of Bt toxin had been added.

In the case of Bt176, the trials were repeated using Bt maize pollen collected from the field as well as pollen which was mixed with double the toxin quantity.

In the case of Mon810, only freshly collected pollen was tested.

Post-market monitoring

For monitoring purposes, nine bee colonies were located in the immediate vicinity of a Bt maize field in 2003. Nine control colonies were placed next to a non-transgenic maize field at a distance of approx. five kilometres to prevent any overlap in the foraging areas, whilst at the same time ensuring that the climatic conditions were as similar as possible. During the flowering period, the frequency of honeybee visits to the maize plants was recorded. The proportion of maize pollen was recorded using pollen traps and microscopic tests. The number of bees and their brood activity were established. Since the colonies remained at the location close to the respective trial plots, the bee colonies were also observed during the winter months.

Results

Testing the effect of Bt maize pollen in the laboratory

Toxicity for adult honeybees. The Bt toxin (Bt176) was found not to be acutely toxic even when the dose was 100 times higher than in the pollen. There was no negative impact on mortality or food consumption at different stages of the bees’ development.

Larvae that survived feeding with Bt-toxin under laboratory conditions. Number of adult bees over a period of 6 weeks, Number of colonies =9 Weight of worker bees at pupation. Population at end of experiment: Bt=126, Control=146 Monitoring: Development of bee colonies in maize fields after overwintering Number of colonies =9

Chronic toxicity for honeybee larvae. No difference was found in the number of larvae surviving. Laboratory test for testing chronic toxicity in mini colonies. It was not possible to rear an adequate number of at least 200 larvae in cages. On the other hand, it was possible to rear more than 200 larvae in free-flying mini colonies with 300 bees. Factors such as flight and foraging activity as well as pollen supply may account for the inadequate brood care performance in the cage trials. Testing the effect of Bt maize pollen in the field  In the first year the bee colonies happened to be infested with parasites (microsporidia). This infestation led to a reduction in the number of bees and subsequently to reduced broods in the Bt-fed colonies as well as in the colonies fed on Bt-toxin-free pollen. The trial was therefore discontinued at an early stage. This effect was significantly more marked in the Bt-fed colonies. (The significant differences indicate an interaction of toxin and pathogen on the epithelial cells of the honeybee intestine. The underlying mechanism which causes this effect is unknown.) It was not possible to investigate the influence of microsporidia further since the attempt to breed them failed, making targeted infection of bee colonies impossible. When the trial was repeated the colonies were treated prophylactically with antibiotics to prevent re-infection. No differences in brood care behaviour or in larva development were established in this trial. In the first four weeks the number of adult bees fell more sharply than in the control colonies. However, no further differences were established after this initial phase.

Testing for chronic toxicity as a result of Bt176 and Mon810 maize pollen. No negative effects were observed with the single and double dose of Bt176 or with the single dose of Mon810. There were no differences between the trial groups in terms of the number of bees or the foraging and brood care activity of the colonies. The weight of young bees on emergence did not differ either. This indicates that healthy bee colonies are not impaired in any way by the toxin in any of the tested vital functions of colony size, foraging activity, brood care activity or development, even when exposed to extreme levels of Bt maize pollen over a period of six weeks.

Post-market monitoring

During the flowering period the bees collected only a very small proportion of maize pollen amounting to less than three percent (both Bt and non-transgenic pollen). The maize pollen had no negative effects on the number of bees or the development of the brood in the colonies. The two groups did not differ in their development during late summer or in their ability to survive the winter.