Jun 1, 2011

Research Projects

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Effects of Bt maize on honeybees

(2008 – 2011) University of Würzburg, Dep. of Animal Ecol. & Trop. Biology (Zoology III)

Topic

The aim of the project was to investigate the potential effects of the genetically modified Bt maize cultivar MON89034xMON88017 on honeybees. This maize produces three Bt proteins, making it resistant to both the European corn borer and the Western corn rootworm.

The project examined the following questions:

• Does ingestion of Bt maize pollen have undesirable effects on the development of bee colonies or on individual honeybee larvae?
• Does ingestion of Bt proteins reduce the bees’ resistance to other stress factors, e.g. pathogens?
• How much maize pollen is actually carried into beehives as a function of the structure of the surrounding agricultural landscape?

Experiment dedcription

To distinguish between varietal effects and Bt effects, two further conventional strains of maize were grown in the field trials besides the genetically modified variety and the isogenic parent variety..

Effects of Bt proteins on honeybees

Two bee colonies with an initial size of approx. 1000 worker bees were placed in a flight cage on each of 32 plots on the trial field (eight plots for each of four maize strains) at the beginning of the maize-flowering season. As a control, nine free-flying bee colonies were placed on a field with Phacelia tenuifolia (a plant that attracts bees).

A dead bee trap was used to count the number of dead bees each day. Changes to the size of the colony were documented throughout the trial period using a special method (Liebefelder estimation method).

At the end of the trial, laboratory experiments were carried out with individual bees:

• Newly emerged worker bees were taken from the trial colonies raised in the flight tents and moved to the laboratory to assess their weight on emergence and individual lifespans.
• Bees were fed with different concentrations and combinations of pure Bt proteins to test whether the three Bt proteins produced by the maize variety under investigation have undesirable effects on honeybees when combined.
• For in vitro breeding of larvae, the young bee larvae were raised under standardised laboratory conditions on synthetic feed to which pollen from the different maize varieties or pure Bt protein could be added. In this experimental approach, it is possible to measure exactly how much Bt protein the larvae consume. After these artificially raised bees emerge they can be further examined for sublethal effects.

Combined effects of Bt proteins and pathogens on honeybees

Colonies on trial plots planted with Bt maize and the isogenic variety were infected with a bee disease pathogen – either with varroa mites or the single-celled intestinal parasite Nosema. The development of the bee colonies was monitored and documented as described above. In addition, experiments are being conducted in the laboratory to investigate the interaction of Bt pollen and Nosema.

Potential exposure risk of honeybees in relation to Bt maize

Twelve landscape areas were selected that differ from one another in terms of the proportion of land area under maize cultivation and the availability of alternative pollen sources during the maize-flowering period. At the beginning of the flowering period, trial colonies with pollen traps were set up in these areas. With the pollen traps it is possible to measure the pollen spectrum used by the bees each week. In this way, it should be possible to draw conclusions about how much maize pollen is actually carried into beehives as a function of the structure of the surrounding agricultural landscape, and the potential exposure risk of bees in relation to Bt maize.

Results

Effects of Bt proteins on honey bees

Field trial: In the first two years of the trial, the dead bee traps developed by the team were used successfully to measure the mortality rate in the bee colonies. Various colony development parameters were measured repeatedly during the flowering season: the number of open and capped brood cells, the number of bees and a special brood index. In addition, during the second year of the trial, the amount of pollen gathered daily was recorded by observing the forager bees, and the corresponding flowering stage was documented to determine the availability of pollen. Statistical analysis of the data from both years is currently underway.

In collaboration with Prof. Christoph Tebbe’s team (vTI Braunschweig), the project investigated the digestibility of maize pollen, the breakdown of Bt proteins in the bee intestine and the influence of Bt maize on the composition of the gut microflora under semi-field conditions. To achieve this, newly emerged bees were marked and placed in the trial colonies. After nine days, they were removed, deep-frozen and later used for gut dissections in the laboratory. Measurements were taken of the amount and digestion rate of the maize pollen grains eaten and DNA sequences were used to investigate the bacterial communities in the different gut sections. A final statistical evaluation of the results has yet to be carried out.

Feeding trials with adult bees
Feeding with maize pollen: Bees were fed both singly and in groups of 30 bees with pollen from the different maize varieties on the trial field. The feeding trials were continued until all the bees had died. The transgenic Bt maize variety was not found to have any negative effect on the life expectancy of worker bees.

Feeding with pure Bt-proteins: The maximum amount of Bt protein to which bees are exposed when consuming Bt maize pollen was calculated (pollen consumed × amount of protein in pollen). The different Bt proteins and a protein mix were fed to the bees in a sugar solution. This also covered a worst-case scenario, i.e. concentrations were tested that were far above those to which a bee would theoretically be exposed. The results show no dose-effect relationship between the Bt proteins tested and bee mortality.

Feeding trials with bee larvae
Since Bt proteins target insect larvae, the in vitro larvae test is used at a potentially sensitive phase in honeybee development and can therefore deliver particularly revealing results. It is recommended as a standard method for the approval of pesticides and transgenic crop plants.

Feeding with maize pollen: Pollen from various maize strains was mixed directly with the feed of the larvae (L3 stage). It was shown that the pollen in the feed is eaten by the bee larvae. The final statistical analysis of the results is still to come.

Feeding bees with pure Bt-protein: The individual Bt proteins and the protein mix were added to the larval feed (L2 stage) in five different concentrations. A worst-case scenario was once again tested. The weight before pupation, the weight at emergence, and the mortality rate were investigated for a possible dose-effect relationship. The preliminary results show no significant correlation between test dose and bee mortality for the Bt proteins tested.

Potential risk of exposure of honeybees to Bt maize

One honeybee colony was placed at each of twelve selected locations during the maize-flowering period of 2009. Over a period of 22 days, pollen collected by the bees was removed with the help of pollen traps and the maize pollen ratio was calculated for each location. The colony’s development was documented before and after the trial period. In order to quantify the amount of pollen eaten by nurse bees in the different landscapes, bee intestines were dissected. The results show that under the experimental conditions, exposure to transgenic maize pollen at colony level and for individual nurse bees is generally low.

In order to analyse the actual foraging distance of honeybees for maize pollen, four observation hives were used and moved around all 12 locations. 662 bee dances were observed and used to calculate the coordinates of the pollen sources visited by the forager bees. The foraging distances for maize pollen were significantly shorter than those for other types of pollen. In other words, in the observation hives less effort was spent on foraging for maize pollen than for other type of pollen.