Med. Weter. 2012, 68 (10) 630
Praca oryginalna Original paper
Pollinators play a crucial role in natural ecosystems by maintaining both the biodiversity of wild plants and agricultural activity (19, 29), the estimated yearly value of which is US-$ 217 billion (10). Bees are important pollinators of many crops (9, 19). Besides honey bees, bumblebees and solitary bees provide pollination around the world. In modern agriculture health problems affecting honey bee populations have resulted in an increased exploitation of alternative pollinators such as bumblebees. Bumblebees and bees might be exposed to different products used in plant protection, such as pesticides and proteins toxic to pests produced by genetically modified plants. The research results regarding the effects of plant protection on honey bees or other bees are insufficient despite growing concerns about the suspected decline of pollinators (1, 19). The
research conducted in the United Kingdom has shown that because of the intensification of agriculture the number of bumblebees has decreased (13). Such data are also important for other European countries having a similar agricultural production system. Thus, to guarantee successful pollination, it is a prerequisite that plant protection products cause no lethal or sublethal effects on pollinating insects, because any impairment could result in lower reproduction and colony mortality. Currently, products based on the soil bacterium Ba-cillus thuringiensis (Berliner) (Bt) and its Cry crystal toxins are widespread in the form of spray agents and transgenic crops in use in the biological control of plant pests (18, 33). In susceptible insects, Cry proteins bind to border membrane receptors in the insect midgut, causing midgut cell lysis and finally lead to the insects
Evaluation of the impact of the toxic protein Cry1Ab
expressed by the genetically modified cultivar
MON810 on honey bee (Apis mellifera L.) behavior
MARCIN GRABOWSKI, ZBIGNIEW T. D¥BROWSKI*
*Department of Applied Entomology Faculty of Horticulture and Landscape Architecture Warsaw University of Life Sciences, Nowoursynowska Street 159, 02-776 Warsaw
Grabowski M., D¹browski Z. T.
Evaluation of the impact of the toxic protein Cry1Ab expressed by the genetically modified cultivar MON810 on honey bee (Apis mellifera L.) behavior
Summary
Pollinators play a crucial role in natural ecosystems by maintaining both the biodiversity of wild plants and agricultural activity. In modern agriculture health problems affecting honey bee populations have resulted in an increased exploitation of alternative pollinators such as bumblebees. When Bt products are used, either as sprays or transgenic crops, non-target organisms such as pollinators may be exposed to the Cry proteins via contact or through ingestion of contaminated pollen and nectar. The cultivation of transgenic crops, e.g. maize, raises concerns among different social groups. Even in scientific bee journals there have appeared articles saying that bees might avoid collecting pollen and/or nectar from such plants. Therefore, the aim of our research was to assess the potential negative influence of the genetically modified maize MON810 expressing Cry1Ab a protein toxic to the most serious lepidopteran pest, the European corn borer (Ostrinia nubilalis) [Hübner] (Lepidoptera: Crambidae) on the behavior of the honey bee in conditions of the limited possible choice of a host plant. The insect material was one honey bee (Apis mellifera L.) family in a hive, which included a queen and approximately 800 worker bees. The honey bees were from the Apiculture Division, Faculty of Animal Sciences, Warsaw University of Life Sciences. The plant material consisted of transgenic maize (Zea mays L.) plants DKC3421 Yield Gard (event MON810) from the Monsanto Company (St. Louis, MO, USA), expressing a gene encoding a truncated form of the Cry1Ab protein derived from B. thuringiensis Berliner, and the corresponding near-isogenic hybrid (DKC 3420). This research has not revealed any significant differences in the number of honey bees visiting the two maize cultivars that would be caused by the potential negative impact of the genetically modified maize MON810.
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death (2, 7, 8). Toxins derived from Bt are recognized as selective against insects from the Lepidoptera, Coleoptera and Diptera. The B. thuringiensis strains that are used most often against Lepidoptera belong to the subspecies kurstaki (containing Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab and Cry1Ia) and aizawai (containing Cry1Ab, Cry1C and Cry1D) (11). When Bt products are used, either as sprays or transgenic crops, non-target organisms such as pollinators may be exposed to the Cry proteins via contact or through ingestion of contaminated pollen and nectar (20). It has been demonstrated that the pollen from Bt crops (e.g. genetically modified maize) can contain signifi-cant amounts of the Cry toxin (32). Therefore, much research has been undertaken in which Cry proteins have been added to the diet of the larval and adult stages of the honey bee to evaluate lethal effects (14, 16, 22-24). The Cry proteins have not been reported to induce mortality in honey bees. Nevertheless it is believed that Bt toxins can pose sublethal effects reducing the population of pollinators in the field. Until now, only a few studies concerning pollinators have focused on the sublethal effects of Cry proteins (4, 25, 30, 31).
The cultivation of transgenic crops, e.g. maize, raises concerns among different social groups. Even in scien-tific bee journals there have appeared articles saying that bees might avoid collecting pollen and/or nectar from such plants. Therefore, the aim of our research was to assess the potential negative influence of the genetically modified maize MON810 expressing Cry1Ab, protein toxic to the most serious lepidopteran pest, the European corn borer (Ostrinia nubilalis) [Hübner] (Lepidoptera: Crambidae), on the behavior of the honey bee in conditions of a limited possible choice of a host plant.
Materials and methods
The insect material was one honey bee (Apis mellifera L.) family in a hive which included a queen and approxi-mately 800 worker bees. The honey bees were from the Apiculture Division, Faculty of Animal Sciences, Warsaw University of Life Sciences.
The plant material consisted of transgenic maize (Zea mays L.) plants DKC3421 Yield Gard (event MON810) from the Monsanto Company (St. Louis, MO, USA) expres-sing a gene encoding of a truncated form of the Cry1Ab protein derived from B. thuringiensis Berliner and the corresponding near-isogenic hybrid (DKC 3420). In the DKC3421 maize the toxin is produced continuously by the plant, because cryIA(b) gene is linked under of the strong enhanced constitutive CaMV 35S promoter. Plants were cultivated in a garden tunnel (6 m × 1.5 m) covered with white fabric, which facilitated air circulation, but cut off the accidental escape of the bees. The pots with the plants were arranged in two rows (Fig. 1), one for each of the two cultivars (DKC3421, DKC3420). They were grown in garden soil (Kronen). The hive with the bees was intro-duced when the plants reached BBCH stage 63. Initially,
when the plants had flowers with-out pollen, the bees were provided with artificial food (Fon-dant) as a substi-tute for the natural food source.
The honey bees were observed during the blos-soming of the two maize cultivars. The dates of the completed observa-tions are shown in the figures (Fig. 2, Fig. 3, Fig. 4, Fig. 5). The number of the bees visiting the flowers of the two
maize cultivars during BBCH stage 69 was counted. The observations were made at 8.00 a.m. and 15.00 p.m. The experiment was carried out in two vegetation seasons, 2010 and 2011.
Results and discussion
At statistical analysis was made using the One-Way Anova test with significant differences at P £ 0.05. The experiment results shown were not clear and simple. Furthermore, this research has not revealed any significant differences in the number of the honey bees visiting the two maize cultivars that would be caused by the potential negative impact of the genetically modified maize MON810. The main conclusion is that a more natural scenario should be consider. The research should perhaps be conducted in open areas, such as an agroecosystem with maize cultivation and the pos-sibility of a free choice of food source (e.g. pollen and/ or nectar) extending to other plants occurring near the cultivated maize plants. Also to be considered is the way pollen is transported to other flowers that grow on field edges: areas where honey bees usually collect pollen, finding those plants more attractive than the maize ones.
This research investigated the effects of the geneti-cally modified maize MON810 on the behavior of the honey bee (A. mellifera L.) and has shown no signifi-cant impact on the choice between the flowers coming from the two maize cultivars, the genetically modified variety and its isogenic line without the Cry1Ab protein. Floral pollen is the sole protein source for A. melli-fera L. colonies (6) and the pollen of a variety of im-portant crops is collected by bee foragers (21). Adults and larvae of the honey bee might be directly exposed to transgenic material via pollen consumption from genetically modified crops if the latter are planted in mass monocultures. On average, a worker consumes 3.4 to 4.3 mg of pollen per day and a colony can accu-Fig. 1. The scheme of the experiment in the garden tunnel where the honey bees were observed
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mulate up to 55 kg per year (34). Honey bees exposed to the MON810 maize pollen did not transmit quanti-fiable amounts of the Bt-proteins via their hypopha-ryngeal glands into the larval food they secrete (4). Nevertheless, the pollen is also added directly to the larval food by nurse bees (15). It was reported that larvae consumed 1720-2310 maize pollen grains under semi-field exposure conditions, which shows a worst case scenario maize pollen exposure of 1.52-2.04 mg (3). Studies on the effects of corn pollen expressing Cry proteins, DKC7565 and three other proteins from transgenic varieties (MON89034 × MON88017) did not show a negative impact on the development of honeybee larvae. However, they demonstrated that current tests concerning the effects of Bt varieties on A. mellifera should always be part of biosafety analyses for these varieties (16). Although there was no effect on the mortality of honey bees, it is believed that one cannot exclude the possibility that Cry toxins can
cause sublethal effects by reducing the number of pol-linating insects in the field. Unfortunately, only a few studies on the effects of Bt toxins have focused on sub-lethal effects. The Ramirez-Romero et al. (30) experi-ment showed a significant impact on the honey bees ability to obtain food after exposure to 1 mg · kg1
Cry1Ab toxin in sugar syrup. Similar results were ob-tained after exposing honeybees to 5 mg of kg1 Cry1Ab
toxin, which resulted in confusion among individuals. In honey bees, the process of orientation is important for long-term memory and the exploitation of food. Mommaerts et al. (26) showed lethal and sublethal effects of proteins produced by B. thuringiensis on small colonies, whose workers were subjected to three different methods of exposure: direct treatment sprays, pollen treated with a product containing Bt, and a water solution treated with a product containing Bt. These experiments demonstrated high mortality caused by the application of an aqueous sugar solution treated with Fig. 2. The numbers of the honey bees noticed on the flowers
of the two maize cultivars (DKC3421, DKC3420) at 8.00 a.m. in 2010
Fig. 3. The numbers of the honey bees noticed on the flowers of the two maize cultivars (DKC3421, DKC3420) at 3.00 p.m. in 2010
Fig. 4. The numbers of the honey bees noticed on the flowers of the two maize cultivars (DKC3421, DKC3420) at 8.00 a.m. in 2011
Fig. 5. The numbers of the honey bees noticed on the flowers of the two maize cultivars (DKC3421, DKC3420) at 3.00 p.m. in 2011
Med. Weter. 2012, 68 (10) 633
a product containing the toxin. The authors concluded that this may be related to the food used in the experi-ment: an aqueous solution of sugar and not pollen, which can be associated with the preferences of bum-blebees. Goulson (12) showed that adult bumblebees prefer nectar to pollen. Mommaerts et al. (26) confirm this observation. Fertility was reduced by 30% when using pollen as food and by 100% when using an aqueous sugar solution. In addition, it should be noted that bumblebee larvae feed mainly on pollen, which makes possible a direct analysis of the effect of Cry toxins (12). Another important observation of the study conducted by Mommaerts et al. (26) revealed that the high toxicity is caused by an aqueous sugar solution whose formula contain Cry1C toxins Cry1C and Cry1D. These are unfortunately the only data on the toxicity of these toxins to bumblebees and honey bees. The experience of other authors has shown that most varieties of transgenic corn and cotton that pro-duce the Cry1Ab toxin are safe for natural bumblebees (Bombus terrestris) and honey bees (5, 30). It is the-refore important to determine how Cry toxins reach their insecticidal activity in the organisms of bumblebees. The activity of Cry toxins in insects of the Hymeno-ptera order may indicate specific binding sites and new receptors. Further research should be more focused on understanding the mechanisms of the action of Bt toxins and receptors as well as their impact on various aspects of the biology of bees. All studies on the effects of GM plants on bees and bumblebees concerned only one toxin from the B. thuringiensis bacterium. The future of agricultural biotechnology belongs to geneti-cally modified varieties with multiple resistant traits. An example would be SmartStaxTM, a Bt corn variety
introduced into cultivation in the United States in 2010. It contains six different genes responsible for resistance to pests damaging the underground and aboveground parts of plants as well as two additional herbicide resi-stance genes (18).
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Author's adress: Marcin Grabowski, Nowoursynowska Street 161/708, 02-787 Warsaw, e-mail: grabo55@yahoo.pl
