IOBCOILB WPRS / SROP

International Organisation for Biological and Integrated Control of Noxious Animals and PlantsOrganisation Internationale de Lutte Biologique et Integrée contre les Animaux et les Plantes Nuisibles

West Palaearctic Regional Section / Section Régionale Ouest Paléarctique

IOBC/wprs Working Group ‘GMOs in Integrated Plant Production’

3rd EIGMO Meeting "Ecological Impact of Genetically

Modified Organisms (EIGMO)"

23-25 May 2007, Warsaw, Poland Under honorary patronage of Prof. dr hab. Tomasz Borecki

President of Warsaw Agricultural University

Program and abstracts

Warsaw, 2007

Program committee Jörg Romeis (Chairman) Agroscope Reckenholz-Tänikon ART, Switzerland

Ramon Albajes Universitat de Lleida, Spain

Detlef Bartsch Federal Office of Consumer Protection and Food Safety, Germany

Franz Bigler Agroscope Reckenholz-Tänikon ART, Switzerland

Zbigniew T. Dabrowski Warsaw Agricultural University, Poland

Alan Raybould Syngenta, UK

Local organizing committee Zbigniew T. Dabrowski (Chairman)

Malgorzata Kielkiewicz

Mariusz Lewandowski

Julia Górecka

Krzysztof Woźniak

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Wednesday, May 23 Warsaw Agricultural University (WAU), Szkoła Główna Gospodarstwa Wiejskiego (SGGW), Nowoursynowska 166 (Old SGGW Campus – Ursynów), The Crystal Hall (building no. 9)

8:00-9:00 Registration and poster mounting 9:00-9:20 Welcome address and introduction

Zbigniew T. Dąbrowski – Local organizer Jörg Romeis – WG Convenor

Prof. Dr. Tomasz Borecki, Rector, Warsaw Agricultural University 9:20-9:30 Short presentation on IOBC/WPRS (Bigler, F.) 9:30-10:05 Anioł, A. (KEYNOTE) (page 8) Research, politics and farmers' needs for GM crops in Poland

10:05-10:50 COFFEE

Report from a special activity on “Non-target risk assessment and regulation” 10:50-11:00 Introduction/background (Jörg Romeis) 11:00-11:45 Presentation of the outcome of this activity

Problem formulation, risk assessment framework (Alan Raybould) Species selection (Franz Bigler) Study design (Joe Huesing) 11:45-12:00 Implementation (Jörg Romeis) 12:00-12:45 Discussion Moderator: Sabine Eber Protocol: Elisabeth Schulte

12:45-14:15 LUNCH (building no. 6) Session: Non-target effects of insecticidal GM crops I Chair: Louise A. Malone 14:15-14:55 Naranjo, S.E., Hellmich, R.L (KEYNOTE) (page 9)

Integrating GM crops in IPM with emphasis on biological control systems 14:55-15:15 Smith, S., Raybould, A., Poppy, G.M. (page 10)

Quality or toxicity – Comparative toxicity of the prey mediated and direct effects of Cry1Ab toxin in the parasitoid wasp Cotesia marginiventris.

15:15-15:35 Li, Y., Romeis, J. (page 11) Assessing the impact of Bt maize pollen on adult green lacewings

15:35-16:20 COFFEE

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Session: Impact of Bt crops on soil organisms Chair: Silvia Fernandez 16:20-16:40 Zurbrügg, C., Nentwig, W. (page 12)

Cry1 and Cry3 toxins show different degradation patterns and thus exposure risk for soil organisms is higher for Cry1

16:40-17:00 Hönemann, L., Nentwig, W. (page 13) Effects of Bt-corn on the soil macro- and mesofauna – A litter bag field study

17:00-17:20 Büchs, W., Prescher, S., Schlein, O. (page 14) Effects of Bt-maize with Diabrotica-resistance and other maize cultivars on saprophagous Diptera larvae

17:20-18:00 Poster presentations (The Cristal Hall, building no. 9) 18:00-20:00 Cocktail party! (The Cristal Hall Annex)

Thursday, May 24 Lecture room 112 (building no. 8) Session: Non-target risk assessment Chair: Marco M.C. Gielkens 9:00-9:40 Sears M.K. (KEYNOTE) (page 15)

Risk assessment of non-target arthropods: the monarch butterfly and Bt maize pollen – a retrospective view

9:40-10:00 Todd, J.H., Ramankutty, P., Malone, L.A. (page 16) A method for selecting non-target organisms for testing the biosafety of GM plants

10:00-10:20 Raybould, A., Stacey, D., Vlachos, D., Joseph, R., Graser, G., Mead-Briggs, M. (page 17) Environmental risk assessment of maize expressing mCry3A for control of forn rootworm

10:20-11:10 COFFEE Session: Non-target effects of insecticidal GM crops II Chair: Salvatore Arpaia 11:10-11:30 Sweet, J. (page 18)

Application of ERA to different environments 11:30-11:50 Schlein, O., Büchs, W. (page 19)

Effects of transgenic maize with Diabrotica v. virgifera - resistance on the feeding rates and development of predatory beetles after consumption of Bt-contaminated prey larvae

11:50-12:10 Priesnitz, K.U., Benker, U., Roß-Nickoll, M. (page 20) Impact of Coleopteran-specific Bt maize on Carabid beetles: results after two years of field and laboratory research

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12:10-12:30 Konrad, R., Babendreier, D. (page 21) Effects of insect-resistant transgenic plants on solitary bees

12:30-14:00 LUNCH (building no. 6) Session: Impact of GMHT crops on biodiversity Chair: Jeremy Sweet 14:00-14:40 Haughton, A.J., Bohan, D.A. (KEYNOTE) (page 22)

The impacts of novel management on ecosystem dynamics; tales from the UK Farm Scale Evaluations of GMHT crops

14:40-15:00 Szekere, D., Kádár, F., Dorner, Z. (page 23) Ground beetles (Coleoptera: Carabidae) in herbicide tolerant (HT) maize hybrid test fields: Impact of HT crop or of weed control practice?

15:00-15:20 Albajes, R., Eizaguirre, M., Casado, D., Pérez, M., López, C., Lumbierres, B., Pons, X. (page 24) Impact of glyphosate use on arthropods related to the cultivation of transgenic herbicide-tolerant maize

15:20-16:10 COFFEE Session: Non-target effects of insecticidal GM crops III Chair: Jozsef Kiss 16:10-16:30 Thu Nguyen, H., Jehle, J. (page 25)

Monitoring the Cry3Bb1 expression of corn line Mon88017 at the field trial in Germany

16:30-16:50 Rauschen, S., Schuphan, I., Eber, S. (page 26) Assessment of possible non-target effects of the novel Bt-corn variety MON88017 resistant to the Western corn rootworm Diabrotica virgifera virgifera (LeConte)

16:50-17:10 Habuštová, O., Doležal, P., Hussein, H.M., Spitzer, L., Turanli, F., Růžička, V., Sehnal, F. (page 27)

Lack of effect of maize expressing bacterial toxin Cry1Ab on the composition of insect communities

17:10-17:30 Husáková, J., Svobodová, Z., Doležal, P., Habuštová, O., Sehnal, F. (page 28)

Effect of Bt toxin Cry3Aa on Spodoptera littoralis (Boisd.)

18:30 Conference dinner (Resteurant Limba Hotel, building no. 38)

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Friday, May 25 Lecture room 112 (building no. 8) Session: Resistance management Chair: Sabine Eber 9:00-9:35 Ferré, J., González-Cabrera, J., Bel, Y., Escriche, B. (KEYNOTE) (page

29) Exploring the potential of corn borers to develop resistance to Bt-corn in Europe

9:35-9:55 Moeser, J., Vidal, S. (page 30) Possible resistance development of Diabrotica virgifera virgifera against transgenic MON 88017 Bt-maize

9:55-10:15 Engels, H., Schuphan, I., Eber, S. (page 31) F2-Screen and field sampling with light trap cages, two methods for a resistance monitoring in transgenic crops

10:15-10:35 Brevault, T., Prudent, P., Vaissayre, M. (page 32)

Baseline susceptibility of Helicoverpa armigera (Hübner) to Bt toxins in West and Central Africa

10:35-11:15 COFFEE Session: Free topics Chair: Zbigniew T. Dabrowski 11:15-11:35 Zaritsky, A., Ben-Dov, E. (page 33)

Transgenic bacteria expressing combinations of genes from Bacillus thuringiensis

11:35-11:55 Hussein, H.M., Procházková, M., Habuštová, O., Sehnal, F. (page 34) Effect of GNA potatoes on the Egyptian armyworm, Spodoptera littoralis

11:55-12:15 Sanvido, O., Stark, M., Romeis, J., Bigler, F. (page 35) Ecological impacts of genetically modified crops: experiences from ten years of experimental field research and commercial cultivation

12:15-12:35 Ammann, K. (page 36) Farming organically and with transgenic plants: a comparison of environmental impact

12:15-12:35 Judziński B. (page 37) Activities and objectives of the Grain and Feed Chamber: actions on objective evaluation and decisions related to GMO

12:35-14:00 LUNCH (building no. 6)

14:00-14:45 Discussion on future activities of the working group; working-group business

14:45 Closure of the meeting 6

Poster Presentations 1. Faragová, N., Faragó, J. (page 38)

Interaction of rhizospheric nitrogenic bacteria with roots of transgenic alfalfa plants containing different transgenes

2. Ferré, J., MacIntosh, S.C. (page 39)

Insect species of importance to currently deployed Bt-crops that have developed resistance to B. thuringiensis toxins in the laboratory

3. Gaspers, C., Engels, H., Schuphan, I., Eber, S. (page 40) Geographic and host plant distribution of pheromone races of the European Corn Borer (Ostrinia nubilalis)

4. Meissle, M., Romeis, J. (page 41) Assessing the risk of Diabrotica-resistant Bt maize for the web building spider Theridion impressum (Ara.: Theridiidae)

5. Górecka, J., Dąbrowski, Z.T. (page 42) Validation of some techniques used in the evaluation of GM plants on tri-trophic relations

6. Twardowski, J.P., Hurej, M. (page 43) European corn borer (Ostrinia nubilalis Hübner) on transgenic Bt corn in Lower Silesia, Poland. Preliminary results

7. Garcia-Alonso, M., Raybould, A. (page 44) Agrisure™ RW: Syngenta’s solution to the corn rootworm pest

8. Lawo, N.C., Romeis, J. (page 45) Performance of Aphis gossypii on Indian Bt cotton varieties

9. Dhillon, M.K., Lawo, N.C., Sharma, H.C., Romeis, J. (page 46) Assessing the direct effects of Galanthus nivalis agglutinin (GNA) and avidin on the ladybird beetle Coccinella septempunctata

10. Farinós, G.P., De la Poza, M., Hernández-Crespo, P., Ortego, F., Castañera, P. (page 47) Diversity and temporal phenology of above-ground fauna in transgenic and conventional maize fields of Central Spain

11. Kiełkiewicz, M., Gajc-Wolska, J., Szwacka, M., Maleszy, S. (page 48)

Unintended changes in biochemistry of cucumber carrying the thaumatin II gene that can affect insect/mite pests

12. Zemek, R., Vávrová, Z. (page 49) Assessing the effects of Bt-maize pollen on Typhlodromus pyri (Acari: Phytoseiidae)

13. Güllü, M., İslamoglu, M., Kanat, A.D. (page 50) Effects of transgenic DK626 Bt (MON810) corn variety to corn stalk borer, Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae) on second corn crop under natural infestation conditions in Turkey

14. Li, Y., Romeis, J. (page 51) Impact of snowdrop lectin (GNA) on adult Chrysoperla carnea

15. Thieme, R., Griess, H., Thieme, T. (page 52) The influence of somaclonal variation on agronomic traits in potato

16. Żurawska, M., Linkiewicz, A.M. (page 53) Cry 1Ab expression in non-target organisms as an element for the correct environmental risk assessment of MON810 in Polish ecosystems

17. Arpaia, S., Di Leo, G.M. (page 54) Foraging behaviour of Bombus terrestris L. on Bt-expressing plants

18. Bereś, P.K., Gabarkiewicz, R. (page 55) Preventing expansion of Ostrinia nubilalis Hbn. by cultivation of Bt transgenic maize – First field experiments in southeastern Poland

19. Tekiela, A., Gabarkiewicz, R. (page 56) Reduction of a threat caused by mycotoxins to mammals and birds by growing Bt maize cultivars in Poland

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KEYNOTE Research, politics and farmer’s needs for GM crops in Poland Anioł, A. Plant Breeding and Acclimatization Institute, Radzików, 05-870 Błonie, Poland The development of green biotechnology indicates that agrobiotechnology can be an important element in improving global agriculture productivity. Its importance is evident in situation when other means of increase in food production exhausted its potential. The decreased potential of traditional agriculture is expressed by decrease in the rate of cereal yield increase from 2.1 per cent per year during 80-ties to less than 1 per cent in 90-ties of XX century. But the utilization of the potential of “gene revolution” depends very much on public acceptance of its methods and products, which is unfortunately very low. And this is a social fact despite that almost all hazards and risks formulated in case of agrobiotechnology are not supported by scientific data. If not overcome in the process of public dispute social rejection of “gene revolution” will make almost impossible its use as a technology in food production and this will have a grave social and economic consequences.

The scientific developments described are proven facts. The main advantage in breeding with the application of transgenesis as compared to traditional methods of recombination and selection is the precision of gene transfer. Transgenesis opens new possibilities for development of new combination of genes, which were not possible to obtain in natural recombination processes therefore opens possibilities of creation of novel cultivars not only with improved agronomic characteristics and better quality as food and feed but also suitable for non-food use.

These new possibilities of novel gene combinations which are so promising for plant breeders at the same time induced strong opposition to this technology based on potential fears of misuse and unpredictable hazards. In order to minimize the risk, the well defined policy and the system of legal and administrative measures was elaborated for control and monitoring of utilization of modern biotechnology in food /feed production and other economic activities. . At the same time it should be stressed that it is necessary to ensure the conditions for co-existence of different types of agriculture which will allow full exploitation of various methods and technologies in food production and by this will give a consumer real possibility of choice.

Present government in Poland is very skeptical to green biotechnology and declared Poland as a “GMO – free country”. The consequences of this attitude and its social and economic consequences are discussed

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KEYNOTE Integrating GM crops in IPM with emphasis on biological control systems Naranjo, S.E.1, Hellmich, R.L.2 1 USDA–ARS, Arid-Land Agricultural Research Center, Maricopa, Arizona, USA 85239 (snaranjo@wcrl.ars.usda.gov); 2 USDA–ARS, Corn Insects and Crop Genetics Research Unit, Ames, Iowa, USA 50011 Modern pest control is guided by the principles of integrated pest management (IPM) that has been articulated for over 50 years. Kogan defines IPM as “a decision support system for the selection and use of pest control tactics, singly or harmoniously coordinated into a management strategy, based on cost/benefit analyses that take into account the interests of and impacts on producers, society, and the environment.” The use of genetically modified (GM) crops, which have activity against insect pests, qualifies as one of the many tactics that can be integrated into IPM strategies for pest insects. Here we will focus primarily on the role of Bacillus thuringiensis (Bt) maize and cotton for managing pests of these crops over the past decade. Worldwide, 32.1 million hectares of Bt and Bt plus herbicide-tolerant crops were cultivated in 2006 and adoption and use of GM crops continues to grow rapidly. For most maize and cotton pests susceptible to Bt toxins, these GM crops are an extremely successful form of host plant resistance. As a result, they substantially alter target pest populations and can potentially influence non-target pests and beneficial arthropods, mainly through indirect paths. Given the prevalence of Bt crops, such changes have, and are, altering the implementation, utility and impact of other IPM tactics like insecticides, biological control, and decision aids. Numerous studies have examined the non-target impacts of Bt maize and cotton and have generally demonstrated, with the exception of specialist natural enemies, that negative effects are minimal. These general patterns have been confirmed recently through meta-analyses utilizing a large number of studies on maize and cotton. Some non-target pest problems, however, have arisen in GM crops, for example, whiteflies and plant bugs on Bt cotton, and aphids and mites in Bt maize. This rise of such pests usually is attributed to reductions in broad-spectrum pesticides that controlled these pests prior to Bt technology. Traditional IPM tactics like scouting and use of thresholds also can be altered by Bt crops whereby different strategies are applied to Bt and non-Bt crops. Given the apparent selectivity of Bt crops towards targeted pests, perhaps one of their greatest assets is the facilitation of biological control, particularly on non-target pests unaffected by Bt toxins. Many studies have clearly demonstrated enhanced natural enemy abundance in Bt crops compared with conventional crops subject to broad-spectrum chemical insecticides. A much smaller number of studies have focused on understanding the functional contribution of this conservation or considered the more subtle indirect effects which may alter natural enemy biology and behavior. For example, studies in the USA revealed that biological control function is unchanged in Bt cotton even though populations of some generalist natural enemies may decline slightly due to target prey reductions. Other studies in Europe have shown that parasitoid/host interactions are not altered in Bt rape and that caterpillar herbivory on Bt and non-Bt maize alters the amount of volatiles produced by the maize but does not change the behavior of parasitoids attracted to the odors. Overall, existing data generally support the beneficial role of Bt crops in providing selective control of key pests and generally reducing the need for disruptive broad-spectrum pesticides enabling more sustainable IPM tactics such as biological control to operate more effectively.

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Quality or toxicity – Comparative toxicity of the prey mediated and direct effects of Cry1Ab toxin in the parasitoid wasp Cotesia marginiventris. Smith, S.1, Raybould, A.2, Poppy, G.M.1 1University of Southampton, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, England; 2Syngenta, Bracknell, Berkshire, UK Plant crops expressing the insecticidal Cry1Ab protein derived from the Bacillus thuringiensis bacteria are widely used in crop protection, therefore there is a potential route of exposure for non-target beneficial insects.

First tier laboratory feeding experiments were carried out to study the prey-mediated effects of artificial diet containing purified Cry1Ab protein on Cotesia marginiventris parasitoids. Activated Cry1Ab toxin was added to Spodoptera littoralis artificial diet at the following concentrations 100, 20, 10, 2 and 1µg g-1. Typical expression levels in the plant being between 7 and 16µg g-1 dependent on age and developmental stage of the plant. Additional experimental trials were also conducted to establish direct toxicity by adding purified Cry1Ab to distilled water at 1mg ml-1 and injecting directly into parasitized hosts.

The objectives of the studies was to establish if C. marginiventris development and fitness was affected due to the direct effects of Cry1Ab toxin (injecting trials) or due to the sub-optimal nature of the host as a result of Cry1Ab incorporation into the host diet.

Parasitoids reared on hosts at the highest concentration of 100ug g-1 had a significantly longer pupation time than at all other doses, tibia length of C. marginiventris raised on hosts at this concentration were also significantly shorter when compared to the control diet.

Preliminary findings suggest that C. marginiventris are adversely affected by prey mediated doses of 20 and 100ug g-1 and that this extension of development time and reduction in fitness is likely to be the result of suboptimal hosts, as hosts reared at these had a significantly reduced mass when compared to controls.

These findings demonstrate the necessity to access whether comprised fitness in beneficial insects occurs due to poor quality hosts or direct toxicity to the juvenile parasitoid. The adverse dose response effect seen in feeding trials relates to toxin levels that are in excess of five times the challenge dose encountered in the field environment, therefore the parasitoid is unlikely to be exposed to these levels. Dose response analysis also indicates the threshold levels at which negative effects can occur, which can be useful in determining regulation.

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Assessing the impact of Bt maize pollen on adult green lacewings Li, Y., Romeis, J.

Agroscope Reckenholz-Tänikon Research Station ART, Reckenholzstrasse 191, CH-8046 Zürich, Switzerland ( yunhe.li@art.admin.ch) The planting of transgenic crops in the world has increased dramatically since the commercialization of Bacillus thuringiensis (Bt) maize in the mid 1990s. An advantage of Bt plants is the reduced need for conventional insecticides with benefits for the environment and human health. Nevertheless, concerns about potential effects of Bt crops on non-target organisms have been raised due to the high and consistent expression of Bt toxin in most plant parts throughout the whole plant growth period.

The green lacewing, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae), has been well studied in respect to potential Bt maize effects. However, previous studies have focused on the predatory larval stage of C. carnea and not on the adults that mainly feed on pollen and honeydew. Pollen from certain Bt maize events such as Event176 and Mon88017 are known to contain considerable amounts of toxin, Cry1Ab and Cry3Bb1, respectively. Maize pollen is abundant during anthesis and readily consumed by adult C. carnea. Therefore, adult lacewings are exposed to the Bt toxin in the pollen grains.

In order to establish a laboratory bioassay to study the potential dietary effects of transgenic pollen on adult green lacewings, the utilization of maize pollen by adult C. carnea was investigated. The results revealed that maize pollen is readily consumed by adult lacewings, and provides, when offered together with sucrose solution, sufficient nutrients for normal egg production comparable to a commonly used artificial diet. Adult C. carnea were fed with pollen from transgenic maize (expressing Cry1Ab or Cry3Bb1) or from corresponding non-transformed maize varieties together with sucrose solution. Over a period of four weeks, different life-table parameters (fecundity, fertility, survival) were recorded. The results revealed no negative effects. To confirm that adult C. carnea are not sensitive to Cry1Ab or Cry3Bb1, additional studies were conducted where the insects were fed with an elevated dose of the two purified Cry proteins incorporated into artificial diet. Again no effects on the recorded life-table parameters were detected.

This study shows that both Bt-transgenic maize events (Event176 and Mon88017) seem to pose a negligible risk to adult C. carnea.

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Cry1 and Cry3 toxins show different degradation patterns and thus exposure risk for soil organisms is higher for Cry1 Zurbrügg, C., Nentwig, W. University of Bern, Zoological Institute, 3012 Bern, Switzerland (corinne.zurbruegg@zos.unibe.ch) Bt-toxins can enter the soil by plant residues or by faeces containing Bt-toxins. Thus, Bt-toxins are a potential risk for the soil ecosystem, and their potential impacts on soil organisms may depend on their persistence. To test how Bt-toxins degrade and how long they persist, we investigated how fast two different Bt-toxins (Cry1Ab, Cry3Bb1) are decomposed in corn fields. Furthermore, we studied how much of up taken Bt-toxin slugs digest, and how much is released via slug faeces into the soil. Degradation patterns of the two Bt-toxins differed significantly from each other in both the corn field and via slug faeces. Initial Cry3Bb1 toxin concentrations in leaves were four times higher compared to leaves containing the Cry1Ab toxin in both green leaves and senescent leaves. However, Cry1Ab was still detectable in higher amounts than Cry3Bb1 towards the end. In the first month, Cry3Bb1 concentration in senescent leaves decreased about 80%, whereas Cry1Ab concentration decreased about 40%. This indicates that Cry3Bb1 is degraded faster by soil organisms than Cry1Ab, suggesting that Cry3Bb1 has a shorter persistence time in soil. Similar degradation patterns of Cry3Bb1 and Cry1Ab were found in the slug feeding experiment. Slugs feeding on transgenic corn leaves excreted Cry3Bb1 toxin at higher concentrations than Cry1Ab toxin. Moreover, we were not able to detect Cry3Bb1 toxin in slug faeces two days after changing diet to dandelion leaves, whereas Cry1Ab was still detectable five days after diet change. Indeed, Cry3Bb1 toxin is expressed in higher amounts in plant tissues than Cry1Ab toxin, but due to faster degradability of Cry3Bb1, soil organisms seem to be exposed for a shorter period to CryBb1 toxin than to Cry1Ab toxin. Thus, to understand the impact and risk of Bt-toxins on soil organisms it is important to know how the different Bt-toxins vary in their degradation patterns.

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Effects of Bt-corn on the soil macro- and mesofauna – A litter bag field study Hönemann, L., Nentwig, W. Zoological Institute, University of Bern, Baltzerstasse 6, CH-3012 Bern, Switzerland (linda.hoenemann@zos.unibe.ch) Bt-corn expresses the Bt-toxin during the whole vegetation period. After the harvest plant residuals are mixed into the upper soil layers by ploughing. The residuals of Bt-corn then still contain the toxin and thus present a potential risk for soil organisms. There are some studies (laboratory and field studies) on Nematodes, woodlice, collembola, soil mites and earthworms which did not show any or no consistent effect of Bt-corn (Saxena & Stotzky 2001, Griffiths et al. 2005, Escher et al. 2000, Wandeler et al. 2002, Pont & Nentwig 2005, Sims & Martin 1997Zwahlen et al. 2003). But currently especially long term experiments under natural field conditions are rare. From October 2005 to June 2006 a litter bag field study was conducted in Switzerland to examine possible effects of transgenic corn litter on the faunistic settlement (including macro- and mesofauna) in the upper 5 cm of agricultural fields We compared litter bags filled with dry leaf material of three transgenic corn varieties (MON88017, MON810, N4640 Bt), the corresponding isolines and three conventional varieties (LG 22.65, LG 22.75, Birko). During the experiment once per month one bag per variety was taken back from each of the 10 study fields. From the remaining leaf material and adherent soil the meso-and macrofauna was extracted by using the MacFadyen method.

If the Bt-toxin has an effect on soil organisms this could get obvious in their abundances, what led to our hypothesis that taxa and/or individual numbers are different for the examined corn varieties. Within the nine month lasting study overall no significant differences in the abundance of the determined taxa were observed for the nine corn varieties. Thus an effect of Bt-corn on soil organisms could not be approved for the examined varieties on the determined taxa level.

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Effects of Bt-maize with Diabrotica-resistance and other maize cultivars on saprophagous Diptera larvae Büchs, W., Prescher, S. Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Pflanzenschutz in Ackerbau und Grünland, Messeweg 11/12, 38115 Braunschweig

In the project „Post-market safety research on transgenic maize with new Bt genes (supported by BMFT) the effects of the cultivation of Bt-maize with Diabrotica-resistence on the saprophagous Diptera is investigated. Larvae of saprophagous Diptera are important for the litter decomposition in agricultural soil and are potentially affected by uptake of Bt-toxine from plant parts. The Bt-maize MON 88017, its origin DKC 5143, the early cultivar Benicia and the late cultivar DK 315 grow on the experimental field and are used for laboratory trials.

From the beginning of the project to the first maize harvest (29.6.-25.9.05) on average 213 Ind./m² Brachycera with saprophagous larvae were caught. Phoridae (60 % of the total catch) and Drosophilidae (30 %) were high abundant. Most Phoridae (abundant species: Metopina oligoneura) emerged in the treatments with Bt-maize, while the highest number of Drosophilidae (abundant species: Scaptomyza pallida) was found in the early cultivar Benicia.

A feeding trial with maize roots of the four treatments and Sciaridae larvae of the species Lycoriella castanescens has been conducted. The most larvae pupated after consuming Bt-maize MON 88017 or DKC 5143 (50 % resp. 48 % of the individuals), while the mortality after feeding Benicia or DK 315 was high (only 35 % resp. 28 % of the larvae pupated). The number of hatched Sciaridae, the duration of the development of larvae and the pupation time will be compared and evaluated.

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KEYNOTE Risk assessment of non-target arthropods: the monarch butterfly and Bt maize pollen – a retrospective view Sears, M.K. University of Guelph, Guelph, Ontario Canada N1G2W1 The purported impact of Bt maize pollen to larvae of monarch butterflies as reported in 1999 has been evaluated by extensive research from 1999-2004 in Canada and the United States. This collaborative effort resulted in a risk analysis that indicated the impact of Bt maize pollen on populations of monarch butterflies in North America was negligible. Since these data were published in 2001 and 2004, little concern regarding Bt pollen and monarch populations has been reported in popular media, but the study has become a touchstone for risk analysis of transgenic insecticidal crops and their risk to non-target organisms. Regulatory agencies around the world are keenly interested in developing a practical, tiered system of risk analysis of genome transformations to non-targets and this example serves as a model for such an approach. Details of the risk analysis approach used in the monarch butterfly issue and developments towards a unified process of non-target impact from such pesticide-incorporated plants will be presented.

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A method for selecting non-target organisms for testing the biosafety of GM plants Todd, J.H., Ramankutty, P., Malone, L.A. Horticulture and Food Research Institute of New Zealand Ltd, Auckland, New Zealand An essential requirement for GM plant risk assessment is the determination of impacts on non-target invertebrates. However, the potential list of non-target invertebrates in any agro-ecosystem is vast, and only a sub-set of these can be tested. We aimed to develop a rational, repeatable method for selecting non-target species for testing the biosafety of GM plants.

Protocols for GM biosafety testing suggest a variety of non-target organism selection criteria, all of which are encompassed by the following five questions: could this crop pose a hazard to this organism?; will this organism be exposed to the hazard?; will there be impacts on ecosystems if this organism is affected?; do people value this organism?; can we perform tests with this organism? We combined these criteria using the following equation to give each candidate species a numerical score corresponding to its suitability as a test organism:

Species suitability score =

where: H = hazard; E = exposureeffects of the hazard); (B + F + web links + special ecosystem fuethical concerns); T = ease with w

+ (B + F + S) + V + T

To test the model, we used thcompiled a database of biologinvertebrate species found in NZGM Bt-pine plant. The databasand each answer was given a scothe invertebrate species susceptibanswer “no” a score of 0. Know5. The database was then interroR, B, F, S, V, T) for each speciesin a ranked list of non-target spetop.

The model offers a flexible, lof organisms. It can easily be adplant modifications. If the user cthe ranking system can be alteecological knowledge gaps byidentifying potential limitations GM plants.

H x E R

; R = resilience (ability of organism to resist or recover from the S) = status of species in ecosystem (biomass + number of food nction); V = anthropocentric value (economic, social, cultural, hich tests may be performed.

e example of a hypothetical Bt-pine forest in New Zealand and ical and ecological information on 80 randomly selected

pine plantations. This was combined with information about a e was composed of answers to 100 questions for each species, re out of 10. For example: the answer “yes” to the question “Is le to the expressed protein?” was given a score of 10 and the ledge gaps (“unknown” answers) were given a middle value of gated to derive values for each of the model’s parameters (H, E, . Final species scores were generated using the model, resulting cies with the most suitable candidate for biosafety testing at the

ogical and transparent way of applying selection criteria to a set apted for use in other agro-ecosystems and with other kinds of onsiders that some criteria are more important than others, then

red to reflect this. It is also possible to expose significant assigning a higher rank to the “unknown” answers, thus to our ability to conduct the best possible biosafety tests with

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Environmental risk assessment of maize expressing mCry3A for control of corn rootworm Raybould, A.1, Stacey, D.1, Vlachos, D. 2, Joseph, R.2, Graser, G.2, Mead-Briggs, M.3 1 Syngenta, Jealott’s Hill, Bracknell, UK; 2 Syngenta, Research Triangle Park, NC, USA; 3 Mambotox Ltd., Southampton, UK (alan.raybould@syngenta.com) Event MIR604 maize expresses a modified Cry3A protein (mCry3A) for control of corn rootworm. As part of the environmental safety assessment of MIR604 maize, risks to non-target organisms of mCry3A were assessed. The potential exposure of non-target organisms to mCry3A following cultivation of MIR604 maize was determined, and the hypothesis that such exposure is not harmful was tested. The hypothesis was tested rigorously by making worst-case or highly conservative assumptions about exposure, along with laboratory testing for hazards using species taxonomically related to the target pest, species expected to have high exposure to mCry3A, or both. Further rigour was introduced by study designs incorporating long exposures and measurements of sensitive endpoints. No adverse effects were observed in any study, and in most cases exposure to mCry3A in the study was higher than the worst-case expected exposure. In all cases, exposure in the study was higher than realistic, but still conservative, estimates of exposure. These results indicate minimal risk of MIR604 maize to non-target organisms.

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Application of environmental risk assessments of pest resistant crops in different environments Sweet, J.B. Environmental Consultant, Cambridge, UK (jeremysweet303@aol.com) Environmental Risk Assessments (ERA) of genetically modified (GM) pest and disease resistant (PR) crops naturally focus on the specificity of the PR genes, lethal and sub-lethal effects on non-targets and food chain effects. Studies conducted in the laboratory (e.g. tier 1 and 2 tests) will identify hazards to particular groups of species and the potential risks to populations. Field experiments and monitoring of crops will identify exposure and hence risks to populations in fields. However concerns remain about the effects of GMPR crops at the landscape scale and the effects that large scale continuous or rotational GMPR cropping may have on species identified as sensitive to PR gene products. Some of these sensitive species may be rare with populations already threatened by agricultural practices and so the GMPR crops may add to the likelihood of localised or regional extinction. This paper reviews some of the recent work in this area and the conclusions for risk assessment and risk management.

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Effects of transgenic maize with Diabrotica v. virgifera - resistance on the feeding rates and development of predatory beetles after consumption of Bt-contaminated prey larvae Büchs, W., Schlein, O. Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Plant Protection in Field Crops and Grassland, Messeweg 11 / 12, 38104 Braunschweig, Germany (o.schlein@bba.de) Polyphagous, carnivorous beetles and their larvae are potentially affected by the coleoptera-specific Bt-toxin Cry3Bb1 in maize fields of the transgenic, Diabrotica v. virgifera- resistant cultivar Mon 88017, when consuming saprophagous insect larvae contaminated with the toxic protein. The present study is part of the project „Post-market safety research on transgenic maize with new Bt genes“ (supported by the BMBF). We conducted laboratory feeding trials with carabid and staphylinid beetles by offering larvae of the fungus gnat Lycoriella castanescens (Diptera:Sciaridae) to the adults and larvae of the predators. The prey larvae were reared in maize plant material, taken from roots and leaves of Mon 88017 as well as of 3 other cultivars (1 origin to Mon 88017, 2 conventional). Possible effects on parameters like the mean daily consumption rate, the developmental time until pupation, the hatching rate of adults and their longevity were evaluated. For an extended feeding trial with the staphylinid Atheta coriaria, a living larvae of L. castanescens was offered daily to young L3 larvae of the rove beetle. Tested predator larvae were divided into 4 separate groups, each fed with prey larvae reared in one of the 4 maize cultivars. Before conducting the experiment, the toxin content of the used Mon 88017 root material as well as of the L. castanescens larvae after root tissue consumption were determined by ELISA. The mean daily consumption rate of A. coriaria larvae, feeding on Bt-contaminated prey larvae, was significantly lower in comparison to feeding rates for prey larvae which have been reared in maize root material from the origin and conventional cultivars. This result was similar to previous findings from feeding trials performed with carabid adults and their larvae as predators. No significant difference between the 4 experimental groups could be found concerning the mortality of A. coriaria larvae and pupae. However, the time until pupation was significantly extended in the A. coriaria larvae group that was consuming Bt-contaminated prey when compared to groups who fed on Lycoriella larvae out of non-Bt-maize cultivars. Therefore, we assume that the Bt toxine Cry3Bb1 has influence on the feeding ecology and developmental duration of some predatory beetles that are common in German maize fields.

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Impact of Coleopteran-specific Bt maize on Carabid beetles: results after two years of field and laboratory research Priesnitz, K.U.1, Benker, U.1, Roß-Nickoll, M.2

1Bayerische Landesanstalt für Landwirtschaft, IPS 2d, Freising, Germany; 2RWTH Aachen University, Institute of Environmental Research (Biology V), Aachen, Germany (Kai.Priesnitz@rwth-aachen.de) Transgenic maize expressing the Coleopteran-specific Bt protein Cry3Bb1 is known as the most effective protection against the Western Corn Rootworm (Diabrotica virgifera virgifera).

In our study we investigated the potential impact of this Bt maize variety on non-target organisms. Ground beetles (Carabids) are important predators in the maize biocenosis and may be exposed to the Bt protein either directly by ingesting plant material or indirectly by eating Bt contaminated prey.

We monitored the relative densities of ground beetles in a field study comprising four maize varieties including transgenic MON88017, the isogenic line and two conventional varieties, each planted in eight plots in a randomised plot design. Weekly samples were taken in each plot using pitfall traps. We identified all Carabid species and performed statistical analyses. The comparison of the four maize varieties showed no significant differences in relative densities of ground beetles.

Exposure of ground beetles to Cry3Bb1 was tested by Enzyme-Linked Immunosorbent Assay (ELISA). We measured the content of Bt protein in different Carabid beetles and quantified the disposition of the Bt-Protein through the food chain. The potential hazard of Cry3Bb1 to four different ground beetle species was tested in bioassays. No acute toxic or behavioral effect could be observed.

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Effects of insect-resistant transgenic plants on solitary bees Konrad, R., Babendreier, D. Agroscope Reckenholz-Tanikon Research Station ART, Reckenholzstrasse 191, 8046 Zurich, Switzerland The cultivation of genetically modified (GM) crop plants carrying insect-resistance traits may pose a risk for beneficial insects such as bees. The honey bee is a key species in non-target testing. Solitary bees are also important pollinators but differ substantially from the honey bee in their biology. Therefore, we tested whether solitary bees are affected by insect-resistant GM crops. We conducted feeding assays with larvae and adults of the red mason bee Osmia bicornis ( = O. rufa) and either a transgenic oilseed rape or purified insecticidal proteins. Larvae were reared on bee-collected pollen provisions from an oilseed rape expressing the protease inhibitor oryzacystatin (OC-I) or on provisions from control rape plants where insecticidal proteins had been added to the provision (i.e. OC-I 0.1%, the snowdrop lectin GNA 0.01% or 0.1%, and the Bt-toxin Cry1Ab 0.01%). The transgenic OC-1 pollen had no significant effect on larval performance during development and subsequent hibernation. A high dose of GNA (0.1%) in the larval diet resulted in significantly longer development time and lower efficiency in converting pollen food into larval body weight. In all other treatments, the measurements taken on different developmental stages were similar to the control. Adult bees were tested in another experiment for potential effects of insecticidal proteins provided in sucrose solution (i.e. GNA, OC-I, the soybean trypsin inhibitor SBTI at 0.01% or 0.1%, and Cry1Ab at 0.01%). GNA (0.1% and 0.01%) and SBTI (only 0.1%) had a significant negative effect on longevity. When bees were allowed to forage on caged transgenic OC-I oilseed rape or control plants no significant difference in longevity was found. The bioassays we developed are well suited for testing transgenic plants or their purified transgene products for effects on larvae and adults of solitary bees.

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KEYNOTE The impacts of novel management on ecosystem dynamics; tales from the UK Farm Scale Evaluations of GMHT crops Haughton, A.J., Bohan, D.A. Ecosystem Dynamics & Biodiversity Group, Division of Plant & Invertebrate Ecology, Rothamsted Research, West Common, Harpenden, Herts. AL5 2JQ. UK (alison.haughton@bbsrc.ac.uk) Concerns about the possible negative impact of the novel herbicide management associated with GM herbicide-tolerant (GMHT) crops on British farm wildlife led to the establishment of the Farm Scale Evaluations (FSEs). This series of field trials evaluated wildlife changes by comparing the wildlife of a GM crop, with its associated herbicide management, against a conventional variety and current ‘best practice’ herbicide management. Using a half-field design, in some 65 fields per GMHT crop distributed across the arable growing areas of Great Britain, the abundance, biomass and diversity of weed plant and invertebrate species or taxa was assessed.

The results showed that there were marked changes in some groups of weed plants and invertebrates with GMHT management. A basic assumption for the trials was that there were no direct effects of GMHT herbicide management on the invertebrates, and that any effects on invertebrates were caused by changes in the weed plants due to differences in herbicide management. Although the experimental approach taken was scientifically rigorous, the repeated testing of the null hypothesis for each species or taxa was bound to generate significant effects just by chance. What was not clear was whether all the observed changes indicated a broad risk to wildlife in farmland. Were the changes important?

The aim of the Ecosystem Dynamics and Biodiversity group is to understand whether there are better approaches to understanding changes in ecosystems with management, and the risk posed. Using analyses of data from the FSEs, I outline some hypotheses for agro-ecosystem structuring, the impact of management on this, and the wider risks to wildlife of these changes. Specifically, I ask: can we detect changes in the agro-ecosystem with novel management; how are invertebrates linked to plants, and can we observe novel management effects on these links; and can we extrapolate to biodiversity groups of social importance, such as birds, but which are difficult to measure in the field?

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Ground beetles (Coleoptera: Carabidae) in herbicide tolerant (HT) maize hybrid test fields: Impact of HT crop or of weed control practice? Szekeres, D.1, Kádár, F.2, Dorner, Z.1

1Szent István University, Department of Plant Protection, H-2100 Gödöllő, Hungary; 2Plant Protection Institute of HAS, Department of Zoology, H-1022 Budapest, Hungary Ground beetles as ground dwelling predators are often used as indicators in field studies when assessing the impact of genetically modified plants on non-target organisms. If genetically modified herbicide tolerant (HT) maize plants are compared to non-modified (non-herbicide tolerant) maize plants as to their impact on non-target arthropod carabids, the comparison is often conducted as glyphosate treated HT maize compared to non-glyphosate herbicide treated non-HT maize. In this case the genetic modification and the weed control practice may have a combined impact on carabids. Activity and density of carabid adults in field crops depend among others on humidity, shadow and temperature at and slightly above the soil surface, prey density (and food plants of potential preys) thus on weed coverage. We aimed at comparing carabid assemblage structure and activity densities in various but all genetically modified HT hybrids treated with glyphosate or with herbicide used in common practice. As part of a larger non-target organism field study with maize hybrids containing various events, thus coleoptera resistant and herbicide tolerant hybrids (CRxHT), lepidoptera resistant and herbicide tolerant ones (LRxHT), coleopteran and lepidoptera resistant and herbicide tolerant ones (CRxLRxHT) were involved in our test. Experimental design:

Above hybrids were planted on plots sized 25x25m arranged in randomized complete block design in four replications (for CRxHT, LRxHT and CRxLRxHT each) in 2006. Hybrid plots were treated as follows: - glyphosate (1,06 kg ai/ha) at V4 and at V7 stage each - non-glyphosate (commercially available registered herbicide used in maize) at V4 stage

Carabid beetles were sampled with three pitfall traps per plots at four different growth stages of maize (mid-V stage, pre-pollination, during pollination and post-pollination). Pitfall traps were placed at a distance from each other near the center of each plot and operated for seven days after setting them. (Traps were filled up with soil when not in use. Killing and preservative agent was ethylene glycol). Carabids sampled by traps were identified to species level. Dominance and Shannon diversity values were calculated. Weed species and density was surveyed during the first week of September on each plot.

The total of 44 carabid species was collected during the maize growing season. Dominant species were Harpalus rufipes, Poecilus sericeus, Dolichus halensis, Calathus ambiguus, H. calceatus and Agonum dorsale in both treatments. Species number and Shannon diversity values did not differ between treatments.

There were significantly higher numbers of individuals and higher activity densities in the second part of the growing season of maize (last two samplings) on non-glyphosate (normal herbicide) treated plots compared to the glyphosate treated ones. Parallel to this, weed densities were significantly higher in almost each plot treated with normal herbicide compared to those treated with glyphosate. Thus there is a good reason to assume that in case of HT maize, the weed control practice and its efficacy impacts the individual densities of carabids in test plots. In other words, less efficient weed control or higher tolerance level by farmer to weeds has an impact on carabid densities instead of HT crop itself.

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Impact of glyphosate use on arthropods related to the cultivation of transgenic herbicide-tolerant maize Albajes, R., Eizaguirre, M., Casado, D., Pérez, M., López, C., Lumbierres, B., Pons, X. Universitat de Lleida, Centre UdL-IRTA, Rovira Roure 191, E-25198 Lleida, Spain Major effects than may be expected from cultivating genetically modified herbicide-tolerant (GMHT) crops are via the alteration of composition, abundance, or phenology of weed flora by herbicide sprayings. In contrast with transgenic Bt crops, in which the expression of the transgenic trait and its repercussions on arthropods cannot be altered to great extent by grower cultural practices, GMHT impact on arthropods (and other organisms) greatly depends on how herbicides are managed.

In 2006, a 4-year farm scale study, sponsored by the Spanish Ministry of Environment, on potential impacts of glyphosate-tolerant maize on maize arthropods was intiated in Spain. A four-block complete randomised design with 0.5 ha elementary plots and two treatments (treated with gliphosate twice at 4/5 and 8/9 leaves growth stages and untreated) was used. Plots were sampled 7 times along the season with visual inspections (25 plants per plot and sampling date) and pitfall and yellow sticky traps (3 traps of each type per plot and sampling date). Comparison of glyphopsate-treated with untreated plots may allow the identification of those arthropods groups that are more dependent on herbicide management and may help to interpret posterior comparisons between broad spectrum and conventional herbicides. Soil insect pests, corn borers and pollinators were not considered for estimations in this first year.

As expected, weeds in untreated plots were more abundant and different in species composition in comparison with treated plots. On-plant countings revealed that leafhoppers and aphids –among the most common herbivores in the trial- were significantly more abundant in glyphosate-treated plots, the same result shown by some predatory groups like Orius spp. nymphs, spiders and trombidids whereas only adults of Nabis sp. were significantly more abundant in treated plots; the rest of groups of predators–coccinellids, carabids, staphylinids, chrysopids, thrips and syrphids were not significantly affected. Leafhoppers and aphids (these not significantly) were more abundant in the yellow sticky traps located in untreated plots but differences were lower than in on-plant countings. Mymarids were more abundant in treated plots contrarily to that found for other parasitoid families such as brachonids and ichneumonids which were more abundant in untreated plots; chalcidids did not show differences. In contrast with that concluded in visual samplings, there were significantly more Orius spp., staphylinids and thrips (both predatory and herbivore ones) on yellow traps placed in untreated plots than in treated ones. Among soil dwelling predators, spiders and carabids showed significantly higher catches in pitfall traps of untreated plots whereas rove beetles and earwigs were not diferent in both kinds of plots. To be noted that catches of elongate Collembola were 2.5 times higher in untreated plots but differences were not significant due to the high variability in catches.

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Monitoring the Cry3Bb1 expression of corn line Mon88017 at the field trial in Germany Thu Nguyen, H., Jehle, J. Biotechnologischer Pflanzenschutz, Abteilung Phytomedizin, Dienstleistungszentrum Ländlicher Raum - Rheinpfalz, 67435 Neustadt/Wstr, Germany Transgenic corn line Mon88017 expressing the insecticidal Cry3Bb1 protein was developed to protect the plants from the insect feeding damage caused by corn rootworm (Diabrotica virgifera). The gene encoding for the transgenic Cry3Bb1 shares more than 99% sequence identity with the wild type protein in Bacillus thuringiensis subsp. kumatomoensis. Mon88017 is also tolerant to glyphosate-based herbicides. From 2001 to 2003, Mon88017 had been field tested in USA. In Europe it was tested for the first time in a field trial as the project “Biosafety research of transgenic Diabrotica-resistante Bt-maize: Develop and evaluation of monitoring methods”.

The field study was conducted during the growing seasons 2005 and 2006. Our major objective is to develop immunological methods (Western Blot, ELISA) for detecting Cry3Bb1 and monitoring the expression of Cry3Bb1 in transgenic corn during three following growing seasons, in order to answer the following questions: Is the expression of Cry3Bb1 protein tissues specific and to which levels is Cry3Bb1 expressed during the vegetation period? Is the expression of Cry3Bb1 stable during different seasons when environmental parameters, such as weather etc. change?

The presentation will focus on results of Cry3Bb1 quantification of different plant tissues taken during the seasons 2005 and 2006. It provides the basic data for ecological studies. Cry3Bb1 levels of more than 600 tissue samples as roots, stalk, under leaf, upper leaf, anthesis, pollen and grain from 4 growth stages (BBCH 21, BBCH 30, BBCH 61, BBCH 83) were quantified using Double Anti-Serum – Enzyme Linked Immuno Sorbent Assay (DAS – ELISA) method. Results show that Cry3Bb1 toxin expression was highest in young leaves (145 - 233 µg/g dry weight). Low levels of Cry3Bb1 protein were detected in the grain (19 - 40 µg/g dw). Cry3Bb1 contents in roots and stalks obviously decreased at later growth stages (BBCH 61 - 83). The estimated total amount of Cry3Bb1 per ha will be also presented.

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Assessment of possible non-target effects of the novel Bt-corn variety MON88017 resistant to the Western corn rootworm Diabrotica virgifera virgifera (LeConte) Rauschen, S., Schuphan, I., Eber, S.

RWTH Aachen University, Institute of Environmental Research, Aachen, Germany (Rauschen@Bio5.RWTH-Aachen.de) The Western corn rootworm (WCR) Diabrotica virgifera virgifera (LeConte) is regarded as a major threat to European corn cultivation since it has been introduced to the Balkans in the early 1990s. So far it has spread very rapidly, reaching economically significant levels in Serbia, Croatia and Hungary.

The novel Bt-corn variety MON88017, which expresses the coleopteran-specific protein Cry3Bb1, is resistant to WCR. In a pre-market risk assessment we investigated possible non-target effects of this corn variety under field conditions. The occurrence and abundance of non-target arthropods, e.g. herbivores, pollen feeders, generalist predators, was assessed in a 4 hectare field experiment with MON88017, its isogenic line DKC5143 and the two conventional hybrids DK315 and Benicia. The four maize lines were planted in a randomized plot design with eight replicates each. Several sampling methods were tested for monitoring the non-target species.

Two arthropod species were selected for detailed studies, based on their prevalence and density in the field, and their exposure to Cry3Bb1 as documented by ELISA tests: the mirid bug Trigonotylus caelestialium (Kirkaldy) and the cicadellid leafhopper Zyginidia scutellaris (Herrich-Schäffer). Results from two cultivation periods did not show significant differences between the densities of these species in plots planted with Bt-corn and the isogenic line. Significant differences exist, however, between these two hybrid lines and the two conventional hybrids. Analyses of the abundance data of T. caelestialium in terms of data gathered on the soil characteristics of individual plots showed no obvious influence of particular soil parameters. For Z. scutellaris the recorded densities varied strongly for different sampling methods and sampling dates.

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Lack of effect of maize expressing bacterial toxin Cry1Ab on the composition of insect communities Habuštová, O.1, Doležal, P.1, Hussein, H.M.2, Spitzer, L.1, Turanli, F.3, Růžička, V.1, Sehnal, F.1

1 Biology Centre AV CR, Institute of Entomology, Branišovská 31, 370 05 České Budějovice, Czech Republic (habustova@entu.cas.cz); 2Nationale Research Centre, Pests and Plant Protection Department, Al Tahrir St., Dokki-Cairo, Egypt; 3 Ege University, Faculty of Agriculture, Department of Plant Protection, 35100 Bornova, Turkey Bacillus thuringiensis is the oldest and most successful microbial agent used in the control of insect pests. This bacterium produces several toxins, of which the crystalline proteins (Cry) are most important. Differences in the type of Cry specify the activity of B. thuringiensis on various insect groups. The gene encoding the protein Cry1Ab was introduced into the genome of maize to provide protection against the stem boring caterpillars such as the European corn borer, Ostrinia nubialis.

The aim of our study was to assess environmental impact of Bt maize MON 810 in comparison with the parental non-transgenic cultivar. Enzyme-linked immunosorbent assay (ELISA) was used to quantify the amount of Cry1Ab in the plants. Selected components of the ecosystem were followed during the vegetation season in three successive years, with focus on the plant-dwelling insects and on the communities of epigeic insects and spiders. The examined species included target and non-target herbivores and predators. The results were evaluated for each sample date and finally for the entire experimental period. The study revealed no significant negative effect of Bt maize on the plant dwelling non-target insects and on the epigeic beetles and spiders.

This study was supported by project KJB6007304 provided by Grant Agency of the Czech Academy of Sciences.

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Effect of Bt toxin Cry3Aa on Spodoptera littoralis (Boisd.) Husáková, J.1, Svobodová, Z. 1, Doležal, P. 1,2, Habuštová, O. 2, Sehnal, F.1,2 1Faculty of Biology, University of South Bohemia, Branišovská 31, České Budějovice; 2Biology Centre AVČR, Institute of Entomology, , Branišovská 31, České Budějovice, Czech Republic (sehnal@entu.cas.cz) Caterpillars of the Egyptian armyworm (EAW), Spodoptera littoralis, were fed a semi-artificial diet supplemented either with a recombinant or with the natural Bt toxin Cry3Aa. Toxin concentration in the diet was verified with ELISA and with a bioassay based on the newly hatched larvae of the Colorado potato beetle (CPB), Leptinotarsa decemlineata. The growth of CPB was hampered with Cry3Aa concentrations below 0.1 ppm, and the survival was reduced with 1 ppm. Since no larval mortality was observed in case of EAW, our study focused on the possible effects on the developmental rate, the pattern of larval growth, female fecundity, and egg hatchability. Neither the developmental rate nor the larval growth was affected by toxin concentrations as high as 8 ppm. However, the fecundity was reduced to 70% of the control value (846 eggs per female) and the egg hatchability dropped from 81% in the controls to 31%.

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KEYNOTE Exploring the potential of corn borers to develop resistance to Bt-corn in Europe Ferré, J., González-Cabrera, J., Bel, Y., Escriche, B. Universitat de València, Department of Genetics, Faculty of Biology, Burjassot, Spain (Juan.Ferre@uv.es) After 10 years of Bt-corn planting in the US, no development of resistance in corn borer populations to Bacillus thuringiensis (Bt) insecticidal proteins has been found in the field. One reason is for sure the strict resistance management imposed by the EPA to the seed companies: a high expression of the Bt protein in the plant and the use of refuges. Europe has started with the adoption of Bt-corn much more cautiously and most of the countries have been reluctant to approve Bt-corn planting. Spain has been the exception, and it has been always the European country with the largest area planted to Bt-corn (over 60,000 ha in 2006).

One of the main threats of adopting Bt-corn is the high selection pressure imposed to the corn borer populations which can lead to the development of resistance. It was for this reason that the EU funded a project with the title “Protecting the benefits of Bt-toxins from insect resistance development by monitoring and management (ProBenBt)” (contract number QLK3-CT-2002-01969), which covered the period from November 2002 till April 2006. The project was coordinated by Ingolf Shuphan from Aachen University (Aachen, Germany) and involved 11 research groups. The objectives of this project were, among others, to characterise the corn borer populations in Europe to determine the genetic diversity and the frequency of resistance alleles. Our group was involved in the mode of action of Bt insecticidal proteins in the corn borers Ostrinia nubilalis and Sesamia nonagrioides, the biochemical characterisation of the resistance mechanism in laboratory-selected resistant strains of O. nubilalis, and the characterisation of major candidate genes for the future application to molecular monitoring of resistance alleles.

In collaboration with the group of Blair D. Siegfried, (University of Nebraska, Lincoln, Nebraska, US), we determined that the mechanism of resistance in a laboratory-selected resistant strain of O. nubilalis, derived from insects collected in Italy, was most likely due to a reduction in the cadherin protein, one of the membrane receptors for the Bt proteins. We have determined the genomic structure of the cadherin gene in this species and have found that it has 34 introns, one of them in the 5’-UTR region, one feature that we also found in the cadherin ortologous genes in other lepidopteran species. The sequence variability in some regions of this gene will be used to design molecular probes for monitoring of resistance alleles in this species.

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Possible resistance development of Diabroica virgifera virgifera against transgenic MON 88017 Bt-maize Moeser, J., Vidal, S.

Georg-August-University, DNPW, Agricultural Entomology, Grisebachstr. 6, 37077 Goettingen, Germany (jmoeser@gwdg.de) We investigate the possible mechanisms, by which the invasive maize pest Diabrotica v. virgifera LeConte could become resistant to the newly developed transgenic maize MON 88017 expressing the Cry3Bb protein. As this event is believed to have a strong detrimental effect only on the first larval stage, we investigated the possibilities of the larvae to first feed on alternative hosts and later switch to transgenic maize. This would allow the larvae to develop relatively normal and selects for individuals that are able to cope with certain amounts of Cry Bb3 during their developmental stages. This in turn could lead to partially resistant individuals, which could aid in a build of a resistant beetle population.

The movement capabilities of the larvae under different soil conditions were investigated as a prerequisite in order to understand the extent of larval mobility. A screening for suitable alternative host plants was carried out using a bioassay. According to the results of the bioassay we chose three alternative hosts (foxtail, wheat and rye) which were used in container trials where we mimicked natural conditions in a quarantine situation. There we planted 1m strips of MON 88017 together or without alternate hosts. These containers we infested with Diabrotica eggs and the larvae extracted 20 days later from the soil and root masses using a high-gradient Kempson extraction. This setup was used to measure the number of surviving larvae and the developmental status in a Bt-maize field with alternate hosts being present or absent.

The results are discussed with regard to resistance development, possible refuge strategies and insect resistance management plans for MON 88017.

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F2-Screen and field sampling with light trap cages, two methods for a resistence monitoring in transgenic crops Engels, H., Schuphan, I., Eber, S. Institute for Environmental Research (Biologie 5), Aachen University, Aachen, Germany

Large-scale cultivation of Bt crops will exert high selection pressure on the target pest, which may consequently evolve resistance. So far, no resistance of the European corn borer Ostrinia nubilalis to Bt crops has been reported. As yet, no anticipatory resistance monitoring plan has been established for Europe. Routine target pest susceptibility screens or the routine use of F2-screens for different Lepidopteran species are in discussion.

When resistance alleles are assumed to be rare, the most efficient method is the F2-screen. This method preserves the genetic variation among isofemale lines and concentrates potential resistance alleles into homozygous genotypes of the F2-generation. This way it is possible to test whether they are recessive or dominant. In our study 450 isofemale lines, started from 650 females, captured in several maize fields in four German regions, were screened over three cultivation periods.

As the F2-screen is time consuming and labour intensive, a simpler long-term monitoring method has been developed and tested. Target pest insects are thereby attracted to light-trap cages containing insect-resistant crop plants. Trapped target insects lay their eggs onto these plants during the cultivation period. Sensitive neonates feeding on the transgenic plants, will die, resistant neonates will survive and develop on the plants. At the end of the cultivation period these larvae can be counted and used for further analyses on the mechanisms of resistance. As a basis for modelling egg masses can be quantified in the light-trap cages throughout the season, so that the number of females can be estimated. So far about 1670 egg masses and thus 50,000 larvae were screened in test trials during one cultivation period in one cage. Neither of the methods revealed any resistant corn borer larvae.

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Baseline susceptibility of Helicoverpa armigera (Hübner) to Bt toxins in West and Central Africa Brevault, T.1,2, Prudent, P.1,3, Vaissayre, M.1

1 CIRAD, UPR 10: Cotton Farming Systems, Montpellier, France; 2 IRAD, Cotton Programme, Garoua, Cameroon; 3 INRAB, ARC-Cotton & Fibers, Cotonou, Benin Burkina Faso will be the first country in West Africa to introduce Bt cotton on a commercial scale in 2007. Undoubtedly other cotton producing countries in this region will adopt the technology soon thereafter. These countries will directly introduce Bt cotton expressing two toxins (Cry1Ac and Cry2Ab)—unlike cotton producing countries elsewhere in the world that began by introducing Bt cotton expressing just one toxin.

The susceptibility of African populations of the bollworm Helicoverpa armigera (Hübner) to Bt toxins is still unknown, and nothing has been published regarding resistance management measures required as a follow-up to the introduction of Bt cotton in Africa. CIRAD is currently building a model to help local authorities develop a strategy for a sustainable use of Bt cotton in small farming systems. Key input factors for the model are: 1) the efficacy of Bt toxins against the bollworm, and 2) the frequency of resistant individuals in naive bollworm populations.

The purpose of the present study was to determine the susceptibility of bollworm strains collected in various cotton growing areas of western and central Africa to Cry1Ac and Cry2Ab and combined, in terms of mortality or, according to the specificity of Bt toxins, in terms of larval growth inhibition.

The results were obtained during the 2006 growing season. They demonstrate the difficulty in evaluating larval mortality (LD50), as well as the advantages of using a growth inhibition index (GI50) for bioassays with Bt toxin. Despite these methodological considerations, the LD50 range as well as the GI50 values obtained in West Africa were quite similar to data published elsewhere.

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Transgenic bacteria expressing combinations of genes from Bacillus thuringiensis Zaritsky, A.1, Ben-Dov, E.2 Departments of Life Sciences1 and Biotechnology Engineering2 Ben-Gurion University of the Negev, POB 653, Beer-Sheva 84105, Israel To avoid resistance in insect pests against Bacillus thuringiensis (Bt), combinations of several toxin genes are cloned for expression in microorganisms that protect the toxins from sunlight inactivation. All 15 combinations of cry4Aa, cry11Aa, cyt1Aa and p20 from subsp. israelensis (Bti) were cloned in Escherichia coli, and the most mosquito larvicidal (with all 4) was transferred into the nitrogen-fixing, filamentous cyanobacterium Anabaena PCC 7120. It displays the highest toxicity against larvae of Aedes aegypti and Anopheles sps. ever reached in transgenic cyanobacteria. This gene combination in E. coli is highly toxic against larvae of Culex quinquefasciatus as well; larval mortality of lines that had been selected for resistance against various combinations of the toxins was only 2.5-3-fold lower. The combination without cyt1Aa is 1000-fold less toxic, except the line selected with Cyt1Aa. The results confirm our notion that recombinant cyanobacteria producing all Bti’s mosquito larvicidal toxins would be as effective as Bti itself, but with additional advantages, and may replace Bti for field delivery. Being of a laboratory origin, it will eventually disappear from the natural habitat by competition with endogeneous species, thus furnish temporal refugia. It is cheaply mass-produced and its activity persists longer than Bti thus anticipated to be highly cost-effective. The transgenic Anabaena endured exposure to sunlight and silt conditions longer than Bti powder (Bactimos) did.

cry1Ac and cry1Ca from subsps kurstaki HD-73 and aizawai 4J4 respectively, were similarly added to E. coli with cyt1Aa and p20. Toxicity of the clone expressing all 4 genes was very toxic against susceptible larvae of Cotton Bollworm (Helicoverpa armigera), displaying substantial synergy between cyt1Aa and both cry genes. Cyt1Aa may thus be an important asset for management of resistant insects pest. The Lepidopteran and Coleopteran species that are not responding to Cyt1Aa may differ in the phospholipids composition of their epithelial midgut cell membranes.

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Effect of GNA potatoes on the Egyptian armyworm, Spodoptera littoralis Hussein, H. M.1,2, Procházková, M.1, Habuštová, O.1, Sehnal, F.1

1Biology Centre AVČR, Institute of Entomology,Branišovská 31, České Budějovice, Czech Republic (sehnal@entu.cas.cz); 2National Research Centre, Dokki, Cairo, Egypt The gene encoding snowdrop lectin GNA (Galanthus nivalis agglutinin) was introduced into the genome of several crops to increase their resistance to insect pests. We compared performance of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, on the GNA-potato and on the parental non-transgenic cultivar. CPB larvae of the penultimate (3rd) and last (4th) larval instars were fed potato leaves freshly cut from the non-flowering plants higher than 20 cm. The leaves (only the pinnate ones) were collected from the base to the top of each plant. GNA expression reduced CPB performance. The biomass increment in course of the experiment and the conversion efficiency of the ingested food were reduced. The adults were smaller and their fecundity was suppressed. However, some of their eggs yielded viable larvae that produced apparently normal beetles when returned to the control potatoes. The larvae grew poorly and produced very few adults when fed on the GNA potatoes. It was not possible to get any insects of a third generation maintained on the GNA plants. (The study was supported by grant 522/02/1507 from the Grant Agency of the Czech Republic).

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Ecological impacts of genetically modified crops: experiences from ten years of experimental field research and commercial cultivation Sanvido, O., Stark, M., Romeis, J., Bigler, F. Agroscope Reckenholz Tänikon Research Station ART, CH-8046 Zürich, Switzerland The worldwide commercial cultivation of genetically modified (GM) crops has raised concerns about potential adverse effects on the environment, which could result from the use of these crops. Consequently, the risks of GM crops for the environment, and especially for biodiversity, have been extensively assessed before and during their commercial cultivation. Substantial scientific data on environmental effects of the currently commercialized GM crops is available today. We have been commissioned by the Swiss Expert Committee for Biosafety to review this scientific knowledge deriving from the past ten years of worldwide experimental field research and commercial cultivation. The sources of information included peer-reviewed scientific journals, scientific books, reports from countries with extensive GM crop cultivation, as well as reports from international organizations. Although there is currently no general consensus on the notion of environmental damage, the data available so far provides no scientific evidence that the commercial cultivation of GM crops has caused significant environmental impacts that would be judged as drastic changes in environmental quality. Nevertheless, a number of issues related to the interpretation of scientific data on effects of GM crops on the environment are debated controversially. The ongoing debate is not primarily due to a lack of scientific data, but more to a lack of clear definitions on how to put a value on effects of GM crops on biodiversity in the context of current agricultural systems. The study highlights these scientific debates and discusses the effects of GM crop cultivation on the environment considering the impacts caused by cultivation practices of modern agricultural systems.

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Farming organically and with transgenic plants: a comparison of environmental impact Ammann, K. Technical University of Delft, Faculty of Applied Sciences, Department of Biotechnology, Julianalaan 67, 2628 BC Delft, The Netherlands In the years since the Convention on Biological Diversity was adopted, issues of traditional knowledge have come to affect the legitimacy of the multilateral trading system, in general, and its IP (intellectual property) aspects, in particular. In order to engage indigenous knowledge in furthering socio-economic development, policy-makers will need to reconsider the prevailing notion of a fundamental dichotomy between indigenous and scientific knowledge and begin to challenge both types of knowledge. This contribution concentrates on traditional knowledge – and how it relates to the ecology of agriculture, in all of its variants – and compares it to recent advances in scientific knowledge and the resulting applications of biotechnology and organic farming in global agriculture.

Deeper examination of the genetic integrity of plants used within organic and biotechnology-based agricultural systems shows that the respective crop varieties being used under each system are more similar than they are different. Increasingly, organic farming is building on scientific knowledge, and agricultural biotechnology is seeking to draw on traditional knowledge.

This contribution challenges policy-makers and scientists to examine and, ultimately, to move beyond those conceptual worldviews, or constructs, that maintain the current divide between traditional knowledge/organic agriculture and scientific knowledge/agricultural biotechnology.

By building the bridge between traditional knowledge and science and becoming free to draw upon the best existing ideas and practices from both, a larger palate is available to draw from. But, more importantly, by integrating the innovation systems of both traditional and scientific communities, a much larger range of new ideas and practices could be generated. The contribution calls such dynamic integration the “participatory approach” to agricultural innovation, building upon the “unifying power of sustainable development” and leading to balanced choices in agricultural production chains and rural land use. For the full account on the topic see: Ammann, K. (2007) Reconciling Traditional Knowledge with Modern Agriculture: A Guide for Building Bridges. In: Intellectual Property Management in Health and Agricultural Innovation a Handbook of Best Practices (eds A. Krattiger, R.T.L. Mahoney, L. Nelsen, G.A. Thompson, A.B. Bennett, K. Satyanarayana, G.D. Graff, C. Fernandez & S.P. Kowalsky), pp. 1539-1559. MIHR, PIPRA, Oxford, U.K. and Davis, USA www.ipHandbook.org. (as of September 2007), Flyer: http://www.botanischergarten.ch/TraditionalKnowledge/ipHandbook-Flyer.pdf and the definite chapter free of copyrights: http://www.botanischergarten.ch/TraditionalKnowledge/Ammann-Traditional-Biotech-2007.pdf

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Activities and objectives of the Grain and Feed Chamber: actions on objective evaluation and decisions related to GMO Judziński, B. The Grain and Feed Chamber, Grzybowska str. 2/49, 00 – 131 Warszawa, Poland (grain@can.waw.pl) The Grain and Feed Chamber is an economic self-governmental organisation which has been functioning since 1997 on the basis of the Act on economic chambers (dated May 30, 1989) entered in the National Court Register and hence constituting a legal entity. There are almost 90 membership companies affiliated to the Chamber, from the sectors of feed production, oilseed crushing industry, milling and storage and the national and international turnover of feeds and feed raw materials, and recently also bio-fuel industry. Among our members there are also companies who provide services for the agricultural sector, such as investments, transport or quality control. Considering that affiliated companies of the Chamber constitute around 70% of the Polish national potential in individual sectors, it can be stated that our organisation covers the largest and widest spectrum of companies operating in the widely understood agricultural market. On the national level we closely cooperate with the National Board of Poultry, National Board of Poultry and Feed Producers, the Polish Association of Breeders and Swine Producers POLSUS, the Polish Association of Breeders and Producers of Meat Cattle, the Polish Association of Corn Producers and the Polish National Seed Chamber. We are also members of the Food Economics Council and the Polish Federation of Food Producers. On an international arena we are members of such organisations as COCERAL (grain companies) and FEFAC (feed producers) with their head offices in Brussels, where our representatives serve in committees of both organisations and participate in the activities of EU bodies (Advisory and Working Committees).

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Poster 1 Interaction of rhizospheric nitrogenic bacteria with roots of transgenic alfalfa plants containing different transgenes. Faragová, N., Faragó, J. Slovak Agricultural Research Center, Research Institute of Plant Production, Dept. of Agricultural Biotechnology, Bratislavská cesta 122, SK-92168 Piešťany, Slovak Republic (faragova@vurv.sk) The aim of our study was to evaluate the nodulation ability and the effect of inoculating by nitrogenic bacteria of three types of transgenic alfalfa (Medicago sativa L.) plants grown in conditions of low soil pH. Transgenic lines, containing the genes Ov from Japanese quail (Coturnix coturnix) coding for ovalbumine, AMVcp from Alfalfa Mosaic Virus (AMV), coding for a coat protein, and the marker genes uidA, coding for β-glucuronidase, and nptII, coding for neomycinphosphotransferase II, respectively, were inoculated by 7 native strains of Sinorhizobium meliloti. These strains showed high tolerance to low pH in an in vitro assay system. The experiment also included a non-transgenic isogenic line SE/22-GT2, plants inoculated with a standard commercial strain of S. meliloti, and a non-inoculated control. Plants were grown in a climatic chamber in plastic pots containing either acidic soil (pH = 4.0) supplemented with P, K, Mg, or neutral soil (pH = 7.0), respectively. The pots were arranged in a completely randomized design. The highest total dry weight (DW) of green matter in low pH soil was obtained in transgenic line GTL4/402-2 (containing the AMCcp gene). Plants grown in neutral soil produced 28% more green matter DW in comparison with those grown in the acidic soil. Inoculation with N2-fixing bacteria increased the DW of green matter from 40% (neutral soil) to 87% (acidic soil). The highest DW of green matter and the highest average shoot number, respectively, in the acidic soil was observed in clones inoculated with the strain 7T4 of S. meliloti. The highest agronomic efficiency, defined as the ratio of difference between the total aboveground biomass DW in inoculated and non-inoculated variant to non-inoculated control, was observed in S. meliloti strains 7T4 and 4T8. The number of nodules on the roots of plants was significantly affected by the genotype and inoculation with rhizobial strain (Statgraphics V., ANOVA, p < 0.05). The highest number of nodules was observed in transgenic clones containing the AMVcp gene, i.e. 85% more than in clones with Ov gene, and 77% more than in marker genes only containing clones of alfalfa. Inoculation with strains of S. meliloti increased the number of nodules from 1.8-fold (commercial strain D113) to 3.3-fold (native strain 7T11). The number of nodules in non-transgenic lines was 8% higher in comparison with Ov-gene containing lines and 3% higher then clones containg the marker genes. Contrary, transgenic alfalfa lines containing the AMVcp gene had in average 71% higher number of nodules than the isogenic non-transgenic line. The highest activity of nodules was also recorded in alfalfa lines with the introduced gene for AMV coat protein, i.e. 70-81% active nodules of the total number of nodules. Inoculation with strain 4T5 increased the N-content in aboveground biomass (stems plus leaves) by 12% in comparison with non-inoculated control. The highest N2-fixation efficiency (11.5%), defined as the ratio of increase in the total N content in aboveground biomass of inoculated variant to total N content in non-inoculated variant, was found in strain 4T5. In conclusion, our results showed a transgene-dependent (positive) interaction of rhizospheric nitrogenic microorganisms with roots of transgenic alfalfa plants. Further research is needed to elucidate the increased nodulation ability of AMVcp transgene-containing alfalfa plants.

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Poster 2

Insect species of importance to currently deployed Bt-crops that have developed resistance to B. thuringiensis toxins in the laboratory Ferré, J.1, MacIntosh, S.C.2

1Universitat de València, Department of Genetics, Faculty of Biology, Burjassot, Spain; 2MacIntosh & Assoc. Inc., Saint Paul, MN, USA (Juan.Ferre@uv.es) Bt-crops have been adopted by many countries and so far, after 11 years of their adoption, no case of insect resistance has been reported in the field. However, laboratory selection has long shown that insects have the potential to develop resistance against Bacillus thuringiensis (Bt) insecticidal proteins (Cry proteins). Here we list and give details of the strains that have been successfully selected for resistance, in the laboratory, of species of importance to current Bt-crops: Heliothis virescens (4 strains), Helicoverpa armigera (9 strains), Pectinophora gossypiella (2 strains), and Ostrinia nubilalis (8 strains).

Resistance levels against Cry1Ab or Cry1Ac have been particularly high in two strains of H. virescens (400 and >10,000-fold), both from North Carolina (USA); 5 strains of H. armigera (over 200-fold) from India, Australia and China; two strains of P. gossypiella (over 100-fold) from USA; and 3 strains of O. nubilalis (>500-fold) from Kansas (USA), Italy, and a mixed population of insects collected in Italy and Nebraska (USA). Selection for resistance to Cry2A protoxins has also been successful, reaching very high levels in H. armigera from New South Wales (Australia) (6800-fold to Cry2Ab and 9600-fold to Cry2Aa) and with O. nubilalis from Kansas (USA)(>640-fold). Resistance is, in all cases, autosomically inherited. However, depending on the strain considered, resistance can be monogenic or due to more than one gene, and its inheritance pattern can be from completely recessive to partially dominant.

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Poster 3

Geographic and host plant distribution of pheromone races of the European Corn Borer (Ostrinia nubilalis) Gaspers, C., Engels, H., Schuphan, I., Eber, S. RWTH Aachen University, Institute for environmental research, Biologie 5, Aachen, Germany

(claudia.gaspers@bio5.rwth-aachen.de) The European corn borer Ostrinia nubilalis (ECB) is one of the most damaging pests of maize in Europe and North America. Two pheromone races of ECB have been identified. Both use 11-tetradecenyl acetate (11-14:OAc) isomers (E and Z) as sex pheromones. Females of the Z-race produce and males respond to a 97:3 ratio of Z11-14:OAc to E11-14:OAc, whereas the opposite E-blend in E-races ranges from 97:3 to 99:1. Small numbers of hybrid females producing an intermediate E/Z pheromone blend of 65:35 were found in regions where the two races occur sympatrically. It is assumed that also in laboratory studies hybrid moths are produced only at a low rate.

Regarding the distribution of different pheromone races on various host plants the E-race is commonly associated with hop and mugwort, and in only a few European countries (Switzerland and Italy) and Eastern North America with maize. The Z-race is associated solely with maize plants. For a resistance management in transgenic crops it would therefore not be advisable to use hop and mugwort as refuges because susceptible E-individuals from these refuges would not mate with resistant individuals from Bt-maize fields.

The pheromone composition of several European ECB populations sampled in maize fields, and from one population each sampled in hop and mugwort was analysed by gaschromatography. Pheromones were extracted from ECB tips incubated in hexan. E-; EZ- and Z- females were identified by comparing retention times and masses of natural compounds with those of synthetic external standards of the isomers. GC-analyses were performed with the parental or the 2nd / 3rd generation, but mostly with advanced laboratory populations.

Populations from Germany, France, Spain and Serbia showed the pheromone composition of the Z-race, but with varying frequencies of hybrids (EZ). In Italy and Greece we also found E-individuals and a considerable number of hybrids. Contradictory to the literature we found Z-individuals in hop and all pheromone types (E, EZ, Z) in mugwort both in an advanced laboratory population and in a freshly sampled field population (both from the same area).

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Poster 4

Assessing the risk of Diabrotica-resistant Bt maize for the web building spider Theridion impressum (Ara.: Theridiidae)

Meissle, M., Romeis, J.

Agroscope Reckenholz-Tänikon Research Station ART, Reckenholzstrasse 191, CH-8046 Zürich, Switzerland, michael.meissle@art.admin.ch Since its introduction in the late 1980s, the Western Corn Rootworm (Diabrotica virgifera virgifera LeConte, Coleoptera, Chrysomelidae) is spreading rapidly over Europe. Especially maize growing regions not following crop rotation are vulnerable to this pest. Larvae feeding on maize roots ultimately cause plant lodging due to reduced nutrient and water supply. Adults feeding on silk and ears cause further damage.

One alternative to chemical pesticides is to grow transgenic maize (event MON88017), which expresses the coleopteran specific Cry3Bb1 protein, originating from the bacterium Bacillus thuringiensis (Bt). The potential impact of insecticidal transgenic crops on naturally occurring beneficial species like generalist predators has to be evaluated in the environmental risk assessment prior to commercial release. In our project, we assess the potential risk of Cry3Bb1-expressing maize for the spider Theridion impressum L. Koch (Araneae: Theridiidae). This species is common all over Europe and reproduces successfully in maize. Prey spectrum analyses conducted in Germany as well as published literature have shown that the spiders are contributing to natural pest regulation. Among the most important groups that they catch in their space webs are aphids, leafhoppers, bugs and other pest species.

As a first step for the risk evaluation, we estimated the potential exposure of the spider to the toxin in an experimental Bt maize field in Germany. Toxin measurements of potential prey species using enzyme linked immunosorbant assays (ELISA) revealed that the prey spectrum consists of taxa containing negligible (e.g. aphids) as well as those with relatively high (e.g. Trigonotylus sp. bugs) toxin levels. Individual T. impressum adults collected in Bt plots also showed a large variation between no detectable toxin and moderate Cry3Bb1 concentrations. In conclusion, exposure of the spider to the insecticidal protein is given, but the exposure level seems to be highly variable depending on the prey consumed.

In a second step, we investigated the hazard of T. impressum being exposed to the Cry protein. Adult spiders were collected in the field before they produced egg sacks and kept individually in the laboratory. Prey species that are known to contain relatively high amounts of Cry protein, i.e. D. v. virgifera beetles fed Bt maize silk and lacewing adults (Chrysoperla carnea (Steph.)) fed Bt maize pollen, were offered alternately for 8 weeks to simulate a realistic worst case situation. No effect on longevity and offspring production was observed. When juvenile spiders were fed with toxin-containing spider mites (Tetranychus urticae Koch) for 8 weeks, both mortality and weight were unaffected.

In summary, T. impressum is likely to be exposed to a highly variable amount of Cry3Bb1 protein in Corn Rootworm resistant Bt maize fields. However, laboratory assays indicate that the hazard of being exposed and consequently the risk for the spider appears to be low.

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Poster 5

Validation of some techniques used in the evaluation of GM plant’s effect on tri-trophic relations. Górecka, J., Dąbrowski, Z.T., Godzina, M., Kubis, K. Department of Applied Entomology, Warsaw Agricultural University, Nowoursynowska St., 02 – 776 Warsaw, Poland In spite of the present position of the Polish Government forbidding commercial plantation of GM crops, independent methodological studies on the effect of GM plants on environment should be carried out. It could not be excluded that the pressure from the farmers’ community may change the present restrictive position of the Government in Poland. Already farmers have witnessed significant advantages of growing resistant GM maize in southern Poland, where the infestation of conventional susceptible cultivars by European corn borer have reached up to 70% on some fields. At the same time, various pro-ecological groups are expressing their anxiety on potential negative unintended effects of GM crops on non-target organisms.

Our greenhouse and laboratory experiments initiated in 2005 have included DKc307, maize cultivar with cry 1Ab gene (insert MON 810) as the GM reference crop and cv. Monumental, its isogenic line as representatives of the fist trophic level and: (a) Anagasta kühniella Zell. and its parasitoid - Venturia canescens; (b) Rhopalosiphum padi L. and parasitic Aphidius colemani (Viereck); and (c) two-spotted spider mite (Tetranychus urticae Koch) and its predator Phytoseiulus persimilis A. H. as the second and third trophic level, respectively. In this paper we report only on two first experiments.

Because our preliminary qualitative chemical analysis indicated some level of Cry 1 Ab toxin in ground DKc307 kernels, therefore our experiment have included Mediterranean flour moth (Anagasta kühniella) as phytophagous pest and its parasitoid - Venturia canescens. No significant differences in larval survival of A. kühniella on MON 810 flour in comparison to its non-GM isogenic cultivar was observed, however, the average weight of larvae reared on MON 810 flour was significant lower. This effect did not affected a level of parasitation by V. canescens. In spite of our conclusion that V. canescens should not be used as a sensitive indicator of food quality change of its host, the results clearly showed that grain and flour made of MON 810 during their storage should be less infested by the moth larvae and the role of V. canescens parasitoid even stronger.

In our greenhouse studies on the role of transgenic crops on tritrophic relations we have included the bird cherry-oat aphid, Rhopalosiphum padi L., the common phytophagous species of cereals in Poland, as the second level and Aphidius colemani (Viereck), as the representative of the third level. A. colemani, an exotic parasitoid commonly used in controlling aphids in greenhouses in Europe was chosen as the bio-indicator. From technical point of view the availability of a species used in experiments on unintended effects due to the genetic modification was mentioned as an important factor in increasing the reproducibility of the tests in different laboratories (Romeis 2004).

It was confirmed that Cry 1 Ab toxin did not showed a toxic effect on R. padi aphids feeding on MON 810. In addition they developed higher (but unsignificantly) populations on the transgenic plants, both in the winter and summer greenhouse tests. Higher aphids’ parasitation by A. colemani was observed on MON 810 in the summer tests and on cv. Monumental in the winter bio-assays, indicating an effect of season on the tri-trophic relations.

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Poster 6

European corn borer (Ostrinia nubilalis Hübner) on transgenic Bt corn in Lower Silesia, Poland. Preliminary results Twardowski, J.P., Hurej, M.

Wroclaw University of Environmental and Life Sciences, Poland, Departament of Plant Protection (jtwardow@ozi.ar.wroc.pl) The substantial discrepancies in risk assessment of transgenic corn cultivars use in Europe indicate that there is an urgent need to study this problem. Till now, there is no sufficient information concerning insect occurrence on Bt corn in Poland. In 2006 in our country corn was grown on approximately 700 thousands hectares, mainly for grain. It is supposed that the corn area will substantially increase in the foreseeable future. This situation can cause a greater corn pests incidence as well. In Lower Silesia, the European Corn Borer (ECB) Ostrinia nubilalis Hübner is the major lepidopteran pest of corn (Zea mays (L.) with necessity of chemical plant protection. Lately, the corn damage caused by larvae rose to 50-80% or even 100%, locally. Losses of grain yield were from 20 to 30%. In the chemical control of the ECB at least twice insecticide treatments are recommended. At the proper time of chemical application plants are very high and the efficient and appropriate chemical intervention causes huge inconvenience. It is important that the transgenic corn with a gene expressing the δ-endotoxin Cry 1Ab from Bacillus thuringensis Berliner var. kurstaki can protect plants against Ostrinia nubilalis throughout the whole growing season.

The aim of the study was to determine the long term impact of Bt corn cultivar on ECB and on non-target organisms in comparison to conventional plants. In this preliminary study only effect of Bt corn on ECB was taken into account.

The preliminary study was conducted on corn field near Wrocław, in Lower Silesia, Poland in 2006. Field of 0.4 ha was divided into two parts. On the first part, transgenic MON 810 corn cultivar (Monsanto Company) with Bt toxin was grown. On the second one, MON 810 conventional form was used. The abundance of pests was monitored once a week, since the last decade of June (beginning of moth flight) till the end of the corn growing season. The observations were done in five places of each treatment (transgenic and conventional) in ten consecutive plants (50 plants in each treatment), along the field diagonal. The visible plant damage done by ECB was recorded on different parts of the plant (stem, cob). At the end of the growing season 100 plants were taken to the lab-analysis from each treatment. The length of the tunnels bored by the larvae and the number of holes were recorded. In addition, presence of Fusarium spp. on corncobs was noted.

During the first year of our study significantly higher level of damage caused by ECB larvae was recorded on conventional plants. The apparent differences were noticed through the whole growing season. Also in the lab-analysis, positive effects of transgenic cultivar were found on different parameters of plant damage caused by larvae. Stem injuries caused by larvae were recorded on 55% of conventional plants and only on 3% of transgenic ones. Similar results were achieved in case of injured cobs, i.e. 14 and 1%, respectively. The number of tunnels bored by ECB larvae reached 48 (mean length 3.5 cm) in conventional and 3 (mean length 0.2 cm) in transgenic. Almost the same proportion of cobs was infected by Fusarium spp. (50% conventional, 46% transgenic).

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Poster 7

Agrisure™ RW: Syngenta’s Solution to the Corn Rootworm Pest

Garcia-Alonso, M., Raybould, A. Syngenta, Jealotts Hill International. Research Center, Bracknell, Berkshire, RG42 6EY, UK Syngenta has produced a genetically modified maize, Agrisure™ RW (MIR604), which expresses a modified Cry3A protein (mCry3A) that controls corn rootworms (CRW), like the Western Corn rootworm (WCRW) (Diabrotica virgifera virgifera, Le Conte) and related species. This maize also expresses the enzyme PMI (phosphomannose isomerase) that acts as a selectable marker.

The mCry3A protein is produced from the mcry3A gene, a synthetic gene related to a cry gene from the naturally occurring soil bacterium, Bacillus thuringiensis (Bt). Syngenta has conducted numerous studies to demonstrate that Agrisure™ RW maize is not materially different in composition, safety, or any relevant parameter from conventional maize now grown, marketed and consumed. Studies have also shown that expression levels and CRW activity are stable across generations, and that the newly produced proteins (mCry3A and PMI) are not toxic to mammals even at very high doses and that their allergenic potential is very low. An environmental risk assessment was also conducted where risks to non-target organisms were assessed. The potential exposure of non-target organisms to mCry3A following cultivation of MIR604 maize was determined, and the hypothesis that such exposure is not harmful was tested. These results indicate minimal risk of Agrisure™ RW to non-target organisms.

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Poster 8

Performance of Aphis gossypii on Indian Bt cotton varieties

Lawo, N.C., Romeis, J. Agroscope Reckenholz-Tänikon Research Station ART, 8046 Zürich, Switzerland (nora.lawo@art.admin.ch) Insect-resistant transgenic cotton plants expressing Cry proteins derived from Bacillus thuringiensis (Bt) are cultivated on an increasing area in India since 2002, reaching 3.8 million hectares in 2006. Bt cotton provides a very effective control of the main pest, the pod borer Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae).

In recent years, several studies examined the effect of Bt crops on non-target organisms. This includes aphids which are not targeted by the Cry proteins expressed by the Bt-transgenic varieties but occasionally reach pest status and play an important role in arthropod food-webs. Since little is known about the interaction of Indian Bt cotton varieties with aphids we have conducted laboratory experiments to investigated the performance of cotton aphids, Aphis gossypii Glover (Hemiptera: Aphididae), on three Indian Bt cotton varieties (MECH 12, MECH 162, MECH 184) expressing Cry1Ac, and their non-transformed near isolines. Bt protein and cotton variety effects on different aphid life-table parameters were determined. Furthermore we have investigated whether the Bt toxin can be detected in the aphids. This would indicate that the protein is transported in the phloem sap on which the aphids feed on.

Our study revealed no significant differences between Bt and non-Bt cotton varieties for any of the different aphid life-table parameters assessed. However, some small differences were observed among the three cotton varieties. None of the aphid samples contained Cry1Ac. As a consequence, natural enemies that feed on aphids are not exposed to the toxin. This finding is consistent with precious studies from other Bt-transgenic plants (maize, oilseed rape, rice and cotton) which have reported only trace amounts of Cry proteins in sap-feeding insects.

Our study allows to conclude that Bt cotton poses a negligible risk for aphid specific antagonists.

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Poster 9

Assessing the direct effects of Galanthus nivalis agglutinin (GNA) and avidin on the ladybird beetle Coccinella septempunctata Dhillon, M.K.1, Lawo, N.C.2, Sharma, H.C.1, Romeis, J.2

1International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India. 2Agroscope Reckenholz-Tänikon Research Station ART, Reckenholzstrasse 191, 8046, Zürich, Switzerland Genes encoding Galanthus nivalis agglutinin (GNA) and avidin have been incorporated in several crops to enhance their resistance to a range of insect pests. The ladybird beetle, Coccinella septempunctata (L.) (Coleoptera: Coccinellidae) is an important predator of aphids and other soft-bodied insects in different crops. Thus, C. septempunctata is likely to ingest insecticidal proteins expressed by transgenic plants either directly by feeding on pollen or through its prey organisms. The present study was conducted to test the direct effects of GNA and avidin on a range of life-table parameters of C. septempunctata. The insecticidal proteins were provided dissolved in a 2M sucrose solution at a concentration of 1% (weight per volume). Neonate C. septempunctata larvae were fed either a pure sucrose solution (control) or a sucrose solution containing GNA or avidin. Every alternate day, predator larvae were fed exclusively with aphid prey. Ingestion of avidin resulted in a significant reduction in larval survival, adult emergence, and adult weight as compared to C. septempunctata receiving pure sucrose solution. Larvae of C. septempunctata appeared to be even more sensitive to GNA since this insecticidal protein caused a 100 % mortality. The results indicate that both GNA and avidin pose a hazard for larvae of the predatory beetle, C. septempunctata.

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Poster 10

Diversity and temporal phenology of above-ground fauna in transgenic and conventional maize fields of Central Spain Farinós, G.P.1, De la Poza, M.1,2, Hernández-Crespo, P.1, Ortego, F.1, Castañera, P.1 1Departamento de Biología de Plantas, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain; 2Centro Nacional de Biotecnología, Cantoblanco, Madrid, Spain (gpfarinos@cib.csic.es) One of the major concerns regarding the release of Bt maize is its potential negative impact on nontarget organisms present in the crop. In this study we compare the temporal phenology and community structure of the above-ground community of arthropods in Bt and non-Bt commercial maize crops of Central Spain, from 2000 to 2002

Spiders, harvestmen, centipedes, ground beetles, rove beetles, carrion beetles, click beetles, earwigs and damsel bugs were captured every year in sufficient number to provide meaningful phenological data. The variability found in activity-density patterns of the above-ground fauna was mostly affected by the year, but no detrimental effects could be associated to transgenic maize.

The three major taxa collected were spiders, ground beetles and rove beetles. One predator spider, Pardosa occidentalis, and three omnivorous species of ground beetles, Poecilus cupreus, Pseudophonus rufipes and Pseudophonus griseus, were consistently present in the maize fields. Rove beetles were catched in a lesser extent, with three dominant species: Acrotona aterrima, Philonthus varians and Platystethus nitens. No shifts in richness and diversity indices of these groups were found between Bt and non-Bt maize.

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Poster 11

Unintended changes in biochemistry of cucumber carrying the thaumatin II gene that can affect insect/mite pests Kiełkiewicz, M.1, Gajc-Wolska, J.2, Szwacka, M.3, Maleszy, S.3

1Department of Applied Entomology, 2Department of Plant Genetics, Breeding and Biotechnology, 3Department of Vegetable and Medicinal Plants, Warsaw Agricultural University, 02-776 Warsaw, Nowoursynowska 159, Poland, (malgorzata_kielkiewicz@sggw.pl)

Thaumatin II gene codes for sweet tasting protein, which shares a significant amino acid homology to PR-5 (pathogenesis-related) proteins, also known as TL (thaumatin-like) proteins. Like other PR proteins, TL proteins when expressed in plants may contribute to endogenous resistance to pathogen/pest attack. They can also be involved in resistance and stress response in plants, although their precise functions are still unknown. Our recent studies showed that transgenic cucumber plants expressing the thaumatin II gene affect the abundance of some piercing-sucking pests but not natural enemies. However, there was no clear relationship between pests density and leaf thaumatin level. Therefore, to better understand the consequence of the presence of thaumatin II protein for chemical composition of transgenic cucumber plants we investigated unintended changes in biochemistry of modified cucumber plants carrying the thaumatin II gene that can influence insect/mite pests.

Four lines of GM-cucumbers (T 224 09, T 225 03, T 212 01, T 210 06) and non-GM inbred line of Cucumis sativus L. cv. Borszczagowski (line B) were grown under field conditions. During the vegetative period, leaf samples were collected and chosen primary and secondary metabolites essentially in host plant – pest interactions were analysed. Additionally, fruit samples at the stage of full fruiting of plants were collected and evaluated for the content of the same compounds as assayed in the leaf samples.

Biochemical analyses revealed that among examined cucumber lines the constitutive level of leaf/fruit biochemical components differs. In some of GM-cucumber lines the concentration of leaf total soluble phenolics and lignin content as well as the value of the lignin:phenolics ratio were altered significantly as compared to the controls. The concentration of glucose, fructose and soluble proteins in GM-cucumber lines was similar to the concentration of leaf primary metabolites in non-transformed line. The changes in the quality of fruits of transformed cucumber lines could be a result of changes in biochemistry of leaves. Disturbance in chemical composition of both leaves and fruits of GM-cucumber lines strongly suggests the alternations of existing biosynthetic pathways in response to transformation. The importance of biochemical properties of cucumbers carrying the thaumatin II gene in herbivorous pests settling, feeding and development is discussed. Further studies are necessary to determine, whether unexpected metabolic changes in GM-cucumber organs are existed in each of the subsequent generation.

Acknowledgements

This work was supported by grant No. 2P06R01729 from the Ministry of Science and Higher Education, Poland.

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Poster 12

Assessing the effects of Bt-maize pollen on Typhlodromus pyri (Acari: Phytoseiidae) Zemek, R.1, Vávrová, Z.2

1Biology Centre AS CR, Institute of Entomology, Branišovská 31, České Budějovice, Czech Republic; 2University of South Bohemia, Branišovská 31, České Budějovice, Czech Republic (rosta@entu.cas.cz) Typhlodromus pyri Scheuten is an omnivorous predatory mite which is considered important for biological control in orchards and vineyards. Its ability to utilize wide range of food including pollen of various plant and tree species has an important role for its success at low prey density.

We assessed the impact of Bt maize (MON 810) expressing Cry1Ab toxin on the performance of T. pyri in laboratory experiments when predatory mites were offered Bt or non-Bt maize pollen as a food source. Various parameters including mortality, development time and fecundity were measured.

The obtained results revealed that predatory mites survived, developed and reproduced well on Bt maize pollen and no significant differences compared to mites reared on non-Bt maize pollen were found. We can conclude that pollen from Bt maize has no detrimental effect on T. pyri.

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Poster 13

Effects of transgenic DK626 Bt (MON810) corn variety to corn stalk borer, Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae) on second corn crop under natural infestation conditions in Turkey Güllü, M., İslamoglu, M., Kanat, A.D. Plant Protection Research Institute, Department of Entomology, P. O. BOX: 21, 01321 Adana, Turkey (mgullu83@hotmail.com) This study has been conducted in Cukurova, Adana where is a place having the Mediterranean climate, during the years 2001-2003 in Turkey. The effects of transgenic DK626 Bt (MON 810) corn variety, that contains insecticidal protein Cry1Ab d-endotoxin from Bacillus thuringiensis, to corn stalk borer, Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae) under second crop maize conditions has been researched by the study. The DK626 and P3394 isogenic hybrid varieties have been used of as comparison varieties.

The experiments were designed as randomized complete blocks with four replications. The infestation rate, number of holes/ stem, number of larva + pupa /stem, infected ears rate, number of larva + pupa / infected ears, and number of larva + pupa/ plant have been recorded by cutting 25 plants/plot on from the surface of land 15-20 days before the harvesting. The 25 plants in the middle of each plot were located at the 5 different points on which there were 5 plants on each point and located near by near as a sequence.

According to the results; the infestation rate with S. nonagrioides, the number of larva + pupa on stem, the rate of infected ears, the number of larva + pupa infected ears and the number of larva+pupa per plant were found as very low in DK626 Bt transgenic corn variety than comparison varieties of years 2001, 2002, and 2003. In the same years these parametric values have been found as so much higher in isogenic DK626 and P3394 varieties. The DK626 Bt hybrid corn has shown a higher level of protection to all three-four generations of S. nonagrioides in second crop under natural field infection conditions.

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Poster 14

Impact of snowdrop lectin (GNA) on adult Chrysoperla carnea Li, Y., Romeis, J. Agroscope Reckenholz-Tänikon Research Station ART, Reckenholzstrasse 191, CH-8046 Zürich, Switzerland ( yunhe.li@art.admin.ch) The gene encoding for snowdrop lectin (Galanthus nivalis agglutinin, GNA) has been engineered successfully into a variety of crops including wheat, rice, tobacco and potatoes to control homopteran pests such as aphids. Since GNA has a broad range of activity and only provides partial control of the target pest(s), the impact of GNA on biological control organisms needs to be assessed.

The common green lacewing, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae), is an important natural predator of aphids in different crops. A previous study by Hogervorst et al. (2006) showed that the longevity of C. carnea larvae was directly affected by GNA. In addition, it was shown that the GNA could not be digested. Since C. carnea larvae are unable to excrete faeces, the protein remained in their bodies.

We investigated whether GNA-feeding during the larval stages has consequences for the performance of adult lacewings. In addition, the direct impact of GNA on adult performance was assessed by providing adults with an artificial diet that contained different amounts of GNA. Subsequently a number of important life-table parameters (survival, fecundity and fertility) were recorded. In addition, we examined the fate of GNA after ingestion by lacewing larvae.

Adult C. carnea were found to be sensitive to GNA and also affected when larvae were sublethally damaged by consumption of this insecticidal protein. Western-blots revealed that GNA ingested by larvae of C. carnea is (partly) transferred to the adult stage and was subsequently excreted or digested within a few days.

The established bioassay was found to be suitable to assess the impact of orally active insecticidal proteins on adult C. carnea. Reference: Hogervorst PAM, Ferry N, Gatehouse AMR, Wäckers FL & Romeis J (2006) Direct effects of snowdrop lectin (GNA) on larvae of three aphid predators and fate of GNA after ingestion. Journal of Insect Physiology 52: 614-624.

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Poster 15

The influence of somaclonal variation on agronomic traits in potato Thieme, R.1, Griess, H.2, Thieme, T.3

1Federal Centre for Breeding Research on Cultivated Plants, Institute of Agricultural Crops, Groß Lüsewitz, Germany (r.thieme@bafz.de); 2Groß Lüsewitz, Germany; 3BTL Bio-Test Labor GmbH Sagerheide, Germany Somaclonal variation is a term used to describe a phenomenon in plants caused by genetic or epigenetic changes induced during callus formation on plant tissues cultured in vitro. Typical genetic alterations are changes in the number and structure of chromosomes, and in DNA sequence, typical epigenetic related events are gene amplifications and gene methylation. The regenerated plants can show genotypic and phenotypic variations. Current techniques used in genetic transformation experiments, involving callus culture and plant regeneration, can result in other traits varying in addition to the expected changes caused by gene transfer.

In order to improve one or more agronomic traits of potato cultivars or breeding clones for potato breeding programmes a study of the induction and analysis of somaclonal variation in plants from in vitro callus cultures of stem and leaf explants, on medium supplemented with different phytohormones, was initiated. About 13,000 somaclones of seventeen cultivars and clones of potato were studied. For this purposes a somaclone is defined as a clone that differs from the donor genotype in at least one or more agronomic traits, induced, by the disorganised cell growth associated with the in vitro procedure. The somaclones were planted in a greenhouse, followed by tuber generations grown in the field. These plants were subjected to the multistage selection procedure commonly used in potato breeding. Over a period of five years and three field generations the haulm growth, earliness, yield, tuber number, size, shape, eye depth, starch content, starch yield and tuber appearance of these somaclones were assessed and compared with that of the controls, the donor varieties. In addition to the frequencies of positive and negative variants the percentage of invariant and positively varying somaclones among the total number of potted somaclones (gain rate) was used to define the effectiveness of this method of potato breeding.

The results indicate that the somaclones of potato vary in haulm growth, earliness, yield and tuber traits. This variation persists for several generations in the field. The degree of variation depends on the genotype and specific characters of the donor. After selection there is a small proportion of desirable aberrants, with better performance in terms of foliage, earliness, yield and tuber characters. Depending on trait the average gain rate for all donor genotypes ranged between 0.2 and 2.3% for –deviants, between 12.2 and 15.5% for invariants and between 0.1–1.4% for +deviants. Therefore, this method could be exploited in potato breeding programmes for improving cvs. or breeding clones specifically for breeding targets such as increasing tuber number per plant, starch content or earliness of one maturity type. For most of the traits tested, 67-98% of the somaclones were indistinguishable from the donor genotype in the first to third field generations. Nevertheless, depending on genotype, up to 15%, 15% and 20% of the somaclones of the second generation in the field consisted of negative variants, in terms of haulm growth, length of the vegetation period and tuber yield per plant, respectively. In the third field generation 1.2–5.5% of the somaclones showed negative variants in tuber number per plant, size of tubers, starch content and yield per plant, tuber shape and eye depth for on average 5 donor cvs. and breeding clones. The unpredictable and uncontrollable nature of somaclonal variation has to be taken into consideration when using in vitro culture for the production of transgenic potato material. THIEME, R.; GRIESS, H.: Somaclonal variation in tuber traits of potato. Potato Research 48, 2005, 153-165.

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Poster 16

Cry 1Ab expression in non-target organisms as an element for the correct environmental risk assessment of MON810 in Polish ecosystems Żurawska, M., Linkiewicz, A.M. Plant Breeding and Acclimatization Institute Radzików, Poland One of the significant components of risk assessment of the insect resistance transgenic plants is to determine the exposure of non-target organism to the Bt toxin. We have focused on non-target arthropods as a major organisms being in contact with transgenic maize. As a numerous factors contribute to gene expression and the level of gene product may vary, studies to assess Bt concentration in plants were conducted using ELISA tests. Maize was grown under different environmental conditions (glasshouse, semi-field conditions). Following arthropod species were taken to the experiments: two-spotted spider mite (Tetranychus urticae) and its predator Phytoseiulus persimilis, aphids (Rhopalosiphum padi, Sitobion avenae, Metopolophium dirhodum), predatory ladybird (Coccinella septempunctata) and stored product pest (Ephestia kuehniella). Quantification of Bt protein in arthropods was determined by immunological assays. ELISA test showed that the amount of toxin in their body varied substantially. Possible impacts of MON810 on herbivores populations and their natural enemies are discussed.

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Poster 17

Foraging behaviour of Bombus terrestris L. on Bt-expressing plants Arpaia, S., Di Leo, G.M.

ENEA – Italian National Agency for New Technologies, Energy and Environment. Research Centre Trisaia, Rotondellan (MT), Italy (salvatore.arpaia@trisaia.enea.it) The introduction at an increasing scale of genetically modified plants (GMP) resistant to insects has fostered numerous studies aimed at monitoring the possible impact of such insecticidal plants on non target organisms. During a previous workshop of this IOBC working group, it has been suggested that the study of ecological functions in the agro-ecosystems through the analysis of arthropod functional groups, could be one of the leading criteria for monitoring GMP (Arpaia, 2004. IOBC-WPRS Bullettin, 27(3): 205-208).

The role of pollinators in agro-ecosystems is very important for most of the high income crops such as fruits and vegetables; many other plants used for animal feed or for industrial scopes are also dependent upon, or benefit from, insect pollination.

Activated Bacillus thuringiensis (Bt) toxins do not seem to have acute toxic effects on honey bees or bumble bees (see Malone and Pham-Delègue, 2001. Apidologie 32: 287–304). Bee-plant relationships though, are rather complex and more biological indicators (e.g. colony development, plant attractiveness, bees feeding behaviour) may be necessary to conduct a thorough risk assessment for flower visiting insects in transgenic agro-ecosystems (Arpaia et al., 2006. in: Environmental Risk Assessment of Transgenic Organisms: A Case Study of Bt cotton in Brazil. CABI International, Wallingsford. U.K.: 155-174).

Any plant transformation event might produce phenotypic modification other than the desired transgene effect. For instance, volatile emissions can be rather different between GMPs and their near isogenic controls (e.g. Uberlacker et al., 1996. Plant Cell 8: 349-362).

In order to detect possible interferences with the normal pollination activity on GMPs, greenhouse experiments were set up to evaluate bumble bee foraging behaviour on Bt-expressing plants and near isogenic controls. Observations were independently made on Bt-expressing canola, eggplants and tomato, in confined cultivations. A commercial colony of Bombus terrestris L. was introduced in the greenhouse and represented the only available pollinator species.

Direct observations were made in order to compare foraging behaviour and frequency of flower visits. Bt-plants were generally accepted as suitable food source by the bumblebees. Nevertheless, the rate of flower visits were often different suggesting that the newly introduced plants were perceived differently from their respective controls.

The possible implications of these findings for Bt crop management will be discussed.

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Poster 18 Preventing expansion of Ostrinia nubilalis Hbn. by cultivation of Bt transgenic maize – First field experiments in southeastern Poland. Bereś, P.K.1, Gabarkiewicz, R.2

1Regional Experimental Station, Institute of Plant Protection, , Langiewicza 28, 35-001 Rzeszów, Poland ( p.bereś@ior.poznan.pl), 2Monsanto Poland, Domaniewska 41, 02-672 Warszawa, Poland (robert.gabarkiewicz@monsanto.com) During last ten years area under maize cultivation is steadily increasing from traditional southern region to central or even northern regions of Poland. The main pest species of maize, the European corn borer (Ostrinia nubilalis Hbn.), used to damage 1 – 25% of plants in the 1957 – 94-ties only in southern, warmer parts of Poland, presently up to 60 – 80%, with 100% on some fields. The serious infestation level has also been recently reported in central regions with infestation of 10 – 15% on its range boundry and there are predictions than the pests will soon cover the whole country.

The staff of the Rzeszów Experimental Station has carried out long term observations and experiments on abundance, biology, economic importance and control methods (including biological control) of maize pests, including O. nubilalis. In the 2005 and 2006 growing season the project has included observations on Bt transgenic cultivars and their parent non-Bt cultivars grown in three locations in the vicinity of Rzeszów .

Tested Bt maize cultivars demonstrated high resistance to the European corn borer infestation. The reduction of stem damage was equal to 97.8 – 100 % in comparison to the non-Bt plants. At the same time other infestation symptoms affecting yield quality and quantity as: broken stem below cob and gnawing up a cob base by larvae were also reduced on Bt plants. The low damage level was due to the sporadic establishment of O. nubilalis larvae in maize stems of Bt cultivars. Comparing economic (cost of equipment) and technical problems with chemical control of maize borers (needs for high wheel tractors and sprayers) and relatively unpredictable efficacy of biological control using Trichogramma spp. we believe that only cultivation of Bt maize cultivars may reduce and delay presently observed expansion of European corn borer, which may bring intolerable yield losses by farmers in Poland. 55

Poster 19 Reduction of a threat caused by mycotoxins to mammals and birds by growing Bt maize cultivars in Poland Tekiela, A.1, Gabarkiewicz, R.2 1Regional Experimental Station, Institute of Plant Protection, Langiewicza 28, 35-001 Rzeszów, Poland (a.tekiela@ior.poznan.pl), 2Monsanto Poland, Domaniewska 41, 02-672 Warszawa, Poland (robert.gabarkiewicz@monsanto.com) In spite of maize diseases of fungal or bacterial origin infecting in lower or higher degree all plantations, their importance is often underestimate by farmers in Poland. The Fusarium group of fungi are consider as the main pathogens attacking maize seedlings, roots, base of stems and kernels in cobs. During the milky and dough stage of kernels the cottony, later pink or red mycelium, pending on a fungus species, is apparent on infected, often disintegrated kernels. The Fusarium kernel rot only occasionally causes substantial yield reduction but affects quality of kernels used for human consumption or animal feed. The other fungi infected kernels belong to: Trichoderma, Penicillium and Trichothecium.

The maize ears infested by stalk borers are usually infected by ear rots, producing such organic compounds as: T-2 toxin, diacetoxyscirpenol, ochratoxin, zearalenone and deoxynivalenol toxic to mammals and birds. Up to now monitoring of the content of these toxic mycotoxins in maize kernels was carried out only on irregular bases. A new directive of European Union on mycotoxic contamination in agricultural products shall give attention to this problem not only by institutions responsible for food safety and processing industry but also farmers.

Our own analysis shows that pending on the plant infestation level by European corn borer and presence of Fusarium fungi, and weather conditions, the average contamination level higher than approved limits was noted from several to 30% of kernel tonnage, eliminating them from processing on animal feed or human consumption.

Presently fungicide treatments are restricted only to seed dressing in Poland, not preventing later infection by pathogenic fungi during the growing season. One of the alternative preventive method would be fungicide sprayings in July and August. However inappropriately carried out treatments may lead to the presence of higher level of pesticide residues in corn flakes or animal feed and presently only a few farmers can afford a specialized spraying equipment.

Our recent field experiments on selected MON 810 maize cultivars has confirmed results obtained in other countries that growing Bt insect – resistant cultivars, substantially reduced a cob infestation by larvae of Ostrinia nubilalis, securing a higher yield and lower ear rots infection of kernels. The chemical analysis of Fusarium originated mycotoxin contamination in kernels of Bt maize cultivars grown in southern region of Poland showed the “O” level or only traces. These facts confirmed farmers opinion that in the Ostrinia infestation regions only growing Bt maize cultivars may guarantee competition of the Polish producers in cultivating maize crop.

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Author index Albajes, R. 24 Ammann, K. 36 Anioł, A. 8 Arpaia, S. 54 Babendreier, D. 21 Bel, Y. 29 Ben-Dov, E. 33 Benker, U. 20 Bereś, P.K. 55 Bigler, F. 35 Bohan, D.A. 22 Brevault, T. 32 Büchs, W. 14, 19 Casado, D. 24 Castañera, P. 47 Dabrowski, Z.T. 42 De la Poza, M. 47 Dhillon, M.K. 46 Di Leo, G.M. 54 Doležal, P. 27, 28 Dorner, Z. 23 Eber, S. 26, 31, 40 Eizaguirre, M. 24 Engels, H. 31, 40 Escriche, B. 29 Faragová, N. 38 Faragó, J. 38 Farinós, G.P. 47 Ferré, J. 29, 39 Gabarkiewicz, R. 55, 56 Gajc-Wolska, J. 48 Garcia-Alonso, M. 44 Gaspers, C. 40 Godzina, M. 42 González-Cabrera, J. 29 Górecka, J. 42 Graser, G. 17 Griess, H. 52 Güllü, M. 50

Habuštová, O. 27, 28, 34 Haughton, A.J. 22 Hellmich, R.L 9 Hernández-Crespo, P. 47 Hönemann, L. 13 Hurej, M. 43 Husáková, J. 28 Hussein, H.M. 27, 34 İslamoglu, M. 50 Jehle, J. 25 Joseph, R. 17 Judziński, B. 37 Kádár, F. 23 Kanat, A.D. 50 Kiełkiewicz, M. 48 Konrad, R. 21 Kubis, K. 42 Lawo, N.C. 45, 46 Li, Y. 11, 51 Linkiewicz, A.M. 53 López, C. 24 Lumbierres, B. 24 MacIntosh, S.C. 39

Maleszy, S. 48

Malone, L.A. 16 Mead-Briggs, M. 17 Meissle, M. 41 Moeser, J. 30 Naranjo, S.E. 9 Nentwig, W. 12, 13 Ortego, F. 47 Pérez, M. 24 Pons, X. 24 Poppy, G.M. 10 Prescher, S. 14 Priesnitz, K.U. 20

Procházková, M. 34 Prudent, P. 32 Ramankutty, P. 16 Rauschen, S. 26 Raybould, A. 10, 17, 44 Romeis, J. 11, 35, 41, 45,

46, 51 Roß-Nickoll, M. 20 Růžička, V. 27 Sanvido, O. 35 Schlein, O. 19 Schuphan, I. 26, 31, 40 Sears, M.K. 15 Sehnal, F. 27, 28, 34

Sharma, H.C. 46 Smith, S. 10 Spitzer, L. 27 Stacey, D. 17 Stark, M. 35 Svobodová, Z. 28 Sweet, J. 18 Szekere, D. 23 Szwacka, M. 48 Tekiela, A. 56 Thieme, R.52 Thieme, T. 52 Thu Nguyen, H. 25 Todd, J.H. 16 Turanli, F. 27 Twardowski, J.P. 42 Vaissayre, M. 32

Vávrová, Z. 49 Vidal, S. 30 Vlachos, D. 17 Zaritsky, A. 33 Zemek, R. 49 Zurbrügg, C. 12 Żurawska, M. 53

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