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1An evaluation of methods for assessing the impacts of Bt-maize MON810 2cultivation and pyrethroid insecticide use on Auchenorrhyncha (Planthoppers 3and Leafhoppers) 4 5Stefan Rauschen1*, Jörg Eckert1, Frank Schaarschmidt2, Ingolf Schuphan1, Achim 6Gathmann1 7 81 RWTH Aachen University, Institute of Environmental Research (Biology V), Chair of 9Ecology, Ecotoxicology, Ecochemistry, Worringerweg 1, D-52074 Aachen, Germany 102 Leibniz Universität Hannover, Faculty of Natural Sciences, Institute of Biostatistics, 11Herrenhaeuser Straße 2, D-30419 Hannover, Germany 12 13*Corresponding author: 14 15Stefan Rauschen 16email: [email protected] 17telephone: +49 (0) 241 80 26676 18fax: +49 (0) 241 80 22182 19 20 21Running title: Assessing the impacts of Bt-maize and insecticide use 22 23Keywords: Bt-maize; MON810; non-target organisms; Zyginidia scutellaris; 24insecticide effect 2 2

25Abstract: 1 Auchenorrhyncha (Planthoppers and Leafhoppers) are not only pests of 26many crops, but they are also non-target organisms with respect to Bt-protein 27expressing genetically modified plants. As herbivorous arthropods, planthoppers and 28leafhoppers ingest Cry proteins depending on their feeding behaviour. Consequently, 29they are directly exposed to these entomotoxic proteins and can also serve as a 30source of Cry protein exposure to predatory arthropods. Auchenorrhyncha, therefore, 31can be reasonably used in the risk assessment of genetically modified crops. 322 During a 2 year field study, we evaluated four different methods in terms of their 33feasibility to assess the impacts of plant-incorporated protectants from Bt-maize and 34of insecticide use on this group of arthropods. Visual assessment of plants, sweep 35netting, yellow traps and custom made sticky traps were utilised in field plots of Bt- 36maize MON810, untreated near-isogenic maize and insecticide treated near-isogenic 37maize and compared in their capability to reflect the diversity and abundance of 38Auchenorrhyncha species. 393 Zyginidia scutellaris (Herrich-Schäffer) (Cicadomorpha: Cicadellidae) represented 40more than 94% of all captured individuals in both years. The analysis of Z. scutellaris 41data showed no consistent differences between Bt-maize MON810 and the untreated 42near isogenic hybrid, showing no negative impact of MON810 on this species. The 43insecticide treatment, on the other hand, was not equivalent to the isogenic maize in 44terms of Z. scutellaris densities. Based on the collected data and on practical 45considerations we recommend the combined use of transect-wise sweep netting and 46sticky traps for the sampling of Auchenorrhyncha in maize. 47 48 49 50 51Introduction 52 53Since their introduction, Bacillus thuringiensis (Bt) modified crop plants have been 54rapidly adopted in many parts of the world and their use is steadily increasing 55(Fishhoff, 1996; James, 2006). These plants express different Cry proteins that are 56toxic to specific arthropod groups and serve as “plant incorporated protectants” 57(PIPs). Until now, crops have been introduced that express Lepidopteran specific 58Cry1 proteins or Coleopteran specific Cry3 proteins. The single most important Bt- 59crop is Bt-maize, with an acreage of 11.1 million ha, equivalent to 10% of the global 60biotech crop area (James, 2006). 61 Planthoppers and leafhoppers are highly abundant and diverse in maize fields 62(Schmitz & Bartsch, 2001; Kiss et al., 2002). Their prevalence and high densities 63make them likely and probably important prey for generalist predators, although their 64role in food-webs is not well understood. Auchenorrhyncha are interesting candidates 65as representative indicators for the non-target risk assessment of Bt-crops (Scholte & 66Dicke, 2005): They will likely take up Cry proteins when feeding on Bt-plants, since 67these proteins are mainly expressed in the green plant tissues. Based on the known 68specificity of Cry proteins it seems unlikely that any insects, other than the specific 69target organism and any closely related species will be negatively affected, including 70Auchenorrhyncha. Notwithstanding, the possible impact on non-target organisms that 71can be assumed to be directly exposed to the entomotoxic protein of a Bt-crop must 72be thoroughly evaluated in the risk assessment. Moreover, when exposed to such 73novel proteins planthoppers and leafhoppers may carry them over to higher trophic 74levels. This may be to predators and parasitoids specialised on Auchenorrhyncha 75(Diptera: Pipunculidae; Hymenoptera: Dryinidae; Strepsiptera: Elenchidae) (Nickel, 3 3

762003) and/or to generalist predators. The mode of feeding differs in taxonomic 77groups (Nickel, 2003); uptake of novel proteins therefore has to be established for 78each species or group individually. So far, Dutton et al. (2004) reported detection of 79Cry1Ab in Zyginidia scutellaris (Herrich-Schäffer) that fed on Bt-maize Bt-11. 80Harwood et al. (2005) showed that Scaphoideus species from Bt-11 maize fields also 81tested positive, but only in 2.2% of all individuals. Different transgenic crops express 82their entomotoxic protein in different and varying amounts (Dutton et al., 2003), 83consequently leading to variable amounts of transgenic proteins in Auchenorrhyncha. 84 To perform a risk assessment of the possible non-target effects of Bt-maize on 85Auchenorrhyncha, methods are needed that reliably and conveniently survey 86planthopper and leafhopper species in the field. Such survey data might also provide 87a basis for the assessment of the food-web significance of Auchenorrhyncha in the 88maize arthropod community and their relevance in trophic interactions. 89 To address this requirement, we evaluated four different methods with regard 90to their sampling efficiency for Auchenorrhyncha in plots with Bt-maize MON810, 91untreated near-isogenic maize and insecticide treated near-isogenic maize. The 92methods were compared based on their costs, sampling efficiency and the number of 93species and individuals caught. The use of multiple methods during the same 94sampling period allows for conclusions about the comparability and robustness of the 95results obtained with them. Many studies rely on only one sampling method (Daly & 96Buntin, 2005; Pons et al., 2005; Whitehouse et al., 2005), which may be a drawback 97if the method and sampling time are not tailored to the organism/s sampled. 98Additionally to the practical comparison of sampling methods, the recorded 99abundance data were statistically analysed in order to quantify the effects of Bt- 100maize and insecticide application on Auchenorrhyncha species. The insecticide 101treatment served both as a negative control and as a standard of comparison for 102potential differences between Bt-maize and the near-isogenic line. The hands-on 103experience, practical considerations and the results of the statistical analyses lead to 104a recommendation of methods for the future use in studies of Auchenorrhyncha in 105the risk assessment of genetically modified plants. 106 107Methods 108 109Three maize treatments were compared in a field study in 2002 and 2003: (1) Bt- 110maize (Bt; transformation event MON810, cultivar Novelis) expressing the Cry1Ab 111protein, (2) the near-isogenic maize line without insecticide treatment (ISO, cultivar 112Nobilis) and (3) the isogenic maize treated with a synthetic pyrethroid insecticide 113(INS; cultivar Nobilis treated with 750 ml Baythroid 50 per ha; active ingredient 50 g 114Cyfluthrin/l; emulsifiable concentrate) representing the conventional cultivation 115strategy. The experimental site was located near Bonn (North Rhine-Westphalia, 116Germany) and consisted of two maize fields approximately 500 m apart. One field 117measured 182 by 248 m with 15 plots (five replications of each treatment arranged in 118three rows of five plots), the other measured 178 by 186 m with nine plots (three 119replications arranged in three rows of three plots). The size of each plot was 0.25 ha. 120The plots were arranged in a randomized complete block design. Planting of maize 121was between mid-April and early May. A mixture of Callisto (900 ml per ha; active 122ingredient Mesotrione) and Gardobuc (900 ml per ha; active ingredients 123Terbuthylazin and Bromoxynil) herbicides was applied 4 weeks after planting. The 124insecticide treatment was in the first half of July. Plants were in growth stages BBCH 12567 - 71 (Meier, 2001), maize stages R1-R2 according to Ritchie & Hanway (1992), at 126the time of sampling. 4 4

127 128Visual assessments of the Auchenorrhyncha assemblages on plants and sweep 129netting were applied in both experimental years and performed as follows. Four 130plants per plot were randomly chosen and assessed once in August. Different parts 131of the plants – leaves, stalks, cobs and panicles – were scanned individually. Plants 132were neither damaged, nor were insects removed from them during the assessment. 133All insects that were deemed to belong to the Auchenorrhyncha based on their habit 134and behaviour were counted, regardless of their developmental stage. 135Catching with a sweep net (diameter 40 cm, mesh width 1.5 mm) was carried out in 136each plot in the same time-frame as the visual assessments. Beginning at about 5 m 137into the gap between two rows of maize plants, thirty one-meter steps (approx. 1383 ms-1) were taken while holding the sweep net vertically in front of the body at a 139height of approximately 90 cm above ground. Sweep netting was performed at four 140transects in 2002 and five transects in 2003 in each plot. Captured insects were 141directly transferred into glass vials with snap-on lids and stored in 70% ethanol until 142identification. 143The use of yellow traps and custom made sticky traps was restricted to 2003. One 144plastic yellow Moericke trap (bowl measuring 340 mm by 260 mm) was mounted in 145the middle of each plot onto a wooden pole at a height of approximately 1.2 m. Traps 146were filled with water and a small amount of detergent and were set up for 3 days in 147August. After this time, the caught insects were immediately transferred to glass vials 148with snap-on lids and stored in 70% ethanol until being sorted and identified in the 149laboratory under a stereomicroscope. 150Sticky traps were constructed from bamboo sticks (length approx. 1.22 m, diameter 151around 10 mm), a rectangular wire frame and a clear office plastic folder (300 mm by 152240 mm, DIN A4) that was slipped over the frame. The surfaces of the folder were 153covered with insect glue (Temmen GmbH, Hattersheim, Germany). One glue trap 154was placed within 5 m distance from each yellow trap, the top of the folder reaching a 155height of around 1 m above ground. Traps were set up in the same time interval as 156the yellow traps. After this time, the folders were covered with cling film, removed 157from the wire frame and stored frozen at -20°C, until the Auchenorrhyncha were 158carefully detached from the glued surfaces with a fine needle. The insects were 159transferred to Eppendorf tubes with 70% ethanol and identified in the laboratory 160under a stereomicroscope. 161The sampling time at the beginning of August was chosen as it had proven to yield 162the highest abundances of Auchenorrhyncha at the experimental site. 163 164Statistical analysis 165One objective was the quantitation of the effect of Bt-maize on selected non-target 166Auchenorrhyncha species. In this respect, the comparison of abundance between the 167Bt-maize MON810 (Bt) and the untreated near-isogenic control variety (ISO) is of 168primary interest. Additionally, the insecticide treatment (INS) is compared with the 169near-isogenic control (ISO). This aimed at verifying that the setting of the trial and the 170different sampling methods were sufficiently sensitive to reveal a severe impact on 171the abundances of species. 172Since the experiment was arranged in a randomized complete block design with 8 173blocks, the mean abundance was modelled in a generalized linear model with log- 174link, including block effect (1-8) and treatment effects (Bt, INS, ISO). For each 175taxonomic group, sampling method and year a separate analysis was performed. 176The counts were assumed to follow a negative binomial distribution, with the 177dispersion parameter estimated from the data (McCullagh & Nelder, 1989). The 5 5

178model was fitted using the R library MASS (Venables & Ripley, 2002). Approximate 17990% confidence intervals were calculated based on estimates for means and 180dispersion from the fitted model, using the R library multcomp (Hothorn et al., 2007). 181For completeness, an Analysis of Deviance Tables is presented for each analysis, 182showing the residual deviance when sequentially adding the block and treatment 183effects to the model, and the p-values for the treatment effect, based on a Chi-square 184test. Second, estimates and 90% confidence intervals for the ratio of mean 185abundances in Bt relative to mean abundances in ISO are presented, denoted 186Bt/ISO. Besides merely describing the effect size of Bt on abundance relative to ISO, 187such intervals can be used to assess equivalence of Bt to ISO with approximately 5% 188error probability (Hothorn & Oberdoerfer, 2006; Altman & Bland, 1995; Chow & Shao, 1892002). E.g., if ratios of mean abundance Bt/ISO of 0.5 to 2.0 are still considered 190acceptable based on ecological reasoning, one could conclude for equivalence when 191both the lower and upper confidence bounds estimated from the data lie within 0.5 192and 2.0. However, such ranges of irrelevant change are usually difficult to define or 193are the subject of controversial discussion. 194Finally, confidence intervals for the ratio of mean abundance in INS relative to ISO 195are presented. Here, the objective is to illustrate that both trial and sampling methods 196were sufficiently sensitive to show detrimental effects on the abundances. One can 197conclude with approximately 5% comparison wise error level that abundances in INS 198are lower than in ISO, if the upper 95% confidence limit for INS/ISO excludes the 199value 1. 200 201The second important issue was the comparison of sampling methods. For each 202method employed, the material cost per trap, the time needed for installation of traps 203and for sampling and processing were recorded. Since labour costs will most often 204be a decisive factor, an estimation of the time associated with a given method will be 205a direct indicator of the cost-efficiency of a method. The level of identification 206possible for a caught specimen and the amount of unwanted bycatch was also taken 207into account. To quantify the variability of the abundance data they yielded, we 208computed the coefficient of variation (CV) as the ratio of the standard deviation σ to 209the mean μ where reasonable, as a measure of a method’s consistency in 210quantitating insects. 211 212Results 213 214Composition of the Auchenorrhyncha assemblage in maize 215In 2002, three different Auchenorrhyncha taxa were caught and identified, while in 2162003 five taxa were detected in the maize plots (Table 1). The leafhoppers 217(Cicadomorpha) were represented by members of two different Cicadellidae 218subfamilies: Zyginidia scutellaris (Herrich-Schäffer) and Empoasca pteridis 219(Dahlbom) from the mesophyll-feeding Typhlocybinae, and Psammotettix alienus 220(Dahlbom) and Macrosteles spec. from the phloem-feeding Deltocephalinae. 221Laodelphax striatella (Fallén), also a phloem-feeder, from the family Delphacidae 222was the only planthopper (Fulgoromorpha) species found. A total of 2612 223Auchenorrhyncha individuals was caught using three of the four methods over the 224two experimental years (Table 1). Identification to species level was not possible with 225visual assessments, so the actual number of specific species is not given for this 226method. However, Z. scutellaris was the most abundant species, representing over 22794% of all individuals caught with the other three methods. The species P. alienus 228and E. pteridis were less abundant, each with a percentage of around 2%. 6 6

229In 2002, sweep netting yielded the highest mean abundance for Z. scutellaris in ISO 230plots (46 ± 38.46 individuals), followed by Bt plots (20.5 ± 10.13) and INS plots (3.5 ± 2312.2). Plot occupancy (the percentage of plots in which a specific species was found) 232of Z. scutellaris was 100% in all treatments. Densities for the other two species were 233too low to be meaningfully interpreted. With the visual assessments a higher total 234number of Auchenorrhyncha was recorded in Bt plots (150 specimens over 8 plots), 235compared to ISO (95) and INS plots (7). Plot occupancy was 100% in Bt and ISO 236plots and 62.5% in INS plots. 237In the multi-method comparison in 2003, the sticky traps detected the different 238Auchenorrhyncha species in a higher number of cases than the other two methods 239and yielded the highest total abundances (Table 1, Table 2). The most abundant 240species Z. scutellaris was caught with the yellow traps and sweep netting in a 241comparable frequency, but with overall lower numbers. For all other species, yellow 242traps and sweep netting caught fewer individuals. Plot occupancies of the 243Auchenorrhyncha species were consistently underestimated by these two methods 244when compared with the sticky traps, except for Z. scutellaris for which plot 245occupancy as measured with the sweep nets was 100% in all treatments. 246 247Impact of Bt-maize MON810 and insecticide use on Zyginidia scutellaris 248Only the data for Z. scutellaris and the visual assessment of Auchenorrhyncha could 249be statistically analysed, since all other species were found in numbers too low for a 250meaningful analysis. In 2002, the Block effect (i.e. differences between the plots 1-8 251of one treatment) accounted for about 33% and 20% of the variability of the data, for 252Z.scutellaris and Auchenorrhyncha, respectively. The overall treatment effect (Bt, 253INS, ISO) in the analysis of deviance was highly significant (Table 3). The confidence 254intervals in Table 4 show, that these differences can be mainly attributed to the 255strong effect of the insecticide treatment (INS). 256The 2002 sweep net data of Z.scutellaris (Table 4, Figure 1) indicate a lower mean 257abundance of the maize leafhopper in Bt plots compared to ISO plots. The 258comparison between the insecticide treated plots and the near-isogenic line shows, 259however, a much stronger impact of the pyrethroid insecticide on Z. scutellaris. While 260the abundance of the maize leafhopper in Bt plots corresponded to between 38% 261and 78% of the abundance in ISO plots, it was down to between 6% and 14% in INS 262plots. For the visual assessment of Auchenorrhyncha, the estimated ratio of mean 263abundance in Bt was 137% of that observed in the ISO plots, with a corresponding 264confidence interval ranging from 78% to 243%. Also here, the insecticide treatment 265resulted in a marked decrease of abundance relative to ISO, mean abundance 266decreased to 3% to 16% of the abundance in ISO. 267For the 2003 data (Table 3, Table 4, Figure 1), similar overall results were obtained: 268The block effects accounted for 6–24% of the overall variation in the data, the 269treatment effect was highly significant in all 4 analyses. The point estimate for the 270ratio Bt/ISO computed for the yellow traps indicates a mean reduction of Z. 271scutellaris in Bt-plots to 72% of the densities in ISO plots. The confidence interval 272indicates that the true ratio lies with 90% confidence probability between 53% and 27397%. The observed reduction is not corroborated by the data from the other 274methods. In fact, sticky traps and the sweep netting in the Bt-plots resulted in more 275individuals than in the near-isogenic plots: sticky traps yielded over 1.21-fold more 276individuals in the Bt-maize; sweep nets yielded 1.22 fold more, when compared to 277the isogenic control. The visual assessments indicated a 1.46 fold increase in total 278Auchenorrhyncha in Bt-maize versus the near-isogenic maize. In summary, there 279was no consistent negative impact of Bt-maize on Z. scutellaris numbers observed in 7 7

2802003. Although there were abundance differences, three out of four methods 281identified no difference between Bt-maize and the untreated near-isogenic maize. In 282contrast, all methods in unison highlighted a reduction of Z. scutellaris and total 283Auchenorrhyncha due to the insecticide treatment. The comparisons INS/ISO show 284that Z. scutellaris was reduced in INS plots to between 1% and 25% of the 285abundance in ISO plots. While the magnitude of the reduction varied among the 286methods, significant differences between INS/ISO were clearly identified by all four 287methods. 288 289Comparison of the four methods used in 2003 290An evaluation of the efficiency and practicability of the used methods are presented 291in Table 5. 292The yellow traps have the highest material cost. The sticky traps are made of 293commercially available parts that can be bought cheaply at most utilities stores. The 294visual assessment of plants and sweep nets have no “per trap” cost, except the cost 295of purchased material. 296Yellow and sticky traps need to be assembled and installed on the field, a process 297that consumes a considerable amount of time per trap. However, once set up, these 298traps can be used repeatedly at the same location and are therefore ideal for long- 299term sampling. 300Time for sampling insects from the yellow and the sticky traps is a matter of minutes. 301Using the sweep nets and visually assessing plants is more time consuming, but the 302time needed depends very much on the experience of the researcher. On the other 303hand, yellow traps require a huge amount of time for sample processing, since a 304variety of insects is caught. If only a specific group of insects is the focus of interest, 305as in this study, this bycatch is wasted effort. Sticky traps also catch a variety of 306insects, but Auchenorrhyncha can be sorted from the plastic folders more specifically 307and hence more quickly. The use of a sweep net also requires a rather limited time 308for sorting insects. 309Identification down to the species level is possible without difficulty with the yellow 310traps and the sweep netting, but takes some experience with the sticky traps. Visual 311assessment does not allow species identification, because detailed observations are 312not possible in most cases. 313The coefficient of variation (CV) was calculated for the abundance of Z. scutellaris 314pooled over plots with Bt- and the near-isogenic maize for every method. The 315variation of the abundance data is similar among the different methods with only little 316less variation for the sticky traps. This suggests that all methods reflect the densities 317of this species equally consistently. 318 319Discussion 320 321Z. scutellaris was found in all plots and mostly in very high abundances in both years. 322This leafhopper species is reported to be a mesophyll feeder (Nickel, 2003) and has 323been shown to ingest Cry1Ab when feeding on Bt-maize plants (Dutton et al., 2004; 324Obrist et al., 2006). Despite the ingestion of Cry1Ab from the genetically modified 325maize, we found no evidence for a negative impact of MON810 on Z. scutellaris. 326 In 2002, the abundance of Z. scutellaris was reduced in Bt plots when 327compared to ISO plots. This reduction was not corroborated by the counts of 328Auchenorrhyncha with the visual assessments, which yielded a higher total amount 329of planthoppers and leafhoppers in Bt-maize plots. Since Z. scutellaris proved to be 330the most prevalent species of the Auchenorrhyncha assemblage in maize, it may be 8 8

331concluded that most of the specimens visually observed belonged in fact to that 332species. The reason for the higher mean abundance of Z. scutellaris in ISO plots as 333detected with the sweep netting may be based on the natural variability, evidenced 334by the Block effect, enhanced by local microclimatic conditions because of 335differences in Ostrinia nubilalis infestation: the higher mean abundance is due to 336higher counts in plots of one field only, namely the one with 3 out of the 8 replicates. 337The remaining five replicates, arranged in the other field, yielded a mean abundance 338of 18.8 Z. scutellaris per plot, which is comparable to the mean abundance in the Bt- 339maize plots. This fact is reflected in the high variability in the ISO treatment in 2002 340compared to the other treatments in both years (Fig. 1). The other plots in the small 341field, with Bt and INS treatments, showed only slightly higher abundances when 342compared to their respective remaining replicates. It cannot be ruled out that 343differences in Ostrinia nubilalis infestation, which are only manifested in ISO plots, 344between the two fields is causal to these differences. According to the proof of safety 345approach, one could conclude for equivalence of Bt to ISO with respect to 346Z.scutellaris and Auchenorrhyncha if abundance in Bt between 38% and 243% of the 347abundance in ISO is still judged as a non-relevant change. Whether abundance 348differences of this order are of ecological relevance in the maize agroecosystem is 349unclear, however. 350 In 2003, the abundance of Z. scutellaris was slightly increased in Bt-maize 351plots when compared to the near-isogenic untreated control, as evidenced by the 352results of three out of the four methods. This increase was not significant, however, 353based on the rationale of the statistical approach, if a range of abundance in Bt-plots 354of 53% to 209% of the abundance in ISO-plots can be still regarded as an acceptable 355range. Hence, considering results of both years, differences between Bt and ISO 356indicate no adverse effects of Bt-maize on Z. scutellaris. 357 On the other hand, the effect of the pyrethroid insecticide treatment on 358Auchenorrhyncha was severe. The occurrence and abundance of these arthropods 359was highly reduced, down to between 1% and 25% of the abundance in the 360untreated near-isogenic control. Similar impacts have been reported by many 361studies, including some studies that only used one method to enumerate specific 362groups of insects (Bhatti et al., 2005; Daly & Buntin, 2005; Dively, 2005; Eckert et al., 3632006; Gathmann et al., 2006; Whitehouse et al., 2005). 364 Pons et al. (2005) reported an increase of Z. scutellaris in a Bt176 Bt-maize 365hybrid and attributed this to changes in the Bt-maize plants that favour this species. 366Daly & Buntin (2005) found a genotype x Bt interaction for leafhoppers while 367assessing the possible impacts of two different MON810 derived Bt-maize varieties. 368This might hint to some influence of different hybrid lines or genotypes on these 369arthropods. Other studies have investigated the possible impact of different Bt-crops 370on planthoppers and leafhoppers, but found no statistically significant negative 371effects (Dively, 2005; Chen et al., 2006). Whitehouse et al. (2005) on the other hand 372found lower densities of Cicadellidae in two Bt-cotton hybrids when compared to a 373conventional unsprayed control. 374 Most of the cited studies relied on only one method to collect their data: 375Whitehouse et al. (2005) used a suction sampling method, Pons et al. (2005) a leaf 376sampling method and Daly & Buntin (2005) visual counts. Chen et al. (2006) 377employed three different methods (yellow sticky cards, Malaise traps and a vacuum- 378suction machine), to assess the Auchenorrhyncha phenology, adult dispersal and 379seasonal patterns in population dynamics. They propose the vacuum-suction method 380as the method of choice, depending on the prevalent species. As can be seen from 381the work on hand, data from different methods can lead to different results, leading to 9 9

382different interpretations of the abundance and occupancy data. Accordingly, studies 383that try to assess possible impacts of Bt-crops should consider using methods that 384complement each other to thoroughly evaluate their test plants. In addition, risk 385assessment should cover more than one growing season. Conclusions based on one 386year studies could possibly lead to wrong decisions as shown by our results on 387sweep netting. The reduction of Z. scutellaris in the first year could not be confirmed 388in the second year. In contrast, in the second year the number of individuals was 389higher in the Bt-plots compared to the control. 390 Most Auchenorrhyncha taxa found in this study suck on phloem vessels and 391therefore are not anticipated to take up transgenic proteins from Bt-maize, since is 392has been repeatedly shown that Cry proteins are not translocated into the phloem of 393Bt-maize plants (Head et al., 2001; Raps et al., 2001; Dutton et al., 2003). 394 A species occurring in such high densities as Z. scutellaris will be a likely prey 395of, at least, generalist predators. The assessment, therefore, of the occurrence and 396abundance of Auchenorrhyncha serves two purposes: (1) to investigate possible 397negative impacts of Bt-transgenic plants on this group of non-target arthropods, and 398(2) to elucidate potential effects on the trophic chain where Auchenorrhyncha are 399prey of predatory and parasitic arthropods. To accomplish such an assessment, 400robust methods are needed that reliably and conveniently detect Auchenorrhyncha 401species and quantitate their abundance. The four methods described here in unison 402detected the severe negative impact of the insecticide treatment on the 403Auchenorrhyncha assemblage, while no consistent negative impact of the Bt-maize 404MON810 on Z. scutellaris could be found. This leafhopper species sampled with any 405of the four methods can thus be used to assess the impact of environmental changes 406due to agricultural practice. Based on the data collected with the different methods, 407the estimated costs and the hands-on experience we recommend the combined use 408of custom made sticky traps and the transect-wise catching with sweep nets as a 409good means for the sampling of Auchenorrhyncha. While the former method yields 410the highest abundances and thus a good data basis for statistical analyses, the latter 411can be used to quickly survey for the presence of other notable Auchenorrhyncha 412species and to capture individuals for accurate identification and reference. 413 414Acknowledgements 415 416The authors thank D. Bartsch, S. Eber and R. Hellmich for comments on earlier 417versions of the manuscript, H. Nickel for help in identifying the Auchenorrhyncha 418species, and the Federal Ministry of Education and Research for financial support 419(0312631.c). 420 421References 422 423Altman, D.G. & Bland, J.M. (1995) Absence of evidence is not evidence of absence. 424British Medical Journal, 311, 485. 425 426Bhatti, M.A., Duan, J., Head, G., Jiang, C., McKee, M.J., Nickson, T.E., Pilcher, C.L., 427Pilcher, C.D. (2005) Field evaluation of the impact of Corn Rootworm (Coleoptera: 428Chrysomelidae) – protected Bt corn on ground-dwelling invertebrates. Environmental 429Entomology, 34, 1325-1335. 430 10 10

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533Figure 1 Box-whisker plots of the abundances of Z.scutellaris and Auchenorrhyncha 534quantified by different sampling methods in 2002 and 2003. The bold horizontal line 535of the box plot gives the median, the box represents 50% of the data, upper and 536lower dashes or dots (circles) represent the maximal and minimal values observed in 537the sample, where dots represent the maximal or minimal values if these are further 538away from the box than 1.5 times the interquartile range. (BT: Bt-maize MON810; 539ISO: near-isogenic line; INS: insecticide treatment) 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555Table 1 Overview of the Auchenorrhyncha species caught with the respective 556methods, their abundances summed over all treatments and their percentage of all 557individuals. 2002 total percent yellow sticky sweep visual traps traps nets assessments Zyginidia scutellaris 560 97.6 - - 560 - Empoasca pteridis 12 2.1 - - 12 - Macrosteles spec. 2 0.3 - - 2 - subtotal 574 100 - - 574 0

Auchenorrhyncha 252 - - - 252 total 826 - - 574 252

2003 total percent yellow sticky sweep visual traps traps nets assessments Zyginidia scutellaris 1920 94.2 400 1078 442 - Psammotettix alienus 45 2.2 1 39 5 - Empoasca pteridis 42 2.1 26 11 5 - Macrosteles spec. 29 1.4 2 24 3 - Laodelphax striatella 2 0.1 1 0 1 - subtotal 2038 100 430 1152 456 0 14 14

Auchenorrhyncha 173 - - - 173 total 2211 430 1152 456 173 558 15 15

559Table 2 Descriptive statistics of abundances recorded with the specified methods in 560the different maize treatments Bt-maize MON810 (Bt), insecticide treated near- 561isogenic maize (INS) and untreated near-isogenic maize (ISO). Occupancy (Occ.) 562gives the percentage of plots in which a species was recorded, arithmetic mean 563(Mean), standard deviation (SD), median (Median) and interquartile range (IQR), 564defined as the difference between the q0.25 and q0.75. Bt INS ISO Yellow traps Species Occ. Mean SD Median IQR Occ. Mean SD Median IQR Occ. Mean SD Median IQR Zyginidia scutellaris 100 20.4 11.8 19 14.5 50 0.6 0.7 0.5 1.0 100 29.0 18.2 28.5 33.3 Macrosteles spec. 12 0.1 0.4 0 0.0 0 0.0 0.0 0 0.0 12 0.1 0.4 0 0.0 Psammotettix alienus 0 0.0 0.0 0 0.0 0 0.0 0.0 0 0.0 12 0.1 0.4 0 0.0 Empoasca pteridis 38 1.5 2.5 0 2.3 25 0.6 1.4 0 0.3 38 1.1 2.1 0 1.3 Laodelphax striatella 0 0.0 0.0 0 0.0 12 0.1 0.4 0 0.0 0 0 0 0 0.0 Sticky traps Occ. Mean SD Median IQR Occ. Mean SD Median IQR Occ. Mean SD Median IQR Zyginidia scutellaris 100 69.9 27.6 69.5 45.8 88 2.4 1.6 2 1.5 100 62.5 41.4 50.5 26.3 Macrosteles spec. 88 1.4 0.9 1 1.0 38 0.4 0.5 0 1.0 88 1.3 0.9 1 0.3 Psammotettix alienus 62 1.6 2.3 1 2.0 62 0.8 0.7 1 1.0 62 2.5 3.7 1.5 3.0 Empoasca pteridis 38 0.8 1.2 0 1.3 25 0.3 0.5 0 0.3 25 0.4 0.7 0 0.3 Laodelphax striatella 0 0.0 0.0 0 0.0 0 0 0 0 0.0 0 0 0 0 0 Sweep nets Occ. Mean SD Median IQR Occ. Mean SD Median IQR Occ. Mean SD Median IQR Zyginidia scutellaris 100 26.6 10.8 30.5 13.5 100 3.3 3.0 2 1.8 100 25.4 22.0 20.5 24.8 Macrosteles spec. 0 0.0 0.0 0 0.0 12 0.1 0.4 0 0.0 25 0.3 0.5 0 0.3 Psammotettix alienus 25 0.4 0.7 0 0.3 0 0.0 0.0 0 0.0 25 0.3 0.5 0 0.3 Empoasca pteridis 0 0.0 0.0 0 0.0 0 0.0 0.0 0 0.0 38 0.6 0.9 0 1.3 Laodelphax striatella 12 0.1 0.4 0 0.0 0 0.0 0.0 0 0.0 0 0 0 0 0 565 566 567 568 569 570 571 572 573Table 3 Analysis of Deviance Tables for the generalized linear models fitted for the 574most abundant taxonomic groups and sampling methods in 2002 and 2003. 575Presented are the degrees of freedom of the effects (Df), as well as the degrees of 576freedom of the residual error (Resid. Df) and the residual deviance for sequentially 577adding the effects to the model, and the p-value of a Chi-square test for the treatment 578effect. Year, Taxon, Method Analysis of Deviance Tables Effect Df Resid. Df Resid. Dev p-value (Chi2) 23 153.2 2002, Z.scutellaris, Sweep netting Block 7 16 102.8 Treatment 2 14 26.2 <0.001 23 80.9 2002, Auchenorrhyncha, Visual Block 7 16 64.1 assessments Treatment 2 14 23.8 <0.001 23 209.9 2003, Z.scutellaris, Yellow traps Block 7 16 172.0 Treatment 2 14 27.4 <0.001 16 16

23 202.2 2003, Z.scutellaris, Stick traps Block 7 16 190.3 Treatment 2 14 24.5 <0.001 23 100.2 2003, Z.scutellaris, Sweep netting Block 7 16 76.1 Treatment 2 14 27.4 <0.001 23 152.7 2003, Auchenorrhyncha, Visual Block 7 16 124.2 assessments Treatment 2 14 18.9 <0.001 579 17 17

580Table 4 Estimated effect sizes for the most abundant taxonomic groups and 581sampling methods. Presented are the estimates and approximate 90% confidence 582intervals for the quotients of mean abundance in maize MON810 to the mean 583abundance in untreated near-isogenic maize (Bt/ISO) and the quotient of mean 584abundance in insecticide treated near-isogenic to the abundance in near-isogenic 585(INS/ISO).

Year, Taxon, Method Comparison Lower Cl Ratio estimate Upper Cl Bt/ISO 0.38 0.55 0.78 2002, Z.scutellaris, Sweep netting INS/ISO 0.06 0.09 0.14 2002, Auchenorrhyncha, Bt/ISO 0.78 1.37 2.43 Visual assessments INS/ISO 0.03 0.07 0.16 Bt/ISO 0.53 0.72 0.97 2003, Z.scutellaris, Yellow traps INS/ISO 0.01 0.02 0.05 Bt/ISO 0.88 1.21 1.66 2003, Z.scutellaris, Stick traps INS/ISO 0.03 0.04 0.07 Bt/ISO 0.81 1.22 1.83 2003, Z.scutellaris, Sweep netting INS/ISO 0.09 0.15 0.25 2003, Auchenorrhyncha, Bt/ISO 1.03 1.46 2.09 Visual assessments INS/ISO 0 0 Infinite* 586*) Confidence interval not computable because no Auchenorryncha were detected in INS 587 18 18

588Table 5 Comparison of different practical aspects of the four methods used in this 589study. All values are estimates and given per trap and plot. Note: € 1 ≈ $ 1.40

Material Time for Level of Bycatch Coefficient cost installation sampling processing identification of variation Yellow traps € 20 30 min 5 min 480 min species high 0.63

Sticky traps € 1 30 min 5 min 30 min genus/ species high 0.52

Sweep netting € 401 none 20 min 10-15 min species depending 0.64

Visual assessments none none 20 min none order none 0.66 5901non-recurrent expense, direct costs depending on number of replications 19 19

591Figure 1 592

593