Epigeic spiders are not affected by the genetically modified maize MON 88017

Transcription

1 J. Appl. Entomol. ORIGINAL CONTRIBUTION Epigeic spiders are not affected by the genetically modified maize MON Z. Svobodová 1,2, O. Habuštová 1, F. Sehnal 1,2, M. Holec 3 & H. M. Hussein 1 1 Biology Centre ASCR, Entomological Institute, České Budějovice, Czech Republic 2 Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic 3 Faculty of Environment, Jan Evangelista Purkyně University, Ústí nad Labem, Czech Republic Keywords Bt maize, HT maize, Cry3Bb1, environmental risk assessment, glyphosate, insecticide Correspondence Oxana Habuštová (corresponding author), Biology Centre AS CR, Entomological Institute, Branišovská 31, České Budějovice, Czech Republic. habustova@entu.cas.cz Received: January 11, 2012; accepted: March 20, doi: /j x Abstract The genetically modified (GM) maize MON facilitates weed management owing to its tolerance to glyphosate, and resists western corn rootworm (WCR), Diabrotica virgifera virgifera, owing to the expression of Cry3Bb1 toxin. MON could therefore contribute to the solution of two major problems of European agriculture: continuous WCR spreading and high use of herbicides. To assess possible unwanted environmental impacts of MON 88017, we compared communities of spiders in plots planted in three successive years with this maize, its near isogenic non-gm cultivar treated or not treated with an insecticide and two unrelated maize cultivars. Each of the five treatments was applied on five 0.5 ha plots in a 14 ha field. Spiders were collected in five pitfall traps per plot five times per year. Upon reaching the waxy ripening stage, all plants of first-year cultivation were shredded to small pieces and ploughed into the soil in the respective plot, whereas in the 2nd and 3rd year the harvest was used for biogas production and only digestate was returned to the field. Out of 79 spider species, Pardosa agrestis, Pachygnatha degeeri and Oedothorax apicatus made up 28%, 25% and 23% of the total spider count in the 1st year of study; 2%, 8% and 84% in the 2nd; and 40%, 8% and 35% in the 3rd year. Statistical analysis did not reveal any influence of GM maize on the spider abundance and biodiversity. The abundance, and in two years also the species diversity, was insignificantly higher on the plots with GM maize than on plots with the insecticide-treated non-gm maize. The composition and size of spider community varied year to year, probably reflecting weather conditions and differences in field fertilization with organic matter. Introduction Genetic modifications (GM) of crops represent a modern way of plant breeding. Genetically modified crops exploited today on a large scale harbour transgenes that render them either insect resistant or herbicide tolerant (HT), and/or provide a combination of these traits. The insect-resistant GM crops express one or more genes encoding insecticidal Cry (crystalline) proteins from different strains of the soil bacterium Bacillus thuringiensis (Bt), which is widely used as a bioagent against diverse agricultural and forest pests and also against mosquito larvae (Lee et al. 2003). The expression of Cry proteins in plants provides resistance to specific herbivorous insects. The HT crops were engineered to tolerate the herbicide glyphosate that kills plants by inhibiting their vital enzyme EPSPS. The tolerance was achieved by introducing into plants a gene isolated from the CP4 56 J. Appl. Entomol. 137 (2013) ª 2012 Blackwell Verlag, GmbH

2 Z. Svobodová et al. Genetically modified maize MON strain of Agrobacterium tumefaciens encoding functionally identical but glyphosate-insensitive enzyme CP4 EPSPS. The HT trait allows farmers to reduce the number of herbicide treatments (in case of maize mostly from 2 to 1) and to use glyphosate-based universal herbicides that are rapidly degraded in the soil (Hawes et al. 2003). The era of practical exploitation of GM crops began in 1996 when a Bt maize resistant to the European corn borer, Ostrinia nubilalis, and a glyphosate-tolerant soybean were commercialized and grown in small areas in USA. The worldwide spreading of GM crops, mainly soya, maize, cotton and canola, at an annual rate close to 10% to 148 million ha planted around the globe in 2010 testifies to the economic advantages of this technology (James 2010). Europe is the only region without a boom in GM crops deployment owing to the complexity of environmental risk assessment needed for the permit to cultivate GM crops and to the thread of post-market environmental monitoring (PMEM) that has not been clearly defined. Sanvido et al. (2009) proposed surveys of herbivores as indicators of the impact of GM crops on their natural enemies. This approach is practical but does not reveal possible effects on biodiversity. It is obviously impossible to monitor many species in the field but it may be doable to identify taxa indicative of the sustainability of agrocoenoses. With our study of epigeic spiders in maize, we wish to contribute to this goal. Epigeic spiders are a highly diversified arthropod group present in all agricultural ecosystems. They may be affected by plant-expressed toxins not only through the prey but in several other routes (Peterson et al. 2011). The ubiquitous presence and the complex role of spiders in the food web justify their use for environmental impact assessment of the GM crops. Several studies (Meissle and Lang 2005; Ludy and Lang 2006a; Řezáč et al. 2006; Toschki et al. 2007, etc.) showed that spiders were not affected by maize expressing Cry1Ab, whereas meta-analysis combining data on different types of Cry showed in some crops a positive and in others a negative influence on spider abundance. In our investigations, we used maize MON that is resistant to the Western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) owing to expression of the Cry3Bb1 toxin, and is also tolerant to glyphosate. Only few studies have so far addressed effects of Cry3Bb1 on spiders (e.g. Bhatti et al. 2005; Meissle and Romeis 2009). We chose MON because of continuous WCR spreading, trends to reduce herbicide applications, crop cultivation for biogas and economic pressures on farmers (increasing cost of fuels), may lead to the adoption of such maize in Europe. We designed a field trial that allowed us to examine spider responses to (i) plantation of maize MON in comparison with its isogenic non-gm cultivar and with two unrelated cultivars and (ii) insecticide treatment currently used in WCR control. Practical aim of our research was assessment of possible environmental risks associated with the deployment of MON or similar GM maize to control WCR in Central Europe. Materials and Methods Field site A field of 15 ha located near the city of České Budějovice (48 59 N, E, altitude about 420 m a.s.l.) was selected for the trial. Average annual precipitation in the area amounts to 550 mm and average temperature to about 11 C. The profiles of daily temperatures and precipitations measured in a meteorological station about 13 km from the field are shown in fig. 1. Medium-weight, mildly humid clay-loam brown soil characterized the field. Field uniformity was disturbed by a hunting lookout and twelve drainage wells. The field, sloping with a 14& south westerly inclination, was flanked on the southwest side by forest and on the three remaining sides by fields planted with winter wheat in 2009 and 2010, and with oilseed rape in Precipita on totals (mm) Precipita on profile ( ) Month Average day temperat ( C) Average temperatures ( ) Month Fig. 1 Monthly temperature and precipitation profiles in the study years. J. Appl. Entomol. 137 (2013) ª 2012 Blackwell Verlag, GmbH 57

3 Genetically modified maize MON Z. Svobodová et al. Experimental design Stripes 10 m wide on the sides adjacent to the surrounding fields and a 20 m wide stripe on the side of the forest were planted with early maize variety DKC 2870 to reduce the edge effect of the surrounding land and to distract deer and boars from the inner experimental area, which was separated from the buffer zone by a 3-m-wide passageway, and was divided into 25 plots, each of about 0.5 ha (63 81 m), arranged into five rows and five columns. Three-metre passageways were left between the rows and 1-m gaps between the columns. Fig. 2 Distribution of plots with different treatments. C: GM maize cultivar MON 88017, N: isogenic non-gm cultivar DK 315, I: DK 315 treated with insecticide, A: unrelated cultivar KIPOUS, B: unrelated cultivar PR38N86. The following maize (Zea mais) cultivars were used in the trial: the glyphosate-tolerant and WCRresistant GM cultivar MON (referred to as treatment C), isogenic, non-gm cultivar DK 315 (treatment N), DK 315 treated with the insecticide Dursban 10G (a.i. chlorpyrifos, treatment I), reference cultivar KIPOUS (treatment A) and reference cultivar PR38N86 (treatment B). The reference cultivars had similar FAO numbers but were otherwise unrelated to MON and DK 315. Each treatment (C, N, I, A, B) was used on five plots (fig. 2). The entire experimental field was treated with a pre-emergence and post-emergence dose of the herbicide Guardian Extra (a.i. acetochlor and terbuthylazine) in the first year, MaisTer (a.i. iodosulfuron-methyl-na and foramsulfuron) in the second year and Adengo (a.i. isoxaflutole and thiencarbazone-methyl) in the last year of experiment. Fertilizers Amofos (N : P 12 : 52, 100 kg/ha) and DAM390 (w = 0.3 N, 325 kg/ha) were applied. The maize was planted at density seeds per hectare in early May in 2009 and 2011 but 1 month later in the exceptionally rainy spring of 2010 (table 1). Fertilizers, and in the I treatment also the insecticide (20 kg/ha, application on soil), were applied simultaneously with the seed sowing. Maize was harvested in October (table 1) and cut by a field chopper to pieces about 1 cm long. In 2009, they were evenly scattered on the harvested plot, whereas in 2010 and 2011 they were transported to silage silos and used for biogas production. In the first years, cattle manure (20 t/ha) and the maize shredding were turned under when the field was ploughed 45 cm deep in about 1 week after the harvest. Field was ploughed in similar way in the second and third year but liquid manure with digestate from the biogas station was applied instead of the solid cattle manure. Table 1 Dates of sowing and harvest, and periods of pitfall traps exposure (sampling 1 5) Sampling 1 14 April 30 April 6 April 20 April 13 April 27 April Sowing 11 May 10 June 6 May Sampling 2 18 May 4 June 25 June 2 July 7 May 13 May Sampling 3 29 July 5 August 25 August 1 September 29 July 5 August Sampling 4 11 September 18 September 5 October 12 October 31 August 7 September Harvest 12 October 18 October 17 October Sampling 5 5 November 19 November 26 October 10 November 14 November 28 November 58 J. Appl. Entomol. 137 (2013) ª 2012 Blackwell Verlag, GmbH

4 Z. Svobodová et al. Genetically modified maize MON Capture and determination of spiders Five pitfall traps, 9 cm in diameter (volume 0.5 l, supplied with about 300 ml 10% NaCl and 2 3 drops of a detergent), were placed on each experimental area (i.e. 125 traps in total). One trap was in the plot centre and four others were placed in the middle of diagonals connecting centre with the plot corners. The traps were placed in the field for about fortnight prior to sowing, for 1-week intervals at the time of maize sprouting, blooming and grain ripening, and for a fortnight after the harvest (see table 1 for exact days). Collection dates and the length of trap exposure slightly varied, especially in 2009 because of irregular torrential rains in May and June (fig. 1). For the purpose of quantitative evaluation of spider abundance, the data were expressed per trap and 1 day exposure time (activity abundance), consistently with other authors (Volkmar et al. 2003; Toschki et al. 2007). This method also allows comparison of different arachnocoenosis (Růžička 1987). Adult spiders collected in the traps were identified to species level using a recent determination key (Nentwig et al. 2010) and recommended nomenclature (Platnick 2011). Statistical processing The relationships between spider occurrences were probed with multivariate analysis. Detrended correspondence analysis (DCA: detrending by segments, log transformed) confirmed linear character of spider distribution. As environmental characteristics dependent on the plot position could cause variability, we examined the effects of rows and columns. Possible influence of the drainage wells and the hunting lookout was checked because small unmanaged areas around them might be a source of species heterogeneity (Venturino et al. 2008). In total, we considered the following environmental characteristics: column, row (owing to the slight field inclination, the row nearest to the forest was drier than the following ones), drainage wells and hunting lookout, collection date (time series: the sowing date was marked with number 1 and the collections by the number of days since then), dummy variables (treatment types: C, N, I, A, B) and covariate (numbers of pitfall traps). The impact of listed variables was examined with the Monte Carlo permutation tests (MCPT) within multivariate linear redundancy analysis (RDA) suitable for studying homogenous agroecosystem: log transformations, 999 permutations, split-plot according to the positions, partial shape (covariate s influence subtracted trap numbers) and forward selection of environmental characteristics. The date of sampling was left intact because the basis for its permutation is less definite than for the permutation of position (Lepš and Šmilauer 2003). This method allows studying the abundance of individual species, which might by more sensitive indicators than the overall population (Bitzer et al. 2005). The multivariate analyses were executed using the program Canoco for Windows 4.5 (Plant Research International, Wageningen, The Netherlands). Graph Pad Prism 4 (GraphPad Software, Inc., San Diego, CA 92037) was employed for descriptive statistics. Variance analyses were computed with the StatSoft Statistica 8 program (Statsoft, Inc., Tulsa, OK 74104). All the tests were carried out at significance level a = The heavy rain and wild boars destroyed 30 (4.8%) of 625 pitfall traps in 2009, mostly during the 3rd trap exposure (15 of 125). Collection loss in 2010 was only 0.9% in the last trap exposure and only 0.4% during The missing values were in all instances substituted with arithmetical averages of spider catches in all undamaged traps at the respective collection time across all types of treatment. Ecological indices To compare the diversity aspects of communities and to assess their similarities, the following ecological indices were applied: Berger-Parker index (d), Simpson dominance index (D), Evenness index (E), Margalef index (D Mg ) and Shannon index (H ) (May 1975; Ryan et al. 1995; Krebs 1999; Magurran 2004; Gamito 2009). Results Spider abundance In total, 4823 spiders were collected in 2009, in 2010 and 9919 in The difference in spider abundance in 2010 in comparison with other years was confirmed by anova (F = 29.56, P 1 2 = , P 1 3 = 0.06, P 2 3 = ). Changes in spider abundance during season were also remarkably different among the years (fig. 3). At the time of maize sprouting in 2009, an average trap contained about 15 spiders, in 2010 as many as 105 and in 2011 only two spiders (but 60 in the first collection). Spider abundance was relatively moderate before sowing, high at the time of maize sprouting and then J. Appl. Entomol. 137 (2013) ª 2012 Blackwell Verlag, GmbH 59

5 Genetically modified maize MON Z. Svobodová et al. Fig. 3 Seasonal changes in spider abundance showed as average numbers of specimens caught per trap and day. SD values are also shown. For sampling times see table 1. declined to less than one quarter of the maximal value for the rest of the season in 2009 and In 2011, the highest number of individuals was captured before sowing and was low in all subsequent collections. Species composition Fifty-seven species belonging to 17 families were found in the field in 2009, 44 species from 15 families (representatives of Corinnidae and Mimetidae were wanting) in 2010 and 43 species from 12 families (Clubionidae, Corinnidae, Liocranidae, Mimetidae and Philodromidae were absent) in The initial samplings carried out before maize planting disclosed presence of 23 species in 2009 and 2010 and 26 species in Forty from the 79 species detected in the field in course of 3 years never occurred before sowing, whereas Cicurina cicur, Pardosa paludicola, Pisaura mirabilis, Porrhomma pygmaeum, Tallusia experta and Zora spinimana were found only at this time. Most spider species were present in all 60 J. Appl. Entomol. 137 (2013) ª 2012 Blackwell Verlag, GmbH

6 Z. Svobodová et al. Genetically modified maize MON collections but often at very low population densities. Araeoncus humilis, Centromerita bicolor, Centromerus sylvaticus, Coelotes terrestris and Ostearius melanopygius consistently occurred in more than 10 individuals per plot. Spider community was dominated by Pardosa agrestis, Pachygnatha degeeri and Oedothorax apicatus, which made up 28%, 25% and 22% of all spiders in The representation of these dominant species changed to 2%, 8% and 84% in 2010, and 40%, 8% and 35% in Overall spider communities in any plot showed quite high degree of diversity, but most species were represented by <10 individuals (table 2). Among the rare species, Mermessus trilobatus, represented by single males in 2009 and 2010 and by two males and one female in 2011, was found in our study for the first time in Southern Bohemia. This species is currently spreading in Central Europe. Although the number of specimens was several times higher in 2010 and 2011 than in 2009, the overall species representation decreased. The number of species per trap and day increased slightly because the probability of catching rarer species increased with the higher number of captured individuals. Species diversity was highest after sowing in twofirst years but in the fourth sampling in anova showed that difference in the species number between 2009 and other years was significant (F = 4.15, P 1 2 = 0.002, P 1 3 = 0.004, P 2 3 = 0.97). The differences in both abundance and species diversity of spiders between 2009, 2010 and 2011 might have been owing to the unusual precipitation pattern during the growing season (fig. 1) and to specific post-harvest field management in Partial field flooding in June 2009 probably killed many spiders allowing field colonization by some less common species. While in the autumn of 2009 the field was heavily supplied with organic matter (maize shredding and cattle manure), in the preceding and in the next years it was organically fertilized only with liquid manure and the digestate left after maize fermentation in the process of biogas production. The large volume of organic matter probably provided food to saprophytic organisms preyed upon by spiders. Biodiversity indexes As field management in the autumn is likely to affect primarily the first collection in the next season, the indexes of biodiversity and dominance were calculated for individual plots using the analysis of variance separately for the 1st collections (bare land before sowing) and the remaining collections combined (fig. 4). The Shannon and the Margalef indexes disclosed significantly higher biodiversity in the first samplings than in those collected during the vegetation period. Lower biodiversity in 2010 than in the foregoing and following years reflected very high abundance of common spider species in Both the Berger-Parker and the Simpson dominance indexes showed that dominant species represented approximately 70% of spiders on all plots in 2009 and 2011 but 80% in 2010 during the vegetation period. Oedothorax apicatus was particularly abundant before and after sowing in 2010, possibly due to abundant prey developing in the maize debris ploughed into soil in the autumn of All used indexes revealed that spider biodiversity was low and the dominance of a few species was high during the growing season of all three years. Effects of plot treatment on abundance and diversity The plots differed from one another by the type of treatment from the 1st sowing until the end of the trial. Statistical analysis of species abundance and diversity carried out for the 1st sampling in 2009 was devoid of this bias and was therefore used to detect spider preferences or avoidance of plot positions alone. Analyses of the plot and treatment effects in other collection times were employed in attempt to find out if inter-annual differences, such as organic fertilization, did not affect spider community this effect could be manifested only in certain season. One-way anova disclosed no effect of plot position and the type of treatment on the abundance of spiders before sowing and during the vegetation period in any of the study years with the exception of fourth sampling in 2011 when we established statistical parameters F = 2.97 and P = Significant difference in the fourth sampling in 2011 was owing to lower number of individuals in the I plots. The numbers of spiders in I plots were mostly lower in the first sampling after sowing (and insecticide treatment). In 2009 and 2010, we collected on I plots 278 and 2031 specimens, respectively, compared with 449 and 2545 spiders collected in the C plots planted with GM maize and not treated with the insecticide. By contrast, the number of individuals in the first collection of 2011 was low in both C and I plots (28 specimens in each). Similar numbers in the I and C plots were also found in the 3rd and 4th samplings in 2009, 1st sampling in 2010, and 1st, 3rd and 4th sampling in 2011 (fig. 3). Summation of data for the whole season disclosed overall lower J. Appl. Entomol. 137 (2013) ª 2012 Blackwell Verlag, GmbH 61

7 Genetically modified maize MON Z. Svobodová et al. Table 2 Total (five plots of each type of treatment) catches of common spider species per year. Maize cultivars: C: MON 88017, N: DK 315, I: DK 315 treated with insecticide, A: KIPOUS, B: PR38N86 C N I A B Family Species Amarobiida Coelotes terrestris Gnaphosidae Drassyllus lutetianus Unidentified Linyphiidae Araeoncus humilis Bathyphantes gracilis Centromerita bicolor Centromerus sylvaticus Diplostyla concolor Erigone atra Erigone dentipalpis Meioneta rurestris Oedothorax apicatus Ostearius melanopygius Porrhomma microphthalmum Stemonyphantes lineatus Walckenaeria vigilax Unidentified Lycosidae Pardosa agrestis Pardosa palustris Pardosa prativaga Trochosa ruricola Unidentified Tetragnathidae Pachygnatha clercki Pachygnatha degeeri Pachygnatha listeri Unidentified Theridiidae Robertus arundineti Unidentified Thomisidae Xysticus cristatus Xysticus kochi Unidentified Zoridae Unidentified Number of specimens Number of species Only species represented at least by 10 individuals are listed in the table. The following species were caught in lower numbers in random fashion throughout the experimental field and are included in the specimen and species counts at the bottom of the table [Correction added on 20 November 2012, after first online publication: The above sentence has been corrected by replacing the text Not included as included.]. Araneidae: Araniella opisthographa (1 specimen), Gibbaranea gibbosa (1), Mangora acalypha (1), Mastigusa arietina (1), unid. (6); Clubionidae: Clubiona leucaspis (2), unid. (4); Corinnidae: Phrurolithus festivus (1); Dictynidae: Cicurina cicur (5), Lathys humilis (2), unid (1); Gnaphosidae: Drassyllus pusillus (1), Haplodrassus silvestris (1), Micaria pulicaria (1); Linyphiidae: Araeoncus crassiceps (4), Bathyphantes parvulus (1), Centromerus brevivulvatus (2), Dicymbium nigrum (5), Diplocephalus picinus (3), Diplostyla concolor (8), Hypomma bituberculatum (1), Lepthyphantes flavipes (3), Leptorhotrum robustum (1), Mansuphantes mansuetus (1), Mermessus trilobatus (5), Micrargus herbigradus (1), Microlinyphia pusilla (1), Moebelia penicillata (2), Neriene clathrata (3), Oedothorax fuscus (1), Pelecopsis parallela (1), Pocadicnemis juncea (1), Porrhomma pygmaeum (4), Tallusia experta (2), Tenuiphantes tenuis (7), Walckenaeria nudipalpis (1); Liocranidae: Agroeca brunnea (3); Lycosidae: Alopecosa pulverulenta (5), Pardosa amentata (4), Pardosa paludicola (9), P. pullata (4), Trochosa terricola (4); Mimentidae: Ero furcate (2); Philodromidae: Philodromus cespitum (1), P. margaritatus (1), Tibellus oblongus (1), Pisauridae: Pisaura mirabilis (2), Salticidae: Ballus chalybeius (2), Euophrys frontalis (1), Heliophanus dubius (1), Salticus zebraneus (1); Theridiidae: Neottiura bimaculata (7), Robertus lividus (2), R. neglectus (1), Theridion impressum (2); Thomisidae: Diaea dorsata (2); Zoridae: Zora spinimana (5). number of individuals on plots treated with the insecticide than on the plots with GM maize in all years. The total number of individuals collected in 2009 and 2011 on plots I was the lowest from all types of treatment (table 2). However, statistical analysis did not confirm significance of these differ- 62 J. Appl. Entomol. 137 (2013) ª 2012 Blackwell Verlag, GmbH

8 Z. Svobodová et al. Genetically modified maize MON Fig. 4 The average SD diversity indexes calculated for first collections in each year and for all remaining collections combined (vegetation season). ences. Spiders were most abundant in the stand of GM maize in 2010 and 2011 (only four more individuals were found on plots with reference cultivar B in 2009 (table 2), but the difference from other treatments was not statistically significant. The type of treatment also had no effect on the species numbers as proven by anova analysis for all years and sampling times, except for the 4th collection in 2010 (F = 4.46, P = 0.01). The significant effect found in this sampling was caused by lower number of species on plots with reference cultivar A. The number of species was lower on plots C than on plots I in 2009, but in two other years the species diversity was higher on plots C, notably in the 1st and 3rd samplings in 2010 and in the 1st, 3rd and 5th samplings in Analysis of data dependence on all variables The impact of all variables on the abundance of single species was examined with the linear limited method (RDA). The lengths of gradients established in advance with the DCA method supported application of RDA. The specimens found in the first collection in 2009 were evaluated separately from the remaining data that were affected by both plot position and treatment. In the first collection, the forward selection method within RDA (DCA length of gradient: 2.14) proved significant influence of plot position in the row (F = 5.19, P = 0.01) and column (F = 4.8, P = 0.03) on the spider community. Maximal numbers of the most abundant species occurred in both marginal rows (first is near the forest) and maximal numbers of specimens in the first column (counting from northwest field margin). First collections made in each year were also analysed (DCA: length of gradient: 1.82). The forward selection method proved significant influence of the year (table 3) that cumulatively explained 31.9% of the species abundance. The abundance was also affected by plot position in the row (marginally significant). Other examined variables had no significant influence (table 3). Ecological preferences (first collection in 2009 was excluded) were studied using a multivariate RDA analysis (DCA: length of gradient: 2.35) that identified years 2009 and 2010, the date of sampling and the column as significant factors affecting spider abundance. No influence of other environmental factors and of the type of treatment was shown (table 3). The type of treatment explained only 0.1% data variability. The cluster of centroids at the intersection of the ordination space (the first and second axis) revealed similarity of species composition and abundance in plots with different treatments (fig. 5). The assembly of factors year, date and treatment (C, N, I, A, B) explained 11.2% of the variability in spider community, while the first canonical axis Table 3 Results of redundancy analysis of data from the first collections (before sowing) and from all remaining collections combined. The F and P values reveal significance of environmental variables Environmental variables Monte Carlo permutation test First collections F P % explained variability F P Remaining collections Date C N I A B Row Column Well Hunting lookout % explained variability J. Appl. Entomol. 137 (2013) ª 2012 Blackwell Verlag, GmbH 63

9 Genetically modified maize MON Z. Svobodová et al. Fig. 5 Redundancy analysis of the spatial and temporal distribution of epigeic spiders (only species represented by more than 10 individuals were included and those with more than 1% explained variability are shown). Horizontal line indicates years and sampling dates and vertical line the types of treatment. The results reveal that the species did not prefer any type of treatment (cluster of the centroids at the intersection of ordination space indicate the similarity of species composition and abundance in diverse plots). explained 6.8% and the second 3.5% variability. Figure 5 demonstrates that P. agrestis and Xysticus kochi were most common in 2009 but in 2010 they were exceeded manifold by O. apicatus. The population density of other displayed species such as P. degeeri was not affected by year. Pardosa agrestis and Xysticus kochi were negatively correlated, while C. bicolor, C. sylvaticus and Stemonyphantes lineatus were positively correlated with the date of sampling. The results of RDA analysis accentuated differences between 2009 and 2010 and similarity of either of these years with 2011 shown in the ordination diagram where the position of 2011 centroid is approximately on the flowline between centroids for 2009 and The most common species occurred in highest abundance in the last two plot columns confirming the result of RDA, but the effect of hunting lookout was insignificant. Discussion Our data and previous studies on spiders in agrocoenoses (extreme biotops) show that very few species usually prevail (Luczak 1979) disregarding the geographic position (Řezáč et al. 2006) and that these species are usually small (Růžička 1987). In the present study, more than three quarters of spiders collected on any plot at any time belonged to the species P. degeeri, O. apicatus and P. agrestis that are common in agricultural ecosystems (Řezáč et al. 2006; Toschki et al. 2007). The dependence of spider communities on plot position in the first sampling of 2009 is best explained by the impact of adjacent forest and the surrounding fields planted with winter wheat. Especially the forest is likely to enhance spider diversity (Venturino et al. 2008). Hence, the pre-seasonal collection revealed trends in the field colonization by spiders owing to surroundings but disclosed no preference of specific plots. Inter-annual differences of the effect of adjacent ecosystems probably also affected species dominance. Pachygnatha degeeri dominated in spider collections before sowing in all study years but other most common species differed. We assume that they were affected by moisture gradient from the forest edge. Soil moisture varied from year to year depending on the precipitation. The influence of forest was more pronounced in early spring when bare soil was exposed than during the vegetation season. Seasonal fluctuations in the population density of spiders reflected their life cycles: most spiders mature and reproduce in spring and their catches are then maximal (Růžička 1987). Some spiders then perish and many of the remainders become prey of larger spiders, beetles and other predators. Only few spider species (O. apicatus, Meioneta rurestris, genus Erigone) exhibit more than one abundance maximum during the year. Selective predation of larger pray by birds and small mammals contributes to the reduction of big spiders (P. agrestis, Pardosa palustris, Trochosa ruricola) (Růžička 1987) and prevalence of the small species during season, as shown by RDA analysis (fig. 5). Most species illustrated in RDA chart exhibit negative relationship to the date of sampling, showing that their abundance declined during season. On the contrary, C. bicolor, C. sylvaticus and Stemonyphantes lineatus were slightly more common in late season. The number of species was highest in 2009, in contrast to spider abundance which was lowest in 2009 (fig. 3). Dry April followed by rainy May and especially June in 2009 (fig. 1) were probably the cause of reduced spider population in this year. By contrast, the deposition of maize biomass into the soil in 2009 probably caused the sharp increase of spider abundance in the next years. In addition to 64 J. Appl. Entomol. 137 (2013) ª 2012 Blackwell Verlag, GmbH

10 Z. Svobodová et al. Genetically modified maize MON cattle manure, about 6-kg raw organic matter per 1m 2 was ploughed into the soil and provided rich substrate for many decomposers at the bottom of the food chain (oribatids and other mites, springtails and other apterygotes, and larvae of Diptera and some other insects). We suppose that abundant prey supported subsequent expansion of the spider population. Results of RDA analysis of species composition and abundance in relation to ecological variables (first collection in 2009 was excluded) accentuated differences between 2009 and 2010 (fig. 5). However, none of the studied species showed a preference or avoidance of certain treatment in any year (table 3). The analysis of variance also demonstrated lack of differences in the number of species and the overall abundance among plots subjected to different treatments. Large variability of spider communities across the field, including identically treated plots, remains mostly unexplained. The population dynamics of soil organisms is influenced by many factors, primarily by weather condition connected with prey abundance and farming practices (Debeljak et al. 2007). Spiders, especially the wolf spiders, are also sensitive and often do not survive flooding (Růžička 1987). Two types of treatment were in the focus of our study: the insecticide treatment (I) and the GM maize cultivation (C). Spider collections after sowing were lower in I than C plots in all years but the number of individuals in the second sampling of 2011 was extremely low (225 in total). The number of species was lower on plots C than on plots I in 2009, but it was higher in the other years. All differences in spider communities between the I plots and either C plots or N plots (control) were statistically not significant, except for the fourth sampling in We are aware that the insecticide could be washed out, especially in 2009 when the locality experienced intensive torrential rains just after sowing. The influence of other environmental conditions on insecticide persistence or insecticide binding to soil particles cannot be excluded either. However, some published data also demonstrated spider resistance to the insecticides chlorpyrifos (Al-Deeb and Wilde 2003), tefluthrin (Ahmad et al. 2005), imidacloprid (Bhatti et al. 2005) and cyfluthrin (Toschki et al. 2007). Venturino et al. (2008) reported that spiders survive low levels of insecticide poisoning. On the other hand, Meissle and Lang (2005) registered reduced density of foliage spiders after application of a pyrethroid and this toxicity was verified in a laboratory bioassay (Ludy and Lang 2006b). Bhatti et al. (2005) recorded negative effect of permethrin applied on foliage and tefluthrin applied on soil on spiders abundance. It is obvious that we do not know enough about the action of insecticides on spiders (Pekár 1999). We can conclude, however, that the soil insecticide treatment, which is currently used to control CWR, does not irreversibly damage spider community. The lack of differences between spider communities in plots planted with the GM maize and the remaining plots is consistent with our previous experience (Sehnal et al. 2004) and the reports of other authors studying possible impact of GM maize on the epigeic (e.g. Volkmar et al. 2004; Dively 2005; Řezáč et al. 2006; Rose and Dively 2007; Toschki et al. 2007) and foliage spiders (Meissle and Lang 2005; Ludy and Lang 2006a). We emphasize consistence of our conclusions with the data published by Al-Deeb and Wilde (2003), Ahmad et al. (2005) and Bhatti et al. (2005), who used GM maize expressing Cry3Bb1 toxin. However, these studies were carried on in small areas and did not identify spiders to the species level. Spiders resistance to the Cry toxins was confirmed in laboratory studies, for example with Pirata subpiraticus (Jiang et al. 2004) and Ummeliata insecticeps (Tian et al. 2010). These species proved insensitive to the Cry toxin present in rice which was consumed by their prey. The toxin was ingested and found in spiders bodies at different levels but without affecting their performance. Experiments with the spider Theridon impressum, which contained various concentrations of Cry 3Bb1 in the body in dependence of the prey species, led to similar results (Meissle and Romeis 2009). It can be concluded that spiders resist Cry toxins similarly to other predators such as the rove beetles shown to tolerate Cry1Ab (García et al. 2010) and ladybirds that were not affected by Cry1Ab and Cry3Bb1 (Álvarez-Alfageme et al. 2011). Spiders could be affected by MON indirectly, by possible changes in the availability and nutritional quality of their prey (Yang et al. 2005). However, lack of any differences between the GM and non-gm plots indicates absence of serious indirect effects. The results of our study show that the GM maize MON has no negative influence on epigeic spiders that also tolerate application of the insecticide Dursban 10G at the time of sowing. Acknowledgements This work was financed by grants QH91093 and QI91A229 from the National Agency for Agricultural J. Appl. Entomol. 137 (2013) ª 2012 Blackwell Verlag, GmbH 65

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