Consequences for Protaphorura Armata (Collembola: Onychiuridae) Following Exposure to Genetically Modified Bacillus Thuringiensi
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Environmental Pollution 142 (2006) 212e216 www.elsevier.com/locate/envpol Consequences for Protaphorura armata (Collembola: Onychiuridae) following exposure to genetically modified Bacillus thuringiensis (Bt) maize and non-Bt maize Lars-Henrik Heckmann a,1, Bryan S. Griffiths b, Sandra Caul b, Jacqueline Thompson b, Marianne Pusztai-Carey c, William J. Moar d, Mathias N. Andersen e, Paul Henning Krogh a,* a National Environmental Research Institute, Department of Terrestrial Ecology, Vejlsøvej 25, PO Box 314, DK-8600 Silkeborg, Denmark b Scottish Crop Research Institute, Department of Soil Plant Dynamics, Invergowrie, Dundee DD2 5DA, UK c Case Western Reserve University, Cleveland, OH 44106, USA d Auburn University, Department of Entomology and Plant Pathology, Auburn, AL 36849, USA e Danish Institute of Agricultural Sciences, Research Centre Foulum, PO Box 50, DK-8830 Tjele, Denmark Received 2 June 2005; received in revised form 5 October 2005; accepted 13 October 2005 Protaphorura armata performed equally well when reared on two Bt and three non-Bt maize varieties. Abstract Studies on the effect of genetically modified Bacillus thuringiensis (Bt) crops on true soil dwelling non-target arthropods are scarce. The objective of this study was to assess the influence of a 4-week exposure to two Bt maize varieties (Cry1Ab) Cascade and MEB307 on the collembolan Protaphorura armata. For comparison three non-Bt maize varieties, Rivaldo (isogenic to Cascade), Monumental (isogenic to MEB307) and DK242, and two control diets based on baker’s yeast (uncontaminated and contaminated with Bt toxin Cry1Ab) were also tested. Due to a lower C:N ratio, individuals reared on yeast performed significantly better in all of the measured endpoints than those reared on maize. P. armata performed equally well when reared on two Bt and three non-Bt maize varieties. Although there were no negative effects of Bt maize in this experiment, we recommend future studies on Bt crops to focus on species interactions in long-term, multi-species experiments. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: GMO; Cry1Ab; Collembola; Life history traits; Population 1. Introduction dwelling (i.e. euedaphic) arthropods that may be directly affected by the traits of many GM crops (Yu et al., 1997). While the use of genetically modified (GM) crops is Bacillus thuringiensis (Bt) insecticidal Cry toxin (e.g. Cry1Ab) currently at debate in Europe, several countries (e.g. USA, is one of the traits that have been genetically engineered into Argentina, China and Australia) are growing GM crops com- crops. Cry toxins are active in insects when ingested orally as mercially on a large scale. Numerous studies concerning the their mode of action is in the midgut. Genetically modified Bt impact of GM crops on terrestrial organisms have been per- crops express the Cry toxin constitutively throughout the en- formed (e.g. Losey et al., 1999; Saxena and Stotzky, 2001a). tire plant (Groot and Dicke, 2002). In the insect midgut Cry But only few have investigated the impact on true soil toxins synthesised by B. thuringiensis, need to be proteolyti- cally cleaved to form the active d-endotoxin before binding to insect midgut receptors (Schnepf et al., 1998). Binding of * Corresponding author. Tel.: þ45 89 20 15 88; fax: þ45 89 20 14 13. E-mail address: [email protected] (P.H. Krogh). the d-endotoxin to the receptors is a requirement for insecti- 1 Present address: The University of Reading, School of Biological Sciences, cidal activity, although binding alone is not sufficient for Environmental Biology, PO Box 228, Reading RG6 6AJ, UK. toxicity. When binding is followed by pore formation in the 0269-7491/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2005.10.008 L.-H. Heckmann et al. / Environmental Pollution 142 (2006) 212e216 213 midgut membrane, the midgut cells are disrupted and the in- maize varieties (Table 1); dry baker’s yeast contaminated with Bt toxin sect dies. The toxicity of different Cry toxins is specific, e.g. (Cry1Ab); and uncontaminated dry baker’s yeast (Table 1). Each replicate con- Cry1A mainly targets lepidopterans, whereas Cry3A targets sisted of a Petri dish (5.4 cm diam., 1.2 cm height, with a plaster of Paris and activated charcoal (8:1 weight:weight) covering the bottom) with 10 adult Chrysomelidae (Coleoptera). Thus, Cry toxins are toxic to P. armata (w4e10 weeks old). Synchronisation of P. armata is very difficult, some species whilst being more or less harmless to others and thus the body area of the individuals in each Petri dish was measured using (Schnepf et al., 1998). Digital Image Processing (DIP). The experimental duration was four weeks Below ground, the rhizosphere is a hot spot with respect to during which P. armata were kept in a climate room with a constant temper- abundance, species diversity and biological interactions of the ature of 20 Æ 1 C and a light:dark regime of 12:12 h. The relative humidity was w100%, which was adjusted weekly by adding demineralised water. soil biota (Coleman et al., 1988). The presence of Cry1Ab toxin P. armata were starved for 2 d prior to the experiment, after which they has been observed in the rhizosphere as root exudate from Bt were fed one of the above-mentioned diets once a week (w4 mg DW per rep- maize (Saxena et al., 1999). No apparent effects have been licate). Somatic growth was measured as body surface area weekly using DIP identified in earthworms (Vercesi et al., in press), nematodes, (Krogh et al., 1998). During DIP, the individuals were anaesthetised with CO2. protozoa, bacteria and fungi (Saxena and Stotzky, 2001a) since The total number of juveniles produced was counted manually after six weeks, two weeks after removal of the surviving adults, thus estimating the total re- the Cry toxins primarily target insects. Some studies have been production during the four weeks as mean egg development takes w14 d at performed on the effects of Bt crops on non-target arthropods 19 C(Bengtsson et al., 1985). Survival and population growth rate (pgr) (NTA), but they mainly focused on aboveground insects (e.g. was estimated after four weeks. Jasinski et al., 2003). So, as concluded by Groot and Dicke The maize root material was obtained from frozen plants harvested after (2002), studies on the effect of Bt crops on euedaphic NTA field trials performed in Denmark in 2001 (varieties Rivaldo (non-Bt) and Cascade (Bt)) and 2002 (DK242 (non-Bt), Monumental (non-Bt) and MEB307 are scarce. (Bt)) (Griffiths et al., in press). The roots were washed, dried and lyophilised Collembolans comprise a large group of NTA, and play over night at ÿ40 C under a vacuum of w30 mTorr. Subsequently, the root a key role in the functioning of the soil environment (Larink, tissue was finely ground in a mortar and kept dry, dark and cold (w5 C) until 1997). Thus, our main objective was to examine the impact of use. Treatments with baker’s yeast were processed according to the method of root tissue from Bt (Cry1Ab) and non-Bt maize varieties Sims and Martin (1997). One gram of dry baker’s yeast was suspended in a Petri dish in 5 ml of distilled water containing 500 mg of purified Cry1Ab toxin. offered as food to the collembolan Protaphorura armata The suspension was quickly frozen in a ÿ80 C freezer, and subsequently (Onychiuridae). Moreover, two diets based on dry baker’s lyophilised as described above. Solubilized, trypsinized (i.e. proteolytically ac- yeast were included as a series of controls to evaluate the over- tivated) and HPLC purified Cry1Ab1 toxin was applied. Protein activity of all performance of P. armata on maize root tissue. Cry1Ab1 was verified at Rothamsted Research (Harpenden, UK) using a bio- assay mortality test with Plutella xylostella (Lepidoptera: Yponomeutidae). Quantification of Cry1Ab in yeast, Bt and non-Bt root tissue was performed 2. Materials and methods with QuantiPlate Kit for Cry1Ab/Cry1Ac (Envirologix Inc., Portland, ME, USA). Carbon:Nitrogen (C:N) ratios were obtained according to the method 2.1. Test species described in Scrimgeour and Robinson (2003). About 1 mg of the samples were weighed into 6 Â 4 mm tin cups for analysis by continuous flow Dumas P. armata Tullberg (formerly Onychiurus armatus) is a euedaphic NTA combustion using a Roboprep CN sample converter (Europa Scientific, Crewe, and a taxonomic representative of the Collembola. It is a known pest on the UK). Nitrogen and carbon were selectively detected as N2 and CO2 using roots of sugar beet (Joosse and Koelman, 1979), and as such is one of the a Tracermass mass spectrometer (Europa Scientific, Crewe, UK). Samples few Collembola known to be herbivorous. P. armata is a slow-growing, were quantified relative to a leucine/citric acid mixture of known C and N long-lived springtail (maximum age w2 y) that reproduces parthenogenetically content. Reference gravimetric samples of wheat flour were used for quality (Hopkin, 1997). The individuals used in the experiment were taken directly control. The experimental part was performed at the National Environmental from the stock culture at the National Environmental Research Institute, Research Institute, Denmark, while the Bt and C:N analyses were completed Silkeborg, Denmark. at the Scottish Crop Research Institute, UK. 2.2. Experimental design 2.3. Data analysis and statistical methods The experimental set-up consisted of 7 diet treatments (5 replicates each): All data were log-transformed. One-way ANOVAwas performed in Enter- 5 treatments with dried ground root tissue, 2 Bt maize varieties and 3 non-Bt prise GuideÒ Version 1 (SAS Institute Inc, Cary, NC, USA), to test for Table 1 Different diets fed to Protaphorura armata Diet Cry1Ab (mg/g)* C:N ratio Diet group Remarks Cascade 1.37 Æ 0.34 32.5 Æ 0.50a Bt maize Transformation event MON810 MEB307 1.01 Æ 0.23 31.1 Æ 1.48a Bt maize Transformation event MON810 Rivaldo N.d.