- Email: [email protected]
Are survival and reproduction of Enchytraeus albidus (Annelida: Enchytraeidae) at risk by feeding on Bt-maize litter?
European Journal of Soil Biology 45 (2009) 351–355
Contents lists available at ScienceDirect
European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Are survival and reproduction of Enchytraeus albidus (Annelida: Enchytraeidae) at risk by feeding on Bt-maize litter? Linda Ho¨nemann a, *, Wolfgang Nentwig b a b
¨rlitzer Platz 1, 06844 Dessau, Germany Federal Environment Agency, Division IV 1.3, Wo Community Ecology, Institute of Ecology and Evolution, University of Bern, Baltzerstrasse 6, 3012 Bern, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 June 2008 Received in revised form 12 March 2009 Accepted 17 March 2009 Available online 7 April 2009 Handling editor: Stefan Schrader
Enchytraeids are saprophagous soil organisms, appearing in high abundances and contributing to ecological processes within the soil. For decades they have been used as model species for biological research. In the framework of research on genetically modified plants, however, they have not been considered to date. Following the ISO/DIS guideline, survival and reproduction of Enchytraeus albidus, fed with diets containing Bt-maize (N4640Bt Cry1Ab, DKC5143Bt Cry3Bb1) leaf material were analysed. For comparison, diets with the corresponding untransformed near-isolines (N4640, DKC5143) were examined. Additionally a high quality control diet (oat flakes) was included. Survival and reproduction showed no significant differences between the Cry3Bb1 treatment and the treatment with the untransformed counterpart. For the Cry1Ab treatment survival was significantly higher than for the treatment with the corresponding near-isoline. In contrast, reproduction was significantly lower for the Cry1Ab treatment compared to that for the isoline. For the Cry3Bb1 treatment, no effect was shown on survival or reproduction. For the Cry1Ab variety and its untransformed counterpart, a contrasting result was detected, which is unlikely to be caused by the Bt-protein but rather by differences in other plant components. Overall survival and reproduction were highest for the control. Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Bt-maize Non-target soil organisms Enchytraeids Feeding test Life history traits
1. Introduction Soil organisms contribute strongly to vital processes within the soil system. Especially, earthworms are known for their high impact on soil properties and influences on the availability of resources for other organisms (animals, micro-organisms, plants) and therefore are regarded as ecosystem engineers [20]. But earthworms are not the only group of annelid worms influencing soil processes. Enchytraeids are saprophagous, colourless to white worms, 2–40 mm long, inhabiting the upper 5–10 cm of the soil [6]. About 900 species are described worldwide, appearing in different soils with high, seasonally fluctuating abundances of a few thousands up to more than 100,000 individuals m2. By their feeding activities enchytraeids support remineralization processes and improve the fine structure of the soil [3,19]. An enchytraeid worm lives on average 2–9 months and sexual maturity is reached 5–7 weeks after hatching from the egg. Enchytraeids are hermaphrodites, but most species show sexual reproduction [6,19]. Due to their contribution to ecological processes, their worldwide appearance and
* Corresponding author. Tel.: þ49 340 2103 2380. E-mail address: [email protected] (L. Ho¨nemann). 1164-5563/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejsobi.2009.03.001
their easy handling and breeding, enchytraeids, in particular Enchytraeus albidus, have been used widely as model species in ecotoxicology, physiology, biochemistry and genetics for more than 50 years [24]. In recent decades national and international regulations to protect the environment against detrimental effects of pollutants have improved steadily. Genetically modified (GM) plants are regarded as a potential source of environmental risks, which have to be adequately assessed [26]. However GM plants have the potential to offer a more efficient and, compared to conventional pesticides, environmentally less harmful protection against pests, thus also enhancing crop yields. Maize, one of the most frequently cultivated crops in Europe [21], is mainly attacked by two insect pests, the tortricid moth Ostrinia nubilalis (European corn borer) and the chrysomelid beetle Diabrotica virgifera virgifera (Western corn rootworm). Larvae of O. nubilalis feed in the stems and D. virgifera virgifera attacks the root system, both are hard to antagonize efficiently with insecticides [29,34]. At present GM maize varieties, expressing Cry proteins of the bacterium Bacillus thuringiensis (Bt), toxic to specific insect groups, present the best protection against these two pests. Cry1 proteins target lepidopterans like O. nubilalis rather selectively and Cry3 proteins antagonize coleopterans like D. virgifera virgifera [10,17]. Due to
352
L. Ho¨nemann, W. Nentwig / European Journal of Soil Biology 45 (2009) 351–355
the physiological specificity of Bt-proteins, effects on target organisms are restricted to a certain protein group of Cry proteins [11,25,26]. Nevertheless non-target organisms may also ingest the Cry proteins expressed by Bt-maize. They can feed on GM plant material, they may predate on prey items containing Bt-protein, or they may come into contact with Cry proteins via root exudates in the soil [11]. The actual amount of research performed on potential side effects of Bt-plants on non-target organisms is quite impressive and allows the general statement that effects are highly improbable [5,25,17]. However, effects like changes in growth, fertility, development time or life span, may be difficult to detect. Such effects must not be attributed to the expression of Bt-proteins they more likely appear due to changes in other plant components. Studies by Escher et al. [7], Saxena and Stotzky [27], Flores et al. [9], Poerschmann et al. [22] and Zurbru¨gg et al. [unpublished results] have shown that Bt- and non-Bt-maize differ not only in the ability to express Bt-proteins but also in the content of important plant components like lignin, cellulose, carbon and nitrogen. These plant components could affect the degradability of plant litter for saprophagous organisms and thus have an impact on their fitness. So far no effect of Bt-maize on soil organisms has been shown, either at the species [7,23,4,15,35] or at the community level [38,16]. Annelid worms are key organisms in soil systems but investigations on them are rare and only earthworms have been examined so far [28,32,37,1,4,33,17]. Now there are no data on potential impacts of Bt-maize on enchytraeids. The presented study here intends to close this gap. In a laboratory feeding experiment we analysed potential effects on survival and reproduction of the enchytraeid E. albidus on leaf diets of two Bt-maize varieties, their untransformed corresponding near-isolines and a high quality control food. Since no Bt-effects on lumbricids have been detected so far, our null hypothesis was that there are also no effects on the closely related enchytraeids.
2. Materials and methods The feeding experiment was designed following the ISO/DIS guideline 16387 [18] for the testing of effects of pollutants on Enchytraeidae (Enchytraeus sp.). The design of the ISO guideline was slightly modified to establish the effect of feeding on different maize varieties on E. albidus. Food diets containing transgenic and non-transgenic maize varieties respectively were used as test substances. Additionally a control diet without maize was used.
2.1. Maize plants Leaf material for the transgenic maize diets was taken from the varieties N4640Bt (transformation event Bt11, Syngenta, Switzerland), expressing Cry1Ab and DKC5143Bt (event MON88017, Monsanto, USA), expressing Cry3Bb1. For the nontransgenic diets leaves of the untransformed corresponding nearisolines (N4640 and DKC5143 respectively) were used. All plants were cultivated in a climatic chamber with 16 h daylight at 25 C and 8 h darkness at 20 C. Plants were grown in 20 L buckets 3⁄4 filled with geranium and balcony plant soil (Mioplant, Zurich, Switzerland). Before sowing 35 g of a slow-release fertilizer (Tardit–Langzeitdu¨nger from Hauert, Grossaffoltern, Switzerland; 14% N, 7% P, 14% K, 1.5% Mg) was added to each bucket. Once per week each bucket received 0.5 L of a 0.2% liquid fertilizer (Wuxal Universal Du¨nger from Maag Agro, Dielsdorf, Switzerland; 10% N, 10% P, 7.5% K, 1.24% B). Water was given as required.
2.2. Enchytraeids The test species E. albidus was bred in our lab, originally deriving from a culture purchased from the Diskus-Laden in Du¨ren, Germany. Following the ISO/DIS guideline, adult worms (with eggs in the clitellum region and approximately 1 cm size) were separated from the breeding culture before the experiment started. Separated enchytraeids were acclimatized to test conditions for 48 h, no food was offered during this time.
2.3. Food diets Senescent maize leaves of the four varieties and oat flakes were ground to a fine powder with an ultra centrifugal mill, type ZM1 (Retsch KG; Haan, Germany). Corresponding to the food in the breeding culture, only oat flake powder moistened with water was used for the high quality control diet. The maize diets contained 70% maize leaf powder of one variety and 30% oat flake powder, moistened with water. Diets were prepared and kept at 25 C until their use in the experiment. From the prepared diets samples were taken for the quantification of the Bt-protein content.
2.4. Soil Plant soil (universal soil for room, balcony and garden plants from Coop, Switzerland), consisting of 40% high bog peat, 40% bark compost and 20% wood fibres, with a pH 5.9 (measured according to ISO/DIS guideline 16387, n ¼ 10) was used. The soil was dried and afterwards sieved to a standard particle size of 2 mm.
2.5. Test design The feeding experiment lasted 6 weeks and included 5 treatments, 4 maize treatments and one control, each replicated 10 times. The test was performed in a climatic closet at 20 C with a day–night cycle of 16 h light and 8 h darkness. We used test vessels of 8 cm height and 6 cm diameter, with the opening closed with fine gauze. Two days before starting the experiment each test vessel was nearly half filled (20 g dry weight) with the prepared soil. The water content of the soil was gravimetrically syntonized to 60%. At the beginning of the experiment 10 adult worms and 0.3 g food diet were added to each test vessel. The food was placed in a hollow in the soil surface, allowing aeration to prevent the diets from molding. Every third day, remaining food was replaced by new supplies and soil was remoistened if required. To avoid losses of eggs or juvenile worms, food remains were separately stored until the end of the experiment. Three weeks after the start of the experiment all adult worms were removed and the number of surviving worms was recorded for each test vessel. To quantify the amount of Bt-proteins (see below) taken up by the worms, all adults removed were lyophilized and frozen at 25 C. After removing the adults the experiment continued for another three weeks. At the end the number of offspring, representing the reproductive output, was determined for each vessel and the associated food remains. For the determination of the number of offspring the soil of each test vessel was divided into portions of 1–2 g. In a Petri dish one of these small portions was bloated with water. Using a binocular microscope, the movement of the juvenile worms was visible and they could be separated with tweezers for counting. Additionally the offspring was visually assessed for obvious changes in appearance or deformations.
¨ nemann, W. Nentwig / European Journal of Soil Biology 45 (2009) 351–355 L. Ho
2.6. Bt-protein quantification
11
a
ab
10
2.7. Data analyses Statistical analyses were conducted using SPSS 14.0 for Windows. To detect a potential impact of the diet on mortality and reproduction in general, differences in the mean ranked performance between all treatments were compared with the Kruskal– Wallis-H-test. The Kruskal–Wallis-H-test was also used to analyse a potential difference among the four maize treatments. Using the Mann–Whitney-U-test, pairwise comparisons were conducted for the mean number of surviving adults and the mean number of offspring between the treatments with Bt-maize (DKC5143Bt, N4640Bt) and the treatments with their corresponding nearisolines (DKC5143, N4640). The Mann–Whitney-U-test was also used to compare the number of survivors and the number of offspring between the two treatments with the non-transgenic maize varieties. 3. Results 3.1. Survival and offspring Eight to ten of the 10 adult worms initially added survived three weeks feeding on the different diets (Fig. 1). For the maize treatments we recorded a mean number of 8 or 9 surviving adults and
bc
ab c
adults/ treatment
9 8 7 6 5 4 3 2 1 0
Control
DKC5143Bt DKC5143
N4640Bt
N4640
treatment Fig. 1. Mean number of adult enchytraeids per treatment (n ¼ 10, þSE) after three weeks exposure to different maize diets and a high quality control diet of oat flakes. Different letters indicate significant differences (Mann–Whitney-U-test, p < 0.05).
the mean number of survivors was higher on the Bt-maize diets compared to the non-transgenic maize diets. A significant impact of the diets on survival of E. albidus was found when all treatments, means also the control with the high quality diet, were included in the analysis (H-test, p < 0.001). This diet impact still was shown if only the maize treatments were analysed (H-test, p < 0.01). Comparing the DKC5143Bt (Cry3Bb1) treatment to the treatment with the corresponding near-isoline, no significant difference was found for the mean number of survivors. In contrast a significant difference was shown for the comparison of the N4640Bt (Cry1Ab) treatment with its corresponding near-isoline treatment (Mann– Whitney-U-test, p < 0.01). A significantly higher number of adults survived in the N4640Bt treatment. The two treatments with nontransgenic maize showed no significant difference. Comparing the control treatment with the maize treatments, a significant difference was shown between the control and the two treatments with non-transgenic maize. For both comparisons the number of surviving adults was significantly higher in the control than in the maize treatments (U-test, p < 0.01). The mean number (n ¼ 10) of offspring (Fig. 2) was highest in the control treatment (mean of 142 juveniles), followed by the DKC5143Bt (Cry3Bb1) treatment (mean of 81 juveniles) and the two treatments with the non-transgenic maize varieties (mean of 79 juveniles for N4640 and 69 juveniles for DKC5143). The lowest reproduction appeared in the N4640Bt (Cry1Ab) treatment (mean of 51 juveniles). Overall the mean number of offspring was 220 200
offspring/ treatment
The ELISA (enzyme-linked immunosorbent assay) method was used to quantify the Bt-protein content in the different maize diets and in adult worms. Cry1Ab proteins were determined as described by Gugerli [12,13] and Cry3Bb1 proteins as described by Agdia (Indiana, USA). The substrates for the tests were extracted from lyophilized samples of food diets and from adult worms. For the extraction samples with a dry weight of 15–20 mg were used, and each of these samples was put into an extraction bag (type universal, Bioreba, Switzerland) with 3 mL extraction buffer. Samples and buffer were homogenized by using a hand model homogenizer (Faust Laborbedarf AG, Schaffhausen, Switzerland), fixed to an electric drill for laboratory use to extract Bt-proteins. Extracts were centrifuged at 600 g for 10 min. Afterwards the supernatants of the food diet samples were diluted 20-fold in extraction buffer. The supernatants of the adult worms and the food diet with the N4640 corresponding near-isoline stayed undiluted. To create the calibration curve of the Cry1Ab, reference samples of purified Cry1Ab protein were suspended in extracts of control leaves (N4640) at concentrations between 0.01 and 1000 ng protein mL1. For all samples with N4640 (transgenic and nontransgenic) the optical density was measured at 405 nm. For the quantification of the Cry3Bb1 proteins a PathoScreen kit for Bt Cry3Bb1 protein by Agdia (Indiana, USA), following the Agdia product documentation, was used. Samples were extracted in PBST washing buffer by Agdia as described above, but this time the food diets with transgenic DKC5143 were diluted 50-fold in PBST washing buffer. Extracts of adult worms and the food diet with nontransgenic DKC5143 again stayed undiluted. The calibration curve was determined with reference samples of purified Cry3Bb1, suspended in PBST washing buffer, diluted to concentrations between 0.313 and 20 ng protein mL1. The optical density of the samples DKC5143 (transgenic and non-transgenic) was measured at 630 nm. To quantify the Cry1Ab and the Cry3Bb1 content, the concentrations of the calibration curves and the optical density of all samples were log-transformed. Afterwards a linear regression was made to calculate the Bt-protein concentration of the samples (GraphPad Software Inc. 2000). All protein concentrations were calculated as microgram Cry protein per gram dry weight.
353
a
180 160 140 120
bc
100
bc
cd
80
d
60 40 20 0
Control
DKC5143Bt DKC5143
N4640Bt
N4640
treatment Fig. 2. Mean number of offspring per treatment (n ¼ 10, þSE) after 6 weeks. Parental generations were exposed to different maize diets for three weeks, and after their removal, F1-generations were kept on equal diets for three weeks. Different letters indicate significant differences (Mann–Whitney-U-test, p < 0.05).
354
L. Ho¨nemann, W. Nentwig / European Journal of Soil Biology 45 (2009) 351–355
significantly different among the treatments (H-test, p < 0.001). Including only the maize treatments the result still was significant (H-test, p < 0.01). As for the number of survivors, no significant difference of the mean number of offspring was shown between the DKC5143Bt (Cry3Bb1) treatment and the treatment with the corresponding near-isoline. The comparison of the N4640Bt (Cry1Ab) treatment with the treatment containing its corresponding near-isoline showed a significant difference in the mean number of offspring (U-test, p < 0.001). The mean number of offspring was significantly lower in the treatment with the N4640Bt as in the treatment with its isoline. No significant difference was shown between the two diets with non-transgenic maize. The visual control showed no changes in appearance or deformations for any treatment, either for adults (removed after 3 weeks) or juveniles (removed after 6 weeks). 3.2. Bt-protein concentration The diet with N4640Bt showed a mean Cry1Ab protein concentration of 0.5 0.04 mg g1 dry weight. The DKC5143Bt containing diet had a mean Cry3Bb1 protein concentration of 10.0 0.73 mg g1 dry weight. Enchytraeids were observed to feed on the diets. However removed adult worms contained no Cry1Ab proteins after three weeks feeding on the N4640Bt diet. In worms fed with the DKC5143Bt diet Cry3Bb1 proteins were detected in a mean concentration of 0.1 0.01 mg g1 dry weight. Neither the diets with non-transgenic maize nor the worms fed with it contained any Bt-protein. 4. Discussion Due to the specificity of Bt-proteins [11,26] we acted on the assumption that feeding on Bt-maize containing diets has no impact on E. albidus. This expectation was supported by the results of our study. The number of survivors and offspring of E. albidus showed no significant differences between the treatment with the Cry3Bb1 maize (DKC5143Bt) and the treatment with the corresponding near-isoline (DKC5143). Thus there was no effect of the Cry3Bb1 expressing maize leaf material. For the Cry1Ab variety there was a different result. Significantly more individuals survived in the treatment with the Bt-maize (N4640Bt) than in the treatment with the corresponding near-isoline (N4640). In contrast a significantly higher number of offspring was shown for the treatment with the isoline compared to the N4640Bt treatment. Due to the inconsistencies in these results, it seems rather improbable that the differences in survivorship and offspring refer to an effect of the Cry1Ab expressing maize leaf material in the food diet. Our results correspond with the findings of several laboratory studies [30,36,28,1,4,15,33,35] and three field studies [2,38,16] on non-target soil organisms (among earthworms, collembolans, mites and diplopods) which did not find any consistent Bt-maize effect. As described by several studies ([7,27,9,22], Zurbru¨gg et al. unpublished results), maize varieties can exhibit considerable quantitative differences in plant components like carbohydrates, cellulose, lignin, carbon and nitrogen. These differences are naturally caused by a high variation within the gene pool of a plant species [8]. Variability in such plant components could influence the degradability and consequently the quality of plant material as food resource [31,14]. In our experiment the control treatment (moistened oat flake powder) offered the best resources for surviving and reproduction. The quality of the maize diets varied but was generally lower. From degradation patterns of the transgenic varieties N4640Bt and
DKC5143Bt and their untransformed corresponding near-isolines in the field Zurbru¨gg et al. [unpublished results] concluded that the C:N ratio has major impact on degradability. They detected the lowest median C:N ratio for DKC5143Bt, followed by the nontransgenic N4640, whereas the highest median C:N ratio was found for N4640Bt and the non-transgenic DKC5143 which were nearly equal. More easily degradable varieties present a better food resource, and therefore feeding on them should positively affect survival and reproduction. The highest number of offspring was produced on the diet with the transgenic variety DKC5143Bt, followed by the diet with the non-transgenic N4640. According to the findings of Zurbru¨gg et al. [unpublished results] the transgenic variety N4640Bt and the non-transgenic variety DKC5143 are the least degradable. Thus feeding on these two varieties could have an impact on fitness parameters, like survival and reproduction, of E. albidus. Indeed the lowest number of offspring was counted on the diet with N4640Bt. The diet with the non-transgenic DKC5143 showed only a slightly higher number of offspring. For survival, this was not the case. The treatment with N4640Bt showed the highest number of living adults after three weeks feeding on the maize diet. The second least degradable variety, according to Zurbru¨gg et al. [unpublished results] DKC5143, showed lower number of survivors but there was no significant difference from the other maize diets. Indigenous plant components of the maize varieties were not analysed within our experiment. However previous studies (e.g. [27,22]) have established differences in the quantities of these plant components between transgenic varieties and their untransformed counterparts and among maize varieties in general. The suggested relationship between E. albidus performance and the degradability of the plant material was not directly tested in the experiment but our results on reproduction were in line with the results of plant component analyses by Zurbru¨gg et al. [unpublished results]. Therefore effects of different maize diets on the fitness of E. albidus are probable. Effects of Bt-proteins on non-target soil organisms were neither observed in previous studies nor in ours. Referring to the expression of Bt-proteins, we therefore conclude that the cultivation of Btmaize is very unlikely to be a risk for E. albidus or other soil organisms which do not belong to the target group of the expressed Bt-proteins. In contrast differences in plant components of maize, as reported by several studies (e.g. [7,27,9]), could impact soil organisms like E. albidus. Our results suggest a variety effect on life history traits of E. albidus although it is unlikely that this effect would appear under natural conditions. Naturally enchytraeids do not feed on a single food but take up all degradable organic matter of adequate size which they find in the soil. Thus the cultivation of maize in general, regardless of whether transgenic or non-transgenic varieties are grown, does not endanger the survival or reproduction of E. albidus as long as higher quality organic matter is available in the soil.
Acknowledgements This study was funded by the NCCR (National Centers of Competence in Research) Plant Survival. For providing the corn seeds our thanks go to Monsanto. We especially thank Juliane Filser and her team (UFT Zentrum fu¨r Umweltforschung und – technologie, Bremen, Germany) for giving background information on the breeding of enchytraeids and the development of the test design. Finally we thank Corinne Zurbru¨gg (Zoological Institute, Bern, Switzerland) and Jo¨rg Romeis (Agroscope Reckenholz-Ta¨nikon Research Station ART, Zurich, Switzerland) for helpful advice and Phil Lambdon for improving the manuscript.
¨ nemann, W. Nentwig / European Journal of Soil Biology 45 (2009) 351–355 L. Ho
References [1] A. Ahmad, G.E. Wilde, K.Y. Zhu, Evaluation of effects of coleopteran-specific Cry3Bb1 protein on earthworms exposed to soil containing corn roots or biomass, Environ. Entomol. 35 (2006) 976–985. [2] M.A. Al-Deeb, G.E. Wilde, J.M. Blair, T.C. Todd, Effect of Bt corn for corn rootworm control on nontarget soil microarthropods and nematodes, Environ. Entomol. 32 (2003) 859–865. [3] M.J.B. Anorim, J. Ro¨mbke, A.M.V.M. Soares, Avoidance behaviour of Enchytraeus albidus: effects of benomyl, Carbendazim, phenmedipham and different soil types, Chemosphere 59 (2005) 501–510. [4] B.W. Clark, J.R. Coats, Subacute effects of Cry1Ab Bt corn litter on the earthworm Eisenia fetida and the springtail Folsomia candida, Environ. Entomol. 35 (2006) 1121–1129. [5] G.P. Dively, Impact of transgenic VIP3A Cry1Ab lepidopteran-resistant field corn on the nontarget arthropod community, Environ. Entomol. 34 (2005) 1267–1291. [6] W. Dunger, H.J. Fiedler (Eds.), Methoden der Bodenbiologie, Gustav Fischer, Jena, 1997. [7] N. Escher, B. Ka¨ch, W. Nentwig, Decomposition of transgenic Bacillus thuringiensis maize by microorganisms and woodlice Porcellio scaber (Crustacea: Isopoda), Basic Appl. Ecol. 1 (2000) 161–169. [8] A.R. Fernie, Y. Tadmor, D. Zamir, Natural genetic variation improving crop quality, Curr. Opin. Plant Biol. 9 (2006) 196–202. [9] S. Flores, D. Saxena, G. Stotzky, Transgenic Bt plants decompose less in soil than non-Bt plants, Soil Biol. 37 (2005) 1073–1082. [10] T.R. Glare, M. O’Callaghan, Bacillus thuringiensis: Biology, Ecology and Safety, John Wiley & Sons, Chichester, UK, 2000. [11] A.T. Groot, M. Dicke, Insect-resistant transgenic plants in a multi-trophic context, Plant J. 31 (2002) 387–406. [12] P. Gugerli, Le test immuno-enzymatique (ELISA) et son application pour le diagnostic rapide des viruses de la pomme de terre, Revue Suisse d‘Agriculture 11 (1979) 253–260. [13] P. Gugerli, Potato viruses, in: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, vol. XI, VCH Verlagsgesellschaft, Weinheim, Germany, 1986, pp. 430–446. [14] C. Halpin, M.E. Knight, G.A. Foxon, M.M. Campbell, A.M. Boudet, J.J. Boon, B. Chabbert, M.-T. Tollier, W. Schuch, Manipulation of lignin quality by downregulation of cinnamyl alcohol dehydrogenase, Plant J. 6 (1994) 339–350. [15] L.H. Heckmann, B.S. Griffiths, J. Thompson, M. Pusztai-Carey, W.J. Moar, M.N. Andersen, P.H. Krogh, Consequences for Protaphorura armata (Collembola: Onychiuridae) following exposure to genetically modified Bacillus thuringiensis (Bt) maize and non-Bt maize, Environ. Pollut. 142 (2006) 212–216. [16] L. Ho¨nemann, C. Zurbru¨gg, W. Nentwig, Effects of Bt-corn decomposition on the composition of the soil meso- and macrofauna, Appl. Soil Ecol. 40 (2008) 203–209. [17] I. Icoz, G. Stotzky, Fate and effects of insect-resistant Bt crops in soil ecosystems, Soil Biol. Biochem. 40 (2008) 559–586. [18] ISO/DIS 16387, Soil Quality – Effects of Pollutants on Enchytraeidae (Enchytraeus sp.) – Determination of Effects on Reproduction and Survival URL: International Organization for Standardization, 2002, http://www.iso.org/iso/ store.htm (accessed May 2002). [19] S. Ja¨nsch, J. Ro¨mbke, W. Didden, The use of enchytraeids in ecological soil classification and assessment concepts, Ecotoxicol. Environ. Saf. 62 (2005) 266–277.
355
[20] P. Jouquet, J. Dauber, J. Lagerlo¨f, P. Lavelle, M. Lepage, Soil invertebrates as ecosystem engineers: intended and accidental effects on soil and feedback loops, Appl. Soil Ecol. 32 (2006) 153–164. [21] C. Ollier, H. Utz, Statistics in focus: main crop areas in the European Union in 2007, URL: http://epp.eurostat.ec.europa.eu/cache/ITY_OFFPUB/KS-SF-07086/EN/KS-SF-07-086-EN.PDF (accessed October 2007). [22] J. Poerschmann, A. Gathmann, J. Augustin, U. Langer, T. Go´recki, Molecular composition of leaves and stems of genetically modified Bt and near-isogenic non-Bt maize – characterization of lignin patterns, J. Environ. Qual. 34 (2005) 1508–1518. [23] B. Pont, W. Nentwig, Quantification of Bt-protein digestion and excretion by the primary decomposer Porcellio scaber, fed with two Bt-corn varieties, Biocontrol Sci. Technol. 15 (2005) 341–352. [24] J. Ro¨mbke, Th. Moser, Validating the enchytraeid reproduction test: organization and results of an international ringtest, Chemosphere 46 (2002) 1117–1140. [25] J. Romeis, M. Meissle, F. Bigler, Transgenic crops expressing Bacillus thuringiensis toxins and biological control, Nat. Biotechnol. 24 (2006) 63–71. [26] O. Sanvido, J. Romeis, F. Bigler, Ecological impacts of genetically modified crops: ten years of field research and commercial cultivation, Adv. Biochem. Eng. Biotechnol. 107 (2007) 235–278. [27] D. Saxena, G. Stotzky, Bt corn has a higher lignin content than non-Bt corn, Am. J. Bot. 88 (2001) 1704–1706. [28] D. Saxena, G. Stotzky, Bacillus thuringiensis (Bt) toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa bacteria, and fungi in soil, Soil Biol. Biochem. 33 (2001) 1225–1230. [29] A.M. Shelton, J.-Z. Zhao, R.T. Roush, Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants, Ann. Rev. Entomol. 47 (2002) 845–881. [30] S.R. Sims, J.W. Martin, Effect of the Bacillus thuringiensis insecticidal proteins CryIA(b), CryIA(c), CryIIA and CryIIIA on Folsomia candida and Xenylla grisea (Insecta: Collembola), Pedobiologia 41 (1997) 412–416. [31] B.R. Taylor, D. Parkinson, W.F.J. Parsons, Nitrogen and lignin content as predictors of litter decay rates: a microcosm test, Ecology 70 (1989) 97–104. [32] US Environmental Protection Agency (EPA), Modified Cry3A corn – Biopesticide Registration Action Document (BRAD) URL: http://www.epa.gov/ oppbppd1/biopesticides/ingredients/tech_docs/brad_006509.pdf (2007) (accessed November 2007). [33] M.L. Vercesi, P.H. Krogh, M. Holmstrup, Can Bacillus thuringiensis (Bt) corn residues and Bt-corn plants affect the life-history traits in the earthworm Aporrectodea caliginosa? Appl. Soil Ecol. 32 (2006) 180–187. [34] S. Vidal, U. Kuhlmann, C.R. Edwards (Eds.), Western Corn Rootworm – Ecology and Management, CABI Publishing, Wallingford, UK, 2005. [35] M. Weber, W. Nentwig, Impact of Bt corn on the diplopod Allajulus latestriatus, Pedobiologia 50 (2006) 357–368. [36] L. Yu, R.E. Berry, B.A. Croft, Effects of Bacillus thuringiensis toxins in transgenic cotton and potato on Folsomia candida (Collembola: Isotomidae) and Oppia nitens (Acari: Oribatidae), J. Econ. Entomol. 90 (1997) 113–118. [37] C. Zwahlen, A. Hilbeck, R. Howald, W. Nentwig, Effects of transgenic Bt corn litter on the earthworm Lumbricus terrestris, Mol. Ecol. 12 (2003) 1077–1086. [38] C. Zwahlen, A. Hilbeck, W. Nentwig, Field decomposition of transgenic Bt maize residue and the impact on non-target soil invertebrates, Plant Soil 300 (2007) 245–257.
Contents lists available at ScienceDirect
European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Are survival and reproduction of Enchytraeus albidus (Annelida: Enchytraeidae) at risk by feeding on Bt-maize litter? Linda Ho¨nemann a, *, Wolfgang Nentwig b a b
¨rlitzer Platz 1, 06844 Dessau, Germany Federal Environment Agency, Division IV 1.3, Wo Community Ecology, Institute of Ecology and Evolution, University of Bern, Baltzerstrasse 6, 3012 Bern, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 June 2008 Received in revised form 12 March 2009 Accepted 17 March 2009 Available online 7 April 2009 Handling editor: Stefan Schrader
Enchytraeids are saprophagous soil organisms, appearing in high abundances and contributing to ecological processes within the soil. For decades they have been used as model species for biological research. In the framework of research on genetically modified plants, however, they have not been considered to date. Following the ISO/DIS guideline, survival and reproduction of Enchytraeus albidus, fed with diets containing Bt-maize (N4640Bt Cry1Ab, DKC5143Bt Cry3Bb1) leaf material were analysed. For comparison, diets with the corresponding untransformed near-isolines (N4640, DKC5143) were examined. Additionally a high quality control diet (oat flakes) was included. Survival and reproduction showed no significant differences between the Cry3Bb1 treatment and the treatment with the untransformed counterpart. For the Cry1Ab treatment survival was significantly higher than for the treatment with the corresponding near-isoline. In contrast, reproduction was significantly lower for the Cry1Ab treatment compared to that for the isoline. For the Cry3Bb1 treatment, no effect was shown on survival or reproduction. For the Cry1Ab variety and its untransformed counterpart, a contrasting result was detected, which is unlikely to be caused by the Bt-protein but rather by differences in other plant components. Overall survival and reproduction were highest for the control. Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Bt-maize Non-target soil organisms Enchytraeids Feeding test Life history traits
1. Introduction Soil organisms contribute strongly to vital processes within the soil system. Especially, earthworms are known for their high impact on soil properties and influences on the availability of resources for other organisms (animals, micro-organisms, plants) and therefore are regarded as ecosystem engineers [20]. But earthworms are not the only group of annelid worms influencing soil processes. Enchytraeids are saprophagous, colourless to white worms, 2–40 mm long, inhabiting the upper 5–10 cm of the soil [6]. About 900 species are described worldwide, appearing in different soils with high, seasonally fluctuating abundances of a few thousands up to more than 100,000 individuals m2. By their feeding activities enchytraeids support remineralization processes and improve the fine structure of the soil [3,19]. An enchytraeid worm lives on average 2–9 months and sexual maturity is reached 5–7 weeks after hatching from the egg. Enchytraeids are hermaphrodites, but most species show sexual reproduction [6,19]. Due to their contribution to ecological processes, their worldwide appearance and
* Corresponding author. Tel.: þ49 340 2103 2380. E-mail address: [email protected] (L. Ho¨nemann). 1164-5563/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejsobi.2009.03.001
their easy handling and breeding, enchytraeids, in particular Enchytraeus albidus, have been used widely as model species in ecotoxicology, physiology, biochemistry and genetics for more than 50 years [24]. In recent decades national and international regulations to protect the environment against detrimental effects of pollutants have improved steadily. Genetically modified (GM) plants are regarded as a potential source of environmental risks, which have to be adequately assessed [26]. However GM plants have the potential to offer a more efficient and, compared to conventional pesticides, environmentally less harmful protection against pests, thus also enhancing crop yields. Maize, one of the most frequently cultivated crops in Europe [21], is mainly attacked by two insect pests, the tortricid moth Ostrinia nubilalis (European corn borer) and the chrysomelid beetle Diabrotica virgifera virgifera (Western corn rootworm). Larvae of O. nubilalis feed in the stems and D. virgifera virgifera attacks the root system, both are hard to antagonize efficiently with insecticides [29,34]. At present GM maize varieties, expressing Cry proteins of the bacterium Bacillus thuringiensis (Bt), toxic to specific insect groups, present the best protection against these two pests. Cry1 proteins target lepidopterans like O. nubilalis rather selectively and Cry3 proteins antagonize coleopterans like D. virgifera virgifera [10,17]. Due to
352
L. Ho¨nemann, W. Nentwig / European Journal of Soil Biology 45 (2009) 351–355
the physiological specificity of Bt-proteins, effects on target organisms are restricted to a certain protein group of Cry proteins [11,25,26]. Nevertheless non-target organisms may also ingest the Cry proteins expressed by Bt-maize. They can feed on GM plant material, they may predate on prey items containing Bt-protein, or they may come into contact with Cry proteins via root exudates in the soil [11]. The actual amount of research performed on potential side effects of Bt-plants on non-target organisms is quite impressive and allows the general statement that effects are highly improbable [5,25,17]. However, effects like changes in growth, fertility, development time or life span, may be difficult to detect. Such effects must not be attributed to the expression of Bt-proteins they more likely appear due to changes in other plant components. Studies by Escher et al. [7], Saxena and Stotzky [27], Flores et al. [9], Poerschmann et al. [22] and Zurbru¨gg et al. [unpublished results] have shown that Bt- and non-Bt-maize differ not only in the ability to express Bt-proteins but also in the content of important plant components like lignin, cellulose, carbon and nitrogen. These plant components could affect the degradability of plant litter for saprophagous organisms and thus have an impact on their fitness. So far no effect of Bt-maize on soil organisms has been shown, either at the species [7,23,4,15,35] or at the community level [38,16]. Annelid worms are key organisms in soil systems but investigations on them are rare and only earthworms have been examined so far [28,32,37,1,4,33,17]. Now there are no data on potential impacts of Bt-maize on enchytraeids. The presented study here intends to close this gap. In a laboratory feeding experiment we analysed potential effects on survival and reproduction of the enchytraeid E. albidus on leaf diets of two Bt-maize varieties, their untransformed corresponding near-isolines and a high quality control food. Since no Bt-effects on lumbricids have been detected so far, our null hypothesis was that there are also no effects on the closely related enchytraeids.
2. Materials and methods The feeding experiment was designed following the ISO/DIS guideline 16387 [18] for the testing of effects of pollutants on Enchytraeidae (Enchytraeus sp.). The design of the ISO guideline was slightly modified to establish the effect of feeding on different maize varieties on E. albidus. Food diets containing transgenic and non-transgenic maize varieties respectively were used as test substances. Additionally a control diet without maize was used.
2.1. Maize plants Leaf material for the transgenic maize diets was taken from the varieties N4640Bt (transformation event Bt11, Syngenta, Switzerland), expressing Cry1Ab and DKC5143Bt (event MON88017, Monsanto, USA), expressing Cry3Bb1. For the nontransgenic diets leaves of the untransformed corresponding nearisolines (N4640 and DKC5143 respectively) were used. All plants were cultivated in a climatic chamber with 16 h daylight at 25 C and 8 h darkness at 20 C. Plants were grown in 20 L buckets 3⁄4 filled with geranium and balcony plant soil (Mioplant, Zurich, Switzerland). Before sowing 35 g of a slow-release fertilizer (Tardit–Langzeitdu¨nger from Hauert, Grossaffoltern, Switzerland; 14% N, 7% P, 14% K, 1.5% Mg) was added to each bucket. Once per week each bucket received 0.5 L of a 0.2% liquid fertilizer (Wuxal Universal Du¨nger from Maag Agro, Dielsdorf, Switzerland; 10% N, 10% P, 7.5% K, 1.24% B). Water was given as required.
2.2. Enchytraeids The test species E. albidus was bred in our lab, originally deriving from a culture purchased from the Diskus-Laden in Du¨ren, Germany. Following the ISO/DIS guideline, adult worms (with eggs in the clitellum region and approximately 1 cm size) were separated from the breeding culture before the experiment started. Separated enchytraeids were acclimatized to test conditions for 48 h, no food was offered during this time.
2.3. Food diets Senescent maize leaves of the four varieties and oat flakes were ground to a fine powder with an ultra centrifugal mill, type ZM1 (Retsch KG; Haan, Germany). Corresponding to the food in the breeding culture, only oat flake powder moistened with water was used for the high quality control diet. The maize diets contained 70% maize leaf powder of one variety and 30% oat flake powder, moistened with water. Diets were prepared and kept at 25 C until their use in the experiment. From the prepared diets samples were taken for the quantification of the Bt-protein content.
2.4. Soil Plant soil (universal soil for room, balcony and garden plants from Coop, Switzerland), consisting of 40% high bog peat, 40% bark compost and 20% wood fibres, with a pH 5.9 (measured according to ISO/DIS guideline 16387, n ¼ 10) was used. The soil was dried and afterwards sieved to a standard particle size of 2 mm.
2.5. Test design The feeding experiment lasted 6 weeks and included 5 treatments, 4 maize treatments and one control, each replicated 10 times. The test was performed in a climatic closet at 20 C with a day–night cycle of 16 h light and 8 h darkness. We used test vessels of 8 cm height and 6 cm diameter, with the opening closed with fine gauze. Two days before starting the experiment each test vessel was nearly half filled (20 g dry weight) with the prepared soil. The water content of the soil was gravimetrically syntonized to 60%. At the beginning of the experiment 10 adult worms and 0.3 g food diet were added to each test vessel. The food was placed in a hollow in the soil surface, allowing aeration to prevent the diets from molding. Every third day, remaining food was replaced by new supplies and soil was remoistened if required. To avoid losses of eggs or juvenile worms, food remains were separately stored until the end of the experiment. Three weeks after the start of the experiment all adult worms were removed and the number of surviving worms was recorded for each test vessel. To quantify the amount of Bt-proteins (see below) taken up by the worms, all adults removed were lyophilized and frozen at 25 C. After removing the adults the experiment continued for another three weeks. At the end the number of offspring, representing the reproductive output, was determined for each vessel and the associated food remains. For the determination of the number of offspring the soil of each test vessel was divided into portions of 1–2 g. In a Petri dish one of these small portions was bloated with water. Using a binocular microscope, the movement of the juvenile worms was visible and they could be separated with tweezers for counting. Additionally the offspring was visually assessed for obvious changes in appearance or deformations.
¨ nemann, W. Nentwig / European Journal of Soil Biology 45 (2009) 351–355 L. Ho
2.6. Bt-protein quantification
11
a
ab
10
2.7. Data analyses Statistical analyses were conducted using SPSS 14.0 for Windows. To detect a potential impact of the diet on mortality and reproduction in general, differences in the mean ranked performance between all treatments were compared with the Kruskal– Wallis-H-test. The Kruskal–Wallis-H-test was also used to analyse a potential difference among the four maize treatments. Using the Mann–Whitney-U-test, pairwise comparisons were conducted for the mean number of surviving adults and the mean number of offspring between the treatments with Bt-maize (DKC5143Bt, N4640Bt) and the treatments with their corresponding nearisolines (DKC5143, N4640). The Mann–Whitney-U-test was also used to compare the number of survivors and the number of offspring between the two treatments with the non-transgenic maize varieties. 3. Results 3.1. Survival and offspring Eight to ten of the 10 adult worms initially added survived three weeks feeding on the different diets (Fig. 1). For the maize treatments we recorded a mean number of 8 or 9 surviving adults and
bc
ab c
adults/ treatment
9 8 7 6 5 4 3 2 1 0
Control
DKC5143Bt DKC5143
N4640Bt
N4640
treatment Fig. 1. Mean number of adult enchytraeids per treatment (n ¼ 10, þSE) after three weeks exposure to different maize diets and a high quality control diet of oat flakes. Different letters indicate significant differences (Mann–Whitney-U-test, p < 0.05).
the mean number of survivors was higher on the Bt-maize diets compared to the non-transgenic maize diets. A significant impact of the diets on survival of E. albidus was found when all treatments, means also the control with the high quality diet, were included in the analysis (H-test, p < 0.001). This diet impact still was shown if only the maize treatments were analysed (H-test, p < 0.01). Comparing the DKC5143Bt (Cry3Bb1) treatment to the treatment with the corresponding near-isoline, no significant difference was found for the mean number of survivors. In contrast a significant difference was shown for the comparison of the N4640Bt (Cry1Ab) treatment with its corresponding near-isoline treatment (Mann– Whitney-U-test, p < 0.01). A significantly higher number of adults survived in the N4640Bt treatment. The two treatments with nontransgenic maize showed no significant difference. Comparing the control treatment with the maize treatments, a significant difference was shown between the control and the two treatments with non-transgenic maize. For both comparisons the number of surviving adults was significantly higher in the control than in the maize treatments (U-test, p < 0.01). The mean number (n ¼ 10) of offspring (Fig. 2) was highest in the control treatment (mean of 142 juveniles), followed by the DKC5143Bt (Cry3Bb1) treatment (mean of 81 juveniles) and the two treatments with the non-transgenic maize varieties (mean of 79 juveniles for N4640 and 69 juveniles for DKC5143). The lowest reproduction appeared in the N4640Bt (Cry1Ab) treatment (mean of 51 juveniles). Overall the mean number of offspring was 220 200
offspring/ treatment
The ELISA (enzyme-linked immunosorbent assay) method was used to quantify the Bt-protein content in the different maize diets and in adult worms. Cry1Ab proteins were determined as described by Gugerli [12,13] and Cry3Bb1 proteins as described by Agdia (Indiana, USA). The substrates for the tests were extracted from lyophilized samples of food diets and from adult worms. For the extraction samples with a dry weight of 15–20 mg were used, and each of these samples was put into an extraction bag (type universal, Bioreba, Switzerland) with 3 mL extraction buffer. Samples and buffer were homogenized by using a hand model homogenizer (Faust Laborbedarf AG, Schaffhausen, Switzerland), fixed to an electric drill for laboratory use to extract Bt-proteins. Extracts were centrifuged at 600 g for 10 min. Afterwards the supernatants of the food diet samples were diluted 20-fold in extraction buffer. The supernatants of the adult worms and the food diet with the N4640 corresponding near-isoline stayed undiluted. To create the calibration curve of the Cry1Ab, reference samples of purified Cry1Ab protein were suspended in extracts of control leaves (N4640) at concentrations between 0.01 and 1000 ng protein mL1. For all samples with N4640 (transgenic and nontransgenic) the optical density was measured at 405 nm. For the quantification of the Cry3Bb1 proteins a PathoScreen kit for Bt Cry3Bb1 protein by Agdia (Indiana, USA), following the Agdia product documentation, was used. Samples were extracted in PBST washing buffer by Agdia as described above, but this time the food diets with transgenic DKC5143 were diluted 50-fold in PBST washing buffer. Extracts of adult worms and the food diet with nontransgenic DKC5143 again stayed undiluted. The calibration curve was determined with reference samples of purified Cry3Bb1, suspended in PBST washing buffer, diluted to concentrations between 0.313 and 20 ng protein mL1. The optical density of the samples DKC5143 (transgenic and non-transgenic) was measured at 630 nm. To quantify the Cry1Ab and the Cry3Bb1 content, the concentrations of the calibration curves and the optical density of all samples were log-transformed. Afterwards a linear regression was made to calculate the Bt-protein concentration of the samples (GraphPad Software Inc. 2000). All protein concentrations were calculated as microgram Cry protein per gram dry weight.
353
a
180 160 140 120
bc
100
bc
cd
80
d
60 40 20 0
Control
DKC5143Bt DKC5143
N4640Bt
N4640
treatment Fig. 2. Mean number of offspring per treatment (n ¼ 10, þSE) after 6 weeks. Parental generations were exposed to different maize diets for three weeks, and after their removal, F1-generations were kept on equal diets for three weeks. Different letters indicate significant differences (Mann–Whitney-U-test, p < 0.05).
354
L. Ho¨nemann, W. Nentwig / European Journal of Soil Biology 45 (2009) 351–355
significantly different among the treatments (H-test, p < 0.001). Including only the maize treatments the result still was significant (H-test, p < 0.01). As for the number of survivors, no significant difference of the mean number of offspring was shown between the DKC5143Bt (Cry3Bb1) treatment and the treatment with the corresponding near-isoline. The comparison of the N4640Bt (Cry1Ab) treatment with the treatment containing its corresponding near-isoline showed a significant difference in the mean number of offspring (U-test, p < 0.001). The mean number of offspring was significantly lower in the treatment with the N4640Bt as in the treatment with its isoline. No significant difference was shown between the two diets with non-transgenic maize. The visual control showed no changes in appearance or deformations for any treatment, either for adults (removed after 3 weeks) or juveniles (removed after 6 weeks). 3.2. Bt-protein concentration The diet with N4640Bt showed a mean Cry1Ab protein concentration of 0.5 0.04 mg g1 dry weight. The DKC5143Bt containing diet had a mean Cry3Bb1 protein concentration of 10.0 0.73 mg g1 dry weight. Enchytraeids were observed to feed on the diets. However removed adult worms contained no Cry1Ab proteins after three weeks feeding on the N4640Bt diet. In worms fed with the DKC5143Bt diet Cry3Bb1 proteins were detected in a mean concentration of 0.1 0.01 mg g1 dry weight. Neither the diets with non-transgenic maize nor the worms fed with it contained any Bt-protein. 4. Discussion Due to the specificity of Bt-proteins [11,26] we acted on the assumption that feeding on Bt-maize containing diets has no impact on E. albidus. This expectation was supported by the results of our study. The number of survivors and offspring of E. albidus showed no significant differences between the treatment with the Cry3Bb1 maize (DKC5143Bt) and the treatment with the corresponding near-isoline (DKC5143). Thus there was no effect of the Cry3Bb1 expressing maize leaf material. For the Cry1Ab variety there was a different result. Significantly more individuals survived in the treatment with the Bt-maize (N4640Bt) than in the treatment with the corresponding near-isoline (N4640). In contrast a significantly higher number of offspring was shown for the treatment with the isoline compared to the N4640Bt treatment. Due to the inconsistencies in these results, it seems rather improbable that the differences in survivorship and offspring refer to an effect of the Cry1Ab expressing maize leaf material in the food diet. Our results correspond with the findings of several laboratory studies [30,36,28,1,4,15,33,35] and three field studies [2,38,16] on non-target soil organisms (among earthworms, collembolans, mites and diplopods) which did not find any consistent Bt-maize effect. As described by several studies ([7,27,9,22], Zurbru¨gg et al. unpublished results), maize varieties can exhibit considerable quantitative differences in plant components like carbohydrates, cellulose, lignin, carbon and nitrogen. These differences are naturally caused by a high variation within the gene pool of a plant species [8]. Variability in such plant components could influence the degradability and consequently the quality of plant material as food resource [31,14]. In our experiment the control treatment (moistened oat flake powder) offered the best resources for surviving and reproduction. The quality of the maize diets varied but was generally lower. From degradation patterns of the transgenic varieties N4640Bt and
DKC5143Bt and their untransformed corresponding near-isolines in the field Zurbru¨gg et al. [unpublished results] concluded that the C:N ratio has major impact on degradability. They detected the lowest median C:N ratio for DKC5143Bt, followed by the nontransgenic N4640, whereas the highest median C:N ratio was found for N4640Bt and the non-transgenic DKC5143 which were nearly equal. More easily degradable varieties present a better food resource, and therefore feeding on them should positively affect survival and reproduction. The highest number of offspring was produced on the diet with the transgenic variety DKC5143Bt, followed by the diet with the non-transgenic N4640. According to the findings of Zurbru¨gg et al. [unpublished results] the transgenic variety N4640Bt and the non-transgenic variety DKC5143 are the least degradable. Thus feeding on these two varieties could have an impact on fitness parameters, like survival and reproduction, of E. albidus. Indeed the lowest number of offspring was counted on the diet with N4640Bt. The diet with the non-transgenic DKC5143 showed only a slightly higher number of offspring. For survival, this was not the case. The treatment with N4640Bt showed the highest number of living adults after three weeks feeding on the maize diet. The second least degradable variety, according to Zurbru¨gg et al. [unpublished results] DKC5143, showed lower number of survivors but there was no significant difference from the other maize diets. Indigenous plant components of the maize varieties were not analysed within our experiment. However previous studies (e.g. [27,22]) have established differences in the quantities of these plant components between transgenic varieties and their untransformed counterparts and among maize varieties in general. The suggested relationship between E. albidus performance and the degradability of the plant material was not directly tested in the experiment but our results on reproduction were in line with the results of plant component analyses by Zurbru¨gg et al. [unpublished results]. Therefore effects of different maize diets on the fitness of E. albidus are probable. Effects of Bt-proteins on non-target soil organisms were neither observed in previous studies nor in ours. Referring to the expression of Bt-proteins, we therefore conclude that the cultivation of Btmaize is very unlikely to be a risk for E. albidus or other soil organisms which do not belong to the target group of the expressed Bt-proteins. In contrast differences in plant components of maize, as reported by several studies (e.g. [7,27,9]), could impact soil organisms like E. albidus. Our results suggest a variety effect on life history traits of E. albidus although it is unlikely that this effect would appear under natural conditions. Naturally enchytraeids do not feed on a single food but take up all degradable organic matter of adequate size which they find in the soil. Thus the cultivation of maize in general, regardless of whether transgenic or non-transgenic varieties are grown, does not endanger the survival or reproduction of E. albidus as long as higher quality organic matter is available in the soil.
Acknowledgements This study was funded by the NCCR (National Centers of Competence in Research) Plant Survival. For providing the corn seeds our thanks go to Monsanto. We especially thank Juliane Filser and her team (UFT Zentrum fu¨r Umweltforschung und – technologie, Bremen, Germany) for giving background information on the breeding of enchytraeids and the development of the test design. Finally we thank Corinne Zurbru¨gg (Zoological Institute, Bern, Switzerland) and Jo¨rg Romeis (Agroscope Reckenholz-Ta¨nikon Research Station ART, Zurich, Switzerland) for helpful advice and Phil Lambdon for improving the manuscript.
¨ nemann, W. Nentwig / European Journal of Soil Biology 45 (2009) 351–355 L. Ho
References [1] A. Ahmad, G.E. Wilde, K.Y. Zhu, Evaluation of effects of coleopteran-specific Cry3Bb1 protein on earthworms exposed to soil containing corn roots or biomass, Environ. Entomol. 35 (2006) 976–985. [2] M.A. Al-Deeb, G.E. Wilde, J.M. Blair, T.C. Todd, Effect of Bt corn for corn rootworm control on nontarget soil microarthropods and nematodes, Environ. Entomol. 32 (2003) 859–865. [3] M.J.B. Anorim, J. Ro¨mbke, A.M.V.M. Soares, Avoidance behaviour of Enchytraeus albidus: effects of benomyl, Carbendazim, phenmedipham and different soil types, Chemosphere 59 (2005) 501–510. [4] B.W. Clark, J.R. Coats, Subacute effects of Cry1Ab Bt corn litter on the earthworm Eisenia fetida and the springtail Folsomia candida, Environ. Entomol. 35 (2006) 1121–1129. [5] G.P. Dively, Impact of transgenic VIP3A Cry1Ab lepidopteran-resistant field corn on the nontarget arthropod community, Environ. Entomol. 34 (2005) 1267–1291. [6] W. Dunger, H.J. Fiedler (Eds.), Methoden der Bodenbiologie, Gustav Fischer, Jena, 1997. [7] N. Escher, B. Ka¨ch, W. Nentwig, Decomposition of transgenic Bacillus thuringiensis maize by microorganisms and woodlice Porcellio scaber (Crustacea: Isopoda), Basic Appl. Ecol. 1 (2000) 161–169. [8] A.R. Fernie, Y. Tadmor, D. Zamir, Natural genetic variation improving crop quality, Curr. Opin. Plant Biol. 9 (2006) 196–202. [9] S. Flores, D. Saxena, G. Stotzky, Transgenic Bt plants decompose less in soil than non-Bt plants, Soil Biol. 37 (2005) 1073–1082. [10] T.R. Glare, M. O’Callaghan, Bacillus thuringiensis: Biology, Ecology and Safety, John Wiley & Sons, Chichester, UK, 2000. [11] A.T. Groot, M. Dicke, Insect-resistant transgenic plants in a multi-trophic context, Plant J. 31 (2002) 387–406. [12] P. Gugerli, Le test immuno-enzymatique (ELISA) et son application pour le diagnostic rapide des viruses de la pomme de terre, Revue Suisse d‘Agriculture 11 (1979) 253–260. [13] P. Gugerli, Potato viruses, in: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, vol. XI, VCH Verlagsgesellschaft, Weinheim, Germany, 1986, pp. 430–446. [14] C. Halpin, M.E. Knight, G.A. Foxon, M.M. Campbell, A.M. Boudet, J.J. Boon, B. Chabbert, M.-T. Tollier, W. Schuch, Manipulation of lignin quality by downregulation of cinnamyl alcohol dehydrogenase, Plant J. 6 (1994) 339–350. [15] L.H. Heckmann, B.S. Griffiths, J. Thompson, M. Pusztai-Carey, W.J. Moar, M.N. Andersen, P.H. Krogh, Consequences for Protaphorura armata (Collembola: Onychiuridae) following exposure to genetically modified Bacillus thuringiensis (Bt) maize and non-Bt maize, Environ. Pollut. 142 (2006) 212–216. [16] L. Ho¨nemann, C. Zurbru¨gg, W. Nentwig, Effects of Bt-corn decomposition on the composition of the soil meso- and macrofauna, Appl. Soil Ecol. 40 (2008) 203–209. [17] I. Icoz, G. Stotzky, Fate and effects of insect-resistant Bt crops in soil ecosystems, Soil Biol. Biochem. 40 (2008) 559–586. [18] ISO/DIS 16387, Soil Quality – Effects of Pollutants on Enchytraeidae (Enchytraeus sp.) – Determination of Effects on Reproduction and Survival URL: International Organization for Standardization, 2002, http://www.iso.org/iso/ store.htm (accessed May 2002). [19] S. Ja¨nsch, J. Ro¨mbke, W. Didden, The use of enchytraeids in ecological soil classification and assessment concepts, Ecotoxicol. Environ. Saf. 62 (2005) 266–277.
355
[20] P. Jouquet, J. Dauber, J. Lagerlo¨f, P. Lavelle, M. Lepage, Soil invertebrates as ecosystem engineers: intended and accidental effects on soil and feedback loops, Appl. Soil Ecol. 32 (2006) 153–164. [21] C. Ollier, H. Utz, Statistics in focus: main crop areas in the European Union in 2007, URL: http://epp.eurostat.ec.europa.eu/cache/ITY_OFFPUB/KS-SF-07086/EN/KS-SF-07-086-EN.PDF (accessed October 2007). [22] J. Poerschmann, A. Gathmann, J. Augustin, U. Langer, T. Go´recki, Molecular composition of leaves and stems of genetically modified Bt and near-isogenic non-Bt maize – characterization of lignin patterns, J. Environ. Qual. 34 (2005) 1508–1518. [23] B. Pont, W. Nentwig, Quantification of Bt-protein digestion and excretion by the primary decomposer Porcellio scaber, fed with two Bt-corn varieties, Biocontrol Sci. Technol. 15 (2005) 341–352. [24] J. Ro¨mbke, Th. Moser, Validating the enchytraeid reproduction test: organization and results of an international ringtest, Chemosphere 46 (2002) 1117–1140. [25] J. Romeis, M. Meissle, F. Bigler, Transgenic crops expressing Bacillus thuringiensis toxins and biological control, Nat. Biotechnol. 24 (2006) 63–71. [26] O. Sanvido, J. Romeis, F. Bigler, Ecological impacts of genetically modified crops: ten years of field research and commercial cultivation, Adv. Biochem. Eng. Biotechnol. 107 (2007) 235–278. [27] D. Saxena, G. Stotzky, Bt corn has a higher lignin content than non-Bt corn, Am. J. Bot. 88 (2001) 1704–1706. [28] D. Saxena, G. Stotzky, Bacillus thuringiensis (Bt) toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa bacteria, and fungi in soil, Soil Biol. Biochem. 33 (2001) 1225–1230. [29] A.M. Shelton, J.-Z. Zhao, R.T. Roush, Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants, Ann. Rev. Entomol. 47 (2002) 845–881. [30] S.R. Sims, J.W. Martin, Effect of the Bacillus thuringiensis insecticidal proteins CryIA(b), CryIA(c), CryIIA and CryIIIA on Folsomia candida and Xenylla grisea (Insecta: Collembola), Pedobiologia 41 (1997) 412–416. [31] B.R. Taylor, D. Parkinson, W.F.J. Parsons, Nitrogen and lignin content as predictors of litter decay rates: a microcosm test, Ecology 70 (1989) 97–104. [32] US Environmental Protection Agency (EPA), Modified Cry3A corn – Biopesticide Registration Action Document (BRAD) URL: http://www.epa.gov/ oppbppd1/biopesticides/ingredients/tech_docs/brad_006509.pdf (2007) (accessed November 2007). [33] M.L. Vercesi, P.H. Krogh, M. Holmstrup, Can Bacillus thuringiensis (Bt) corn residues and Bt-corn plants affect the life-history traits in the earthworm Aporrectodea caliginosa? Appl. Soil Ecol. 32 (2006) 180–187. [34] S. Vidal, U. Kuhlmann, C.R. Edwards (Eds.), Western Corn Rootworm – Ecology and Management, CABI Publishing, Wallingford, UK, 2005. [35] M. Weber, W. Nentwig, Impact of Bt corn on the diplopod Allajulus latestriatus, Pedobiologia 50 (2006) 357–368. [36] L. Yu, R.E. Berry, B.A. Croft, Effects of Bacillus thuringiensis toxins in transgenic cotton and potato on Folsomia candida (Collembola: Isotomidae) and Oppia nitens (Acari: Oribatidae), J. Econ. Entomol. 90 (1997) 113–118. [37] C. Zwahlen, A. Hilbeck, R. Howald, W. Nentwig, Effects of transgenic Bt corn litter on the earthworm Lumbricus terrestris, Mol. Ecol. 12 (2003) 1077–1086. [38] C. Zwahlen, A. Hilbeck, W. Nentwig, Field decomposition of transgenic Bt maize residue and the impact on non-target soil invertebrates, Plant Soil 300 (2007) 245–257.