Decomposition of transgenic Bacillus thuringiensis maize by microorganisms and woodlice Porcellio scaber (Crustacea: Isopoda)

Basic Appl. Ecol. 1, 161–169 (2000) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/baecol

Basic and Applied Ecology

Decomposition of transgenic Bacillus thuringiensis maize by microorganisms and woodlice Porcellio scaber (Crustacea: Isopoda) Natalie Escher, Barbara Käch, Wolfgang Nentwig1 Zoological Institute, University of Bern, Bern, Switzerland

Received January 6, 2000 · Accepted August 2, 2000

Abstract Foliage of transgenic maize Zea mays L., expressing a Cry1Ab protein derived from Bacillus thuringiensis (Berliner) subsp. kurstaki, was compared with foliage of the corresponding non-transgenic maize variety in laboratory feeding and decomposition experiments to study the effects of the B. thuringiensis protein on the chemical composition of the maize leaves, on the decomposer Porcellio scaber (Crustacea: Isopoda), and on leaf-litter-colonising microorganisms. Initial contents of fructose and soluble carbohydrates were significantly higher in non-transgenic maize. Lignin was decomposed more quickly in transgenic maize. Starch, cellulose, hemicellulose and ash content did not differ. Bacterial growth on faeces of P. scaber fed on non-transgenic maize was up to 60% higher than on faeces of the transgenic-fed woodlice, but bacterial growth on leaves and fungal growth on faeces were equal on both maize varieties. P. scaber showed no significant difference in its consumption rate of transgenic and non-transgenic maize. The number of offspring did not differ between the two treatment groups, but the mortality of juveniles reared on non-transgenic maize leaves was significantly higher. During the first 131 days weight increase of the offspring was significantly higher in the non-transgenic group, but weight increase of adult P. scaber was higher in the transgenic group. Due to a slightly lower C:N ratio, a lower lignin content, and a higher content of soluble carbohydrates, the nutritional quality of transgenic maize leaves was better than that of the non-transgenic variety. This explains the lower mortality of P. scaber offspring and the faster weight gain of adult P. scaber on the transgenic diet. Blätter von transgenem Mais Zea mays L., die ein Cry1Ab Protein von Bacillus thuringiensis (Berliner) subsp. kurstaki exprimieren, wurden in Laborversuchen mit Blättern der nicht-transgenen korrespondierenden Maissorte verglichen, um den Einfluss des B. thuringiensis Proteins auf die chemische Zusammensetzung der Maisblätter, auf den Detritophagen P. scaber und auf streubesiedelnde Mikroorganismen zu untersuchen. Anfänglich wies nicht-transgener Mais einen signifikant höheren Gehalt an Fruktose und löslichen Kohlehydraten auf. Der Ligningehalt war in transgenem Mais während des Abbaus signifikant niedriger, Stärke, Hemicellulose und Aschegehalt waren gleich. Auf dem Kot von P. scaber war das bakterielle Wachstum, wenn den Tieren nichttransgener Mais gefüttert wurde, bis zu 60% höher als auf dem Kot transgen gefütterter Tiere, aber auf den Maisblättern selbst waren Bakterien- und Pilzwachstum bei beiden Maisvarietäten gleich. Die Nahrungsaufnahme von P. scaber war bei beiden Maissorten ebenfalls gleich. Die Fortpflanzungsrate unterschied sich bei beiden Versuchsgruppen nicht, aber die Mortalität der Jungen war auf nicht-transgenem Mais signifikant höher. Während der ersten 131 Lebenstage nahm das Körpergewicht der Jungen bei nicht-transgenem Mais signifikant schneller zu, adulte P. scaber wuchsen 1

Corresponding author: Wolfgang Nentwig, Zoological Institute, University of Bern, Baltzerstr. 3, CH 3012 Bern, Switzerland, Phone +0041-31-631 4520, Fax +0041-31-631 4888, E-mail [email protected]

1439-1791/2000/1/02-161 $ 15.00/0

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Escher et al. jedoch bei transgener Ernährung schneller. Aufgrund des geringfügig niedrigeren C:N-Verhältnisses, des niedrigeren Ligningehaltes und der größeren Verfügbarkeit von löslichen Kohlehydraten war die Nahrungsqualität von transgenem Mais besser als die der nicht-transgenen Vergleichssorte. Dies erklärt die niedrigere Mortalität des Nachwuchses und das schnellere Wachstum der adulten Tiere bei transgener Nahrung. Key words: decomposition – Bt corn – Bt maize – litter – agroecosystem – transgenic plant – food choice

Introduction The effect of transgenic plants expressing insecticidal proteins of Bacillus thuringiensis on nontarget organisms has become an area of interest in recent years (e.g. Riddick & Barbosa 1998). The genetically engineered plants produce the insecticidal proteins until plant senescence begins (Koziel et al. 1993) and may therefore prolong the environmental persistence and increase the bioavailability of the toxins to both target and nontarget invertebrates (Sims & Martin 1997). The protein may have an impact on different trophic levels: nontarget herbivores (primary consumers) are directly affected by feeding on transgenic plant material (e.g. Losey et al. 1999), predators by feeding on intoxicated prey (e.g. Hilbeck et al. 1998, Zwahlen et al. 2000) and, finally, decomposers by feeding on litter containing the toxins (e.g. Sims & Martin 1997). Decomposer organisms live not only on decaying plant material but also on dead animal bodies and faeces. As a consequence, all food types could be a direct (plant material) or indirect (tissue and faeces of intoxicated animals) source of B. thuringiensis toxins for decomposers if the toxin is still in an active form. An important aspect of the risk assessment of pesticidal transgenic plants is therefore the potential for detrimental effects on the soil ecosystem from residual plant material following harvesting and tillage (Donegan et al. 1997). Relatively few studies concern the decomposer fauna and flora, which is astonishing, considering the importance of decomposer organisms and the persistence of Bt toxins in the soil (e.g. Sims & Ream 1997, Saxena et al. 1999). The activity of soil organisms unlocks mineral sources such as phosphorus and nitrogen that are fixed in dead organic matter, and the speed of this process determines the rate at which such resources are released to growing plants. Additionally, litter decomposition rates are also regulated by environmental conditions and the chemical composition of the litter. N content (or, more usually, the C:N ratio) is of critical importance for the litter decay rate (Berg & Staaf 1980, Flanagan & Van Cleve 1983, Taylor et al. 1989, Cadish & Giller 1997). Other studies found limitations of decomposition rates through lignin content or C:lignin ratio (Schlesinger & Hasey 1981). Basic Appl. Ecol. 1, 2 (2000)

So far, no study has compared the nutritional value of transgenic Bt-maize, one of the most abundantly grown transgenic plants, with its correspondent nontransgenic variety. Therefore, we analysed some nutritional parameters of both maize varieties and investigated the decomposition processes. Since in most European countries it is not legally possible to grow transgenic maize in the field, we performed laboratory studies using the isopod Porcellio scaber as a model organism to ascertain the equal nutritional values of both maize varieties. Consequently the decomposition of Bt-maize by microorganisms and non-target woodlice should be comparable to that of the nontransgenic variety. This hypothesis was tested by analysis of food selection, growth and reproduction of P. scaber.

Materials and methods Plants and nutritional value analyses The transgenic maize variety (Bt+) (X4334-EPR, Novartis, previously Northrup King) containing the synthetic version of a gene from B. thuringiensis subsp. kurstaki coding for the expression of the insecticidal B. thuringiensis-δ-endotoxin Cry1Ab was compared to the corresponding non-transgenic variety (Bt–) not carrying the Cry1Ab-gene (isoline). The plant material of both varieties was kindly provided by Angelika Hilbeck, Swiss Federal Research Station for Agroecology and Agriculture, Zurich, Switzerland. All plants were cultivated in plastic pots in a greenhouse in Zurich. The freshly cut plants were sent to Bern where the stalks and leaves were dried separately at 45–50 °C until weight constancy was achieved. The dried material was stored at –20 °C until use. Dried leaf samples of Bt+ and Bt- maize (2 g per sample) were soaked with distilled water for 2 min. and placed for 0, 2, 4, 6 and 8 weeks respectively (= decomposition times) in plastic boxes (15.5 × 11 × 6 cm) (15 °C, dark). A small gap between box and lid (0.2 mm) allowed air circulation. The samples were then re-dried at 40 °C for two days, ground first with a mixer (Braun HM), then by mortar and pestle to obtain a fine, homogeneous powder, and stored until

Decomposition of transgenic Bacillus thuringiensis maize by microorganisms

analysis at –20°C. Concentrations of Cry1Ab in the ground dry leaf material were measured at the Federal Agricultural Research Station in Changins, Switzerland, using enzyme-linked immunosorbent assays (ELISA). Energy content (kJ) of the powdered samples was measured on 3 replicates of Bt+ and Bt- maize for each decomposition time in an oxygen bomb calorimeter (Parr 1341 plain oxygen bomb calorimeter, Parr Instr. Comp., Illinois, USA). Soluble sugars were analysed following the method of Schnyder & Nelson (1987). D-fructose (Fluka Nr. 47739, 0–75 mµg) was used as a standard. Starch was analysed with the method of Brändle (1985) using D-glucose (Fluka Nr. 49139) as a standard (8 replicates each). Hemicellulose, cellulose, lignin and ash content were determined quantitatively by sequential analysis using the methods of Scehovic (1975) (3 replicates each). Total nitrogen was determined on a NA 2000 Nitrogen Analyser (CE Instruments, Milano, Italy) using subsamples of 9–12 mg ground leaf material (8 replicates). Decomposition by P. scaber The woodlouse Porcellio scaber (Latreille) (Crustacea: Isopoda: Porcellionidae) was chosen for the feeding experiments as a representative of the soil-inhabiting detritophagous macrofauna of considerable indicator importance and as a nontarget organism to test the possible impact of a transgenic crop (e.g. Alberti et al. 1998, Sutton 1980, Ullrich et al. 1991). The woodlice were collected from a compost heap near Bern in summer 1997 and kept in plastic boxes with a transparent lid (15.5 × 11 × 6 cm) in a climate chamber (15 ± 1 °C, 80 ± 5% r.h., 7 h light : 17 h dark). For the experiments, boxes were covered 1.5 cm high with plaster of Paris containing neutralised activated charcoal (1% v/v), soaked with water, to ensure constant humidity. Four food-choice laboratory trials were carried out. Before the experiments, P. scaber was starved for 48 h. Single woodlice of 30–50 mg individual fresh weight (weighed to the nearest 0.1 mg) were placed into plastic boxes. A wire mesh (mesh size 7 × 7 mm) divided the box in the middle, allowing the woodlice to pass but not to drag whole leaves from one side of the box to the other. Two pieces of crock on each side provided hiding places for P. scaber. Leaves that had been decomposed for three weeks (at which state previous experiments had shown the consumption rate by P. scaber to be highest) were dried, weighed, remoistened and offered to the woodlice (800 mg remoistened material). Bt+ maize was placed on one side of the wire mesh and Bt- maize on the other, allowing P. scaber to choose. The experiments lasted for eight days, after which the woodlice were weighed,

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the remaining leaves carefully dried at 40 °C for at least 48 h and also weighed. Consumption was estimated as mg dry weight of leaves consumed per mg live weight of woodlouse. In each trial, 10 experiments with P. scaber (only non-moulting woodlice and woodlice without a brood pouch were used) and 10 control experiments without woodlice were carried out. Each woodlouse was tested only once. The control experiments were used to estimate the weight loss caused by microorganisms, which was then subtracted from the consumption by the woodlice. To determine reproduction, growth and survival of juveniles, P. scaber were separated by sex and held for one month on Bt+ or Bt- maize leaves. After this pretrial time, 60 individuals each were placed in pairs into boxes and maintained under conditions designed to induce reproduction (21 ± 1 °C, 16 h light : 8 h dark, 80 ± 5% r.h.) for seven months. Trial diet corresponded to the pre-trial diet. The animals were controlled twice a week for food supply and for the development of a marsupium, a brood pouch in which the pregnant female carries the eggs and juveniles. Every two weeks the adults’ weights were recorded. The difference between beginning weight and final weight was used for statistical analysis. Juveniles were kept on the same diet as the adults immediately after hatching and checked once a week for mortality. Percentage of survival was calculated after 120 days. The weight of juveniles between the age of 5 and 131 days was recorded once and the data used for regression analysis. Decomposition by microorganisms For the quantification of microorganisms on the faeces of P. scaber, three replicate populations of ten individuals each were tested. At the beginning of the experiment, the woodlice were offered 5 g re-moistened Bt+ and Bt– maize leaves, which lasted for at least 12 weeks. Faeces were collected and tested every two weeks. Three days before the faecal pellets were collected, each population was transferred into another plastic box containing only some pieces of crock but no food. 50 fresh faecal pellets (total dry weight 2.8 mg) were suspended in 100 ml sterile 0.9% NaCl solution and incubated under gentle shaking for 30 minutes. Simultaneously to the treatment of the woodlice, maize leaves of either variety were decomposed: dry leaf samples of approx. 0.5 g were remoistened and left to decay for 0, 2, 4, 6, 8, 10 and 12 weeks (3 replicates each). For the preparation of the samples the decayed plant material was transferred into 49.5 ml sterile 0.9% NaCl solution and incubated under gentle shaking for 30 minutes. To determine bacterial and Basic Appl. Ecol. 1, 2 (2000)

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fungal densities on maize leaves and faeces, three different growth media were used: 1. CASO agar (Merck 1.05458) as a complex medium to isolate a wide range of microorganisms. 2. Cellulose agar, prepared with Avicel® PH-101 (Fluka 11365) instead of carboxymethyl-celluloseremazol brilliant blue (CMC-RBB), to cultivate microorganisms that are capable of decomposing cellulose. Bacterial and fungal growth was counted (as colony-forming units) separately on this medium. 3. Sabouraud 4% maltose agar (Merck 1.05439) to count fungal growth. It incorporated 0.1 g/l streptomycin and 0.04 g/l chloramphenicol to inhibit bacterial growth (Anonymous 1996/97). Methods described by Alberti et al. (1998) were followed to prepare different media, ten-fold dilution series, streaking-out of samples and incubation of plates. Counts of colony-forming units were expressed in colony-forming units per g dry weight faeces or leaf respectively.

Fig. 1. Fructose content of transgenic maize (Bt+) and non-transgenic maize (Bt–) in percentage of dry weight. Columns show median, 25% and 75% quartiles. Different letters indicate significant differences in fructose content (Mann-Whitney U-test, n = 8, Bonferroni-Holm corrected, t = 5, p < 0.05); n. s. = not significant.

Data analysis Analyses were performed using Systat software (SPSS Inc. 1997). For parametric data (consumption rates, number of offspring per female) t-tests were used, for non-parametric data (all chemical compound data, weight increase of adults) Mann-Whitney U-tests, Bonferroni-Holm corrected, were used. Kruskal-Wallis one-way analysis of variance was applied to test for differences in decomposition times. Survival data as percentages were arcsine square-root transformed before analysis. To analyse the growth of the juveniles, a regression model was used. Data were ln transformed to obtain a linear regression that was used to perform the F-tests on non-parallelism, incline and distance between the two regression lines.

Results Nutritional value of litter The concentrations of fructose (Fig. 1) and soluble carbohydrates (data not shown) were significantly higher in Bt– foliage (p = 0.03). The fructose content decreased during eight weeks of decomposition to about half the original content. This effect is significant for both varieties and the degradation of the carbohydrates of Bt– maize was faster (Kruskal-Wallis one-way analysis of variance, p < 0.001). The percentage of starch did not significantly differ between Bt+ and Bt– maize at any decomposition time (Fig. 2). In Basic Appl. Ecol. 1, 2 (2000)

Fig. 2. Starch content of transgenic maize (Bt+) and non-transgenic maize (Bt–) in percentage of dry weight. Columns show median, 25% and 75% quartiles (Mann-Whitney U-test, n = 8, p < 0.05).

addition, no decrease of the starch content could be found during 8 weeks of decomposition, neither for the Bt+ maize nor for the Bt– maize (Kruskal-Wallis one-way Analysis of Variance, p > 0.05, Fig. 2). The hemicellulose and cellulose contents were similar for both varieties (Fig. 3). The hemicellulose content of dry leaves increased slightly over four weeks and then slightly decreased. The temporal course of the cellulose content was similar to that of hemicellulose. The lignin content differed between Bt+ and Bt– maize (Fig. 4): Both varieties showed the lowest lignin content in the first week, which increased to a maximum at four weeks for Bt– plants and at six weeks for Bt+ plants. The content of control plants remained high (4.4%, eight weeks) but decreased in Bt+ maize (2.7%, eight weeks). Concerning the ash content, val-

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Fig. 3. Changes in hemicellulose and cellulose content of Bt+ maize (closed symbols) and Bt– maize (open symbols) over eight weeks of decomposition. Each value shows the mean percentage of initial total dry weight of three replicates.

Fig. 4. Changes in lignin and ash content of Bt+ maize (closed symbols) and Bt– maize (open symbols) over eight weeks of decomposition. Each value shows the mean percentage of initial total dry weight of three replicates.

ues and time course were nearly identical for Bt+ and Bt– maize. It changed only slightly in the course of decomposition and dropped at four weeks to a minimum of 1.0% for Bt+ plants and 0.8% for Bt– plants (Fig. 4). No significant difference in nitrogen content was found between the hybrids at any decomposition time. Nevertheless, total nitrogen content was always higher in Bt+ maize. It increased slightly but steadily from 1.2 ± 0.2% to 2.0 ± 0.3% (8 weeks) in Bt+ maize over time. In Bt– plants, N content increased from 1.1 ± 0.3% to 1.5 ± 0.5% (8 weeks). Concentrations of Cry1Ab protein in the leaves were between 0.46 and 0.51 mg/g dry weight. Within a decomposition time of eight weeks the protein concentration only decreased by about 25%.

Fig. 5a–d. Counts of colony-forming units on Bt+ (closed symbols) and Bt– (open symbols) maize leaves and faeces of P. scaber fed with either variety of foliage. Circles and squares represent medians of three replicates (a = CASO agar, bacterial growth; b = cellulose agar, bacterial growth; c = cellulose agar, fungal growth; d = Sabouraud agar, fungal growth).

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The energy content of the Bt+ foliage was 1.5% lower (16.5 ± 0.1 vs. 16.75 ± 0.01 kJ/g dry matter) than that of the Bt– foliage (not significant) and decreased by 2.2 ± 0.3% (Bt– leaves) and 2.8 ± 0.4% (Bt+ leaves) within eight weeks. The energy content of Bt+ maize was always 0.2–0.4 kJ/g lower (but never significant). Microbial growth on maize leaves and P. scaber faeces The bacterial density on faeces of woodlice was much higher than on leaves, and population growth was more constant on faeces than on maize foliage (Fig. 5a and b). Bacterial growth decreased only on cellulose agar during the first two weeks (Fig. 5b). Bacterial density on faeces of Bt+-fed P. scaber was up to 60% lower than on faeces of Bt–fed individuals (Fig. 5a and b). Fungal growth was less constant, and the density on faeces of Bt+-fed P. scaber was not constantly lower or higher than that of the control group (Fig. 5c and d). The same was found for the microbial density on maize foliage. The bacterial growth seemed to be less susceptible to variations in density than fungal growth. During 8 weeks, decomposition of Bt+ leaves was always 2–5% faster than decomposition of Bt– leaves (mass losses e.g. after 4 weeks 56 ± 5% and 52 ± 4%, n. s.).

Fig. 6. Weight increase (mg) of P. scaber juveniles fed on transgenic (Bt+) (solid line) or non-transgenic (Bt–) (dotted line) maize foliage for 131 days (F-test, p < 0.05, df = 1, 39, after ln transformation).

Bt– maize leaves gained weight faster than those kept on Bt+ foliage (Fig. 6). There was also a significant difference in weight increase of adult P. scaber (p = 0.034, Table 1). Adult woodlice fed on the Bt+ foliage diet gained 33% more weight than the control group within 22 weeks.

Discussion

Leaf consumption by P. scaber

Biochemical analysis of litter quality

Weekly consumption (expressed in mg dry weight leaf material per mg live weight of P. scaber) did not differ significantly between Bt+ and Bt– plants (paired samples t-test, p > 0.05, two-tailed) (0.348 ± 0.15 mg dry matter / mg fresh weight for Bt+ leaves vs. 0.313 ± 0.08 mg for Bt– leaves). There were no significant differences in the number of juveniles per female between Bt+ and Bt– diets (16.05 ± 1.52 vs. 13.33 ± 1.52, p = 0.214, Table 1), but a significantly higher mortality of the juveniles reared on Bt– foliage was observed (p = 0.03, Table 1). During the first 131 days, the juveniles reared on

The major part of the energy content of maize leaves consists of different carbohydrates. Cellulose, hemicellulose and lignin, which are structural polysaccharides, constitute the plant tissue whereas starch and fructose are storage carbohydrates. Minor components of energy content are lipids and proteins (Lüttge et al. 1994). Energy content decreases with increasing decomposition time due to microbial activity. The progress of material loss was the same for both varieties. However, the energy content of the two varieties differed, Bt– maize having a (non-significant but constant) 1.5% higher energy content than the Bt+ variety.

Table 1. Reproduction, proportion of surviving juveniles after 120 days and weight increase in percent of adult P. scaber fed on transgenic (Bt+) or non-transgenic (Bt–) maize foliage. a t-test (independent, two-tailed), b t-test (independent, two-tailed, after arcsine square-root transformation), c Mann-Whitney U-test. Mean ± SE response for Bt–

Bt+

T

p

df

number of juveniles per female

13.33 ± 1.52 (n = 21)

16.05 ± 1.52 (n = 21)

1.265

0.214a

37.9

proportion of surviving juveniles after 120 days

0.507 ± 0.052

0.697 ± 0.066

2.27

0.03b

35.0

weight increase of adult P. scaber (%)

23.5

31.3

0.034c

1

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Decomposition of transgenic Bacillus thuringiensis maize by microorganisms

The main factor controlling the decomposition rate of litter is the activity of bacteria and fungi. The greenhouse where the maize plants had been cultivated was not a controlled and closed environment and therefore the colonisation of the maize leaves was very heterogeneous. Patches with a high density of sugardegrading fungi and bacteria could have undergone faster decomposition of fructose. This heterogeneous colonisation could explain the difference in the fructose content. After four weeks of decomposition, the fructose content was significantly higher in Bt+ maize foliage, whereas at weeks zero and six it was significantly higher in Bt– leaves. However, the degradation of starch did not seem to be affected by this heterogeneity; there was only a tendency towards differences, but of no significance. Therefore, if soluble carbohydrates and starch determine early stages of litter decomposition, there should be no difference in decomposition rates of the two varieties. If nitrogen is considered to be one of the crucial substances responsible for the decomposition rate (Taylor et al. 1989), a tendency for faster decomposition of Bt+ maize could be assumed because it contained more nitrogen at all decomposition times, although this difference was not significant. This tendency corresponds with mass losses over decomposition time, where Bt+ maize lost more weight than the control. However, only total nitrogen was measured and it cannot be concluded from which component the difference in nitrogen content resulted. It is also not clear why Bt+ maize contained more nitrogen than Bt– maize even if a certain amount of the nitrogen was probably used to synthesise the B. thuringiensis protein. These results may indicate that the B. thuringiensis protein is responsible for hitherto unknown changes in the plant's metabolism. Regarding the C:N ratio as a decomposition factor, we found only a tendency towards a different nitrogen content, but no significant difference in carbohydrate content between the two maize varieties. Cellulose, hemicellulose and ash content were nearly identical, but a lower lignin content was found in Bt+ maize. Since both a high nitrogen content and a low lignin content facilitate a rapid decomposition process, this indicates that the nutritional quality of the two tested maize varieties may have differed to some degree. Decomposition by microorganisms Bacterial biomass plays an important role in isopod feeding activity. Bacteria make the consumed nutrients more readily available by decomposition and by supplementing food with essential nutrients such as vitamins, enzymes and amino acids (Drobne 1995). The total contribution of bacterial biomass ingested

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with food and faeces to the energy requirement is estimated to be quite low, but the quality of bacterial biomass seems to be very important for the animal (Ullrich et al. 1991). Woodlice are able to utilise the by-products of ingested cellulolytic microorganisms, which are the only source of cellulase present in the alimentary system of woodlice (Drobne 1995, Carefoot 1984, Hassall & Jennings 1975). Ineson & Anderson (1985) showed that the number of bacteria increases dramatically from leaf litter to faeces, after passing the gut of Oniscus asellus and Glomeris marginata. These observations are in accordance with the results of this study (Fig. 5a-d). Bacterial as well as fungal density increased vastly with gut passage of ingested leaf litter although the bacterial increase was higher than that of the fungi. Bacterial and fungal density is more constant over time on faeces than on maize foliage. This is easily explicable because faeces provide a more homogeneous microhabitat for microorganisms. We see, however, also the limits of our counting methods, which only indicate the number of colony-forming units and do not allow to draw conclusion on the activity of these microorganisms in situ. Leaf consumption by P. scaber Food choice of P. scaber was not significantly different between Bt+ and Bt– foliage. Henke (1960) showed that woodlice are sensitive to chemical vapours, e.g. ammonia and carbon dioxide. It is not known if the B. thuringiensis protein, a rather large molecule, can be perceived by P. scaber. Our results do not support this hypothesis. A comparison of the ingestion rate of P. scaber when fed with an optimal diet (Palissa 1964) suggests that maize foliage is a suboptimal diet for woodlice. One reason might be the high cellulose and lignin content of the maize litter, which renders the leaves very hard and unpalatable for woodlice. The other reason that renders maize foliage suboptimal as a diet could be its nutritional value. An energy content of 16–17 kJ is comparable to other leaf material, but the sugar and soluble carbohydrate content of 3–4% is quite low as compared to other leaf litter. The nitrogen content of 1–2% is also low, thus rendering the C:N ratio high, which is not favoured by woodlice (Sutton 1980). Starch and lactose were the most important food components of P. scaber, followed by glucose and sucrose. Cellulose, however, if too abundant in the diet, caused increased mortality (Carefoot 1984). Our results are consistent with these findings. The number of woodlice offspring did not differ between the two treatment groups. However, the mortality of juveniles reared on Bt– maize leaves was significantly higher, whereas the same diet yielded a sigBasic Appl. Ecol. 1, 2 (2000)

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nificantly faster weight increase of the offspring during the first 131 days. Weight increase of adult P. scaber, however, was faster in the Bt+ treatment group. Such obviously contradictory results may be correct since different life stages of P. scaber react differently to different environmental factors and may simply reflect the different susceptibility to food quality. These results strongly suggest an analysis of the effect of transgenic plant material on population level. This can be done in a computer simulation, but should also be supported by data obtained in natural or semi-natural field experiments.

Conclusion This study detected no detrimental effects of Bt+ maize on the nontarget organism P. scaber. Woodlice did not differentiate between transgenic and nontransgenic maize leaves in their food preference. Several explanations are possible. One is the heterogeneous colonisation of leaves by microorganisms that metabolise nutrients and act themselves as a food source for woodlice. An important factor for the food choice is the nutritional quality, in which the two varieties slightly differ. We do not know if such differences are caused by the Bt–toxin and are therefore possible physiological side-effects of the genetic transformation. The general poor nutritional quality of maize leaves may interfere with our results. Therefore, additional experiments with several maize varieties (of different leaf quality) and several transgenic varieties (which should by then be available on the market) may be a suitable next step in the evaluation of the effects of transgenic maize on macrodecomposers. In this study we used the isopod Porcellio scaber as a model macrodecomposer though it does not belong to the common decomposer arthropods of maize fields. Though this is widely accepted in the context of ecotoxicological research, further studies should rather include other groups of decomposers typical of agroecosystems such as Collembola or earthworms. Because of the use of a soil-free test system, many soil-borne microorganisms were absent in our degradation assays and therefore, our results are likely to differ from the situation in the soil. Rather than simulating the situation of a maize leaf covered by soil (i.e. after soil treatments such as ploughing), our study simulates the situation of a maize leaf that has fallen to the ground and decomposes there (i.e. during plant growth or harvest). Our test system has two main advantages: it is a simple and easy to produce, highly reduced laboratory system that allows a better understanding of the results and it avoids the high variations that derive from using natural soil, since no stanBasic Appl. Ecol. 1, 2 (2000)

dardisation technique or internationally accepted “artificial soil” exists. Our results, however, need to be confirmed by exposure tests in the field. Surface-living animals deposit large quantities of faecal matter on the soil. These faecal pellets of primary decomposers are utilised not only by fungi and bacteria, but also by a legion of small arthropods, such as Collembola, and nematodes (McE Kevan 1968). Since the Cry1Ab protein is rather persistent in decomposing foliage and is attached to soil particles (Sims & Ream 1997, Saxena et al. 1999) and the new generation of transgenic Bt–crops will have a much higher Bt toxin expression (Mc Bride et al. 1995, Kota et al. 1999), possible side effects will much more pronouncedly occur when such plants are grown. The residual biological activity of insecticidal proteins in soil or litter originating directly or indirectly from transgenic plant material may therefore become one of the most important parameters for the assessment of environmental fate and possible risk of transgenic plants. Acknowledgements: We would like to thank A. Hilbeck for providing the plant material, E. Lehmann, R. Brändle and P. Gugerli for valuable help with the laboratory work, S. Bacher and L. Heer for assistance with the statistical analysis, C. Zwahlen and two anonymous reviewers for their helpful discussion and S. Zingg and H. Boyle for editing this manuscript.

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