Heiri Wandeler 2002

Consumption of two Bt and six non-Bt corn varieties by the woodlouse Porcellio scaber

Heiri Wandeler, Jana Bahylova, Wolfgang Nentwig*

Zoological Institute, University of Bern, Bern, Switzerland

Received December 11, 2001 · Accepted February 21, 2002

Abstract

Studies of the degradation of transgenic Bacillus thuringiensis corn were limited to date, to a com-parison between one Bt corn variety and its isogenic control line. Laboratory experiments using sixnon-transgenic and two transgenic Bt corn varieties were carried out to study the effect of Bt proteinCry1Ab and corn variety on the consumption of the decomposer Porcellio scaber (Latreille). TheCry1Ab toxin concentration in the Bt corn leaves was quantified at the beginning and at the end ofthe trial. Further, P. scaber and their faeces were analysed for presence of the Cry1Ab toxin afterfeeding on Bt corn using ELISA.During a feeding period of 20 days, P. scaber fed significantly less on the transgenic Bt corn (Bt+)than the control corn variety (Bt–). Comparing all eight corn varieties, the consumption dependedsignificantly on the corn variety. The transgenic corn variety N4640Bt equalled the poorly con-sumed corn varieties; the second transgenic variety, Max88, which contained much less of theCry1Ab protein, was one of the most consumed varieties. No differences in the nitrogen content butvarying energy content were detected across the eight corn varieties. Neither the nitrogen, nor theenergy content showed a significant correlation to the consumption rate. The Cry1Ab toxin concen-tration decreased in both Bt corn varieties during the time period of 20 days, but only significantlyin one variety. The Cry1Ab protein could be detected in both the body of P. scaber and its faeces,showing that P. scaber ingested and excreted the Cry1Ab protein only to some extent. These resultssuggest that corn varieties, including conventional ones, differ with respect to degradation. There-fore, it is difficult to draw conclusions about the effective consequences from just one isogene testsystem. This study also supports earlier reports on the slow degradation of Bt corn.

Bisherige Studien über den Abbau von transgenem Bacillus thuringiensis Mais beruhen auf demVergleich einer Bt-Maissorte mit ihrer isogenen Kontrolllinie. In Laborexperimenten mit sechsnicht-transgenen und zwei transgenen Bt-Maissorten wurde der Effekt des Bt-Protein Cry1Ab sowieein möglicher Sorteneffekt auf die Konsumption des Destruenten Porcellio scaber untersucht. DieKonzentration von Cry1Ab in den Bt-Maisblättern wurde am Anfang und am Ende des Experi-mentes gemessen, außerdem wurde P. scaber sowie der Kot untersucht.Während einer Versuchsdauer von 20 Tagen fraß P. scaber signifikant weniger von Bt-Mais (Bt+) alsvon dessen Kontrolllinie (Bt–). Über alle acht Maissorten gesehen hatte der Faktor Sorte auf dieKonsumption einen signifikanten Einfluss. Die hoch exprimierende transgene Maissorte N4640Btwurde im Vergleich wenig gefressen, die niedrig exprimierende transgene Maissorte Max88 hinge-gen war eine der am meisten konsumierten Maissorten. Der Stickstoffgehalt der acht Sorten unter-schied sich nicht, im Energiegehalt gab es jedoch signifikante Unterschiede. Es konnte keine Sig-nifikanz zwischen der Konsumption und dem Stickstoffgehalt, respektive der Konsumption und

*Corresponding author: Wolfgang Nentwig, Zoological Institute, University of Bern, Baltzerstr. 6, CH 3012 Bern, Switzerland,Phone +41-31-631 4520, Fax +41-31-631 4888, E-mail: [email protected]

1439-1791/02/03/04-357 $ 15.00/0

Basic Appl. Ecol. 3, 357–365 (2002)© Urban & Fischer Verlaghttp://www.urbanfischer.de/journals/baecol

Basic and Applied Ecology

dem Energiegehalt gefunden werden. Die Cry1Ab Konzentration nahm während des Experimentesin beiden Bt-Maissorten ab, die Abnahme war jedoch nur in einer Sorte signifikant. Das Cry1AbProtein konnte sowohl im Körper wie auch im Kot nachgewiesen werden.Diese Resultate zeigen, dass Unterschiede im Abbau von verschiedenen Maissorten existieren,sowohl zwischen transgenen und konventionellen Sorten. Der Vergleich von mehreren Sorten er-laubt bessere Aussagen über die Relevanz der erhaltenen Unterschiede als wenn die Resultate nurauf einem isogenen System basieren. Diese Studie stützt bereits früher gemachte Aussagen, dassBt-Mais einen langsamen Abbau aufweist.

Key words: transgenic plant – Bt corn – decomposition – variety effect – ingestion – degradation

Woodlice belong to the soil macro-fauna. Usuallythey feed on dead plant material and are therefore re-garded as primary decomposers. Since they also feedon their own faeces to whatever extent, woodlice aresecondary decomposers as well. It is generally accept-ed that a ‚softening up‘ process has to be carried outby microorganisms before woodlice will eat litter.However, they will also feed on animal remains anddung, while they may, to some measure, gain nourish-ment from living bacteria and fungi (Sutton 1980).They accelerate the process of humification by frag-menting dead plant material and enriching it with mi-croorganisms in their intestinal tract. Therefore theirfaeces may contain in excess of 500 times more bacte-ria than the food materials (Ineson & Anderson 1985).

So far, only a few investigations about the impact ofBt toxin on non-target soil organisms exist. In an ex-periment using P. scaber, the consumption rate and thenumber of offspring did not differ between transgenicand non-transgenic corn. Weight increase of the off-spring was significantly higher in the non-transgenicgroup, but weight increase of adult P. scaber was high-er in the transgenic group (Escher et al. 2000). WhenFolsomia candida (Collembola) was fed residues oftransgenic cotton, oviposition and egg productionwere unaffected (Yu et al. 1997). Saxena & Stotzky(2001b) did not find significant differences in mortali-ty and weight of Lumbricus terrestris after 40 days insoil planted with Bt or non-Bt corn or after 45 days insoil amended with biomass of Bt or non-Bt corn.

In the currently available transgenic corn lines, theexpression of Bt endotoxin is usually under the controlof the constitutive cauliflower mosaic virus promoterCaMV35S. At present it is unclear whether the expres-sion of this foreign gene causes potential modificationsof a plant’s physiology (pleiotrophic effects) whichcould be one reason for different plant residue qualityand digestibility. Some studies point out such differ-ences with respect to nitrogen and energy content(Escher et al. 2000) or lignin content (Saxena & Stotzkyin press). Thus the observed differences in response toBt corn or its control variety by P. scaber (Escher et al.2000) may be based not only on the Bt toxin itself but

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Basic Appl. Ecol. 3, 4 (2002)

Introduction

From 1996 to 2000, global adoption rates for trans-genic crops were unprecedented. In the year 2000, theglobal area of transgenic crops has almost reached 45million ha (James 2001). One of the major transgeniccrops is Bt corn which is transformed with a syntheticgene encoding a truncated version of the Cry1Ab in-secticidal protein from Bacillus thuringiensis (Bt)(Fearing et al. 1997) to control Ostrinia nubilalis, theEuropean Corn Borer. Bt proteins cause toxicity uponingestion and the target organisms are killed whenfeeding on plant tissues containing the Bt toxins.Though this system works on a relatively high level oftarget selectivity, non-target herbivores and even carni-vores may be impacted by feeding directly on the Btplant (tissues and pollen) or by consuming the tissuesof intoxicated target pests or non-targets feeding onthe plant (Riddick & Barbosa 1998, Hilbeck et al.1998, Losey et al. 1999, Hansen & Obrycki 2000,Wraight et al. 2000). Larvae of foliar-feeding insectsdefinitely ingest the Bt toxins when feeding on leaf tis-sues. For example, Cry1Ab toxin could be detected inthe larvae of Spodoptera littoralis and in their faeceswhen fed with transgenic (Bt+) corn (Raps et al.2001).

Only a few studies have focused on the decomposi-tion of Bt maize residues, although some estimated 24tons (dry weight) leaf residues per ha remain in thefield after harvesting of grain maize and corn-cob-mix(Wehrli et al. 1986). Plant residues containing the Bttoxin could be a potential risk for soil organisms in-gesting the transgenic corn material. So far the persis-tence of the Bt toxin in corn tissue in soil was under-valued on the basis of a laboratory study (Sims &Holden 1996). According to a field study, the dissipa-tion of the Bt toxin in corn residues in soil takes longerthan assumed (Zwahlen et al. submitted). Further,Saxena & Stotzky (2001a) showed in a field trial thatthe toxin in soil is still detectable and retained insecti-cidal activity several months after the cutting of theplants. Therefore soil organisms are exposed to thetoxin for long time periods.

also on differences in general plant physiology. To ourknowledge, nobody has as yet compared the trans-genic variety to its control and additionally to otherconventional varieties, which would be a useful way toobtain a reference to this question.

In the research project presented here, laboratorystudies were performed using the isopod Porcellioscaber as a representative of the soil living detri-tophagous macrofauna. The objectives of this studywere: (i) to determine the common range of consump-tion by P. scaber when feeding on several corn varietiesin order to compare the impact of the Cry1Ab proteinwith the variety effect (ii) to analyse the digestibility ofCry1Ab protein by P. scaber after feeding on Bt corn.

Materials and methods

Two separate experiments were performed. Firstly, theconsumption of eight corn varieties by P. scaber wasdetermined (herein after referred to as ‘Food consump-tion’) and secondly, P. scaber and its faeces were anal-ysed for the presence of Bt toxin after feeding on Btcorn (herein after referred to as ‘Analysis of Bt toxin inP. scaber’).

Plants

Food consumption. Eight corn hybrids were used inthe experiment, two genetically modified and six con-ventional varieties. The transgenic hybrids (Bt+) usedwere Max88 (event 176) and N4640Bt (event Bt11)both from Syngenta (previously Novartis). They con-tain truncated, synthetic versions of a gene from Bacil-lus thuringiensis var. kurstaki HD-1 coding for the ex-pression of the insecticidal d-endotoxin Cry1Ab(Koziel et al. 1993). Transcription of the Cry1Ab genein Max88 is controlled by the phosphoenolpyruvatecarboxylase and a pollen-specific promoter, whereas inN4640Bt, the cauliflower mosaic virus 35S promoteris used to control the transcription (Koziel et al 1993).The non-transgenic hybrids (Bt–) used in the experi-ment were N4640, the corresponding isogenic controlline to N4640Bt, and five corn varieties planted com-monly in Switzerland: Magister, Banguy, LG2265,LG2275 and Attribut (Landi & UFA 2000). Theplants were cultivated in plastic pots (20 cm diameter)in an climate chamber at an average temperature of23.3 °C (25 °C during the photophase of 16 h, and 20 °C for the remaining 8 h). Seven plants per pot andthree pots per variety were planted. The plant materialused for the experiment was taken from plants whichhad reached a height of 100–120 cm (after pollenshed). Only senescent (brown) leaves were used (leaves1–5). After cutting them into approximately 12 cm

pieces, they were dried at 40 °C for 48 h and stored at–20 °C until they were used for the experiment.

Analysis of Bt toxin in P. scaber. For food plant ma-terial for P. scaber, only the two hybrids N4640 (Bt–)and N4640Bt (Bt+) were cultivated under the sameconditions as described above. The leaves of the plantswere used in the 5–7 leaf stages.

Animals

Adult individuals of Porcellio scaber (Latreille) (Isopo-da, Oniscidea) were collected from a compost-heapnear Bern in September 2000. Animals with a mini-mum weight of at least 15 mg fresh weight were cho-sen and sexed under a binocular. Either fifty males orfifty females were kept in a plastic box (16 × 11 × 5cm), filled with a 1 cm layer of moistened garden soil.The boxes were closed with a transparent plastic lidwhich sealed incompletely to allow for air circulation.For maintenance diet, corn leaves (a mix of the vari-eties Banguy and DK250) from a field near Bern weredried at 40 °C for 48 h, stored at –20 °C, until theywere added after re-moistening to the animals as thefood source. They were kept in a climate chamber atan average temperature of 11.5 °C (15 °C during thephotophase of 7 h, and 10 °C for the remaining 17 h)until they were used for the experiments.

Food consumption

The experiment was also carried out in the same cli-mate chamber where the animals were reared. Each P. scaber was placed individually in a clear plastic box(7 × 6 × 3 cm) closed with a lid that had a hole (20 mmdiameter) covered with fine mesh netting to allow forair circulation. The bottom was covered with a 1 cmlayer of plaster of Paris to maintain sufficient humidi-ty. The boxes were allocated to random positions andprotected against direct lighting by cardboard. All ani-mals were starved for 36 h and weighed immediatelybefore the beginning and after the end of the experi-ment. The frozen plant material was dried again for 12h at 40 °C in order to weigh it. Approximately 100 mgdry weight was added to each box after re-moistening.After 20 days, the remaining plant material was driedat 40 °C for 48 h and re-weighed. The amount of con-sumed plant material was estimated by subtracting thedry weight of food remains from the dry weight offood offered. 15 female and 15 male P. scaber wereused per treatment, resulting in a total of 240 individ-uals. Each animal was used once only. In order to as-sess the degradation of plant material due to the mi-croorganisms’ activity only, a control with ten leavesper treatment was carried out under identical condi-tions except for the absence of isopods. The control

Consumption of the Bt and six non-Bt 359


was taken into account by subtracting the percentageloss of plant material by microorganisms from thetotal plant material reduction.

Herbivore Bioassays

Bioassays were conducted using the susceptible targetspecies Ostrinia nubilalis (Hübner) (Lepidoptera,Pyralidae) to determine whether the Cry1Ab toxin inthe transgenic plant material used for the food con-sumption experiment was still insecticidally active.Only Max88 (Bt+), N4640Bt (Bt+) and N4640 (Bt–)were used for the bioassays. Plant material which hadbeen exposed to the same conditions as in the foodconsumption experiment but without woodlice, wasthen homogenised with a mortar and pestle and mixedwith artificial diet (a mixture of agar-agar, cornsemolina, wheat germs, torula yeast and water), beforeit was put into each of 10 vials (5 cm diameter by 2.2 cm height) per treatment. Ten neonate larvae wereplaced into each vial (resulting in a total of 100 larvaeper treatment) and the vials were subsequently sealedwith parafilm. The vials were kept in a climate cham-ber at 25 °C. The numbers of dead larvae and theweight of surviving larvae were recorded after sixdays.

Nitrogen and energy content

Total nitrogen content was determined on a NA 2000Nitrogen Analyser (CE Instruments, Milano, Italy)using subsamples of 20 mg dry weight leaf materialreduced to small pieces with scissors. Ten samples percorn variety were analysed. Ten leaf samples of eachcorn variety (dry weight = 0.4 g) were cut into slides of3 cm to measure the energy content in an oxygenbomb calorimeter (Parr 1341 plain oxygen bombcalorimeter, Parr Instr. Comp., Illinois, USA).

Analyses of Bt toxin in P. scaber

After starving for 72 h, 26 adult P. scaber were placedpairwise into boxes as described above. Five pairswere then fed leaves of N4640 (Bt–), whereas eightpairs were fed leaves of N4640Bt (Bt+) corn for 72 h.From each box, the faeces and one adult were weighedand stored at –20 °C. The gut (hindgut and the twopairs of lobes of hepatopancreas) of the other one wasremoved, weighed and stored at –20 °C.

Enzyme-linked immunosorbent assay (ELISA) of Bt toxin

Food consumption. The Cry1Ab toxin concentrationin the N4640Bt (Bt+) and Max88 (Bt+) leaves fromthe beginning and the end of the experiment was

quantified by ELISA as described by Gugerli (1979,1986). From the initial material, ten leaves of 15 mgdry weight were used per treatment. Furthermore,from plant material which was exposed to the food ex-periment conditions except for the absence ofP. scaber, ten leaves per treatment were cut into twohalves, and both were weighed. One part was used forthe analysis, the other one was dried at 40 °C for 48 hto calculate the dry weight of the test material. Undi-luted samples were used for the analysis. The test ma-terial was put into universal bags (Bioreba, Reinach,Switzerland) and was homogenised with a hand modelhomogeniser (Bioreba AG) in 5 ml extraction buffer(10 mM phosphate, 137 mM NaCl, 2.7 mM KCl,3 mM NaN3, 2% (v/v) polyvinylpyrrolidone (Mr =25000), 0.05 % (v/v) Tween®20, pH 7.4) to extractthe Bt toxin.

In order to determine the calibration curve, refer-ence samples of purified Cry1Ab toxin were suspend-ed in pooled extracts of control leaves (N4640) at aconcentration of 1000, 100, 50, 20, 10, 5, 2, 1, 0.5,0.2, 0.1, 0.01 ng Cry1Ab toxin/ml plant extractionbuffer. Microtitre immunoassay plates Immunolon®4(Dynatech Laboratories Inc., Virginia, USA) wereanalysed with a MRX microplate reader operated withthe Revel software package, Version G 3.2. (DynexTechnologies Inc.). Polyclonal coating IgG and alka-line phosphatase-conjugated IgG against the Cry1Abtoxin were purchased from Bioreba AG. Diethanol-amine and 4-nitrophenylphosphate for the substratebuffer were obtained from Merck, Darmstadt,Germany. All samples, including the calibration curve,were analysed in duplicate. The arbitrary detectionthreshold was defined as the optical density mean ofthe control leaf samples plus three times its standarddeviation (SD).

Analysis of P. scaber. Each single body and gut washomogenised in 2 ml extraction buffer, the faeces ofeach box were homogenised in 3 ml extraction buffer.Afterwards, all samples were centrifuged, and the su-pernatants were stored at –20 °C until the analyseswere carried out. The food plant material was anal-ysed with the same ELISA-method described above ex-cept for using the fresh weight. The purified Cry1Abtoxin for the reference curve was suspended in pureextraction buffer. All samples were analysed undilutedand in duplicate. The detection threshold was definedas described above.

Data analysis

The data of the food consumption experiment were log-transformed to maintain homogeneous variances. Anal-ysis of variance was performed to compare the con-sumption of the eight plant varieties using the general

360 Wandeler et al.


linear model (GLM) procedure in Systat( 9.0 with theassociated Tukey HSD Multiple Comparisons. The in-dividual weight of P. scaber was serving as covariate. AMann-Whitney-U-test was carried out to show whethertoxin concentration in transgenic leaves had decreasedsignificantly during the experiment (Systat® 9.0).

For the statistical analysis of mortality and weightof O. nubilalis, a Kruskal-Wallis-test was performed.Mean mortality and mean individual weight of thetwo (Bt+)-treatments were compared to the (Bt–)-treatment using Dunn’s Multiple Comparison Test(GraphPad Software Inc 2000). The nitrogen and en-ergy content of the eight corn varieties were comparedusing the Kruskal-Wallis-test with the associated Dun-n’s Multiple Comparison Test (GraphPad Software Inc2000).

For the quantitative analysis of ELISA, data of thereference curve were log-transformed before a linearregression was carried out to calculate the Cry1Abtoxin concentration in the samples (GraphPad Soft-ware Inc 2000).

Results

Food consumption

Analysis of the two (Bt+) plant varieties by enzyme-linked immunoabsorbent assay (ELISA) indicated aninitial Cry1Ab toxin concentration of 19.7 ± 6.6 µg/gdry weight in N4640Bt (Fig. 1a) and 2.9 ± 1.9 µg/g dryweight in Max88 (Fig. 1b). After 20 days, the toxinconcentration decreased to 15.5 ± 7.9 µg/g dry weightin N4640Bt (Fig. 1a) and 1.1 ± 0.7 µg/g dry weight inMax88 (Fig. 1b). No Cry1Ab toxin was detected inthe control leaves (N4640). The decrease of 21% ofthe toxin in N4640Bt was not significant (U = 70.0;p > 0.1), but the decrease in concentration of thetoxin in Max88 of 62% after 20 days was significant(U = 75.0; p < 0.05).

The food consumption of corn leaves differed sig-nificantly between the eight corn varieties (p < 0.0001)and was related to the individual weight of P. scaber(serving as covariate, p < 0.001) (R2 = 0.43) (Table 1).P. scaber fed significantly less from N4640Bt (Bt+)corn leaves than from its control N4640 (Bt–) (p <0.01). The second transgenic variety, Max88, was con-sumed significantly more than N4640Bt (p < 0.001),but there was no significant difference to N4640.Within the six non-transgenic corn varieties, a widerange of consumption was detected (Fig. 2a). N4640Btcoincided with the poorly consumed corn varieties,whereas Max88 was one of the most consumed vari-eties. Magister, a non-transgenic variety, was con-sumed the least (Fig. 2a). However, in general no dif-ference could be detected between consumed trans-

genic (two varieties) and non-transgenic (six varieties)plant material.

The mean nitrogen content varied from 0.20 ±0.02% (LG2265) to 0.25 ± 0.05% (Attribut) and themean energy content ranged between 14.50 ± 0.30 kJ(Max88) and 16.28 ± 0.37 kJ (Magister) (Fig. 2b,c).One transgenic corn variety, N4640Bt ranged in theupper, the other one, Max88, in the lower level of ni-trogen and energy content. No significant correlationcould be found either between the consumption andthe nitrogen content (Pearson; p > 0.09, r = –0.63,n = 8) or between the consumption and the energycontent (Pearson; p > 0.1, r = –0.61, n = 8).



Fig. 1. The initial Cry1Ab concentration and the concentration after 20 daysin leaves of the two (Bt+) varieties a) N4640Bt and b) Max88. Means ± stan-dard deviations (SD) are given, sample size was 10 per variety and date. Barswith different letters represent means that are significant different at P =0.05 (Mann-Whitney-U-test).

Table 1. Analysis of variance to estimate the effects of variety, sex and indi-vidual weight (serving as covariable) on consumption of P. scaber (N = 231;R2 = 0.43).

df F P

sex 1 0.55 > 0.05variety 7 17.23 < 0.0001sex × variety 7 3.65 < 0.001weight 1 11.91 < 0.001

Herbivore bioassays

The bioassay results with the target pest of transgeniccorn, O. nubilalis, confirmed the continuous insectici-dal activity of the transgenic corn plants used in thefood consumption experiment. Mean mortality andweight of O. nubilalis were 53 ± 50% and 0.21 ± 0.26mg when feeding on N4640Bt (Bt+), 82 ± 13% and0.27 ± 0.65 mg when feeding on Max88 (Bt+) and 0%

and 0.66 ± 0.23 mg when feeding on N4640 (Bt–).Both the medians of mortality (H = 12.72; p < 0.001)and the medians of weight (H = 10.01; p < 0.005) weresignificantly different. O. nubilalis fed on Max88 (Bt+)showed a significantly higher mortality (p < 0.01) andreduced weight (p < 0.01) than O. nubilalis larvae fedon non-transgenic corn (N4640). No significant differ-ence in mortality (p > 0.05) but a significantly reducedlarval weight (p < 0.05) was observed in the N4640Bt-treatment (Bt+) compared to the N4640-treatment(Bt–).

Analysis of P. scaber body, gut and faeces

Cry1Ab could be detected in the gut and body ofP. scaber and in their faeces after feeding on transgenic

362 Wandeler et al.


Fig. 3. Detection of Cry1Ab toxin in body, gut and faeces of P. scaber (blackbars) after a 72-h feeding period on N4640Bt (Bt+) or on its control N4640(Bt–). Bars show the extinction measured as optical density (OD) at 405 nmwavelength. Means ( SD are given, sample size was eight for the (Bt+) sam-ples and five for the (Bt–) samples. The white bars show purified Cry1Ab inextraction buffer at concentrations of 0 (pure extraction buffer as a negativecontrol), 0.01, 0.1, 0.5 and 1 ng Cry1Ab toxin/ml buffer.

Fig. 2. a) Consumption (mean + standard deviation) of six non-transgenic(white bars) and two transgenic corn varieties (striped bars) by P. scaber. b)Mean nitrogen and c) mean energy content of these eight corn varieties.Columns with different letters are significantly different at P = 0.05 .

Table 2. ELISA of P. scaber, their feeding plants, guts and faeces when fedon control (N4640) or (Bt+) corn (N4640Bt). Given is the number of repli-cates (n), mean ( SD of the amount material used, the amount of extractionbuffer used and the calculated concentrations of Cry1Ab in the respectivetissues (mean ( SD).

Tissue analysed N Material (mg) Buffer (ml) µg Cry1Ab/gfresh weig

Feeding plants (Bt–) 5 105 ± 23.6 5 0Feeding plants (Bt+) 5 132 ± 11.9 5 2.43 ± 1.14Body (Bt–) 5 36.0 ± 6.2 2 0Body (Bt+) 8 36.4 ± 9.4 2 0.03 ± 0.02gut (Bt–) 5 5.1 ± 2.2 2 0gut (Bt+) 8 5.5 ± 1.2 2 0.10 ± 0.02faeces (Bt–) 5 7.3 ± 3.8 3 0faeces (Bt+) 8 7.3 ± 1.6 3 0.32 ± 0.30

corn (N4640Bt) (Fig. 3). The mean Cry1Ab toxin con-centration in the food plants was 2.43 µg/g freshweight. Mean toxin concentrations in the body andgut were very low (0.03 and 0.1 µg/g fresh weight, re-spectively), whereas the mean concentration of thetoxin in the faeces was higher (0.32 µg/g fresh weight)(Table 2).

Discussion

Food consumption

P. scaber fed on significantly more N4640 (Bt–) thanN4640Bt (Bt+) plant material, confirming the resultsof Escher (1998). Escher et al. (2000) found no differ-ence in the consumption between these two corn vari-eties, but did not use homogeneous, senescent cornleaves but rather a mixture of green and brown leaves.This result shows that the Bt toxin affects the con-sumption of corn leaves by P. scaber, which may wellindicate a Bt-effect. Another explanation could be thehigher lignin content in Bt corn as compared to non-Btcorn (Saxena & Stotzky in press), which persists forseveral weeks during degradation (Escher et al. 2000).The significantly lower consumption of N4640Bt(Bt+) residues compared to Max88 could be explainedby the higher Cry1Ab content in the N4640Bt leaves.The available amount of Bt toxin in Max88 couldhave been too low to affect the consumption. Anotherreason for the difference in consumption of the two Btvarieties could be varying microbial activity or coloni-sation, as microbial conditioning increases the palata-bility of the plant litter (Hassal & Rushton 1984).However, Müller (1999) could not find any differencesin bacterial and fungal communities between N4640Btand Max88 residues. These results show that variousBt corn varieties can induce different feeding responsesand therefore every Bt variety needs to be assessed ona case-by-case basis.

The corn variety had the strongest effect on the con-sumption by P. scaber. Likewise, individual weight ofP. scaber affected the consumption significantly,wherefore the relative wide range of weight used wasaccounted for. Comparing the consumption across alleight corn varieties, there were two non-transgeniccorn varieties, namely Magister and Banguy, fromwhich P. scaber also ate very little. The amount ofN4640Bt consumed ranked among the least fed uponvarieties such as Magister and Banguy, whereasN4640, compared to the other non-transgenic cornvarieties, was one of the most preferred. The consump-tion of N4640Bt coincides with the range of the non-transgenic varieties, but it cannot be equated with itscontrol N4640 pertaining to decomposition. These re-sults suggest a general variety-dependent consumption

of corn residues by P. scaber. The causes of this are un-known, but differences in physical and chemical prop-erties could be one possibility. It is well known that theconsumption by P. scaber is an indirect measure forthe residues quality which depends, among otherthings, on the energy and nitrogen content, the tough-ness of the leaves, humidity and pH value, and on themicrobial colonisation (Sutton 1980, Hassal & Rush-ton 1984, Zimmer & Topp 1997). The nitrogen andthe energy content of the corn leaves varied across thedifferent varieties used and both can potentially con-tribute to the differences in the consumption observed.N4640Bt ranked among the corn varieties with a highnitrogen and energy content whereas Max88 was inthe lower range. Woodlice might adapt the amount ofconsumption to the quality of food as a pre-ingestivecompensatory mechanism (Lavy et al. 2001). The con-sumption was negatively correlated (but not signifi-cantly) to the nitrogen and energy content. Magisterand N4640Bt showed a higher energy and nitrogencontent than Max88 (Fig. 2b,c) and both were fedupon significantly less than Max88 which wouldspeak in favour of the pre-ingestive compensatorymechanism.

The mean expression level of Cry1Ab toxin inN4640Bt (Event Bt11) quantified by ELISA was nearlytwice as high as that reported by EPA for field growncorn (EPA 2000). This level of Bt toxin may be due tothe fact that the level varies between different stagesand tissues of the plant (Fearing et al. 1997). Further-more, the corn variety described by EPA originatesfrom the similar event (Bt11) as N4640Bt, but it doesnot have exactly the same genotype. In the other Btcorn variety used in our experiment, Max88 (Event176), the concentration of the toxin approximatelyequalled the lowest level described by Fearing et al.(1997).

Herbivore bioassays with O. nubilalis confirmedthe insecticidal activity of the toxin in the plant mate-rial used in the consumption experiment. Compared toN4640, the larvae showed a significant higher mortali-ty and reduced weight when feeding on Max88,whereas when feeding on N4640Bt, only the larvalweight was significantly reduced. Generally, only alow amount of plant material could be mixed with theartificial diet. Otherwise the larvae did not accept themixture because they do not like senescent plant mate-rial. Therefore O. nubilalis did not necessarily ingestthe toxin in a lethal dose, which might be responsiblefor the high variance in the mortality data of the larvaefeeding on N4640Bt. Due to this high variance, feed-ing on N4640Bt caused only a sublethal effect for thelarvae, based on the evidence of the significantly re-duced larval weight, despite its very high expressionlevel.



Analysis of Bt toxin in P. scaber

The detection of Cry1Ab toxin in P. scaber and in itsfaeces showed that Cry1Ab toxin is still available afteringestion and excretion. The concentration of Cry1Abprotein in the faeces is approximately tenfold higherthan in the gut. The food plant material again has thetenfold higher toxin concentration than the faeces. Be-cause the data are based on fresh weight, the faecesshowed a wide range in the measured Cry1Ab toxinconcentration, as faeces can lose water very quicklydue to its large surface. As the trial was carried out at21 °C for 72h, it might be possible that the originallyavailable quantity of the toxin in the faeces had al-ready been degraded by microorganisms. Obviously, P. scaber ingests the Cry1Ab toxin and degradation ofthe toxin is observable, however uncertain to what ex-tent. Being excreted by soil organisms is one possibili-ty as to how the Bt toxin from plant residues can reachthe soil where it is available to non-target soil organ-isms not ingesting the plant material itself. TheCry1Ab protein in root exudates and biomass of Btcorn appears not to be toxic to earthworms, but thetoxin was present in the gut (Saxena & Stotzky2001b). If Bt corn-fed earthworms excrete the Bt toxinwith their faeces too, substantial concentrations of Bttoxin would be present in soil, what could potentiallypose a hazard to faeces-feeding or faeces-living organ-isms that are important in the decomposition of thesefaeces.

Conclusions

1) Due to the wide range of consumption of the differ-ent non-transgenic corn varieties, it is shown thatthe consumption is not only a question of Bt or not.Further investigations should take into account thatit is difficult to draw conclusions from just one iso-gene test system.

2) The Cry1Ab toxin was still detectable in the faecesof P. scaber in considerable amounts. Thus, the Bttoxin is available continuously after a first ingestionof an unsusceptible organism.

Acknowledgements: We gratefully thank J. Müller, Dr. L.Kuhn-Nentwig and P. Wandeler for their technical assistance,P. Gugerli and C. Zwahlen for help with the ELISA and herhelpful discussions, Dr. J.-P. Airoldi and Dr. S. Bacher fortheir great support in the statistical analysis, K. Ruchti andR. Howald for assistance with the food consumptionexperiment, K. Lüdi and C. Klingler for improving themanuscript and two referees for valuable comments.

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