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 six
non-transgenic and two transgenic Bt corn varieties were carried out to study the effect of Bt protein
Cry1Ab and corn variety on the consumption of the decomposer Porcellio scaber (Latreille). The
Cry1Ab toxin concentration in the Bt corn leaves was quantified at the beginning and at the end of
the trial. Further, P. scaber and their faeces were analysed for presence of the Cry1Ab toxin after
feeding 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 depended
significantly 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 the
Cry1Ab protein, was one of the most consumed varieties. No differences in the nitrogen content but
varying energy content were detected across the eight corn varieties. Neither the nitrogen, nor the
energy 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 significantly
in 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 results
suggest 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 test
system. This study also supports earlier reports on the slow degradation of Bt corn.
Bisherige Studien ber den Abbau von transgenem Bacillus thuringiensis Mais beruhen auf dem
Vergleich einer Bt-Maissorte mit ihrer isogenen Kontrolllinie. In Laborexperimenten mit sechs
nicht-transgenen und zwei transgenen Bt-Maissorten wurde der Effekt des Bt-Protein Cry1Ab sowie
ein mglicher Sorteneffekt auf die Konsumption des Destruenten Porcellio scaber untersucht. Die
Konzentration von Cry1Ab in den Bt-Maisblttern wurde am Anfang und am Ende des Experi-
mentes gemessen, auerdem wurde P. scaber sowie der Kot untersucht.
Whrend einer Versuchsdauer von 20 Tagen fra P. scaber signifikant weniger von Bt-Mais (Bt+) als
von dessen Kontrolllinie (Bt). ber alle acht Maissorten gesehen hatte der Faktor Sorte auf die
Konsumption einen signifikanten Einfluss. Die hoch exprimierende transgene Maissorte N4640Bt
wurde 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: wolfgang.nentwig@zos.unibe.ch
1439-1791/02/03/04-357 $ 15.00/0
Basic Appl. Ecol. 3, 357365 (2002)
Urban & Fischer Verlag
http://www.urbanfischer.de/journals/baecol
Basic and Applied Ecology
dem Energiegehalt gefunden werden. Die Cry1Ab Konzentration nahm whrend des Experimentes
in beiden Bt-Maissorten ab, die Abnahme war jedoch nur in einer Sorte signifikant. Das Cry1Ab
Protein konnte sowohl im Krper 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 ber die Relevanz der erhaltenen Unterschiede als wenn die Resultate nur
auf einem isogenen System basieren. Diese Studie sttzt bereits frher gemachte Aussagen, dass
Bt-Mais einen langsamen Abbau aufweist.
Key words: transgenic plant Bt corn decomposition variety effect ingestion degradation
Woodlice belong to the soil macro-fauna. Usually
they feed on dead plant material and are therefore re-
garded as primary decomposers. Since they also feed
on their own faeces to whatever extent, woodlice are
secondary decomposers as well. It is generally accept-
ed that a softening up process has to be carried out
by microorganisms before woodlice will eat litter.
However, they will also feed on animal remains and
dung, 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 their
faeces 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 of
Bt toxin on non-target soil organisms exist. In an ex-
periment using P. scaber, the consumption rate and the
number of offspring did not differ between transgenic
and non-transgenic corn. Weight increase of the off-
spring was significantly higher in the non-transgenic
group, but weight increase of adult P. scaber was high-
er in the transgenic group (Escher et al. 2000). When
Folsomia candida (Collembola) was fed residues of
transgenic cotton, oviposition and egg production
were unaffected (Yu et al. 1997). Saxena & Stotzky
(2001b) did not find significant differences in mortali-
ty and weight of Lumbricus terrestris after 40 days in
soil planted with Bt or non-Bt corn or after 45 days in
soil amended with biomass of Bt or non-Bt corn.
In the currently available transgenic corn lines, the
expression of Bt endotoxin is usually under the control
of the constitutive cauliflower mosaic virus promoter
CaMV35S. At present it is unclear whether the expres-
sion of this foreign gene causes potential modifications
of a plants physiology (pleiotrophic effects) which
could be one reason for different plant residue quality
and digestibility. Some studies point out such differ-
ences with respect to nitrogen and energy content
(Escher et al. 2000) or lignin content (Saxena & Stotzky
in press). Thus the observed differences in response to
Bt corn or its control variety by P. scaber (Escher et al.
2000) may be based not only on the Bt toxin itself but
358 Wandeler et al.
Basic Appl. Ecol. 3, 4 (2002)
Introduction
From 1996 to 2000, global adoption rates for trans-
genic crops were unprecedented. In the year 2000, the
global area of transgenic crops has almost reached 45
million ha (James 2001). One of the major transgenic
crops is Bt corn which is transformed with a synthetic
gene encoding a truncated version of the Cry1Ab in-
secticidal protein from Bacillus thuringiensis (Bt)
(Fearing et al. 1997) to control Ostrinia nubilalis, the
European Corn Borer. Bt proteins cause toxicity upon
ingestion and the target organisms are killed when
feeding on plant tissues containing the Bt toxins.
Though this system works on a relatively high level of
target selectivity, non-target herbivores and even carni-
vores may be impacted by feeding directly on the Bt
plant (tissues and pollen) or by consuming the tissues
of intoxicated target pests or non-targets feeding on
the plant (Riddick & Barbosa 1998, Hilbeck et al.
1998, Losey et al. 1999, Hansen & Obrycki 2000,
Wraight et al. 2000). Larvae of foliar-feeding insects
definitely ingest the Bt toxins when feeding on leaf tis-
sues. For example, Cry1Ab toxin could be detected in
the larvae of Spodoptera littoralis and in their faeces
when 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 24
tons (dry weight) leaf residues per ha remain in the
field after harvesting of grain maize and corn-cob-mix
(Wehrli et al. 1986). Plant residues containing the Bt
toxin 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 longer
than assumed (Zwahlen et al. submitted). Further,
Saxena & Stotzky (2001a) showed in a field trial that
the toxin in soil is still detectable and retained insecti-
cidal activity several months after the cutting of the
plants. Therefore soil organisms are exposed to the
toxin for long time periods.
also on differences in general plant physiology. To our
knowledge, nobody has as yet compared the trans-
genic variety to its control and additionally to other
conventional varieties, which would be a useful way to
obtain a reference to this question.
In the research project presented here, laboratory
studies were performed using the isopod Porcellio
scaber as a representative of the soil living detri-
tophagous macrofauna. The objectives of this study
were: (i) to determine the common range of consump-
tion by P. scaber when feeding on several corn varieties
in order to compare the impact of the Cry1Ab protein
with the variety effect (ii) to analyse the digestibility of
Cry1Ab protein by P. scaber after feeding on Bt corn.
Materials and methods
Two separate experiments were performed. Firstly, the
consumption of eight corn varieties by P. scaber was
determined (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 Bt
corn (herein after referred to as Analysis of Bt toxin in
P. scaber).
Plants
Food consumption. Eight corn hybrids were used in
the experiment, two genetically modified and six con-
ventional varieties. The transgenic hybrids (Bt+) used
were 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 gene
in Max88 is controlled by the phosphoenolpyruvate
carboxylase and a pollen-specific promoter, whereas in
N4640Bt, the cauliflower mosaic virus 35S promoter
is used to control the transcription (Koziel et al 1993).
The non-transgenic hybrids (Bt) used in the experi-
ment were N4640, the corresponding isogenic control
line to N4640Bt, and five corn varieties planted com-
monly in Switzerland: Magister, Banguy, LG2265,
LG2275 and Attribut (Landi & UFA 2000). The
plants were cultivated in plastic pots (20 cm diameter)
in an climate chamber at an average temperature of
23.3 C (25 C during the photophase of 16 h, and
20 C for the remaining 8 h). Seven plants per pot and
three pots per variety were planted. The plant material
used for the experiment was taken from plants which
had reached a height of 100120 cm (after pollen
shed). Only senescent (brown) leaves were used (leaves
15). 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 same
conditions as described above. The leaves of the plants
were used in the 57 leaf stages.
Animals
Adult individuals of Porcellio scaber (Latreille) (Isopo-
da, Oniscidea) were collected from a compost-heap
near 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 or
fifty females were kept in a plastic box (16 11 5
cm), filled with a 1 cm layer of moistened garden soil.
The boxes were closed with a transparent plastic lid
which 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 were
dried at 40 C for 48 h, stored at 20 C, until they
were added after re-moistening to the animals as the
food source. They were kept in a climate chamber at
an average temperature of 11.5 C (15 C during the
photophase 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 mm
diameter) covered with fine mesh netting to allow for
air circulation. The bottom was covered with a 1 cm
layer of plaster of Paris to maintain sufficient humidi-
ty. The boxes were allocated to random positions and
protected against direct lighting by cardboard. All ani-
mals were starved for 36 h and weighed immediately
before the beginning and after the end of the experi-
ment. The frozen plant material was dried again for 12
h at 40 C in order to weigh it. Approximately 100 mg
dry weight was added to each box after re-moistening.
After 20 days, the remaining plant material was dried
at 40 C for 48 h and re-weighed. The amount of con-
sumed plant material was estimated by subtracting the
dry weight of food remains from the dry weight of
food offered. 15 female and 15 male P. scaber were
used 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 leaves
per 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
Basic Appl. Ecol. 3, 4 (2002)
was taken into account by subtracting the percentage
loss of plant material by microorganisms from the
total plant material reduction.
Herbivore Bioassays
Bioassays were conducted using the susceptible target
species Ostrinia nubilalis (Hbner) (Lepidoptera,
Pyralidae) to determine whether the Cry1Ab toxin in
the 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 had
been exposed to the same conditions as in the food
consumption experiment but without woodlice, was
then homogenised with a mortar and pestle and mixed
with artificial diet (a mixture of agar-agar, corn
semolina, wheat germs, torula yeast and water), before
it was put into each of 10 vials (5 cm diameter by
2.2 cm height) per treatment. Ten neonate larvae were
placed into each vial (resulting in a total of 100 larvae
per treatment) and the vials were subsequently sealed
with parafilm. The vials were kept in a climate cham-
ber at 25 C. The numbers of dead larvae and the
weight of surviving larvae were recorded after six
days.
Nitrogen and energy content
Total nitrogen content was determined on a NA 2000
Nitrogen Analyser (CE Instruments, Milano, Italy)
using subsamples of 20 mg dry weight leaf material
reduced to small pieces with scissors. Ten samples per
corn variety were analysed. Ten leaf samples of each
corn variety (dry weight = 0.4 g) were cut into slides of
3 cm to measure the energy content in an oxygen
bomb calorimeter (Parr 1341 plain oxygen bomb
calorimeter, Parr Instr. Comp., Illinois, USA).
Analyses of Bt toxin in P. scaber
After starving for 72 h, 26 adult P. scaber were placed
pairwise into boxes as described above. Five pairs
were then fed leaves of N4640 (Bt), whereas eight
pairs were fed leaves of N4640Bt (Bt+) corn for 72 h.
From each box, the faeces and one adult were weighed
and stored at 20 C. The gut (hindgut and the two
pairs of lobes of hepatopancreas) of the other one was
removed, weighed and stored at 20 C.
Enzyme-linked immunosorbent assay (ELISA) of Bt toxin
Food consumption. The Cry1Ab toxin concentration
in the N4640Bt (Bt+) and Max88 (Bt+) leaves from
the 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 mg
dry weight were used per treatment. Furthermore,
from plant material which was exposed to the food ex-
periment conditions except for the absence of
P. scaber, ten leaves per treatment were cut into two
halves, and both were weighed. One part was used for
the analysis, the other one was dried at 40 C for 48 h
to 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 model
homogeniser (Bioreba AG) in 5 ml extraction buffer
(10 mM phosphate, 137 mM NaCl, 2.7 mM KCl,
3 mM NaN
3
, 2% (v/v) polyvinylpyrrolidone (M
r
=
25000), 0.05 % (v/v) Tween

20, pH 7.4) to extract

the 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 a
concentration of 1000, 100, 50, 20, 10, 5, 2, 1, 0.5,
0.2, 0.1, 0.01 ng Cry1Ab toxin/ml plant extraction
buffer. Microtitre immunoassay plates Immunolon

4
(Dynatech Laboratories Inc., Virginia, USA) were
analysed with a MRX microplate reader operated with
the Revel software package, Version G 3.2. (Dynex
Technologies Inc.). Polyclonal coating IgG and alka-
line phosphatase-conjugated IgG against the Cry1Ab
toxin were purchased from Bioreba AG. Diethanol-
amine and 4-nitrophenylphosphate for the substrate
buffer were obtained from Merck, Darmstadt,
Germany. All samples, including the calibration curve,
were analysed in duplicate. The arbitrary detection
threshold was defined as the optical density mean of
the control leaf samples plus three times its standard
deviation (SD).
Analysis of P. scaber. Each single body and gut was
homogenised in 2 ml extraction buffer, the faeces of
each box were homogenised in 3 ml extraction buffer.
Afterwards, all samples were centrifuged, and the su-
pernatants were stored at 20 C until the analyses
were 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 Cry1Ab
toxin for the reference curve was suspended in pure
extraction buffer. All samples were analysed undiluted
and in duplicate. The detection threshold was defined
as 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.
Basic Appl. Ecol. 3, 4 (2002)
linear model (GLM) procedure in Systat
(
9.0 with the
associated Tukey HSD Multiple Comparisons. The in-
dividual weight of P. scaber was serving as covariate. A
Mann-Whitney-U-test was carried out to show whether
toxin concentration in transgenic leaves had decreased
significantly during the experiment (Systat

9.0).
For the statistical analysis of mortality and weight
of O. nubilalis, a Kruskal-Wallis-test was performed.
Mean mortality and mean individual weight of the
two (Bt+)-treatments were compared to the (Bt)-
treatment using Dunns Multiple Comparison Test
(GraphPad Software Inc 2000). The nitrogen and en-
ergy content of the eight corn varieties were compared
using the Kruskal-Wallis-test with the associated Dun-
ns Multiple Comparison Test (GraphPad Software Inc
2000).
For the quantitative analysis of ELISA, data of the
reference curve were log-transformed before a linear
regression was carried out to calculate the Cry1Ab
toxin 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 an
initial Cry1Ab toxin concentration of 19.7 6.6 g/g
dry weight in N4640Bt (Fig. 1a) and 2.9 1.9 g/g dry
weight in Max88 (Fig. 1b). After 20 days, the toxin
concentration decreased to 15.5 7.9 g/g dry weight
in N4640Bt (Fig. 1a) and 1.1 0.7 g/g dry weight in
Max88 (Fig. 1b). No Cry1Ab toxin was detected in
the control leaves (N4640). The decrease of 21% of
the toxin in N4640Bt was not significant (U = 70.0;
p > 0.1), but the decrease in concentration of the
toxin 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) (R
2
= 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 wide
range of consumption was detected (Fig. 2a). N4640Bt
coincided 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 the
mean 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 the
upper, the other one, Max88, in the lower level of ni-
trogen and energy content. No significant correlation
could be found either between the consumption and
the nitrogen content (Pearson; p > 0.09, r = 0.63,
n = 8) or between the consumption and the energy
content (Pearson; p > 0.1, r = 0.61, n = 8).
Consumption of the Bt and six non-Bt 361
Basic Appl. Ecol. 3, 4 (2002)
Fig. 1. The initial Cry1Ab concentration and the concentration after 20 days
in 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. Bars
with 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;
R
2
= 0.43).
df F P
sex 1 0.55 > 0.05
variety 7 17.23 < 0.0001
sex variety 7 3.65 < 0.001
weight 1 11.91 < 0.001
Herbivore bioassays
The bioassay results with the target pest of transgenic
corn, O. nubilalis, confirmed the continuous insectici-
dal activity of the transgenic corn plants used in the
food consumption experiment. Mean mortality and
weight of O. nubilalis were 53 50% and 0.21 0.26
mg when feeding on N4640Bt (Bt+), 82 13% and
0.27 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) were
significantly different. O. nubilalis fed on Max88 (Bt+)
showed a significantly higher mortality (p < 0.01) and
reduced weight (p < 0.01) than O. nubilalis larvae fed
on non-transgenic corn (N4640). No significant differ-
ence in mortality (p > 0.05) but a significantly reduced
larval 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 of
P. scaber and in their faeces after feeding on transgenic
362 Wandeler et al.
Basic Appl. Ecol. 3, 4 (2002)
Fig. 3. Detection of Cry1Ab toxin in body, gut and faeces of P. scaber (black
bars) 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 nm
wavelength. 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 in
extraction buffer at concentrations of 0 (pure extraction buffer as a negative
control), 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 fed
on control (N4640) or (Bt+) corn (N4640Bt). Given is the number of repli-
cates (n), mean ( SD of the amount material used, the amount of extraction
buffer used and the calculated concentrations of Cry1Ab in the respective
tissues (mean ( SD).
Tissue analysed N Material (mg) Buffer (ml) g Cry1Ab/g
fresh weig
Feeding plants (Bt) 5 105 23.6 5 0
Feeding plants (Bt+) 5 132 11.9 5 2.43 1.14
Body (Bt) 5 36.0 6.2 2 0
Body (Bt+) 8 36.4 9.4 2 0.03 0.02
gut (Bt) 5 5.1 2.2 2 0
gut (Bt+) 8 5.5 1.2 2 0.10 0.02
faeces (Bt) 5 7.3 3.8 3 0
faeces (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 g/g fresh
weight. Mean toxin concentrations in the body and
gut were very low (0.03 and 0.1 g/g fresh weight, re-
spectively), whereas the mean concentration of the
toxin in the faeces was higher (0.32 g/g fresh weight)
(Table 2).
Discussion
Food consumption
P. scaber fed on significantly more N4640 (Bt) than
N4640Bt (Bt+) plant material, confirming the results
of 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 corn
leaves 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 well
indicate a Bt-effect. Another explanation could be the
higher lignin content in Bt corn as compared to non-Bt
corn (Saxena & Stotzky in press), which persists for
several weeks during degradation (Escher et al. 2000).
The significantly lower consumption of N4640Bt
(Bt+) residues compared to Max88 could be explained
by the higher Cry1Ab content in the N4640Bt leaves.
The available amount of Bt toxin in Max88 could
have been too low to affect the consumption. Another
reason for the difference in consumption of the two Bt
varieties could be varying microbial activity or coloni-
sation, as microbial conditioning increases the palata-
bility of the plant litter (Hassal & Rushton 1984).
However, Mller (1999) could not find any differences
in bacterial and fungal communities between N4640Bt
and Max88 residues. These results show that various
Bt corn varieties can induce different feeding responses
and therefore every Bt variety needs to be assessed on
a case-by-case basis.
The corn variety had the strongest effect on the con-
sumption by P. scaber. Likewise, individual weight of
P. scaber affected the consumption significantly,
wherefore the relative wide range of weight used was
accounted for. Comparing the consumption across all
eight corn varieties, there were two non-transgenic
corn varieties, namely Magister and Banguy, from
which P. scaber also ate very little. The amount of
N4640Bt consumed ranked among the least fed upon
varieties such as Magister and Banguy, whereas
N4640, compared to the other non-transgenic corn
varieties, 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 its
control 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 the
consumption by P. scaber is an indirect measure for
the residues quality which depends, among other
things, on the energy and nitrogen content, the tough-
ness of the leaves, humidity and pH value, and on the
microbial colonisation (Sutton 1980, Hassal & Rush-
ton 1984, Zimmer & Topp 1997). The nitrogen and
the energy content of the corn leaves varied across the
different varieties used and both can potentially con-
tribute to the differences in the consumption observed.
N4640Bt ranked among the corn varieties with a high
nitrogen and energy content whereas Max88 was in
the lower range. Woodlice might adapt the amount of
consumption to the quality of food as a pre-ingestive
compensatory mechanism (Lavy et al. 2001). The con-
sumption was negatively correlated (but not signifi-
cantly) to the nitrogen and energy content. Magister
and N4640Bt showed a higher energy and nitrogen
content than Max88 (Fig. 2b,c) and both were fed
upon significantly less than Max88 which would
speak in favour of the pre-ingestive compensatory
mechanism.
The mean expression level of Cry1Ab toxin in
N4640Bt (Event Bt11) quantified by ELISA was nearly
twice as high as that reported by EPA for field grown
corn (EPA 2000). This level of Bt toxin may be due to
the fact that the level varies between different stages
and tissues of the plant (Fearing et al. 1997). Further-
more, the corn variety described by EPA originates
from the similar event (Bt11) as N4640Bt, but it does
not have exactly the same genotype. In the other Bt
corn variety used in our experiment, Max88 (Event
176), the concentration of the toxin approximately
equalled the lowest level described by Fearing et al.
(1997).
Herbivore bioassays with O. nubilalis confirmed
the insecticidal activity of the toxin in the plant mate-
rial used in the consumption experiment. Compared to
N4640, the larvae showed a significant higher mortali-
ty and reduced weight when feeding on Max88,
whereas when feeding on N4640Bt, only the larval
weight was significantly reduced. Generally, only a
low amount of plant material could be mixed with the
artificial diet. Otherwise the larvae did not accept the
mixture because they do not like senescent plant mate-
rial. Therefore O. nubilalis did not necessarily ingest
the toxin in a lethal dose, which might be responsible
for the high variance in the mortality data of the larvae
feeding on N4640Bt. Due to this high variance, feed-
ing on N4640Bt caused only a sublethal effect for the
larvae, based on the evidence of the significantly re-
duced larval weight, despite its very high expression
level.
Consumption of the Bt and six non-Bt 363
Basic Appl. Ecol. 3, 4 (2002)
Analysis of Bt toxin in P. scaber
The detection of Cry1Ab toxin in P. scaber and in its
faeces showed that Cry1Ab toxin is still available after
ingestion and excretion. The concentration of Cry1Ab
protein in the faeces is approximately tenfold higher
than in the gut. The food plant material again has the
tenfold higher toxin concentration than the faeces. Be-
cause the data are based on fresh weight, the faeces
showed a wide range in the measured Cry1Ab toxin
concentration, as faeces can lose water very quickly
due to its large surface. As the trial was carried out at
21 C for 72h, it might be possible that the originally
available quantity of the toxin in the faeces had al-
ready been degraded by microorganisms. Obviously,
P. scaber ingests the Cry1Ab toxin and degradation of
the 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 reach
the soil where it is available to non-target soil organ-
isms not ingesting the plant material itself. The
Cry1Ab protein in root exudates and biomass of Bt
corn appears not to be toxic to earthworms, but the
toxin was present in the gut (Saxena & Stotzky
2001b). If Bt corn-fed earthworms excrete the Bt toxin
with their faeces too, substantial concentrations of Bt
toxin would be present in soil, what could potentially
pose a hazard to faeces-feeding or faeces-living organ-
isms that are important in the decomposition of these
faeces.
Conclusions
1) Due to the wide range of consumption of the differ-
ent non-transgenic corn varieties, it is shown that
the consumption is not only a question of Bt or not.
Further investigations should take into account that
it is difficult to draw conclusions from just one iso-
gene test system.
2) The Cry1Ab toxin was still detectable in the faeces
of P. scaber in considerable amounts. Thus, the Bt
toxin is available continuously after a first ingestion
of an unsusceptible organism.
Acknowledgements: We gratefully thank J. Mller, Dr. L.
Kuhn-Nentwig and P. Wandeler for their technical assistance,
P. Gugerli and C. Zwahlen for help with the ELISA and her
helpful discussions, Dr. J.-P. Airoldi and Dr. S. Bacher for
their great support in the statistical analysis, K. Ruchti and
R. Howald for assistance with the food consumption
experiment, K. Ldi and C. Klingler for improving the
manuscript and two referees for valuable comments.
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