Biocontrol Science and Technology, June 2005; 15(4): 341 /352
Quantification of Bt-protein digestion and excretion by
the primary decomposer Porcellio scaber, fed with two
Bt-corn varieties
BENOÎT PONT & WOLFGANG NENTWIG
Zoological Institute, University of Bern, Bern, Switzerland
(Received 6 January 2004; returned 25 February 2004; accepted 14 September 2004)
Abstract
Despite increasing use and fabrication of new transgenic plants, there are still concerns about a
potential accumulation of Bt-proteins in the soils. Even previous studies have revealed that some
phytophagous animals ingest and excrete Bt-protein. Neither the digested proportion of Btprotein nor the potential amount of an insecticidal activity of the feces excreted by soil
arthropods is known. For a period of 15 days, we fed the primary decomposer woodlouse
Porcellio scaber (Latreille) with leaves of two transgenic Bt-corn varieties (N4640Bt and
Max88Bt) and one non-transgenic control variety (N4640). The Bt-protein in the leaves and
in feces was quantified using ELISA. The Bt-protein digestion rate was obtained as a ratio of the
Bt-protein ingested to the Bt-protein excreted. Additionally we quantified the naturally
occurring Bt-protein dissipation in the leaves of the two transgenic corn varieties over a period
of 15 days. Finally bioassays using the susceptible species Ostrinia nubilalis (Hübner)
(Lepidoptera, Pyralidae) were carried out to determine if the Bt-protein in the decomposer
feces is still active. We calculated that P. scaber feeding on N4640Bt corn leaves digests a mean of
61.19/16.8% of the Bt-protein they ingest, while P. scaber feeding on Max88Bt corn leaves
digests 80.59/14.4%, which is significantly more (P B/0.05). At an average temperature of
18.38C, the Bt-protein concentration in the leaves shows a rapid and constant Bt-protein
decrease of approximately 409/15% per 3 days in both transgenic corn plants. This accumulates
to a Bt-protein loss of more than 90% after 15 days. The bioassays indicate that the Bt-protein
excreted with the feces is still insecticidally active. Our study suggests that a part of the Btprotein taken up by primary decomposers is not digested and is released in its active form into
the soils. Under the much cooler field conditions in autumn and winter, it stays active and
remains available to soil organisms until the next field season.
Keywords: Bacillus thuringiensis, transgenic corn, ELISA, Bt-protein activity, Bt-protein
digestion rate
Introduction
Advances in genetic engineering technology over the past few years have permitted
altering the physiology of plants, conferring different qualities on them. Among
others, several species of crop plants (including cotton, corn and potato) have been
genetically modified to express genes of various strains of Bacillus thuringiensis Berliner
(Bt) encoding insecticidal proteins (d-endotoxins) (see Lewellyn et al. 1994; Perseley
Correspondence: Wolfgang Nentwig, Zoological Institute, University of Bern, Balzerstr. 6. CH 3012 Bern,
Switzerland. Tel: 41 31 631 4520. Fax: 41 31 631 4888. E-mail: [email protected]
ISSN 0958-3157 print/ISSN 1360-0478 online # 2005 Taylor & Francis Group Ltd
DOI: 10.1080/09583150400016969
342
B. Pont & W. Nentwig
1996; Federici 1998). Most varieties of Bt-corn express the cry1Ab protein (here
named Bt-protein), targeting the pest insect Ostrinia nubilalis , the European corn
borer. Despite their increasing use in agriculture, it is still prohibited to grow
transgenic plants in many countries, due to environmental or other concerns.
Although each different insecticidal protein is considered to be specific to one target
group of pest insects, some studies using Bt-corns showed that non target species can
be affected as well. The studies on monarch butterflies and black swallowtails also
showed how different results can be interpreted (Losey et al. 1999; Wraight et al.
2000; Hansen Jesse & Obrycki 2000). Moreover, Bt-plants may enhance the selection
and enrichment of Bt-protein resistant target insects (Ferre & Van Rie 2002). Bt-crops
produce the insecticidal Bt-protein until plant senescence begins. This was not the
case with the Bt-sprays farmers were previously using. They may therefore prolong the
environmental persistence of Bt-protein and increase the bio-availability of the Btprotein to non target organisms (Sims & Martin 1997). Indeed, dead organic matter
provides a direct source of food for many soil animals. Some estimated 24 tons (dry
weight) of leaf residues per hectare remain in the field after harvesting of grain maize
and corn-cob-mix (Wehrli et al. 1986).
Studies on Bt-protein dissipation have obtained contradictory results, probably due
to the different experimental conditions (especially the temperature) under which they
had been performed. Sims and Holden (1996) tested decomposition at 24 /278C.
They observed that 50% of the Bt-protein contained in lyophilized transgenic plant
material was dissipated 1.6 days after incorporation of the material into soil and 90%
was dissipated after 15 days. The results of Head et al. (2002) also argue for a rapid
dissipation and non persistence of Bt-protein in soils. They could not detect any
Cry1Ac protein in six different soils of Mississippi and Alabama (between 338 and 358
North, continental climate) after multiple years of transgenic Bt-cotton use. On the
other hand, a field study in the temperate region of Switzerland (488 North) showed
dissipation times of the Bt-protein from corn residues in soil longer than 150 days
(Zwahlen et al. 2003). Another field trial conducted by Saxena and Stotzky (2001)
showed that the Bt-protein can still be detectable in its active form several months
after cutting the plants. Therefore, concerns about a possible accumulation of Btproteins in soils deserve serious consideration, particularly in temperate regions.
Although some studies have focused on the Bt-protein dissipation in decomposing
plant material, only limited information is known on the fate of Bt-protein taken up by
decomposers. Recent studies revealed that Bt-protein is present in some decomposer
and herbivorous animals feces. This could be another means by which Bt-protein ends
up in the soil: in a study where the primary decomposer, the woodlouse Porcellio scaber
(Latreille) (Isopoda: Porcellionidae), was fed on dry Bt-corn leaves, Wandeler et al.
(2002) observed a 10-time lower Bt-protein concentration in feces than in the leaves.
Raps et al. (2001) showed that Bt-protein could be detected in the larvae of Spodoptera
littoralis and in their feces when fed on transgenic Bt corn leaves. They also observed
that the Bt-protein concentration in the feces was comparable to that of fresh leaves,
showing that no digestion seemed to occur. To summarize, these studies revealed that
Bt-protein originating from transgenic plants is present in the feces of various animals,
but they failed to quantify the amounts and the remaining insecticidal activity.
Woodlice, such as the presently considered common Porcellio scaber are among the
most important representatives of the soil living detritophagous macrofauna and can
therefore be considered as model decomposer organisms. As primary decomposers,
Bt-protein excretion by Porcellio
343
isopods feed mainly on dead plant material. They accelerate the process of
humidification by fragmenting dead plant material and enriching it with microorganisms in their intestinal tract. The feces of soil animals, such as millipeds, woodlice and
earthworms may contain in excess of 500 times more bacteria than the initial food
material (Ineson & Anderson 1985). Escher et al. (2000) did not observe a negative
impact of Bt-corn litter on consumption, reproduction and growth of P. scaber. But
in their study, Wandeler et al. (2002) noticed high concentrations of Bt-protein in
P. scaber feces. They concluded that such an excretion could make the Bt-protein
available to many non-herbivorous organisms, posing a hazard especially for fecesfeeding or feces-living organisms.
Since a first experiment with Bt-corn showed that a period of 15 days is sufficient to
analyze uptake and complete excretion of the ingested Bt-protein, we restricted our
main experiment to this duration. The objectives of the present study is (i) to analyze
Bt-protein dissipation in the decaying leaves, (ii) to calculate, for both transgenic corn
varieties N4640Bt and Max88Bt, the Bt-protein digestion rate after ingestion by the
primary decomposer P. scaber , and (iii) to test if this Bt-protein, after excretion by the
woodlouse, is still insecticidally active.
Material and methods
Plants
Two genetically modified corn varieties (Max88Bt and N4640Bt, both from Syngenta,
previously Novartis), and one isogenic control line (N4640) were used. The
transgenic plants contain a truncated, synthetic version of the gene from Bacillus
thuringiensis var. kurstaki coding for the expression of the insecticidal d-endotoxin
Cry1Ab (Koziel et al. 1993). The transcription of the Cry1Ab gene in Max88Bt 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).
Plants were cultivated in plastic pots (30 cm diameter) in a climate chamber at an
average temperature of 23.38C (258C during the photophase of 16 h and 208C for the
remaining 8 h). Three plants per pot and 10 pots per variety were planted. We used
commercial garden soil (Landi, Switzerland) and added liquid fertilizer every 2 weeks
during the growing season (1.2 g N, 0.9 g P2O5, 1.1 g K2O in 1 l per pot). The plant
material used for the experiment was taken from plants which had reached a height of
120 cm (after pollen shed). Only senescent, brown leaves were used (leaves 5 /7) and
cut into pieces of approximately 12 cm length. They were dried at 408C for 48 h and
stored at /258C until use. We restricted our experiments to such leaves because
(i) woodlice do not feed on green leaves, (ii) after harvest, most plant material turns
brown within a few days, and (iii) this guarantees a minimum standardization of leaf
quality.
Animals
Adult individuals of the woodlouse Porcellio scaber were collected from a garden near
Sierre (Southern Switzerland) in March 2002. They were maintained in a plastic box
(16 /11 /5 cm), filled with a 1-cm layer of moistened garden soil with an
incompletely sealed transparent plastic lid to allow air circulation. As a maintenance
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diet, brown leaves of Salix sp. were added into the box. Until they were used in the
following experiments, the animals were kept in the same climate chamber, at an
average temperature of 11.58C (158C during the photophase of 7 h, and 108C for the
remaining 17 h).
Uptake and dissipation of Bt-protein by Porcellio scaber setup of experiment
Each P. scaber was placed individually in a transparent plastic box (7/6/3 cm). The
lid had a hole (30 mm diameter) covered with a fine mesh netting to allow air
circulation. The bottom was covered with a 1-cm layer of plaster of Paris to maintain
sufficient humidity. The boxes were allocated to random positions in the climate
chamber and protected with cardboard against direct lighting. At the initiation of the
experiment, we fed 200 adult P. scaber with non-transgenic corn (N4640) for 4 days.
The animals with the highest food uptake were selected and starved for 48 h prior to
feeding with transgenic or control corn leaves.
Feeding procedure. In the final experiment which is only reported here, each of our
three treatment groups was constituted of 12 subgroups of two or three individuals.
For 15 days, the three treatment groups were fed on three different diets: N4640
Bt-corn leaves, Max88Bt-corn leaves, and the non transgenic N4640 as control.
After 15 days, the first two groups switched to the non-transgenic diet (N4640) and
all three groups continued on this diet for another 15 days. Prior to this final
experiment, three other series were run to optimize the conditions of the final
experiment. In a first experiment, we kept three large groups of woodlice on the three
different diets, collected the feces after 14 days and measured the remaining Btprotein content by ELISA. In a second experiment, we tested the daily feces
production of different group sizes of woodlice (1, 2/3, 10) during 30 days to find
the lowest group size with a suitable feces production for the ELISA analyses. In a
third experiment, the diet was changed twice between Bt-corn and non-transgenic
corn to identify the number of days the food passage lasts and how many days after
diet change the previous food is still excreted. In all three pre-experiments, all feces
were collected and analyzed by ELISA. These data were all in the range of the final
experiment presented here but we report only on the results of the most elaborated
final experiment.
Food material. For use as food, the frozen leaves were dried again for 48 h at 408C and
cooled to room temperature for 30 min in an dessicator filled with silica gel. Each leaf
was cut into equal sized pieces (5 /1 cm, 10 /20 pieces per leaf) which were weighed
up to the next tenth of a mg. One piece was analyzed for Bt-protein content by an
enzyme-linked immunosorbent assay (ELISA) as discussed below. The others were
remoistened by laying for 2 min in water and offering to each animal of the same
subgroup. Before switching to the non-transgenic diet (N4640), the remaining noneaten leaf material from the first 15 days was removed, dried for 48 h at 408C and reweighed. The amount of ingested plant material was estimated by subtracting the dry
weight of the remaining leaves from the dry weight of the leaves initially offered.
Bt-protein dissipation in leaves. In order to know how much Bt-protein the leaves were
losing naturally over time, the following test was performed. From each corn variety,
Bt-protein excretion by Porcellio
345
12-cm pieces were selected from 10 leaves and cut into six pieces of 2 cm each. The
first piece was immediately analyzed for its Bt-protein content by ELISA. The other
pieces were put into boxes under the same conditions as in the feeding experiment (see
above). At 3-day intervals, the next piece was analyzed, and the last piece after
15 days. With these ELISA data, we generated a detailed Bt-protein dissipation curve
over 15 days. This percentage loss of Bt-protein was used as a correction when
calculating the effective amount of Bt-protein taken up by P. scaber.
Feces. Every 24 h, the feces of each isopod was collected in 2-ml Eppendorf vials and
immediately frozen to avoid natural Bt-protein dissipation. Due to the small quantity
of feces, probes of three successive days had to be lumped together into one probe
which was analyzed by ELISA. Thus, the 30-day experiment yielded ten 3-day probes.
Analogous to corn leaves, a parallel assay was carried out to determine the natural
Bt-protein loss in feces. The percentage of natural Bt-protein loss was taken into
account for the average time between feces production and feces collection (/24/2 h),
assuming a linear degradation process for this short time interval.
ELISA of Bt-protein
Corn leaves. Following the general descriptions of the ELISA test system presented by
Gugerli (1979) and Zwahlen et al. (2003), the test material was dried to be weighed,
put into universal bags (Bioreba, Reinach, Switzerland) and then homogenized for
2 min with a hand model homogenizer (Bioreba AG) in 5-ml extraction buffer
(10 mM phosphate, 137 mM NaCl, 2.7 mM KCL, 3 mM NaN3, 2% (v/v)
polyvinylpyrrolidon (Mr /25 000), 0.05% (v/v) Tween† 20, pH 7.4) to extract the
Bt-protein. The obtained liquid was then centrifuged (20 min, 108C, 5000 rpm), and
the supernatant used for analysis.
In order to determine the calibration curve, reference samples of purified Cry1Ab
Bt-protein (gift of P. Gugerli) were suspended in pooled extracts of control leaves
(N4640) at a concentration of 1000, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.01 ng Cry1Ab
protein/ml plant extraction buffer. Microtitre immunoassay plates, Immunolon† 4
(Dynatech Laboratories Inc., Virginia, USA) were analyzed with an MRX microplate
reader operated with the Revel software package, Version G 3.2 (Dynex Technologies
Inc.). Polyclonal coating IgG and alkaline phosphatase-conjugate IgG against
the Cry1Ab Bt-protein were purchased from Bioreba AG. Diethanolamine and
4-nitrophenylphosphate for the substrate buffer were obtained from Merck, Darmstadt, Germany. All samples, including the calibration curve, were analyzed in
duplicate. The arbitrary detection threshold was defined as the mean of the optical
density of the control leaf samples plus three times its standard deviation.
Feces. Frozen feces were exposed for 35 min in an dessicator filled with silica gel to a
vacuum until constant weight was reached. The dried feces were weighed. We added
1.8 ml extraction buffer per probe and allowed 5 min for rehydration prior to
homogenization. After centrifugation, the supernatant was analyzed for Bt-protein
content by ELISA. We analyzed the feces with the same method as described above
but the purified Bt-protein for the reference curve was now suspended in pure
extraction buffer.
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Herbivore bioassays
To test if the Bt-protein in the transgenic plants leaves and in the feces was
insecticidally active, bioassays using the susceptible target species Ostrinia nubilalis
(Hübner) (Lepidoptera, Pyralidae) were conducted as following. Leaves of the three
different corn varieties or feces excreted by animals fed with the three different
corn varieties (N4640, N4640Bt and Max88Bt) were homogenized with a mortar
and pestle and added with 20% to an artificial diet (a mixture of 2% agar /agar, 5%
corn semolina, 5% wheat germs, 5% torula yeast, 0.5% ascorbic acid in water,
Wandeler et al. 2002). The mixture was put into each of 10 vials (22 mm diameter
by 53 mm 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 chamber at 208C. The larvae mortality
was recorded after 10 days. This bioassay was performed with 15 and 50% of feces
or leaf material in the diet to test the willingness of the larvae to feed on this diet.
Fifty percent of feces was not accepted by many larvae and we had problems with
the consistency of the diet. Therefore, we took 15% in the final test as described
above.
Data analysis
For the quantitative analysis of ELISA, data of the reference curve were logtransformed and a non-linear regression (curve fit: sigmoidal dose response) was
carried out to calculate the Bt-protein concentration in the samples (GraphPad
Software Inc. 2000). Mean mortality of the two transgenic treatments was compared
to the control treatment with non transgenic plants using a Tukey HSD multiple
comparisons test. The same test was used to compare the Bt-protein dissipation in
leaves. The Bt-protein digestion rate of P. scaber was calculated as ratio of the ingested
Bt-protein to the excreted Bt-protein (both data obtained according to ELISA
measurements).
Results
Bt-protein dissipation in leaves and in feces
The Bt-protein concentration in the leaves obtained by ELISA after each period of
3 days showed a rapid and constant decrease of around 409/15% in the leaves of both
transgenic corn varieties (Figure 1). Except between days 9 and 12 where it was only
13.5%, the Bt-protein dissipation for the four other periods of 3 days in N4640Bt corn
leaves was quite constantly 469/5.1%. In Max88Bt corn leaves, except between days
6 and 9 where the dissipation was only 23%, the mean dissipation for the remaining
four periods of 3 days was 44.59/10.3%, which is very comparable to the leaves of
N4640Bt. Of the initial Bt-protein, 42.39/20.6% for N4640Bt and 42.99/14.7% for
Max88Bt was degraded after 3 days. After 15 days, 92.69/3.8 and 92.19/8.6 of the
initial Bt-protein content was dissipated in N4640Bt and Max88Bt corn leaves,
respectively.
Bt-protein concentration in feces of the N4640Bt treatment group was 2.769/1.2
mg/g DW, and in feces of the Max88Bt treatment group it was 0.39/0.1 mg/g DW. After
Bt-protein excretion by Porcellio
30
347
a
µg Bt-protein / g leave
25
20
1
b
N4640Bt corn
Max88Bt corn
15
c
2,1
10
cd
5
cd
2
2
2
d
2
0
0
0-3
3-6
6-9
9-12
12-15
days
Figure 1. Bt-protein dissipation during 15 days in two varieties of transgenic corn leaves (at 18.38C, 959/5
humidity). Bt-protein content (mean/standard deviation) is shown for periods of 3 days and for dry weight
leaves. Columns with different letters (for N4640Bt) or different numbers (for Max88Bt) are significant at
P/0.05 (n /10).
24 h, the Bt-protein concentration was 2.29/0.9 and 0.39/0.1 mg/g DW, respectively.
The decrease was not significant (P/0.15).
Bt-protein digestion rate
Bt-protein ingested. During a period of 15 days, a total mean of 419/7.9 mg DW was
eaten by each woodlouse subgroup fed on N4640Bt and 31.69/5.7 mg DW fed on
Max88Bt. The difference was statistically significant (P B/0.05). Based on the data
collected at 3-day intervals, we calculated the food intake over the 15 days. The food
intake for every period of 3 days is shown in Figure 2. The curves look quite similar for
both treatment groups and show three different phases. During the first 3 days, the
food uptake was very low. Between days 3 and 12 the curves rise rapidly and then
decrease between days 12 and 15.
Bt-protein excretion. Total feces production per animal during 15 days was 10.59/4.3
mg DW for the Max88Bt group and 14.09/6.5 mg DW for the N4640Bt group. The
difference was significant (P B/0.05). Feces production was highest between days 6
and 12 (6.09/1.8 mg DW for Max88Bt and 8.79/4.1 mg DW for N4640Bt group) and
lower before and after this period (Figure 3).
Woodlice eating N4640Bt corn excreted significantly more Bt-protein than the ones
fed with Max88Bt (P 5/0.005). Each P. scaber subgroup fed with N4640Bt excreted
during the experiment a total mean of 0.0679/0.02 mg Bt protein while each
woodlouse subgroup on Max88Bt excreted a total mean of 0.00849/0.006 mg Bt
protein. The total Bt-protein excreted by all animals (24/36 woodlice) together
during the experiment was 0.8 mg for the N4640Bt treatment group and 0.1 mg for
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B. Pont & W. Nentwig
20
mg food intake / 100 mg isopod
18
16
14
12
10
8
6
N4640Bt
Max88Bt
4
2
0
0
0-3
3-6
6-9
9-12
12-15
days
Figure 2. Mean (/SD) mg food uptake per 120 mg fresh weight of P. scaber for each period of 3 days during
15 days (n/12).
Max88Bt treatment group, i.e., on average 1.8 ng for N4640Bt and 0.2 ng for
Max88Bt per animal per day.
Bt-protein was excreted in small amounts up until day 18, despite the fact that
animals received non-transgenic food after day 15. After day 18, no Bt-protein could
be traced in the feces with our methods, and therefore we assume that food passage in
P. scaber takes up to 3 days.
Bt-protein digested by P. scaber. Over 15 days, the P. scaber group feeding on N4640Bt
corn leaves digested a mean of 61.19/16.8% of the Bt-protein they ingested, while the
Max88Bt group digested 80.59/14.4% of the Bt-protein which is significantly more
(P B/0.05).
Herbivore bioassays
Mean mortality of O. nubilalis was 769/26% when feeding on N4640Bt, 519/39%
when feeding on Max88Bt, and 169/7% when feeding on the control N4640.
A significantly higher mortality was observed when O. nubilalis fed on Max88Bt
(P B/0.05) and on N4640Bt (P B/0.005) leaf diet than when they fed on the nontransgenic N4640 leaf diet. No significant difference between the two transgenic
treatments was observed (P /0.05). Mean mortality of O. nubilalis was higher when
feeding on transgenic Max88Bt (499/19%) and on N4640Bt (519/25%) feces diet
than when feeding on control N4640 (159/16%). Compared to the control group,
Bt-protein excretion by Porcellio
349
7
mg dry weight faeces
6
5
Max88Bt
N4640Bt
4
3
2
1
0
0
0-3
3-6
6-9
9-12
12-15
15-18
days
Figure 3. Mg dry weight feces produced (mean/standard deviation) by individual P. scaber (53.7 mg fresh
weight) for periods of 3 days (n/30).
O. nubilalis mortality was significantly higher when reared on both transgenic feces
diet (P B/0.005 for both groups). No significant difference (P /0.97) between both
transgenic treatments was observed. These results are summarized in Table I.
Discussion
Bt-protein dissipation in leaves
The Bt-protein dissipation in corn leaves was rapid and constant (every 3 days about
409/15%) in both transgenic corn varieties at 18.38C. After 3 days, more than 40%
and after 15 days, more than 90% of the initial Bt-protein content of the leaves was
degraded. Even if the experimental conditions were not the same as far as temperature
and plant material are concerned, our results have much in common with the
laboratory studies of Palm et al. (1994) or Sims and Holden (1996). The latter
estimated that 50% of Bt-protein in sieved and lyophilized transgenic plant material
was dissipated after 1.6 days and 90% after 15 days. In their 30-day study using two
different transgenic cotton lines expressing Cry1Ac or Cry1Ab protein, Palm et al.
Table I. Mean (9/SD) mortality percentage of Ostrinia nubilalis in a bioassay and significance between the
mortality (P value) when feeding on leaves and feces from three different corn varieties.
Mortality (%)
N4640Bt
Max88Bt
N4640
769/26
519/39
169/7
N4640 vs Max88Bt
N4640 vs N4640Bt
Max88Bt vs N4640Bt
0.048
0.001
0.124
519/25
499/19
59/16
N4640 vs Max88Bt
N4640 vs N4640Bt
Max88Bt vs N4640Bt
0.003
0.002
0.97
Fed on leaves
Fed on feces
Significance (P )
350
B. Pont & W. Nentwig
(1994) measured with ELISA a rapid Bt-protein dissipation (at 24 /278C) for the first
7 days and then a slower Bt-protein dissipation. These results indicate generally a
rapid Bt-protein dissipation under laboratory conditions.
However, under natural field conditions, Zwahlen et al. (2003) investigated the
Bt-protein dissipation of Bt-corn material in the soil and explained that the rapid
dissipation under laboratory conditions cannot be extrapolated to field conditions.
Due to much lower average soil temperatures, Bt-protein could still be detected
5 months after the exposure of leaf material in the soil. This result is not in conflict
with the recent study of Head et al. (2002) who found no Bt-protein in the soil after
multiple years of growing transgenic Bt-cotton (Bollgard) since these two studies
cannot be compared for two main reasons. The average annual temperature in the
fields of Mississippi and Alabama (USA, 338 to 358 North) is about 168C while in
Central Europe (Dijon, France and Munich, Germany, both at 488 North) the annual
average is about 98C. The temperature difference between these two regions is about
98 in summer and 68 in winter. An increase of 108C leads on average to a 2/3-times
higher microbial activity. Secondly, the C:N ratio of cotton is by a factor of 2 /3 more
in favor of a rapid decomposition than corn (Nentwig et al. 2004). Therefore, cotton
in Alabama is likely to be dissipated 4 /6 times faster than Bt-corn in Central Europe.
Bt-protein digestion by P. scaber
Food intake during the experiment was very low during the first 3 days for both
treatment groups. This delay was observed every time we fed P. scaber with corn
leaves. After a new kind of food had been offered, woodlice seem to wait till the leaf
piece is a bit more palatable before they really start to eat it. As expected, the feces
curve is similar to the food intake curve, describing the same phases. Most of the feces
production collected in the first 3-day period was probably due to food intake prior to
the experiment. This is corroborated by the low amount of Bt-protein found in feces
during the first 3 days. As explained above, the replacement of the transgenic food
by the control diet again caused a decrease in feces production after day 15. The
Bt-protein excretion curve follows both the food intake and feces curves. Moreover,
despite the fact that animals received non-transgenic food only from day 15 onwards,
they kept excreting Bt-protein in low amounts till day 18. No Bt-protein could be
traced in the feces after this. This confirms that the gut passage in P. scaber takes no
longer than 3 days. Woodlice eating N4640Bt excreted significantly more Bt-protein
than the other transgenic treatment group, probably due to the much higher Btprotein content of N4640Bt leaves.
The Bt-protein digestion rates we calculated indicate that not all the ingested Btprotein is digested by P. scaber. About 40% of the ingested Bt-protein was found again
in the feces when N4640Bt was fed, while significantly less was excreted when
Max88Bt was fed. We do not know whether this incomplete digestion is due to
inadequate mechanical processing of the corn leaves by the isopods, or due to some
physiological insufficiencies (e.g., not enough digesting enzyme available) of the
isopod or its symbiotic microorganisms. Therefore, incompletely digested food still
containing Bt-protein is excreted. Some connection between lignin content and
digestibility could also be possible since the lignin content is higher in N4640Bt than
in Max88Bt (Saxena & Stotzky 2001).
Bt-protein excretion by Porcellio
351
In general, hardly any information about Bt-protein uptake by decomposing
animals is available in the literature. Wandeler et al. (2002) measured the Bt-protein
concentration in feces of P. scaber fed with an identical diet as in the present study.
In agreement with our results, Wandeler et al. (2002) observed a ten times lower
Bt-protein concentration in feces than in the leaves. The following studies investigated
the Bt-protein ingestion by herbivorous animals: Raps et al. (2001), who fed the
herbivore Spodoptera littoralis on N4640Bt corn leaves, estimated that the Bt-protein
concentration in the feces was comparable to that in fresh leaves, thus indicating that
no digestion seemed to occur. Howald et al. (2002) who fed the herbivore Athalia
rosae (Hymenoptera: Tenthredinidae) with Bt-rape cultivars expressing the Cry1Ac
protein also assumed that the Bt-protein was still present in larval body and feces of
these animals. However, in both studies the Bt-protein concentrations in food ingested
and feces produced were not taken in such a detailed manner that the exact ratio of
ingested to excreted Bt-protein could be determined.
Herbivore bioassays
Our herbivore bioassays confirmed the insecticidal activity of the Bt-protein in the
plant material we used and they pointed out that the remaining Bt-protein in the
transgenic corn leaves is still lethal for Ostrinia nubilalis. Some studies (Tapp & Stotzky
1995; Saxena & Stotzky 2001) have already indicated that Bt-protein (in corn root
exudates, in Bt-corn biomass and purified Bt-protein) can stay active for more than
200 days in certain soils. Our results suggest that even after digestion by a primary
decomposer such as P. scaber, Bt-protein is still active.
Conclusion
P. scaber , considered here as a model decomposer organism, is shown in our study to
only incompletely digest the Bt-protein from corn leaves. When excreted in the feces,
this leads to a depositing of still active Bt-protein in the upper soil of corn fields. It is
not trivial to quantify Bt-protein bound by soil particles (Crecchio & Stotzky 2001)
and so far an accumulation of Bt-protein in the soil of corn fields had not been
detected. Under the temperate temperature conditions of Central Europe, it must be
expected that Bt-protein degradation and dissipation rates are reduced (Hilbeck &
Meier 2002). This leads to the assumption that such an accumulation is possible when
corn is cultivated for several years at the same location. This also sheds new light on
several groups of the soil fauna, an area which so far has been rather neglected with
respect to side effects and long-term exposure. We strongly recommend studies in this
area of concern.
Acknowledgements
We thank Lucia Kuhn-Nentwig and Heiri Wandeler (University of Bern, Switzerland)
for their technical assistance and helpful discussions. Many thanks to P. Gugerli
(Federal Research Station for Plant Production of Changins (RAC), Nyon, Switzerland) for his support with ELISA. We also like to thank Christoph Schmid (Swiss
Institute of Bioinformatik, Lausanne) for improving the manuscript, Claudia
Zwahlen, Benno Wullschleger and Patrik Kehrli (University of Bern, Switzerland)
for multiple help, and two anonymous reviewers for their advice.
352
B. Pont & W. Nentwig
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