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European Journal of Soil Biology 47 (2011) 160e164

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European Journal of Soil Biology


journal homepage: http://www.elsevier.com/locate/ejsobi

Original article

Fragmentation of Cry1Ab protein from Bt-maize (MON810) through the gut


of the earthworm species Lumbricus terrestris L.
Christoph Emmerling a, *, Heiko Strunk a, Ulrike Schöbinger a, Stefan Schrader b
a
University of Trier, FB VI e Department of Soil Science, Campus II, Behringstraße, D e 54286 Trier, Germany
b
Johann Heinrich von Thünen-Institute (vTI) e Federal Research Institute for Rural Areas, Forestry and Fisheries, Institute of Biodiversity,
Bundesallee 50, D e 38116 Braunschweig, Germany

a r t i c l e i n f o a b s t r a c t

Article history: A microcosm experiment was performed to study the possible fragmentation of Cry1Ab protein from Bt
Received 12 July 2010 maize (MON810) in the earthworm gut and cast after ingestion by Lumbricus terrestris L. using a Western
Received in revised form Blot technique. The gut was further differentiated into foregut, midgut, and hindgut. Additionally, we
30 November 2010
investigated the concentration of Cry1Ab in these samples by using DAS-ELISA. The results revealed
Accepted 8 December 2010
Available online 22 December 2010
a significantly greater decrease in Cry1Ab concentration in Bt maize litter during the 14-day experiment
Handling editor: Bryan Griffiths in the earthworm treatment than in the control without earthworms. Concentrations were further
significantly decreased in the foregut compared to Bt maize litter, and from foregut to midgut. The
Keywords:
digestive system of the foregut can be localized to be the most important compartment along the whole
Bt maize gut for the fate of Cry1Ab protein during digestion. Cast material showed a slight increase in Cry1Ab
Earthworms concentration relative to the hindgut. We found a considerable fragmentation of Cry1Ab protein in the
Gut direction Bt maize e foregut e midgut from 65 kDa size to 31, 23 and 17 kDa size, respectively, whereas
ELISA fragments of 23 kDa appeared to be the most abundant. No fragments were found in the hindgut and
Cry1Ab fragmentation casts. We suggest, that fragments smaller than 17 kDa size were quantified by DAS-ELISA which in turn
Western Blot could not be detected in the Western Blot. It is concluded that detritivorous earthworms accelerate the
fragmentation of Cry1Ab protein from MON810 maize litter.
Ó 2010 Elsevier Masson SAS. All rights reserved.

1. Introduction anaerobic soils as well as under dry, wet, frozen or thawed soil
conditions [25]. Crecchio and Stotzky [9] found no correlation
Possible pathways of Cry1Ab protein from genetically modified between the adsorption of Cry1Ab-protein on organo-mineral-
maize (Bt maize) into soil have been recognized as root exudates complexes and the amount of soil organic carbon. In contrast,
produced during the vegetation period [35], input of pollen during Pagel-Wieder et al. [31] demonstrated that the adsorption of
flowering [28], herbivore faeces [12], and post-harvest crop resi- Cry1Ab-protein decreased with increasing amount of soil organic
dues [1,41]. carbon.
Highest amounts of Cry1Ab in soil have been found from living Much work has been done in order to investigate the effects of
root fragments compared to one year old maize residues in soil [22]. Cry1Ab toxin derived from post-harvest maize residues on non-
Adsorption experiments revealed that the pro-toxin as well as the target organisms in soil [7,24]. Baumgarte and Tebbe [1] for
Cry1Ab toxin were adsorbed onto clay minerals (montmorillonite example, found no accumulation of Cry1Ab in Bt maize rhizosphere
and kaolinite), humic acids and clay-humic-complexes within during the vegetation period in soil. Additionally, no adverse effects
30 min [40,42]. Interestingly, the adsorbed toxins retained their on microbial community due to Bt maize cultivation were found.
structure and insecticide activity whereas biodegradability When soil was incubated with Bt cotton tissue enzyme activities,
decreased. For example, the Cry1Ab toxin was still detectable after such as urease, acid phophomonoesterase, invertase, and cellulase
234 days [38,42]. Moreover, persistence of insecticide activity was were stimulated, whereas arylsulphatase activity was inhibited
higher in acid soils whereas it was comparable in aerobic or [39]. Moreover, various field investigations on soil microbiota and
nematodes revealed no changes in the bacterial and micro-faunal
communities, as well as enzyme activities and pH levels in soil after
* Corresponding author. Tel.: þ49 (0)651 201 2238; fax: þ49 (0)651 201 3809. Bt maize cultivation [13,20,23]. Toxicity tests by Dubelman et al.
E-mail address: emmerling@uni-trier.de (C. Emmerling). [13] detected no growth inhibition for larvae of the European corn

1164-5563/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejsobi.2010.12.003
C. Emmerling et al. / European Journal of Soil Biology 47 (2011) 160e164 161

borer (Ostrinia nibilalis) and thus, no active Cry1Ab toxin in soil. collected from the Ap-horizon (0e30 cm depth) of an arable field
Also, decomposition of Bt maize straw did not differ from non-Bt near Trier (Germany) and sieved less than 2 mm. Soil chemical
maize [8,11]. Results from Hopkins and Gregorich [22], however, properties were as follows: pH (CaCl2) 5.9, Nt 0.11%, TOC 1.50%, and
suggest that much of the d-endotoxin in crop residues is highly CEC 11.5 cmolc kg1 dry mass (dr.m.).
labile and quickly decomposed in soil, but that a small fraction may Each microcosm was filled with 1000 g moist soil which was
be protected from decay in relatively recalcitrant residues. In wetted previously to a water content of 20% by weight, equivalent
addition, Zwahlen et al. [44] showed in two field studies that to 70% of water holding capacity. Soil was compacted to a dry bulk
degradation of Cry1Ab toxin was inconsistent and showed density of approximately 1.2 g cm3. Earthworms were purchased
temporal variation. from a local earthworm culturing plant (Mosella, Laufeld, Germany)
It is generally accepted, that earthworms strongly affect the and were stored in the experimental soil for one week prior to the
turnover of soil organic matter (SOM) in agricultural ecosystems. experiment. After this period earthworms were washed with
Due to their high feeding and burrowing activity they expand the distilled water, carefully dried on cellulose paper and then weighed.
surface of SOM, redistribute it vertically in the soil profile, change Afterwards, each earthworm was tagged with a visible implant
the size and activity of the microbial biomass in soil [4,10,16], and elastomer (VIE, Northwest Marine Technology, Shaw Island, USA) at
therefore strongly modify the fertility and nutrient availability in an individual position according to Butt and Lowe [5]. Subse-
soil [21]. Additionally, earthworms appear to change the enzymatic quently, earthworms were placed on the soil surface. Mean fresh
degradation of SOM due to an enhancement of the reactive surface biomass of earthworms (n ¼ 3) per microcosm varied from 13.5
of SOM and an active release of extracellular enzymes through gut (1.7) g fresh mass (fr.m.) to 17.2 (0.5) g. After all earthworms had
passage [34]. Generally, litter breakdown by earthworms is strongly entered the soil, 1e3 cm long fragments of 5 g (dr.m.) Bt-maize
related to the chemical properties of the litter [17,18,30] and most litter equivalent to 12.7 g (fr.m.) was placed on the soil surface and
earthworm species feed on simple soluble organic compounds rewetted by spraying 5 ml tap water. Bt maize material was
because of their apparently poor enzyme systems within their provided from the Agricultural Technology Centre Augustenberg,
intestinal tract [27]. Germany, field office Forchheim at harvest. Whole senescent maize
Concerning the impact of genetically modified crops on earth- plants (except roots) of the cultivar Kuratus (Bt; MON810) were
worms there have been relatively few studies, which indicate Bt harvested, chopped and stored at -20  C until use. The C-to-N ratio
maize does not cause any detrimental effects on earthworm health of Bt maize litter was 41.62 and amount of Cry1Ab was 8.8 mg g1
[24]. Krogh et al. [26] for example studied the effects of soil tillage fresh matter.
and cultivation of genetically modified maize on earthworm pop- The microcosms were covered with a gauze (0.5 mm diameter
ulation. The most profound effects were due to tillage practise but mesh) to prevent possible escape of earthworms and then incu-
not to Bt maize cultivation. In a 200-day study with immature and bated at 15  C and 65% relative humidity for 2 weeks. During this
adult Lumbricus terrestris in the field and in the laboratory no time, the soil in each microcosm was moistened with tap water to
significant effects of transgenic Bt maize on biomass and mortality even out loss of moisture, when necessary.
of earthworms was found [44]. According to Vercesi et al. [43] Bt
maize residues had no deleterious effects on survival, growth, 2.2. Sampling of maize, gut content and casts
development or reproduction of Aporrectodea caliginosa, even at
high Cry1Ab concentrations. However, a significant negative effect At the end of the experiment the remaining Bt maize material
was found for cocoon hatchability. A microcosm experiment from and earthworm casts were carefully sampled from the soil surface
Schrader et al. [36] on the fate of Cry1Ab protein from Bt maize and immediately lyophilized and pulverized. For gut analysis,
residues (leaves and roots) in the presence of different earthworm earthworms were taken out of the microcosms at the end of the
species revealed a significant higher decay relative to treatments experiment and weighed individually again according to their tag
without earthworms. For both species tested (L. terrestris and position and transferred into liquid nitrogen (196  C) to prevent
A. caliginosa), no Cry1Ab was found in earthworm tissue [36]. protein decay. All earthworms were dissected to sample their
Immunoreactive Cry1Ab protein concentrations in casts and compartments: Foregut, midgut and hindgut. The midgut was also
earthworm affected soil were detectable only in traces but seemed divided into three cross-sections of equal size (midgut 1 to 3 from
to be protected from biodegradation for at least four months [36]. anterior to posterior). The particular sections of the three individ-
The present experiment was designed to follow the fate of the uals from each microcosm were pooled in order to get sufficient
Cry1Ab protein from genetically modified Bt maize (MON810) litter quantity of gut material for DAS-ELISA (see 2.4) and Western Blot
as food source in different compartments of the gut of the detri- (see 2.5) analysis. The gut content of each compartment was
tivorous earthworm species L. terrestris after Bt maize ingestion, ground by hand in a porcelain mortar.
and in its casts. Furthermore, we asked if a distinct compartment
along the alimentary canal can be localized where the Cry1Ab 2.3. Extraction and quantification of Cry1Ab by DAS-ELISA
protein is primarily digested to its greatest extent.
A commercially available double antibody sandwich-enzyme-
2. Material and methods linked immunosorbent assay (DAS-ELISA) was used to determine
the concentration of Cry1Ab protein (PathoScreen kit for Bt-
2.1. Experimental design Cry1Ab/1Ac ELISA, peroxidase label, Agdia, USA).
For protein extraction from Bt maize material, 1 ml of ice cold
A soil microcosm experiment was performed to assess the fate PBST (Phosphate Buffered Saline Tween; Roche Diagnostics, Man-
of Cry1Ab protein during Bt-maize litter ingestion by earthworms. nheim, Germany) buffer with complete mini protease inhibitor
The experiment consisted of microcosms (n ¼ 4) each containing (complete mini protease Inhibitor Cocktail Tablets, Roche) were
three individuals of L. terrestris (Linnaeus, 1758) and a control added to 100 mg of a lyophilized Bt maize and 500 mg Lysing
treatment without earthworms. The soil within each microcosm Matrix D (MP Biomedicals, Ilkirch, France) and homogenized in the
was covered with Bt-maize (MON810) litter. Fast Prep extractor (BIO101, Thermo Savant, USA) for 30 s and
For the experiment polyethylene containers (2 L) were used as 5.0 m s1, with 4 replicates and a 10 min incubation on ice in-
microcosms. The experimental soil, a sandy loamy Fluvisol, was between. A homogenate was then centrifuged at 15,000  g for
162 C. Emmerling et al. / European Journal of Soil Biology 47 (2011) 160e164

15 min at 4  C and the supernatant was transferred into a new tube. Table 1
For the extraction of Cry1Ab from gut and cast material, 300 mg Mean ( S.D.; n ¼ 4) Bt maize litter weight (dry mass) in the control and earthworm
treatments, and individual earthworm biomass (fresh mass) at the beginning (start)
samples were extracted with 1 ml PBST buffer, then homogenized of the experiment and after two weeks (end), and percentage difference during the
for 30 s and centrifuged at 16,000  g for 15 min at 4  C [36]. Soil experimental period.
material could not been sampled because a differentiation between
Experiment Difference (%)
soil and casts was impossible due to earthworm bioturbation.
For DAS-ELISA, 100 ml of the protein extract was used. The ELISA Start End

was conducted following the instructions of the manufacturer. The Bt maize [g] control 5.00 4.79 4.20 (1.05)
Bt maize [g] earthworm treatmernt 5.00 1.21 75.80 (3.17)
color development was stopped after 15 min with 50 ml 3 M sulfuric
Earthworm biomass [g] 5.28 (0.88) 5.55 (0.92) þ 5.17 (2.26)
acid and detection was carried out with an Multilabel Reader
(Victor3, Perkin Elmer, Germany) measuring the OD at 450 nm. To
quantify the samples, a standard curve (4e3e2.5e2e1.5e1e0.5e
0.25 ng ml1) was also measured in the same ELISA. Each of the fresh weight at the start of the experiment and from 3.5 to 6.7 g at
four samples were analysed in duplicate. the end, individuals increasing by 0.1 to 0.5 g fresh weight. This was
a mean percentage increase in earthworm biomass of 5.18% during
2.4. SDS- polyacrylamide gel electrophoresis (SDS-PAGE) the experimental period (Table 1).
and Western Blotting
3.2. Quantification of Cry1Ab
For SDS-PAGE, 4 to 12% Bis-Tris mini gels (NuPAGE, Invitrogen
GmbH, Germany) were used. To generate enough material for Initial concentration of Cry1Ab of maize litter was 8.8 (0.09)
analysis, the gut sections from each of the three worms were mg g1 dry mass (Fig. 1). At the end of the experiment Cry1Ab
pooled to give a composite sample for each replicate. A random concentration of the remaining litter was significantly decreased to
three of the four replicates were subsequently analysed (because of 2.32 (0.15) mg g1 dr.m. in the control without earthworms and
restricted availability of the Cry1 Ab antibody). Samples were 0.77 (0.10) mg g1 dr.m. in the earthworm treatment (Fig. 1). This
amended with NuPAGE sample buffer and reducing agent (both was a decrease of Cry1Ab of approximately 74% in the control and
Invitrogen GmbH, Germany) at the recommended concentrations. 91% in the earthworm treatment, respectively, relative to the initial
Samples were denatured for ten minutes at 70  C, briefly annealed concentration.
and subsequently applied together with a positive control (Cry1Ab Analyses of Cry1Ab protein concentration in earthworm gut
protein of ELISA-Kit; Agdia, USA) and 2 protein standards (Pre- compartments revealed a clear decrease from foregut to midgut
stained; Fermentas GmbH, Germany and Magic Mark XP; Invi- and hindgut (Fig. 2). In the foregut mean Cry1Ab concentration was
trogen GmbH, Germany), and separated for 40 min by 200 V in 6.47 (0.53) ng g1 dry mass. This was a significant decrease of
a MES-SDS- buffer (Invitrogen GmbH, Germany). Subsequently, nearly 99% compared to the mean Cry1Ab concentration of surface
proteins were transferred electrophoretically on a polyvinyliden maize litter at the end of the experiment (770 ng g1). Compared to
fluoride (PVDF) membrane in a wet-blotting system with a constant the foregut Cry1Ab concentration was further significantly
voltage of 200 mA (Enduro Page System, Labnet Intern.). For decreased to 2.45 (0.13) ng g1 dr.m. in midgut 1, 2.59 (0.46)
immune-detection membranes were blocked in a PBST-buffer for ng g1 in midgut 2, and 2.24 (0.13) ng g1 in midgut 3 on average,
one hour with 1% BSA at room temperature and then incubated for respectively. Mean concentration of Cry1Ab in the hindgut was 2.05
2 h with a primary Cry1Ab antibody (provided by Dr. V. Paul, (0.19) ng g1, and this was a significant decrease compared to
Technical University Munich, Germany). After repeated washing midgut 1. Cast material revealed a slight increase in Cry1Ab
with PBST-buffer the membrane was incubated for 1 h with a HRP concentration of 2.11 (0.19) ng g1 compared to the hindgut.
labelled secondary antibody (goat anti rabbit IgG- HRP; Santa Cruz
Biotechnology, Germany) and finally with SuperSignalÒ West Pico 3.3. Cry1Ab fragmentation
chemo-luminescent substrate (Perbio Science GmbH, Germany)
following the instructions of the manufacturer. The expression of Western Blot analyses revealed a considerable fragmentation of
the proteins was detected with a Lumi-Imager (Roche Diagnostics, proteins from Bt maize during the passage through the gut of
Germany) by using the software LumiAnalyst. L. terrestris individuals. Cry1Ab protein from Bt maize had
a molecular weight of approximately 65 kDa (Fig. 3). In the foregut
2.5. Statistical analysis and the anterior part of the midgut protein fragments of about 31,
23 and 17 kDa were found, whereas a fragment of 23 kDa appeared
All results are presented as means  S.D. of four replicates per to be the most intensive. In the remaining two parts of the midgut
treatment. Statistical comparison of Bt maize, gut and cast material, a fragment of 17 kDa size was detected. No protein fragments were
and gut compartments (foregut, midgut, hindgut) were done by found in the hindgut or in the cast material (Fig. 3).
a non-parametric KruskaleWallis H-test and subsequently pairwise
ManneWhitney-U-tests using SPSS 17.01 software package.
12 a
Cry1Ab [µg g -1]

10
3. Results
8
6 b
3.1. Litter loss and earthworm biomass 4 c
2
0
After two weeks 75.80% of Bt maize litter disappeared from the Maize Start Maize Control Maize
soil surface of the microcosms of the earthworm treatment on End Earthworm
average (Table 1). In the control treatment without earthworms Treatment End
maize litter loss was 4.2%. However, this result included the Fig. 1. Mean ( S.D.; n ¼ 4) Cry1Ab concentration of Bt maize at the start and final
decrease in litter weight due to microbial, e.g. fungal, growth, concentrations in the control and earthworm treatment at the end of the experiment.
respectively. Individual earthworm biomass varied from 3.3 to 6.4 g Significant differences between treatments are marked by different letters.
C. Emmerling et al. / European Journal of Soil Biology 47 (2011) 160e164 163

A further 99% decrease in Cry1Ab concentration occurred


between surface litter at the end of the experiment and in the
earthworm foregut. This agrees with results of Bewley and DeVillez
[2] who pointed out that protein digestion occurs in the foregut.
The earthworm digestive system consists of pharynx, oesophagus,
crop and gizzard followed by an intestine and a proctodeum [14].
This system is a hot spot of microbiota and enzyme activity since
there is a dramatic increase in numbers of microorganisms of up to
1000 times [15,32,37]. The anterior part of the intestine secretes
Fig. 2. Mean ( S.D.; n ¼ 4) Cry1Ab concentrations in different compartments of
earthworm gut (foregut [FG], midgut [MG], hindgut [HG]) and cast material. Significant enzymes while the posterior part absorbs nutrients [15]. The
differences between intestinal compartments and cast material are marked by midgut fluid may suppress growth of bacteria and inhibit the
different letters. germination of spores [6]. Sampedro and Whalen [33] emphasised,
that the microbial community in the earthworm gut was different
from bulk soil and that significant changes occurred at very small
4. Discussion spatial scales between midgut, hindgut and proctodeum.
The amount of Cry1Ab was further significantly decreased
During the experimental period all earthworms increased in between foregut and midgut of the earthworms. Compared to this,
their biomass which indicates that the experimental conditions only a relatively small decrease occurred from the anterior part of
were favorable for the earthworms [19]. the midgut to the hindgut and cast. No Cry1Ab was found in
In the present experiment we found a decrease in the concen- earthworm tissue (data not shown). Cry1Ab concentration was
tration of Cry1Ab protein in the direction Bt maize e foregut e nearly completely degraded, from 770 ng g1 in surface Bt maize
midgut (1e3) e hindgut and casts, which is in accordance to the litter at the end of the experiment to approx. 2 ng g1 in cast
investigation of Schrader et al. [36]. Mean initial concentration of Bt material, respectively. In the cast material, thus only 0.26% of
maize was 8.8 mg g1 dr.m., and this was a typical concentration of ingested Bt maize derived Cry1Ab was found. In a previous inves-
a whole Bt maize plant on average [8]. The most profound differ- tigation [36], Cry1Ab decline was also significantly higher in
ence in Cry1Ab concentration was found between the initial earthworm treatments relative to a control with less than 10% of
concentration in Bt maize and in surface Bt maize litter after the the initial Cry1Ab concentration remaining after 5 weeks. Cry1Ab
two week experiment. This result underlines that microbial activity concentrations in casts were only 0.1% of those found in remaining
was of major importance for the decomposition of Bt maize litter plant material.
and the decay of Bt maize derived Cry1Ab protein. According to The molecular weight of Cry1Ab protein decreased gradually
Hopkins and Gregorich [22] much of the d-endotoxin in crop resi- from Bt maize material (w65 kDa) to foregut and midgut (approx.
dues is highly labile and quickly decomposable in soil. Additionally, 17, 23 and 31 kDa). This is at least circumstantial evidence that the
Cry1Ab concentration in surface Bt maize litter was significantly Cry1Ab protein was fragmented during the passage through the gut
lower in the earthworm treatment relative to the control which of the earthworm. No protein fragments were blotted in hindgut
might be attributed in turn to an increase in microbial activity and cast material although very small amounts of Cry1Ab could be
when earthworms are present [15,16]. This phenomenon is the detected with DAS-ELISA. Thus it seems likely that fragments
priming effect due to mucus secretion by earthworms, which smaller than 17 kDa size could be quantified with the immuno-
promotes microbial activity [3] and thus leads to an increase in assay but then were too small for our Western Blot technique. This
microbial decay of proteins. However, it is unknown how much of corresponds to results from Lutz et al. [29] who found a significant
surface litter loss can be attributed to earthworm burial activity. degradation of Cry1Ab protein and a fragmentation to approx. 17

Fig. 3. Molecular weight [kDa] of Cry1Ab protein fragments in maize (M) and three replicates of pooled samples, see text for details, from the foregut (FG), midgut 1 (MG1), midgut
2 (MG2), midgut 3 (MG3), hindgut (HG) and cast material (CA) after passage through the intestinal tract of Lumbricus terrestris. For protein mass control two protein ladders were
used: Page Ruler (PR; not visible after blotting) and Magic Mark XP (MM, visible with Lumi Imager).
164 C. Emmerling et al. / European Journal of Soil Biology 47 (2011) 160e164

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