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Bt rice expressing Cry2Aa does not cause direct detrimental
effects on larvae of Chrysoperla sinica
Yunhe Li
•
Yuanyuan Wang
•
Jo
¨
rg Romeis
•
Qingsong Liu
•
Kejian Lin
•
Xiuping Chen
•
Yufa Peng
Accepted: 11 September 2013 / Published online: 22 September 2013
Ó Springer Science+Business Media New York 2013
Abstract To assess the potential effects of Cry2Aa-
expressing insect-resistant Bt rice on Chrysoperla sinica
larvae, we conducted two tritrophic bioassays using a non-
target (Laodelphax striatellus) and a target herbivore
(Chilo suppressalis) as prey. None of the tested life-table
parameters of C. sinica did differ when fed with L. stria-
tellus nymphs reared on either Bt or control rice plants.
Similarly, C. sinica larval survival and development were
not affected when fed C. suppressalis larvae that were
reared on Cry2Aa-contained artificial diet compared to
those fed control diet. However, the 7-day larval weight
was significantly decreased in the Bt treatment and none of
the C. sinica larvae developed to the adult stage. To clarify
whether the observed effects were due to the direct toxicity
of Cry2Aa or prey-quality mediated, we conducted a die-
tary exposure assay in which the toxicity of Cry2Aa to C.
sinica larvae was tested. Potassium arsenate (PA) was
included as a positive control. None of the tested life-table
parameters of C. sinica was adversely affected when fed
Cry2Aa at 500 lg/ml sucrose solution. In contrast, C. si-
nica larvae were adversely affected by feeding on sucrose
solution containing PA. In the feeding assays, exposure of
C. sinica larvae to Cry2Aa was confirmed by ELISA. Our
results demonstrate that C. sinica larvae are not sensitive to
Cry2Aa at concentrations exceeding the levels that the
larvae may encounter in Bt rice fields. Consequently the
detrimental effects observed in the tritrophic studies using
Bt rice-fed C. suppressalis as prey can be attributed to the
decreased prey quality due to the sensitivity of C. sup-
pressalis larvae to Cry2Aa.
Keywords Non-target effects Chilo suppressalis
Laodelphax striatellus Tritrophic bioassay T2A-1
Genetically modified rice
Introduction
In rice production, insect pests are an important restraining
factor for yield (Chen et al. 2011). Many rice pests can
cause economic losses, among which a group of lepi-
dopteran species such as Chilo suppressalis Walker (Lep-
idoptera: Crambidae), Scirpophaga incertulas Walker
(Lepidoptera: Pyralidae) and Cnaphalocrocis medinalis
Guene
´
e (Lepidoptera: Pyralidae) are the most serious pests
causing large reductions in yields in almost all rice-grow-
ing regions in China (Cohen et al. 2008). In 2002, these
pests occurred on 15 million hectares in China, represent-
ing half of the rice area and resulting in significant yield
losses (Sheng et al. 2003). To control rice pests, a large
amount of broad-spectrum insecticides is used. This not
only increases the production costs, but also potentially
harms the health of rice farmers and deteriorates the
environment (Pingali and Roger 1995; Matteson 2000).
In recent years, insect-resistant genetically engineered
(GE) crops have widely been planted in the world and
Yunhe Li and Yuanyuan Wang contribute equally to this work.
Y. Li (&) Y. Wang J. Romeis Q. Liu K. Lin X. Chen
Y. Peng (&)
State Key Laboratory for Biology of Plant Diseases and Insect
Pests, Institute of Plant Protection, Chinese Academy of
Agricultural Sciences, Beijing, China
e-mail: yunhe.li@hotmail.com
Y. Peng
e-mail: yfpeng@ippcaas.cn
J. Romeis
Agroscope Reckenholz-Ta
¨
nikon Research Station ART, Zurich,
Switzerland
123
Ecotoxicology (2013) 22:1413–1421
DOI 10.1007/s10646-013-1127-0
provide an efficient measure for insect pest control that has
led to a significant reduction in insecticide use (James
2012; Brookes and Barfoot 2011). Since the first report of
an insect-resistant GE rice line expressing an d-endotoxin
from Bacillus thuringiensis (Bt) in 1993 (Fujimoto et al.
1993), China in particular has devoted great efforts to
develop GE rice. Although no GE rice variety has been
commercialized so far, many lines expressing Bt proteins
have been obtained to protect against lepidopteran pests
(Chen et al. 2011). Most of the Bt rice lines, for example
those expressing Cry1Ac, Cry1Ab or Cry1Ab/Ac, have
been extensively evaluated for their resistance to the target
pests (Tu et al. 2000; Bashir et al. 2004; Han et al. 2006,
2007; Zhang et al. 2011), and also for potential risks to the
environment and human health (Cohen et al. 2008; Chen
et al. 2011; Li et al. 2007; Zhang et al. 2011). A rice line
transformed with the cry2Aa gene is a relatively new
development and its efficient resistance against stem borers
such as C. suppressalis and S. incertulas has been con-
firmed (Chen et al. 2008). However, the potential envi-
ronmental risks of this rice line, in particular the effects on
non-target organisms, have received little attention.
Green lacewings (Neuroptera: Chrysopidae), such as
Chrysoperla carnea (Stephens) and Chrysoperla sinica
(Tjeder), are known to contribute to the biological control
of crop pests. They are important insect predators, having a
wide geographic distribution, and occurring in many dif-
ferent cropping systems including maize, cotton and rice
(Brooks 1994; Bai et al. 2005; Jiang and Xiao 2010). Since
the species can be easily reared and manipulated in the
laboratory, they are regarded as suitable surrogate species
for laboratory studies to support the environmental risk
assessment of plant protection products, such as insecti-
cides and fungicides (Nasreen et al. 2007; Sabry and Ei-
sayed 2011; Nasreen et al. 2005; Romeis et al. 2013). In
recent years, lacewings have been tested in laboratory
studies to support the risk assessment of GE plants (Hil-
beck et al. 1998a, b; Dutton et al. 2002; Romeis et al. 2004;
Obrist et al. 2006; Rodrigo-Simo
´
n et al. 2006
; Lawo and
Romeis 2008; Lawo et al. 2010; Li et al. 2008; Wang et al.
2012; Tian et al. 2013).
Chrysoperla sinica is a prevalent predator species in
Chinese rice fields (Bai et al. 2005; Wang et al. 2012). The
predatory larval stage preferentially feeds on aphids but
may consume planthoppers, mites and eggs and young
larvae of lepidopteran insects, while C. sinica adults feed
primarily on pollen, honeydew and nectar (Jervis and Kidd
1996;Xu2001). Thus both larvae and adults are likely to
be exposed to Cry proteins in rice fields by prey or pollen
consumption. The potential effect of Cry2Aa-expressing
rice pollen on adult C. sinica has been evaluated by Wang
et al. (2012). Adult C. sinica were fed with Cry2Aa-
expressing rice pollen or with artificial diet containing
300 lg Cry2Aa/g dry weight of diet for 26 days, and no
negative effect on important life-table parameters was
detected. However, no such study has been conducted with
C. sinica larvae so far.
In the current study, two tritrophic experiments were
conducted to assess whether Cry2Aa-expressing rice plants
have potential prey-mediated effects of on the larvae of C.
sinica, when the plant hopper L. striatellus (Falle
´
n) (Ho-
moptera: Delphacidae), a non-target species of Cry2Aa-
expressing Bt rice, and the rice stem borer C. suppressalis,
a target pest, were used as prey. To further verify whether
Cry2Aa protein has direct toxicity to C. sinica larvae, a
feeding bioassay was carried out in which C. sinica larvae
were fed sucrose solution incorporated with purified
Cry2Aa protein at a level much higher than that likely to be
encounter under field conditions.
Materials and methods
Plant materials
Transgenic rice line T2A-1 and its corresponding non-
transformed near isoline Minghui 63 were used for the
experiments. T2A-1 plants express a synthesized cry2Aa
gene driven by the maize ubiquitin promoter. Minghui 63
is an elite indica restorer line for cytoplasmic male-sterile
in China. Both rice lines were obtained from Huazhong
Agricultural University, Wuhan (Chen et al. 2005).
The two rice lines were simultaneously planted in two
separate climatic chambers at 26 ± 2 °C, 75 ± 5 %RH
and 16:8 h L: D. Plants were raised in plastic cups (5 cm
diameter, 40 cm height) containing rice nutrient solution
(see Supporting information) and used for the experiments
when they were 2 weeks old.
Insects
Chrysoperla sinica were collected at the experimental field
station of the Institute of Plant Protection, CAAS, near
Langfang city, Hebei Province, China (39.5°N, 116.7°E) in
2010 and since then maintained in the laboratory without
introductions of field-collected insects. Larvae of C. sinica
were reared on soybean seedlings infested with Aphis
glycines Masumura (Homoptera: Aphididae). Adults were
fed an artificial diet containing sucrose and brewers yeast at
a ratio of 1:1. Water was supplied using saturated cotton
balls. Freshly hatched C. sinica larvae (\12 h after
hatching) were used for the experiments.
Laodelphax striatellus were retrieved from a laboratory
colony that has been maintained on conventional rice
plants for over 40 generations in a climatic chamber at
27 ± 1 °C, 65 ± 5 % RH and 16:8 L:D.
1414 Y. Li et al.
123
Chilo suppressalis used in this study were retrieved
from a laboratory colony that has been maintained on an
artificial diet for over 30 generations (Han et al. 2012).
Insecticidal chemicals
Potassium arsenate (PA, KH2AsO4) was purchased from
Sigma-Aldrich (St. Louis, MO). Cry2Aa protein was
bought from EnviroLogix Inc. (Portland, Maine). The
Cry2Aa protoxin from Bacillus thuringiensis had been
expressed as single gene products in Escherichia coli, then
the protoxin inclusion bodies were dissolved and trypsini-
zed, subsequently the toxins were isolated using high-per-
formance liquid chromatography by EnviroLogix.
Bioactivity of the Cry2Aa proteins was confirmed in a
sensitive bioassay in our laboratory using neonate larvae of
C. suppressalis that were fed for 7 days with artificial diet
containing a range of Cry protein concentrations. The EC
50
(toxin concentration resulting in 50 % weight reduction)
was estimated to be 1.31 lg/ml diet (Yunhe Li et al.
unpublished data).
Prey-mediated effects of Bt rice on C. sinica larvae
Testing with L. striatellus as prey
Neonates of C. sinica were individually confined in glass
tubes (1 cm diameter 9 10 cm height) that were closed
with a cotton plug and fed soybean aphids during the first
instar. When C. sinica larvae reached the 2nd instar they
received L. striatellus that had fed on Bt rice or the cor-
responding non-Bt rice plants. For aphid feeding, soybean
seedlings fully covered with aphids were cut into 2 cm
segments before being provided to C. sinica larvae. For
planthopper feeding, L. striatellus were transferred to Bt
rice or non-Bt rice plants in the climatic chambers where
the plants were grown. After 7 days of feeding, young
nymphs of L. striatellus were collected and provided to the
C. sinica larvae (approximately ten per larva). The prey
insects were replaced daily to ensure that they were
available ad libitum. The experiment started with a total of
30 lacewing larvae per treatment. The larvae were indi-
vidually kept, resulting in 30 replications per treatment.
The lacewings were observed daily until adult emergence.
Larval survival and developmental time, 7 days larval
weight and weight of freshly emerged adults were mea-
sured on an electronic balance (CPA224S, Sartorius, Ger-
many; d = 0.1 mg).
To investigate the transfer of Cry2Aa from Bt rice-
reared L. striatellus to C. sinica larvae, a separate set of
insects was prepared. Second instar C. sinica larvae were
fed with Bt rice-reared or non-Bt rice-reared planthoppers.
After 5 days of feeding, the lacewing larvae were
collected. In addition, L. striatellus were collected for the
analyses. Five to ten insects were pooled per sample, and
five samples were collected for each treatment. In addition,
five Bt and non-Bt rice leaf samples (about 30 mg) were
collected. All samples were kept at -80 °C for later
Cry2Aa measurement.
Testing with C. suppressalis as prey
Similar to the bioassay with L. striatellus, C. sinica were
fed with soybean aphids during the first instar, and then
subsequently received C. suppressalis larvae from the 2nd
instar until pupation. Neonate C. suppressalis were fed
with pure artificial diet (diet without Cry protein incorpo-
ration) for 7 days. Subsequently half of them were trans-
ferred to artificial diet containing Cry2Aa (100 lg/g diet)
and the half remained on pure artificial diet. Seven days
later, 10 C. suppressalis larvae were randomly selected
from each treatment and weighted to check whether the
were negatively affected by feeding on Cry2Aa-contained
diet. Subsequently the C. suppressalis larvae were provided
to C. sinica. Prey larvae were replaced daily to ensure that
they were available ad libitum. The experiment started with
a total of 30 C. sinica larvae per treatment. The larvae were
individually kept, resulting in 30 replications per treatment.
Larval survival and developmental time, 7 days larval
weight and weight of freshly emerged adults were recorded
using an electronic balance.
To clarify the exposure of C. sinica larvae to Cry2Aa in
the bioassay, a separate set of insects was prepared. Samples
of C. suppressalis larvae that had fed Cry2Aa-contained
artificial diet or pure artificial diet for 7 days were collected.
Similarly, C. sinica larvae that had been fed with Cry2Aa-
containing or non-Bt treated C. suppressalis larvae for
5 days were collected. For each treatment, five samples were
collected and each sample contained 5–10 insects. All
samples were kept at -80 °C for later Cry2Aa measurement.
Direct feeding bioassay
Assay validation
A dietary exposure system was established in which C. si-
nica larvae were alternatively fed with soybean aphids and
2 M sucrose solution. In the dietary system, sucrose solution
was used as a carrier for the test substance, while soybean
aphids provided the nutrients required for normal develop-
ment of C. sinica larvae. PA was used as model compound to
test whether the test system can detect toxicity as the com-
pound is toxic to C. sinica (Yunhe Li et al. unpublished
data). Different amounts of PA were dissolved into 2 M
sucrose solution to attain the target concentrations of 0, 16,
32, 64 and 128 lg/ml sucrose solution. Chrysoperla sinica
Bt rice expressing Cry2Aa does not affect C. sinica larvae 1415
123
larvae were fed with soybean aphids during the first instar.
Afterward, they were fed exclusively with PA-containing or
pure sucrose solution during the first day of each instar.
Subsequently the soybean aphids were provided in addition
to the sucrose solution until the next ecdysis. When adults
emerged, they were fed on artificial diet (sucrose and
brewers yeast at a ratio of 1:1) without incorporation of PA.
Thirty-five individually kept insects were tested per treat-
ment. The experiment was terminated after 20 days when
almost all C. sinica had reached the adult stage or died in the
control treatment. The survival rates of C. sinica during the
whole feeding period were calculated based on daily sur-
vival records.
Testing with Cry2Aa
To test the toxicity of the Cry2Aa protein to C. sinica
larvae, we conducted a bioassay including three treatments:
(a) 2 M sucrose solutions (negative control) ? aphids;
(b) 2 M sucrose solution containing PA at 64 lg/ml
(positive control) ? aphids; (c) 2 M sucrose solution
containing Cry2Aa protein at 500 lg/ml ? aphids.
Chrysoperla sinica larvae were fed as described above. To
guarantee the bioactivity of Cry2Aa protein, newly pre-
pared Cry2Aa-containing sucrose solution was provided to
each instar of C. sinica. The experiment was started with
30 individually kept insects per treatment. The experiment
was terminated after 20 days when almost all C. sinica had
reached the adult stage or died in the negative control
treatment. Larval survival, larval development time and
weight of freshly emerged adults were recorded. In addi-
tion, the survival rates of C. sinica during the whole
feeding period were calculated based on daily survival
records.
For confirmation of the ingestion of Cry2Aa by C. sinica
larvae, larvae were fed with Cry2Aa-contained sucrose
solution or pure sucrose solution as described above. The
3rd instar larvae were collected just before they were
transferred to aphid prey. Five insect samples were col-
lected per treatment and each sample contained 5–10
insects. The samples were kept at -80 °C for later Cry2Aa
measurement.
ELISA measurements
The concentrations of Cry2Aa proteins in rice leaves and
insect samples were measured by double-antibody sand-
wich enzyme-linked immunosorbent assays using the
Cry2Aa detection kits from EnviroLogix (Portland, Maine,
USA). Prior to the analyses, all insects were washed in
PBST buffer to remove any Bt protein from their outer
surface. After adding PBST to the samples at a ratio of at
least 1:10 (mg sample: ml buffer) in 1.5 ml centrifuge
tubes, the samples were fully ground by hand using a
plastic pestle. After centrifugation and appropriate dilution
of the supernatants, ELISA was performed according to the
manufacturer’s instructions. The measured optical density
values were calibrated to a range of concentrations of
Cry2Aa standards provided with the kit.
Data analyses
In all the experiments, statistical comparisons were made
between each treatment and the control. Chi square and
Mann–Whitney U tests were used for the parameters larval
survival and larval development time, respectively, since
the assumptions for parametric analyses were not fulfilled.
In the direct feeding experiment, Bonferroni correction was
applied to correct for two pair-wise comparisons, leading to
an adjusted a = 0.025. Student’s t test was used to com-
pare larval weight between the two treatments in the tri-
trophic experiments. Adult weight was compared using
Student’s t test. Sexes were pooled since fresh weight of
freshly emerged males and females did not differ.
The survival response of C. sinica to different dietary
treatments was analyzed using the Kaplan–Meier proce-
dure and Logrank test.
All statistical analyses were conducted using the soft-
ware package SPSS (version 13 for windows, 2004).
Retrospective power analyses were conducted on non-
significant (p
[ 0.05) results using PASS 12 (Hintze, 2013)
to avoid committing type II errors. Based on the observed
control means and standard deviations and the true sample
sizes, the detectable differences (percentage difference of
treatment means relative to control means) were calculated
for a = 0.05 and a power of 80 %. Depending on the
statistical method that was applied, detectable differences
for means were calculated based on t tests or v
2
-tests.
Results
Tritrophic experiment with L. striatellus as prey
Survival, development time, and weight of C. sinica larvae
did not differ between lacewings fed planthoppers reared
on Bt or non-Bt rice (Table 1). Similarly, the weight of
freshly emerged adults did not differ between treatments
(Table 1). Retrospective power analyses revealed that the
detectable effect size for the different parameters ranged
from 7 to 32 %.
Cry2Aa concentration (mean ± SE) in rice leaves was
3.53 ± 0.29 lg/g fresh weight (FW). The concentration of
Cry2Aa in planthoppers that had fed on Bt rice for 5 days
was just 0.4 % of the concentration in leaves (14.6 ±
2.01 ng/g FW). No Cry2Aa was detected in larvae of C.
1416 Y. Li et al.
123
sinica that were provided with Bt rice fed L. striatellus.No
Cry2Aa was detected in the control samples.
Tritrophic experiment with C. suppressalis as prey
After 7-day feeding on Cry protein-contained artificial diet,
the C. suppressalis larval weight was significantly reduced
compared to larvae fed on pure diet (Student’s t test;
t = 10.95, df = 18, p \ 0.001). Survival of larvae and
7-day larval weight of C. sinica were not affected when fed
C. suppressalis larvae that had been reared on Cry2Aa-
containing artificial diet compared to those in the control
(Table 2). Larval development time, however, was signifi-
cantly prolonged (by 8 %) for C. sinica larvae in the Cry2Aa
treatment (Table 2). In addition, no adults emerged in the
Cry2Aa treatment (Table 2). For the two non-significant
parameters (i.e. larval survival and 7-day larval weight),
power analyses revealed a detectable effect size of 38 %.
The Cry2Aa concentration (mean ± SE) in C. sup-
pressalis larvae that fed on Cry2Aa incorporated artificial
diet was 1.11 ± 0.12 lg/g FW. The mean concentration in
larvae of C. sinica that were exclusively fed with Cry2Aa-
containing C. suppressalis larvae for 5 days was
0.09 ± 0.02 lg/g FW, which was 8.1 % of the concen-
tration found in the prey. No Cry2Aa protein was detected
in the insects that had not been exposed to Cry2Aa.
Direct feeding experiment
Testing with PA
In the control treatment, over 90 % of C. sinica survived after
20 days feeding exposure (Fig. 1). With increasing con-
centration of PA in the sucrose solution, the survival rates of
C. sinica were steadily reduced and no larvae survived to
pupal stage at the highest PA concentration (128 lg/ml)
(Fig. 1). Survival analysis revealed no statistical difference
between the treatment containing PA at 16 lg/ml and the
control (v
2
= 3.28, p = 0.07). In contrast, survival rates
were significantly decreased compared to the control for
insects fed sucrose solution containing PA at 32 lg/ml
(v
2
= 4.59, p = 0.03), 64 lg/ml (v
2
= 24.30, p \0.001),
and 128 lg/ml (v
2
= 70.65, p \ 0.001) (Fig. 1).
Table 1 Prey-mediated effects on Chrysoperla sinica larvae fed Laodelphax striatellus nymphs that were reared on Cry2Aa-expressing Bt rice
plants or the corresponding non-transformed rice plants
Parameters Non-Bt rice Bt rice Statistics Detectable difference (%)
d
Larval survival (%)
a
93.3 (30) 96.7 (30) V
2
= 0.35,p= 0.55 32
Larval development time (days ± SE)
b
11.11 ± 0.19 (27) 11.46 ± 0.21 (28) U = 326.50,p= 0.37 7
7-day larval fresh weight (mg ± SE)
c
6.04 ± 0.37 (11) 5.31 ± 0.36 (10) t = 1.40,p= 0.18 26
Adult fresh weight (mg ± SE)
c
4.23 ± 0.22 (25) 4.20 ± 0.23 (20) t = 1.11,p= 0.92 21
Number of replicates is given in parentheses
a
Chi square test
b
Mann–Whitney U test
c
Student’s t test
d
Detectable difference for means at a = 0.05 and a power of 80 %
Table 2 Prey-mediated effects on Chrysoperla sinica larvae fed Chilo suppressalis larvae that were reared on pure artificial diet or artificial diet
incorporated with purified Cry2Aa (100 lg/g diet)
Parameters Control Bt treatment Statistics Detectable difference (%)
d
Larval survival (%)
a
76.7 (30) 70.0 (30) V
2
= 0.34,p= 0.55 38
Larval development time (days ± SE)
b
8.62 ± 0.27 (23) 9.32 ± 0.23 (21) U = 63.5,p= 0.004 nc
7-day larval fresh weight (mg ± SE)
c
4.72 ± 0.46 (23) 4.02 ± 0.35 (28) t = 1.22, p = 0.23 38
Adult fresh weight (mg ± SE)
c
4.16 ± 0.51 (7) – – nc
Number of replicates is given in parentheses
– no adult emerged, nc not calculated
a
Chi square test
b
Mann–Whitney U test
c
Student’s t test
d
Detectable difference for means at a = 0.05 and a power of 80 %
Bt rice expressing Cry2Aa does not affect C. sinica larvae 1417
123
Testing with Cry2Aa
Pair-wise comparisons revealed that the ingestion of
Cry2Aa did not significantly affect C. sinica larval survival
(Chi square test; v
2
= 0.11, p = 0.74), larval development
time (Mann–Whitney U test; U = 273.50, p = 0.23) and
adult weight (Student’s t test; t = 0.17, df = 11, p = 0.87)
(Table 3). In contrast, C. sinica fed PA had a significantly
decreased survival rate (v
2
= 21.70, p \ 0.001) and a
longer larval development time (U = 0.00, p \ 0.001)
than those fed pure sucrose solution and did not reach the
adult stage (Table 3). Depending on the parameter recor-
ded, the detectable effect size ranged from 10 to 38 %.
Survival analysis revealed no significant differences
between the Cry2Aa treatment and the control (v
2
= 0.06,
p = 0.81), while the survival rate of C. sinica was signif-
icantly reduced when fed sucrose solution containing PA
(v
2
= 20.20, p \ 0.001) (Fig. 2).
The concentrations of Cry2A detected in the C. sinica
larvae ranged from 512.6 to 883.3 with a mean of 640.5 ng/
g FW. As expected, no Cry2Aa protein was detected in C.
sinica larvae that were collected from the control
treatment.
Discussion
Assessments of the effects of insecticidal GE crops on non-
target organisms follow a tiered framework that is con-
ceptually similar to that used to assess the environmental
impact of conventional pesticides (Hill and Sendashonga
2003; Garcia-Alonso et al. 2006; Romeis et al. 2008). In
early-tiers, experiments are conducted under confined
conditions that aim to identify the potential effects of the
insecticidal factor on selected surrogate species at worst-
case exposure conditions (Romeis et al. 2011). For these
assessments, three types of studies are commonly
conducted: (1) direct feeding experiments in which purified
insecticidal proteins are fed to the test species by the use of
artificial diets; (2) bitrophic experiments in which test
species are fed with plant tissues such as pollen; and (3)
tritrophic experiments in which the insecticidal proteins are
delivered to the test species indirectly through their prey or
hosts (Romeis et al. 2011; Li et al. 2008, 2011a, b;A
´
lvarez-
Fig. 2 Survival rates of Chrysoperla sinica when larvae fed pure
sucrose solution (negative control) or sucrose solution containing
insecticidal proteins. Per ml sucrose solution, 500 lg Cry2Aa and
64 lg PA (positive control) were incorporated. An asterisk denotes a
significant difference between a Cry treatment or a positive control
and the negative control (N = 30)
Table 3 Impact of sucrose solution containing purified Cry2Aa (500 lg/ml) or potassium arsenate (PA) (64 lg/ml) on Chrysoperla sinica
larvae
Parameters Control Cry2Aa PA Detectable difference (%)
d
Larval survival (%)
a
83.3 (30) 80.6 (30) 22.2 (27)* 38
Larval development time (days ± SE)
b
9.14 ± 0.23 (25) 8.89 ± 0.27 (24) 13.17 ± 0.23 (6)* 10
Adult fresh weight (mg ± SE)
c
4.95 ± 0.38 (7) 4.85 ± 0.45 (6) – 34
Number of replicates is given in parentheses. Each toxin treatment was compared to the control. Asterisks denote a significant difference to the
control
– no adult emerged
a
Chi square test with Bonferroni corrections (adjusted a = 0.025)
b
Mann–Whitney U test with Bonferroni corrections (adjusted a = 0.025)
c
Student’s t test
d
Detectable difference for means at a = 0.05 and a power of 80 %
Fig. 1 Survival rates of Chrysoperla sinica when larvae fed sucrose
solution containing different concentrations of PA. Pure sucrose
solution served as a negative control. An asterisk denotes a significant
difference between a PA treatment and the control (N = 35)
1418 Y. Li et al.
123
Alfageme et al. 2011). When tritrophic experiments are
conducted, great care has to be taken to ensure that the
herbivores themselves are not adversely affected by the
ingested protein to avoid the impact of prey/host-quality
mediated effects (Romeis et al. 2006, 2011; Naranjo 2009;
Shelton et al. 2009).
In the current study, two tritrophic experiments were
conducted to assess the prey-mediated effects of Cry2Aa-
expressing Bt rice on C. sinica larvae. Two herbivores that
commonly occur in rice fields were used as prey, the non-
target pest species L. striatellus and a target pest species C.
suppressalis. No detrimental effect was found on the sur-
vival and development of C. sinica larvae when fed Bt rice-
reared planthoppers vs. non-Bt rice-reared planthoppers.
However, C. sinica larval development was significantly
prolonged and all insects died during the pupal stage when
C. suppressalis larvae that had consumed Cry2Aa-contained
diet were used as prey.
ELISA analyses showed that the Cry2Aa concentration
in C. suppressalis larvae that had consumed Bt-containing
artificial diet was over 75 times higher than in L. striatellus
that were reared on Bt rice. Consequently, the Cry2Aa
concentration in C. sinica larvae was 90 ng/g FW when fed
Cry2Aa-containing C. suppressalis larvae, while no
Cry2Aa was detected in the predator when fed Bt rice-
reared L. striatellus. Thus, the adverse effects observed in
the experiment with C. suppressalis as prey could be due to
the fact that C. sinica larvae ingested much more Cry2Aa
compared to the experiment where L. striatellus were used
as prey. Alternatively, the adverse effects could be due to
the fact that C. suppressalis was sublethally affected by the
Cry2Aa because of its susceptibility to the toxin.
To separate potential prey-quality mediated effects from
direct toxic effects of the Cry2Aa protein, a dietary exposure
experiment was conducted, in which the purified Cry2Aa
protein was directly fed to C. sinica larvae by incorporation
into a 2 M sucrose solution. Sucrose solution has been suc-
cessfully used as a toxin carrier in previous studies with other
non-target species such as C. carnea (Romeis et al. 2004;
Lawo and Romeis 2008) and the ladybirds (Coleoptera:
Coccinellidae) Adalia bipunctata (A
´
lvarez-Alfageme et al.
2011) and Coccinella septempunctata (A
´
lvarez-Alfageme
et al. 2012). In the current study, we have used the oral poison
PA to validate our dietary exposure assay. Chrysoperla si-
nica larvae were fed with 2 M sucrose solutions containing
PA at different doses and dose-dependent responses were
observed for larval survival. This confirmed that the test
system used in the current study can detect dietary effects of
insecticidal compounds.
No toxicity was found to any of the tested life-table
parameters of C. sinica larvae that were provided with
sucrose solution containing Cry2Aa at a concentration of
500 lg/ml. This can be regarded as worst-case exposure
given the fact that the concentration is [140-fold higher
than what we measured in rice leaves. Our tritrophic
experiment also confirmed the low exposure of C. sinica to
Cry proteins when consuming planthoppers that are com-
mon prey for C. sinica larvae in rice fields. In contrast,
feeding on sucrose solution containing PA caused signifi-
cant adverse effects on the survival and development of C.
sinica larvae.
ELISA measurements confirmed the uptake of Cry2Aa
by C. sinica larvae when feeding on the sucrose solution. In
addition, the bioactivity of the Cry proteins before they
mixed into sucrose solution was confirmed by sensitive-
insect bioassays. Therefore, we can ensure that the C. si-
nica larvae were exposed to high concentrations of bioac-
tive Cry proteins during the duration of the direct feeding
bioassay. In addition, our bioassay set-up was found to be
sensitive enough to show a 38 % lower survival, a 10 %
prolongation in larval development time and a 34 %
reduction in adult fresh weight. In regulatory risk assess-
ments, a 50 % effect typically is defined as a threshold that
would trigger additional studies (Rose 2007). This together
with the fact that we have exposed the lacewing larvae to
unrealistically high concentrations of Cry2Aa demonstrates
that the Cry protein is unlikely to cause toxicity to C. sinica
larvae at the concentrations that may be encounter in Bt
crop fields.
Due to their ecological importance as biological control
agents, green lacewings have received much attention in
the risk assessment of GM plants especially after negative
effects of Cry1Ab on C. carnea larvae were reported by
Hilbeck et al. (1998a, b). However, subsequent studies
could not confirm these adverse effects when lacewing
larvae were either directly fed with sucrose solution con-
taining purified Cry1Ab protein or fed with Cry1Ab-fed
prey organisms that were not susceptible to the Cry1Ab
protein (Romeis et al. 2004; Dutton et al. 2003; Obrist et al.
2006; Lawo and Romeis 2008). Furthermore a study indi-
cated that Cry1A toxins do not show specific binding to
brush border membrane vesicles from the midgut of C.
carnea larvae, which is a prerequisite for toxicity (Rodrigo-
Simo
´
n et al. 2006). Thus the reported effects of Cry1Ab
protein on lacewing larvae appear to be caused by a
reduction in the nutritional quality of toxin-affected preys
(Romeis et al. 2006; Naranjo 2009; Shelton et al.
2009).
The importance of such indirect prey-quality mediated
effects has been demonstrated in two studies which have
used Bt-resistant and Bt-susceptible Lepidoptera strains as
prey for C. carnea (Lawo et al. 2010) and C. rufilabris
(Tian et al. 2013). Based on this previous experience, the
detected hazardous effects in the tritrophic experiment with
C. suppressalis larvae as prey thus appear to be an indirect
effect of the reduced prey quality of this Cry2Aa-sensitive
species.
Bt rice expressing Cry2Aa does not affect C. sinica larvae 1419
123
Conclusions
Our results demonstrate that C. sinica larvae are not sen-
sitive to Cry2Aa. Thus, Bt rice expressing Cry2Aa should
have no direct negative effects on C. sinica larvae. Com-
bining with our previous data that C. sinica adults were not
negatively affected when fed with Cry2Aa-expressing rice
pollen or an artificial diet containing 300 lg Cry2Aa/g dry
weight of diet for 26 days (Wang et al. 2012), we conclude
that growing of Cry2Aa-expressing rice should not affect
C. sinica population and its ecological function in the field.
Acknowledgments We thank Prof. Yongjun Lin (Huazhong Agri-
cultural University) for kindly providing transgenic rice seeds. The
study was supported by the National GMO New Variety Breeding
Program of PRC (2012ZX08011-001) and (2013ZX08011-001).
Conflict of interest The authors declare that they have no conflict
of interest.
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