Biological Control 35 (2005) 134–141

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EVects of Bt transgenic cotton lines on the cotton bollworm parasitoid

Microplitis mediator in the laboratory
Xiao-xia Liu a, Qing-wen Zhang a,¤, Jian-Zhou Zhao b, Jian-cheng Li c, Bao-liang Xu d,
Xiao-mu Ma a
a
Department of Entomology, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100094, China
b
Department of Entomology, Cornell University-NYSAES, Geneva, NY 14456, USA
c
Institute of Plant Protection, Hebei Academy of Agriculture and Forestry. Baoding 071001, China
d
Chinese Academy of Inspection and Quarantine, Beijing 100025, China

Received 2 February 2005; accepted 19 August 2005

Abstract

Microplitis mediator (Haliday) is an important endoparasitoid of the cotton bollworm, Helicoverpa armigera (Hübner) in northern
China. Interactions among H. armigera, its larval parasitoid M. mediator, and insect–resistant transgenic cotton lines were evaluated
under laboratory conditions. Two major transgenic cotton cultivars used in Hebei province of northern China, DP99B (Bollgard), carry-
ing the cry1Ac gene, and SGK321, carrying both cry1A and CpTI (Cowpea trypsin inhibitor) genes, were used in the experiments. The
results indicated that there was signiWcant growth inhibition of the H. armigera larvae when they were fed a diet containing Bt transgenic
cotton powder. The parasitoid oVspring developed more slowly and pupal and adult weight was reduced signiWcantly when the parasit-
ized host larvae fed on the Bt cotton powder leaf diet compared with non-Bt treatment. With an increase of Bt cotton leaf powder concen-
tration in the host larvae diet, the parasitism rate and adult emergence of the parasitoid decreased and the abnormal pupal rate increased.
There was no evident diVerence in the eVects on M. mediator between the transgenic single- and two-gene cotton cultivars; however, the
parasitized host larval mortality was higher than that of unparasitized larvae in most treatments. The observed eVects on M. mediator
were probably host-quality mediated rather than direct eVects of transgenic cotton because the H. armigera larvae which fed on diet with
leaf powder of both transgenic cotton cultivars also experienced a signiWcant decrease in weight, particularly when the host larvae were
parasitized.
 2005 Elsevier Inc. All rights reserved.

Keywords: Microplitis mediator; Braconidae; Helicoverpa armigera: Noctuidae; Bacillus thuringiensis; Bt cotton; Transgenic plant; Parasitism rate;
Non-target insect

1. Introduction 2003 to 3.7 million hectares in China, equivalent to 66% of

The adoption of transgenic insect–resistant plants as a
mean of crop protection in agriculture has proceeded rap-
idly since advances in recombinant DNA technology have
enabled their use (James, 2004). The area of transgenic cot-
ton expressing Cry1A insecticidal protein from Bacillus
thuringiensis (Bt) increased from 2.8 million hectares in
*
Corresponding author. Fax: +86 10 62734946.
E-mail addresses: liuxiaoxia611@sohu.com (X. Liu), zhangqingwen@
263.net (Q. Zhang).
the total cotton producing area of 5.6 million hectares in
2004 (James, 2004). Research from both the laboratory and
the Weld indicates that Bt plants are eVective in killing the
larvae of a number of species of Lepidoptera, including the
cotton bollworm, Helicoverpa armigera (Hübner) (Deng
et al., 2003; Shelton et al., 2002; Zhao et al., 1998), a key
crop pest in China that caused large production losses prior
to the introduction of Bt (Wu et al., 1997). The eYcacy of
Bt cotton cultivars developed both by Monsanto in the US
and by Chinese institutions was excellent for the control of
H. armigera (Zhao et al., 2000) and pink bollworm

1049-9644/$ - see front matter  2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.biocontrol.2005.08.006
 X. Liu et al. / Biological Control 35 (2005) 134–141
(Pectinophora gossypiella) (Wan et al., 2004). Coincident
with the increased pest control, there has been a signiWcant
reduction in the amount of insecticide used, with the aver-
age number of pesticide applications per season declining
from 20 to seven in Hebei and Shangdong Province (Pray
et al., 2001).
Theoretical models and experimental data suggest that
plants expressing two dissimilar insecticidal proteins in the
same plant have the potential to delay insect resistance
more eVectively than single toxin plants (Zhao et al., 2003).
A range of crops has been modiWed to express diVerent
plant-derived genes encoding insecticidal proteins, includ-
ing enzyme inhibitors, lectins, and hydrolytic enzymes (Bell
et al., 2001). Cowpea trypsin inhibitor (CpTI), a plant-
derived gene, was expressed successfully in plant and dem-
onstrated enhanced levels of insect resistance (Bell et al.,
2001). In recent years, transgenic cotton line SGK321
expressing two insecticidal proteins (Cry1A and CpTI)
(Guo et al., 1999) was commercialized in northern China
(Shelton et al., 2002) and demonstrated increased insecti-
cidal activity on H. armigera relative to single gene Bt cot-
ton (Fan et al., 2001). Transgenic cotton plants with two Bt
genes (Cry1Ac and Cry2Ab2) were approved for commer-
cial use in Australia and the US in 2002 with superior con-
trol of cotton pests (Jackson et al., 2004).
Laboratory results suggest that H. armigera is capable of
developing resistance to Bt protoxins or Bt cotton over
long-term selection (Akhurst et al., 2003; Liang et al., 2000;
Lu et al., 2004; Meng et al., 2003; Zhao et al., 1998). At the
same time, the deployment of transgenic crops must be
compatible with other pest management strategies, such as
biological control, if their use is to be sustainable. Addition-
ally, it has been reported that a proportion of H. armigera
larvae fed on transgenic cotton leaves in the late season can
survive (Zhao and Rui, 2004), and that the surviving larvae
may remain as potential hosts for parasitoid. As a result,
the parasitoids could be aVected by the presence of Bt pro-
tein in food ingested by the host larvae. Accordingly, there
is a need to evaluate the potential non-target eVects associ-
ated with the use of transgenic plants. A number of studies
have addressed the eVect of Bt transgenic crops and Bt pro-
toxin on beneWcial non-target insects, both parasitoids and
predators. Some results showed no apparent negative
eVects of transgenic plants on parasitoids (Atwood et al.,
1998; Johnson, 1997; Schuler et al., 1999) some reported
lower parasitoid survival rates due to premature host death
(Blumberg et al., 1997), lower parasitoid emergence rates
(Atwood et al., 1997), increased parasitoid larval develop-
ment times (Liu et al., 2005a), or reduced longevity and
fewer female ova (Baur and Boethel, 2003). However, there
have been only a few reports on eVects of transgenic plants
expressing two insecticidal proteins on parasitoids (Liu
et al., 2005b; Ren et al., 2004).
Microplitis mediator (Haliday) (Hymenoptera: Braconi-
dae) is an important endoparasitoid of H. armigera in
northern China (Liu et al., 2005a). Historically, the average
parasitism rate on H. armigera was 22.9% in the cotton
135
Welds of Hebei Province (Wang et al., 1984) where insecti-
cidal transgenic cotton cultivars have been used in almost
100% of cotton Welds in recent years. The technology neces-
sary for the mass propagation of the parasitoid has been
developed since 2002 in China, and release of the wasps to
control H. armigera on non-Bt cotton Welds in Xinjiang
cotton belt showed promising results (Q.-W. Zhang et al,
unpublished data). The overall goal of this study was to
determine the potential host-mediated eVects of Bt cotton
on M. mediator under laboratory conditions. SpeciWcally,
this study quantiWed the eVects of transgenic cotton
expressing either one or two insecticidal proteins on
selected life history parameters of M. mediator, including
egg-larval period, development rate, immature stage mor-
tality, cocoon weight, cocoon duration, adult weight and
longevity.
2. Materials and methods
2.1. Insects
Helicoverpa armigera was reared in the laboratory at
26 § 1 °C, 70% RH and a photoperiod of 14:10 (L:D). This
species had been cultured for more than 10 years without
exposure to Bt or other insecticides. The adults were fed a
10% honey solution and the larvae were reared on an artiW-
cial diet (Liu et al., 2004a, 2005a,b).
Microplitis mediator was obtained from the Institute of
Plant Protection, Hebei Academy of Agriculture and For-
estry in China. The parasitoid emerges from the host as a
mature larva and spins a silk cocoon inside which it
pupates. Parasitized hosts do not pupate, but remain in
their larval instar until the parasitoid emerges. The host
subsequently dies 2–11 days after parasitoid emergence
(Wang et al., 1984). The parasitoid adults fed on a 10%
honey solution and mated in culture cages
(20 £ 20 £ 10 cm). Females from three to eight days old
were used in the experiments. The mated female wasps were
allowed to parasitize H. armigera larvae, and the parasit-
ized larvae were then reared in the laboratory on an artiW-
cial diet at 26 § 1 °C and 14L:10D (Liu et al., 2005a,b).
2.2. Cotton plants
The transgenic cotton cultivars DP99B (Bollgard), car-
rying cry1Ac gene developed by Monsanto in the US, and
SGK321, carrying both cry1A and CpTI (Cowpea trypsin
inhibitor) gene developed by the Agri-Biotechnology
Research Institute of CAAS in China (Guo et al., 1999;
Zhang et al., 2004), were used in the experiments. Both cul-
tivars are major insecticidal transgenic cotton lines used in
Hebei Province in northern China. The Cry1A expression
level (mean § SEM) of SGK321 (90.4 § 8.3 ng/g) was simi-
lar to that of DP99B (80.8 § 14.5 ng/g) in the cotton leaf
powder used in the study ground under liquid nitrogen as
tested by ELISA method (Fan et al., 2001) using the com-
mercial ELISA QuantiPlate kit (Envirologix, USA) for
136 X. Liu et al. / Biological Control 35 (2005) 134–141
Cry1Ab/Cry1Ac quantiWcation. Shiyuan321, the parental
cultivar of SGK321 and a major commercial cotton culti-
var originally used in northern China, was used as the con-
ventional cotton. The three cotton cultivars used in the
experiments were provided by the Chinese Academy of
Inspection and Quarantine. All plants were cultivated in
greenhouses. When the plants reached 20–30 cm (7-leaf
stage) in height, their leaves were taken for use in the
experiments.
In the preliminary bioassays using fresh transgenic cot-
ton leaf and neonates or second instars of H. armigera,
almost 100% mortality of H. armigera was observed. Lar-
vae of M. mediator could not complete their development
in host fed on fresh transgenic cotton leaf because of host
death. To test for possible eVects of transgenic cotton on
M. mediator, the cotton leaves were ground to powder
under liquid nitrogen. The powder was stored at ¡20 °C
and then added to artiWcial diet of cotton bollworm for the
experiments.
2.3. Bioassays using leaf powder of transgenic cotton
expressing Cry1Ac
2.3.1. EVects of Bt cotton on host larvae
The Bt cotton cultivar DP99B and non-transgenic culti-
vars Shiyuan321 were used. H. armigera neonates were
reared individually on artiWcial diets containing leaf pow-
der of Bt or non-Bt cotton at concentrations of 0.5, 1, 3, and
5%, respectively. Each of the treated H. armigera larvae
were then weighed at growth intervals of 4, 6, 8, and 10
days. Determination of the larval growth curve Wt was per-
formed using the exponential regression function in Micro-
soft Excel, with time (t, day) as the dependent variable and
larval weight (W, mg) as the independent variable (W D a eb, t)
(Liu et al., 2005a). Larval period, pupal rate and pupal
weight were all recorded, with 10 larvae serving as a replica-
tion. Six replications per treatment were performed for a
total of 60 larvae per treatment.
2.3.2. EVects of Bt cotton on M. mediator
Helicoverpa armigera neonates were reared individually
for 4 or 5 days on the control diet containing 5% non-Bt
cotton leaf powder, for 6 days on diets containing Bt cotton
leaf powder at 0.5 and 1%, for 7 days at 3%, and for 8 days
at 5%. Most of the treated host larvae reached the second
instar and then were provided to the parasitoid. Each larva
was observed until the parasitoid made one successful ovi-
position into the larva. Parasitized larvae were removed
immediately, and were reared on the same diet they had
previously. Then the next treated larva was given to M.
mediator. Adult M. mediator females used in the experiment
were mated and between 3 and 8 days old. The same group
of females was allowed to parasitize H. armigera larvae fed
on diets of diVerent treatments. At least 20 larvae from each
treatment were parasitized daily, with 100 H. armigera lar-
vae parasitized per treatment. Twenty larvae comprised a
replicate and each treatment had at least Wve replicates.
The egg and larval duration period, pupal period,
pupal, and fresh adult weight, and the longevity of adults
(males and females) were all recorded. Adults were fed
daily on 10% honey solution. Host mortality rate (HMR),
successful parasitism rate (SPR), calculated based on the
number of hosts still alive on the day when mature larvae
of M. mediator emerged and the larvae formed a normal
cocoon (Weseloh and Andreadis, 1982), abnormal cocoon
rate (ACR, when the mature M. mediator larvae can
emerge from host larvae, but they cannot successfully
form a cocoon), and emergence rate (ER) were calculated
as follows:
HMR% D M/N0 £ 100
SPR% D N1/(N0 ¡ M) £ 100
ACR% D N2/(N1 + N2) £ 100
ER% D E/N1 £ 100
N0, total number of parasitized larvae; M, number of para-
sitized larvae that died during development; N1, number of
successful cocoons; N2, number of abnormal cocoons; and
E, number of emerged adults
2.4. Comparison of the eVects of transgenic cotton expressing
Cry1Ac and Cry1A + CpTI
To keep the size of the parasitized host larvae the same,
H. armigera neonates were reared on normal artiWcial diets
until they reached second instars. They were then intro-
duced to M. mediator for parasitization according to the
same procedure used in 2.3 with three cotton varieties,
DP99B (single gene cotton), SGK321 (two-gene cotton)
and Shiyuan321 (Control). The diet concentrations of cot-
ton leaf powder were 2, 4, 8, and 12%, respectively. In each
treatment, 80 larvae were parasitized. Half of the parasit-
ized larvae (40) were weighed daily, mortality was calcu-
lated on the other half (40) after 8 days. A total of sixty
larvae from each treatment were left unparasitized as a neg-
ative control. Thirty larvae were weighed daily after treat-
ment, and mortality of H. armigera after 8 days was
estimated from the other 30 larvae. The egg and larval
duration period of the oVspring, cocoon period, cocoon
weight, and adult fresh weight were recorded.
2.5. Statistical analysis
One-way ANOVA was performed using SPSS (SPSS
Institute, 1998). H. armigera mortality within groups
(same concentration, same variety) between hosts treated
by the combination of Bt-diet plus parasitism and hosts
treated by Bt-diet alone and some parameters between
Cry1Ac and Cry1A + CpTI cotton cultivars were com-
pared by independent t tests at the 0.05 level. Treatment
means, including abnormal cocoon rate, successful para-
sitism rate, adult emergence, egg-larvae duration, cocoon
weight, fresh wasp weight, and wasp longevity, were com-
pared and separated by Duncan’s Multiple Range test at
P D 0.05.
 X. Liu et al. / Biological Control 35 (2005) 134–141 137

3. Results concentrations compared to the control (F D 7.81, df D 314,

3.1. EVects of transgenic cotton expressing Cry1Ac
3.1.1. EVects of Bt cotton on host larvae
Bt cotton leaf powder signiWcantly aVected the develop-
ment of H. armigera larvae. According to the growth equa-
tion, inhibition of larval growth increased when the
concentration of Bt cotton leaf powder was higher. Larval
period was also signiWcantly longer compared to the con-
trol (F D 83.76, df D 191, P < 0.001), while both pupal rate
(F D 3.25, df D 47, P D 0.008) and weight (F D 5.30, df D 155,
P < 0.001) were signiWcant lower than the control (Table 1).
No signiWcant diVerence among concentrations was
observed in larval period, pupal rate and pupal weight
(F D 0.15, df D 96, P D 0.927; F D 0.259, df D 23, P D 0.854;
F D 77, df D 1.284, P D 0.286; respectively) when the H.
armigera larvae fed on diets containing conventional cotton
leaf powder. However, there were signiWcant diVerences
among concentrations in larval period (F D 54.98, df D 94,
P < 0.001) and pupal rate (F D 3.49, df D 23, P D 0.035) when
H. armigera larvae fed on the diet containing Bt cotton leaf
powder, but pupal weight was not also signiWcantly diVer-
ent among concentrations (F D 2.330, df D 77, P D 0.081)
(Table 1).
3.1.2. EVects of Bt cotton on M. mediator
The eVects of Bt cotton leaf powder diets on the develop-
ment of the parasitoid M. mediator are presented in Table
2. Egg-larval development was prolonged signiWcantly at all
P < 0.001). Cocoon weight in all treatments also decreased
compared to the control (F D 13.05, df D 298, P < 0.001).
When host larvae fed on diets containing 3% Bt cotton
powder, cocoon weight decreased to 3.5 mg, while that for
the control was 4.1 mg. Adult weight also decreased signiW-
cantly (F D 13.41, df D 243, P < 0.001). At concentrations of
1 and 3%, adult male longevity decreased signiWcantly in
comparison with the control (F D 2.44, df D 171, P D 0.049),
although there were no signiWcant diVerences in male and
female cocoon duration times under all treatments in com-
parison with the control (F D 0.68, df D 172, P D 0.610 and
F D 0.98, df D 71, P D 0.424, respectively). Female adult lon-
gevity was not aVected signiWcantly (F D 1.31, df D 71,
P D 0.276).
The host mortality varied from 12.49 to 29.82% among
all treatments (Table 3). There was a signiWcant diVerence
between the 3% treatment and the control (F D 8.15, df D 22,
P < 0.001). When the concentration of Bt cotton leaf pow-
der was 5%, the successful parasitism rate decreased signiW-
cantly compared to that observed at concentrations of 0.5%
and 1%, but there were no obvious diVerences compared
with the control (F D 1.95, df D 22, P D 0.145). The abnor-
mal cocoon rate increased signiWcantly when host larvae
were treated with higher concentrations of 3 and 5%
(F D 7.36, df D 22, P < 0.001). The emergence rate was also
signiWcantly lower under diets with 3 and 5% concentra-
tions of Bt cotton leaf powder, but the diVerence was not
signiWcant with 0.5 and 1% concentrations when compared
to control (F D 2.35, df D 22, P D 0.093) (Table 3).

Table 1
Bioassay of H. armigera on diet containing non-Bt (CK) and Bt (DP 99B) cotton leaf powder
Treatment (%) Growth equationa Larval period (days)b Pupal rate (%)b Pupal weight (mg)b
0.0267t
CK-0.5 W D 0.3269 e 15.2 § 0.4 a 71.7 § 7.0 ab 241.0 § 10.9 a
CK-1 W D 0.3264 e0.0264t 14.9 § 0.4 a 70.0 § 5.8 ab 224.4 § 7.3 ab
CK-3 W D 0.2426 e0.0284t 15.1 § 0.4 a 70.0 § 7.3 ab 231.7 § 6.2 abc
CK-5 W D 0.2827 e0.0272t 15.3 § 0.4 a 76.7 § 4.2 a 220.5 § 6.5 abc
Bt-0.5 W D 0.1925 e0.0243t 17.7 § 0.4 b 68.3 § 6.0 ab 212.7 § 7.2 bc
Bt-1 W D 0.1531 e0.0229t 19.9 § 0.5 c 66.7 § 7.6 ab 202.0 § 7.5 cd
Bt-3 W D 0.1047 e0.0247t 22.0 § 0.4 d 51.7 § 7.9 bc 185.4 § 9.6 d
Bt-5 W D 0.1899 e 0.0181t 25.4 § 0.5 e 41.7 § 5.4 c 185.1 § 12.7 d
a
W, larval weight (mg); t, time (day).
b
Means (§SEM) within the same column followed by diVerent letters are signiWcantly diVerent (P < 0.05, Duncan’s multiple range test).

Table 2
The development of M. mediator in hosts fed on diet containing non-Bt (CK) and Bt (DP99B) cotton leaf powder
Treatment Egg-larval Pupal Male pupal Female pupal Adult Male adult Female adult
(%) period (days) weight (mg) period (days) period (days) weight (mg) longevity (days) longevity (days)
CK-5 7.61 § 0.05 (75) a 4.1 § 0.06 (72) a 4.37 § 0.08 (46) a 5.00 § 0.15 (20) a 1.8 § 0.04 (66) a 11.85 § 0.57 (46) a 17.95 § 1.60 (20) a
Bt-0.5 8.06 § 0.10 (54) b 3.8 § 0.07 (54) b 4.45 § 0.10 (24) a 4.88 § 0.12 (17) a 1.6 § 0.04 (40) b 12.00 § 0.78 (23) ac 15.06 § 1.58 (17) a
Bt-1 7.89 § 0.06 (65) b 3.9 § 0.06 (64) b 4.52 § 0.09 (42) a 5.19 § 0.13 (8) a 1.6 § 0.04 (50) b 10.10 § 0.61 (42) b 20.75 § 2.27 (8) a
Bt-3 8.35 § 0.17 (62) c 3.5 § 0.06 (56) c 4.46 § 0.10 (26) a 5.16 § 0.09 (19) a 1.4 § 0.04 (45) c 10.04 § 0.61 (26) bc 15.00 § 1.96 (19) a
Bt-5 7.92 § 0.07 (59) b 3.7 § 0.05 (53) b 4.54 § 0.09 (35) a 5.13 § 0.13 (8) a 1.6 § 0.03 (43) b 11.83 § 0.61 (35) a 18.88 § 3.18 (8) a
Means (§SEM) within the same column followed by diVerent letters are signiWcantly diVerent (P < 0.05, Duncan’s multiple range test). Numbers in
brackets refer to the total number of insects used.
138 X. Liu et al. / Biological Control 35 (2005) 134–141

Table 3
EVects of diet containing non-Bt (CK) and Bt (DP 99B) cotton leaf powder on M. mediator and host larvae
Treatment (%) No. of parasitized larvae Host larval mortality (%) Parasitism rate (%) Abnormal pupal rate (%) Adult emergence (%)
CK-5 157 14.99 § 1.27 a 60.81 § 2.41 ab 11.35 § 0.77 a 88.15 § 1.93 a
Bt-0.5 101 12.49 § 1.81 a 64.24 § 1.41 a 17.71 § 0.60 ab 77.78 § 1.96 ab
Bt-1 120 15.92 § 1.64 a 64.97 § 6.68 a 13.44 § 4.79 a 75.90 § 6.02 ab
Bt-3 154 29.82 § 3.65 b 57.85 § 3.46 ab 34.95 § 8.41 bc 71.46 § 6.03 b
Bt-5 146 21.14 § 2.49 a 50.34 § 5.40 b 48.26 § 7.95 c 71.44 § 5.04 b
Means (§SEM) within the same column followed by diVerent letters are signiWcantly diVerent (P < 0.05, Duncan’s multiple range test).

3.2. Comparison of the eVects of transgenic cotton expressing

Cry1Ac and Cry1A + CpTI
CK CK + P
DP99B DP99B + P
3.2.1. EVects of transgenic cotton lines and parasitoid on host SGK321 SGK321 + P
larvae A
In most of the treatments, the weight of unparasitized H. 2% cotton leaf powder
armigera larvae was more than those parasitized (Fig. 1).
100
Seven days after treatment, the diVerence was statistically
signiWcant (2%: F D 43.99, df D 69, P < 0.001; 4%: F D 49.11,
df D 79, P < 0.001; 8%: F D 75.06, df D 71, P < 0.001; 12%:
F D 69.70, df D 97, P < 0.001). For example, the weight of 10
parasitized host larvae was less than 40 mg until the devel-
opment of M. mediator larvae was complete, while the
weight of the unparasitized larvae fed on diet containing
transgenic single gene cotton (DP99B) was >100 mg. For B
4% cotton leaf powder
parasitized host larvae, the diVerence of host weight was
signiWcant among three cotton varieties seven days after 100
treatment at 4, 8, and 12% concentrations (4%: F D 7.37,
df D 48, P < 0.001; 8%: F D 18.50, df D 37, P < 0.001; 12%:
Mean weight per larva (mg)

F D 9.17, df D 57, P < 0.001). For unparasitized larvae, the 10

diVerence of larval weight seven days after treatment was
statistically signiWcant in all treated concentrations (2%:
F D 17.30, df D 30, P < 0.001; 4%: F D 14.71, df D 30,
P < 0.001; 8%: F D 46.56, df D 33, P < 0.001; 12%: F D 26.73, C
df D 39, P < 0.001). Higher growth inhibition was observed 8% cotton leaf powder
in the two-gene cotton treatment compared with the single- 100
gene, especially when the leaf powder concentrations were
higher (8 and 12%, Figs. 1C and D).
The mortality of unparasitized H. armigera larvae fed 10
on transgenic cotton diet was not signiWcantly diVerent
from the control for the concentrations of 2 and 4% (2%:
F D 0.16, df D 12, P D 0.853; 4%: F D 0.65, df D 12,
P D 0.543), but it was signiWcantly higher than the control D
in transgenic cotton diet for the concentration of 8% 12% cotton leaf powder
(F D 4.396, df D 12, P D 0.043) and in the two-gene cotton 100
treatment at 12% (F D 5.24, df D 14, P D 0.023) (Table 4).
There was no signiWcantly increased mortality of parasit-
ized host larvae in the treatments of transgenic cotton
10
lines compared with the control at the concentrations of 2
and 4% (2%: F D 0.03, df D 13, P D 0.975; 4%: F D 0.26,
df D 13, P D 0.775). Mortality of parasitized hosts was sig-
0 2 4 6 8
niWcantly lower in transgenic single cotton diet than in
transgenic two-gene cotton diet and control at the concen- Days after treatment
trations of 8 and 12%, but the diVerence was not signiW- Fig. 1. Weight of H. armigera larvae fed on diet containing diVerent con-
cant among three cotton cultivars (8%: F D 3.38, df D 13, centrations (A–D) of non-Bt (CK), Cry1Ac (DP99B) or Cry1A + CpTI
P D 0.072; 12%: F D 3.38, df D 13, P D 0.061). For most (SGK321) cotton leaf powder either unparasitized or parasitized
treatments within groups (same concentration, same vari- (treatment + P) by M. mediator.
 X. Liu et al. / Biological Control 35 (2005) 134–141 139

Table 4
EVects of diet containing non-Bt (CK), Cry1Ac (DP99B) or Cry1A + CpTI (SGK321) cotton leaf powder on M. mediator and host larvae
Concentration of Cotton Egg-larval Cocoon Cocoon Adult Host larval
cotton leaf powder (%) varieties period (d) weight (mg) period (d) weight (mg) mortality (%)
Transgenic cotton Transgenic
plus parasitism cotton alone
2 CK 7.8 § 0.1 (15) a 4.4 § 0.2 (12) a 4.7 § 0.2 (12) a 1.7 § 0.1 (12) ab 37.5 § 6.3 a 13.3 § 8.8 a
SGK321 8.5 § 0.1 (35) b 3.7 § 0.1 (29) b 5.1 § 0.2 (23) a 1.5 § 0.0 (23) a 35.8 § 7.1 a 16.0 § 5.1 a
DP99B 8.6 § 0.2 (15) b 3.8 § 0.1 (14) b 5.1 § 0.3 (7) a 1.7 § 0.1 (7) b 37.5 § 2.5 a 12.0 § 3.7 a**
4 CK 8.0 § 0.2 (12) a 4.1 § 0.1 (11) a 4.8 § 0.2 (10) a 1.8 § 0.1 (9) a 35.8 § 4.8 a 10.0 § 5.8 a*
SGK321 8.3 § 0.1 (36) a 3.8 § 0.1 (32) a 5.1 § 0.2 (20) a 1.5 § 0.1 (20) b 30.8 § 4.9 a 16.0 § 4.0 a*
DP99B 9.0 § 0.2 (19) b 4.1 § 0.1 (16) a 5.0 § 0.3 (12) a 1.6 § 0.1 (12) ab 32.5 § 4.8 a 16.0 § 2.5 a*
8 CK 8.3 § 0.1 (20) a 4.2 § 0.1 (15) a 4.4 § 0.2 (10) a 1.7 § 0.1 (9) a 25.0 § 6.5 ab 13.3 § 3.3 a
SGK321 8.9 § 0.2 (28) b 3.5 § 0.1 (27) b 4.9 § 0.2 (22) a 1.5 § 0.1 (22) a 36.7 § 4.9 b 24.0 § 2.5 b
DP99B 8.9 § 0.2 (19) b 4.0 § 0.1 (19) a 4.7 § 0.2 (17) a 1.5 § 0.1 (17) a 17.5 § 4.8 a 28.0 § 2.4 b
12 CK 8.0 § 0.1 (34) a 4.1 § 0.1 (25) a 4.6 § 0.1 (19) a 1.7 § 0.0 (19) a 25.2 § 2.7 a 8.0 § 5.8 a*
SGK321 8.6 § 0.1 (32) b 3.6 § 0.1 (25) b 4.8 § 0.2 (15) a 1.5 § 0.1 (15) b 23.3 § 6.7 a 28.0 § 3.7 b
DP99B 8.7 § 0.1 (27) b 3.7 § 0.1 (26) b 4.6 § 0.1 (20) a 1.5 § 0.1 (20) ab 7.5 § 4.8 b 20.0 § 3.2 ab*
Means (§SEM) within the same column and same concentration followed by diVerent letters are signiWcantly diVerent among cotton varieties (P < 0.05,
Duncan multiple range test). Means within the same concentration followed by * indicates signiWcant diVerence between parasitized and unparasitized lar-
vae (P < 0.05, independent t test). Numbers in brackets refer to the total number of insects used.

ety), parasitized larval mortality was greater than unpara-
sitized mortality. At the 4% concentration level, the
diVerence was statistically signiWcant by an independent
sample test (t D 0.05 level). Parasitized host mortality was
higher than that of unparasitized larvae at concentrations
of 2 and 4% (t < 0.05), but signiWcantly less than that for
unparasitized larvae at 12% concentration of DP 99B
(t < 0.05) (Table 4).
3.2.2. EVects of transgenic cotton lines on M. mediator
When the host larvae fed on diet containing leaf powder
of transgenic cotton expressing Cry1Ac or Cry1A + CpTI,
M. mediator oVspring had longer egg-larva duration than
the control (2%: F D 6.96, df D 64, P D 0.010; 4%: F D 5.86,
df D 66, P D 0.005; 8%: F D 3.58, df D 66, P D 0.034; 12%:
F D 8.87, df D 92, P < 0.001) (Table 4). There was no signiW-
cant diVerence between the transgenic cotton lines express-
ing Cry1Ac and Cry1A + CpTI based on independent t test
at the concentrations of 2, 8, and 12% (t D 0.05 level). At the
4% level, the egg-larval period in single gene cotton diet was
longer than that in double gene cotton diet treatment
(t < 0.05). For the same cotton variety, there was also no
signiWcant diVerence among concentrations (CK: F D 2.38,
df D 80, P D 0.076; SGK321: F D 2.16, df D 130, P D 0.096;
DP 99B: F D 0.08, df D 79, P D 0.458) (Table 4).
In the treatments of 2, 8, and 12% concentrations of
transgenic cotton leaf powder, the cocoon weight of M.
mediator was signiWcantly lower compared to the control
(2%: F D 8.62, df D 54, P < 0.001; 8%: F D 18.8, df D 60,
P < 0.001; 12%: F D 7.46, df D 75, P < 0.001). However, the
diVerence was not statistically signiWcant at 4% (F D 2.17,
df D 58, P D 0.124). The cocoon weight in 8% two-gene cot-
ton diet treatment was signiWcantly less than that in single
gene cotton diet (t < 0.05). There was no signiWcant diVer-
ence between transgenic two-gene and single gene cotton at
2, 4, and 12% based on t tests at t D 0.05 (Table 4). For the
same cotton variety, there were no signiWcant diVerences
among the various concentrations for any of the measured
parameters. Cocoon period was also not aVected when the
host larvae fed on diet with leaf powder of both transgenic
cotton varieties in most of the treatments. The diVerence
was statistically signiWcant at 2% for adult weight among
cotton cultivars (F D 4.12, df D 41, P D 0.024). Adult weight
in transgenic two-gene cotton was signiWcantly lower than
control at 4 and 12%, but there was no signiWcant diVerence
when compared to transgenic single cotton(4%: F D 2.89,
df D 40, P D 0.068; 12%: F D 2.78, df D 53, P D 0.071). The
diVerence was also no statistically signiWcant at 8% among
three cotton cultivars (F D 2.04, df D 47, P D 0.142).
4. Discussion
Helicoverpa armigera larvae are highly susceptible to
intoxication from Bt transgenic cotton, making it diYcult
to study the eVect of Bt cotton on the development of
parasitoids if using Bt cotton leaf directly because of high
mortality of host larvae fed transgenic cotton and the para-
sitoid larvae not being able to complete their development
in dead hosts. To overcome this problem, we used ground
cotton leaves added to cotton bollworm diets (Greenplate,
1999; Ren et al., 2004), then tested a range of concentra-
tions in a preliminary experiment. Larvae of H. armigera
can survive on diet containing Bt cotton leaf powder in the
tested concentrations, but their larval period was longer,
and pupal rate and pupal weight decreased compared with
non-Bt treatment.
In the present study, adult M. mediator female parasitized
the host larvae fed on Bt-diet in a no-choice situation, and
M. mediator larvae can complete their development in such
hosts. However, immature parasitoids required more time to
develop completely, both pupal and adult weight were
decreased, and abnormal pupal rate increased when the con-
centration of Bt cotton leaf powder was higher in the diet.
Further, when M. mediator developed inside host larvae fed
140 X. Liu et al. / Biological Control 35 (2005) 134–141
Bt-diets, the adult emergence rate decreased. There was no
evident diVerence in the eVects on M. mediator between
transgenic single- and two-gene cotton cultivars. A study
with Bt cotton showed that the parasitoid Cotesia margini-
ventris (Cresson) developed more slowly in the soybean
looper (Pseudoplusia includens Walker) fed Bt cotton than
non-Bt cotton. OVspring of C. marginventris developed inside
P. includens larvae fed a NuCotn 33B diet suVered reduced
longevity and the females had fewer ova (Baur and Boethel,
2003). In a previous study, we used a susceptible laboratory
strain and a strain collected from Bt cotton Welds as hosts to
study the eVect of Bt protein on the development of M. medi-
ator (Liu et al., 2004a). The results showed that larval period
of M. mediator was delayed, and pupal weight, newly
emerged adult weight and adult longevity decreased signiW-
cantly within the range of concentrations tested. Salama et al.
(1991) reported that emergence rate and adult longevity
decreased when the parasitoid Bracon brevicornis fed on host
larvae treated with the diVerent concentrations of Bt.
Ren et al. (2004) reported similar results that transgenic
Cry1A + CpTI cotton suppressed the growth and develop-
ment of host no matter whether it was parasitized or not.
The pupal rate and pupal weight of M. mediator and Cam-
poletis chlorideae Uchida (Hymenoptera: Ichneumonidae)
parasitizing the host fed on transgenic cotton declined
greatly. Duration of egg and larvae stage was prolonged,
pupal and adult weight of C. chlorideae was decreased
when H. armigera fed on transgenic Cry1A + CpTI cotton
leaves compared to those fed on traditional cotton leaves
for 12–48 h (Liu et al., 2005b). In the present study, there
was no evident diVerence in the eVects on M. mediator
between transgenic single and two-gene cotton cultivars.
However, with increasing area of transgenic two-gene culti-
vars in China, further research, for example, host location,
success with oviposition etc., is needed to study eVects on
parasitoids in the future.
Within the limited range of concentrations tested, H.
armigera was capable of surviving on the Bt-diet, but their
development was delayed signiWcantly. The delayed growth
allows more time for M. mediator to parasitize the host,
increasing the parasitism rate because M. mediator prefers
to parasitize low instar host larvae (Wang et al., 1984).
Johnson and Gould (1992) and Johnson (1997) demon-
strated a synergism between transgenic plants and parasit-
oids, and suggested that the synergism may be the result of
increased development time in Heliothis virescens fed Bt-
tobacco. However, there is some evidence suggesting that
parasitoid populations may be lower in transgenic cotton
Welds compared to conventional Welds because of reduced
host density (Cui and Xia, 1999). There is no general con-
clusion about the compatibility between Bt transgenic crop
and parasitoids.
Atwood et al. (1997) reported H. virescens larvae para-
sitized by C. marginiventris were more susceptible to Bt
mixed into diet. Survival of P. includens parasitized by C.
marginventris was reduced about 15% (Baur and Boethel,
2003). In our study, mortality of host larvae exposed to the
combination of Bt-diet and M. mediator parasitism was
higher than that of Bt-diet only in most treatments. How-
ever, we could not conclude that parasitism by M. mediator
increased the susceptibility of H. armigera to the endotoxin
of transgenic cotton because of high mortality of parasit-
ized larvae in the non-Bt treatment. Moreover, mortality of
hosts in the combination of 12% Bt (DP99B) and parasit-
ism was signiWcantly lower than in parasitism alone.
The performance of parasitoids is inXuenced by both
behavioral and physiological factors. Physiological aVects
of transgenic plants on the third trophic level can include
both direct toxic eVects of the heterogeneous protein and
indirect eVects due to changes in host quality (Schuler et al.,
2001). In this study, transgenic cotton had a negative eVect
on the development of M. mediator, with the weight of H.
armigera larvae fed Bt-diets being signiWcantly less than
that of larvae fed the control diet (Fig. 1). Because host size
is known to inXuence the development of parasitoids (Har-
vey et al., 1999; Liu et al., 2004b), we believe the observed
eVects were most probably host-quality mediated rather
than the direct eVects of the Bt cotton.
Chilcutt and Tabashnik (1997) reported that interac-
tions between the parasitoid Cotesia plutellae and Bt
depended on host phenotype of Bt-resistance. In susceptible
hosts of diamondback moth (Plutella xylestella), the para-
sitoid did not aVect the performance of Bt, with the patho-
gen having a signiWcant eVect on the parasitoid. In
moderately resistant hosts, the interaction between Bt and
the parasitoid had a roughly equal negative impact on each
other, with no interaction occurring in highly Bt-resistant
hosts. In this study, a highly susceptible host, H. armigera
larvae, was used. Indeed, some cases of long-term selection
in the laboratory indicate that H. armigera has the poten-
tial to develop resistance to Bt transgenic cotton and Bt
protein (Liang et al., 2000; Meng et al., 2003; Zhao et al.,
1998). Similar studies using Bt-resistant H. armigera larvae
in the future will help to understand interactions of Bt cot-
ton and parasitoids.
Acknowledgments
We are grateful to Yong Zhong and Jie Dong for techni-
cal assistance. We also thank Richard Dawson and Hilda
Collins for helpful comments on an earlier version of the
manuscript. This research was funded by “973” projects
(G2000016209) and the State Key Research Programs of
Ministry of Science and Technology (2001BA507A-11-02)
of PR China.
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