Effects of Transgenic Bt Rice on the Food Consumption, Growth and Survival of Cnaphalocrocis medinalis (Guenee) Larvae

202 Rice Science, 2005, 12(3): 202?206
http://www.ricescience.org


Effects of Transgenic Bt Rice on the Food Consumption, Growth and
Survival of Cnaphalocrocis medinalis (Guenée) Larvae
LI Fang-fang 1, YE Gong-yin 1, CHEN Xue-xin 1, PENG Yu-fa 2
(1 Institute of Applied Entomology and State Key Laboratory of Rice Biology, Zhejiang University, Hangzhou 310029, China;
2Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100094, China)
Abstract: The transgenic rice KMD1, expressing a synthetic Cry1Ab gene from Bacillus thuringiensis, showed effective resistance to
the older (third- to fifth-instar) larvae of the rice leaf-folder (RLF), Cnaphalocrocis medinalis (Guenée) in laboratory bioassay.
Significant declines were revealed in food consumption and growth of the older RLF nymphs fed on the cut-leaves of transgenic
KMD1 plants. The increase rate of food consumption by larvae fed on KMD1 was drastically lower than those on Xiushui 11. Food
consumption was varied with different instars when the larvae fed on the Bt rice. Those of fourth- and fifth-instar larvae were different
compared to the third-instar, lower than those on the non-transgenic rice but still increased a little when the feeding time prolonged. It
is indicated that younger RLF larvae are more sensitive to Bt rice than older ones. Also, about 81%, 78% and 68% of the third-, fourth-
and fifth-instar RLF larvae died within 72 hours bioassay period on KMD1 leaves, respectively. These results demonstrated that
Bt-transgene in KMD1 rice confers substantial protection against infestations with older RLF larvae.
Key words: Caphalocrocis medinalis; cry1Ab gene; transgenic rice; resistance to insects

Bacillus thuringiensis is a bacterium that occurs
cloning and expression of insecticidal Bt-cry genes in
naturally in the soil. It was first discovered in Japan in
crop plants serves as an effective strategy for protection
1902 and has been used for more than 50 years as a
of crops against insect damage. The crystal proteins are
biopesticide [1–3]. The most distinctive feature of this
specifically toxic to lepidopteran insect pests [4–9].
bacterium is protein crystals formed during
The transgenic Bt rice line, KMD1, produced by
sporulation. The crystal proteins are dissolved by the
Agrobacterium-mediated transformation, is an elite
alkaline gut juices in the insect’s midgut lumen, and
line resistant to lepidopteran rice pest species [10].
converted into toxic core fragments by gut proteases.
Shu et al [10] had assessed the resistance of this line to
However, the biopesticide could not be popularized
eight lepidopteran pest species in paddy field,
due to some of the defects such as lack of stability,
including the rice leaf folder, and observed that 100%
mortality of this insect when its newly hatched or
narrow specificity and penetration in the specific plant
third-instar larvae were fed KMD1 leaf tissues in
tissues.
laboratory bioassays whereas only 9.65% of the
Rice is one of the most important staple food
neonates and none of the third-instar larvae died when
crops and is grown extensively in different parts of the
fed the leaf tissues of the non-transgenic control.
world. The rice leaf-folder (RLF), Cnaphalocrocis
The current experiments were undertaken to
medinalis (Guenée) (Lepidoptera: Pyralidae), is one of
evaluate the resistance of the transgenic line KMD1
the most serious destructive pests of rice. Among the
against the older (from the third- to fifth-instar) RLF
alternative tactics for its control, transgenic plants
larvae, including the effect on the food consumption
expressing toxic proteins of Bacillus thuringiensis not
and larvae weight, and the larvae mortality.
only offer an effective method to prevent the crop
losses caused by lepidopteran insect pests, but also
MATERIALS AND METHODS
break the bottlenecks of biopesticides. In short, the
Experimental materials
Received: 4 March 2005; Accepted: 5 July 2005
The transgenic rice KMD1 and the non-transgenic
Corresponding author: CHEN Xue-xin (xxchen@zju.edu.cn)

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LI Fang-fang, et al. Effects of Transgenic Bt Rice on the Food Consumption, Growth and Survival of C. medinalis Larvae 203
parental commercial cultivar Xiushui 11 were employed
leaves to the larvae, also the water losses of the leaves
in the experiment. The transgenic line harbored the
would take into consideration by using leaves only
cry1Ab gene under the control of the maize
without larvae as a control. The relative growth of the
ubiquitin-1 promoter, linked in tandam with gus
larvae was measured before and after the exposure of
(encoding the ?-glucuronidase), hpt (encoding the
the larvae to the Bt rice and the control Xiushui 11.
hygromycin phosphotransferase), and npt (encoding
Also the corrected mortality of the larvae was
the neomycin phosphotransferase) genes [11, 12].
calculated as the difference between the tested
mortality (TM) (the rate death of the larvae fed on Bt
Culturing of the leaf-folder larvae
rice) and control mortality (CM) (the rate death of the
The initial colony of the RLF was obtained from
larvae fed on Xiushui 11) divided by 100 minus the
the experimental farm of Zhejiang University. Adults
control mortality, i.e. [(TM?CM)/(100?CM)].
were maintained in a nylon net cage (50 cm × 50 cm ×
50 cm) for mating and oviposition. Eggs were then
RESULTS
inoculated on the rice leaf, and then kept in the nylon
Effects of transgenic Bt rice on food consumption
net cage (50 cm × 50 cm ×50 cm) at 26±1?, 70–80%
of RLF larvae
relative humidity, and lighting cycle of 16 h light / 8 h
dark until hatching.
The bioassay results revealed that the food
consumption of the RLF larvae on Bt rice at different
Laboratory tests
ages was significantly lower than that on the
The tests were conducted with the transgenic and
non-transgenic rice considered as a control (Table 1).
non-transgenic rice leaves inside a tube (1.5 cm
RLF larvae fed normally on control plants,
diameter, 7.5 cm long).
Xiushui 11, and the accumulated consumption
For each test, the newly emerged young rice
increased as the feeding period prolonged. However,
leaves were freshly excised from the plants and
the increase rate of food consumption by larvae fed on
brought to the laboratory in the labeled containers.
KMD1 was drastically lower than those on Xiushui 11.
Some wet cotton was used inside the tube to keep
Food consumption was varied with different instars
leaves fresh. Each leaf was 4 cm long and was placed
when the larvae fed on the Bt rice. Those of the
separately inside a tube. It was artificially infested
fourth- and fifth-instar larvae were different compared
with one leaf folder larva, which has been starved for
to the third-instar: lower than those on the non-
two hours. Every 30 larvae at the same age was
transgenic rice but still increased a little when the
treated as a group. Each group was repeated three
feeding time prolonged.
times. The Bt rice KMD1 and the control Xiushui 11
were artificially infested with the third- to fifth-instar
Effects of transgenic Bt rice on growth of RLF
larvae. Food consumption and larvae weight were
larvae
measured individually after 24, 72 and 240 h. Larva
mortality was also recorded. The rice leaves without
It is evident that the growth and development of
larvae were also weighed to correct weight change due
the RLF larvae feeding on KMD1 were distinctly
to evaporation.
retarded as compared to those on Xiushui 11 (Table 2).
The relative increase of the body weights of the RLF
Statistical analysis
larvae feeding on KMD1 was negative while those on
The data obtained were subjected to analysis of
Xiushui 11 were positive.
variance (ANOVA) using DPS [13]. Duncan’s Multiple
Effects of transgenic Bt rice on corrected mortality
Range Test was conducted for comparison of means at
of RLF larvae
P?0.05 to evaluate the level of significance. The food
consumption by larvae was calculated as losses of the
The high mortality of RLF larvae was observed
leaves weight before and after being exposed the
when they fed on the leaf tissue of transgenic Bt rice

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204 Rice Science, Vol. 12, No. 3, 2005
Table 1. Effects of KMD1 (Bt rice) and Xiushui 11(check) on food consumption of RLF at different ages.
Instar
Rice cultivar
Time after inoculation (h)
Sample size of the insects
Food consumption ( x ± SE)(mg/larva)
3rd KMD1
24
90
219.5 ± 17.5 cC

72
90
40.1 ± 18.5 eE

240
90
76.6 ± 1.2 dDE
Xiushui
11
24
90
106.8 ± 8.0 dD

72
90
395.3 ± 8.7 bB

240
90
933.7 ± 5.6 aA
4th KMD1
24
90
53.0 ± 25.5 eD

72
90
69.9 ± 40.4 eD

240
90
3.9 ± 2.3 cC
Xiushui
11
24
90
226.2 ± 33.3 dC

72
90
524.1 ± 11.2 bB

240
90
956.7 ± 0.7 aA
5th KMD1
24
90
6.2 ± 0.5 eD

72
90
138.2 ± 18.5 eE

240
90
82.8 ± 5.1 deCD
Xiushui
11
24
90
200.4 ± 39.4 cC

72
90
467.3 ± 69.0 bB

240
90
991.3 ± 1.0 aA
Means within column followed by the same letters are not significantly different based on ANOVA (Duncan’s Multiple Range Test, P?0.05).
Mean food consumption (± SE) was calculated from data of only three groups (each group consists of 30 individuals, the tested larvae didn’t
include the dead because of age).

Table 2. Effects of KMD1 (Bt rice) and Xiushui 11(check) on the relative growth of RLF at different ages.
Instar
Rice cultivar
Time after inoculation (h)
Sample size of the insects
Relative growth ( x ± SE) (mg/h)
3rd KMD1
24
90
-34.4 ± 7.1 dD

72
90
-9.5 ± 2.0 cC

240
90
-5.3 ± 0.3 cC
Xiushui
11
24
90
9.0 ± 0.1 bB

72
90
12.8 ± 0.4 bB

240
90
66.7 ± 0.5 aA
4th KMD1
24
90
-100.3 ± 1.1 fE

72
90
-89.2 ± 5.8 dE

240
90
-73.8 ± 3.2 dD
Xiushui
11
24
90
183.9 ± 3.1 cC

72
90
301.3 ± 0.3 bB

240
90
998.9 ± 0.7 aA
5th KMD1
24
90
-38.5 ± 31.9 bC

72
90
-47.8 ± 30.5 cC

240
90
-6.8 ± 0.5 bC
Xiushui
11
24
90
45.5 ± 6.21 cB

72
90
200.1 ± 0.8 bA

240
90
253.9 ± 4.3 aA
Means within column followed by the same letter are not significantly different based on ANOVA (Duncan’s Multiple Range Test, P ?
0.05). Mean body weight (± SE) was calculated from data of only three groups (each group consists of 30 individuals, the tested larvae didn’t
include the dead because of age).

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LI Fang-fang, et al. Effects of Transgenic Bt Rice on the Food Consumption, Growth and Survival of C. medinalis Larvae 205
as compared to those on the non-transgenic control
(Table 3). About 81, 77 and 68% of RLF larvae of the
Table 3. Effects of Bt rice KMD1 on the corrected mortality of
RLF at different ages.
third-, fourth and fifth-instar died by 72 h after they
fed on the Bt rice as compared to the non-transgenic
Time after
Sample size of
Corrected mortality
Instar
control, respectively. It is also indicated that younger
inoculation (h)
the insects
( x ± SE) (%)
RLF larvae are more sensitive to Bt rice than elder
3rd
24

90
17.78 ± 1.11 cC
ones.

72

90
81.11 ± 1.11 bB

240

90
96.58 ± 0.85 aA
DISCUSSION
4th
24

90
11.90 ± 1.19 cC

72

90
77.78 ± 1.11 bB

240

90
93.33 ± 1.67 aA
Many Asian countries have reported the serious
5th
24

90
17.78 ± 1.11 cC
RLF infestations on rice, including China, India, Japan,

72

90
68.06 ± 0.14 bB
Korea, Malaysia, Sri Lanka and Vietnam. The RLF

240

90
72.22 ± 1.11 aA
larvae damage plants by folding the leaves and
Means within column followed by the same letters are not
scraping the green leaf tissues within the fold, causing
significantly different based on ANOVA (Duncan’s Multiple Range
yield loss by reducing leaf photosynthetic activity.
Test, P?0.05).
Transgenics are an additional tool to supplement
Mean corrected mortality (± SE) was calculated from data of
conventional pest resistance programs. It is important
only three groups (each group consists of 30 individuals).
to integrate transgenic technology into the on-going
KMD1, which was the transgenic line harbored
insect pest management (IPM) programs. Advances in
the cry1Ab gene under the control of the maize
molecular biology have opened new opportunities to
ubiquitin-1 promoter, linked in tandam with gus
introduce transgenes into crop plants from diverse
(encoding the ?-glucuronidase), hpt (encoding the
systems.
hygromycin phosphotransferase), and npt (encoding
Fujimoto et al [14] bred rice variety Nipponbare
the neomycin phosphotransferase) genes. Shu et al [10]
with cry1Ab gene under the control of 35S promoter,
had already showed that KMD1 caused high mortality
revealing the increase of resistance to the striped stem
of the newly hatched or third-instar larvae of eight
borer and RLF [14]. Many laboratories have produced
lepidopteran pest species, including the RLF, the
transgenic rice carrying Bt genes for tolerance to the
yellow stem borer and the striped stem borer, when
stem borers [15–18]. Wünn et al [18] introduced cry1Ab
they were fed on this transgenic rice line. Our results
gene into rice variety IR58 through particle
revealed that KMD1 has also highly toxic to the older
bombardment, and the transgenic plants showed
RLF larvae.
significant insecticidal effect on several lepidopteran
When the older RLF larvae fed on the Bt rice, the
insect pests. Feeding studies showed up to 100%
rate of increases in food consumption of larvae fed on
mortality for the yellow stem borer and the striped
KMD1 was drastically lower than those on XS11.
stem borer. Nayak et al transformed IR64 through
Food consumption was varied with different instars.
particle bombardment using cry1Ac gene placed under
Those of the fourth- and fifth-instar larvae were
the control of the maize ubiquitin-1 promoter [16]. The
different compared to the third-instar: lower than
transferred synthetic cry1Ac gene was stably
those on the non-transgenic rice but still increased a
expressed in the T2 of these lines and the transgenic
little when the feeding time prolonged. It is also
IR64 were highly toxic to larvae of the yellow stem
evident that the growth and development of the RLF
borer, which caused little feeding damage. Ghareyazie
larvae feeding on KMD1 were distinctly retarded as
et al [15] transformed aromatic rice variety “Tarom
compared to those on the non-transgenic rice. The
Molali” carrying a synthetic truncated cry1Ab toxin
high mortality of older RLF larvae was observed when
gene. The transgenic lines showed high levels of
they were fed on the leaf tissues of the transgenic Bt
tolerance to the yellow stem borer. Alam et al [19]
rice, about 81, 78 and 68% of RLF larvae of the third-,
introduced cry1Ab gene driven by 35S promoter
fourth and fifth-instar died by 72 h after they fed on Bt
through biolistic method. The integration of the
rice, respectively, indicating that younger RLF larvae
cry1Ab gene in the transgenic rice (IR68899B)
are more sensitive to Bt rice than older ones. This
showed resistant to the yellow stem borer.

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206 Rice Science, Vol. 12, No. 3, 2005
situation is in accordance with the results as were
proteins of Bacillus thuringiensis Berliner on growth and
observed with the rice stem borers and European corn
survival of noctuid (Lepidoptera) larvae. J Econ Entomol,
borer [14–20].
2001, 94: 752-760.
10
Shu Q Y, Ye G Y, Cui H R, Cheng X, Xiang Y B, Wu D X,
From the results above, it can be concluded that
Gao M, Xia Y W, Hu C, Sardana R, Altosaar I. Transgenic
the development of KMD1 can serve as an effective
rice plants with a synthetic cry1Ab gene from Bacillus
strategy for protection of rice. But long-term impact of
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11
Cheng X, Sardana R, Altossar I. Agrobacterium-transformed
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12
Xiang Y B, Cheng X, Liang Z, Shu Q Y, Ye G Y, Gao M,
Altosaar I. Agrobacterium-mediated transformation of
ACKNOWLEDGEMENT
insecticidal Bacillus thuringiensis cry1Ab and cry1Ac genes
and their expression in rice. Chin J Biotechnol, 1999, 15:
This work is funded by the National Basic
494-500. (in Chinese with English abstract)
13
Tang Q Y, Feng M G. Practical statistics and DPS Data
Research Program (001CB109004) from the Ministry
Processing System. Beijing: China Agricultural Press, 1997.
of Science and Technology of China.
407pp. (in Chinese)
14
Fujimoto H, Itoh K, Yamamoto M, Kyozuka J, Shimamoto K.
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