Temporal allocation of metabolic tolerance in the body of beet armyworm in response to three gossypol-cotton cultivars

Science in China Series C: Life Sciences

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Citation: WU G, Marvin K HARRIS, GUO J Y, et al. Temporal allocation of metabolic tolerance in the body of beet armyworm in response to three gossypol-cottoncultivars. Sci China Ser C-Life Sci, 2009, 52(12): 1140-1147, doi: 10.1007/s11427-009-0157-6

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Temporal allocation of metabolic tolerance in the body of beet armyworm in response to three gossypol-cotton cultivars

WU Gang1,2, Marvin K HARRIS3, GUO JianYing1† & WAN FangHao1† 1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural

Sciences, Beijing 100081, China; 2 College of Plant Science, Huazhong Agricultural University, Wuhan 430070, China; 3 Department of Entomology, Texas A&M University of USA, TX, USA

The nutrient composition and enzyme activities in larvae of the beet armyworm, Spodoptera exigua (Hübner), fed on high, medium or low gossypol cotton cultivars were examined at different time inter-vals. Significantly lower free fatty acid was observed in larvae fed for 6 h on high gossypol ‘M9101’ compared to larvae fed on the low (ZMS13) and intermediate (HZ401) gossypol cultivars. Significantly higher trypsin activity was observed in larvae fed on high gossypol ‘M9101’ for 24 h compared to those fed for 1, 4 and 6 h. Significantly higher catalase and total superoxide dismutase enzyme activities were observed in larvae of S. exigua fed on high gossypol ‘M9101’ compared with low gossypol cultivars ‘ZMS13’ and ‘HZ401’ for 1, 4, 6 and 24 h. However, significantly lower carboxylesterase and acetylcho-linesterase enzyme activities were found in larvae fed on high gossypol ‘M9101’ compared with the other cultivars for 1, 4, 6 and 24 h. The interaction between cotton variety and beet armyworm infesta-tion time significantly affected the carboxylesterase enzyme activity in S. exigua. The characterization of the effects of plant allelochemicals on herbivorous larvae is important for aiding understanding of plant-insect interaction as well as in devising solutions to pest problems by breeding plant resistance, identifying metabolic targets for insecticide development, etc.

Spodoptera exigua, gossypol, free fatty acid, detoxification enzyme, acetylcholinesterase, total superoxide dismutase

Crop losses due to insect pests, estimated at 10%—20% for major crops, are a significant factor in limiting food production[1]. Insecticide application is the primary method of controlling insect herbivores, but high resis-tance to most insecticides and associated environmental problems may jeopardize their continued use[2−4]. Therefore, control programs should rely to the extent possible on alternative tactics that prevent the develop-ment of damaging infestations or reduce the cost of management[5].

Plant secondary substances are useful and desirable tools in most pest management programs because they can be effective and often complement the actions of insecticides and natural enemies[5−9]. Cotton cultivars

with a high gossypol level are anecdotally considered resistant to herbivorous insects and have been adopted by cotton growers solely for the purpose of herbivorous insects’ management[10]. Gossypol, a phenolic sesquiter- penoid aldehyde, is an important allelochemical occur-ring in glanded cotton cultivars. This allelochemical ex-hibits antibiosis to many pests and contributes to the host plant resistance of glanded cotton varieties[11]. Sev- Received July 16, 2008; accepted April 29, 2009 doi: 10.1007/s11427-009-0157-6 †Corresponding author (email: [email protected]; [email protected]) Supported by the National Basic Research Program of China (Grant No. 2006CB102004), the National Natural Science Fund of China (Grant No. 30800724), International Foundation for Sciences (Grant No. C/4559-1) and New Detecting Tech- nology of Exogenous Gene Protein (Grant Nos. 2008ZX08012-005, 2009ZX08011- 013B and 2009ZX08012-005B)

Article

WU G, et al. Sci China Ser C-Life Sci | Dec. 2009 | vol. 52 | no. 12 | 1140-1147 1141

eral researchers have investigated the relationship be-tween the gossypol level and aphid abundance[12,13]. Du et al.[14] and Gao et al.[10] indicated that high gossypol in the cotton plant had an antibiotic effect on Aphis gos-sypii (Glover) and produced a positive effect on growth and development of Propylaea japonica (Thunberg) at the third trophic level. Chen et al.[15,16] reported that wheat plants highly resistant to aphids contain high con-tents of phenolics and tannins.

Beet armyworm, Spodoptera exigua (Hübner), is a polyphagous pest of numerous crops and causes eco-nomic damage in China. Historically, beet armyworm, S. exigua (Hübner) has been managed as part of a “worm” complex of cotton (Gossypium hirsutum) and initially received little attention following adoption of integrated pest management (IPM) for cotton varieties in China. S. exigua was added to the insect pest prediction list in 2001 in China[17,18]. The subsequent failure of chemical measures to control S. exigua has accelerated interest in developing new ways to bolster the IPM program to manage S. exigua. The antagonistic compounds of plants mainly affect insect detoxifying enzymes, and this effect is closely related to the level of secondary metabolites in host plants[19]. Most published documents focus on en-zyme activities in S. exigua in response to insecti-cides[20,21]. To date, a few experiments have been con-ducted to evaluate effects of secondary substances of plants on the enzyme activities of S. exigua, and espe-cially focus on the temporal variation of protection and detoxification enzymes in S. exigua. The temporal varia-tion of efficacy by plant secondary metabolites may lead to insufficient control of herbivorous insects and evolu-tion of resistance or tolerance to secondary substances of plants against S. exigua.

Protection enzymes (e.g. catalase, peroxidase and to-tal superoxide dismutase) and detoxification enzymes (e.g. carboxylesterase and acetylcholinesterase) in beet armyworm larvae fed on three cotton cultivars (low, middle and high gossypol contents, respectively) for different infestation intervals were examined to address the following objectives: (i) To quantify the effects (di-rect and indirect) of secondary substances of cotton plants on the nutrient composition (protein, glucose and total amino acid) and enzyme activities in S. exigua larvae; (ii) to measure these effects after different periods of feeding by S. exigua larvae; (iii) to evaluate the interac-tion between cotton variety and beet armyworm infesta-

tion time (1, 4, 6 and 24 h) on the protection and detoxi-fication enzymes activities of S. exigua.

1 Materials and methods

1.1 Cotton variety and growth condition

Three cotton cultivars used in the study included ‘ZMS13’, ‘HZ401’, and ‘M9101’, with gossypol content of 0.06%, 0.44%, and 1.12%, respectively[10,14]. The three cotton cultivars were planted in white plastic pots (15 cm diameter, 13 cm height) in a thermostatic cham-ber. The temperature was maintained at (28±1)℃ and relative humidity was maintained at 70%—80% in the thermostatic chamber. One hundred and sixty pots for each cotton cultivar were randomly placed in the ther-mostatic chamber and re-randomized once a week to minimize position effects. No chemical fertilizers or insecticides were used during the experiment.

1.2 Beet armyworm stocks

Egg masses of beet armyworm, S. exiguity were ob-tained from the Insect Virology Laboratory, Institute of Zoology, Chinese Academy of Sciences (CAS), and hatched in a growth chamber (PRX-500D-30; Haishu Safe Apparatus, Ningbo, China). The chamber was maintained at (75±5)% RH, (28±0.5)℃, and 14︰10 (L︰D) at 30000 Lx of active radiation supplied by 39, 26 W fluorescent lamps.

1.3 Beet armyworm feeding treatments

The beet armyworm feeding experiment was carried out in the thermostatic chamber maintained at (28±1)℃ and relative humidity at 70%—80%.

The 4th instar larvae of S. exigua were randomly col-lected from cotton that had grown to the seven-leaf stage (about 35—40 d after planting) in the stock growth chamber (control conditions as described above), and placed on cotton grown in the experimental chamber; larval placement was on the fourth leaf from the bottom with one larva per pot. Each pot was covered with net-ting to exclude other insects. There were 10 pots with 4 replications per cotton cultivar for 4 treatments of S. exigua infestation (e.g. infestation time for 1, 4, 6 and 24 h), respectively (total of 40 insects per infestation treat-ment for each cotton cultivar). After S. exigua inocula-tion for 1, 4, 6 and 24 h, beet armyworm larvae were collected and analyzed for nutrient composition, diges-

1142 WU G, et al. Sci China Ser C-Life Sci | Dec. 2009 | vol. 52 | no. 12 | 1140-1147

tive enzymes and detoxification enzymes.

1.4 Beet armyworm nutrient compositions and en-zyme activities assays

Biochemical assays were conducted to test whether there was a biochemically significant change in chemical composition of S. exigua in response to different gossy-pol content among cotton cultivars. Four types of nutri-ent composition (protein, total amino acid, free fatty acid and glucose), 3 types of digestive enzymes (lipase, trypsin, amylase) and 5 protection and detoxification enzymes (carboxylesterase, acetylcholinesterase, total superoxide dismutase, total superoxide dismutase and peroxidase) were used to test changes in chemical com-position in S. exigua. Carboxylesterase activity was de-termined per Van Asperen[22]. Reagent Kit protocols were followed to measure nutrient composition and en-zyme activity (Nanjing Jiancheng Ltd. Co., Nanjing, Jiangsu Province, China). Enzyme activities were pre-sented relative to protein concentration, which was de-termined using the method of Bradford[23] with bovine serum albumin (Nanjing Jiancheng Ltd. Co., Nanjing, Jiangsu Province, China) as the standard.

1.5 Data Analysis

One-way ANOVAs (SAS 6.12, SAS Institute Inc. USA, 1996[24]) were used to analyze the 4 nutrient composi-tions, 3 digestive enzymes and 5 detoxification enzymes in the larvae. Two-way ANOVAs were used to analyze the impacts of cotton cultivars and beet armyworm in-festation time and their interactions on 4 nutrient com-positions, 3 digestive enzymes and 5 detoxification en-zymes in the larvae. Difference between means was compared with the least significant difference (LSD) test.

2 Results

2.1 Changes in nutrient composition of beet army-worm larvae after feeding on three cotton cultivars

(1) Protein and total amino acid. From Table 1, the cotton variety significantly affected the contents of pro-tein (P<0.05) and total amino acid (P<0.01) in beet armyworm larvae. Beet armyworm infestation time sig-nificantly influenced the total amino acid in the larvae (P<0.001). The interaction between cotton variety and S. exigua infestation time has no effect on the protein and total amino acid in the beet armyworm (P>0.05).

Significantly lower protein content was found in the larvae fed on ‘M9101’ compared with ‘ZMS13’ and

Table 1 Effects of cotton variety, beet armyworm infestation time and their interactions on nutrient compositions in larvae of beet armyworm, S. exigua

Measured indexes Varietya) Timeb) Variety × Time

Protein 0.0181 0.3548 0.3153

Total amino acid 0.005 0.0008 0.0999

Free fatty acid 0.0169 0.0483 0.8810

Glucose 0.0310 0.3028 0.0759

a) ZMS13, HZ401 and M9101; b) beet armyworm infestation time for 1, 4, 6 and 24 h. * P<0.05, ** P<0.01 and *** P<0.001.

‘HZ401’ for 24 h (F=6.51, df=2,9, P=0.0178). Signifi-cantly lower total amino acid was observed in the larvae fed on ‘HZ401’ (F=8.15, df =3,12, P=0.0032) for 6 and 24 h than those fed for 1 and 4 h. Significantly higher total amino acid was observed in larvae fed on ‘ZMS13’ compared with ‘M9101’ and ‘HZ401’ for 6 h (F=5.50, df =2,9, P=0.0275). However, significantly lower total amino acid was observed in larvae fed on ‘M9101’ compared with ‘ZMS13’ and ‘HZ401’ for 4 h (F=6.11, df=2,9, P=0.0211) (Table 2).

(2) Free fatty acid and Glucose. Cotton variety sig-nificantly affected the contents of free fatty acid (P<0.05) and glucose (P<0.05) in the larvae of beet armyworm (Table 1). Beet armyworm infestation time significantly influenced the free fatty acid in the larvae of beet army-worm (P<0.001).

Significantly lower free fatty acid was observed in the larvae fed on ‘M9101’ compared with ‘ZMS13’ and ‘HZ401’ for 6 h (F=7.31, df=2,9, P=0.0130). Signifi-cantly lower free fatty acid was found in larvae fed on ‘HZ401’ (F=4.01, df=3,12, P=0.0343) for 24 h com-pared to larvae fed for 1 h, 4 h and 6 h. Significantly lower glucose was observed in larvae fed on ‘M9101’ compared with‘HZ401’ and ‘ZMS13’ for 1 h (F=4.61, df=2,9, P=0.0419) and 6 h (F=7.00, df=2,9, P= 0.0419). Significantly lower glucose was observed in larvae fed on ‘HZ401’ for 4 h compared to those fed for 1 h, 6 h and 24 h (F=4.53, df=3,12, P=0.0241) (Table 2).

2.2 Changes in digestive enzymes of beet armyworm larvae after feeding on three cotton cultivars

Cotton variety significantly affected the lipase (P<0.001) and trypsin (P<0.0001) contents of larvae (Table 3). Beet armyworm infestation time significantly influenced trypsin activity in the larvae (P<0.001).

Significantly lower lipase activity was observed in beet armyworms after feeding on ‘M9101’ compared


with ‘ZMS13’ and ‘HZ401’ for 1 h (F=121.68, df=2,9, P=0.0001), 4 h (F=196.10, df=2,9, P=0.0001), 6 h (F=192.96, df=2,9, P=0.0001) and 24 h (F=287.22, df=2,9, P=0.0001). Significantly lower lipase contents were observed in the larvae fed on ‘M9101’ (F=4.01, df=3,12, P=0.0344) for 24 h compared to those fed for 1 h, 4 h and 6 h (Table 4).

Significantly higher trypsin activity was observed in the larvae after feeding on ‘M9101’ compared with ‘ZMS13’ and ‘HZ401’ for 1 h (F=145.95, df=2,9, P=0.0001), 4 h (F=128.24, df=2,9, P=0.0001), 6 h

(F=114.71, df=2,9, P=0.0001) and 24 h (F=89.90, df=2,9, P=0.0001). Significantly lower trypsin activity was found in larvae fed on ‘M9101’ (F=11.34, df=3,12, P=0.0008) and ‘ZMS13’ (F=3.71, df=3,12, P=0.0426) for 24 h compared to those fed for 1, 4 and 6 h (Table 4).

2.3 Changes in detoxification enzymes of beet armyworm larva after feeding on three cotton culti-vars

Cotton variety significantly affected the carboxylesterase (P<0.001), acetylcholinesterase (P<0.0001), catalase

Table 2 Changes (mean±SD) in nutrient composition in larvae of S. exigua after feeding on three cottons cultivars for different time intervals (1, 4, 6 and 24 h)a)

Cotton variety Infestation time Nutrient compositions

M9101 HZ401 ZMS13

Protein (g/L) 0.83 ± 0.04a, A 0.83 ± 0.04a, B 0.85 ± 0.05a, A

Total amino acid (umol/mL) 51.4 ± 2.5a, A 53.8 ± 2.1a, A 53.6 ± 2.0a, A

Free fatty acid (umol/mL) 508 ± 5a, A 505 ± 22a, A 519 ± 16a, A

1 h after ingestion

Glucose (mg/dl) 4.07 ± 0.10b, A 4.36 ± 0.07a, A 4.29 ± 0.21ab, A

Protein (g/L) 0.84 ± 0.04a, A 0.88 ± 0.01a, A 0.86 ± 0.03a, A

Total amino acid (umol/mL) 48.8 ± 1.7 b, A 51.6 ± 0.6a, A 52.2 ± 1.9a, AB

Free fatty acid (umol/mL) 505 ± 22a, A 516 ± 9a, A 524 ± 33a, A

4 h after ingestion

Glucose (mg/dl) 4.19 ± 0.21a, A 4.07 ± 0.10a, B 4.15 ± 0.14a, A

Protein (g/L) 0.83 ± 0.01a, A 0.87 ± 0.01a, AB 0.85 ± 0.05a, A

Total amino acid (umol/mL) 49.6 ± 1.5b, A 49.0 ± 2.4b, B 53.0 ± 1.4a, A

Free fatty acid (umol/mL) 493 ± 19b, A 513 ± 9a, A 519 ± 8a, A

6 h after ingestion

Glucose (mg/dl) 4.00 ± 0.14b, A 4.31 ± 0.03a, A 4.31 ± 0.18a, A

Protein (g/L) 0.81 ± 0.01b, A 0.85 ± 0.01ab, AB 0.89 ± 0.05a, A

Total amino acid (umol/mL) 49.9 ± 2.5a, A 48.7 ± 0.9a, B 50.1 ± 1.8a, B

Free fatty acid (umol/mL) 492 ± 27a, A 490 ± 15a, B 509 ± 10a, A

24 h after ingestion

Glucose (mg/dl) 4.22 ± 0.16a, A 4.26 ± 0.20a, A 4.24 ± 0.15a, A

a) Means within a row indicated by different lowercase letters are significantly different (LSD test, P<0.05, d.f = 2, 9); means of each nutrient composi-tion across different infestation time within a column indicated by different uppercase letters are significantly different (LSD test, P<0.05, d.f = 3, 12).

Table 3 Effects of cotton variety, beet armyworm infestation time and their interactions on the enzymes found in beet armyworm, S. exigua

Measured indexesa) Varietyb) Timec) Variety × Time

Lipase 0.0001 0.0689 0.9797

Trypsin 0.0001 0.0001 0.2505

Amylase 0.0957 0.6365 0.5206

CarE 0.0001 0.0001 0.0386

AChE 0.0001 0.0005 0.8809

CAT 0.0001 0.0019 0.0302

T-SOD 0.0001 0.0001 0.2436

POD 0.0001 0.0001 0.3312

a) CarE, Carboxylesterase; CAT, Catalase; AchE, Acetylcholinesterase; T-SOD, Total superoxide dismutase; POD, Peroxidase. b) ZMS13, HZ401 and M9101; c) beet armyworm infestation time for 1, 4, 6 and 24 h. P<0.05, ** P<0.01 and *** P<0.001.


(P<0.0001), total superoxide dismutase (P<0.0001) and peroxidase (P<0.001) activities in the larvae of beet armyworm (Table 3). Beet armyworm infestation time significantly influenced carboxylesterase (P<0.001), acetylcholinesterase (P<0.001), catalase (P<0.01), total superoxide dismutase (P<0.0001) and peroxidase (P< 0.0001). The interaction between cotton variety and S. exigua infestation time significantly affected the car-boxylesterase (P<0.05) and catalase (P<0.05) activities in the larvae of beet armyworm.

Significantly lower carboxylesterase activity was ob-served in the larvae fed on ‘M9101’ compared with ‘ZMS13’ and ‘HZ401’ for 1 h (F=307.60, df=2,9, P=0.0001), 4 h (F=412.13, df=2,9, P=0.0001), 6 h (F= 425.16, df=2,9, P=0.0001) and 24 h (F=308.82, df= 2,9, P=0.0001). Significantly higher carboxylesterase activ-ity was observed in the body of beet armyworms fed on ‘HZ401’ (F=9.45, df=3,12, P=0.0017) for 24 h than those for 1, 4 and 6 h (Table 5).

Significantly lower acetylcholinesterase activity was observed in the body of beet armyworms after feeding on ‘M9101’ compared with ‘ZMS13’ and ‘HZ401’ for 1 h (F=127.65, df=2,9, P=0.0001), 4 h (F=137.86, df=2,9, P=0.0001), 6 h (F=254.48, df=2,9, P=0.0001) and 24 h (F=171.42, df=2,9, P=0.0001). Significantly higher ace-tylcholinesterase activity was found in the larvae of S. exigua fed on ‘HZ401’ (F=4.06, df=3,12, P=0.0332) for 24 h than those for 1, 4 and 6 h (Table 5).

Significantly higher catalase activity was observed in the larvae of beet armyworm after feeding on ‘M9101’ compared with ‘ZMS13’ and ‘HZ401’ for 1 h (F=190.44, df=2,9, P=0.0001), 4 h (F=273.88, df=2,9, P=0.0001), 6 h (F=321.62, df=2,9, P=0.0001) and 24 h (F=263.31, df=2,9, P=0.0001). Significantly higher catalase activity was found in the larvae of S. exigua fed on ‘HZ401’ (F=10.35, df=3,12, P=0.0012) for 1 h than those for 4, 6 and 24 h (Table 5).

Significantly higher total superoxide dismutase activ-ity was observed in the larvae of S. exigua fed on ‘M9101’ compared with ‘ZMS13’ and ‘HZ401’ for 1 h (F=264.15, df=2,9, P=0.0001), 4 h (F=140.78, df=2,9, P=0.0001), 6 h (F=326.11, df=2,9, P=0.0001) and 24 h (F=94.92, df=2,9, P=0.0001). Significantly higher total superoxide dismutase activity was observed in the larvae of the beet armyworm fed on ‘M9101’ (F=11.99, df=3,12, P=0.0006), ‘HZ401’ (F=20.12, df=3,12, P= 0.0001) and ‘ZMS13’ (F=19.50, df=3,12, P=0.01) for 24 h than those for 1, 4 and 6 h (Table 5).

Significantly higher peroxidase activity was observed in the larvae of the beet armyworm after feeding on ‘M9101’ compared with ‘ZMS13’ and ‘HZ401’ for 1 h(F=271.97, df=2,9, P=0.0001), 4 h (F=275.84, df=2,9, P=0.0001), 6 h (F=148.31, df=2,9, P=0.0001) and 24 h (F=106.57, df=2,9, P=0.0001). Significantly higher peroxidase activity was found in the larvae of S. exigua

Table 4 Changes (mean ± SD) in digestive enzymes (lipase, trypsin and amylase) in larvae of S. exigua after feeding on three cottons cultivars for differ-ent time intervals (1, 4, 6 and 24 h)a)

Cotton variety Infestation time Digestive enzymes

M9101 HZ401 ZMS13

Lipase (U/gprot) 1434 ± 31c, A 1608 ± 27b, A 1786 ± 45a, A

Trypsin (U/mL) 1764 ± 28c 1497 ± 41b, A 1412 ± 15a, A

1 h after ingestion

Amylase (u/dl) 140 ± 3a, A 138 ± 4a, A 138 ± 6a, A

Lipase (U/gprot) 1419 ± 8c, AB 1589 ± 11b, A 1758 ± 40a, A

Trypsin (U/mL) 1746 ± 14c, A 1513 ± 43b, A 1428 ± 23a, A

4 h after ingestion


Lipase (U/gprot) 1413 ± 14c, AB 1590 ± 19b, A 1758 ± 36a, A

Trypsin (U/mL) 1736 ± 23c, A 1485 ± 38b, A 1404 ± 33a, AB

6 h after ingestion


Lipase (U/gprot) 1402 ± 13c, B 1579 ± 34b, A 1761 ± 4a, A

Trypsin (U/mL) 1658 ± 39c, B 1468 ± 27b, A 1371 ± 24a, B



a) Means within a row indicated by different lowercase letters are significantly different (LSD test, P<0.05, d.f = 2, 9); means of each digestive enzyme across different infestation time within a column indicated by different uppercase letters are significantly different (LSD test, P<0.05, d.f = 3, 12).


fed on ‘M9101’ (F=8.19, df=3,12, P=0.0031), ‘HZ401’ (F=7.80, df=3,12, P=0.0037) and ‘ZMS13’ (F=10.77, df=3,12, P=0.001) for 24 h than those for 1, 4 and 6 h (Table 5).

3 Discussion

Plant resistance is the bedrock of integrated pest man-agement[19,25]. Secondary metabolic compounds of plants form the biochemical basis for plant resistance to insects. Cotton gossypol contents are one of the most important toxic chemicals to herbivorous insects and are considered one of the key allelochemicals to insects. Bottger et al.[12] observed that aphid infestations in-creased in cotton cultivars lacking the gossypol gland. Gao et al.[10] found significantly shorter larval duration and greater adult female weight in P. japonica fed aphids reared on the high gossypol ‘M9101’ compared to those fed aphids reared on the two cultivars with lower gossypol contents. Du et al.[14] reported significantly shorter adult longevity and lower fecundity of A. gossypii when

fed on high gossypol cultivars than on cultivars with lower gossypol content. Correlations showed that nym-phal duration of A. gossypii was positively correlated with nitrogen and gossypol content of cotton, and adult life span and lifetime fecundity were both negatively correlated with gossypol content[10]. These findings show that gossypol content affects the intrinsic rate of increase of the herbivore directly and increases herbi-vore susceptibility to natural enemies. Understanding the metabolic processes involved in the plant/herbivore in-teraction will aid in exploiting this form of resistance in IPM.

In the present study, the free fatty acid showed de-crease in the larvae of beet armyworm after feeding on high gossypol ‘M9101’ compared with the two cultivars with lower gossypol contents for each infestation time. Significantly lower free fatty acid was also observed in the larvae of beet armyworm after feeding on high gossypol cultivar than those feeding on the two lower gos-sypol cultivars for 6 h (P<0.05). However, Gao et al.[10]

Table 5 Changes (mean±SD) in protection and detoxification enzymes in larvae of S. exigua after feeding on three cottons cultivars for different time intervals (1, 4, 6 and 24 h)a)

Cotton variety Infestation time Nutrient compositions

M9101 HZ401 ZMS13

CarE (A600/mgPr/min) 0.42 ± 0.01c, B 0.61 ± 0.02b, BC 0.80 ± 0.02a, B

AChE (U/mgprot) 0.63 ± 0.02c, AB 0.71 ± 0.02b, B 0.88 ± 0.02a, B

CAT (U/gprot) 139 ± 2c, A 117 ± 3b, A 100 ± 3a, A

T-SOD (U/mgprot) 115 ± 3c, B 102 ± 1b, C 100 ± 3a, C

1 h after ingestion

POD 42.8 ± 1.0c, C 34.0 ± 0.9b, C 23.5 ± 1.6a, C

CarE (A600/mgPr/min) 0.45 ± 0.02c, AB 0.60 ± 0.01b, C 0.83 ±0.02a, AB

AChE (U/mgprot) 0.62 ± 0.02c, B 0.72 ± 0.02b, B 0.90 ±0.03a, AB

CAT (U/gprot) 139 ± 4c, A 113 ± 2b, B 99.0 ± 1.4a, A

T-SOD (U/mgprot) 117 ± 1c, B 104 ± 2b, BC 90.8 ± 3.2a, B

4 h after ingestion

POD 43.4 ± 1.2c, BC 34.8 ± 1.0b, BC 26.3 ± 0.9a, B

CarE (A600/mgPr/min) 0.47 ± 0.01c, A 0.63 ± 0.02b, B 0.84 ± 0.01a, A

AChE (U/mgprot) 0.63 ± 0.02c, AB 0.73 ± 0.01b, AB 0.91 ± 0.02a, AB

CAT (U/gprot) 141 ± 1c, A 111 ± 1b, BC 97.7 ± 3.9a, A

T-SOD (U/mgprot) 121 ± 1c, A 106 ± 1b, B 93.4 ± 2.2a, B

6 h after ingestion

POD 45.3 ± 1.4c, AB 35.9 ± 1.4b, AB 26.7 ± 1.8a, B

CarE (A600/mgPr/min) 0.46 ± 0.03c, A 0.67 ± 0.01b, A 0.84 ± 0.02a, A

AChE (U/mgprot) 0.66 ± 0.01c, A 0.76 ± 0.04b, A 0.92 ± 0.02a, A

CAT (U/gprot) 137 ± 2c, A 108 ± 2b, C 96.3 ± 3.1a, A

T-SOD (U/mgprot) 123 ± 3c, A 111 ± 3b, A 98.7 ± 2.4a, A


POD 46.8 ± 1.5c, A 37.5 ± 0.9b, A 30.1 ± 2.2a, A

a) Means within a row indicated by different lowercase letters are significantly different (LSD test, P<0.05, d.f = 2, 9); Means of each digestive enzyme across different infestation time within a column indicated by different uppercase letters are significantly different (LSD test, P<0.05, df = 3, 12).


observed that cotton cultivars with high gossypol ‘M9101’ enhanced free fatty acid content of cotton aphids. The results indicated that beet armyworm, com-pared with cotton aphid (a phloem sap-feeding insect), varied in susceptibility to gossypol (and perhaps other secondary substances) in cotton plants. Leaf-chewing insects (e.g. beet armyworm in this study) and phloem sap-feeding insects (e.g. cotton aphid) displayed differ-ent responses to changes in quantity/quality of plant secondary substances (e.g. gossypol in this study).

Most herbivores are affected by the physiological and nutritional state of their host plants, such as the level of secondary metabolites in their host plants. The insect protection and detoxification enzymes in herbivorous insects are the most important defences against plant secondary metabolites[19]. Acetylcholinesterase and su-peroxide dismutase are two important protection and detoxification enzymes in herbivorous insect larvae. Acetylcholinesterase is a key enzyme that terminates nerve impulses by catalyzing the hydrolysis of the neu-rotransmitter acetylcholine in the nervous system[26]. Superoxide dismutase has an important balance for her-bivorous insects’ oxidation and anti-oxidation effect and protects the cell in the insect larva from negative envi-ronmental conditions. In this study, significantly lower acetylcholinesterase activities were observed in 1 h by 39.99%, 4 h by 45.48%, 6 h by 44.61 % and 24 h by 40.04% of beet armyworms fed on high gossypol ‘M9101’ compared with low gossypol ‘ZMS13’. How-ever, significantly higher total superoxide dismutase was found in 1 h by 34.55%, 4 h by 28.86%, 6 h by 29.08% and 24 h by 24.87% of beet armyworms after feeding on high gossypol ‘M9101’ compared with low gossypol ‘ZMS13’. The results show that total superoxide dismu-tase activity from high gossypol cultivar was higher than that from the low gossypol cultivar. However, acetyl-cholinesterase activity from the high gossypol cultivar

was lower than that from the low gossypol cultivar at each infestation time. The gossypol levels in different cotton varieties significantly correlated with the protec-tion and detoxification enzymes in the larvae of beet armyworm. S. exigua may develop tolerance or resis-tance to gossypol if held under conditions of continuous selection.

Cotton variety significantly affected all digestive en-zymes (e.g. lipase and trypsin in this study), protection enzymes (e.g. catalase, total superoxide dismutase and peroxidase in this study) and detoxification enzymes (e.g. acetylcholinesterase and carboxylesterase in this study) of S. exigua, except for amylase. Gossypol con-tent clearly affects the enzymatic contents of S. exigua. The interaction between cotton variety and beet army-worm infestation time significantly affected the car-boxylesterase and catalase enzyme contents in the larvae of S. exigua (P<0.05). However, the interaction between cotton variety and beet armyworm infestation time did not differentially affect contents of other enzymes in the larvae of beet armyworm (P>0.05). Protection and de-toxification enzyme contents in larvae fed for various times were variously affected by different cotton gossy-pol contents.

Our studies provide a profile to exemplify the direct effects of plant secondary substances on the enzyme activities of S. exigua feeding for various timeintervals. Measuring the temporal allocation of enzyme contents in the larvae of S. exigua in response to gossypol may pro-vide a more meaningful evaluation of metabolic toler-ance of herbivorous insects under continuous selection by plant allelochemicals. Therefore, development and implementation of effective insect tolerance or resis-tance management plans should be viewed as a part of integrated management strategies (Integrated Pest Man-agement, IPM and Integrated Resistance Management, IRM).

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Temporal allocation of metabolic tolerance in the body of beet armyworm in response to three gossypol-cotton cultivars