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The Sublethal Effects of Chlorantraniliprole on Helicoverpa armigera (Lepidoptera: Noctuidae)
Journal of Integrative Agriculture
March 2013
2013, 12(3): 457-466
RESEARCH ARTICLE
The Sublethal Effects of Chlorantraniliprole on Helicoverpa armigera (Lepidoptera: Noctuidae) ZHANG Rui-min1, DONG Jun-feng2, CHEN Jia-hua1, JI Qing-e1 and CUI Jin-jie3 Department of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R.China College of Forestry, Henan University of Science and Technology, Luoyang 471003, P.R.China 3 Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang 455000, P.R.China 1 2
Abstract Sublethal effects of chlorantraniliprole to Helicoverpa armigera (Hübner) larvae were evaluated through exposure of third instar larvae to the insecticide incorporated into an artificial diet. When larvae were fed on the insecticide-containing diet for 7 d, the LC10, LC20, LC40, and LC50 values were 3.790, 7.977, 21.577, and 33.121 g active ingredient L-1, respectively. Chlorantraniliprole at sublethal concentrations significantly reduced the larval body mass, emergence ratio, adult longevity and egg hatching rate in both the parental and offspring generations. The pupation and copulation rate in the parental generation and the pupal mass in the offspring also strongly decreased. Reproduction was seriously disturbed in both the parental and offspring groups even when only one of the partners was exposed to chlorantraniliprole at larval stages. However, at the lowest concentration of exposure (LC 10), offspring fecundity was strongly reduced only when both partners were exposed. The net reproductive rate (R0), intrinsic rate of increase (rm), and gross reproduction rate (GRR) at LC20, LC40 concentrations were significantly lower than that of the control. Post-exposure effects also included an extended larval developmental time and increased male proportion in both generations. The doubling time (Dt) at LC20, LC40 concentrations as well as gross reproduction rate (GRR) at LC 10 concentration were also significantly increased. Chlorantraniliprole might have significant effects on H. armigera population dynamics even at sublethal concentrations on both parental and offspring generations. Key words: Helicoverpa armigera, chlorantraniliprole, sublethal effects, sublethal concentration
INTRODUCTION Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), the cotton bollworm, is a serious pest in Asia, Australia, southern Europe and Africa. It can damage a wide range of agricultural crops, such as cotton, maize, chickpea, pigeonpea, sorghum, sunflower, soybean and groundnuts (Fitt 1989). Due to a long history of chemical control, H. armigera gradually became resistant to many insecticides includ-
ing some new kinds of chemicals such as fipronil, chlorfenapyr, spinosad and indoxacarb, and it is difficult or impossible to control this pest now (Ahmad et al. 2003; Wu 2007). In China, transgenic insecticidal cotton has been used to control this insect pest since 1997 (Gao et al. 2011) and this has been widely successful (Wu and Guo 2005). Although natural refuges play an important role in delaying development of H. armigera resistance (Wu and Guo 2005), evolution of resistance in this pest is still a great threat to the continued efficacy of Bt crops (Alstad and Andow 1995; Gahan et al.
Received 11 June, 2012 Accepted 12 September, 2012 Correspondence CUI Jin-jie, Tel: +86-372-2562217, E-mail: [email protected]
© 2013, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S2095-3119(13)60246-4
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2001). Li et al. (2007) argued a significant increase in the resistance of H. armigera larvae to Cry1Ac in Xiajin County, Henan Province, China, based on laboratory bioassay data. In consequence, the insecticidal activity declined as the cotton plants mature, and some H. armigera are able to complete development on transgenic cotton (Fitt et al. 1994). In addition, since H. armigera is polyphagous and highly mobile (Wu et al. 2008), its density can still be high in some years in China (Gao et al. 2010). Therefore, pesticides are still being used to control this pest. This situation, combined with the resistance problem, necessitates the search for new insecticides. Chlorantraniliprole (5% SC, RynaxypyrTM) is a novel insecticide developed by DuPont Agricultural Chemicals Ltd., Shanghai, China. It belongs to the anthranilic diamides class acting on the insect ryanodine receptors (Lahm et al. 2005). Once ingested, it can lead to an unregulated release of internal calcium stores from the sarcoplasmatic reticulum, the affected insect stops feeding, develops lethargy, muscle paralysis, and ultimately dies (Lahm et al. 2007). RynaxypyrTM shows an exceptional activity on a broad range of Lepidoptera (Lahm et al. 2007) as well as selected other species, but safe to mammals because of its poor intrinsic activity on mammalian ryanodine receptors (Clark 2008). Under field conditions, some target pests receive lethal does and perish, while others are exposed to sublethal doses causing sublethal effects (Singh and Marwaha 2000), and these multiple sublethal effects should be considered as well when testing the total effects of an insecticide. The sublethal effects include latent toxicity, decreased reproductive potential and longevity, changes in behavior and enzyme induction (Moriarty 1969; Lee 2000). Currently, there is no report on the sublethal effects of chlorantraniliprole on H. armigera. In this paper, the sublethal effects of larval exposure were studied on two generations, parent and offspring of H. armigera, including larval, prepual and pupal development, adult emergence, longevity, fecundity, and egg hatch rate. The larval and pupal mass and the copulation behaviour
were also evaluated. Finally, population growth parameters such as the net reproductive rate (R0), intrinsic rate of increase (rm), gross reproduction rate (GRR) and doubling time (Dt) were established.
RESULTS Determination of the sublethal concentrations The susceptibility of the third instars to chlorantraniliprole analyzed by incorporated diet assay, resulted in LC10, LC20, LC40, and LC50 values of 3.790, 7.978, 21.577, and 33.121 g active ingredient L-1, respectively (Table 1). Those values were used as sublethal concentrations during the subsequent experiments. After exposure of each sublethal concentration the total mortalities were 7, 16, 32.33%, and 13, 19, 49%, respectively, on d 7 and 8 (Fig. 1). This demonstrated that the sublethal concentration used for this study was reasonable.
Effects of chlorantraniliprole on survival, larval body mass Mortality increased when sublethal concentrations increased and the mortality of LC40 increased faster compared to other groups (Fig. 1). Larval body mass in the parent generation decreased significantly with increasing sublethal concentrations (F3, 8=578.722, P<0.001), but the offspring generation showed a different trend, with body mass at LC20 being the lowest (F3, 8=7.784, P=0.009) (Fig. 2).
Effects of chlorantraniliprole on life history parameters The larval development duration was significantly prolonged with respect to control and there were also significant differences between sublethal concentrations in the parent groups (F3, 8=92.93, P<0.0001). However,
Table 1 Probit analysis for the concentration-mortality response of chlorantraniliprole on third instars of H. armigera Insecticide Chlorantraniliprole
n 540
df
LC10 (g L-1)
LC20 (g L-1)
LC40 (g L-1)
LC50 (g L-1)
Slope±SE
c2
P value
16
3.790 (2.217-5.496 )
7.977 (5.501-10.433)
21.577 (17.499-26.110)
33.121 (27.374-40.767)
1.362±0.139
14.703
0.546
30 individuals per replicates per concentration, seven concentrations were used including the control.
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The Sublethal Effects of Chlorantraniliprole on Helicoverpa armigera (Lepidoptera: Noctuidae)
Fig. 1 Survival ratio of Helicoverpa armigera larvae after exposure to various concentrations of chlorantraniliprole. Values are the means and standard deviations of three separate experiments. The same as below.
Fig. 2 The parent and offspring weight after 4 d of exposure to various concentrations of chlorantraniliprole. Means marked with different letters are significantly different (P<0.05), comparison are within parent or offspring (parent: F3, 8=578.722, P<0.0001; offspring: F3, 8=7.784, P=0.009)
there were no significant differences in larval development in the offspring except at LC10 (F3, 8=19.59, P<0.0001) (Table 2). No significant difference was found in the duration of the prepupa stage in either the parental or the offspring generations except the LC40 in the offspring groups (parent: F3, 8=3.299, P=0.079; offspring: F3, 8=10.142, P=0.004). Pupal development was longer in all treatments. However, only the pupal period of LC20 in
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parent was significantly different from that of the control. In contrast to this, the pupal period was significantly shorter in the offspring in the LC10 and LC20 treatments compared to control and LC40 (parent: F3, 8=4.778, P=0.04; offspring: F3, 8=13.23, P=0.002). Male longevity was significantly shortened in all treatments of both generations, with respect to the control, but there was no significant difference among the treated groups (parent: F3, 76=25.71, P<0.0001; offspring: F3, 76=12.679, P=0.002). Female longevity also decreased after ingesting the insecticide-containing diet, especially in the offspring. Female longevity was significantly shortened in the parent treatments of LC10 and LC20 and only in the offspring treatments of LC20 (parent: F3, 76=6.212, P=0.017; offspring: F3, 76=6.587, P=0.015) (Table 2). Egg hatching rates were reduced in parent treatments; LC20 and LC40 showed significant difference (F3, 36=17.477, P<0.0001). A similar situation was found in the offspring treatments even when the offsprings were not exposed to chlorantraniliprole at all (F 3, 36 =8.736, P<0.0001) (Table 2). There was no significant difference in pupal mass except the male of LC10 in the parent treatments (F3, 196= 1.92, P=0.204; female: F=0.625, P=0.604; male: F=6.033, P=0.019). In the offspring, the treatments showed no significant difference within each other but all had significant difference with respect to the control (F3, 196=10.43, P=0.004; female: F=16.556, P=0.001; male: F=10.439, P=0.004). All parent treatments had lower pupation percentage compared to the control but there was no significant difference between LC10 and LC20. There was no significant difference in pupation success (F3, 8= 2.587, P=0.126) (Table 3). Further, at the lowest concentration, the adult emergence rate in the parental generation was significantly higher than in the control or other sublethal concentrations (F3, 8=14.889, P=0.001). Adult emergence rates in the offspring of LC20 and LC40 were much lower than that of control or LC10 (F3, 8=13.3, P=0.002) (Table 3). Sex ratio of the parent generation showed more males than females in LC40 than that of the control or LC10 (F3, 8=36.484, P<0.0001). There was a significant male reduction in LC20 offspring group compared with the control or other treatments (F3, 8=10.438, P=0.004) (Table 3).
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Table 2 Larval, prepupal and pupal periods, adult longevity and egg hatch percent of the parent and offspring of H. armigera treated with sublethal doses of chlorantraniliprole and control Development time (d) Parent Control LC10 LC20 LC40 P F df Offspring Control LC10 LC20 LC40 P F df
Longevity (d) Pupa
Male
Egg hatch (%)
Larva
Prepupa
Female
7.90±0.37 d 12.70±0.23 c 15.68±0.59 b 20.19±1.71 a <0.0001 92.93 3, 8
2.47±0.04 a 1.99±0.25 a 2.54±0.26 a 2.33±0.29 a 0.079 3.299 3, 8
11.42±0.17 b 11.56±0.40 ab 12.40±0.27 a 11.84±0.46 ab 0.04 4.778 3, 8
15.53±1.22 a 9.14±0.75 b 8.62±0.96 b 9.56±1.39 b <0.0001 25.71 3, 76
12.60±2.40 a 7.47±0.99 b 7.62±1.06 b 8.70±1.81 ab 0.017 6.212 3, 76
98.04±1.53 a 97.25±2.13 a 92.84±4.46 b 89.20±4.16 b <0.0001 17.477 3, 36
12.00±0.42 b 14.06±0.31 a 12.30±0.58 b 11.67±0.40 b <0.0001 19.59 3, 8
2.57±0.38 a 2.59±0.19 a 2.38±0.09 a 2.00±0.18 b 0.004 10.142 3, 8
11.45±0.19 a 10.89±0.07b c 10.57±0.12 c 11.22±0.29 ab 0.002 13.23 3, 8
15.76±1.17 a 9.11±1.35 b 9.50±2.50 b 9.53±0.50 b 0.002 12.679 3, 76
12.83±2.45 a 8.44±1.64 ab 6.67±1.53 b 8.65±1.18 ab 0.015 6.587 3, 76
98.20±1.00 a 94.73±2.89a b 93.10±5.23 bc 89.91±4.07 c <0.0001 8.736 3, 36
Means marked with different letters within the same column are significantly different (Tukey test; P<0.05), values are the means and standard deviations of three repeats. The larval development time of the parent generation was calculated from the day they were exposed to different doses of chlorantraniliprole. The same as below.
Table 3 Comparison of pupal weight, percentage of pupation, adult emergence, and female percentage of parent and offspring of H. armigera Pupal weight (g) Parent Control LC10 LC20 LC40 P F df Offspring Control LC10 LC20 LC40 P F df
Female pupal weight (g)
Male pupal weight (g)
pupation rate (%)
Adult emergence rate (%)
Female percentage (%)
0.35±0.03 a 0.30±0.01 a 0.31±0.01 a 0.32±0.05 a 0.204 1.92 3, 196
0.35±0.03 a 0.30±0.01 a 0.31±0.01 a 0.32±0.09 a 0.604 0.652 3, 196
0.35±0.03 a 0.30±0.01 b 0.31±0.01 a 0.32±0.01 a 0.019 6.033 3, 196
84.03±2.23 a 68.67±0.92 b 52.92±8.78 b 11.83±4.42 c <0.0001 110.962 3, 8
56.67 ±5.86 b 79.11±5.40 a 47.27±11.23 b 40.43±6.03 b 0.001 14.889 3, 8
53.34 ±1.02 a 51.26 ±1.43 a 47.73 ±5.94 ab 31.00 ±2.00 b <0.0001 36.484 3, 8
0.37±0.04 a 0.28±0.01 b 0.30±0.01 b 0.30±0.01 b 0.004 10.43 3, 196
0.36±0.03 a 0.27±0.01 b 0.29±0.01 b 0.30±0.01 b 0.001 16.556 3, 196
0.35±0.02 a 0.29±0.01 b 0.31±0.02 b 0.31±0.01 b 0.004 10.439 3, 196
85.35±10.01 a 71.32±9.37 a 80.21±5.95 a 78.84±5.85 a 0.126 2.587 3, 8
65.27±3.01 a 72.63±8.96 a 32.77±2.90 b 45.67±10.92 b 0.002 13.3 3, 8
51.10±1.45 a 44.93±5.76 a 43.37±2.23 b 46.67±2..62 a 0.004 10.438 3, 8
Effects of chlorantraniliprole on reproduction and copulation behaviour Reproduction in all the sublethal concentrations and the untreated group in the parental generation were similar, when both sexes were exposed to the treatments (F3, 76=1.248, P=0.355). However, in the offspring generation, the number of eggs per female in the LC10 treatment was significantly reduced (F3, 76=4.148, P=0.048) (Table 4). When only the females were exposed, the reproduction of the parent group of LC20 decreased significantly with respect to that of the control (F 3, 36 =6.431,
P=0.016). Surprisingly, there was a marked reduction in the reproduction of the lowest concentration in the offspring groups when compared with control and LC40 (F3, 36=5.159, P=0.034) (Table 4). When only males were exposed, the sublethal concentration of chlorantraniliprole had no significant effects on the reproduction of the parent generation (F3, 36=13.338, P=0.002), with the single exception of LC40. In the offspring groups, only LC20 had lower number of eggs than the control (F3, 36=7.463, P=0.01) (Table 4). Chlorantraniliprole could also affect the adult copulation: when both adults in the parent were exposed to insecticide at high concentration (LC40), the
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The Sublethal Effects of Chlorantraniliprole on Helicoverpa armigera (Lepidoptera: Noctuidae)
copulation percentage declined significantly (F3, 36=5.313, P=0.026, df=3, 76). When the treated adults of one sex were paired with the control of the other, no such effect was observed (Female only: F3, 36=0.628, P=0.617; Male only: F3, 36=0.471, P=0.711). However, in the offspring treatment, the adult copulation was significantly disturbed when only females were treated in LC20 (Table 5).
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DISCUSSION Chlorantraniliprole is effective to control both Cry1Acsusceptible and resistant populations of H. armigera (Cao et al. 2010). It is also among the fastest-acting insecticides (Hannig et al. 2009). Although it is a new kind of insecticide, it has been demonstrated that field beet armyworm populations collected from different geographic areas have different resistance levels due to the different insecticide use histories (Lai et al. 2011). Chlorantraniliprole has been proved to be hightly effective against pest Lepidoptera (Knight and Flexner 2007; Sial and Brunner 2010), and Lai et al. (2011b) and Han et al. (2012) also reported its sublethal effects on Spodoptera exigua and Plutella xylostella, respectively. The current study denonstrated that chlorantraniliprole was effective against H. armigera larvae. The LC50 value of chlorantraniliprole after 7 d of exposure in third instar larvae of H. armigera was 31.5 g active ingredient L-1. In the present study, the multiple sublethal effects of chlorantraniliprole were revealed on both of the parent and offspring generations of H. armigera. In general, the effects of chlorantraniliprole on biological parameters were greater in the treatment of LC20, and the impacts on the offspring were much lower than on the parent generation.
Sublethal effects on population growth parameters The population growth parameters of H. armigera were strongly affected by chlorantraniliprole (Table 6). The net reproduction rate (R0), intrinsic rate of increase (rm), and gross reproduction rate (GRR) at LC20, LC40 concentrations were significantly lower compared to the control. However, the gross reproduction rate (GRR) at LC10 significantly increased. In addition, the net reproductive rate, intrinsic rate of increase in LC40 was significantly lower than LC20 (R0: F3, 76=137.386, P<0.0001; rm: F3, 76=91.500, P<0.0001; GRR: F3, 76=33.054, P<0.0001). The doubling time (Dt) at LC20, LC40 concentrations were significantly higher than that of control and LC10. Similarly to R0 and rm, doubling time in LC40 concentration was significantly higher than in LC20 (F3, 76=1 020.180, P<0.0001).
Table 4 Number of eggs laid by per female H. armigera of parent and offspring among various mating type Treatment LC 10 LC 20 LC 40 Control P F df
Parent
Offspring
Female only
Male only
Both
1 093.50±52.12 a 590.17±116.53 b 939.07±219.93 ab 1 096.67±202.80 a 0.016 6.431 3, 36
1 263.17±102.53 a 960.67±44.97 a 618.00±117.69 b 1 096.67±202.80 a 0.002 13.338 3, 36
1 126.00±288.71 874.10±106.13 929.83±106.42 1 096.67±202.80 0.355 1.248 3, 76
Female only a a a a
428.50±131.43 909.00±702.12 1 284.00±396.26 1 177.67±207.19 0.034 5.159 3, 36
Male only b ab a a
780.22±138.38 554.33±165.17 809.06±104.44 1 177.67±207.19 0.01 7.463 3, 36
Both ab b ab a
607.33±169.63 b 854.50±293.24 ab 912.47±29.26ab 1 177.67±207.19 a 0.048 4.148 3, 76
Table 5 Copulation percentage of parent and offspring of H. armigera among various cross-combination Treatment LC 10 LC 20 LC 40 Control P F df
Parent Female only
Male only
83.01±14.12 a 74.67±24.52 a 58.01±38.22 a 80.06±8.42 a 0.617 0.628 3, 36
67.35±8.04 a 67.71±23.92 a 81.03±17.26 a 79.85±7.93 a 0.711 0.471 3, 36
Offspring Both 80.01±7.43 a 68.42±17.16 ab 50.42±7.03 b 80.42±8.32 a 0.026 5.313 3, 76
Female only
Male only
75.00±25.02 a 59.62±4.99 a 67.38±34.06 a 81.13±6.05 a 0.699 0.49 3, 36
67.43±14.01 a 32.84±13.79 b 75.00±25.01 a 81.42±6.03 a 0.029 5.099 3, 36
Both 56.31±10.18 a 88.01±11.03 a 71.13±17.42 a 81.01±6.39 a 0.395 1.124 3, 76
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Table 6 Comparison of biological parameters of H. armigera treated with chlorantraniliprole and control Gross reproduction rate (GRR) Control LC 10 LC 20 LC 40 P F df
600.10±20.84 b 787.17±62.09 a 423.09±41.83 c 469.23±55.57 c <0.0001 33.054 3, 8
Net reproduction rate (R0 ) 290.12±25.13 a 334.20±36.81 a 72.41±10.72 b 17.66±2.57c <0.0001 137.386 3, 8
The larval body mass of the parent generation treated with sublethal concentrations of chlorantraniliprole showed a significant decline as well as the first offspring generation. H. zea larvae stopped feeding after exposure to chlorantraniliprole for 20.3 min (Hannig et al. 2009). After treatment with tebufenozide, Chrysodeixis chalcites larvae suffered gut alterations, and stopped feeding, and finally led to weight loss (Smagghe et al. 1997). It is speculated that long time starvation may be the reason for the larval mass decline. Starvation in the larval stage might cause damage to the intestinal absorption system, leading to a reduced mass of offspring larvae. As the insects grow, the residues of insecticide can be diluted, and this could be responsible for the reduction effect on the offspring of treated parents. We noticed that the larval body mass of LC20 declined most sharply among the first offspring generation. This might because the high concentration (LC 40 ) of chlorantraniliprole killed most of the susceptible larvae and the resistant ones survived. However, in the treatment of LC20, some susceptible individuals still survived even though their metabolic systerm got damaged. After exposure to sublethal concentrations of chlorantraniliprole for 72 h, S. exigua neonate larvae showed some sublethal effects including the increase of larval mortality, extension of larval development, reduction of pupation success, and improvement of males ratio (Lai ans Sun 2011). Those phenomena were all found in the current experiments on the parent generation of H. armigera. However, Lai and Sun (2011) did not report the sublethal effects on the offspring generation. The present study found that there was a small decrease in the fecundity when both parents were treated with the sublethal dose of chlorantraniliprole (LC20 and LC40) in the parent generation, however, it declined significantly at LC10 in the offspring of both paired couples originated from treated groups. The similar phenom-
Intrinsic rate of increase (rm)
Doubling time (Dt)
0.14±0.01 a 0.15±0.01 a 0.10±0.01 b 0.07±0.02 c <0.0001 91.5 3, 8
4.68±0.17 c 4.50±0.13 c 6.84±0.07 b 10.40±0.20 a <0.0001 1 020.18 3, 8
enon was observed in the experiment of Adamski et al. (2009) using a different insecticide on beet armyworm. The ovulation of S. exigua females is interrupted after feeding with tebufenozide (Smagghe and Degheele 2004) and even low concentration can cause potent effects (Adamski et al. 2009; Han et al. 2012). This leads us to speculate that the large offspring effect on the low concentration might because the high concentration of chlorantraniliprole killed most of the susceptible larvae and only the resistant ones survived. The low concentration treatment did not exert such a “selection pressure”, however, it did cause some underlying damage. This damage could be serious in the embryo, causing delay in the development and the reduction on the reproduction of the offspring. Previous studies showed chlorantraniliprole at the sublethal concentration had no significant effect on the pupal period in the offspring of Plutella xylostella (Han et al. 2012), but in this study the pupa duration was extended in LC20 both of the parent and offspring generations of H. armigera with the same insecticide. Our data also showed that the pupal mass did not increase as Lai ans Sun (2011) reported. In contrast, it decreased slightly in the parent treatment groups and the offspring showed a significant decline at all concentrations. Han et al. (2012) also argued that the pupal mass of P. xylostella was sharply reduced in the parent generation at sublethal concentrations of chlorantraniliprole but similar results were not found in the offspring. We found that the emergence rate of H. armigera could be enhanced in the parent at the LC10 concentration. Most of the insecticides could inhibit emergence and reduce adult longevity (Wang et al. 2009; Wei et al. 2010; Mahmoudvand et al. 2011a). However, the mechanism of adult emergence underlying this dramatic change is unknown. Those differences might be due to different insect species, different exposure methods
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The Sublethal Effects of Chlorantraniliprole on Helicoverpa armigera (Lepidoptera: Noctuidae)
and the concentrations used (Han et al. 2012). Our results also indicated male but not female longevity significantly shortened. Those results were also different from Lai et al. (2011b) and Han et al. (2012). However, they agreed with Knight et al. (2006), who indicated that adult males were more sensitive than females to the same insecticide. The present results also showed that high concentration of chlorantraniliprole could disturb copulation of H. armigera. Wei et al. (2004) reported that after exposure to insecticide, males were less likely to locate a sex pheromone source, chemical communication between the sexes could be interrupted (Yang et al. 2003), and success mating significantly decreased (Wei et al. 2004). Kerns and Stewart (2000) observed that bifenthrin at sublethal concentrations increased the net reproductive rate of aphids. Dong et al. (2011) reported that emamectin benzoate significantly decreased the net reproductive rate and intrinsic rate of increase of H. armigera at sublethal doses. In this study, the net reproductive rate (R 0), intrinsic rate of increase (rm) and the gross reproduction rate (GRR) were significantly decreased except LC10 compared with control, when both of the mated moths were exposed to chlorantraniliprole, due to the reduction in survival, the shortness of life span and the decline in fecundity. Insecticides at sublethal doses enhance fecundity and reproduction parameters of insects in insecticide hormoligosis. Sublethal doses of fanvalerate (LC12.5, LC25) increased the fecundity and intrinsic rate of increase in P. xylostella (Sota et al. 1998). Similar to Mahmoudvand et al. (2011b), our work indicated that chlorantraniliprole at LC10 significantly increased the gross reproduction rate in H. armigera. However, whether hormoligosis is involved or not need further clarification. In conclusion, the present results verified chlorantraniliprole had a lethal and sublethal effect on larval stages of H. armigera. Also this compound had sublethal effects on the biotic performance of both parent and offspring of H. armigera, such as reduction in emergence ratio and reproduction. In all, we found that chlorantraniliprole had both direct and indirect effects on H. armigera, but the effects on the offspring were much smaller than those on the parent. It follows that an overall effect of chlorantraniliprole on the pest
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should not only be estimated from the exposed generation but also from their offspring. Because of the new mode of action and high effectiveness on Lepidoptera, chlorantraniliprole might be used as an important insecticide in the field, and the sublethal effects should also be considered while building pest control strategies. Further work could clarify if such sublethal effects also occur on subsequent generations, because this could contribute to the eventual development of resistance.
MATERIALS AND METHODS Insect rearing and insecticide treatments The susceptible strain of H. armigera was obtained from a laboratory colony maintained at the Institute of Plant Protection, Nanjing Agricultural University, China. All larvae were reared on an artificial diet (Wu and Gong 1997), and adults were fed on a 15% solution of honey and water. The colony was maintained under laboratory conditions of (27±1)°C, (60±5)% RH and 16 h L:8 h D. The insecticide tested was commercially available chlorantraniliprole (5% SC, RynaxypyrTM, DuPont Agricultural Chemicals Ltd., Shanghai, China).
Experimental design Bioassay and determination of sublethal concentrations Toxicity of chlorantraniliprole to H. armigera was determined by using incorporated diet with newly molted third instar larvae (within 1 h). Six concentrations of chlorantraniliprole were used (7.813, 10.417, 15.625, 31.250, 62.500 and 125.000 g active ingredient L-1). 1 mL of the desired concentrations of chlorantraniliprole in a water solution were mixed with 282 g diet to yield the insecticide-treated diet (the total distilled water in the artificial diet was 200 mL), while the temperature of the preparing diet was under 40°C. Then the insecticide-treated diet was agitated for 30-40 s to distribute insecticide evenly, and then poured into 300 mL Petri dishes. Control diet contained 1 mL distilled water instead of the chlorantraniliprole. Diet prepared before the bioassay and was kept at 4°C for no longer than 48 h before being used or discarded. A small portion of the diet (approximately 1 cm3) was placed in a clean glass tube (10.0 cm length×1.8 cm diameter), into which one third newly molted larva was transferred. About 30 tubes were prepared for each concentration, in three replicates; thus the total number of larvae of each concentration was 90. All the tubes were closed with cotton pads and kept under laboratory condition of (27±1)°C, (60±5)% RH and 16 h L:8 h D. Larval
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mortality was evaluated on d 7 and 8. Larvae not responding with head movements or peristaltic contractions when touched with a fine brush were recorded as dead; moribund larvae were scored as alive (Loriatti et al. 2009). According to the bioassay results, LC10, LC20 and LC40 concentrations of chlorantraniliprole were selected as sublethal dosages for the subsequent experiments. Sublethal treatments and effects on the parent generation Newly molted third instar larvae obtained from the stock culture were separated into groups of 100. For the LC40 treatment, 13 groups were included (1 300 larvae); there were 7 groups (700 larvae) and 3 groups (300 larvae) for LC20 and LC10 treatments, respectively, to allow for the differential mortality, and still obtain sufficient number of alive adults. These groups were fed with the insecticide incorporated diet of each concentration for 7 d as described. After that, all of the survived larvae were kept on diet free of the pesticide. On d 4, 50 larvae selected randomly were weighed (0.001 g precision) from each treatment and the survived larvae from each group were transferred to fresh, insecticide-free artificial diet until pupation on d 7 and 8. Pupae were individually placed into small plastic cups (15 cm in diameter, 20 cm in depth) to emerge. After emergence, individuals from the same day were paired randomly to form pairs with both or only one of the sexes sublethally treated. A piece of absorbent cotton soaked in a 10% honey solution, changed daily, was provided for adults. Ten pairs were used in each group except the “both sexes exposed” group (20 pairs). The females were dissected as soon as they died to check the number of matings which can be detected by the number of trichoqyne receptive hypha. Mortality per day, pupation time, fresh body mass on d 4, pupal mass on d 3, the sex of adults, as well as the ratio of pupation and adult emergence were recorded. The numbers of eggs laid by each pair were counted daily until females died; if the male died first, the survived female was given a new mate of the same treatment. A total of 150 eggs (repeated 3 times) were taken randomly from each pair of adult moths, and the egg hatching rate was determined. Male and female adult longevities were recorded, as well as the mating time were checked. Pairs that failed to mate or lay eggs were discarded.
Sublethal effects on the offspring F1 generation To e v a l u a t e t h e p o s s i b l e c a r r y - o v e r a c t i v i t y o f chlorantraniliprole to their offspring, the F1 generation eggs laid by adults with both partners exposed to the same concentration were collected to hatch. 300 larvae that hatched from each treatment were maintained on untreated artificial diet until pupation. However, the F1 generation was not exposed to the insecticide directly. The diet was changed on the 4th d of the third instar, after that it was changed when necessary. The same data were recorded as on the parent generation. The adults were paired as the same way
as their parents were. Here, one of the paired moths treated, means one of parents of the offspring were exposed to sublethal concentration of chlorantraniliprole.
Data analysis Data obtained from the experiments were statistically analyzed using one-way analysis of variance (ANOVA). Means were compared by Tukey’s studentized range test (equal variance), if the variance was unequal after transformation, Games-Howell corrections were applied (Adamski et al. 2009), both accepting significant differences at P<0.05. All percentage data were arcsine transformed before being subjected to ANOVA. SPSS ver. 17.0 software was used for all analyses.
Acknowledgements The authors sincerely thank the Institute of Plant Protection, Nanjing Agricultural University, China, for providing the susceptible strain of H. armigera, and and Prof. Lovei G L from Department of Agroecology, Aarhus University, Denmark, for comments on the manuscript. This study was supported by the National 863 Program of China (2012AA101502) and the Special Fund for Agro-Scientific Research in the Public Interest, China (200903033).
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2013, 12(3): 457-466
RESEARCH ARTICLE
The Sublethal Effects of Chlorantraniliprole on Helicoverpa armigera (Lepidoptera: Noctuidae) ZHANG Rui-min1, DONG Jun-feng2, CHEN Jia-hua1, JI Qing-e1 and CUI Jin-jie3 Department of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R.China College of Forestry, Henan University of Science and Technology, Luoyang 471003, P.R.China 3 Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang 455000, P.R.China 1 2
Abstract Sublethal effects of chlorantraniliprole to Helicoverpa armigera (Hübner) larvae were evaluated through exposure of third instar larvae to the insecticide incorporated into an artificial diet. When larvae were fed on the insecticide-containing diet for 7 d, the LC10, LC20, LC40, and LC50 values were 3.790, 7.977, 21.577, and 33.121 g active ingredient L-1, respectively. Chlorantraniliprole at sublethal concentrations significantly reduced the larval body mass, emergence ratio, adult longevity and egg hatching rate in both the parental and offspring generations. The pupation and copulation rate in the parental generation and the pupal mass in the offspring also strongly decreased. Reproduction was seriously disturbed in both the parental and offspring groups even when only one of the partners was exposed to chlorantraniliprole at larval stages. However, at the lowest concentration of exposure (LC 10), offspring fecundity was strongly reduced only when both partners were exposed. The net reproductive rate (R0), intrinsic rate of increase (rm), and gross reproduction rate (GRR) at LC20, LC40 concentrations were significantly lower than that of the control. Post-exposure effects also included an extended larval developmental time and increased male proportion in both generations. The doubling time (Dt) at LC20, LC40 concentrations as well as gross reproduction rate (GRR) at LC 10 concentration were also significantly increased. Chlorantraniliprole might have significant effects on H. armigera population dynamics even at sublethal concentrations on both parental and offspring generations. Key words: Helicoverpa armigera, chlorantraniliprole, sublethal effects, sublethal concentration
INTRODUCTION Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), the cotton bollworm, is a serious pest in Asia, Australia, southern Europe and Africa. It can damage a wide range of agricultural crops, such as cotton, maize, chickpea, pigeonpea, sorghum, sunflower, soybean and groundnuts (Fitt 1989). Due to a long history of chemical control, H. armigera gradually became resistant to many insecticides includ-
ing some new kinds of chemicals such as fipronil, chlorfenapyr, spinosad and indoxacarb, and it is difficult or impossible to control this pest now (Ahmad et al. 2003; Wu 2007). In China, transgenic insecticidal cotton has been used to control this insect pest since 1997 (Gao et al. 2011) and this has been widely successful (Wu and Guo 2005). Although natural refuges play an important role in delaying development of H. armigera resistance (Wu and Guo 2005), evolution of resistance in this pest is still a great threat to the continued efficacy of Bt crops (Alstad and Andow 1995; Gahan et al.
Received 11 June, 2012 Accepted 12 September, 2012 Correspondence CUI Jin-jie, Tel: +86-372-2562217, E-mail: [email protected]
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2001). Li et al. (2007) argued a significant increase in the resistance of H. armigera larvae to Cry1Ac in Xiajin County, Henan Province, China, based on laboratory bioassay data. In consequence, the insecticidal activity declined as the cotton plants mature, and some H. armigera are able to complete development on transgenic cotton (Fitt et al. 1994). In addition, since H. armigera is polyphagous and highly mobile (Wu et al. 2008), its density can still be high in some years in China (Gao et al. 2010). Therefore, pesticides are still being used to control this pest. This situation, combined with the resistance problem, necessitates the search for new insecticides. Chlorantraniliprole (5% SC, RynaxypyrTM) is a novel insecticide developed by DuPont Agricultural Chemicals Ltd., Shanghai, China. It belongs to the anthranilic diamides class acting on the insect ryanodine receptors (Lahm et al. 2005). Once ingested, it can lead to an unregulated release of internal calcium stores from the sarcoplasmatic reticulum, the affected insect stops feeding, develops lethargy, muscle paralysis, and ultimately dies (Lahm et al. 2007). RynaxypyrTM shows an exceptional activity on a broad range of Lepidoptera (Lahm et al. 2007) as well as selected other species, but safe to mammals because of its poor intrinsic activity on mammalian ryanodine receptors (Clark 2008). Under field conditions, some target pests receive lethal does and perish, while others are exposed to sublethal doses causing sublethal effects (Singh and Marwaha 2000), and these multiple sublethal effects should be considered as well when testing the total effects of an insecticide. The sublethal effects include latent toxicity, decreased reproductive potential and longevity, changes in behavior and enzyme induction (Moriarty 1969; Lee 2000). Currently, there is no report on the sublethal effects of chlorantraniliprole on H. armigera. In this paper, the sublethal effects of larval exposure were studied on two generations, parent and offspring of H. armigera, including larval, prepual and pupal development, adult emergence, longevity, fecundity, and egg hatch rate. The larval and pupal mass and the copulation behaviour
were also evaluated. Finally, population growth parameters such as the net reproductive rate (R0), intrinsic rate of increase (rm), gross reproduction rate (GRR) and doubling time (Dt) were established.
RESULTS Determination of the sublethal concentrations The susceptibility of the third instars to chlorantraniliprole analyzed by incorporated diet assay, resulted in LC10, LC20, LC40, and LC50 values of 3.790, 7.978, 21.577, and 33.121 g active ingredient L-1, respectively (Table 1). Those values were used as sublethal concentrations during the subsequent experiments. After exposure of each sublethal concentration the total mortalities were 7, 16, 32.33%, and 13, 19, 49%, respectively, on d 7 and 8 (Fig. 1). This demonstrated that the sublethal concentration used for this study was reasonable.
Effects of chlorantraniliprole on survival, larval body mass Mortality increased when sublethal concentrations increased and the mortality of LC40 increased faster compared to other groups (Fig. 1). Larval body mass in the parent generation decreased significantly with increasing sublethal concentrations (F3, 8=578.722, P<0.001), but the offspring generation showed a different trend, with body mass at LC20 being the lowest (F3, 8=7.784, P=0.009) (Fig. 2).
Effects of chlorantraniliprole on life history parameters The larval development duration was significantly prolonged with respect to control and there were also significant differences between sublethal concentrations in the parent groups (F3, 8=92.93, P<0.0001). However,
Table 1 Probit analysis for the concentration-mortality response of chlorantraniliprole on third instars of H. armigera Insecticide Chlorantraniliprole
n 540
df
LC10 (g L-1)
LC20 (g L-1)
LC40 (g L-1)
LC50 (g L-1)
Slope±SE
c2
P value
16
3.790 (2.217-5.496 )
7.977 (5.501-10.433)
21.577 (17.499-26.110)
33.121 (27.374-40.767)
1.362±0.139
14.703
0.546
30 individuals per replicates per concentration, seven concentrations were used including the control.
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The Sublethal Effects of Chlorantraniliprole on Helicoverpa armigera (Lepidoptera: Noctuidae)
Fig. 1 Survival ratio of Helicoverpa armigera larvae after exposure to various concentrations of chlorantraniliprole. Values are the means and standard deviations of three separate experiments. The same as below.
Fig. 2 The parent and offspring weight after 4 d of exposure to various concentrations of chlorantraniliprole. Means marked with different letters are significantly different (P<0.05), comparison are within parent or offspring (parent: F3, 8=578.722, P<0.0001; offspring: F3, 8=7.784, P=0.009)
there were no significant differences in larval development in the offspring except at LC10 (F3, 8=19.59, P<0.0001) (Table 2). No significant difference was found in the duration of the prepupa stage in either the parental or the offspring generations except the LC40 in the offspring groups (parent: F3, 8=3.299, P=0.079; offspring: F3, 8=10.142, P=0.004). Pupal development was longer in all treatments. However, only the pupal period of LC20 in
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parent was significantly different from that of the control. In contrast to this, the pupal period was significantly shorter in the offspring in the LC10 and LC20 treatments compared to control and LC40 (parent: F3, 8=4.778, P=0.04; offspring: F3, 8=13.23, P=0.002). Male longevity was significantly shortened in all treatments of both generations, with respect to the control, but there was no significant difference among the treated groups (parent: F3, 76=25.71, P<0.0001; offspring: F3, 76=12.679, P=0.002). Female longevity also decreased after ingesting the insecticide-containing diet, especially in the offspring. Female longevity was significantly shortened in the parent treatments of LC10 and LC20 and only in the offspring treatments of LC20 (parent: F3, 76=6.212, P=0.017; offspring: F3, 76=6.587, P=0.015) (Table 2). Egg hatching rates were reduced in parent treatments; LC20 and LC40 showed significant difference (F3, 36=17.477, P<0.0001). A similar situation was found in the offspring treatments even when the offsprings were not exposed to chlorantraniliprole at all (F 3, 36 =8.736, P<0.0001) (Table 2). There was no significant difference in pupal mass except the male of LC10 in the parent treatments (F3, 196= 1.92, P=0.204; female: F=0.625, P=0.604; male: F=6.033, P=0.019). In the offspring, the treatments showed no significant difference within each other but all had significant difference with respect to the control (F3, 196=10.43, P=0.004; female: F=16.556, P=0.001; male: F=10.439, P=0.004). All parent treatments had lower pupation percentage compared to the control but there was no significant difference between LC10 and LC20. There was no significant difference in pupation success (F3, 8= 2.587, P=0.126) (Table 3). Further, at the lowest concentration, the adult emergence rate in the parental generation was significantly higher than in the control or other sublethal concentrations (F3, 8=14.889, P=0.001). Adult emergence rates in the offspring of LC20 and LC40 were much lower than that of control or LC10 (F3, 8=13.3, P=0.002) (Table 3). Sex ratio of the parent generation showed more males than females in LC40 than that of the control or LC10 (F3, 8=36.484, P<0.0001). There was a significant male reduction in LC20 offspring group compared with the control or other treatments (F3, 8=10.438, P=0.004) (Table 3).
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Table 2 Larval, prepupal and pupal periods, adult longevity and egg hatch percent of the parent and offspring of H. armigera treated with sublethal doses of chlorantraniliprole and control Development time (d) Parent Control LC10 LC20 LC40 P F df Offspring Control LC10 LC20 LC40 P F df
Longevity (d) Pupa
Male
Egg hatch (%)
Larva
Prepupa
Female
7.90±0.37 d 12.70±0.23 c 15.68±0.59 b 20.19±1.71 a <0.0001 92.93 3, 8
2.47±0.04 a 1.99±0.25 a 2.54±0.26 a 2.33±0.29 a 0.079 3.299 3, 8
11.42±0.17 b 11.56±0.40 ab 12.40±0.27 a 11.84±0.46 ab 0.04 4.778 3, 8
15.53±1.22 a 9.14±0.75 b 8.62±0.96 b 9.56±1.39 b <0.0001 25.71 3, 76
12.60±2.40 a 7.47±0.99 b 7.62±1.06 b 8.70±1.81 ab 0.017 6.212 3, 76
98.04±1.53 a 97.25±2.13 a 92.84±4.46 b 89.20±4.16 b <0.0001 17.477 3, 36
12.00±0.42 b 14.06±0.31 a 12.30±0.58 b 11.67±0.40 b <0.0001 19.59 3, 8
2.57±0.38 a 2.59±0.19 a 2.38±0.09 a 2.00±0.18 b 0.004 10.142 3, 8
11.45±0.19 a 10.89±0.07b c 10.57±0.12 c 11.22±0.29 ab 0.002 13.23 3, 8
15.76±1.17 a 9.11±1.35 b 9.50±2.50 b 9.53±0.50 b 0.002 12.679 3, 76
12.83±2.45 a 8.44±1.64 ab 6.67±1.53 b 8.65±1.18 ab 0.015 6.587 3, 76
98.20±1.00 a 94.73±2.89a b 93.10±5.23 bc 89.91±4.07 c <0.0001 8.736 3, 36
Means marked with different letters within the same column are significantly different (Tukey test; P<0.05), values are the means and standard deviations of three repeats. The larval development time of the parent generation was calculated from the day they were exposed to different doses of chlorantraniliprole. The same as below.
Table 3 Comparison of pupal weight, percentage of pupation, adult emergence, and female percentage of parent and offspring of H. armigera Pupal weight (g) Parent Control LC10 LC20 LC40 P F df Offspring Control LC10 LC20 LC40 P F df
Female pupal weight (g)
Male pupal weight (g)
pupation rate (%)
Adult emergence rate (%)
Female percentage (%)
0.35±0.03 a 0.30±0.01 a 0.31±0.01 a 0.32±0.05 a 0.204 1.92 3, 196
0.35±0.03 a 0.30±0.01 a 0.31±0.01 a 0.32±0.09 a 0.604 0.652 3, 196
0.35±0.03 a 0.30±0.01 b 0.31±0.01 a 0.32±0.01 a 0.019 6.033 3, 196
84.03±2.23 a 68.67±0.92 b 52.92±8.78 b 11.83±4.42 c <0.0001 110.962 3, 8
56.67 ±5.86 b 79.11±5.40 a 47.27±11.23 b 40.43±6.03 b 0.001 14.889 3, 8
53.34 ±1.02 a 51.26 ±1.43 a 47.73 ±5.94 ab 31.00 ±2.00 b <0.0001 36.484 3, 8
0.37±0.04 a 0.28±0.01 b 0.30±0.01 b 0.30±0.01 b 0.004 10.43 3, 196
0.36±0.03 a 0.27±0.01 b 0.29±0.01 b 0.30±0.01 b 0.001 16.556 3, 196
0.35±0.02 a 0.29±0.01 b 0.31±0.02 b 0.31±0.01 b 0.004 10.439 3, 196
85.35±10.01 a 71.32±9.37 a 80.21±5.95 a 78.84±5.85 a 0.126 2.587 3, 8
65.27±3.01 a 72.63±8.96 a 32.77±2.90 b 45.67±10.92 b 0.002 13.3 3, 8
51.10±1.45 a 44.93±5.76 a 43.37±2.23 b 46.67±2..62 a 0.004 10.438 3, 8
Effects of chlorantraniliprole on reproduction and copulation behaviour Reproduction in all the sublethal concentrations and the untreated group in the parental generation were similar, when both sexes were exposed to the treatments (F3, 76=1.248, P=0.355). However, in the offspring generation, the number of eggs per female in the LC10 treatment was significantly reduced (F3, 76=4.148, P=0.048) (Table 4). When only the females were exposed, the reproduction of the parent group of LC20 decreased significantly with respect to that of the control (F 3, 36 =6.431,
P=0.016). Surprisingly, there was a marked reduction in the reproduction of the lowest concentration in the offspring groups when compared with control and LC40 (F3, 36=5.159, P=0.034) (Table 4). When only males were exposed, the sublethal concentration of chlorantraniliprole had no significant effects on the reproduction of the parent generation (F3, 36=13.338, P=0.002), with the single exception of LC40. In the offspring groups, only LC20 had lower number of eggs than the control (F3, 36=7.463, P=0.01) (Table 4). Chlorantraniliprole could also affect the adult copulation: when both adults in the parent were exposed to insecticide at high concentration (LC40), the
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The Sublethal Effects of Chlorantraniliprole on Helicoverpa armigera (Lepidoptera: Noctuidae)
copulation percentage declined significantly (F3, 36=5.313, P=0.026, df=3, 76). When the treated adults of one sex were paired with the control of the other, no such effect was observed (Female only: F3, 36=0.628, P=0.617; Male only: F3, 36=0.471, P=0.711). However, in the offspring treatment, the adult copulation was significantly disturbed when only females were treated in LC20 (Table 5).
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DISCUSSION Chlorantraniliprole is effective to control both Cry1Acsusceptible and resistant populations of H. armigera (Cao et al. 2010). It is also among the fastest-acting insecticides (Hannig et al. 2009). Although it is a new kind of insecticide, it has been demonstrated that field beet armyworm populations collected from different geographic areas have different resistance levels due to the different insecticide use histories (Lai et al. 2011). Chlorantraniliprole has been proved to be hightly effective against pest Lepidoptera (Knight and Flexner 2007; Sial and Brunner 2010), and Lai et al. (2011b) and Han et al. (2012) also reported its sublethal effects on Spodoptera exigua and Plutella xylostella, respectively. The current study denonstrated that chlorantraniliprole was effective against H. armigera larvae. The LC50 value of chlorantraniliprole after 7 d of exposure in third instar larvae of H. armigera was 31.5 g active ingredient L-1. In the present study, the multiple sublethal effects of chlorantraniliprole were revealed on both of the parent and offspring generations of H. armigera. In general, the effects of chlorantraniliprole on biological parameters were greater in the treatment of LC20, and the impacts on the offspring were much lower than on the parent generation.
Sublethal effects on population growth parameters The population growth parameters of H. armigera were strongly affected by chlorantraniliprole (Table 6). The net reproduction rate (R0), intrinsic rate of increase (rm), and gross reproduction rate (GRR) at LC20, LC40 concentrations were significantly lower compared to the control. However, the gross reproduction rate (GRR) at LC10 significantly increased. In addition, the net reproductive rate, intrinsic rate of increase in LC40 was significantly lower than LC20 (R0: F3, 76=137.386, P<0.0001; rm: F3, 76=91.500, P<0.0001; GRR: F3, 76=33.054, P<0.0001). The doubling time (Dt) at LC20, LC40 concentrations were significantly higher than that of control and LC10. Similarly to R0 and rm, doubling time in LC40 concentration was significantly higher than in LC20 (F3, 76=1 020.180, P<0.0001).
Table 4 Number of eggs laid by per female H. armigera of parent and offspring among various mating type Treatment LC 10 LC 20 LC 40 Control P F df
Parent
Offspring
Female only
Male only
Both
1 093.50±52.12 a 590.17±116.53 b 939.07±219.93 ab 1 096.67±202.80 a 0.016 6.431 3, 36
1 263.17±102.53 a 960.67±44.97 a 618.00±117.69 b 1 096.67±202.80 a 0.002 13.338 3, 36
1 126.00±288.71 874.10±106.13 929.83±106.42 1 096.67±202.80 0.355 1.248 3, 76
Female only a a a a
428.50±131.43 909.00±702.12 1 284.00±396.26 1 177.67±207.19 0.034 5.159 3, 36
Male only b ab a a
780.22±138.38 554.33±165.17 809.06±104.44 1 177.67±207.19 0.01 7.463 3, 36
Both ab b ab a
607.33±169.63 b 854.50±293.24 ab 912.47±29.26ab 1 177.67±207.19 a 0.048 4.148 3, 76
Table 5 Copulation percentage of parent and offspring of H. armigera among various cross-combination Treatment LC 10 LC 20 LC 40 Control P F df
Parent Female only
Male only
83.01±14.12 a 74.67±24.52 a 58.01±38.22 a 80.06±8.42 a 0.617 0.628 3, 36
67.35±8.04 a 67.71±23.92 a 81.03±17.26 a 79.85±7.93 a 0.711 0.471 3, 36
Offspring Both 80.01±7.43 a 68.42±17.16 ab 50.42±7.03 b 80.42±8.32 a 0.026 5.313 3, 76
Female only
Male only
75.00±25.02 a 59.62±4.99 a 67.38±34.06 a 81.13±6.05 a 0.699 0.49 3, 36
67.43±14.01 a 32.84±13.79 b 75.00±25.01 a 81.42±6.03 a 0.029 5.099 3, 36
Both 56.31±10.18 a 88.01±11.03 a 71.13±17.42 a 81.01±6.39 a 0.395 1.124 3, 76
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Table 6 Comparison of biological parameters of H. armigera treated with chlorantraniliprole and control Gross reproduction rate (GRR) Control LC 10 LC 20 LC 40 P F df
600.10±20.84 b 787.17±62.09 a 423.09±41.83 c 469.23±55.57 c <0.0001 33.054 3, 8
Net reproduction rate (R0 ) 290.12±25.13 a 334.20±36.81 a 72.41±10.72 b 17.66±2.57c <0.0001 137.386 3, 8
The larval body mass of the parent generation treated with sublethal concentrations of chlorantraniliprole showed a significant decline as well as the first offspring generation. H. zea larvae stopped feeding after exposure to chlorantraniliprole for 20.3 min (Hannig et al. 2009). After treatment with tebufenozide, Chrysodeixis chalcites larvae suffered gut alterations, and stopped feeding, and finally led to weight loss (Smagghe et al. 1997). It is speculated that long time starvation may be the reason for the larval mass decline. Starvation in the larval stage might cause damage to the intestinal absorption system, leading to a reduced mass of offspring larvae. As the insects grow, the residues of insecticide can be diluted, and this could be responsible for the reduction effect on the offspring of treated parents. We noticed that the larval body mass of LC20 declined most sharply among the first offspring generation. This might because the high concentration (LC 40 ) of chlorantraniliprole killed most of the susceptible larvae and the resistant ones survived. However, in the treatment of LC20, some susceptible individuals still survived even though their metabolic systerm got damaged. After exposure to sublethal concentrations of chlorantraniliprole for 72 h, S. exigua neonate larvae showed some sublethal effects including the increase of larval mortality, extension of larval development, reduction of pupation success, and improvement of males ratio (Lai ans Sun 2011). Those phenomena were all found in the current experiments on the parent generation of H. armigera. However, Lai and Sun (2011) did not report the sublethal effects on the offspring generation. The present study found that there was a small decrease in the fecundity when both parents were treated with the sublethal dose of chlorantraniliprole (LC20 and LC40) in the parent generation, however, it declined significantly at LC10 in the offspring of both paired couples originated from treated groups. The similar phenom-
Intrinsic rate of increase (rm)
Doubling time (Dt)
0.14±0.01 a 0.15±0.01 a 0.10±0.01 b 0.07±0.02 c <0.0001 91.5 3, 8
4.68±0.17 c 4.50±0.13 c 6.84±0.07 b 10.40±0.20 a <0.0001 1 020.18 3, 8
enon was observed in the experiment of Adamski et al. (2009) using a different insecticide on beet armyworm. The ovulation of S. exigua females is interrupted after feeding with tebufenozide (Smagghe and Degheele 2004) and even low concentration can cause potent effects (Adamski et al. 2009; Han et al. 2012). This leads us to speculate that the large offspring effect on the low concentration might because the high concentration of chlorantraniliprole killed most of the susceptible larvae and only the resistant ones survived. The low concentration treatment did not exert such a “selection pressure”, however, it did cause some underlying damage. This damage could be serious in the embryo, causing delay in the development and the reduction on the reproduction of the offspring. Previous studies showed chlorantraniliprole at the sublethal concentration had no significant effect on the pupal period in the offspring of Plutella xylostella (Han et al. 2012), but in this study the pupa duration was extended in LC20 both of the parent and offspring generations of H. armigera with the same insecticide. Our data also showed that the pupal mass did not increase as Lai ans Sun (2011) reported. In contrast, it decreased slightly in the parent treatment groups and the offspring showed a significant decline at all concentrations. Han et al. (2012) also argued that the pupal mass of P. xylostella was sharply reduced in the parent generation at sublethal concentrations of chlorantraniliprole but similar results were not found in the offspring. We found that the emergence rate of H. armigera could be enhanced in the parent at the LC10 concentration. Most of the insecticides could inhibit emergence and reduce adult longevity (Wang et al. 2009; Wei et al. 2010; Mahmoudvand et al. 2011a). However, the mechanism of adult emergence underlying this dramatic change is unknown. Those differences might be due to different insect species, different exposure methods
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The Sublethal Effects of Chlorantraniliprole on Helicoverpa armigera (Lepidoptera: Noctuidae)
and the concentrations used (Han et al. 2012). Our results also indicated male but not female longevity significantly shortened. Those results were also different from Lai et al. (2011b) and Han et al. (2012). However, they agreed with Knight et al. (2006), who indicated that adult males were more sensitive than females to the same insecticide. The present results also showed that high concentration of chlorantraniliprole could disturb copulation of H. armigera. Wei et al. (2004) reported that after exposure to insecticide, males were less likely to locate a sex pheromone source, chemical communication between the sexes could be interrupted (Yang et al. 2003), and success mating significantly decreased (Wei et al. 2004). Kerns and Stewart (2000) observed that bifenthrin at sublethal concentrations increased the net reproductive rate of aphids. Dong et al. (2011) reported that emamectin benzoate significantly decreased the net reproductive rate and intrinsic rate of increase of H. armigera at sublethal doses. In this study, the net reproductive rate (R 0), intrinsic rate of increase (rm) and the gross reproduction rate (GRR) were significantly decreased except LC10 compared with control, when both of the mated moths were exposed to chlorantraniliprole, due to the reduction in survival, the shortness of life span and the decline in fecundity. Insecticides at sublethal doses enhance fecundity and reproduction parameters of insects in insecticide hormoligosis. Sublethal doses of fanvalerate (LC12.5, LC25) increased the fecundity and intrinsic rate of increase in P. xylostella (Sota et al. 1998). Similar to Mahmoudvand et al. (2011b), our work indicated that chlorantraniliprole at LC10 significantly increased the gross reproduction rate in H. armigera. However, whether hormoligosis is involved or not need further clarification. In conclusion, the present results verified chlorantraniliprole had a lethal and sublethal effect on larval stages of H. armigera. Also this compound had sublethal effects on the biotic performance of both parent and offspring of H. armigera, such as reduction in emergence ratio and reproduction. In all, we found that chlorantraniliprole had both direct and indirect effects on H. armigera, but the effects on the offspring were much smaller than those on the parent. It follows that an overall effect of chlorantraniliprole on the pest
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should not only be estimated from the exposed generation but also from their offspring. Because of the new mode of action and high effectiveness on Lepidoptera, chlorantraniliprole might be used as an important insecticide in the field, and the sublethal effects should also be considered while building pest control strategies. Further work could clarify if such sublethal effects also occur on subsequent generations, because this could contribute to the eventual development of resistance.
MATERIALS AND METHODS Insect rearing and insecticide treatments The susceptible strain of H. armigera was obtained from a laboratory colony maintained at the Institute of Plant Protection, Nanjing Agricultural University, China. All larvae were reared on an artificial diet (Wu and Gong 1997), and adults were fed on a 15% solution of honey and water. The colony was maintained under laboratory conditions of (27±1)°C, (60±5)% RH and 16 h L:8 h D. The insecticide tested was commercially available chlorantraniliprole (5% SC, RynaxypyrTM, DuPont Agricultural Chemicals Ltd., Shanghai, China).
Experimental design Bioassay and determination of sublethal concentrations Toxicity of chlorantraniliprole to H. armigera was determined by using incorporated diet with newly molted third instar larvae (within 1 h). Six concentrations of chlorantraniliprole were used (7.813, 10.417, 15.625, 31.250, 62.500 and 125.000 g active ingredient L-1). 1 mL of the desired concentrations of chlorantraniliprole in a water solution were mixed with 282 g diet to yield the insecticide-treated diet (the total distilled water in the artificial diet was 200 mL), while the temperature of the preparing diet was under 40°C. Then the insecticide-treated diet was agitated for 30-40 s to distribute insecticide evenly, and then poured into 300 mL Petri dishes. Control diet contained 1 mL distilled water instead of the chlorantraniliprole. Diet prepared before the bioassay and was kept at 4°C for no longer than 48 h before being used or discarded. A small portion of the diet (approximately 1 cm3) was placed in a clean glass tube (10.0 cm length×1.8 cm diameter), into which one third newly molted larva was transferred. About 30 tubes were prepared for each concentration, in three replicates; thus the total number of larvae of each concentration was 90. All the tubes were closed with cotton pads and kept under laboratory condition of (27±1)°C, (60±5)% RH and 16 h L:8 h D. Larval
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mortality was evaluated on d 7 and 8. Larvae not responding with head movements or peristaltic contractions when touched with a fine brush were recorded as dead; moribund larvae were scored as alive (Loriatti et al. 2009). According to the bioassay results, LC10, LC20 and LC40 concentrations of chlorantraniliprole were selected as sublethal dosages for the subsequent experiments. Sublethal treatments and effects on the parent generation Newly molted third instar larvae obtained from the stock culture were separated into groups of 100. For the LC40 treatment, 13 groups were included (1 300 larvae); there were 7 groups (700 larvae) and 3 groups (300 larvae) for LC20 and LC10 treatments, respectively, to allow for the differential mortality, and still obtain sufficient number of alive adults. These groups were fed with the insecticide incorporated diet of each concentration for 7 d as described. After that, all of the survived larvae were kept on diet free of the pesticide. On d 4, 50 larvae selected randomly were weighed (0.001 g precision) from each treatment and the survived larvae from each group were transferred to fresh, insecticide-free artificial diet until pupation on d 7 and 8. Pupae were individually placed into small plastic cups (15 cm in diameter, 20 cm in depth) to emerge. After emergence, individuals from the same day were paired randomly to form pairs with both or only one of the sexes sublethally treated. A piece of absorbent cotton soaked in a 10% honey solution, changed daily, was provided for adults. Ten pairs were used in each group except the “both sexes exposed” group (20 pairs). The females were dissected as soon as they died to check the number of matings which can be detected by the number of trichoqyne receptive hypha. Mortality per day, pupation time, fresh body mass on d 4, pupal mass on d 3, the sex of adults, as well as the ratio of pupation and adult emergence were recorded. The numbers of eggs laid by each pair were counted daily until females died; if the male died first, the survived female was given a new mate of the same treatment. A total of 150 eggs (repeated 3 times) were taken randomly from each pair of adult moths, and the egg hatching rate was determined. Male and female adult longevities were recorded, as well as the mating time were checked. Pairs that failed to mate or lay eggs were discarded.
Sublethal effects on the offspring F1 generation To e v a l u a t e t h e p o s s i b l e c a r r y - o v e r a c t i v i t y o f chlorantraniliprole to their offspring, the F1 generation eggs laid by adults with both partners exposed to the same concentration were collected to hatch. 300 larvae that hatched from each treatment were maintained on untreated artificial diet until pupation. However, the F1 generation was not exposed to the insecticide directly. The diet was changed on the 4th d of the third instar, after that it was changed when necessary. The same data were recorded as on the parent generation. The adults were paired as the same way
as their parents were. Here, one of the paired moths treated, means one of parents of the offspring were exposed to sublethal concentration of chlorantraniliprole.
Data analysis Data obtained from the experiments were statistically analyzed using one-way analysis of variance (ANOVA). Means were compared by Tukey’s studentized range test (equal variance), if the variance was unequal after transformation, Games-Howell corrections were applied (Adamski et al. 2009), both accepting significant differences at P<0.05. All percentage data were arcsine transformed before being subjected to ANOVA. SPSS ver. 17.0 software was used for all analyses.
Acknowledgements The authors sincerely thank the Institute of Plant Protection, Nanjing Agricultural University, China, for providing the susceptible strain of H. armigera, and and Prof. Lovei G L from Department of Agroecology, Aarhus University, Denmark, for comments on the manuscript. This study was supported by the National 863 Program of China (2012AA101502) and the Special Fund for Agro-Scientific Research in the Public Interest, China (200903033).
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