Sublethal effects of Bt toxin and chlorpyrifos on various Spodoptera exigua populations
Abstract
Bt cotton (Cry1Ac) has been commercially grown in China since 1997, saving China's cotton production from attack by Bt-target pests and also tremendously reducing pesticide usage. In recent years, however, Bt cotton, with 4.2 million ha of cultivation, has suffered from a secondary target pest, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). In China, growers have even had to re-adopt conventional pesticides to control the pest, and this practice has already caused serious pesticide residue. In order to clarify the sublethal effects of chemical pesticide, the responses of a Bt-susceptible and a Bt-tolerant (Bt10) S. exigua strain to three treatment combinations were examined, including Bt toxin, sublethal chlorpyrifos, and Bt + sublethal chlorpyrifos. The susceptible and the Bt10 strain responded differently to dual pressure. Bt toxin + sublethal chlorpyrifos treatment lowered larval mortality and stimulated population increase of the susceptible S. exigua, whereas it delayed growth and development of the Bt10 strain. Under dual pressure, although larvae of the Bt10 strain developed faster than larvae of the susceptible strain, the Bt10 population experienced higher larval mortality, prolonged pupal duration, decreased pupal weight, decreased emergence rate, and shortened adult longevity. Compared with the susceptible strain, the Bt10 strain was deleteriously affected by sublethal chlorpyrifos. The Bt-tolerant/resistant S. exigua population was more vulnerable to chemical pesticides like chlorpyrifos regardless of whether it was exposed to Bt toxin or not. Our study provides a reference for increasing the efficacy of control of S. exigua in Bt-cotton planting areas.
Introduction
In 2012, 170.3 million ha of biotech crops were grown in 28 countries globally (James, 2012). The most widespread of these crops, transgenic cotton, has been planted for 18 years. In China, 4.2 million ha of Bt cotton are currently under cultivation with a grower adoption rate of 90% (James, 2012). Initially, target pests such as Helicoverpa armigera Hübner were effectively controlled due to the increasing adoption of Bt cotton (Wu et al., 2008). In addition, the use of insecticide sprays was significantly decreased which led to a marked increase in abundance of arthropod predators (ladybirds, lacewings, and spiders). Those predators are beneficial not only for Bt cotton but also for the neighboring non-Bt crops such as maize, peanut, and soybean (Lu et al., 2012). However, farmers are now being faced with new challenges, such as the resistance of target pests to Bt toxin and the evolution of secondary pests (Li et al., 2007). For example, Zhang et al. (2011) found that susceptibility of H. armigera to Cry1Ac was lower in 13 field populations from northern China (where Bt cotton has been planted intensively) than in two populations from northwestern China (where exposure to Bt cotton has been limited). Additionally, many non-target pests of Bt cotton like mirid bugs have emerged in many crops due to the adoption of Bt cotton and the consequent reduction in conventional insecticide spraying (Lu et al., 2010).
Globally, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) is an important agricultural pest, and its hosts extend to more than 170 species, including vegetables, cotton, maize, soybean, and flowers. Cotton leaves, buds, flowers, and immature bolls can all be damaged by S. exigua (Zheng et al., 2010). It has been demonstrated that S. exigua is not as susceptible to Cry1Ac toxin as H. armigera, and under normal circumstances S. exigua is a secondary target pest of Bt cotton planted in China (Yin et al., 2004; Guo et al., 2010). In recent years, however, S. exigua damage to Bt cotton has become more frequent and widespread. For example, in 1999, S. exigua broke out in the Yellow River area with one larva per plant (Guo & Yang, 2000); in 2001, an infestation on Bt cotton was recorded in Chuzhou city (Wang & Li, 2002); and in 2006, in a coastal region of Jiangsu province, S. exigua caused serious damage to Bt cotton leaves and buds with a density of five larvae per plant, which led to a 15% production loss (Zhu et al., 2008); and in 2009, the pest emerged in Jiangxi province (Jiang et al., 2010). Due to the failure of Bt toxin to control pests in these areas, farmers have been forced to revert to chemical pesticides including chlorpyrifos, methomyl, and beta-cypermethrin (Zhang et al., 2010). Of these, chlorpyrifos is widely used on cotton, vegetables, wheat, and rice. Thus, in these areas S. exigua is currently under dual control pressure: Bt toxin and chemical pesticides. The exact consequences of this dual selection are still unclear. Xue et al. (2002) found that S. exigua larvae fed with Bt cotton became less sensitive to alphamethrin, methomyl, profenofos, and chlorfluazuron which differed from the response of the Bt target pest, H. armigera (Liang et al., 2000). It was also found that host plants significantly influenced the activities of detoxification enzymes in S. exigua and, subsequently, the sensitivity of S. exigua to chemical pesticides such as cyhalothrin, deltamethrin, chlorpyrifos, chlorfenapyr, and tebufenozide (Jiang et al., 2001; Le et al., 2009; Song et al., 2009). Traditionally, determining an acute median lethal dose or concentration of pesticide has been widely adopted to measure the acute toxicity of pesticides to insects. However, measurement of the acute toxicity only reflects some of the deleterious effects of pesticides on insects. Besides direct mortality induced by pesticides, their sublethal effects on insect growth and development parameters must be considered for a complete analysis of their impact (Desneux et al., 2007).
However, currently no research has been conducted regarding the effects of dual Bt toxin and sublethal chemical pesticide pressure on the growth and development of S. exigua strains with different Bt-tolerance levels. The purpose of this study was to explore the responses of a Bt-tolerant and a Bt-susceptible strain to single selection pressure, such as chlorpyrifos or Bt toxin, and to dual pressure (chlorpyrifos + Bt toxin). Such information may help improve the efficacy of control of secondary target pests in Bt cotton.
Materials and methods
Artificial diet preparation
Preparation of the artificial diet for susceptible S. exigua is described in detail by Su et al. (2010). The endotoxin protein used for the Bt-treated artificial diet was Cry1Ac contained in MVPII (a commercial bioinsecticide formulation supplied by Mycogen, San Diego, CA, USA). MVPII was freeze-dried and powdered (Cry1Ac purity was 20%). MVPII powder was dissolved in water and added to the artificial diet to obtain the Bt-treated artificial diet with a final concentration of 10 μg Cry1Ac g−1 diet which was used to develop the Bt-tolerant/resistant strain (Bt10).
Insect rearing
The susceptible S. exigua strain was provided by the Plant Protection Institute of the Chinese Agricultural Academy of Sciences (Beijing, China). This strain had been reared on an artificial diet in the laboratory for over 10 years without any pesticide selection.
The Bt10 S. exigua strain was obtained by rearing the above susceptible S. exigua strain on a Bt-treated artificial diet (10 μg g−1) for more than 15 generations. Rearing was conducted at 27 ± 1 °C, 70 ± 7% r.h., and a L14:D10 photoperiod.
Pesticides tested
Chlorpyrifos (97%) (Pioneer Chemical, Yangzhou, Jiangsu, China) was diluted to 4 mg l−1 solution, resulting in about 15% death of susceptible S. exigua individuals in laboratory assays. Bt-treated diet contained 0.7 μg Cry1Ac g−1: this content in the diet was equal to the average value of that recorded in the top third leaf of transgenic cotton over the entire growth season, as established by enzyme-linked immunosorbent assay (ELISA) in our laboratory.
Pesticide assays
Larvae were dipped into 4 mg l−1 chlorpyrifos solution for 3 s and put on absorbent paper to remove excess solution. The larvae were then reared in 24-well boxes filled with diet. The larvae were recorded as dead when they did not respond to touch by forceps 48 h after pesticide treatment. Twenty-four larvae of each strain were used in each treatment with three replicates – in total, 72 larvae were used in each treatment.
Treatments
Neonates of the susceptible strain and the Bt10 strain were fed the (1) artificial diet (no Bt toxin) until pupation, (2) Bt-treated diet (0.7 μg g−1) until pupation, (3) artificial diet (no Bt toxin) until the third instar, then dipped in 4 mg l−1 chlorpyrifos solution for 3 s and returned to the artificial diet (no Bt toxin) until pupation, or (4) Bt-treated diet (0.7 μg g−1) until the third instar, then dipped in 4 mg l−1 chlorpyrifos solution for 3 s and returned to Bt-treated diet (0.7 μg g−1) until pupation.
Larval mortality, developmental duration of larvae and pupae, emergence rate, adult longevity, total fecundity, and egg hatching rate of the next generation were recorded. In addition, each pupa was weighed. To assess fecundity, newly emerged S. exigua females from each treatment were paired with males from the same treatment and kept in individual plastic boxes (10 cm diameter) and allowed to mate. A honey solution-saturated cotton ball was provided on the bottom of each box to provide nutrition for the adults. Eggs were removed and recorded daily. To examine egg-hatching rates, 20 egg masses from each treatment were randomly selected and individually put into 9-cm-diameter Petri dishes and eggs were counted until they all hatched.
Data analysis
DPS v7.05 was used to conduct the data analysis. T-tests were conducted to analyze differences between the two populations when fed artificial diets (no Bt toxin). Two-factor ANOVA was used to analyze the data of the other three treatments (Bt toxin, chlorpyrifos, Bt toxin + chlorpyrifos) for the two populations, and Tukey's HSD test was used to separate the treatment means. Before analysis, all percentage data (larval mortality, emergence rate) were arcsine √x-transformed, when necessary, but untransformed means are presented.
Results
There were no significant differences in larval mortality, larval duration, pupal duration, pupal weight, adult longevity, total fecundity, and egg hatching rate of the next generation between the susceptible S. exigua population and the Bt10 population when both were fed with an artificial diet containing no Bt toxin and no pesticide (Table 1). Toxin and pesticide treatments were found to have effects on larval duration, pupal duration, pupal weight, emergence rate, and adult longevity that differed between the susceptible strain and the Bt10 strain (Table 2). Larval mortality, pupal duration, and adult longevity differed among the three treatments, whereas there were significant interactions of strain and treatment on larval duration, larval mortality, and adult longevity (Table 2).
| Parameter | Bt-susceptible | Bt10 |
|---|---|---|
| Larval mortality (%) | 2.78 ± 2.41 | 2.78 ± 2.41 |
| Larval duration (days) | 12.47 ± 0.22 | 13.51 ± 0.41 |
| Pupal duration (days) | 6.83 ± 0.14 | 6.81 ± 0.04 |
| Pupal weight (mg) | 117.90 ± 0. 50 | 116.40 ± 4.2 |
| Adult longevity (days) | 9.61 ± 0.87 | 9.90 ± 0.22 |
| No. eggs | 504.67 ± 245.00 | 514 ± 179.82 |
| Egg hatching rate (%) | 84.84 ± 3.13 | 80.44 ± 9.36 |
- Differences between strains were not statistically significant (t tests: P>0.05).
| Strain | Treatment | Strain*treatment | ||||
|---|---|---|---|---|---|---|
| F1,12 | P | F1,12 | P | F2,12 | P | |
| Larval duration | 42.98 | 0.0001 | 2.54 | 0.12 | 14.85 | 0.0006 |
| Larval mortality | 1.78 | 0.21 | 14.84 | 0.0006 | 10.36 | 0.0024 |
| Pupation duration | 29.75 | 0.0001 | 5.38 | 0.022 | 1.96 | 0.18 |
| Pupal weight | 101.43 | 0.0001 | 1.80 | 0.21 | 2.16 | 0.16 |
| Emergence rate | 43.36 | 0.0001 | 2.3 | 0.14 | 3.59 | 0.060 |
| Adult longevity | 17.64 | 0.0012 | 31.21 | 0.0001 | 10.44 | 0.0024 |
Compared with Bt-toxin diet only, susceptible S. exigua larvae treated with Bt toxin + chlorpyrifos, larval duration, pupal duration, and pupal weight did not differ, whereas larval mortality decreased from 57.3 to 15.3%, emergence rate increased from 59.0 to 83.4%, and adult longevity was prolonged by 3.5–10.1 days (Table 3). When susceptible S. exigua larvae were treated with Bt toxin + chlorpyrifos, mean pupal weight decreased by 14.1 mg and adult longevity was prolonged by 2.7 days relative to treatment with chlorpyrifos alone (Table 3).
| Strain | Treatment | Larval duration (days) | Larval mortality (%) | Pupal duration (days) | Pupal weight (mg) | Emergence rate (%) | Adult longevity (days) |
|---|---|---|---|---|---|---|---|
| CK | Bt toxin | 16.68 ± 0.26aA | 57.29 ± 6.45aA | 5.89 ± 0.36cB | 122.30 ± 3.10abA | 58.97 ± 8.97bAB | 6.58 ± 0.28cBC |
| Chlorpyrifos | 15.95 ± 0.12aAB | 12.48 ± 5.10cB | 5.48 ± 0.12cB | 132.90 ± 2.00aA | 83.23 ± 5.30aA | 7.42 ± 0.18bB | |
| Bt toxin + chlorpyrifos | 15.98 ± 0.16aA | 15.27 ± 5.53cB | 5.82 ± 0.11abcB | 118.80 ± 1.60bA | 83.37 ± 2.38aA | 10.13 ± 0.21aA | |
| Bt10 | Bt toxin | 12.60 ± 0.19cC | 33.33 ± 4.41bAB | 6.38 ± 0.23bcB | 92.00 ± 4.10cB | 44.27 ± 5.85bB | 6.76 ± 0.22bcBC |
| Chlorpyrifos | 15.44 ± 0.20aAB | 31.67 ± 1.67bB | 6.54 ± 0.22bAB | 93.40 ± 3.60cB | 39.15 ± 2.07bB | 6.31 ± 0.38cC | |
| Bt toxin + chlorpyrifos | 13.98 ± 0.28bBC | 30.00 ± 2.89bB | 7.89 ± 0.49aA | 94.60 ± 3.20cB | 44.14 ± 7.86bB | 7.61 ± 0.40bB |
- Means within a column followed by different letters are significantly different (Tukey's HSD test: lower case letters: P<0.05, capital letters: P<0.01).
Treatment of Bt10 larvae with Bt toxin + sublethal chlorpyrifos prolonged larval and pupal duration by 1.4 and 1.5 days, respectively, and adult longevity by 0.8 day, compared to larvae fed with the Bt-treated diet only (Table 3). Relative to treatment with chlorpyrifos alone, the dual treatment shortened larval duration by 1.5 days and it prolonged pupal duration and adult longevity by 1.4 and 1.3, days respectively (Table 3).
After treatment with the Bt toxin diet, the Bt10 strain experienced 24% lower larval mortality, 4 days shorter larval duration, and 30 mg lighter pupae than the susceptible strain (Table 3). Treatment with chlorpyrifos led to 19.2% higher larval mortality, 1 day longer pupal duration, 39.5 mg lower pupal weight, and 43.1% lower pupal emergence rate in the Bt10 compared to the susceptible strain (Table 3). Hence, the Bt10 strain suffered more harm from chlorpyrifos than the susceptible strain.
When Bt10 larvae were treated with Bt toxin + sublethal chlorpyrifos, compared with susceptible larvae larval duration was 2 days shorter, pupal duration was 2.1 days longer, pupae were 24.2 mg lighter, emergence rate was 39.2% lower, and adults lived 2.5 days shorter (Table 3).
Discussion
In Bt cotton, the Cry-toxin expression pattern is specific for developmental stage and tissue type. Toxin concentration in cotton leaves decreases with the growth and development of the plant: it is highest in seedlings, intermediate in the bud stage, and lowest in the flowering and boll-forming stage (Chen et al., 2000). In addition, an S. exigua outbreak is favored by high temperatures; hence, from July to September in the Yangtze River area serious damage may occur to cotton, vegetables, and other crops. It is during this period, when weather and crop conditions are favorable for S. exigua population growth, that growers are forced to resort to chemical pesticides. Chemical pesticides degrade gradually and different pesticides have different degradation dynamics. For the same pesticide, seasons and cultivation styles can affect the degradation rates. Half-life periods for chlorpyrifos were found to be 1.4 and 1.8 days on the outdoor vegetables in the summer and autumn, respectively, at recommended dosages (Shi et al., 2005). Thus, the surviving larvae are exposed to sublethal doses of insecticides.
When susceptible S. exigua larvae were treated with Bt toxin + sublethal chlorpyrifos, population growth and development were stimulated significantly compared with susceptible S. exigua larvae fed the Bt-treated diet only. Larval mortality decreased to one-third that of susceptible S. exigua larvae fed the Bt-containing diet only, indicating that after being exposed to Bt toxin, susceptible S. exigua larvae became less sensitive to a chemical pesticide like chlorpyrifos. This is consistent with the finding of Xue et al. (2002), that S. exigua larvae fed with Bt cotton became ca. 1–1.6× less sensitive to alphamethrin, methomyl, profenofos, and chlorfluazuron, compared with S. exigua larvae fed conventional cotton, soybean, Chinese cabbage, green Chinese onion, or redroot amaranth. Enhanced detoxification enzyme activities in insects are often involved in elevated detoxification of allelochemicals and/or insecticides (Brattsten & Wilkinson, 1997; Valles et al., 1999). Guo et al. (2010) found that total superoxide dismutase (T-SOD) played an important role in the rapid detoxification of S. exigua to Bt or other secondary metabolic compounds in Bt cotton, and carboxylesterase (CarE) and acetylcholinesterase (AChE) were involved in the multi-generation host plant adaptation of S. exigua. The lower sensitivity to chlorpyrifos of the Bt-fed susceptible S. exigua larvae might be due to increased activities of detoxification enzymes. In our study it was also shown that when treated with Bt toxin + sublethal chlorpyrifos, emergence rate and adult longevity of susceptible S. exigua increased compared with treatment with Bt toxin alone. These results indicate that a sublethal residue of chemical pesticides in Bt cotton fields will stimulate susceptible S. exigua population increase.
When Bt10 larvae were treated with Bt toxin + sublethal chlorpyrifos, larval mortality, pupal weight, and emergence rate were not significantly different from those of Bt10 larvae treated with Bt toxin only, although it significantly prolonged the duration of larval and pupal stages. The results also indicate that after being exposed to Bt toxin, Bt10 larvae became less sensitive to a chemical pesticide like chlorpyrifos, which is similar with the response to chlorpyrifos of susceptible S. exigua larvae fed with Bt toxin. We adopted the insect-dipping method to treat the larvae with chlorpyrifos, hence the lower sensitivity to chlorpyrifos is not because of less ingestion of it. Possibly, Bt toxin induced the enhancement of detoxification enzyme activities in the Bt10 larvae fed with Bt toxin, and chlorpyrifos is detoxified by those elevated detoxinfication enzymes (Guo et al., 2010).
When susceptible S. exigua larvae and Bt10 larvae were treated with chlorpyrifos, larval mortality of the Bt10 strain was 2.5-fold higher than that of the susceptible strain, indicating that the Bt10 strain is more sensitive to chlorpyrifos. Wu & Guo (2004) also found that Cry1Ac-resistant H. armigera (106-fold resistance to Cry1Ac toxin) were more sensitive to lambda-cyhalothrin, phoxim, and endosulfan. So for both a target pest like H. armigera and a secondary pest like S. exigua, there was no positive cross-resistance between Cry1Ac toxin and these chemical insecticides. It is well-known that resistant insects may display the fitness cost in the absence of selection pressure with higher larval mortality, longer larval and/or pupal developmental time, decreased pupal weight, lower fecundity and fertility, or morphologically abnormal adults (Bird & Akhurst, 2005; Anilkumar et al., 2008; Jakka et al., 2014). Significantly higher larval mortality, longer pupal duration, lower pupal weight, decreased emergence rate, and shorter adult longevity of Bt10 S. exigua after exposed to chlorpyrifos agree with the literature, highlighting fitness costs associated with resistance to Bt toxins in Bt10 S. exigua that have led to its vulnerability to conventional pesticide treatments (Williams et al., 2011; Sosa-Gómez & Miranda, 2012).
When Bt10 S. exigua larvae were fed with Bt toxin (0.7 μg g−1 diet), they grew faster and the larval mortality was lower than the susceptible strain larvae fed with Bt toxin. Yet, the pupal weights were lower compared with the susceptible strain larvae, indicating that resistance to Cry1Ac reduced the fitness of Bt10 larvae. Many Bt resistant pests exhibit a fitness decrease. For example, Ostrinia nubilalis Hübner developed resistance to Cry1Ab in the field, which was associated with reduced pupal weight, slower development, a higher proportion of unsuccessful matings, and lower fertility compared with the susceptible strain (Crespo et al., 2010).
When the Bt10 and the susceptible strain were exposed to the dual selective pressure Bt toxin + sublethal chlorpyrifos, the Bt10 strain exhibited reduced fitness with about 2× higher larval mortality, slower pupal development, decreased pupal weight, lower emergence rate, and shorter adult longevity, although the larval duration of Bt10 was shorter than that of the susceptible strain. We can conclude that under the dual pressure of Bt toxin and a chemical pesticide, Bt10 S. exigua are more vulnerable than the susceptible ones. Under the dual pressure of Bt toxin and lethal chlorpyrifos, the Bt10 strain may be at a serious disadvantage. Thus, Bt tolerant/resistant S. exigua may be easier to control using chemical pesticides.
Acknowledgements
We thank Connie Allison very much for her contribution to the manuscript revision. This research was supported by the Natural Science Foundation – Young Scientific Talent Project in Yangzhou City (YZ2014037), by a major science and technology project to create new crop varieties using gene transfer technology (Biosafety Monitoring Technique of Genetically Modified Organisms: 2014ZX08012004-007), by the Special Fund for Agro-Scientific Research in the Public Interest of China (201103026, 201103021), and by open funding from State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control (2010DS700124-KF1109).
