Effect of Tea Saponin-Treated Host Plants on Activities of Antioxidant Enzymes in Larvae of the Diamondback Moth Plutella xylostella (Lepidoptera: Plutellidae)

Effect of Tea Saponin-Treated Host Plants on Activities of Antioxidant Enzymes in Larvae of the... Abstract Tea saponin (TS) is extracted from the seeds of the tea plant and is generally regarded as a safe compound that has insecticidal properties and can act synergistically with other compounds. In this study, the activities of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and the levels of malondialdehyde (MDA) were compared in midgut tissues of third instar larvae of the diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae). The larvae were fed on three different host plants, cabbage (Brassica oleracea L. var. capitata [Capparales: Brassicaceae]), radish (Raphanus sativus L. var. radiculus Persi [Capparales: Brassicaceae]), or rape (Brassica campestris L. [Capparales: Brassicaceae]), that had been treated with TS. Higher SOD, POD, and CAT activities were found in DBM larvae fed on cabbage after LC20 (concentration that induced 20% larval mortality) or LC50 (concentration that induced 50% larval mortality) treatment than on the control. On rape, TS treatments led to lower SOD and CAT activities than in the control and to higher POD activities after 24 h. MDA content increased in larvae fed on rape but decreased in larvae fed on radish after 12 h. Our results indicated that DBM larvae are more susceptible to TS on rape than on cabbage and radish, suggesting that this treatment may be an economic and effective means of controlling DBM on rape. antioxidant enzyme, host plant Management of the diamondback moth (DBM, Plutella xylostella [Lepidoptera: Plutellidae]), is a major topic of pest research because of its increasing resistance to insecticides. In the past several decades, much attention has been devoted to studying the toxicity of different chemicals in the insect and its adaptation capabilities to natural pest control agents (Talekar and Shelton 1993; Wei et al. 2010, 2015; You et al. 2013; Liu et al. 2015). To date, although many natural compounds have been reported to reduce pest populations, only a few have been widely used in practice, such as nicotine, pyrethrum, and neem (Roychoudhury 2016). One potential alternative control option for DBM is tea saponin (TS), which has insecticidal properties and can act synergistically with other compounds (Wang and Huang 1998). TS is extracted from the seeds of the tea plant (Camellia sinensis L.) and is generally regarded as a safe compound. It has low toxicity to mammals but is toxic to insects, as has been shown, for example, in the butterfly Pieris rapae L. (Lepidoptera: Pieridae) and the housefly Musca domestica L. (Diptera: Muscidae) Kawaguchi et al. (1994) reported that 50–150 mg/kg per d of TS administered orally for 3 mo had no effects on male and female rats. However, TS increased the efficiency of Bacillus thuringiensis infection of Spodoptera exigua Hubner (Lepidoptera: Noctuidae) (Rizwan-ul-Haq et al. 2009) and DBM (Li et al. 2005). Toxic and antifeedant effects are the main ways through which TS exerts its effects on insects (Wang and Huang 1998). However, it was found that some antioxidant enzyme activities in P. rapae (L.) were significantly decreased after TS treatment (Wang and Huang 1999). Hence, it is essential to elucidate the variation in activities of detoxification enzymes in DBM after TS treatment if this compound is to be used as a control agent. The goal of this study was therefore to evaluate the effects of TS on antioxidant enzymes in DBM. Insects have evolved for more than 400 million yr and have developed protective enzyme systems to detoxify insecticides and secondary substances used by plants as defenses against herbivory (Zhou et al. 2011, Zhang et al. 2013). Specifically, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione-S-transferase (GST) are the major antioxidant enzyme systems in insects (Felton and Summers 1995, Wang et al. 2001, Dubovskiy et al. 2008). SOD converts superoxide into oxygen and hydrogen peroxide, the latter being eliminated by CAT and POD activities (Felton and Summers 1995, Wang et al. 2001, Dubovskiy et al. 2008). GSTs are involved in removing the products of lipid peroxidation and hydroperoxides from cells (Wang et al. 2013). The activities of SOD, POD, CAT, and reactive oxygen species (ROS) maintain a physiological equilibrium, but various abiotic and biotic stress factors, such as insecticides, pathogens, and extreme temperature stresses, can induce changes in enzyme activities to protect against damage to the physiology and metabolism of the insect (Dubovskiy et al. 2008, Wang et al. 2013, Zhang et al. 2013). ROS degrade polyunsaturated lipids to form malondialdehyde (MDA) (Pryor and Stanley 1975), which causes toxic stress in cells and forms covalent protein adducts, which are referred to as advanced lipoxidation end products (Marnett 1999, Farmer and Davoine 2007). MDA is commonly used as an indicator that physiological or oxidative stress has occurred (Farmer and Davoine 2007). In this study, we measured the enzymatic activities of SOD, CAT, POD, and also determined the MDA content in the midgut tissues of DBM larvae. The purpose of the present study was to gain insights into the effects of TS on antioxidant enzyme activities in the DBM fed on three widely-grown cruciferous vegetables host plants, namely cabbage (Brassica oleracea L var. capitata), radish (Raphanus sativus L. var. radiculus Persi), and rape (Brassica campestris L.). Materials and Methods Host Plants and Insects The cruciferous species cabbage (B. oleracea L var. capitata [Capparales: Brassicaceae]), radish (R. sativus L. var. radiculus Persi [Capparales: Brassicaceae]), and rape (B. campestris L. [Capparales: Brassicaceae]) were used in this study. All plants were grown in a greenhouse (E119°34′, N26°13′) at our laboratory without insecticide treatment. DBM larvae were originally collected from a cabbage field without insecticide treatment in Fuzhou City, Fujian province, China (E119°18′, N26°07′); this location has cultivated cabbages for over 10 yr. The populations were maintained in a rearing room (E119°34′, N26°13′) at 25 ± 1°C and 70–80% relative humidity, under a 14:10 (L:D) h cycle. The adults were introduced into a chamber containing host plants and absorbent cotton with a 100 g/kg honey solution. Insects were then allowed to mate and lay eggs. Larvae in the experiment were reared continuously on the different host plants for at least five generations to maintain population homogeneity. Reagents TS containing 90% saponin was obtained from Hanqing Biological Technology Co. Ltd., Huaihua, Hunan Province, China. Assay kits for Coomassie brilliant blue staining of enzymes (POD, CAT, and SOD) and MDA were purchased from Nanjing Jiancheng Bioengineer Institute, Jiangsu, China. Toxicity Tests LC20 and LC50 concentrations, the TS concentrations causing 20% and 50% larval mortality, respectively, were determined in toxicity tests using third instar larvae. Six concentrations of TS in distilled water (pH = 6.8) were screened after preliminary mortality tests for each host plant species. For cabbage, TS concentrations of 200, 1,000, 5,000, 25,000, and 125,000 mg liter−1 were used. For radish, TS concentrations of 64, 320, 1,600, 8,000, and 40,000 mg liter−1 were used. For rape, TS concentrations of 125, 250, 500, 1,000, and 2,000 mg liter−1 were used. Freshly prepared leaf disks (diameter = 3.4 cm) from the three host species were dipped into TS solutions for 15 s and then transferred to plastic jars (diameter = 3.4 cm, two leaf disks per jar). Moistened filter paper was placed on the bottom of each plastic jar to prevent withering of the leaf disks. Thirty third-instar larvae were starved for 4 h and then transferred into a plastic jar (30 larvae/bottle, two leaf disks/bottle). When larvae did not respond to a light touch, they were assumed to be dead. After 24 h, the number of dead larvae was recorded to estimate mortality. Each treatment was repeated three times. Assays of Enzyme Activities Tissue Collection Toxicity bioassays were used to determine the LC20 and LC50 concentrations of TS of treated leaf disks as described previously. DBM third-instar larvae (24 h after molt) were transferred to radish, rape, and cabbage leaf disks that had been treated with either the LC20 or LC50 concentration of TS for 2 h. Then 20 live third-instar DBM larvae, with three replications per treatment, were removed from radish, rape, and cabbage leaf disks after feeding for 1, 12, or 24 h and used to prepare enzyme extracts. At each sampling interval, the midgut was dissected from each larva, and the adhering fat body, Malpighian tubules and gut contents were removed, and the tissue was stored at −80°C. Enzyme Activity and MDA Content Determination For SOD activity measurement, midguts from 20 larvae were collected and placed in a chilled Eppendorf tube containing cold homogenization buffer (2.0 ml of 0.01 M Tris–HCl, pH 8.2); the homogenate was centrifuged at 3,000 rpm for 10 min. The supernatant beneath the fat layer was collected and assessed for SOD activity by an improved pyrogallol method; SOD activity was measured at 440 nm on a 752 UV-visible spectrophotometer (Jing and Zhao 1995). For POD activity measurement, midguts from another 20 larvae were collected as described previously and placed in a cold homogenization buffer (2.0 ml 0.05 M phosphate buffer, pH 7.0); the homogenate was treated as described previously, and POD activity was determined at 470 nm on a 752 UV-visible spectrophotometer (Simon et al. 1974). For CAT activity measurement, midguts from another 20 larvae were collected and homogenized in cold homogenization buffer (2.0 ml of 0.05 M phosphate buffer, pH 7.0) as described previously. The CAT activity was assessed in the supernatant after centrifugation by measuring H2O2 consumption, using the decrease in absorbance at 230 nm on a 752 UV-visible spectrophotometer (Aebi 1983). MDA contents were assessed using the midguts of 20 larvae; the midguts were incubated at 95°C in thiobarbituric acid under aerobic conditions (pH 3.4). The MDA content was measured in a thiobarbituric acid assay at 532 nm on a 752 UV-visible spectrophotometer, based on the release of a color complex following reaction of thiobarbituric acid with MDA (Ohkawa et al. 1979). Total protein concentration of extracts was determined using a Lowry protein assay (Fryer et al. 1986). All enzyme extracts were kept in an ice water bath before analysis. Extraction was performed in triplicate for each sampling time. For all enzyme assays, the protein content of samples was measured using bovine serum albumin as a standard, as described by Bradford (1976). Each treatment was repeated three times. Statistical Analysis The mortality data were subjected to probit analysis to determine LC20 and LC50 using the SPSS 15.0 data processing system. Mean values of enzyme activities were compared by a post hoc Duncan’s Multiple Range Test with SAS 6.12 software. Significance was set at P < 0.05. Significant differences between treatment groups on the same host species were analyzed for analysis of variance using the SPSS 15.011.5 software package. Results Mortality in Third-Instar DMB Larvae After TS Treatments The LC20 values for third-instar DBM larvae fed on radish, rape, and cabbage were estimated as 259.98, 162.43, and 272.16 mg liter−1, respectively; LC50 values were 5,148.95, 570.54, and 3,608.56 mg liter−1, respectively (Table 1). Overall, larvae exposed to treated rape showed higher rates of mortality. Table 1. Lethal concentration values of tea saponin against third instar larvae of the diamondback moth Host plant  Slope of regression equation (b) (SE)  LC20 (mg/l) (95% CI)  LC50 (mg/l) (95% CI)  χ2  df  Sig.  Radish  0.649 (0.070)  259.98 (121.52–455.19)  5148.95 (3266.51–8769.71)  1.896  3  0.594  Rape  1.543 (0.159)  162.43 (112.91–211.33)  570.54 (472.78–692.91)  6.152  3  0.104  Cabbage  0.750 (0.017)  272.16 (125.42–483.05)  3608.56 (2393.04–5343.58)  6.140  3  0.105  Host plant  Slope of regression equation (b) (SE)  LC20 (mg/l) (95% CI)  LC50 (mg/l) (95% CI)  χ2  df  Sig.  Radish  0.649 (0.070)  259.98 (121.52–455.19)  5148.95 (3266.51–8769.71)  1.896  3  0.594  Rape  1.543 (0.159)  162.43 (112.91–211.33)  570.54 (472.78–692.91)  6.152  3  0.104  Cabbage  0.750 (0.017)  272.16 (125.42–483.05)  3608.56 (2393.04–5343.58)  6.140  3  0.105  View Large Table 1. Lethal concentration values of tea saponin against third instar larvae of the diamondback moth Host plant  Slope of regression equation (b) (SE)  LC20 (mg/l) (95% CI)  LC50 (mg/l) (95% CI)  χ2  df  Sig.  Radish  0.649 (0.070)  259.98 (121.52–455.19)  5148.95 (3266.51–8769.71)  1.896  3  0.594  Rape  1.543 (0.159)  162.43 (112.91–211.33)  570.54 (472.78–692.91)  6.152  3  0.104  Cabbage  0.750 (0.017)  272.16 (125.42–483.05)  3608.56 (2393.04–5343.58)  6.140  3  0.105  Host plant  Slope of regression equation (b) (SE)  LC20 (mg/l) (95% CI)  LC50 (mg/l) (95% CI)  χ2  df  Sig.  Radish  0.649 (0.070)  259.98 (121.52–455.19)  5148.95 (3266.51–8769.71)  1.896  3  0.594  Rape  1.543 (0.159)  162.43 (112.91–211.33)  570.54 (472.78–692.91)  6.152  3  0.104  Cabbage  0.750 (0.017)  272.16 (125.42–483.05)  3608.56 (2393.04–5343.58)  6.140  3  0.105  View Large Enzyme Activities in Third-Instar Larvae Fed on TS-Treated Host Plants Effect of TS on SOD Activities Overall, except for rape at 24 h, SOD activities in larvae fed on LC50-treated host plants were higher than in those fed on untreated host plants (Fig. 1). TS treatment led to significantly lower SOD activities in rape at 24 h than observed in the control (Fig. 1C). SOD activity in larvae fed TS-treated radish increased continuously from 1 h to the end of the experiment (Fig. 1B). Fig. 1. View largeDownload slide Superoxide dismutase activities in third instar larvae fed on different host plants: (A) cabbage; (B) radish; and (C) rape. The controls were untreated host plants. Different lowercase letters indicate significant differences between treatment groups on the same host species (P < 0.05). ANOVA statistics were: cabbage (F8,27 = 9.310, P < 0.001); radish (F8,27 = 348.910, P < 0.001); and rape (F8,27 = 61.824, P < 0.001). Fig. 1. View largeDownload slide Superoxide dismutase activities in third instar larvae fed on different host plants: (A) cabbage; (B) radish; and (C) rape. The controls were untreated host plants. Different lowercase letters indicate significant differences between treatment groups on the same host species (P < 0.05). ANOVA statistics were: cabbage (F8,27 = 9.310, P < 0.001); radish (F8,27 = 348.910, P < 0.001); and rape (F8,27 = 61.824, P < 0.001). In larvae fed on radish, the final SOD activity in LC20 treatments was 79.30 U/mg protein with an initial value of 60.54 U/mg protein. However, SOD activity in the control was 55.49 U/mg protein at 24 h. The SOD activity from LC50 gradually increased from the initial sampling interval to 24 h (Fig. 1B). In larvae fed on rape, SOD activity in the LC50 treatments rose at 12 h to 16.44 U/mg protein; SOD activity declined at 24 h (Fig. 1C). Effect of TS Treatment on POD Activity POD activities in larvae fed on cabbage and radish treated with LC20 and LC50 concentrations of TS were significantly higher at 12 h and 24 h than in the control (Fig. 2A and B). Enzyme activities in the LC50 treatments were higher than those of LC20 and control and reached a level of 27.55 U/mg protein in the 12-h sample (Fig. 2B). Fig. 2. View largeDownload slide Peroxidase activities in third instar larvae fed on different host plants: (A) cabbage; (B) radish; and (C) rape. Controls were untreated host plants. Different lowercase letters indicate significant differences between concentrations and sampling times in the same host species (P < 0.05). ANOVA statistics were: cabbage (F8,27 = 303.171, P < 0.001); radish (F8,27 = 161.919, P < 0.001); and rape (F8,27 = 280.359, P < 0.001). Fig. 2. View largeDownload slide Peroxidase activities in third instar larvae fed on different host plants: (A) cabbage; (B) radish; and (C) rape. Controls were untreated host plants. Different lowercase letters indicate significant differences between concentrations and sampling times in the same host species (P < 0.05). ANOVA statistics were: cabbage (F8,27 = 303.171, P < 0.001); radish (F8,27 = 161.919, P < 0.001); and rape (F8,27 = 280.359, P < 0.001). In larvae fed on rape, POD activities increased from 1 h and reached their highest levels at 24 h. Overall, TS increased POD activities at both LC20 and LC50 concentrations (Fig. 2C). Effect of TS on CAT Activity Compared with the control, the LC50 treatments led to higher CAT activities in larvae fed on cabbage and radish at 12 h and 24 h (Fig. 3A and B). In larvae fed on rape, CAT activity increased from 1 h to 12 h for the LC50 treatment, but this declined at 24 h (Fig. 3C). In contrast, CAT activity in the LC20 treatment groups rose continuously to 67.62 U mg−1 protein at 24 h (Fig. 3C). Fig. 3. View largeDownload slide Catalase activities in third instar larvae fed on different host plants: (A) cabbage; (B) radish; and (C) rape. Controls were untreated host plants. Different lowercase letters indicate significant differences between concentrations and sampling times on the same host species (P < 0.05). ANOVA statistics were: cabbage (F8,27 = 29.422, P < 0.001); radish (F8,27 = 10.717, P < 0.001); and rape (F8,27 = 20.174, P < 0.001). Fig. 3. View largeDownload slide Catalase activities in third instar larvae fed on different host plants: (A) cabbage; (B) radish; and (C) rape. Controls were untreated host plants. Different lowercase letters indicate significant differences between concentrations and sampling times on the same host species (P < 0.05). ANOVA statistics were: cabbage (F8,27 = 29.422, P < 0.001); radish (F8,27 = 10.717, P < 0.001); and rape (F8,27 = 20.174, P < 0.001). Effect of TS on MDA Content The level of MDA content in the larvae varied with the host plant species. TS treatment inhibited MDA content in larvae fed cabbage and radish for the LC50 treatment at 1 h as compared to the control. In larvae fed on cabbage, lower contents were found after the LC50 treatment than in the LC20 treatment at 1 and 12 h (Fig. 4). At 12 h, a higher MDA content was found in larvae fed on rape; however, larvae fed on radish had a significantly lower MDA content than did the control at 12 h. At 24 h, the TS treatments did not alter MDA content from that in the controls (Fig. 4). Fig. 4. View largeDownload slide Malondialdehyde content in third instar larvae fed on different host plants: (A) cabbage; (B) radish; and (C) rape. Controls were untreated host plants. Different lowercase letters indicate significant differences between concentrations and sampling times on the same host species (P < 0.05). ANOVA statistics were: cabbage (F8,27 = 3.318, P = 0.009); radish (F8,27 = 24.037, P < 0.001); and rape (F8,27 = 20.173, P < 0.001). Fig. 4. View largeDownload slide Malondialdehyde content in third instar larvae fed on different host plants: (A) cabbage; (B) radish; and (C) rape. Controls were untreated host plants. Different lowercase letters indicate significant differences between concentrations and sampling times on the same host species (P < 0.05). ANOVA statistics were: cabbage (F8,27 = 3.318, P = 0.009); radish (F8,27 = 24.037, P < 0.001); and rape (F8,27 = 20.173, P < 0.001). Discussion In recent years, the efficacy of botanical insecticides against DBM has been evaluated (Tang et al. 2013, Nasr et al. 2017). Our study focussed on whether TS might be an effective alternative for the control of P. xylostella larvae, by delaying the resistance levels to a chemical pesticide. We showed that although rape and cabbage are closely related species of the same genus, higher LC20 and LC50 values were found in cabbage. This effect may be because DBM is more adapted to feeding on cabbage than rape. In addition, our results indicated that TS may be an economic and effective means of controlling DBM on rape. Many previous studies have shown that host plants can have different effects on physiological and biochemical traits in herbivores, such as on the activities of protective enzymes and detoxification enzymes (Abd-Elghafar et al. 1989, Wang et al. 2010, Zhou et al. 2011, Deng et al. 2013, Tang et al. 2013, Zhang et al. 2013, Cai et al. 2016, Nasr et al. 2017). For example, in comparison to untreated controls, insects treated with essential oil of Origanum vulgare L. show a significant decrease in alkaline phosphatase and protease activities; however, lipase activity is higher at 24 h after treatment than in the control. Additionally, with regard to detoxification enzymes such as GST and esterase, treated insects show significant differences compared with the controls (Nasr et al. 2017). It has been reported that P. xylostella that have been fed green Chinese cabbage treated with extracts from Ginkgo biloba L. show significant variations in SOD, CAT, and POD activities compared with the controls (Tang et al. 2013). In other examples, some antioxidant enzymes in Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae) and Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) vary among host plants (Zhou et al. 2011, Zhang et al. 2013). Furthermore, antioxidant enzyme activity in P. rapae (L.) significantly decreases after TS treatment (Wang and Huang 1999). In the present study, we found higher SOD, POD, and CAT activities in third-instar DBM larvae that had fed for 12 h on leaf disks from three host plant species treated with an LC50 concentration of TS. Thus, DBM can show some resistance to TS treatment, which may be similar to that found in P. rapae larvae that degrade harmful free radicals (O2−) after treatment with cypermethrin for 10 min (Li et al. 1994). Moreover, larvae exposed to LC50-treated rape leaf disks had higher SOD and CAT activities at 12 h than at 24 h. Similar responses have been reported for larvae of the Chinese pine caterpillar (Dendrolimus tabulaeformis Tsai et Liu) after treatment with the insecticidal fungus Beauveria bassiana (Bals.) (Liu et al. 2005) and for Parasa consocia Walker and Thosea postornata (Hampson) treated with deltamethrin (Li et al. 1994). The lower CAT activities at 24 h might indicate that a high level of free radicals accumulated in larval cells, which would eventually be toxic to the larvae. Overall, our results indicate that TS treatment disrupted the activities of SOD, CAT, and POD and suggested that this disruption might increase the resistance risk of DBM to TS. A possible explanation of the responses of DBM to the defense systems of its cruciferous host plants, particularly to the glucosinolate-myrosinase complex (the so-called ‘mustard oil bomb’), has been suggested by Ratzka et al. (2002). DBM modifies ingested plant glucosinolates by a sulfatase enzyme in the gut to prevent hydrolysis of the glucosinolates by plant myrosinase into a highly toxic product, such as isothiocyanate. In our study, the higher enzyme activities in radish might be due to DBM being more adapted to this species than other cruciferous plants. DBM have evolved various detoxification mechanisms that allow rapid adaptation to new host plants or new pesticides. Our results showed that TS had no effect on MDA contents after 24 h. These results, together with our evidence that lower values of GST were found in larvae fed a susceptible host plant, indicate that the larvae adjusted their enzyme activities to adapt to unfavorable plant substances. There is no doubt that insect resistance mechanisms are associated with adaptation to secondary substances in the host plants. Herbivores gradually evolve and adapt to changes in plant secondary substances by modification of their behavior and physiological processes. The relationship between plants, insects, and pesticides is complex. Our study provides evidence for the use of plant secondary substances in pest control. In practice, farmers do not pay much attention to combining resistant host plant varieties with pesticides to achieve positive synergistic effects and thereby reduce pesticide application. In general, sublethal concentrations of TS increased SOD, POD, and CAT activities, but the values from rape treatments were relatively lower than those from radish and cabbage. This suggests that rape treated with TS may reduce the defenses of DBM against unfavorable environmental factors, including insecticides. It may be that enzyme activities in DBM are more vulnerable to unknown secondary substances in rape. 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Environmental Entomology Oxford University Press

Effect of Tea Saponin-Treated Host Plants on Activities of Antioxidant Enzymes in Larvae of the Diamondback Moth Plutella xylostella (Lepidoptera: Plutellidae)

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Entomological Society of America
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© The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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10.1093/ee/nvy031
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Abstract

Abstract Tea saponin (TS) is extracted from the seeds of the tea plant and is generally regarded as a safe compound that has insecticidal properties and can act synergistically with other compounds. In this study, the activities of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and the levels of malondialdehyde (MDA) were compared in midgut tissues of third instar larvae of the diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae). The larvae were fed on three different host plants, cabbage (Brassica oleracea L. var. capitata [Capparales: Brassicaceae]), radish (Raphanus sativus L. var. radiculus Persi [Capparales: Brassicaceae]), or rape (Brassica campestris L. [Capparales: Brassicaceae]), that had been treated with TS. Higher SOD, POD, and CAT activities were found in DBM larvae fed on cabbage after LC20 (concentration that induced 20% larval mortality) or LC50 (concentration that induced 50% larval mortality) treatment than on the control. On rape, TS treatments led to lower SOD and CAT activities than in the control and to higher POD activities after 24 h. MDA content increased in larvae fed on rape but decreased in larvae fed on radish after 12 h. Our results indicated that DBM larvae are more susceptible to TS on rape than on cabbage and radish, suggesting that this treatment may be an economic and effective means of controlling DBM on rape. antioxidant enzyme, host plant Management of the diamondback moth (DBM, Plutella xylostella [Lepidoptera: Plutellidae]), is a major topic of pest research because of its increasing resistance to insecticides. In the past several decades, much attention has been devoted to studying the toxicity of different chemicals in the insect and its adaptation capabilities to natural pest control agents (Talekar and Shelton 1993; Wei et al. 2010, 2015; You et al. 2013; Liu et al. 2015). To date, although many natural compounds have been reported to reduce pest populations, only a few have been widely used in practice, such as nicotine, pyrethrum, and neem (Roychoudhury 2016). One potential alternative control option for DBM is tea saponin (TS), which has insecticidal properties and can act synergistically with other compounds (Wang and Huang 1998). TS is extracted from the seeds of the tea plant (Camellia sinensis L.) and is generally regarded as a safe compound. It has low toxicity to mammals but is toxic to insects, as has been shown, for example, in the butterfly Pieris rapae L. (Lepidoptera: Pieridae) and the housefly Musca domestica L. (Diptera: Muscidae) Kawaguchi et al. (1994) reported that 50–150 mg/kg per d of TS administered orally for 3 mo had no effects on male and female rats. However, TS increased the efficiency of Bacillus thuringiensis infection of Spodoptera exigua Hubner (Lepidoptera: Noctuidae) (Rizwan-ul-Haq et al. 2009) and DBM (Li et al. 2005). Toxic and antifeedant effects are the main ways through which TS exerts its effects on insects (Wang and Huang 1998). However, it was found that some antioxidant enzyme activities in P. rapae (L.) were significantly decreased after TS treatment (Wang and Huang 1999). Hence, it is essential to elucidate the variation in activities of detoxification enzymes in DBM after TS treatment if this compound is to be used as a control agent. The goal of this study was therefore to evaluate the effects of TS on antioxidant enzymes in DBM. Insects have evolved for more than 400 million yr and have developed protective enzyme systems to detoxify insecticides and secondary substances used by plants as defenses against herbivory (Zhou et al. 2011, Zhang et al. 2013). Specifically, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione-S-transferase (GST) are the major antioxidant enzyme systems in insects (Felton and Summers 1995, Wang et al. 2001, Dubovskiy et al. 2008). SOD converts superoxide into oxygen and hydrogen peroxide, the latter being eliminated by CAT and POD activities (Felton and Summers 1995, Wang et al. 2001, Dubovskiy et al. 2008). GSTs are involved in removing the products of lipid peroxidation and hydroperoxides from cells (Wang et al. 2013). The activities of SOD, POD, CAT, and reactive oxygen species (ROS) maintain a physiological equilibrium, but various abiotic and biotic stress factors, such as insecticides, pathogens, and extreme temperature stresses, can induce changes in enzyme activities to protect against damage to the physiology and metabolism of the insect (Dubovskiy et al. 2008, Wang et al. 2013, Zhang et al. 2013). ROS degrade polyunsaturated lipids to form malondialdehyde (MDA) (Pryor and Stanley 1975), which causes toxic stress in cells and forms covalent protein adducts, which are referred to as advanced lipoxidation end products (Marnett 1999, Farmer and Davoine 2007). MDA is commonly used as an indicator that physiological or oxidative stress has occurred (Farmer and Davoine 2007). In this study, we measured the enzymatic activities of SOD, CAT, POD, and also determined the MDA content in the midgut tissues of DBM larvae. The purpose of the present study was to gain insights into the effects of TS on antioxidant enzyme activities in the DBM fed on three widely-grown cruciferous vegetables host plants, namely cabbage (Brassica oleracea L var. capitata), radish (Raphanus sativus L. var. radiculus Persi), and rape (Brassica campestris L.). Materials and Methods Host Plants and Insects The cruciferous species cabbage (B. oleracea L var. capitata [Capparales: Brassicaceae]), radish (R. sativus L. var. radiculus Persi [Capparales: Brassicaceae]), and rape (B. campestris L. [Capparales: Brassicaceae]) were used in this study. All plants were grown in a greenhouse (E119°34′, N26°13′) at our laboratory without insecticide treatment. DBM larvae were originally collected from a cabbage field without insecticide treatment in Fuzhou City, Fujian province, China (E119°18′, N26°07′); this location has cultivated cabbages for over 10 yr. The populations were maintained in a rearing room (E119°34′, N26°13′) at 25 ± 1°C and 70–80% relative humidity, under a 14:10 (L:D) h cycle. The adults were introduced into a chamber containing host plants and absorbent cotton with a 100 g/kg honey solution. Insects were then allowed to mate and lay eggs. Larvae in the experiment were reared continuously on the different host plants for at least five generations to maintain population homogeneity. Reagents TS containing 90% saponin was obtained from Hanqing Biological Technology Co. Ltd., Huaihua, Hunan Province, China. Assay kits for Coomassie brilliant blue staining of enzymes (POD, CAT, and SOD) and MDA were purchased from Nanjing Jiancheng Bioengineer Institute, Jiangsu, China. Toxicity Tests LC20 and LC50 concentrations, the TS concentrations causing 20% and 50% larval mortality, respectively, were determined in toxicity tests using third instar larvae. Six concentrations of TS in distilled water (pH = 6.8) were screened after preliminary mortality tests for each host plant species. For cabbage, TS concentrations of 200, 1,000, 5,000, 25,000, and 125,000 mg liter−1 were used. For radish, TS concentrations of 64, 320, 1,600, 8,000, and 40,000 mg liter−1 were used. For rape, TS concentrations of 125, 250, 500, 1,000, and 2,000 mg liter−1 were used. Freshly prepared leaf disks (diameter = 3.4 cm) from the three host species were dipped into TS solutions for 15 s and then transferred to plastic jars (diameter = 3.4 cm, two leaf disks per jar). Moistened filter paper was placed on the bottom of each plastic jar to prevent withering of the leaf disks. Thirty third-instar larvae were starved for 4 h and then transferred into a plastic jar (30 larvae/bottle, two leaf disks/bottle). When larvae did not respond to a light touch, they were assumed to be dead. After 24 h, the number of dead larvae was recorded to estimate mortality. Each treatment was repeated three times. Assays of Enzyme Activities Tissue Collection Toxicity bioassays were used to determine the LC20 and LC50 concentrations of TS of treated leaf disks as described previously. DBM third-instar larvae (24 h after molt) were transferred to radish, rape, and cabbage leaf disks that had been treated with either the LC20 or LC50 concentration of TS for 2 h. Then 20 live third-instar DBM larvae, with three replications per treatment, were removed from radish, rape, and cabbage leaf disks after feeding for 1, 12, or 24 h and used to prepare enzyme extracts. At each sampling interval, the midgut was dissected from each larva, and the adhering fat body, Malpighian tubules and gut contents were removed, and the tissue was stored at −80°C. Enzyme Activity and MDA Content Determination For SOD activity measurement, midguts from 20 larvae were collected and placed in a chilled Eppendorf tube containing cold homogenization buffer (2.0 ml of 0.01 M Tris–HCl, pH 8.2); the homogenate was centrifuged at 3,000 rpm for 10 min. The supernatant beneath the fat layer was collected and assessed for SOD activity by an improved pyrogallol method; SOD activity was measured at 440 nm on a 752 UV-visible spectrophotometer (Jing and Zhao 1995). For POD activity measurement, midguts from another 20 larvae were collected as described previously and placed in a cold homogenization buffer (2.0 ml 0.05 M phosphate buffer, pH 7.0); the homogenate was treated as described previously, and POD activity was determined at 470 nm on a 752 UV-visible spectrophotometer (Simon et al. 1974). For CAT activity measurement, midguts from another 20 larvae were collected and homogenized in cold homogenization buffer (2.0 ml of 0.05 M phosphate buffer, pH 7.0) as described previously. The CAT activity was assessed in the supernatant after centrifugation by measuring H2O2 consumption, using the decrease in absorbance at 230 nm on a 752 UV-visible spectrophotometer (Aebi 1983). MDA contents were assessed using the midguts of 20 larvae; the midguts were incubated at 95°C in thiobarbituric acid under aerobic conditions (pH 3.4). The MDA content was measured in a thiobarbituric acid assay at 532 nm on a 752 UV-visible spectrophotometer, based on the release of a color complex following reaction of thiobarbituric acid with MDA (Ohkawa et al. 1979). Total protein concentration of extracts was determined using a Lowry protein assay (Fryer et al. 1986). All enzyme extracts were kept in an ice water bath before analysis. Extraction was performed in triplicate for each sampling time. For all enzyme assays, the protein content of samples was measured using bovine serum albumin as a standard, as described by Bradford (1976). Each treatment was repeated three times. Statistical Analysis The mortality data were subjected to probit analysis to determine LC20 and LC50 using the SPSS 15.0 data processing system. Mean values of enzyme activities were compared by a post hoc Duncan’s Multiple Range Test with SAS 6.12 software. Significance was set at P < 0.05. Significant differences between treatment groups on the same host species were analyzed for analysis of variance using the SPSS 15.011.5 software package. Results Mortality in Third-Instar DMB Larvae After TS Treatments The LC20 values for third-instar DBM larvae fed on radish, rape, and cabbage were estimated as 259.98, 162.43, and 272.16 mg liter−1, respectively; LC50 values were 5,148.95, 570.54, and 3,608.56 mg liter−1, respectively (Table 1). Overall, larvae exposed to treated rape showed higher rates of mortality. Table 1. Lethal concentration values of tea saponin against third instar larvae of the diamondback moth Host plant  Slope of regression equation (b) (SE)  LC20 (mg/l) (95% CI)  LC50 (mg/l) (95% CI)  χ2  df  Sig.  Radish  0.649 (0.070)  259.98 (121.52–455.19)  5148.95 (3266.51–8769.71)  1.896  3  0.594  Rape  1.543 (0.159)  162.43 (112.91–211.33)  570.54 (472.78–692.91)  6.152  3  0.104  Cabbage  0.750 (0.017)  272.16 (125.42–483.05)  3608.56 (2393.04–5343.58)  6.140  3  0.105  Host plant  Slope of regression equation (b) (SE)  LC20 (mg/l) (95% CI)  LC50 (mg/l) (95% CI)  χ2  df  Sig.  Radish  0.649 (0.070)  259.98 (121.52–455.19)  5148.95 (3266.51–8769.71)  1.896  3  0.594  Rape  1.543 (0.159)  162.43 (112.91–211.33)  570.54 (472.78–692.91)  6.152  3  0.104  Cabbage  0.750 (0.017)  272.16 (125.42–483.05)  3608.56 (2393.04–5343.58)  6.140  3  0.105  View Large Table 1. Lethal concentration values of tea saponin against third instar larvae of the diamondback moth Host plant  Slope of regression equation (b) (SE)  LC20 (mg/l) (95% CI)  LC50 (mg/l) (95% CI)  χ2  df  Sig.  Radish  0.649 (0.070)  259.98 (121.52–455.19)  5148.95 (3266.51–8769.71)  1.896  3  0.594  Rape  1.543 (0.159)  162.43 (112.91–211.33)  570.54 (472.78–692.91)  6.152  3  0.104  Cabbage  0.750 (0.017)  272.16 (125.42–483.05)  3608.56 (2393.04–5343.58)  6.140  3  0.105  Host plant  Slope of regression equation (b) (SE)  LC20 (mg/l) (95% CI)  LC50 (mg/l) (95% CI)  χ2  df  Sig.  Radish  0.649 (0.070)  259.98 (121.52–455.19)  5148.95 (3266.51–8769.71)  1.896  3  0.594  Rape  1.543 (0.159)  162.43 (112.91–211.33)  570.54 (472.78–692.91)  6.152  3  0.104  Cabbage  0.750 (0.017)  272.16 (125.42–483.05)  3608.56 (2393.04–5343.58)  6.140  3  0.105  View Large Enzyme Activities in Third-Instar Larvae Fed on TS-Treated Host Plants Effect of TS on SOD Activities Overall, except for rape at 24 h, SOD activities in larvae fed on LC50-treated host plants were higher than in those fed on untreated host plants (Fig. 1). TS treatment led to significantly lower SOD activities in rape at 24 h than observed in the control (Fig. 1C). SOD activity in larvae fed TS-treated radish increased continuously from 1 h to the end of the experiment (Fig. 1B). Fig. 1. View largeDownload slide Superoxide dismutase activities in third instar larvae fed on different host plants: (A) cabbage; (B) radish; and (C) rape. The controls were untreated host plants. Different lowercase letters indicate significant differences between treatment groups on the same host species (P < 0.05). ANOVA statistics were: cabbage (F8,27 = 9.310, P < 0.001); radish (F8,27 = 348.910, P < 0.001); and rape (F8,27 = 61.824, P < 0.001). Fig. 1. View largeDownload slide Superoxide dismutase activities in third instar larvae fed on different host plants: (A) cabbage; (B) radish; and (C) rape. The controls were untreated host plants. Different lowercase letters indicate significant differences between treatment groups on the same host species (P < 0.05). ANOVA statistics were: cabbage (F8,27 = 9.310, P < 0.001); radish (F8,27 = 348.910, P < 0.001); and rape (F8,27 = 61.824, P < 0.001). In larvae fed on radish, the final SOD activity in LC20 treatments was 79.30 U/mg protein with an initial value of 60.54 U/mg protein. However, SOD activity in the control was 55.49 U/mg protein at 24 h. The SOD activity from LC50 gradually increased from the initial sampling interval to 24 h (Fig. 1B). In larvae fed on rape, SOD activity in the LC50 treatments rose at 12 h to 16.44 U/mg protein; SOD activity declined at 24 h (Fig. 1C). Effect of TS Treatment on POD Activity POD activities in larvae fed on cabbage and radish treated with LC20 and LC50 concentrations of TS were significantly higher at 12 h and 24 h than in the control (Fig. 2A and B). Enzyme activities in the LC50 treatments were higher than those of LC20 and control and reached a level of 27.55 U/mg protein in the 12-h sample (Fig. 2B). Fig. 2. View largeDownload slide Peroxidase activities in third instar larvae fed on different host plants: (A) cabbage; (B) radish; and (C) rape. Controls were untreated host plants. Different lowercase letters indicate significant differences between concentrations and sampling times in the same host species (P < 0.05). ANOVA statistics were: cabbage (F8,27 = 303.171, P < 0.001); radish (F8,27 = 161.919, P < 0.001); and rape (F8,27 = 280.359, P < 0.001). Fig. 2. View largeDownload slide Peroxidase activities in third instar larvae fed on different host plants: (A) cabbage; (B) radish; and (C) rape. Controls were untreated host plants. Different lowercase letters indicate significant differences between concentrations and sampling times in the same host species (P < 0.05). ANOVA statistics were: cabbage (F8,27 = 303.171, P < 0.001); radish (F8,27 = 161.919, P < 0.001); and rape (F8,27 = 280.359, P < 0.001). In larvae fed on rape, POD activities increased from 1 h and reached their highest levels at 24 h. Overall, TS increased POD activities at both LC20 and LC50 concentrations (Fig. 2C). Effect of TS on CAT Activity Compared with the control, the LC50 treatments led to higher CAT activities in larvae fed on cabbage and radish at 12 h and 24 h (Fig. 3A and B). In larvae fed on rape, CAT activity increased from 1 h to 12 h for the LC50 treatment, but this declined at 24 h (Fig. 3C). In contrast, CAT activity in the LC20 treatment groups rose continuously to 67.62 U mg−1 protein at 24 h (Fig. 3C). Fig. 3. View largeDownload slide Catalase activities in third instar larvae fed on different host plants: (A) cabbage; (B) radish; and (C) rape. Controls were untreated host plants. Different lowercase letters indicate significant differences between concentrations and sampling times on the same host species (P < 0.05). ANOVA statistics were: cabbage (F8,27 = 29.422, P < 0.001); radish (F8,27 = 10.717, P < 0.001); and rape (F8,27 = 20.174, P < 0.001). Fig. 3. View largeDownload slide Catalase activities in third instar larvae fed on different host plants: (A) cabbage; (B) radish; and (C) rape. Controls were untreated host plants. Different lowercase letters indicate significant differences between concentrations and sampling times on the same host species (P < 0.05). ANOVA statistics were: cabbage (F8,27 = 29.422, P < 0.001); radish (F8,27 = 10.717, P < 0.001); and rape (F8,27 = 20.174, P < 0.001). Effect of TS on MDA Content The level of MDA content in the larvae varied with the host plant species. TS treatment inhibited MDA content in larvae fed cabbage and radish for the LC50 treatment at 1 h as compared to the control. In larvae fed on cabbage, lower contents were found after the LC50 treatment than in the LC20 treatment at 1 and 12 h (Fig. 4). At 12 h, a higher MDA content was found in larvae fed on rape; however, larvae fed on radish had a significantly lower MDA content than did the control at 12 h. At 24 h, the TS treatments did not alter MDA content from that in the controls (Fig. 4). Fig. 4. View largeDownload slide Malondialdehyde content in third instar larvae fed on different host plants: (A) cabbage; (B) radish; and (C) rape. Controls were untreated host plants. Different lowercase letters indicate significant differences between concentrations and sampling times on the same host species (P < 0.05). ANOVA statistics were: cabbage (F8,27 = 3.318, P = 0.009); radish (F8,27 = 24.037, P < 0.001); and rape (F8,27 = 20.173, P < 0.001). Fig. 4. View largeDownload slide Malondialdehyde content in third instar larvae fed on different host plants: (A) cabbage; (B) radish; and (C) rape. Controls were untreated host plants. Different lowercase letters indicate significant differences between concentrations and sampling times on the same host species (P < 0.05). ANOVA statistics were: cabbage (F8,27 = 3.318, P = 0.009); radish (F8,27 = 24.037, P < 0.001); and rape (F8,27 = 20.173, P < 0.001). Discussion In recent years, the efficacy of botanical insecticides against DBM has been evaluated (Tang et al. 2013, Nasr et al. 2017). Our study focussed on whether TS might be an effective alternative for the control of P. xylostella larvae, by delaying the resistance levels to a chemical pesticide. We showed that although rape and cabbage are closely related species of the same genus, higher LC20 and LC50 values were found in cabbage. This effect may be because DBM is more adapted to feeding on cabbage than rape. In addition, our results indicated that TS may be an economic and effective means of controlling DBM on rape. Many previous studies have shown that host plants can have different effects on physiological and biochemical traits in herbivores, such as on the activities of protective enzymes and detoxification enzymes (Abd-Elghafar et al. 1989, Wang et al. 2010, Zhou et al. 2011, Deng et al. 2013, Tang et al. 2013, Zhang et al. 2013, Cai et al. 2016, Nasr et al. 2017). For example, in comparison to untreated controls, insects treated with essential oil of Origanum vulgare L. show a significant decrease in alkaline phosphatase and protease activities; however, lipase activity is higher at 24 h after treatment than in the control. Additionally, with regard to detoxification enzymes such as GST and esterase, treated insects show significant differences compared with the controls (Nasr et al. 2017). It has been reported that P. xylostella that have been fed green Chinese cabbage treated with extracts from Ginkgo biloba L. show significant variations in SOD, CAT, and POD activities compared with the controls (Tang et al. 2013). In other examples, some antioxidant enzymes in Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae) and Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) vary among host plants (Zhou et al. 2011, Zhang et al. 2013). Furthermore, antioxidant enzyme activity in P. rapae (L.) significantly decreases after TS treatment (Wang and Huang 1999). In the present study, we found higher SOD, POD, and CAT activities in third-instar DBM larvae that had fed for 12 h on leaf disks from three host plant species treated with an LC50 concentration of TS. Thus, DBM can show some resistance to TS treatment, which may be similar to that found in P. rapae larvae that degrade harmful free radicals (O2−) after treatment with cypermethrin for 10 min (Li et al. 1994). Moreover, larvae exposed to LC50-treated rape leaf disks had higher SOD and CAT activities at 12 h than at 24 h. Similar responses have been reported for larvae of the Chinese pine caterpillar (Dendrolimus tabulaeformis Tsai et Liu) after treatment with the insecticidal fungus Beauveria bassiana (Bals.) (Liu et al. 2005) and for Parasa consocia Walker and Thosea postornata (Hampson) treated with deltamethrin (Li et al. 1994). The lower CAT activities at 24 h might indicate that a high level of free radicals accumulated in larval cells, which would eventually be toxic to the larvae. Overall, our results indicate that TS treatment disrupted the activities of SOD, CAT, and POD and suggested that this disruption might increase the resistance risk of DBM to TS. A possible explanation of the responses of DBM to the defense systems of its cruciferous host plants, particularly to the glucosinolate-myrosinase complex (the so-called ‘mustard oil bomb’), has been suggested by Ratzka et al. (2002). DBM modifies ingested plant glucosinolates by a sulfatase enzyme in the gut to prevent hydrolysis of the glucosinolates by plant myrosinase into a highly toxic product, such as isothiocyanate. In our study, the higher enzyme activities in radish might be due to DBM being more adapted to this species than other cruciferous plants. DBM have evolved various detoxification mechanisms that allow rapid adaptation to new host plants or new pesticides. Our results showed that TS had no effect on MDA contents after 24 h. These results, together with our evidence that lower values of GST were found in larvae fed a susceptible host plant, indicate that the larvae adjusted their enzyme activities to adapt to unfavorable plant substances. There is no doubt that insect resistance mechanisms are associated with adaptation to secondary substances in the host plants. Herbivores gradually evolve and adapt to changes in plant secondary substances by modification of their behavior and physiological processes. The relationship between plants, insects, and pesticides is complex. Our study provides evidence for the use of plant secondary substances in pest control. In practice, farmers do not pay much attention to combining resistant host plant varieties with pesticides to achieve positive synergistic effects and thereby reduce pesticide application. In general, sublethal concentrations of TS increased SOD, POD, and CAT activities, but the values from rape treatments were relatively lower than those from radish and cabbage. This suggests that rape treated with TS may reduce the defenses of DBM against unfavorable environmental factors, including insecticides. It may be that enzyme activities in DBM are more vulnerable to unknown secondary substances in rape. 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Environmental EntomologyOxford University Press

Published: Mar 20, 2018

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