Effects of Five Pesticides on Toxicity, Detoxifying and Protective Enzymes in Phauda flammans Walker (Lepidoptera: Zygaenidae)

Ficus (Urticales: Moraceae) are important decorative trees which used to line avenues in the urban landscapes of China and Southeast Asian countries (Liu et al., 2016). However, biodiversity descended obviously in an ecosystem or landscape with the increasing of the cultivated area of Ficus resulting in a sharp rise of pest species (Arthurs et al., 2016; Bhandari and Cheng, 2016).

Phauda flammans Walker (Lepidoptera: Zygaenidae) is one of the notorious defoliating pests of Ficus in southern China (Liu et al., 2015a), India (Nageshchandra et al., 1972; Verma and Dogra, 1982), Vietnam (FOF, 2015) and Tailand (Anonymous, 2017). P. flammans develops 2-3 generations per year in Nanning City, Guangxi, southern China. Adults fly in the daytime. Eggs are aggregated on leaves. The larval peaks of the first and second generations occur from mid-May to late June and early August to mid-October, respectively. Most larvae prefer to pupate near roots and tussocks exposed on the ground, and only a few individuals pupate in the topsoil. Larvae of this species can be found on F. microcarpa leaves even during winter (Liu et al., 2014, 2015a). P. flammans is a chill-intolerant species, which larvae can live safely on F. concinna and F. benjamina leaves during winter (Liu et al., 2015b;Zheng et al., 2017).

Although four species of insect parasitoids were identified from P. flammans, chemical control is one of the main ways for the management of P. flammans due to the lower parasitoid rates of their enemies (Zheng et al., 2015). However, much less is known of the effective pesticides and its concentrations to control them, especially during the period of outbreak.

The detoxifying and protective enzymes of insects, removing or dissolving toxic substances in the body for self-protection, mainly include carboxylesterase (CarE), glutathione-s transferase (GST), and superoxide dismutase (SOD), catalase (CAT) (Bolter and Chefurka, 1990; Zou et al., 2017). The activities of detoxifying and protective enzymes varied after different pesticides act on insects due to the different mechanisms (Desneux et al., 2007). Thus, study on the effects of different pesticides on detoxifying and protective enzymes in insects can provide theoretical basis for rational using of pesticides (Zhang et al., 2009; Allen` and Balin, 1989).

The purpose of this study is to explore the effects of several pesticides on toxicity against P. flammans larvae, and their effects on detoxifying and protective enzymes. Addressing this issue is conducive to provide reasonable suggestion to manage the P. flammans population.

Larvae of 1st - 6th instar were collected from Guangxi University (108°29’E, 22°85’N) in Nanning City, Guangxi, southern China. Three P. flammans larvae were reared with fresh leaves of F. microcarpa on each petri dish (d = 9 cm) in laboratory with a light photoperiod of L16:D8 h and 70 ± 5% relative humidity maintained at 27 ± 1 °C. Leaves were renewed daily. Larval instars were determined according to Liu et al. (2015a).

Five pesticides were tested: beta-cypermethrin 3% EW, abamectin 16% SW, chlorpyrifos 30% EW, thiamethoxam 25% SC and 36% bisultap mother liquor. All of the pesticides were provided by Guangxi Tianyuan Biochemical Co., Ltd. (Guangxi, China).

Carboxylesterase (CarE) activity determination kit, glutathione-s transferase (GST) assay kit, superoxide dismutase (SOD) test box, catalase (CAT) test box, coomassie brilliant blue G-250 were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Bovine serum albumin, dibasic sodium phosphate (Na2HP04), sodium dihydrogen phosphate (NaH2PO4), phosphoric acid (analytical reagent, purity ≥ 95%), ethanol (analytical reagent, purity ≥ 95%), acetic acid (analytical reagent, purity ≥ 95%), acetone (analytical reagent, purity ≥ 95%) were obtained from Tianjin Fuyu Fine Chemical Co. Ltd. (Tianjin, China).

The leaf-dipping method was used to determine the toxicity of five pesticides to P. flammans larvae. Based on the preliminary experiments, pesticides were diluted to a series of concentrations as the experimental treatment and the distilled water as the control (Wang et al., 2012; Rong et al., 2016). The leaves of F. microcarpa without any pesticides were punched by puncher (Φ = 2cm), and immersed in the diluted pesticides for 10 s. These leaves were used to rear 1st - 6th instar larvae (3 larvae / petri dish) after natural drying, and maintained at 27 ± 1 °C placed with a light photoperiod of L16: D8 hr and 70 ± 5% relative humidity in laboratory.

This experiment was three replications with total of 60 per instars in each concentration and pesticide. After treatment of 8 h, 12 h, 24 h, 48 h, the number of dead larvae of P. flammans were counted. In this experiment, the volume concentrations of beta-cypermethrin, abamectin, chlorpyrifos, thiamethoxam and bisultap were 6, 7.5, 10, 15 and 30 mg/L; 32, 40, 53.3, 80 and 160 mg/L; 60, 75, 100, 150 and 300 mg/L; 50, 62.5, 83.3, 125 and 250 mg/L; 72, 90, 120, 80 and 360 mg/L.

According to the results of toxicity, the LC50 of each pesticide was selected as sub-lethal metrology to study the effects of five pesticides on detoxifying and protective enzymes in 4th instar larvae of P. flammans. The result of this experiment was expressed as the enzyme specific activity (Rumpf et al., 1997; Quan et al., 2016). The leaf-dipping method was used as mentioned above, and the 4th instar larvae after treatments for 24 h and 48 h were collected as experimental materials.

Larvae (n = 9) treated by different pesticides and times were selected, respectively. All selected larvae were placed in precooled mortar, and then 2 mL phosphate buffer (0.1 mol/L, pH = 7.0) were put to mill in the ice bath. The mixture were centrifugalized with TGL-16 high-speed refrigerated centrifuge (Hunan Xiangyi Laboratory Instrument Development Co. Ltd., Hunan, China) in the 2 ml centrifuge tube (4 °C, 10000 r/min) for 15 min after milling. The supernatant stored at -20°C for the following experiments.

According to Bradford (1976) method, 0.1 ml enzyme, 0.9 ml distilled water and 5 ml Coomassie Brilliant Blue G-250 were added to a test tube and the contents mixed either by inversion or vortexing. The control group without enzyme was added in the same reagents as the experiment group. The absorbance at 595 nm was measured with visible spectrophotometer (Spectrumlab 722 sp, Shanghai Jiguang Technology Co. Ltd., Shanghai, China) after 2-3 min, and this experiment was three replications. The protein content was calculated according to the standard curve of bovine serum protein.

CarE, GST, SOD and CAT activities were determined by using assay kit (Nanjing Jiancheng Biotechnology Institute, Nanjing, China), and the tests were carried out strictly with the assay kit directions. The experiments were replicated three times.

Statistical analysis was performed using SPSS 16.0 (SPSS, Chicago, IL, USA). Duncan’s multiple range tests was used for significance test (P ≤ 0.05).

The LC50 values of the same pesticide increased gradually with increasing of the larval instar of P. flammans. The LC50 values of the same pesticide decreased gradually with increasing of the treatment time (Table I). Beta-cypermethrin showed the highest toxicity for P. flammans larvae in the five pesticides. Abamectin had no significant lethal effect for P. flammans 1st instar larvae at 8 h after treatment in the five pesticides. Bisultap had the lowest toxicity for P. flammans 2th instar larvae at 24h after treatment and 3th - 5th instar larvae at 48h after treatment, while the lowest toxicity for P. flammans 6th instar larvae at 48h after treatment was thiamethoxam. The order of toxicity against P. flammans larvae from high to low for the five pesticides was as follows: beta-cypermethrin, abamectin, chlorpyrifos, thiamethoxam and bisultap.

There were different effects of five pesticides on detoxifying enzymes in P. flammans larvae (Fig. 1A, B). Compared with control, CarE activity in P. flammans 4th instar larvae at 24h (F = 55.938, df = 5, 17, P < 0.001) and 48h (F = 15.148, df = 5, 17, P < 0.001) after treatment were significant difference in the five pesticides. Activity of CarE was promoted by beta-cypermethrin, the effect of which on CarE activity was the most obvious (Fig. 1A). Compared with control, GST activity in P. flammans 4th instar larvae at 24h (F = 6.926, df = 5, 17, P < 0.01) and 48h (F = 21.771, df = 5, 17, P < 0.001) after treatment were significant difference in the five pesticides. Beta-cypermethrin, thiamethoxam and bisultap played a significant role in promoting GST activity. Beta-cypermethrin and bisultap promoted the activity of GST, and weakened slowly, but thiamethoxam always enhanced the activity of GST (Fig. 1B). As the results showed, effects of beta-cypermethrin on the CarE and GST activities were the most obvious among the five pesticides.

Five pesticides had different effects on detoxifying enzymes of P. flammans larvae (Fig. 1C, D). Compared with control, SOD activity in P. flammans 4th instar larvae at 24h (F = 76.773, df = 5, 17, P < 0.01) and 48h (F = 116.253, df = 5, 17, P < 0.001) after treatment were statistical significance in the five pesticides. Beta-cypermethrin and chlorpyrifos promoted the activity of SOD, while abamectin and bisultap inhibited SOD activity; thiamethoxam inhibited the SOD activity at first and promoted it at later (Fig. 1C). Compared with control, CAT activity in P. flammans 4th instar larvae at 24h (F = 3.320, df = 5, 17, P < 0.05) and 48h (F = 57.217, df = 5, 17, P < 0.001) after treatment were statistical significance in the five pesticides. Effects of beta-cypermethrin, chlorpyrifos and bisultap on CAT activity had positive impacts, while abamectin and thiamethoxam showed no significant effects on CAT activity compare with the control (Fig. 1D). As the results, effect of beta-cypermethrin on the SOD activity was the most obvious among five pesticides, and chlorpyrifos had the greatest effect on CAT activity.

Beta-cypermethrin, a pyrethroid pesticide, is an efficient isomer of cypermethrin with high biological activity, which has the characteristics of wide insecticide controlling spectrum, rapid effects and high insecticidal activity (Qu et al., 2017). Wang et al. (2012) found that beta-cypermethrin had high toxicity in the toxicity test of 6 pesticides on Histia rhodope Ceamer. Our results also indicated that low-dose beta-cypermethrin had good control effect on 1st-6th instar larvae of P. flammans. Abamectin, an antibiotic pesticide, had high toxic activity to Achelura yunnanensis Horie&Xue (Rong et al., 2016). In our study, the mortality of larvae rose with treated time delay of abamectin. The changes of detoxifying and protective enzymes in insects are different under the action of pesticides (Li et al., 1994; Ding et al., 2001). In order to achieve detoxification, insects will oxidize, restore, hydrolyze or combine the toxic compounds entered into the body from natural environment by increasing the activity of their detoxifying enzymes, so as to enhance the water-solubility of the toxic compounds, and make it easy to eliminate toxins from the body or turn them into low-toxic or non-toxic substances (Xing et al., 2011; Zhang et al., 2015; Jia et al., 2016a). The enhancement of detoxifying enzymes activity is one of the mechanisms for insects to improve the resistance against pesticides. For example, abamectin and beta-cypermethrin could induce the increasing of GST activity in Plutella xylostella L. (Liang et al., 2003). However, some pesticides killed insects by inhibiting the activity of detoxifying enzymes. For example, sub-lethal concentration of abamectin could obviously inhibit the GST activity of Diadegma semiclausum Hellen (Jia et al., 2016b). Results from the current study showed that beta-cypermethrin promoted the activities of both CarE and GST in P. flammans larvae, while abamectin promoted CarE activity but inhibited GST activity. It indicated that the sub-lethal concentration of beta-cypermethrin induced the increase of detoxifying enzymes activity to enhance the detoxification ability of P. flammans larvae. Therefore, we speculated that P. flammans might resist beta-cypermethrin when it used for a long time. The sub-lethal concentration of abamectin inhibited detoxifying enzymes activity in P. flammans, which reduced the ability of larvae to degrade pesticides and enhanced its toxicity. Furthermore, effect of beta-cypermethrin on SOD activity in Blattella germanica appeared a trend of “rise-decrease-rise”, which was reported by Ma et al. (2009). However, it was different from the positive effects of beta-cypermethrin on SOD and CAT activities in P. flammans larvae. It hinted that P. flammans larvae resisted beta-cypermethrin mainly by enhancing the protective enzymes activity. Abamectin could inhibit SOD activity, but effect of abamectin on CAT activity was not significant, which suggested that abamectin could destroy protective enzymes when it acted on P. flammans larvae for killing them.

Although the toxicity of beta-cypermethrin was the best in this study, it significantly activated the activity of detoxifying enzymes in P. flammans which may lead to insect resistance. Effect of abamectin on toxicity was slightly inferior than beta-cypermethrin, but it effectively inhibited detoxifying and protective enzymes activities. Therefore, we recommend that P. flammans larvae could be effectively controlled by beta-cypermethrin 10 mg/L and abamectin 25 mg/L with alternate spray, which could prevent the resistance to pesticides in this pest.

We are grateful to the anonymous referees for their valuable comments on an earlier version. This work was supported by the National Natural Science Foundation of China (31660206) and Natural Science Foundation of Guangxi (2015GXNSFAA139056).

Authors have declared no conflict of interest.