Effect of heat shock on the susceptibility of Frankliniella occidentalis (Thysanoptera: Thripidae) to insecticides

Journal of Integrative Agriculture 2016, 15(10): 2309–2318 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Effect of heat shock on the susceptibility of Frankliniella occidentalis (Thysanoptera: Thripidae) to insecticides ZHANG Bin1, ZUO Tai-qiang1, LI Hong-gang3, SUN Li-juan1, WANG Si-fang1, ZHENG Chang-ying1, WAN Fang-hao1, 2 1

Key Laboratory of Integrated Crop Pest Management of Shandong, College of Agronomy and Plant Protection, Qingdao Agricultural University, Qingdao 266109, P.R.China 2 Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China 3 Institute for the Control of Agrochemicals of Shandong Province, Jinan 250100, P.R.China

Abstract Currently, insecticides are considered as the primary approach for controlling western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). However, the heavy use of insecticides resulted in high insect resistance and serious environmental pollution. Given its characteristics of ease of operation and environmental friendliness, insect control using high temperature is receiving considerable renewed research interest. However, although the combination of insecticides and high temperature to control F. occidentalis has been studied before, few studies have focused on the short-term effect of such treatment. In a laboratory study, F. occidentalis adults and second-instar nymphs were exposed to 45°C for 2 h. Then, their susceptibility to acetamiprid, spinosad, methomyl, and beta-cypermethrin was tested after different periods of recovery time (2–36 h). Additionally, the specific activity of three detoxification enzymes (esterase, glutathione S-transferase, and cytochrome p450 (CYP) monooxygenase) of the treated insects was determined. The results indicated that the fluctuation of susceptibility to insecticides and detoxification enzyme activity during F. occidentalis recovery from heat shock are related. Furthermore, several recovery time points (2, 30, and 36 h) of significant susceptibility to four tested insecticides compared with the control were found during the treatment of adults that were heat-shocked. Recovery time points of higher susceptibility compared with the control depended on different insecticides during the second-instar nymph recovery from heat shock. Interestingly, the fluctuation of CYP monooxygenase activity exhibited a trend that was similar to the fluctuation of susceptibility to insecticides (especially spinosad) during the recovery from heat shock of adults. In addition, the glutathione S-transferase and CYP monooxygenase activity trend was similar to the trend of susceptibility to spinosad during the recovery from heat shock of second-instar nymphs. Our results provide a new approach for controlling F. occidentalis using the combined heat shock and insecticide. This effectively enhances the control efficiency of heat shock and significantly reduces the application of insecticides. Keywords: Frankliniella occidentalis, heat shock, susceptibility, detoxification enzymes, insecticide

Received 8 March, 2016 Accepted 22 June, 2016 ZHANG Bin, E-mail: [email protected]; Correspondence ZHENG Chang-ying, E-mail: [email protected]

1. Introduction

© 2016, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(16)61431-4

Among the current pest management strategies, insecticides remain one of the most effective prevention and control

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measures (Lee 2000). However, concurrently with the increasing use of insecticides, the resistance to these chemicals is also increasing. Western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), is a ‘6S’ pest, which means small size, short generation time, six developmental stages, strong reproductive ability, serious crop plant damage, significant plant virus vector, which has become one of the most significant pests that affects commercial vegetables, fruits, and ornamental crops worldwide (Kirk and Terry 2003). In China, F. occidentalis was first recorded in Beijing in 2003 (Zhang et al. 2003). Since then, it was reported in Shangdong (Zheng et al. 2007), Yunnan (Liang et al. 2007), Guizhou (Yuan et al. 2008), Hunan (Liu et al. 2010), and Jiangsu provinces (Yan et al. 2010). The potential distribution of F. occidentalis was further extended by the geographical spread of the greenhouse effect, which encouraged the invasion. Currently, insecticides are considered as the primary approach for controlling F. occidentalis. The pest’s short generation time, high female fecundity, and haplodiploid reproduction system enabled F. occidentalis to evolve different levels of resistance to various insecticides including organochlorines, organophosphates, carbamates, pyrethroids, and spinosad (Immaraju et al. 1992; Brødsgaard 1994; Zhao et al. 1995; Broadbent and Pree 1997; Jensen 2000a). General mechanisms for insecticide resistance include metabolic detoxification, reduced penetration of toxicants, alterations of target sites for toxicants, and behavioral resistance (Brattsten et al. 1986). Most documented cases of insecticide resistance in F. occidentalis result from generalized metabolic detoxification. Herbivorous insects, including F. occidentalis, have three general enzymatic systems: cytochrome p450 (CYP)-dependent monooxygenases, esterases, and glutathione S-transferases (GSTs) (Li et al. 2007). The heavy use of insecticides leads to pest resistance, death of natural predators, and environmental pollution. Because food safety issues become increasingly prominent, societal demand for green and organic agricultural products is rapidly increasing. Currently, due to ease of operation and environmental friendliness, the use of high temperature to control pests is receiving renewed attention. Previous studies showed that high temperature was effective for controlling pests (Zhou et al. 2002; Wang and Fan 2003; Mahroof et al. 2005; Kalosaka et al. 2009; Wang et al. 2014). However, because it is difficult to control temperature in the field, this method is rarely used, whereas it is used in greenhouses to prevent plant diseases and insect pests (Ma et al. 2008). To avoid heat damage to plants, the greenhouse temperature should be increased to 40–50°C for only 1.5–2 h, with at least 12–15 d between each temperature increase (Zhou et al. 2004). The combined effect of temperature and insecticides

on insects was studied previously. The research showed that insects had cross-tolerance to high temperature and to insecticides (Liu et al. 2007; Feng et al. 2010). The activity of enzymes was affected, and toxicity of insecticides was increased with the temperature increase (Li et al. 2011). However, most of the previous results about the cross effect were documented using the high temperature and/or resistant strains, which usually developed for at least several days. Few studies focused on determining insect susceptibility to and enzymatic activity against insecticides after short-term (several hours) exposure to high temperatures. Therefore, the aim of this study was to determine the optimal time for insecticide application after exposure to high temperature in a greenhouse system. We measured changes in the susceptibility of F. occidentalis to different insecticides following heat exposure and explored the physiological mechanisms of that susceptibility by determining the specific activity of three detoxifying enzymes. The results provided valuable suggestions for effectively integrating the use of insecticides and high temperature, reducing insecticide dose, enhancing high temperature control efficiency, and for developing F. occidentalis integrated management strategies.

2. Materials and methods 2.1. Insects and treatments F. occidentalis that was used in this study was originally collected from clover (Trifolium repens L.) at the Experimental Station of Qingdao Agricultural University, Shandong, China. The colony was maintained on purple cabbage (Brassica oleracea L.) plants in separate greenhouses under constant conditions ((25±2)°C, 50–60% relative humidity (RH), with 16 h day length). During the summer, the greenhouse temperature was maintained at 40–50°C for 1.5–2 h, which was generally harmless to the crop. A previous study showed that the survival rate of different developmental stages of F. occidentalis was greater than 80% when exposed to 41 or 43°C for 2 h. Whereas, the survival rate of F. occidentalis second-instar nymphs and adults was less than 20 and 70%, respectively, when they were exposed to 45°C for 2 h (Wang et al. 2014). Therefore, we chose a 45°C exposure temperature and a 2 h exposure time to examine the effect of heat shock on the susceptibility of F. occidentalis to insecticides. Thousands of second-instar nymphs and female adults within 1 day of emergence were collected in rearing bottles, which were placed in an incubator (Jiangnan Instrument Factory, Ningbo, China) at 45°C for 2 h. Then, the heat-shocked F. occidentalis (approximately 16 000 second-instar nymphs and approximately 20 000 female adults) were held in

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another incubator at 25°C, 50–60% RH, with a 16 h day length. These heat-shocked thrips were allowed to recover for different periods of time (2, 4, 8, 12, 16, 20, 24, 30, or 36 h). The susceptibility to acetamiprid, spinosad, methomyl, and beta-cypermethrin was tested. Female adults or second-instar nymphs that were allowed to recover from heat shock for different periods of time were immediately stored at –80°C for further detoxification enzyme assay.

2.2. Bioassays The susceptibility of F. occidentalis to different insecticides was determined using the leaf-dip bioassay method, as described by Rueda and Shelton (2003) with slight modifications. The following four insecticides were used in this study: acetamiprid (96% technical, Hebei Veyong Bio-Chemical Co., Ltd., China), spinosad (91% technical, Hebei Veyong Bio-Chemical Co., Ltd., China), methomyl (98% technical, Jiangsu Changlong Chemicals Co., Ltd., China), and beta-cypermethrin (95% technical, Nanjing Red Sun Co., Ltd., China). Five concentrations of each of the above insecticides were prepared by dissolving in acetone (99.5% analytical, Laiyang Kangde Chemical Co., Ltd., China). Further, 220 mL of insecticide solution at each concentration was dropped into one glass vial with a diameter of 2 cm and a height of 8 cm. To accelerate the volatilization of insecticide and to make the solution cover the glass vial surface, each glass vial was slowly rolled by hand for 10 min. Leaf discs with 10 mm diameters were made from B. oleracea. The leaf discs were dipped into solutions with different concentrations for 10 s and then were placed into glass vials after air-drying. All solutions with different concentrations were mixed with a small amount of 0.1% Tween 80 (Tianjin Fuyu Chemical Co., Ltd., China). The control group was treated only with acetone and 0.1% Tween 80. A total of 10–20 adults or second-instar nymphs were transferred from heat-treated colony to leaf discs. Then, the glass vials were sealed with a 200-mesh nylon gauze and placed in an incubator at 25°C, 50–60% RH, with a 16 h day length. Mortality was assessed after 48 h. Adult females that could not move after stimulation with a camel hair brush were considered to be dead.

2.3. Detoxification enzyme assay Sample preparation For esterase and GST assays, 100 adult or second-instar nymph thrips in each microcentrifuge tube were homogenized in a precooled 0.1 mol L–1 phosphate buffer (pH 7.5). Then, the homogenates were centrifuged, and the supernatant was collected for assaying. Each microcentrifuge tube was identical, and each experi-

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ment was replicated five times. For the CYP monooxygenase assay, 150 adult or second-instar nymph thrips in each microcentrifuge tube were homogenized in a precooled 0.1 mol L–1 phosphate buffer (pH 7.8) containing 1 mmol L–1 EDTA, 1 mmol L–1 DTT, 1 mmol L–1 PTU, and 1 mmol L–1 PMSF. After the homogenates were centrifuged, the supernatants were centrifuged again. Then, the supernatants were collected and used for the assay. Each microcentrifuge tube was identical, and each experiment was replicated five times. Enzyme assay The protein content of homogenates was determined using the method of Bradford (1976), with bovine serum albumin as a standard. General esterase activity was measured using the method described by van Asperen (1962) with a modified version of the microplate technique. Briefly, 25 µL of the enzyme preparation and 75 µL of 1.5 mmol L–1 α-NA were added to each well of a Multiscan MK3 microplate (Thermo Fisher Scientific, Waltham, MA, USA). After 5 min, 50 µL of the staining reagent containing Fast Blue B (0.1% final concentration) and sodium dodecyl sulfate (1.0% final concentration) were added to stop the reaction. Then, 5 min later, the optical density at 595 nm (OD595) was recorded. GST activity was assayed using the method of Habig et al. (1974) with a modified version of the microplate technique. Briefly, 20 µL of the enzyme preparation was combined with 100 µL of 2 mmol L–1 1-chloro-2, 4-dinitrobenzene (CDNB) and 80 µL of 12.5 mmol L–1 reduced glutathione in a 0.1 mol L–1 sodium phosphate buffer (pH 6.5). After the microplate was equilibrated for 2 min, absorbance at 340 nm was recorded continuously, and Vmax was computed. CYP monooxygenase activity was determined using the methods of Yu and Nguyen (1992), and 4-nitroanisole was used as a substrate. Briefly, 90 µL of the enzyme preparation was mixed with 10 µL of 9.6 mmol L–1 NADPH and 100 µL of 2 µmol L–1 4-nitroanisole and was reacted for 30 min at 30°C. Then, the optical density at 405 nm (OD405) was recorded.

2.4. Statistical analysis Probit analysis using statistic package for social science (SPSS) 19.0 (IBM, Armonk, NY, USA) was conducted to determine the median lethal concentration (LC50) values, slopes, and 95% confidence intervals (CI). Mortality was corrected using Abbott’s formula for each probit analysis. No overlap between the 95% CI of LC50 values was used as a significant difference criterion. The enzyme assay data were analyzed via one-way analysis of variance and Tukey’s test using SPSS (ver. 19.0). In addition, the data were plotted using Excel 2010 (Microsoft, Redmond, WA, USA). For all tests, statistical significance was defined as P<0.05.

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3. Results 3.1. Susceptibility of heat-shocked adult F. occidentalis to different insecticides There were significant differences in the susceptibility of heat-shocked adult F. occidentalis to different insecticides (Table 1). After recovering from the heat shock for 2 h, the susceptibility of adult F. occidentalis to four insecticides (acetamiprid: LC50=3.789 mg L–1, spinosad: LC50=0.114 mg L–1, beta-cypermethrin: LC50=14.112 mg L–1, and methomyl:

LC50=0.050 mg L–1) was significantly higher than that of the control (95% CI of LC50 non-overlapping). However, no significant difference in susceptibility occurred from 4 to 24 h, except for acetamiprid at 24 h (LC50=4.135 mg L–1, significantly higher susceptibility than that of the control), spinosad at 8 h (LC50=0.376 mg L–1, significantly lower susceptibility than that of the control), and methomyl at 20 h (LC50=0.053 mg L–1, significantly higher susceptibility than that of the control). When the recovery time was increased, the susceptibility of adults to four insecticides was significantly higher than that of the control for the recovery time of

Table 1 Slopes and lethal concentrations of four insecticides against heat-shocked Frankliniella occidentalis adults1) Insecticides Acetamiprid

Spinosad

Beta-cypermethrin

Methomyl

1)

Recovery time (h) Control 2 4 8 12 16 20 24 30 36 Control 2 4 8 12 16 20 24 30 36 Control 2 4 8 12 16 20 24 30 36 Control 2 4 8 12 16 20 24 30 36

n1) 341 365 345 342 337 342 351 361 358 357 351 366 348 358 378 362 373 365 362 357 351 366 348 358 378 362 373 365 362 357 351 366 348 358 378 362 373 365 362 357

Slope±SE 2.19±0.26 1.42±0.23 1.69±0.24 2.01±0.25 2.16±0.26 1.68±0.24 1.41±0.23 1.39±0.22 1.75±0.26 1.48±0.24 2.13±0.26 1.51±0.26 2.24±0.29 1.48±0.22 2.43±0.29 1.88±0.25 1.60±0.24 1.63±0.24 1.46±0.24 1.49±0.24 1.39±0.22 1.65±0.23 1.47±0.22 1.57±0.23 1.43±0.22 1.40±0.22 1.46±0.23 1.71±0.23 1.71±0.24 1.71±0.23 1.64±0.25 1.73±0.26 1.79±0.26 1.67±0.25 1.98±0.27 1.51±0.24 1.47±0.24 1.41±0.24 1.58±0.24 1.45±0.24

LC50 (mg L−1 ) (95% CI) 8.454 (6.803–10.415) 3.789 (2.373–5.183)* 6.247 (4.615–8.039) 10.087 (8.102–12.617) 8.103 (6.531–9.994) 7.407 (5.564–9.524) 5.574 (3.768–7.300) 4.135 (3.035–5.548)* 3.699 (2.455–4.908)* 4.116 (2.642–5.576)* 0.215 (0.173–0.264) 0.114 (0.070–0.158)* 0.288 (0.230–0.361) 0.376 (0.285–0.511)* 0.267 (0.218–0.324) 0.233 (0.181–0.295) 0.250 (0.187–0.327) 0.183 (0.131–0.238) 0.127 (0.082–0.170)* 0.121 (0.078–0.163)* 39.262 (29.400–55.086) 14.112 (10.803–18.250)* 47.164 (35.734–66.329) 62.800 (48.054–83.048) 50.594 (37.963–72.875) 49.378 (35.727–66.229) 42.584 (30.671–56.235) 36.742 (28.837–48.021) 18.652 (14.541–24.431)* 17.250 (13.488–22.419)* 0.094 (0.072–0.126) 0.050 (0.038–0.066)* 0.091 (0.070–0.118) 0.123 (0.093–0.161) 0.096 (0.075–0.122) 0.085 (0.061–0.113) 0.053 (0.036–0.068)* 0.055 (0.038–0.075) 0.047 (0.035–0.062)* 0.044 (0.032–0.060)*

X2 2.59 0.64 0.95 1.11 2.70 1.45 0.59 1.39 1.27 1.04 1.35 0.87 0.67 1.68 1.09 2.27 1.06 1.20 1.26 1.37 0.41 0.78 0.53 1.32 0.49 1.16 1.47 1.22 0.92 0.87 1.01 1.18 0.81 1.39 0.91 1.99 1.11 1.14 0.87 0.53

n, number of insects used in the concentration-mortality response bioassay; SE, standard error; LC, lethal concentration; CI, confidence limits. LC50 values within a row followed by * are significantly different from the control (P<0.05). The same as below.

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30 h (acetamiprid: LC50=3.699 mg L–1, spinosad: LC50=0.127 mg L–1, beta-cypermethrin: LC50=18.652 mg L–1, and methomyl: LC50=0.047 mg L–1) and 36 h (acetamiprid: LC50=4.116 mg L–1, spinosad: LC50=0.121 mg L–1, beta-cypermethrin: LC50=17.250 mg L–1, and methomyl: LC50=0.044 mg L–1).

3.2. Susceptibility dynamics of heat-shocked second-instar nymphs of F. occidentalis to different insecticides After the 4 h recovery, the susceptibility of heat-shocked second-instar nymphs to acetamiprid (LC50=0.942 mg L–1) and spinosad (LC50=0.055 mg L–1) was significantly higher than that of the control. However, a significant increase of susceptibility to acetamiprid occurred after the recovery time of 24 h (LC50=1.579 mg L–1), 30 h (LC50=1.239 mg L–1), and 36 h (LC50=1.186 mg L–1). Meanwhile, no significant difference of susceptibility to spinosad occurred even after the recovery time was increased. In the case of susceptibility to beta-cypermethrin, no significant increase occurred until the recovery time reached 30 h (LC50=17.770 mg L–1) and 36 h (LC50=16.783 mg L–1). A significant increase of susceptibility to methmyl occurred after 8 h of recovery time. No significant difference was observed between 12 and 24 h. With the recovery time increase, the susceptibility of heatshocked second-instar nymphs to methmyl was significantly higher than that of the control after the recovery time of 30 h (LC50=0.039 mg L–1) and 36 h (LC50=0.036 mg L–1) (Table 2).

3.3. Changes in detoxification enzyme activities of heat-shocked adult F. occidentalis During the recovery process of adults from heat-shock, the specific activity of CYP monooxygenase significantly decreased after the 2 h recovery time (0.0056 µmol mg–1 30 min–1) compared with the control. When the recovery time increased, the CYP monooxygenase activity gradually increased from 4 to 16 h (a significant increase occurred after 8 and 12 h compared with the control). However, when the recovery time lasted longer than 20 h, the CYP monooxygenase activity gradually decreased. A significant decline occurred after 30 h (0.0062 µmol mg–1 30 min–1) and 36 h (0.0056 µmol mg–1 30 min–1) (Fig. 1). Interestingly, a similar trend was observed for the LC50 value, which is responsible for the susceptibility of adults to insecticides (similar to spinosad) during the recovery process from heat-shock. For the specific activity of esterase obtained from heatshocked adults, no significant difference was found after 2 h of recovery time compared with the control. When the recovery time lasted longer than 4 h, the esterase activity gradually increased until 12 h (significant increase after 8 h compared with the control). When the recovery time lasted

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longer than 16 h, no significant difference of the esterase activity was observed between the heat-shocked adults and the control (Fig. 1). The trend differed from the change of LC50 value, which was responsible for the susceptibility of adults to any tested insecticide during the recovery process from heat-shock of adults. In the case of GST, the activity of heat-shocked adults significantly increased after 2 and 4 h of recovery time. However, no significant difference occurred between heatshocked adults and the control with an increase of recovery time (Fig. 1). In contrast to CYP monooxygenase, the GST activity trend did not show similarity to the change of LC50 value, which was responsible for the susceptibility of adults to any tested insecticide during the recovery process from heat-shock.

3.4. Change in detoxification enzyme activities of heat-shocked second-instar F. occidentalis nymphs When the heat-shocked second-instar nymphs recovered for less than 4 h, the decline of GST and CYP monooxygenase activities was observed, and a significant decline occurred after 4 h (GST: 0.0736 µmol mg –1 min –1, CYP monooxygenase: 0.0094 µmol mg –1 30 min–1) compared with the control (Fig. 2). When the recovery time was increased, the increase of these two detoxification enzyme activities was observed from 8 to 12 h, which was significantly higher compared with the control. Then, between 16 and 36 h, the activities dropped with no significant difference compared with the control. In the bioassay of heat-shocked second-instar nymph susceptibility to spinosad, the single highest peak of susceptibility occurred after 4 h of recovery time, which was similar to the GST and CYP monooxygenase activity trends. In the case of esterase activity of heat-shocked second-instar nymphs, a significant decline was observed after 4 (1.1501 µmol mg–1 min–1) and 8 h (1.0394 µmol mg–1 min–1) of recovery compared with the control. Subsequently, the esterase activity dropped below the level of the control between 12 and 36 h. The esterase activity trend appeared to have no significant similarity with the susceptibility dynamics of heat-shocked second-instar nymphs for any type of insecticide tested during the heat-shock recovery process of second-instar nymphs.

4. Discussion Both Heugens et al. (2001) and Sokolova and Lannig (2008) reported that organisms could increase susceptibility to chemicals and reduce thermal tolerance when they were exposed to chemicals and elevated temperatures. The results of our study indicated that when F. occidentalis

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Table 2 Slopes, lethal concentrations of four insecticides against heat-shocked F. occidentalis of second-instar nymph Insecticides Acetamiprid

Spinosad

Beta-cypermethrin

Methomyl

Recovery time (h) Control 2 4 8 12 16 20 24 30 36 Control 2 4 8 12 16 20 24 30 36 Control 2 4 8 12 16 20 24 30 36 Control 2 4 8 12 16 20 24 30 36

n 356 370 374 353 355 361 373 380 378 367 359 367 370 381 372 359 366 371 365 370 359 367 370 381 372 359 366 371 365 370 359 367 370 381 372 359 366 371 365 370

underwent heat shock for 2 h, the susceptibility to four insecticides fluctuated during the recovery process. During the treatment on adults, a significantly higher susceptibility to all four insecticides was observed at three recovery time points: after 2, 30, and 36 h. The results indicated that when F. occidentalis adults experienced 45°C for at least 2 h, the four tested insecticides exhibited better control efficiency against F. occidentalis adults after the recovery time of 2, 30 or 36 h at 25°C than without heat-shock. In addition, for the recovery time points during which significant susceptibility occurred, there was little difference between four insecticides. For instance, a significantly higher susceptibility to acetamiprid also occurred after the 24 h recovery time in addition to 2, 30, and 36 h. During the treatment of

Slope±SE 1.52±0.20 1.54±0.19 1.48±0.19 1.56±0.20 2.04±0.22 1.87±0.21 1.67±0.23 2.07±0.23 1.95±0.21 2.30±0.23 1.51±0.20 1.54±0.20 1.29±0.19 1.37±0.19 1.40±0.19 1.77±0.20 1.41±0.20 1.19±0.19 1.24±0.19 1.73±0.21 1.17±0.19 1.24±0.20 1.75±0.20 1.48±0.19 1.92±0.26 1.10±0.20 1.16±0.20 1.36±0.20 1.81±0.20 1.53±0.19 1.36±0.19 1.19±0.18 1.18±0.18 1.98±0.21 1.10±0.18 1.88±0.22 1.60±0.21 1.75±0.21 1.81±0.20 2.00±0.21

LC50 (mg L−1 ) (95% CI) 2.733 (2.132–3.478) 1.882 (1.467–2.406) 0.942 (0.729–1.208)* 2.238 (1.772–2.878) 4.170 (3.450–5.048) 3.410 (2.786–4.219) 2.506 (2.001–3.128) 1.579 (1.303–1.908)* 1.239 (1.014–1.510)* 1.186 (0.996–1.412)* 0.118 (0.093–0.152) 0.098 (0.077–0.124) 0.055 (0.041–0.073)* 0.127 (0.098–0.171) 0.176 (0.134–0.228) 0.139 (0.111–0.171) 0.101 (0.078–0.132) 0.108 (0.080–0.149) 0.077 (0.055–0.102) 0.073 (0.057–0.098) 30.026 (21.937–46.875) 27.312 (20.311–40.700) 22.596(18.331–28.084) 19.792 (15.422–25.294) 26.111 (20.712–34.563) 41.780 (29.978–68.270) 36.378 (26.774–54.906) 27.960 (21.484–37.519) 17.770 (14.396–21.721)* 16.783 (13.278–21.417)* 0.066 (0.051–0.087) 0.048 (0.035–0.064) 0.051 (0.037–0.068) 0.041 (0.034–0.050)* 0.075 (0.054–0.104) 0.098 (0.079–0.119) 0.079 (0.062–0.099) 0.061 (0.049–0.075) 0.039 (0.032–0.048)* 0.036 (0.030–0.044)*

X2 0.71 0.68 0.96 1.21 2.26 2.19 1.50 1.30 1.79 2.81 1.15 0.99 1.68 1.29 0.91 1.43 1.56 1.08 0.88 2.03 0.71 0.69 0.79 0.66 1.16 1.04 0.48 0.90 1.43 1.03 0.70 0.91 0.78 1.64 0.95 2.30 1.84 1.40 0.78 2.88

second-instar nymphs, susceptibility fluctuation was more pronounced during the recovery process between four insecticides compared with the adults. Compared with the treatment of adults, the first peak of significant susceptibility to acetamiprid and methomyl was observed at 4 and 8 h, which was slower than that for the adults. However, the second peak of significant susceptibility to acetamiprid (from 24 to 36 h) and methomyl (from 30 to 36 h) was similar to that of the adults. For spinosad or beta-cypermethrin, only one peak of significant susceptibility was found (at 4 h for spinosad and from 30 to 36 h for beta-cypermethrin). When the second-instar nymphs experienced heat-shock, we chose a suitable time for insecticide application against the nymphs according to the type of insecticide. The results

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ZHANG Bin et al. Journal of Integrative Agriculture 2016, 15(10): 2309–2318 A c

Specific activity (µmol mg–1 min–1)

0.30 bc

0.25

ab

0.20 0.15

a

a

0.10

a

a

a

a

a

24

30

36

0.05 0 Control 2

B

4 8 12 16 20 Recovery time (h)

Specific activity (µmol mg–1 min–1)

A

0.35

d

0.010 0.008

bc

b

B cd

bc

b

ab

a

0.006

a

a

0.004 0.002 0 Control 2

4 8 12 16 20 Recovery time (h)

24

30

36

Specific activity (µmol mg–1 min–1)

3.0 2.5 2.0

ab

ab

bc

c

bc

ab

ab

1.5

a

a

a

1.0 0.5 0 Control 2

4

8 12 16 20 Recovery time (h)

24

30

36

Fig. 1 Activities of three detoxification enzymes in Frankliniella occidentalis adults recovered from heat-shock for different

durations (2–36 h). A, glutathione S-transferases (GST) activity. B, cytochrome p450 (CYP) monooxygenase activity. C, esterase activity. Vertical bars represent the means±SE. The figures sharing the same small letters have no significant difference at 5% level.

The same as below.

indicated that the recovery time from 30 to 36 h was optimal for all tested insecticides in the study except for spinosad.

Specific activity (µmol mg–1 30 min–1)

C

0.012

C

Specific activity (µmol mg–1 min–1)

Specific activity (µmol mg–1 30 min–1)

0.014

0.20 d

0.16 0.12

ab

0.08

c

bc

bc

bc

b

ab

ab

24

30

36

b

b

ab

24

30

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a

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0.04 d

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bc

bc ab

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3.0 2.5 2.0

d cd

cd

c

1.5

ab

cd

cd

bc

24

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abc

a

1.0 0.5 0 Control 2

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36

Fig. 2 Activities of three detoxification enzymes in F. occidentalis second-instar nymphs recovered from heat-shock for different durations (2–36 h). A, GST activity. B, CYP monooxygenase activity. C, esterase activity.

Wu and Jiang (2002, 2004) determined that the resistance level of Plutella xylostella (Fuzhou region, Fujian, China) in spring and autumn was significantly higher than during the summer. The research by Ma et al. (2011) suggested that the susceptibility of Sitobion avenae to phoxim, chlorpyrifos, methomyl, carbosulfan, and imidacloprid was enhanced with the temperature increase of 10–25°C. Unlike long-term monitoring in the field, our study provides a more practical approach for controlling F. occidentalis using the combined action of heat-shock and insecticides. Esterase, glutathione S-transferase, and CYP monooxy-

genase are three major metabolic detoxification enzymes in insects. These enzymes are important for insect resistance and detoxification metabolism (Gao et al. 1997; Rumpf et al. 1997; Feyereisen 1999). For instance, CYP monooxygenase is an important metabolic enzyme that catalyzes a number of lipophilic compounds. It is involved in metabolism of a wide range of endogenous and exogenous chemical substances such as hormones, pheromones, pesticides, and plant secondary compounds (Scott 1999; Ding et al. 2013). In the present study, detoxification enzyme activities

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fluctuated during the process of recovery from heat shock in the tested adults and second-instar nymphs. Interestingly, it was observed that some activity trends were similar to insecticide susceptibility, such as CYP monooxygenase activity and susceptibility to spinosad during the treatment on adults, activities of GST and esterase and susceptibility to acetamiprid and spinosad during the treatment on second-instar nymphs. The results indicated that there potentially was a relationship between detoxification enzymes (especially CYP monooxygenase) and susceptibility to some insecticides (especially spinosad) during the recovery from heat shock. Previous study reported that high temperature could inhibit activity of detoxification enzymes and change the susceptibility to insecticides (Zhao et al. 1995; Broadbent and Pree 1997; Jensen 1998, 2000b; Espinosa et al. 2005; Maymó et al. 2006; Wang et al. 2006; Liu et al. 2007; Wang et al. 2011). In our study, we observed the relationship between the susceptibility to insecticides and the detoxification enzyme activity during the process of recovery from heat shock as well as short-term fluctuation of detoxification enzyme activity after the heat shock. We hypothesized that susceptibility to some types of insecticides was partly attributed to the change of detoxification enzyme activity even for a short term after the heat shock. However, we note that the change of detoxification enzyme activity was not consistent with susceptibility to some types of insecticides such as GST and insecticides, which were tested during the treatment of adults, and esterase and beta-cypermethrin during the treatment of second-instar nymphs. Although the change of detoxification enzyme activity might affect the susceptibility to insecticides after heat shocking F. occidentalis, other physiological changes could also influence the susceptibility to insecticides during the recovery from heat shock. For example, the heat shock proteins (HSPs), which can enable insects to adapt quickly to temperature changes in the environment and protect the body from heat damage (Li et al. 2004), need to be degraded during the recovery from heat shock. Then, normal proteins begin to be synthesized and are recovered to normal levels. The two abovementioned processes consume energy (Koehn and Bayne 1989; Hoffmann 1995) and negatively affect the organism (Feder et al. 1992; Silbermann and Tatar 2000; Miriam 2003; Huang et al. 2007), which may influence susceptibility of organisms to insecticides. In addition, future studies are needed to explore whether detoxification enzymes participate in degrading HSPs during the F. occidentalis recovery from heat shock. In the present study, the fluctuation of susceptibility to insecticides after heat shock provides useful details about individual recovery from heat shock. We suggest to use a more effective combined approach (insecticides and heat shock) to control F. occidentalis. This method enhances control efficiency

and reduces the application of insecticides. The time period for applying insecticides after heat shock is essential for enhancing the control efficiency. In the next study, we will focus on the mechanism of how the fluctuation of susceptibility to insecticides, detoxification enzymes activity, and HSPs expression occur during recovery of F. occidentalis from heat shock.

5. Conclusion The susceptibility to insecticides was related to the detoxification enzymes activity during the recovery of F. occidentalis from heat shock. A significant difference in susceptibilities to four insecticides was observed for several recovery time periods (2, 30, and 36 h) after adults were heat-shocked compared with the control. Furthermore, higher susceptibility to four insecticides was observed after nymphs were heat-shocked compared with the control. A similar fluctuation of CYP monooxygenase activity was found compared with the susceptibility to insecticides (especially spinosad) after adults were heat shocked. In addition, a similar trend of glutathione S-transferase and CYP monooxygenase activities for second-instar nymphs was found compared with the trend of susceptibility to spinosad during recovery from heat shock. Our results provide a new method for controlling F. occidentalis using combined heat shock and insecticides. The approach effectively enhances the heat shock control efficiency and significantly reduces the application of insecticides.

Acknowledgements This study was funded by the National Natural Science Foundation of China (31372003), the Shandong Modern Agricultural Technology and Industry System, China (SDAIT-02-021-11), the Taishan Scholarship Construction Engineering Special Fund, China, the Startup Fund for Distinguished Scholars (631316) supported by the Qingdao Agricultural University, China.

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