Lethal and sublethal effects of the chitin synthesis ...

Pesticide Biochemistry and Physiology 136 (2017) 80–88

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Lethal and sublethal effects of the chitin synthesis inhibitor chlorfluazuron on Bradysia odoriphaga Yang and Zhang (Diptera: Sciaridae) Peng Zhang a,b, Yun-He Zhao a, Qiu-Hong Wang a, Wei Mu a, Feng Liu a,⁎ a b

College of Plant Protection, Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect Pests, Shandong Agricultural University, Tai'an, Shandong 271018, PR China College of Environmental Science and Engineering, Nankai University, Tianjin 300071, PR China

a r t i c l e

i n f o

Article history: Received 20 February 2016 Received in revised form 21 July 2016 Accepted 25 July 2016 Available online 26 July 2016 Keywords: Bradysia odoriphaga Yang and Zhang Chlorfluazuron Two-sex life table Population dynamics Glutathione S-transferase Microsomal mixed function oxidase

a b s t r a c t Bradysia odoriphaga Yang and Zhang is the primary insect pest that affects Chinese chive in northern China. Nevertheless, very few studies have been conducted on the use of chitin synthesis inhibitors (CSIs) for the control of B. odoriphaga. Here, lethal and sublethal effects of the CSI chlorfluazuron on B. odoriphaga were studied to explore the use for integrated pest management (IPM) of B. odoriphaga. A contact and ingestion toxicity bioassay showed that chlorfluazuron was more active against B. odoriphaga than three other CSIs, with a 72 h LC50 of 0.1593 mg/L. Treatment with the LC50 dose of chlorfluazuron decreased both the intrinsic and finite rates of increase of B. odoriphaga, in addition to reproduction rate, survival rate, and fecundity, and the mean generation time, total preovipositional period and larval development duration were shortened, compared with those of the control and LC10 groups. The mean generation time, total preovipositional period and larval development duration were all also markedly decreased by treatment with chlorfluazuron at the LC10. Furthermore, chlorfluazuron inhibited the feeding of the final instar larvae for a short period. Glutathione S-transferase and microsomal mixed function oxidase activities increased after exposure to the chemical. These results showed that chlorfluazuron at the sublethal LC50 treatment inhibited B. odoriphaga population growth, whereas the danger of causing rapid population growth by using a lower sublethal concentration was demonstrated with the sublethal LC10 treatment. Therefore, chlorfluazuron should be used with caution in an IPM program for B. odoriphaga. © 2016 Elsevier Inc. All rights reserved.

1. Introduction The chive gnat Bradysia odoriphaga Yang and Zhang (Diptera: Sciaridae) is the primary insect pest that affects Chinese chive in northern China [1]. This insect feeds on the plants of seven families and more than thirty species, including Chinese chive (Liliaceae), garlic (Liliaceae), onion (Liliaceae), and cucumber (Cucurbitaceae) [2,3] and also causes production losses in mushroom sheds [4]. The gnat larvae live in the roots and stems of Chinese chive, thereby making control difficult using the common strategies. In China, outbreaks of the chive gnat have occurred frequently in recent years. This pest attacks 20–30% of Chinese chive and causes 30–80% of the production losses; in severe cases, new Chinese chive must be replanted [2]. Bradysia odoriphaga produces 4–6 overlapping generations annually in the chive fields of northern China, with the peak damage occurring in the spring and autumn [5]. The application of chemical insecticides, such as organophosphates, carbamates, and neonicotinoids, is one of the most prevalent ⁎ Corresponding authors at: College of Plant Protection, Shandong Agricultural University, 61 Daizong Street, Tai'an, Shandong 271018, PR China. E-mail address: fl[email protected] (F. Liu).

http://dx.doi.org/10.1016/j.pestbp.2016.07.007 0048-3575/© 2016 Elsevier Inc. All rights reserved.

management practices used for B. odoriphaga control in China and elsewhere [6]. Chemical control remains a primary strategy in IPM systems because of the advantages of being rapid, efficient, easy to use, costeffective, and notably recommended, in addition to providing reliable and effective control of the targeted insect [7]. Many laboratory toxicity and field efficacy studies of chemical pesticides such as diazinon, chlorpyrifos, phoxim and imidacloprid are reported [8–10]. These chemical insecticides have been used to irrigate roots to control B. odoriphaga [10], but this practice led to the excessive use of certain insecticides, resulting in environmental pollution and high levels of residues in marketed Chinese chives [11]. CSIs are less toxic insecticides and are compatible with insect pest management protocols that were developed to reduce the pollution of food and the environment. CSIs have a specific mode of action in insects and low toxicity against vertebrates compared with those of conventional insecticides [12]. Chlorfluazuron, which is an effective pesticide against major lepidopteran and coleopteran pests, is an insect growth regulator that acts as an anti-molting agent by inhibiting the biosynthesis of chitin, an important constituent of insect cuticles, leading to the loss of cuticular elasticity and resulting in abortive molting [13]. Application of chlorfluazuron to the adult females of Anagrus nilaparvatae (Pang et Wang) (Hymenoptera: Mymaridae) [14] and Spodoptera litura (F.) (Lepidoptera: Noctuidae)

P. Zhang et al. / Pesticide Biochemistry and Physiology 136 (2017) 80–88

[15] decreases their fecundity. In the only study of chlorfluazuron against B. odoriphaga, Chen et al. observed a control efficacy of 70.2% in the field 24 days after application [16]. However, a comprehensive evaluation of a pesticide requires not only assessment of acute lethal effects but also sublethal effects [17]. For example, following the field application of insecticides, pest organisms are exposed to different doses of an insecticide over time, and these varying dosages can lead to different biological and ecological outcomes [18,19]. Perveen [18,20] and Shahout [21] found that the application of sublethal doses of chlorfluazuron to the fifth instar larvae of S. litura reduces the fertility and fecundity of the resulting adult females and the hatchability of their larvae. These results are consistent with those of You et al. [22]. Perveen [20,23] also found that the size of the eggs is reduced and the number of inseminated eupyrene sperm decreases by 66% and 88% after the LD10 and LD30 treatments, respectively, of S. litura males, whereas no significant reductions are found when both sexes or only females are treated. Moreover, Guo [24] observed that chlorfluazuron increases the biological activity of a nucleopolyhedrovirus through the disruption of the peritrophic matrix of S. litura larvae. However, the effects of sublethal concentrations of chlorfluazuron on B. odoriphaga have not been reported. Life table studies offer a comprehensive description of the effect of insecticides on insect population dynamics and can explain the multiple sublethal effects on insects [25–28]. However, in studies using a traditional female age-specific life table [29–30], the overlap of developmental stages is overlooked, and the age-specific fecundity is calculated only for females, whereas that of males is ignored. These limitations are potentially problematic because the developmental rate of all the individuals in a population may affect population parameters. A marked improvement over the traditional female age-specific life table is an age-stage, two-sex life table. This type of analysis can be applied to the age-stage structure of two-sex populations and can accommodate variations in the preadult developmental periods, resulting in more accurate survival and fecundity curves. Including both sexes is an innovative feature of the age-stage, two-sex life table [31,32]. Two significant types of detoxifying enzymes that play an important role in the resistance and detoxification metabolism of insects are glutathione S-transferase (GST) and microsomal mixed function oxidase (MFO) [33–35]. Yu and Terriere [36,37] found that diflubenzuron changes the titer of molting hormone and kills insects by decreasing the molting hormone enzyme activity and stimulating the activation of MFOs, which decrease the levels of juvenile hormones. Additionally, Sonoda and Tsumuki found that the glutathione S-transferase gene is involved in the detoxification of chlorfluazuron, which led to the development of chlorfluazuron resistance in the diamondback moth [38]. Thus, the variation in activity of these two enzymes is an expression of the biochemical status of B. odoriphaga; however, no studies have examined GST and MFO activity as sublethal effects of chlorfluazuron in B. odoriphaga. Thus, the information on lethal and sublethal effects of chlorfluazuron on B. odoriphaga is limited. In the present study, the objectives were to gain a comprehensive understanding of lethal and sublethal effects of chlorfluazuron on B. odoriphaga, a primary insect pest affecting Chinese chive in northern China, by examining the developmental and demographic parameters represented in an age-stage, two-sex life table, the amount of feeding, and the activities of glutathione S-transferase and microsomal mixed function oxidase. Based on these results, valuable insight was gained into the population dynamics and biochemical characteristics of B. odoriphaga following the application of the pesticide chlorfluazuron, which will assist in the development of novel IPM strategies. 2. Materials and methods 2.1. Insects and insecticides Colonies of B. odoriphaga were originally obtained from a Chinese chive field in Tai'an, Shangdong Province, China (Site location:

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36.18°N, 117.13°E) in September 2012 and were maintained in the Key Laboratory of Pesticide Toxicology and Application Techniques at Shandong Agricultural University, Tai'an. The insects were placed in 90-mm diameter Petri dish rearing containers. To maintain humidity, each Petri dish contained a filter paper soaked with 2.5% agar. Simultaneously with the addition of insects, freshly cut Chinese chive pseudostems (0.5 cm) were placed in the container to provide food for larvae and oviposition sites for females. The rearing containers were maintained in growth chambers at 26 ± 1 °C, 65–75% RH, and a photoperiod of 12:12 h L:D. The 95% chlorfluazuron and 95% hexaflumuron were obtained from Jinan Luba Pesticide Co., Ltd., China. The 96% diflubenzuron and 98% cyromazine were purchased from Tonghua Pesticide Chemical Co., Ltd., and Jiangsu Institute of Ecomones Co., Ltd., China. Four stock solutions were prepared by dissolving the CSIs in acetone with 0.1% Tween-80, which were stored for later use. 2.2. Bioassays Bioassays were conducted on newly emerged 4th instar larvae of B. odoriphaga using standard contact and ingestion bioassay methods [39]. Serial dilutions (mg/L) of the active ingredient of four CSIs (i.e., chlorfluazuron, hexaflumuron, diflubenzuron and cyromazine) were prepared using distilled water. Freshly cut Chinese chive pseudo-stems (0.5 cm) were dipped into a test solution for 30 s with gentle agitation and then air-dried at room temperature. Four treated pseudo-stems were transferred to a 90-mm diameter Petri dish containing a 90-mm filter paper moistened with 1 mL of a diluted solution. One Petri dish was treated as a single replicate. Four replicates (approximately 25 larvae per replicate) of at least seven concentrations were used to determine the mortality rate for each concentration. Distilled water containing 0.1% acetone and 0.1% Tween-80 was used as the control for each insecticide. In all the experiments, the larvae were maintained at a constant temperature of 26 ± 1 °C and a RH of 65–75% with a photoperiod of 12:12 h L:D. Mortality was assessed after eclosion. Insects were considered dead when they were unable to pupate and emerge. 2.3. Sublethal effects of chlorfluazuron on biological parameters and the life cycle At 24 h after oviposition, eggs were collected using a moist writing brush, and 600 eggs were used for the life table study. Two hundred eggs were placed on freshly cut Chinese chive pseudo-stems in individual Petri dishes. The experiment involved three treatments (i.e., LC10, LC50, and control), with 200 eggs exposed per treatment, and each egg considered as one replicate [40,41]. The eggs were maintained in the growth chamber as described above. The number of hatched eggs was recorded daily, and the newly hatched larvae were transferred to 90mm diameter Petri dishes containing 90-mm moist filter papers and Chinese chive. The survival and development of the larvae were evaluated daily, and fresh slices of Chinese chive were supplied to avoid fungal growth. At day 12 after spawning (approximately the 4th instar), the larvae were topically treated with chlorfluazuron at two sublethal concentrations using the bioassay method described above; the LC10 and LC50 treatments were 0.0255 mg/L and 0.1593 mg/L, respectively. The survival and development of B. odoriphaga were also evaluated daily, and fresh slices of Chinese chive were supplied to avoid fungal growth. The feeding amount was also recorded daily from day 11 to 16 after spawning, which was calculated as the feeding amount per 100 insects. The filter papers were moistened daily using deionized water. Immediately after the transformation from larva to pupa, the pupae were moved to new Petri dishes (as described above). After emergence, the adults were sexed and were paired as one female with one male and then were transferred to individual plastic oviposition containers (a

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plastic cup 5 cm in diameter and 2.5 cm in height, containing a moist 50mm diameter filter paper). Because sex is determined by a special mechanism [42,43], Sciaridae can reproduce single-sex or dual-sex offspring. Unsuccessful pairs were provided more sexed adults, and the results of the extra pairings were excluded from the data analysis. Each day, the oviposition of adults was checked, and the adults were transferred to a new Petri dish, as described above, following egg collection. The survival and fecundity of each individual were evaluated until death. In the experimental process, an overview was obtained from a study of the body development of B. odoriphaga using an electron microscope. 2.4. Preparation of samples for biochemical assays Total protein and GST and MFO activity of chive gnat fourth instar larvae were evaluated. Standard contact and feeding bioassay methods were used with LC10 and LC50 treatments with chlorfluazuron. In each experiment, 50 insects were tested with four replicates for each concentration. After 24, 48, and 72 h, live larvae were selected, and the whole body of larvae was used to measure the total protein and the activity of the two biochemical compounds. 2.5. Determination of total protein Protein concentrations were determined using the Bradford method [44]. Larvae and saline (w/v = 1:9) were homogenized at 15,000 g for 20 min at 4 °C. Then, 10 μL of supernatant was mixed with 5000 μL of dye (10 mg powder of Coomassie Brilliant Blue (Beijing Biotopped Science & Technology Co. Ltd.) in 5 mL of 96% ethanol and 10 mL of 85% phosphoric acid (w/w); the solution was brought to 100 mL with distilled water). The absorbance was read at 595 nm. 2.6. Glutathione S-transferase activity GST activity of chive gnat larvae was determined using the method of Deng et al. [45]. In this experiment, 1-chloro-2,4-dinitrobenzene (CDNB) was used as the substrate. First, larvae and saline (w/v = 1:9) were homogenized at 15,000 g for 20 min at 4 °C. Then, 15 μL of supernatant was mixed with 135 μL of phosphate buffer (pH = 7), 50 μL of CDNB and 100 μL of GST. The absorbance was read at 412 nm. One unit of glutathione S-transferase activity was defined as the concentration of reduced glutathione (GSH) required to reduce 1 μmol/L of substrate in 1 min at 37 °C.

trichloroacetic acid was added, and the oil phase was collected after standing for 5 min. Then, 3 mL 0.5 mol/L NaOH was added into the collected liquid, and the aqueous phase was collected after standing for 5 min. The absorbance was read at 400 nm. 2.8. Data analysis The data were corrected for the control mortality using Abbott's formula before analysis, and then the data were analyzed using the SAS/ STAT® statistical software package version 6.12 (SAS Institute Inc., USA). The raw data for all the individuals represented in the life table were analyzed according to the age-stage, two-sex life table theory [31,48]. The means and standard errors of developmental time, longevity, and fecundity were estimated using the bootstrap method [49] that is included in the TWOSEX-MSChart computer program [50]. The statistical significance of the mean values (p b 0.05) was determined using ANOVA, and the means were separated using Duncan's multiple range test (DMRT) in the SAS/STAT® version 6.12 program. For the population parameters, the values were calculated and mean values and standard errors and the significance of the differences were estimated using the bootstrap technique [49] that is included in the TWOSEX-MSChart computer program [50]. The differences among the treatments were compared using the Tukey-Kramer procedure [51]. The curves for the survival rate, fecundity, reproductive value and feeding amount were constructed using SigmaPlot 12.0 software. 3. Results 3.1. Toxicity of four CSIs to fourth instar Bradysia odoriphaga The toxicity of four CSIs (i.e., chlorfluazuron, hexaflumuron, diflubenzuron and cyromazine) in fourth instar B. odoriphaga was investigated (Table 1). The concentrations (95% confidence intervals are shown in parentheses) of chlorfluazuron, hexaflumuron, diflubenzuron and cyromazine that caused 50% mortality were 0.1593 (0.1195– 0.2136), 0.2912 (0.1173–0.4277), 0.7932 (0.3734–1.3198), and 13.0397 (7.0366–22.4439) mg (ai) L− 1, respectively (Table 1). Chlorfluazuron had the highest toxicity of the four CSIs. Otherwise, the concentration (95% confidence intervals are shown in parentheses) that caused 10% mortality was 0.0255 (0.0142–0.0385) mg (ai) L−1 (Table 1). 3.2. Life tables of Bradysia odoriphaga exposed to chlorfluazuron at sublethal concentrations

2.7. Microsomal mixed function oxidase activity To determine MFO activity, the method of Hansen et al. [46] and Yuan et al. [47] was used. Paranitroanisole was used as the substrate. A larva and 0.05 mol/L TriS-HCl buffer solution (pH = 7.8, w/v = 1:9) were homogenized at 15,000 g for 20 min at 4 °C. The reaction was performed by 1 mL 4 × 10−3 mol/L paranitroanisole with 1 mL of the enzyme, 0.5 mL 2 × 10−4 mol/L NADPH, and 0.9 mL 0.05 mol/L TriS-HCl buffer solution (pH = 7.8) for 30 min at 37 °C. MFO activity was stopped by the addition of 1 mL 1 mol/L HCl. As an extract liquid, 5 mL of

The development time, longevity and fecundity of B. odoriphaga exposed to chlorfluazuron at sublethal concentrations are shown in Table 2. At sublethal concentrations, chlorfluazuron did not have any significant effect on the developmental duration of the pupal stages or the mean longevity of adult females; however, the treatments significantly shortened the developmental duration of the larval stages (14.33, 13.69 and 13.60 days for the control, LC10 and LC50 groups, respectively). Additionally, the LC20 treatment also shortened the mean longevity of adult males (3.74, 3.79 and 3.29 days for the control, LC10

Table 1 Toxicity of four chitin synthesis inhibitors on the forth instar larvae of Bradysia odoriphaga. Insecticide

Na

Chlorfluazuron

1012

Hexaflumuron Diflubenzuron Cyromazine

922 893 1091

Concentration mg (ai) litre−1 (95% CL)−1

Slop ± SE

χ2 (df)

p-valueb

1.612 ± 0.1804

1.6911 (7)

0.98

2.485 ± 0.2251 2.111 ± 0.3254 1.178 ± 0.2690

1.8254 (6) 1.6358 (6) 1.2217 (7)

0.99 0.92 0.54

LC50

a b

0.1593 (0.1195–0.2136) 0.0255(0.0142–0.0385) (LC10) 0.2912 (0.1173–0.4277) 0.7932 (0.3734–1.3198) 13.0397 (7.0366–22.4439)

Number of subjects. p-value derived from Chi square test larger than 0.05 indicates the log-logistic model provides acceptable description of data.


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Table 2 Life tables of Bradysia odoriphaga exposed to sublethal concentrations of chlorfluazuron. Stages

CK

Eggs (days) larvae (days) Pupae (days) Eggs-pupae (days) Mean longevity of adult male (days) Mean longevity of adult female (days) APOPb TPOPc Fecundity (eggs/♀) a b c

LC10

LC50

n

Mean ± SEa

n

Mean ± SE

n

Mean ± SE

185 165 159 159 73 86 86 86 86

3.64 ± 0.04 a 14.33 ± 0.07 a 3.35 ± 0.04 a 21.26 ± 0.10 a 3.74 ± 0.12 a 2.10 ± 0.07 a 0.93 ± 0.07 a 22.69 ± 0.16 a 123.1 ± 2.11 b

183 161 136 136 63 73 73 73 73

3.69 ± 0.04 a 13.69 ± 0.07 b 3.26 ± 0.04 a 20.52 ± 0.11 b 3.79 ± 0.12 a 2.09 ± 0.08 a 0.95 ± 0.08 a 21.81 ± 0.15 b 134.7 ± 1.77 a

181 159 79 79 35 44 44 44 44

3.57 ± 0.05 a 13.60 ± 0.10 b 3.25 ± 0.05 a 20.33 ± 0.12 b 3.29 ± 0.17 b 2.05 ± 0.11 a 0.95 ± 0.11 a 21.52 ± 0.19 b 118.2 ± 3.18 b

p

F

df

0.1963 0.0001 0.2708 0.0001 0.0396 0.8825 0.9807 0.0001 0.0001

1.633 35.01 1.311 22.23 3.291 0.1250 0.0190 13.43 13.08

548 484 373 373 170 202 202 202 202

Means in the same row followed by the same letter are not significantly different (p N 0.05) using the DMRT. Adult preovipositional period. Total preovipositional period (from egg to first oviposition).

and LC50 groups, respectively). The adult preoviposition period (APOP) was not significantly different among the control, LC10 and LC50 groups, but the differences for the total preovipositional period (TPOP) of B. odoriphaga among the control, LC10 and LC50 groups were statistically significant (Table 2). Compared with that of the control group (123.1 eggs per female), the number of eggs produced increased significantly in the LC10 treatment (134.7 eggs per female), whereas an increase was not observed with the LC50 treatment (118.2 eggs per female). The sublethal effects of chlorfluazuron on the population parameters were estimated using the bootstrap method (Table 3). The intrinsic rate of increase, finite rate of increase, net reproduction rate, and mean generation time were significantly affected by chlorfluazuron treatments, but no differences were found in the gross reproductive rate (GRR) with LC5 or LC20 treatments (p N 0.05; Table 3). Compared with that of the control larvae (0.1684 d−1), the decrease in the intrinsic rate of increase (r) was obvious in the LC50 treatment (0.1447 d−1), whereas in the LC10 treatment, the intrinsic rate of increase increased but the increase was nonsignificant (0.1713 d−1; p b 0.05; Table 3). The trend for the finite rate of increase (λ) was similar to that of the intrinsic rate of increase. The net reproductive rates (R0) were 52.91, 49.14, and 25.97 offspring/individual for the control, LC10 and LC50 treated larvae, respectively, and the decrease was significant for the LC50 treatment (p b 0.05; Table 3). However, no significant difference was found in the gross reproductive rate (GRR) of the control and treated larvae (164.7, 145.0, and 134.4 offspring/individual for the control, LC10 and LC50 groups, respectively). The mean generation time of the treated individuals was reduced compared with that of the control larvae (23.54, 22.71, and 22.44 days for the control, LC10 and LC50 groups, respectively). In the curves that show the different developmental rates of individuals, obvious overlaps were observed (Fig. 1). Male adults emerged earlier than females and survived longer. The relative number of adults declined after LC10 and LC50 treatments compared with the controls (Fig. 1). The total development time of the LC10 and LC50 treated larvae was also shorter than that of the control larvae; in particular, the larval development time was clearly shorter (Fig. 1). In Fig. 2, the age-specific survival rate (lx), female age-specific fecundity (fx4), age-specific Table 3 The sublethal effects of chlorfluazuron on Bradysia odoriphaga population parameters. Population parameters

Control

LC10

LC50

Mean ± SEa

Mean ± SE

Mean ± SE

r (d−1) λ (d−1) R0 T (d) GRR

0.1684 ± 0.0038 a 1.183 ± 0.0045 a 52.91 ± 4.449 a 23.54 ± 0.1650 a 164.7 ± 26.79 a

0.1713 ± 0.0044 a 1.187 ± 0.0052 a 49.14 ± 4.655 a 22.71 ± 0.1540 b 145.0 ± 26.26 a

0.1447 ± 0.0063 b 1.156 ± 0.0073 b 25.97 ± 3.517 b 22.44 ± 0.2070 b 134.4 ± 28.97 a

a Means in the same row followed by the same letter are not significantly different (p N 0.05) as calculated using the Tukey-Kramer procedure.

Fig. 1. Age-stage specific survival rate (sxj) of B. odoriphaga exposed to sublethal concentrations of chlorfluazuron.

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Fig. 2. Age-specific survival rate (lx), female age-specific fecundity (fx4), age-specific fecundity of the total population (mx), and age-specific maternity (lxmx) of B. odoriphaga exposed to sublethal concentrations of chlorfluazuron.

fecundity of the total population (mx), and the age-specific maternity (lxmx) of the insects are illustrated. The curves of lx declined significantly for the LC50-treated larvae 16 days after treatment. The highest peaks in the fx4 curve of the LC10 group were higher than those of the control and LC50 groups, whereas the highest peaks in the mx curve of the control group were higher than those of the LC10 and LC50 groups. The lxmx value was clearly dependent on lx and mx and declined significantly in LC50-treated larvae. The vxj value represents the devotion to future offspring of individuals from age x to stage j (Fig. 3). The reproductive values of larvae and pupae declined in LC50-treated larvae compared with those of the control and LC10 groups, but the highest peaks for the female reproductive value of the LC10 and LC50 groups were higher

than those of the control group (Fig. 3). The entire preoviposition period of the females in the LC10 and LC50 groups was shortened compared with that of the control group (Fig. 3). 3.3. Sublethal effects of chlorfluazuron on the feeding amount of Bradysia odoriphaga The consumption of Chinese chive pseudo-stems by B. odoriphaga larvae was noted daily from day eleven to sixteen after spawning (Fig. 4). Before the treatment (days eleven and twelve), the feeding amounts of the control group and the other groups were not significantly different. However, on day thirteen, the day of the chlorfluazuron


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treatment, the feeding amount of the LC50- and LC10-treated B. odoriphaga larvae declined significantly compared with that of the control group, and the decline in feeding amount increased as the concentration of chlorfluazuron increased. Food consumption in the LC10and LC50-treated groups gradually returned to the level of the control group in the two days after chlorfluazuron treatment. However, because some of the larvae developed into mature forms that reduced their feeding, the feeding amounts of the LC10 and LC50 groups declined significantly on day sixteen. 3.4. Overview based on body development of Bradysia odoriphaga exposed to chlorfluazuron The numbers of LC50- and LC10-treated B. odoriphaga all declined compared with those of the control group (Table 2), and in the identical treatments, an overview based on the body development of B. odoriphaga also indicated an effect of chlorfluazuron (Fig. 5). After LC50 treatment, treated larvae showed no symptoms and all successfully transformed into pupae, but symptoms of abnormal development, including pupa blackening, head and abdomen distention and wing dysplasia, were observed, which caused death at the pupal stage and the failure of adults to emerge. These symptoms were less prominent in the LC10-treated group. 3.5. Sublethal effects of chlorfluazuron on three biochemical compounds The level of total protein declined in treated larvae, and the decline was significant in LC10 and LC50 treatments of B. odoriphaga from 24 to 48 h compared with the protein level in the control group. However, the amount of total protein increased significantly in LC10 and LC50 treatments from 48 to 72 h (Fig. 6). Differences in GST and MFO activities between the treated and control larvae are shown in Figs. 7 and 8. The LC10 and LC50 of chlorfluazuron increased the activity levels of GST and MFO, and this increase was significant when compared with the activity in the control at 48 and 72 h after treatment. 4. Discussion

Fig. 3. Age-stage specific reproductive values (vxj) of B. odoriphaga exposed to sublethal concentrations of chlorfluazuron.

Chlorfluazuron is an insect-growth regulator that is highly effective against lepidopterous insect pests by disrupting the formation of chitin and is very effective against immature insects with relatively slow but strong activity [18]. In the current study, chlorfluazuron was more active against B. odoriphaga than hexaflumuron, diflubenzuron and cyromazine, and the 72 h LC50 was only 0.1593 mg/L, which showed

Fig. 4. The feeding amount of B. odoriphaga exposed to sublethal concentrations of chlorfluazuron.

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Fig. 5. Overview from body development of B. odoriphaga exposed to chlorfluazuron. a, b: normal pupa in control group; c, d: head and abdomen distention and wing dysplasia; e: pupa blackening; f: molting obstacle.

that chlorfluazuron had the potential to control B. odoriphaga. Additionally, the age-stage, two-sex life tables for B. odoriphaga showed that chlorfluazuron at the sublethal LC50 treatment decreased both the intrinsic (r) and finite rates of increase (λ), the net reproduction rate (R0), the survival rate, and the fecundity. Although the mean generation time, total preovipositional period, and larval development duration were shortened by the LC50 treatment, no significant differences were found between the LC10 treatment and control groups, except for the mean generation time, the total preovipositional period and the larval development duration. These results showed that chlorfluazuron at the sublethal LC50 treatment inhibited B. odoriphaga population growth, whereas at the sublethal LC10 treatment, population growth was stimulated. Furthermore, chlorfluazuron inhibited the feeding of the final instar larvae for a short period and increased GST and MFO activities after exposure to the chemical. Different insects or the identical insects under different conditions have different survival rates, which are a reflection of species-specific

parameters and environmental effects [52]. In the present study, the sxj value was affected by treatment with chlorfluazuron, and this measure of survival rate decreased with increasing concentrations of chlorfluazuron. The relative differences in the numbers of declining larvae, pupae and adults in the different treatment groups were difficult to quantify. The differences in the different developmental stages were not clearly reflected in the age-specific survival rate (lx) because this rate is a simplified version of the age-stage survival rate (sxj; Fig. 2) [53] and because a typical overlap phenomenon was observed in the sxj values due to differences in the developmental rates of individuals. Many other studies report similar phenomena in Spodoptera littoralis [54], S. exigua [55] and Plutella xylostella (L.) [22], and pupal developmental deformities were observed in all those studies and in this study. The feeding amount of B. odoriphaga was inhibited for a short period after chlorfluazuron treatment, but the temporary feeding suppression was not harmful. Based on this result, chlorfluazuron likely requires mixed application with rapidly effective and highly efficient pesticides, which

Fig. 6. Amount of total protein in fourth larvae of B. odoriphaga after treatment with LC10 and LC50 concentrations of chlorfluazuron. Note: The bars between treatments at the same time in figure with different letters significantly differ (p b 0.05).

Fig. 7. Acticity of glutathione S-transferase in in fourth larvae of B. odoriphaga after treatment with LC10 and LC50 concentrations of chlorfluazuron. Note: The bars between treatments at the same time in figure with different letters significantly differ (p b 0.05).


Fig. 8. Acticity of microsomal mixed function oxidase in in fourth larvae of B. odoriphaga after treatment with LC10 and LC50 concentrations of chlorfluazuron. Note: The bars between treatments at the same time in figure with different letters significantly differ (p b 0.05).

is the conclusion also reached by Zhang et al. [56] after studying the seedling maintenance rate when pyriproxyfen and cyromazine were applied. Life tables and the parameters are a useful method to assess the development and reproduction of populations [31]. In this paper, chlorfluazuron at sublethal concentrations significantly shortened larval development time and the total preovipositional period (TPOP) and decreased the feeding amount for a short period. No significant differences were found in the gross reproductive rate (GRR) between the control and treated groups; however, the net reproductive rate (R0) of the control and LC50-treated groups was significantly different. This result was consistent with that of Christian-Luis et al. [57] and Ishaaya et al. [54] who reported that Spodoptera exigua and S. littoralis treated with methoxyfenozide did not show reduced fecundity but their net reproductive value was significantly reduced compared with that of the untreated control. These results indicated that chlorfluazuron treatment at very low concentrations, less than the LC10, could stimulate population growth by promoting the reproduction of a similar number of offspring that consume a similar amount of food in a shorter developmental time. Additionally, these results also indicated that attempts to control B. odoriphaga using an ineffective dose of chlorfluazuron are extremely harmful. However, other reports show that chlorfluazuron treatment at sublethal concentrations prolongs larval developmental time and reduces the weight of all developmental stages [18,21,22]. Therefore, these phenomena clearly require more research. In the current study, the female age-specific fecundity (fx4), agespecific maternity (lxmx) and reproduction value (vxj) of each agestage group of B. odoriphaga were measurably higher in LC10-treated organisms, which indicated that sublethal doses of chlorfluazuron increased the biological productivity of B. odoriphaga. This effect might be due to the pesticide affecting the accumulation of nutrients and/or the growth of ovaries. However, Perveen et al. [18] observed that the ovaries of chlorfluazuron-treated individuals have spaces in the ovarioles and that only immature ovae were found in some of the LD30treated S. litura females. Perveen [23] also reported that sublethal doses of chlorfluazuron decrease the weight of spermatophores and the number of inseminating eupyrene sperm and delay the transfer of the spermatophores by 5–15 min in the common cutworm, S. litura. Therefore, the effect of sublethal doses of chlorfluazuron on the reproductive physiology of B. odoriphaga requires further research. Furthermore, our measurements of the chitin levels after a chlorfluazuron challenge indicated that the chitin content was significantly reduced in LC50-treated B. odoriphaga larvae compared with that in the control

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and LC10-treated group (Table S1). The variable symptoms of abnormally developed pupa (Fig. 5) observed in the LC50-treated group were possibly caused by the decline in chitin levels. Proteins are involved in many biochemical reactions and play a role in the structure of molecules, the structure of enzymes for transformations, the morphology of cuticle sclerotization, and the synthesis of visual pigments and neural transmitters [58]. In the present study, total proteins were reduced after treatment with chlorfluazuron, and insect physiology may be affected by the degradation of proteins into their respective amino acids for involvement in the TCA cycle as a ketoacid to compensate the lacunae of energy and the stress [59]. The reduction of total protein might due to a reduction in feeding, and the total protein rapidly increased after the feeding ability of insects recovered. The activities of GST and MFO increased significantly in the chlorfluazuron treatments compared with those in the control groups, which was consistent with the results of Devorshak [60]. This could indicate that these enzymes could play a role in detoxification processes of the studied chlorfluazuron and made it possible for the treated insects to survive and their activities may lead to the growth of total protein at 72 h [61, 62]. Merzendorfer et al. [63] conducted genomic and proteomic studies on the effects of diflubenzuron and found that only 5 down-regulated and 21 up-regulated proteins (N 2-fold) among 388 proteins and numerous genes encoding cuticle structural proteins were significantly affected, such as the encoding cytochrome P450 4c3 (TcCYP4c3), glutathione S-transferase (TcGST), sulfotransferase (TcSULT), glucosyl/ glucuronosyl-transferase (TcGLCT) and the subfamily C of ABC transporters. This study indicates that the action mechanism of chlorfluazuron on a pest is complicated, and further genomic and proteomic studies are required. Furthermore, Yu and Terriere [37] found that MFOs metabolize and decrease the levels of the juvenile hormone and analogues, which perhaps led to the shortened life span of B. odoriphaga treated with chlorfluazuron. Chlorfluazuron is a high-efficiency and low-toxicity insect growth regulator used to control the primary insect pests of crops. In this study, chlorfluazuron had the highest acute toxicity among four CSIs, causing the death of pupae with developmental deformities. Additionally, the LC50 dose of chlorfluazuron caused pupal death and the failed emergence of B. odoriphaga and decreased both the intrinsic (r) and finite rates of increase (λ), the net reproduction rate (R0), survival rate, and fecundity, which provided useful information for the control of B. odoriphaga. However, the danger of low sublethal doses causing rapid population growth was indicated by the shortened mean generation time, the unaffected net reproductive rate (R0) and intrinsic rate (r), and the finite rates of increase (λ) of the LC10-treated group. Furthermore, chlorfluazuron inhibited the feeding of the final instar larvae for a short period, and the activities of GST and MFO increased after exposure to the chemical, which possibly led to the reduced intake and the increase in metabolism of chlorfluazuron. Therefore, determining how to use the highly effective chlorfluazuron to full advantage but avoid causing rapid population growth of B. odoriphaga will require further research. Moreover, in this study, only the parental populations were investigated, and the effects on the next generation of treated larvae remains to be studied. Acknowledgements Grants from the Special Fund for Agro-scientific Research in the Public Interest from the Ministry of Agriculture of China (201303027) supported this work. We thank Professor Hsin Chi (National Chung Hsing University, Taichung, Taiwan, Republic of China) for his assistance with the data analysis. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.pestbp.2016.07.007.

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References [1] H.J. Li, X.K. He, A.J. Zeng, Y.J. Liu, S.R. Jiang, Bradysia odoriphaga copulatory behavior and evidence of a female sex pheromone, J. Agric. Urban Entomol. 24 (2007) 27–34. [2] H.Q. Feng, F.Q. Zheng, Studies of the occurrence and control of Bradysia odoriphaga Yang et Zhang, J. Shandong Agric. Univ. 18 (1987) 71–80. [3] P. Zhang, F. Liu, W. Mu, Q.H. Wang, H. Li, Comparison of Bradysia odoriphaga Yang and Zhang reared on artificial diet and different host plants based on an agestage, two-sex life table, Phytoparasitica 43 (2015) 102–120. [4] Y.C. Shi, J.Q. Zheng, Y. Zhang, Mushroom major pest survey briefing in suburbs, Plant Pro. Technol. Exten. 21 (2001) 18–19. [5] Z.X. Mei, Q.J. Wu, Y.J. Zhang, L. Hua, Life tables of the laboratory population of Bradysia odoriphaga Yang et Zhang (Diptera: Mycetophilidae) at different temperatures, Acta Entomol. Sin. 47 (2004) 219–222. [6] J. Ma, S.L. Chen, M. Moens, R.C. Han, P.D. Clercq, Efficacy of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) against the chive gnat, Bradysia odoriphaga, J. Pest. Sci. 86 (2013) 551–561. [7] S.H. Zhao, Plant Chemical Protection, China Agricultural Press, Beijing, China, 2000. [8] W. Mu, F. Liu, M.H. He, Bioassay technique of Bradysia odoriphaga and control to larvae and adults by insecticide, Acta Agric. Boreali-Sinica 17 (2002) 12–16 Suppl. [9] W. Mu, F. Liu, Z.M. Jia, D. Zhao, L.Y. Mu, Sublethal effects of eight insecticides on development and reproduction of Bradysia odoriphaga, Acta Entomol. Sin. 48 (2005) 147–150. [10] P. Wang, Y.C. Qin, D. Zhu, Toxicity determination of biopesticide to larvae of Bradysia odoriphaga Yang et Zhang in laboratory and its control effect in fields, J. Plant Prot. 31 (2011) 40–42. [11] H.X. Wang, Y. Sha, J.T. Sun, J.Z. Sun, A report of food poisoning by consuming of organophosphate pesticide contaminated vegetable, Occup. Health 22 (2006) 512–513. [12] S. Grenier, A.M.F. Grenier, A fairly new insect growth regulator, a review of its effects on insects, Ann. Appl. Biol. 122 (1993) 369–403. [13] S.K. Amani, Ultrastructural changes in integument of Tribolium castaneum (Coleoptera: Tenebrionidae) induced by chitin synthesis inhibitor (IGR) chlorfluazuron, Egypt. Acad. J. Biol. Sci. 2 (2009) 237–246. [14] H.Y. Wang, Y. Yang, J.Y. Su, J.L. Shen, C.F. Gao, Y.C. Zhu, Assessment of the impact of insecticides on Anagrus nilaparvatae (Pang et Wang) (Hymenoptera: Mymanidae), an egg parasitoid of the rice planthopper, Nilaparvata lugens (Hemiptera: Delphacidae), Crop. Prot. 27 (2008) 514–522. [15] F. Perveen, Effects of residual chlorfluazuron on haemolymph-borne ovipositionstimulating factors in Spodoptera litura, Entomol. Exp. Appl. 132 (2009) 241–249. [16] D. Chen, S.C. Zhang, L. Zhang, Field test of insect growth regulators and biopesticides against Bradysia odoriphaga, Zhiwu Baohu 31 (2005) 82–84. [17] J.D. Stark, L. Tanigoshi, M. Bounfour, Reproduc-tive potential: its influence on the susceptibility of a species to pesticides, Ecotoxicol. Environ. Saf. 37 (1997) 273–279. [18] F. Perveen, Sublethal effects of chlorfluazuron on reproductivity and viability of Spodoptera litura (F.) (Lep. Noctuidae), J. Appl. Entomol. 124 (2000) 223–231. [19] D.G. James, T.S. Price, Fecundity in two-spotted spider mite (Acari: Tetranychidae) is increased by direct and systemic exposure to imidacloprid, J. Econ. Entomol. 95 (2002) 729–732. [20] F. Perveen, Reduction in egg-hatch after a sublethal dose of chlorfluazuron to larvae of the common cutworm, Spodoptera litura, Physiol. Entomol. 31 (2006) 39–45. [21] H.A. Shahout, J.X. Qiao, Q.D. Jia, Sublethal effects of methoxyfenozide, in comparison to chlorfluazuron and beta-cypermethrin, on the reproductive characteristics of common cutworm Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), J. Entomol. Res. Soc. 13 (2011) 53–63. [22] L. You, G.L. Wang, S.R. Tian, H.Y. Wei, Sublethal effects of four low-toxicity insecticides on the development and reproduction of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), Acta Phytophylacica Sin. 6 (2013) 551–556. [23] F. Perveen, Effects of sublethal doses of chlorfluazuron on insemination and number of inseminated sperm in the common cutworm, Spodoptera litura (F.) (Lepidoptera: Noctuidae), Entomol. Sci. 11 (2008) 111–121. [24] H.F. Guo, J.C. Fang, B.S. Liu, J.P. Wang, W.F. Zhong, F.H. Wan, Enhancement of the biological activity of nucleopolyhedrovirus through disruption of the peritrophic matrix of insect larvae by chlorfluazuron, Pest Manag. Sci. 63 (2007) 68–74. [25] S. Rahmani, A.R. Bandani, Sublethal concentrations of thiamethoxam adversely affect life table parameters of the aphid predator, Hippodamia variegata (Goeze) (Coleoptera: Coccinellidae), Crop. Prot. 54 (2013) 168–175. [26] K. Day, N.K. Kaushik, An assessment of the chronic toxicity of the synthetic pyrethroid fenvalerate to Daphnia galeata mendotae using life tables, Environ. Pollut. 44 (1987) 12–26. [27] Q.Y. Gu, W.B. Chen, L.J. Wang, J. Shen, J.P. Zhang, Effects of sublethal dosage of abamectin and pyridaben on life table of laboratory populations of Tetranychus turkestani (Acari: Tetranychus), Acta Entomol. Sin. 53 (2010) 876–883. [28] H. Yang, Z. Wang, D.C. Jin, Sublethal effect of chlorantraniliprole on the experimental population of non-target insect Ni-laparvata lugens (Stal), Chin. J. Appl. Ecol. 24 (2013) 549–555. [29] E.G. Lewis, On the generation and growth of a population, Sankhya 6 (1942) 93–96. [30] P.H. Leslie, On the use of matrices in certain population mathematics, Biometrika 33 (1945) 183–212. [31] H. Chi, Life-table analysis incorporating both sexes and variable development rate among individuals, Environ. Entomol. 17 (1988) 26–34. [32] Y.B. Huang, H. Chi, Age-stage, two-sex life tables of Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) with a discussion on the problem of applying female agespecific life tables to insect populations, Insect Sci. 19 (2011) 263–273.

[33] L. Lagadic, A. Cuany, J.B. Berge, et al., Purification and partial characterization of glutathione S-transferases from insecticide resistant and lindane induced susceptible Spodoptera littoralis larvae, Insect Biochem. Mol. Biol. 23 (1993) 467–474. [34] I. Kostaropoulos, A.I. Papadopoulos, A. Metaxakis, et al., Glutathione S-transferase in the defence against pyrethroid in insects, Insect Biochem. Mol. Biol. 31 (2001) 313–319. [35] J.G. Vontas, G.J. Small, D.C. Nikou, et al., Purification, molecular cloning and heterologous expression of a glutathione S-transfer involved in insecticide resistance from the rice brown planthopper, Nilaparvata lugens, J. Bio. Chem. 362 (2002) 329–337. [36] S.J. Yu, L.C. Terriere, Activities of hormone metabolizing enzymes in house flies treated with some substituted urea growth regulators, Life Sci. 17 (1975) 619–625. [37] S.J. Yu, L.C. Terriere, Ecdysone metabolism by soluble enzymes from three species of diptera and its inhibition by the insect growth regulator TH-6040, Pestic. Biochem. Physiol. 7 (1977) 48–55. [38] S. Sonoda, H. Tsumuki, Studies on glutathione S-transferase gene involved in chlorfluazuron resistance of the diamondback moth, Plutella xylostella L. (Lepidoptera: Yponomeutidae), Pestic. Biochem. Physiol. 82 (2005) 94–101. [39] W. Mu, F. Liu, Z.M. Jia, M.H. He, G.Q. Xiang, A simple and convenient rearing technique for Bradysia odoriphaga, Entomol. J. East China 12 (2003) 87–89. [40] H. Chi, T. Yang, Two-sex life table and predation rate of Propylaea japonica Thunberg (Coleoptera: Coccinellidae) fed on Myzus persicae (Sulzer) (Homoptera: Aphididae), Environ. J. Entomol. 32 (2003) 327–333. [41] M.I. Schneider, N. Sanchez, S. Pineda, H. Chi, A. Ronco, Impact of glyphosate on the development, fertility and demography of Chrysoperla externa (Neuroptera: Chrysopidae): ecological approach, Chemosphere 76 (2009) 1451–1455. [42] C. Goday, M.R. Esteban, Chromosome elimination in sciarid flies, BioEssay 23 (2001) 242–250. [43] B.B. Normark, The evolution of alternative genetic systems in insects, Annu. Rev. Entomol. 48 (2003) 397–423. [44] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [45] X.Y. Deng, F. Wu, Z. Liu, M. Luo, L. Li, Q.S. Ni, Z. Jiao, M.H. Wu, Y.F. Liu, The splenic toxicity of water soluble multi-walled carbon nanotubes in mice, Carbon 47 (2009) 1421–1428. [46] L.G. Hansen, E. Hodgson, Biochemical characteristics of insect microsomes: N- and O-demethylation, Biochem. Pharmacol. 20 (1971) 1569–1578. [47] J.G. Yuan, G.Y. Sun, G.L. Feng, P. Zhu, K.Y. Gong, The activities for acetylcholinesterase, carboxylesterase and mixed-function oxidase in relation to pesticide resistance in the housefly, Acta Entomol. Sin. 30 (1987) 126–132. [48] H. Chi, H. Liu, Two new methods for the study of insect population ecology, Bull Inst. Zool Acad Sin. 24 (1985) 225–240. [49] B. Efron, R.J. Tibshirani, An Introduction to the Bootstrap, Chapman & Hall Press, New York, 1993. [50] H. Chi, TWOSEX-MSChart: a computer program for the age-stage, two-sex life table analysishttp://140.120.197.173/Ecology/prod02.htm 2012. [51] C.W. Dunnett, Pairwise multiple comparisons in the homogeneous variance, unequal sample size case, J. Am. Stat. Assoc. 75 (1980) 789–795. [52] Z.M. Zhao, X.Y. Zhou, Introduction of Ecology, Scientific and Technological Literature Press, Chongqing, 1984 16–18. [53] R. Farhadi, H. Allahyari, H. Chi, Life table and predation capacity of Hippodamia variegata (Coleoptera: Coccinellidae) feeding on Aphis fabae (Hemiptera: Aphididae), Biol. Control 59 (2011) 83–89. [54] I. Ishaaya, S. Yablonski, A.R. Horowitz, Comparative toxicity of two ecdysteroid agonists, RH-2485 and RH-5992, on susceptible and pyrethroid-resistant strains of the Egyptian cotton leafworm, Spodoptera littoralis, Phytoparasitica 23 (1995) 139–145. [55] T.S. Zhang, Q.L. Zhang, Y.Y. Xi, Y.D. Yuan, H.Y. Teng, D.S. Wang, Sublethal effects of chlorfluazuron on growth and fecundity of beet armyworm (Spodoptera exigua), Acta Agric. Shanghai 29 (2013) 1–4. [56] P. Zhang, C.Y. Chen, H. Li, F. Liu, W. Mu, Selective toxicity of seven neonicotinoid insecticides to Bradysia odoriphaga and Eisenia foetida, Acta Phytophylacica Sin. 41 (2014) 79–86. [57] R. Christian-Luis, S. Pineda, Toxicity and sublethal effects of methoxyfenozide on Spodoptera exigua (Lepidoptera: Noctuidae), J. Econ. Entomol. 103 (2010) 662–667. [58] R.F. Chapman, The Insects, Structure and Function Fourth Ed, Cambridge University Press, Cambridge, 1998. [59] L.M. Schoonhoven, Biological aspects of antifeedants, Entomol. Exp. Appl. 31 (1982) 57. [60] C. Devorshak, R.M. Roe, The role of esterases in insecticide resistance, Rev. Toxicol. 2 (1998) 501. [61] S. Rumpf, S. Hatzel, F.J. Chris, Lacewings and integrated pest management: enzyme activity as biomarker of sublethal insecticide exposure, Econ. Entomol. 90 (1997) 102. [62] S. Dugravot, E. Thibout, A. Abo-Ghalia, J. Huignard, How a specialist and a nonspecialist insect cope with dimethyl disulfide produced by Allium porrum, Entomol. Exp. Appl. 113 (2004) 173. [63] H. Merzendorfer, H.S. Kim, S.S. Chaudhari, M. Kumari, C.A. Specht, S.B. Susan, J. Brown, J.R. Manak, R.W. Beeman, K.J. Kramer, S. Muthukrishnan, Genomic and proteomic studies on the effects of the insect growth regulator diflubenzuron in the model beetle species Tribolium castaneum, Insect Biochem. Molec. 42 (2012) 264–276.