Sublethal effects of bifenazate on biological traits and enzymatic properties in the Panonychus citri (Acari: Tetranychidae)

Panonychus citri, a major citrus pest. In pest management, bifenazate is a novel acaricide with high biological activity against red mites, such as Tetranychus urticae Koch. However, in the field, pests are frequently exposed to sublethal or lethal concentrations of pesticides. At present, its sublethal effects on P. citri have not been reported. Therefore, in order to investigate sublethal effect of bifenazate on biological traits and enzymatic properties of P. citri. The newly emerged females were treated with two concentrations of bifenazate: LC10 and LC30, the development and fecundity were observed. The results showed that female adult duration, fecundity, oviposition days, longevity were decrease compared with control, but pre-oviposition period was longer, net reproductive rate (R0), mean generation (T) were decreased, intrinsic rate of increase (rm), finite rate (λ) were decreased in LC30, however, doubling time was increased. Enzymatic tests showed that CAT, POD, CarE activities were higher in treatments than control. The SOD and GST activities were lower in LC30 than control and LC10, the CYP450 activity was decreased with the increasing concentrations. This study demonstrated that low lethal concentrations of bifenazate adversely affected life table parameters, enzymatic properties in P. citri. Therefore, bifenazate has the potential to control this pest.

www.nature.com/scientificreports/ (GST), and carboxylesterase (CarE) 21 . Superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) are three important protective enzymes in insects that play roles in immunity, preventing free-radical-associated damage, and protecting cells from adverse environmental effects 22 . In some cases, the low lethal concentration of insecticides can inhibit the enzyme activity in insects, while in other cases, the low lethal concentration of insecticides can stimulate the enzyme activity. For example, it has been reported that low lethal concentrations (LC 10 and LC 25 ) of abamectin can promote upregulation of the SOD, POD, and CAT activities in Diadegma semiclausum, with activity increasing with increasing insecticide concentration 23 In contrast, the levels of SOD, POD, and CAT activity in Harmonia axyridis were shown to decrease with an increase in abamectin concentration 24 . Avermectin (LC 10 and LC 25 ) resulted in a significant induction of CarE, GST activities in Sogatella furcifer 25 . Previously, it was found that GST and P450 activities in Aphis craccivora were significantly induced after treatment with cycloxaprid and imidacloprid (LC 50 ), whereas in contrast, the activity of the CarE activity was inhibited. Traditional estimating only by measuring the lethal effect of insecticides may underestimate the total effects of insecticides on the pest 26,27 . Accordingly, in this study, we sought to gain insights into the roles of these enzymes to insecticide-induced stress. To this end, we exposed this insect to low lethal concentrations of bifenazate and subsequently monitored the changes in enzyme activity levels and biological traits. In pest management (IPM), it has a significant influence on the sublethal effects of insecticides. The objective of present study was to obtain a comprehensive understanding of the sublethal effects of bifenazate on P. citri, including its developmental time, fecundity, life table parameters and detoxification enzymes, antioxidant enzyme 28 . The results may be employed to understand the sublethal effects of bifenazate on P. citri, which will contribute to the assessment and rational application of bifenazate for controlling this pest. Overall, research on the effects of acaricides on P. citri populations will be invaluable in evaluating the role that particular acaricides play in sustainable management programs involving the pest mite. Moreover, knowledge on the activity of detoxification enzymes in P. citri may provide a basis for abating or minimizing the development of resistance to effective acaricides 29 .

Materials and methods
Mite and pesticide. The laboratory strains of P. citri were collected from the citrus orchard in Wuning County, Jiujiang City, Jiangxi Province, China, In September 2019. The population was reared on the leaf disc of Aurantii Fructus in climate-controlled chamber, under the conditions of 26 ± 1 °C, relative humidity 70% ± 10%, photoperiod 16 h: 8 h (L:D). Place on a wet sponge in a Petri dish (15 cm in diameter), cotton slivers were placed around each leaf to prevent mites from escaping, without exposure to insecticide. Bifenazate (C17H20N2O3; ≥ 99% Purity) was provided by accustandard (New Haven, USA). All studies involving plants (Fructus Aurantii leaves) have been carried out in accordance with relevant institutional, national or international guidelines.
Bioassay. Bioassay of female P. citri was performed according to the modified leaf dish dipping method of Ken and Yamamoto 30 . The newly emerged third instar female adult mites were transferred to the leaves of Fr. aurantii by small brush. 45 individuals were selected from each leaf. Wet cotton was put around the leaves with mites to prevent mites from escaping. The mites were placed in climate-controlled chamber. After 4 h, the mites were examined by microscope, the dead and inactive individuals were removed. Then, use tweezers to immerse the leaves with mites in different concentrations of acaricide solution for 5 s, and take out the excess liquid and quickly suck it up with absorbent paper. Leaves with female adult mites soaked in 0.1% ethyl acetate solution were set as control treatment. Put the treated leaves into the prepared leaf dish and put them into the climate control chamber. Each treatment was repeated in 4 groups. After 24 h, the mortality was recorded under microscope. If their foot does not move, it will be regarded as death. If the mortality of control group is less than 10%, it will be regarded as effective experiment.
Low lethal concentration of bifenazate effects on the life-history traits of P. citri. In this study, leaf dipping method and leaf dish feeding method were used, and the feeding table was made as shown in the Fig. 1. The third instar female adult mites were selected and placed on each leaf dish. After 4 h, the leaves and female adult mites were soaked with bifenazate (LC 10 , LC 30 ) by leaf dish dipping method. The leaves with female adult mites were soaked with 0.1% ethyl acetate solution as control. Then they were raised in climate-controlled chamber. After 24 h, the survivors were selected to the fresh rearing platform to continue feeding, and the same number of male adult mites were selected at the same time. After mating for 12 h, 100 eggs were taken from each treatment for single feeding. When female mites are mature, male mites are selected for mating, and the fertility and mortality are observed every 24 h until all female adults die.
Preparation of samples for enzyme assay. The female adult mites (as described in "Low lethal concentration of bifenazate effects on the life-history traits of P. citri" section) were exposure to bifenazate for 24 h at concentrations of LC 10 , LC 30 , and the survivors were picked up. 150 individuals were placed in a 1.5 mL centrifuge tube. The collected samples were treated with liquid nitrogen and stored at − 80 °C. There were four treatments in the experiment, and three replicates were set for each treatment.

Measurement of tissue total protein. The purchased total protein extraction kit (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China) was used to determine the total protein content of the sample. PBS buffer (0.05 mol/L, pH 7) was added into the centrifuge tube containing 150 mites. After full homogenization, the homogenate was centrifuged at 4 ℃ and 14,000 rpm for 15 min, and then the supernatant was taken as the

Measurement of antioxidant enzymes activities. Catalase (CAT). According to the instruction of
CAT test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), the activity of CAT was determined by ammonium molybdate method. Absorb 200 μL of reaction mixture and add it into 96 well plate. Read the OD value at 405 nm, repeat 3 times, and take the average value. The activity of CAT was calculated.
Superoxide dismutase (SOD). Refer to the instruction of SOD activity test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Mix the solution well, place it at room temperature for 10 min, absorb 200 μL accurately, and add the reaction solution into 96 well plate. At the wavelength of 550 nm, read the OD value of absorbance, repeat for 3 times, and take the mean value. The activity of SOD was calculated.
Peroxidase (POD). The activity of POD was determined according to the instruction of POD assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). After the solution was mixed, centrifuged at 3500 rpm for 10 min, 200 μL supernatant was added into 96 well plate. Read the OD value at the wavelength of 420 nm, repeat three times, and take the mean value. The activity of pod was calculated.

Measurement of detoxifying enzyme activities. Carboxyl esterase (CarE).
According to the instructions of CarE assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), the activity of CarE was determined by spectrophotometry. Fully mix the solution, take 5 μL supernatant and 1000 μL preheating working solution into 1 mL glass cuvette in turn, quickly mix them, and read the absorbance value after 10 s and 190 s at 450 nm. The change of absorbance value is the measured value, repeat for 3 times, and take the average value. Calculate the vitality of CarE.   www.nature.com/scientificreports/ period (APOP, the length of the preoviposition period beginning from adult emergence), and total preoviposition period (TPOP, the length of the preoviposition period beginning from birth). Population growth parameters including net reproductive rate (R 0 ), intrinsic rate of increase (rm), finite rate of increase (λ), and mean generation time (T) were also calculated as follows: The standard errors of the developmental times, survival, fecundity, longevity, and population parameters were estimated by using the bootstrap method with 100,000× resamplings 33 , the paired bootstrap test was used to compare differences 34 . The computer program TWOSEXMSChart was used to analyze the raw data and calculate population parameters. Both bootstrap and paired bootstrap test routines were included in this program.

Results
Toxicity of bifenazate to the third instar female adults of P. citri. The toxicity of bifenazate to the third instar female adults of P. citri was stronger ( Table 1). The median lethal concentration (LC 50 ) was 11.915 mg/L. the concentrations leading to 10% and 30% mortality were 3.625 mg/L and 7.015 mg/L, respectively.
Low lethal concentration of bifenazate effects on the life-history traits of P. citri. Through bioassay of the third instar female adults of P. citri, we obtained low and median lethal concentrations. Tables 2  and 3 show the effects on the life table parameters   Compared with the control, the net reproductive rate (R 0 ) and average generation time (T) of female adult mites treated with LC 10 and LC 30 were significantly decreased, while the intrinsic rate of increase (rm) of insects treated with LC 10 had no significant difference; however, The intrinsic rate of increase (rm), finite rate of increase (λ) for the LC 30 treated group significantly decreased for the control group (Table 3); the population doubling time (DT) of LC 30 group were prolonged, but there was no significant difference between LC 10 group and control group.
As shown in Fig. 2, the probability of newborn eggs surviving to x age and developing to j stage is expressed by the survival rate of specific age stage (s xj ), and the survival curves of different age stages overlap obviously; Compared with the control group, the survival rate of female adult mites in LC 10 and LC 30 treatment groups is relatively low. www.nature.com/scientificreports/ Age-specific survival rate (l x ), age-specific fecundity (m x ) and age-specific fecundity (l x m x ) can be reflected in Fig. 3. l x is the survival rate of newborn eggs to the age of x, m x is the total fecundity of the population to the age of x, l x m x is the result of the multiplication of l x and m x ; l x and m x of LC 10 and LC 30 treatment are lower than the control, l x and m x show a downward trend with the increase of concentration.

Low lethal concentration of bifenazate effects on protective enzyme activities of P. citri. The
CAT、SOD 、POD activities of the third instar female adults P.citri treated with bifenazate was determined after 24 h. As shown in Fig. 4, the CAT activity of LC 10 and LC 30 treatment increased significantly compared with the control; the SOD activity of LC 10 treatment increased, but the SOD activity of LC 30 treatment group had no significant differences. Moreover, there was no significant difference in POD activity in the LC 10 treated group compared with the control, However, POD activity was significantly higher in the LC 30 treated group.
Low lethal concentration of bifenazate effects on detoxifying enzyme activities of P. citri. The activities of CarE, GSH-ST and CYP450 of the third instar female adults P. citri treated with bifenazate were determined 24 h later. As shown in Fig. 5, the activity of CarE in all treatment groups increased significantly compared with the control; the activity of GSH-ST in LC 10 treatment increased, while in LC 30 treatment decreased significantly; the activity of CYP450 in all treatment decreased compared with the control.

Discussion
In pest control, it is important to assess the sublethal effects of insecticides on pest populations, and life table analysis is an important technique for effective assessment 35 . Fecundity, intrinsic rate of increase (rm), finite rate of increase (λ), and net reproductive rate (R 0 ) are several key parameters for evaluating population growth, development and reproduction 36,37 . www.nature.com/scientificreports/ Our toxicity assay results showed that bifenazate is a potential insecticide for effective control of P. citri; However, insecticide has not only acute toxicity, but also the sublethal effects 38 . In the current study, the fecundity of P. citri was decreased in the LC 10 or LC 30 treated groups, and the same response was observed in pests treated with other insecticides 39 . For example, the low lethal concentrations (LC 20 , LC 30 ) of chlorfenapyr inhibit T. urticae development and reproduction 40 . A low lethal concentration (LC 30 ) of cyantraniliprole significantly inhibited fecundity in Helicoverpa assulta 11 . In contrast, many studies have shown that low concentrations of insecticides promote pest fecundity, for example, treatment with LC 10 and LC 20 of spinetoram reduces the time of development of spotted mites from eggs to adults and promotes their fecundity 20 . Bradysia odoriphaga reproduction was stimulated by chlorfenapyr 40 . In this study, we found that the low lethal concentration of bifenazate can effectively inhibit the increasing population of P. citri. This result showed that the logical application of insecticides is integral to P. citri management.
Insecticide stress has an impact on insect development, fecundity and population parameters 10 . In addition, they can also affect the activities of antioxidant enzymes and detoxification enzymes 41 . Panonychus citri exposure to different sublethal and low lethal concentrations of bifenazate (LC 10 , LC 30 ) resulted in a significant increase in the activity of antioxidant enzymes, which was reduced near death (Fig. 4). Chemical stress causes the body to start its own defense system, produce a lot of reactive oxygen species, increase SOD activity and produce H 2 O 2 , which requires CAT and POD decomposition to achieve the relative balance of the body; when the concentration of chemical continues to rise, the self-defense ability will be weakened, resulting in the inhibition of protective enzyme activity 42,43 ; this is consistent with the previous studies on other pests using other insecticides 44 .
At the same time, it can also affect the activity of detoxification enzymes; CYP450, GSH-ST, and CarE are three key supergene families in insect detoxification metabolism 45 . GSH-ST is a family of enzymes that can catalyze the binding reaction of reduced glutathione (GSH) compounds, which has electrophilic properties and can catalyze the hydrolysis of esters, sulfates and amides. GSH-ST and CarE are the main enzymes involved in pesticide metabolism 46,47 . Previous studies showed that the activities of CarE and GSH-ST were up-regulated in Sogatella furcifera treated with buprofezin at low lethal concentrations (LC 10 and LC 25 ) for 48 h, which increased with insecticide concentration rising 45 . In the present study, the activities of CarE and GSH-ST increased significantly after exposure to different low lethal concentrations (LC 10 , LC 30 ) of bifenazate, and decreased when approaching death (Fig. 5). In addition, CYP450 is a multifunctional enzyme, which plays an important role in the formation of insecticide resistance 5 . In a variety of animal cells, it has been proved that a variety of insecticides can induce the production of reactive oxygen species (ROS), and then induce oxidative stress 48 . ROS attack, protein and lipid lead to oxidative damage, which destroys the integrity of enzyme structure and reduces enzyme activity 49 . At present, the results showed that compared with the control group, the CYP450 enzyme activity in the treatment  (CK served as control group, data are means ± SE of three biological replications; different letters above each bar indicate statistically significant difference by ANOVA followed by the Duncan's multiple range test). *p < 0.05, **p < 0.01, ***p < 0.001. www.nature.com/scientificreports/ group decreased significantly with the increase of the concentration of bifenazate. For example, when locusts were exposed to different sublethal doses of chlorpyrifos, the activity of CYP450 enzyme decreased with insecticide concentration increasing 50 . Therefore, these results provide a basis for further study on the molecular characteristics of antioxidant enzymes and detoxification enzymes of P. citri, and it is of great significance to understand the relationship between insecticides, antioxidant enzymes and detoxification enzymes. In summary, bifenazate not only showed acute toxicity to P. citri, but also had sublethal effect. This study showed that sublethal and low lethal concentration of bifenazate could significantly affect the development duration and fecundity of P. citri. In addition, the activity of CYP450 in P. citri exposed to low lethal concentration was inhibited. Therefore, it is necessary to study bifenazate effects on CYP450 and reproductive related genes in P. citri at mRNA level in the future. It is an effective way to control the growth and development of P. citri. License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.