Introduction
Panonychus citri (Acarina: Tetranychidae) is distributed worldwide and mainly feeds on the leaves of citrus (Pan et al., Reference Pan, Dou, Yuan, Zhou and Wang2020). Presently, the key method for the control of P. citri is chemical control (Cheng et al., Reference Cheng, Hou, Sun, Yu, Li, Liu, Cong and Ran2022). The enhanced application of pesticides prevents further damage, but it has several undesirable consequences, such as impacting natural enemies and non-target organisms. Meanwhile, the increased use of insecticides has led to widespread resistance of P. citri to insecticides (Quesada and Sadof, Reference Quesada and Sadof2019). This emphasises the need to adopt effective acaricides for the control of P. citri (Andrade et al., Reference Andrade, Lorencon, Siqueira, Novelli and Bassanezi2018).
Spirodiclofen is an acaricide that belongs to the spirocyclic tetraelectron acid family and works by interfering with lipid biosynthesis (Amaral et al., Reference Amaral, Melville, Rocha, Della, Prado and Andrade2020). Lipids constitute approximately 30–40% of the dry weight in insects (Ziegler and Antwerpen, Reference Ziegler and Antwerpen2006), with a higher proportion in mites. Consequently, this acaricide is widely used worldwide for controlling P. citri and mites in other crops, making it one of the most commonly used acaricides in China (Claudiane et al., Reference Claudiane, Jaqueline, Patrice, Celso and Daniel2021). However, its extensive use has also increased the frequency of misuse and overuse of insecticides. This improper usage has led to insects being exposed to lethal and sub-lethal concentrations of the insecticides, thereby becoming a key factor in accelerating pest resistance development. Previous research has shown that field populations of P. citri have relatively low resistance levels to spirodiclofen, but there is a possibility of developing higher levels of resistance in the future (Alavijeh et al., Reference Alavijeh, Khajehali, Snoeck, Panteleri, Ghadamyari, Jonckheere, Bajda, Saalwaechter, Geibel, Douris, Vontas, Van and Dermauw2020). Field populations of phytophagous mites have also been confirmed to possess tolerance to spirodiclofen in regions such as the United States, Germany, Brazil, China, Turkey, and Iran (Cheng et al., Reference Cheng, Hou, Sun, Yu, Li, Liu, Cong and Ran2022). Therefore, exposure of P. citri to lethal and sub-lethal concentrations of spirodiclofen in the field cannot be avoided. Assessing the potential lethal and sub-lethal effects of spirodiclofen on P. citri is crucial for a comprehensive analysis of the effectiveness of this insecticide.
Sublethal effects are defined as impacts (either physiological or behavioural) on survival individuals when exposed to a toxicant at low or sublethal concentration/dose (Desneux et al., Reference Desneux, Decourtye and Delpuech2007). The sublethal effects of pesticides on pests are not only reflected in the direct killing, but also affect their reproduction, lifespan, and physiological characteristics (Wang et al., Reference Wang, Zhang, Xie, Wu and Wang2016; Dong et al., Reference Dong, Wang, Li and Wang2017), as well as changes in their detoxification and antioxidant enzymes (Ma et al., Reference Ma, Tang, Xia, Lv and Gao2019). Hormesis, the stimulation of performance when an organism is exposed to low levels of agents, is regarded as the main mechanism for pest population resurgence (Cordeiro et al., Reference Cordeiro, de Moura, Fadini and Guedes2013). For example, some researchers have suggested that the sublethal concentrations of insecticides might incite insect outbreaks (Bartle, Reference Bartle1968). Currently, the lethal and sublethal effects of spirodiclofen on P. citri have not been determined.
Here, we used an age-stage, life table to evaluate the lethal and sublethal effects of the spirodiclofen on the life table parameters of P. citri, including development time, survival rate, longevity, fecundity, and hatchability. The results will help to provide a comprehensive assessment of this new insecticide for integrated pest management (IPM) and chemical applications in the field. Furthermore, understanding the activities of several key antioxidant enzymes and detoxification enzymes in P. citri can provide the basis for mitigating or reducing resistance to potent acaricides (Rasheed et al., Reference Rasheed, Hafeez, Zhao, Islam, Ali, Ur-Rehman, Hani and Zhou2020).
Materials and methods
Mite and pesticide
In 2019, a stable population of P. citri was established by collecting a colony from Nanchang University of Nanchang, Jiangxi Province, China and maintaining them through continuous breeding. The population of P. citri were fed citrus tender leaves and were kept in an artificial climate box with the following environmental conditions: a temperature of 26 ± 1℃, a relative humidity (RH) of 70 ± 5%, and a photoperiod of 16:8 hours. The population was not exposed to any pesticides until the present study (in 2021), which was considered the acaricide-susceptible strain (SS).
The acaricide used in the bioassay was spirodiclofen (24%SC), which was purchased from Bayer Crop Science (China) Co. Ltd.
Bioassay
The modified leaf dipping method was used for the bioassay on the P. citri population (Yamamoto et al., Reference Yamamoto, Yoneda, Hatano and Asada1995). The adult females were transferred to citrus leaves (n = 45, three repetitions per treatment). Based on the pre-experiment, the acaricide was diluted to six concentrations (fig. 1). The control was performed on leaves with adult females soaked in triton X-100 solution. Immerse the leaves with P. citri in the prepared insecticide for 5 s (Wang et al., Reference Wang, Xin, Wang, Zou, Zhong and Xia2021). The treated citrus leaves were placed on the prepared Petri dishes (d = 15 cm) in an air-conditioned room. After 24 h, the mortality of the P. citri population was recorded, and if their legs did not move, they were considered dead. The mortality data were used for LC30 and LC50 determination. All treated-individuals are raised through fresh citrus leaves (not exposed to any insecticides).
(A) Bioassay method schematic diagram (B) An observation of the life history of adult females of Panonychus citri.
Changes in life table parameters
The adult females (n = 100, three repetitions per treatment) were transferred to the leaf dish and soaked with two spirodiclofen concentrations (LC30 and LC50) by the leaf dipping method refer to the method described 2.2 (fig. 1A), the control was performed on leaves with adult females soaked in triton X-100 solution. Following 24 h, survivors (newly emerged 3-day-old adult females) were selected to continue feeding on the fresh-rearing platform. Meanwhile, adult males (without insecticide treated) were selected for mating. Eggs produced were cultured independently 12 h after mating (n = 30). The adult females (with insecticide treatment) were mated with adult males as soon as they reached maturity, and they were observed every 24 h until died (fig. 1B).
Enzyme activity assay
3-day-old adult females were exposed to two concentrations of spirodiclofen (LC30 and LC50) and control for 24 h, the prepared samples (n = 300, three biological repetitions per treatment) were frozen in liquid nitrogen and stored at −80°C in one 1.5 mL centrifuge tube to create the crude enzyme solution.
After calculation of protein concentration (BCA Protein Assay Kit- A045, Nanjing Jiancheng Bioengineering Institute, China), the supernatant of required volume was used to measure the enzyme activities of SOD, CAT, POD, CarE, GST, and CYP450 by commercial assay kits A001, A007, A084, A133, A004, and H303(Nanjing Jiancheng Bioengineering Institute, China). According to the instruction of CAT, SOD, POD test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), antioxidant enzymes activities: CAT, SOD, POD and detoxifying enzyme activities: CarE, GST, CYP450 were measured (Qi et al., Reference Qi, Niu, Wang, Wang, Zhu, Xue, Zhang and Wu2020). Using a photometer set at 450 nm, 550 nm, 420 nm, 450 nm, 412 nm, 450 nm, the amount of CAT, SOD, POD, CarE, GST, CYP450 in P. citri was determined. The change of absorbance value is the measured value, repeat for three times, and take the average value. Calculate the activity of six enzyme activities.
Gene expression analysis
The StepOnePlus real-time quantitative PCR machine was used to determine the mRNA level of some genes (such as Vg and VgR genes) after two concentrations of spirodiclofen exposure.
The 200 adult females were used to extract total RNA by Trizol reagent.(Shenggong Biological Technology Co. Ltd., Shanghai, China) following the manufacturer's specifications, and quantification was then performed using a NanoDrop 2000 spectrophotometer. In the following step, reverse transcription of RNA is performed using PrimeScript RT Reagent Kit with gDNA Eraser in a 20 μl reaction volume (TaKaRa, Shiga, Japan). Every RT-qPCR was carried out in a 20-μL mixture. The following were the qPCR cycling parameters: 95°C for 10 min, then 40 cycles of 95°C for 30 s and 60°C for 30 s. Primers were designed as shown in table 1, with the GAPDH and EF-1FA genes as internal reference genes. Relative quantification was calculated using the comparative 2−△△Ct method (Zhao et al., Reference Zhao, Wang, Wang and Cui2018b).
Primers for real-time quantitative PCR.
Statistical analysis
The life history parameters were counted, including age-stage specific rate (sxj), Age-specific survival (lx), age-specific reproduction (mx), APOP (adult pre-ovipositional period), TPOP total pre-ovipositional period (from newborn egg to first oviposition). Population parameters including R 0, rm, λ, and T were also calculated as follows:
The standard errors of raw data were calculated by using the bootstrap method with 100,000× resamplings, the paired bootstrap test was used to compare differences (Akkopru et al., Reference Akkopru, Atlihan, Okut and Chi2015). The TWOSEXMSChart program was used to analyse the raw data and population parameters (Chi and Liu, Reference Chi and Liu1985).
Results
Toxicity of spirodiclofen in the adult females of Panonychus citri
Acute toxicity of spirodiclofen (24%SC) was observed in adult females P. citri (table 2). Concentrations resulting in 30 and 50% mortality were 3.898 g l−1 and 5.215 g l−1.
Acute toxicity of spirodiclofen to adult females of Panonychus citri.
Responses of spirodiclofen on population parameters of Panonychus citri
Through bioassay of P. citri, the low lethal concentration (LC30) and median concentration (LC50) were obtained. tables 3 and 4 present the lethal and sublethal effects on the F1 generation of P. citri. The average immaturity time (The development stage of adult females before spawning) of P. citri treated with LC30 was not significantly different from the control (table 3). However, LC50 treatment was prolonged in egg duration. Compared to the control, LC30 and LC50 treatments significantly prolonged both oviposition period and total pre-ovipositional period. Compared with control in maturity (13.930 d), longevity (22.310 d), and fecundity (5.350 eggs/female), there were significant reductions in maturity (10.510 d and 5.620 d), longevity (21.430 d and 17.650 d), and fecundity (4.310 eggs/female and 2.680 eggs/female) after exposure two concentrations (LC30 and LC50) of spirodiclofen.
Spirodiclofen effects on Panonychus citri developmental time.
The bootstrap paired test does not detect significant differences between means in a row following the same letter (P > 0.05). APOP, adult pre-ovipositional period; TPOP, total pre-ovipositional period (from newborn egg to first oviposition).
Effects of spirodiclofen on population parameters of Panonychus citri offspring.
By the bootstrap paired test (P > 0.05), means in a row followed by the same letter are not significantly different.
The R 0 and T of adult females were significantly reduced on LC30 and LC50 treatments compared to the control. Adult females P. citri treated with LC30 showed no significant difference in rm and λ from the control, whereas they decreased in the LC50 treatment (table 4). It is clear that the survival rates at different ages are overlapping, as eggs are more likely to survive up to age x and develop to stage j when they occur at different age stages (fig. 2). Compared with the control, adult females survival rates in the LC30 and LC50 groups are relatively low. Panonychus citri survival rate (lx), fecundity (mx), and maternity rates (lxmx) after exposure to two concentrations of spirodiclofen are shown in fig. 3. The lx and mx of the treatment groups are lower than the control. Furthermore, both lx and mx decrease with the increase of concentration.
The age-stage specific rate (sxj) of Panonychus citri at two concentrations of spirodiclofen.
Age-specific survival (lx), age-specific reproduction (mx) and age-specific motherhood (lxmx) of Panonychus citri after exposure to two concentrations of spirodiclofen.
Responses of spirodiclofen on enzymatic activity of Panonychus citri
CAT, SOD, and POD activities were measured 24 h after spirodiclofen treatment. Difference in CAT between control and LC30 group was not significant (fig. 4). However, LC30 treatment significantly increased SOD activity compared with control, but both CAT and SOD activities of the LC50 treatment group showed a significant decline. The POD activity of the LC30 group has no significant difference which was compared with the control, whereas POD activity was significantly higher in the LC50 group. The activities of CarE, GST, and CYP450 of P. citri treated with spirodiclofen were determined. The activity of CarE in LC30 treatment increased compared with control, while it decreased significantly with increasing concentrations (fig. 5). Comparatively to the control group, all treatment groups showed a decrease in GST and CYP450 activities.
Response of protective enzyme activities of Panonychus citri to spirodiclofen stress (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.
Response of detoxification enzyme activities of Panonychus citri to spirodiclofen stress. (data are Means ± SE of three biological replications; different letters above each bar indicate statistically significant difference by ANOVA followed by Duncan's multiple range test). *P < 0.05, **P < 0.01, ***P < 0.001.
Vg and VgR relative expression
To determine how spirodiclofen affects the contents of Vg and VgR in P. citri, mRNA-relative expression of Vg and VgR were measured. Compared with CK, Vg and VgR expression were significantly decreased and showed a more significant decrease with increasing concentrations (fig. 6). In addition, significant concentration effects were also observed between the LC30 and LC50 treatments.
Relative expression level of Vg and VgR genes in adult females of Panonychus citri exposed to two concentrations of spirodiclofen. The bars represent the Means ± SE of three replications.
Discussion
The study of life table is a key aspect of insect population dynamics (Desneux et al., Reference Desneux, Decourtye and Delpuech2007). The field of insect population dynamics is characterised by changes that are affected by many factors, including diet, temperature, light, and especially chemical pesticides (Mousavi et al., Reference Mousavi, Maroofpour and Nariman2020). Fecundity, rm, λ, and R0 are several important parameters for assessing population dynamics (Papachristos and Milonas, Reference Papachristos and Milonas2008; Rahmani and Bandani, Reference Rahmani and Bandani2013).
The toxicity test indicated that fecundity and population parameters including rm, λ and R 0 of P. citri were decreased in the treated groups, and similar findings were observed in other insecticide-treated pests (Zhao et al., Reference Zhao, Wang, Ding, Wang, Zhang, Liu and Mu2018c). Chlorfenapyr, for example, inhibits Tetranychus urticae Koch growth and reproduction at low lethal concentrations (LC20 and LC30) (Sani et al., Reference Sani, Hamid and Elham2018). At low lethal concentrations (LC30), cyantraniliprole significantly inhibits the fertility of Helicoverpa assulta (Dong et al., Reference Dong, Wang, Li and Wang2017). In contrast, there have been several studies showing that low levels of pesticides can stimulate fertility. For instance, treatment with spinetoram LC10 and LC20 shortens the time for T. urticae to develop from egg to adult and increases their fecundity (Wang et al., Reference Wang, Zhang, Xie, Wu and Wang2016). Chlorfenapyr stimulated Bradysia odoriphaga reproduction (Sani et al., Reference Sani, Hamid and Elham2018). However, in the present study, the growth of P. citri can be effectively inhibited by two concentrations of spirodiclofen. As a consequence, different insects react differently to insecticide stress.
The fertility of the P. citri F1 generation decreased significantly. Adverse effects of drugs such as shorter lifespans and reduced fertility are often associated with their development (Zhang et al., Reference Zhang, Mao, Liao, He, Jin, Tang, Wan and Li2018). Adaptive costs of insecticide resistance, or the dominant disadvantage that accompanies the development of insecticide resistance, are associated with susceptible insects in a population. To gain more insight into this mechanism, our study measured the relative expression levels of two genes associated with growth and reproduction. Vitellogenin, a protein associated with reproduction, has traditionally been considered a suitable parameter for assessing the fertility of female insects (Zhao et al., Reference Zhao, Sun, Xiao, Tan, Jiang and Bai2018a). For instance, the down-regulation of Vg levels adversely affected fertility in Chilo suppressalis and Apolygus lucorum (Huang et al., Reference Huang, Lu, Han, Du and Wang2016). The vitellogenin receptor is the main receptor of Vg function; notably, the down-regulation of VgR can inhibit Vg function. There was a significant reduction in mRNA expression of Vg and VgR in the experimental group compared to the control group. Combined with the significantly reduced fecundity of the treatments, these data suggest that the reduced expression of Vg mRNA in spirodiclofen-treatment may bear a significant effect on P. citri fecundity.
Various parameters related to insect population and development are affected by insecticide stress (Zhao et al., Reference Zhao, Wang, Ding, Wang, Zhang, Liu and Mu2018c; Zhang et al., Reference Zhang, Guo, Atlihan, Chi and Chu2019, Reference Zhang, Wang, Gu, Gong, Chen, Zhang, Hasnain, Shen and Jiang2020; Ullah et al., Reference Ullah, Gul, Desneux, Said, Gao and Song2020). When insects are repeatedly exposed to the same pesticide, pesticide resistance will develop. Furthermore, the activities of the protective enzymes are influenced (Van et al., Reference Van, Van and Tirry2006). Antioxidant enzymes are essential components of the insect immune system, preventing oxidative damage caused by foreign organisms. Toxin-induced reactive oxygen species (ROS) are removed by POD, SOD, and CAT antioxidant enzymes. As a result, the SOD-CAT-POD system acts as a first line of defence against ROS. H2O2 is produced when the body responds to chemical stress by generating ROS, upregulating the SOD activity, and activating defence systems of the body. A relative balance of the body needs to be regulated by the decomposition of CAT and POD. An increase in pesticide concentration weakened the self-defence mechanism and inhibited the function of the protective enzymes (Bolter and Chefurka, Reference Bolter and Chefurka1990). At low concentration, avermectin stimulates SOD activity. Conversely, at high concentrations SOD activity was inhibited. The same trend was observed for CAT activity. The study results showed that SOD and CAT have an inhibitory effect on LC50 treatment compared to lower concentrations (Ma et al., Reference Ma, Zhou and Li2014; Liu et al., Reference Liu, Wang, Qi, He and Bai2021).
Insecticide resistance is frequently caused by physiological changes that increase detoxifying enzymes like CarE, GST, and CYP450 (Zhao et al., Reference Zhao, Wang, Ding, Wang, Zhang, Liu and Mu2018c; Zhang et al., Reference Zhang, Wang, Gu, Gong, Chen, Zhang, Hasnain, Shen and Jiang2020). In present study, when exposed to low lethal concentrations (LC30) of spirodiclofen, CarE activity increased significantly, but decreased when near death. As concentrations increased, CYP450 and GST activities decreased (fig. 5). Toxins introduced into the body are dealt with by detoxifying enzymes in arthropods. CarE is phase II detoxification enzyme important in chemical metabolisation and detoxification (Papachristos and Milonas, Reference Papachristos and Milonas2008) The activities of CarE was upregulated after 48 h of treatment with low concentrations of buprofezin, and the activity decreased as insecticide concentration increased (Zhao et al., Reference Zhao, Wang, Ding, Wang, Zhang, Liu and Mu2018c). However, several hydrophobic toxins fail to be modified by CarE, causing lipid peroxidation in cell membranes. Therefore, the CYP450 and GST are important in the emergence of resistance genes (Ullah et al., Reference Ullah, Gul, Desneux, Said, Gao and Song2020). The production of ROS by insecticides induces oxidative stress in a wide variety of animal cells (Döker et al., Reference Döker, Kazak and Ay2021). ROS attack causes oxidative damage to proteins and lipids, destroys structural integrity, and reduces enzyme activity (Goel et al., Reference Goel, Dani and Dhawan2005). For instance, in locusts exposed to different sublethal doses of chlorpyrifos, CYP450 enzyme activity decreased with increasing concentrations of the insecticide (Van et al., Reference Van, Van and Tirry2006). Our study showed a significant decrease in the CYP450 enzyme activity with increasing spirodiclofen concentrations. The study assumed that GST and CYP450 activities in P. citri were a cause of tolerance against spirodiclofen.
In summary, spirodiclofen not only shows acute toxicity to P. citri but also exhibits sublethal effects. The present study showed that the population parameters of P. citri had adverse effects under two concentrations of spirodiclofen stress. Furthermore, reduced expression of Vg mRNA under spirodiclofen markedly affects P. citri reproduction. Thus, as a part of pest management programme, the responses of spirodiclofen stress show an effective way to control P. citri. However, the mechanism should be studied further in the future to clarify the effects against gene expression levels of detoxification enzymes and antioxidant enzymes.
Acknowledgements
This research was supported by the key project of the Ministry of Agriculture and Rural Affairs of China ‘Investigation on the Occurrence, Harm and Diffusion Risk of Agricultural Alien Invasive Species’ (13220141), The Graduate Innovation Foundation of Jiangxi Province, China (YC2022—B024).
Competing interests
None.
