Exploration of Synergistic Pesticidal Activities, Control Effects and Toxicology Study of a Monoterpene Essential Oil with Two Natural Alkaloids

With the increasing development of pest resistances, it is not easy to achieve satisfactory control effects by using only one agrochemical. Additionally, although the alkaloid matrine (MT) isolated from Sophora flavescens is now utilized as a botanical pesticide in China, in fact, its pesticidal activities are much lower in magnitude than those of commercially agrochemicals. To improve its pesticidal activities, here, the joint pesticidal effects of MT with another alkaloid oxymatrine (OMT) (isolated from S. flavescens) and the monoterpene essential oil 1,8-cineole (CN) (isolated from the eucalyptus leaves) were investigated in the laboratory and greenhouse conditions. Moreover, their toxicological properties were also studied. Against Plutella xylostella, when the mass ratio of MT and OMT was 8/2, good larvicidal activity was obtained; against Tetranychus urticae, when the mass ratio of MT and OMT was 3/7, good acaricidal activity was obtained. Especially when MT and OMT were combined with CN, the significant synergistic effects were observed: against P. xylostella, the co-toxicity coefficient (CTC) of MT/OMT (8/2)/CN was 213; against T. urticae, the CTC of MT/OMT (3/7)/CN was 252. Moreover, the activity changes over time of two detoxification enzymes, carboxylesterase (CarE) and glutathione S-transferase (GST) of P. xylostella treated with MT/OMT (8/2)/CN, were observed. In addition, by scanning electron microscope (SEM), the toxicological study suggested that the acaricidal activity of MT/OMT (3/7)/CN may be related to the damage of the cuticle layer crest of T. urticae.


Introduction
Plutella xylostella Linnaeus (diamondback moth; Lepidoptera: Plutellidae) as the major migratory insect pest mainly endangered cruciferous vegetables and caused economic loss in the world of up to US$ 4-5 billion annually [1,2]. P. xylostella has strong reproductive ability and strong stress resistance. Currently, it has been proved that P. xylostella has resistance to a variety of commercial insecticides, which makes it extremely difficult to control [3]. Tetranychus urticae Koch (Acari: Tetranychidae) as the major miscellaneous feeding mite had the characteristics of the wide host and high reproduction rate [4,5]. In recent years, with the continuous expansion of vegetable cultivation area, the damage of T. urticae becomes more and more serious. The role of chemical insecticides in the management of these pests was indisputable; however, the extensive and irrational use of chemical agrochemicals has resulted in increasing resistances and environmental problems [6][7][8]. Cantharidin and (S)-(-)-palasonin showed different toxicity to P. xylostella, with median lethal concentrations, and quinolizidine alkaloids and neem-based products exhibited moderate acaricidal activities against T. urticae [9][10][11]. Therefore, the research and development of potential pesticide alternatives from bioactive plant natural products has received much attention in recent years [12][13][14][15][16].
Matrine (MT, Figure 1) and oxymatrine (OMT, Figure 1) were two main alkaloids isolated from the plant Sophora flavescens (Kushen). Matrine was one of the promising botanical pesticides registered in China due to its good insecticidal, antibacterial, and other agricultural activities [17][18][19][20]. Oxymatrine showed a wide range of biological properties, such as pesticidal [21,22], antiviral [23], anti-inflammatory [24], and anti-tumor effects [25]. The monoterpene 1,8-cineole (CN, Figure 1, eucalyptol), an essential oil isolated from the eucalyptus leaves, exhibited a variety of interesting activities, such as antimicrobial [26,27], anti-inflammatory [28], and insecticidal properties [29,30]. The plant essential oils used as botanical pesticides for pest control will be a trend in the foreseeable future [31][32][33][34][35][36][37][38]. Due to their synergistic effects, a mixture of two or three bioactive compounds could play the vital role for the management of pests [39][40][41][42][43][44][45][46]. The unique advantages of plant essential oils can greatly reduce the dosage of pesticides and effectively improve their efficacy level [47,48]. Due to the problems of poor efficiency of matrine and oxymatrine, the wide application of matrine and oxymatrine as the pesticide is restricted. Therefore, in this paper, the pesticidal activities of the different complexes of 1,8-cineole with matrine and oxymatrine were investigated against P. xylostella and T. urticae. Meanwhile, detoxification enzymes in insects usually play an important role in the process of metabolizing pesticides and producing drug resistance [49]. The change in detoxification activity level may be a response to environmental stress [50]. Carboxylesterase (CarE) and glutathione S-transferase (GST) are two important metabolic detoxification enzymes in insects. Moreover, the epidermis, a common component of insect body walls, is an extracellular matrix released by dermal cells and plays a crucial role in maintaining insect body form, reducing water loss and resisting microbial infection and predation [51]. So, the enzyme activity changes over time of CarE and GST of treated P. xylostella, and the effects of compounds on the cuticles of T. cinnabarinus were also investigated. chemical agrochemicals has resulted in increasing resistances and environmental problems [6][7][8]. Cantharidin and (S)-(-)-palasonin showed different toxicity to P. xylostella, with median lethal concentrations, and quinolizidine alkaloids and neem-based products exhibited moderate acaricidal activities against T. urticae [9][10][11]. Therefore, the research and development of potential pesticide alternatives from bioactive plant natural products has received much attention in recent years [12][13][14][15][16].
Matrine (MT, Figure 1) and oxymatrine (OMT, Figure 1) were two main alkaloids isolated from the plant Sophora flavescens (Kushen). Matrine was one of the promising botanical pesticides registered in China due to its good insecticidal, antibacterial, and other agricultural activities [17][18][19][20]. Oxymatrine showed a wide range of biological properties, such as pesticidal [21,22], antiviral [23], anti-inflammatory [24], and anti-tumor effects [25]. The monoterpene 1,8-cineole (CN, Figure 1, eucalyptol), an essential oil isolated from the eucalyptus leaves, exhibited a variety of interesting activities, such as antimicrobial [26,27], anti-inflammatory [28], and insecticidal properties [29,30]. The plant essential oils used as botanical pesticides for pest control will be a trend in the foreseeable future [31][32][33][34][35][36][37][38]. Due to their synergistic effects, a mixture of two or three bioactive compounds could play the vital role for the management of pests [39][40][41][42][43][44][45][46]. The unique advantages of plant essential oils can greatly reduce the dosage of pesticides and effectively improve their efficacy level [47,48]. Due to the problems of poor efficiency of matrine and oxymatrine, the wide application of matrine and oxymatrine as the pesticide is restricted. Therefore, in this paper, the pesticidal activities of the different complexes of 1,8-cineole with matrine and oxymatrine were investigated against P. xylostella and T. urticae. Meanwhile, detoxification enzymes in insects usually play an important role in the process of metabolizing pesticides and producing drug resistance [49]. The change in detoxification activity level may be a response to environmental stress [50]. Carboxylesterase (CarE) and glutathione S-transferase (GST) are two important metabolic detoxification enzymes in insects. Moreover, the epidermis, a common component of insect body walls, is an extracellular matrix released by dermal cells and plays a crucial role in maintaining insect body form, reducing water loss and resisting microbial infection and predation [51]. So, the enzyme activity changes over time of CarE and GST of treated P. xylostella, and the effects of compounds on the cuticles of T. cinnabarinus were also investigated.

Insects and Chemicals
Tetranychus urticae (female adults) were reared for many generations in our laboratory with cowpea seedlings as the host plants to establish a stable population (temperature: 26 ± 1 °C; RH: 60−80%; photoperiod: light/dark = 14/10 h). Plutella xylostella (3rd instar larvae) were obtained from a laboratory population reared for many generations in the

Insects and Chemicals
Tetranychus urticae (female adults) were reared for many generations in our laboratory with cowpea seedlings as the host plants to establish a stable population (temperature: 26 ± 1 • C; RH: 60-80%; photoperiod: light/dark = 14/10 h). Plutella xylostella (3rd instar larvae) were obtained from a laboratory population reared for many generations in the School Key Laboratory of Applied Entomology, Northwest A&F University. The cabbage net seedlings as the host plants were continuously planted in the greenhouse of our The solutions of matrine (MT), oxymatrine (OMT) and their binary complexes were prepared in acetone at 10 mg/mL (toosendanin as the positive control and acetone as CK) ( Table 1). For each compound, 45 robust third instar larvae of P. xylostella were selected out (15 insects per group). The corresponding solution (1 µL) was evenly spread on a cabbage leaf disc (surface area: 0.25 cm 2 ). One piece of the above discs was added and eaten up by each P. xylostella, which was raised in each well of 12-well culture plates during 48 h (temperature: 25 ± 2 • C; RH: 70 ± 10%; photoperiod: light/dark = 16/8 h). Their corrected mortality rate (CMR) values (%) = (T − C) × 100/(100% − C); C is the mortality rate of CK, and T is the mortality rate of the treated P. xylostella [52].  For each compound, 45 robust third instar larvae of P. xylostella were selected (15 insects per group). The cabbage leaf disc (surface area: 0.25 cm 2 ) was dipped into the corresponding solution for 3 s and taken out. The treated ones were added to three dishes during 48 h (15 insects per dish) (temperature: 25 ± 2 • C; RH: 70 ± 10%; photoperiod: light/dark = 16/8 h). Their CMR values were calculated in the same way as mentioned above [53].
The procedure for the determination of LC 50      The co-toxicity coefficient (CTC) values of the binary complexes were further evaluated according to Sun's formula [54]. The value of CTC is used to determine whether the efficiency is increased: when CTC > 120, it is synergistic; when CTC < 80, it is antagonistic; when 80 < CTC < 120, it is additive. A significant synergistic effect is observed when the value of CTC is 200.

Control Efficiency of MT/OMT (8/2), and MT/OMT (8/2)/CN against P. xylostella in the Greenhouse
The solutions of MT/OMT (8/2), MT/OMT (8/2)/CN and β-cypermethrin were prepared at 0.2 mg/mL in 0.1% aq. Tween-80, respectively (Table 4). Each cabbage seedling was infested with 20 third instar larvae of P. xylostella prior to spraying. One cabbage seedling was chosen for one group, and each treatment was three replicates. An airbrush was used to spray 10 mL of the corresponding solution for each treatment. The cabbage seedlings treated with 0.1% aq. Tween-80 alone were used as CK (temperature: 25 ± 2 • C; RH: 70 ± 10%; photoperiod: light/dark = 16/8 h). Their control effects on the 1st, 3rd, and 5th days were calculated in the same way as mentioned above [55].  (Table 4). Each cabbage seedling was infested with 20 third instar larvae of P. xylostella prior to spraying. One cabbage seedling was chosen for one group, and each treatment was three replicates. An airbrush was used to spray 10 mL of the corresponding solution for each treatment. The cabbage seedlings treated with 0.1% aq. Tween-80 alone were used as CK (temperature: 25 ± 2 °C; RH: 70 ± 10%; photoperiod: light/dark = 16/8 h). Their control effects on the 1st, 3rd, and 5th days were calculated in the same way as mentioned above [55].    The solutions of MT, OMT, their binary complexes (with different mass ratio), and spirodiclofen (a positive control) (treated by 0.1 g/L of aq. Tween-80 as CK) were prepared at 0.5 mg/mL in Tween-80 in water (0.1 g/L), respectively (Table 5). For each compound, 90-120 healthy and size-consistency female adults of mites (30-40 ones per group) were selected out. Slides affixed with mites were dipped into the corresponding solution for 5 s and taken out (temperature: 26 ± 1 • C; RH: 60-80%; photoperiod: light/dark = 14/10 h). Their mortalities at 48 and 72 h were calculated as follows: cor- rected mortality rate (%) = (T − C) × 100/(100% − C); C is the mortality rate of CK, and T is the mortality rate of the treated T. urticae [56]. Table 5. Acaricidal activity of MT, OMT, CN, and their mixtures against T. urticae treated at a concentration of 0.5 mg/mL.

Compound
Corrected Mortality Rate (Mean ± SE, %) According to the above results, the acaricidal activity of CN and MT/OMT (3/7)/CN against T. urticae (Table 5) was tested as follows: The solution of CN was prepared at 0.048 mg/mL in Tween-80 in water (0.1 g/L), and the solution of MT/OMT (3/7)/CN was prepared at 0.5 mg/mL in Tween-80 in water (0.1 g/L) containing CN (C CN = 0.048 mg/mL). The next procedure was completed in the same way as mentioned above. light/dark = 14/10 h). Their control effects on the 1st, 3rd, and 5th days were calculated in the same way as mentioned above (Table 8) [56].

Enzyme Activity Assay against P. xylostella 2.3.1. Sample Preparation Using Leaf-Dipping Method
According to the above-mentioned leaf-dipping method, 180 robust 3rd instar larvae of P. xylostella were treated with CN, MT/OMT (8/2) and MT/OMT (8/2)/CN at 0.4 mg/mL, respectively (treated by acetone as CK). Then, the 30 surviving larvae in the treated group were collected at 12, 24, 36, and 48 h, respectively. They were then snap-frozen in liquid nitrogen and stored at −80 • C for subsequent enzyme activity analysis [53].

Preparation of Homogenous Liquid
The homogenization treatment (including ten larvae: Weight (g)/Volume (mL) = 1/10) was performed in an ice bath. The homogenous liquid was obtained for carboxylesterase (CarE) activity assay when the sample was centrifuged at 12,000× g for 30 min at 4 • C. The homogenous liquid was obtained for glutathione-S-transferase (GST) activity assay when the sample was centrifuged at 8000× g for 10 min at 4 • C.

CarE and GST Activity Assay According to Bradford's Method
The absorption values over time of CarE and GST were tested by using CarE (αnaphthyl acetate (α-NA) as a substrate) and GST (1-chloro-2,4-dinitrobenzene and reduced glutathione as substrates) assay kits (Suzhou Keming Biotechnology Co., Ltd., China), respectively. Total protein concentration was determined according to the Bradford method (using bovine serum albumin (BSA) as a standard). The protein content was tested by a BCA protein quantitative assay kit (Shaanxi Zhonghui Hecai Biomedical Technology Co., Ltd., China). Finally, the enzymes activity values were obtained according to the absorption value and the protein content. Each treatment was replicated three times [57].

Pretreatment of Mites
The complex (MT/OMT (3/7)/CN) was prepared at 0.894 mg/mL in 0.1 g/L of aq. Tween-80 (0.1 g/L of aq. Tween-80 as CK). Female adult mites with the same physiological status and good growth conditions were selected to cowpea leaves (5 cm in length and 3 cm in width; 30 ones/leaf). After 4 h, the leaves were immersed in the above solution for 5 s, the excess solution was absorbed by filter paper, and the leaves were placed in a Petri dish with a moist sponge (three replicates/treatment). Then, Petri dishes were placed in a light incubator (temperature: 26 ± 1 • C; RH: 60-80%; photoperiod: light/dark = 14/10 h). Finally, the dead mites were collected at 24, 48 and 72 h after treatment by the complex for scanning electron microscope (SEM) analysis [56,58].

Observation of Morphology and Structural Changes of Cuticles in Spider Mites
The collected samples were fixed with 2.5% glutaraldehyde under ice bath conditions, incubated at 4 • C for 4 h, rinsed three times with 0.1 mol/L of phosphate buffer saline (PBS), dehydrated with different concentrations of ethanol, and freeze-dried for 3 h. The mites were then placed on a sample table and sprayed with gold under vacuum conditions. The morphology was observed and photographed by S-3400N SEM [59].

Statistic Analysis
Mortality data were corrected with Abbott's formula and analyzed by a multiple range test using Duncan's test (p < 0.05). The median lethal concentration (LC 50 ) values were calculated on log-concentration versus probit (% mortality) regression analysis. The values of r, χ 2 , df, and P were obtained on regression analysis by IBM SPSS Statistics 20.0 (p < 0.05).

Insecticidal Activity
First, the insecticidal activities of two compounds (MT and OMT) and their binary mixtures at different mass ratios against P. xylostella were tested. As shown in Table 1, to binary mixtures, when the mass ratio of MT and OMT was 8:2, the corresponding 48 h corrected mortality rate (CMR) was 27.9%, which was higher than those of MT (20.9%) and OMT (16.2%). The 48 h CMRs were all 20.9% when the mass ratio of MT/OMT was 7:3 or 5:5. The 48 h CMR was 16.2% when the mass ratio of MT/OMT was 1:9, which was similar to that of OMT. However, when the mass ratio of MT and OMT was 3:7, the corresponding 48 h CMR was decreased to 13.9%, which was lower than those of MT and OMT. Obviously, for MT and OMT against P. xylostella, the best mass ratio of MT and OMT was 8:2. As shown in Figure 2 (Table 2). As described in Table 3

Acaricidal Activity
The 48 and 72 h acaricidal results of MT, OMT, and their binary mixtures at 0.5 mg/mL against the female adults of T. urticae are shown in Table 5. Among nine binary complexes, the 72 h CMRs of six complexes were higher than those of MT (23.5%) and OMT (18.2%); especially when the mass ratio of MT to OMT was 3:7, the corresponding 72 h CMR was 34.8%. At 0.048 mg/mL, the 72 h CMR of CN against T. urticae was only 11.2%; interestingly, when MT and OMT (mass ratio: 3/7) were dissolved in the solution of CN (CCN = 0.048 mg/mL) in 0.01% aq. Tween-80, and CMT/OMT (3/7) was 0.5 mg/mL, the corresponding CMR was increased to 38.0%. It may be related to the strong permeability of CN as an essential oil [38]. Then, 72 h LC50 values of MT, OMT, MT/OMT (3/7), and MT/OMT (3/7)/CN against T. urticae were further calculated according to their CMRs at different concentrations ( Table 6). As shown in Table 7

Acaricidal Activity
The 48 and 72 h acaricidal results of MT, OMT, and their binary mixtures at 0.5 mg/mL against the female adults of T. urticae are shown in Table 5. Among nine binary complexes, the 72 h CMRs of six complexes were higher than those of MT (23.5%) and OMT (18.2%); especially when the mass ratio of MT to OMT was 3:7, the corresponding 72 h CMR was 34.8%. At 0.048 mg/mL, the 72 h CMR of CN against T. urticae was only 11.2%; interestingly, when MT and OMT (mass ratio: 3/7) were dissolved in the solution of CN (C CN = 0.048 mg/mL) in 0.01% aq. Tween-80, and C MT /OMT (3/7) was 0.5 mg/mL, the corresponding CMR was increased to 38.0%. It may be related to the strong permeability of CN as an essential oil [38]. Then, 72 h LC 50 values of MT, OMT, MT/OMT (3/7), and MT/OMT (3/7)/CN against T. urticae were further calculated according to their CMRs at different concentrations ( Table 6). As shown in Table 7 Table 8, the control effects of MT/OMT (3/7) and MT/OMT (3/7)/CN after 5 days were 31.3% and 42.3%, respectively. Fifth-day symptoms of asparagus bean seedling leaves treated with MT/OMT (3/7) and MT/OMT (3/7)/CN were described in Figure 4. There were lots of white spots destroyed by T. urticae on the seedling leaves in the CK-treated group, whereas in the MT/OMT (3/7)-and MT/OMT (3/7)/CN-treated groups, almost no small white spots were on the seedling leaves. These findings were the same as our previous report [58]. Photographs of control effects of those complexes against T. urticae are shown in Figures S4-S6.

Changes of Detoxification Enzymes Activities
Subsequently, the changes of detoxification enzymes (CarE and GST) activities in P. xylostella, treated with CN, MT/OMT (8/2) and MT/OMT (8/2)/CN at 0.4 mg/mL after 12, 24, 36, and 48 h were depicted in Figures 5 and 6, respectively. The enzymatic activities of CarE and GST are always in dynamic change. As described in Figure 5, the CarE activity values in the treated groups at 36 and 48 h were much lower than those of the control group. The CarE activity value in the CN-treated group at 12 h (82.1 U/g) was higher than that of the control group (69.4 U/g). It suggested that CN was toxic to P. xylostella and stimulated the detoxification ability of the CarE in P. xylostella at 12 h. At 24 h, the CarE activity value in the MT/OMT (8/2)/CN-treated group (67.7 U/g) was higher than that of the control group (52.4 U/g). It indicated that MT/OMT (8/2)/CN stimulated the detoxifi-

Changes of Detoxification Enzymes Activities
Subsequently, the changes of detoxification enzymes (CarE and GST) activities in P. xylostella, treated with CN, MT/OMT (8/2) and MT/OMT (8/2)/CN at 0.4 mg/mL after 12, 24, 36, and 48 h were depicted in Figures 5 and 6, respectively. The enzymatic activities of CarE and GST are always in dynamic change. As described in Figure 5, the CarE activity values in the treated groups at 36 and 48 h were much lower than those of the control group. The CarE activity value in the CN-treated group at 12 h (82.1 U/g) was higher than that of the control group (69.4 U/g). It suggested that CN was toxic to P. xylostella and stimulated the detoxification ability of the CarE in P. xylostella at 12 h. At 24 h, the CarE activity value in the MT/OMT (8/2)/CN-treated group (67.7 U/g) was higher than that of the control group (52.4 U/g). It indicated that MT/OMT (8/2)/CN stimulated the detoxification ability of the CarE in P. xylostella at 24 h. After that, the detoxification ability of the CarE in P. xylostella in all treated groups was inhibited. The time for reaching the lowest points of the CarE activities in the treated groups was different.   As illustrated in Figure 6, the GST activity value in the MT/OMT (8/2)/CN-treated group at 12 h (3910 nmol/min/g) was higher than that of the control group (2698 nmol/min/g). Similarly, it suggested that the mixture MT/OMT (8/2)/CN stimulated and enhanced the detoxification ability of the GST in P. xylostella at 12 h. At 24 h, the GST activity value in the CN-treated group was slightly higher than that of the control group. Afterwards, the GST activity in the treated groups decreased significantly when compared with that of the control, and it reached the lowest point at 36 h in all treated groups. The GST activity values at 36 h in CN-, MT/OMT (8/2)-and MT/OMT (8/2)/CN-treated groups were 2208, 2591 and 1932 nmol/min/g, respectively, and they were decreased 1.4-1.9 folds of that of the control group (3603 nmol/min/g). Obviously, the GST activity of P. xylostella in the treated groups was largely inhibited at 36 h.

Toxicological Study of Structural Changes of Cuticles by SEM
The penetration of insecticide to pest epidermis is the premise of its toxic effect. The cuticle serves as the initial barrier of defense between the body and the outside world, effectively blocking pesticide penetration. The reduction in epidermal penetration can delay the time of pesticide reaching the target site, and pests have sufficient time and faster speed to metabolize the pesticide entering the body [59]. As shown in Figure 7, in the CKtreated group, the cuticles of T. urticae are flat and have an entire structure, with neat and continuous skin texture; whereas in the MT/OMT (3/7)/CN-treated group, the structure of the cuticles was damaged with visible wrinkles and an uneven arrangement of the inner ridges. The cuticles of wounded mites were thinner, softer, and more asymmetrically bent than those of normal mites, losing their barrier-like function against acaricide penetration. As a result, CN may enhance the penetration ability of this complex on T. urticae [38,60].

Toxicological Study of Structural Changes of Cuticles by SEM
The penetration of insecticide to pest epidermis is the premise of its toxic effect. The cuticle serves as the initial barrier of defense between the body and the outside world, effectively blocking pesticide penetration. The reduction in epidermal penetration can delay the time of pesticide reaching the target site, and pests have sufficient time and faster speed to metabolize the pesticide entering the body [59]. As shown in Figure 7, in the CKtreated group, the cuticles of T. urticae are flat and have an entire structure, with neat and continuous skin texture; whereas in the MT/OMT (3/7)/CN-treated group, the structure of the cuticles was damaged with visible wrinkles and an uneven arrangement of the inner ridges. The cuticles of wounded mites were thinner, softer, and more asymmetrically bent than those of normal mites, losing their barrier-like function against acaricide penetration. As a result, CN may enhance the penetration ability of this complex on T. urticae [38,60].

Toxicological Study of Structural Changes of Cuticles by SEM
The penetration of insecticide to pest epidermis is the premise of its toxic effect. The cuticle serves as the initial barrier of defense between the body and the outside world, effectively blocking pesticide penetration. The reduction in epidermal penetration can delay the time of pesticide reaching the target site, and pests have sufficient time and faster speed to metabolize the pesticide entering the body [59]. As shown in Figure 7, in the CKtreated group, the cuticles of T. urticae are flat and have an entire structure, with neat and continuous skin texture; whereas in the MT/OMT (3/7)/CN-treated group, the structure of the cuticles was damaged with visible wrinkles and an uneven arrangement of the inner ridges. The cuticles of wounded mites were thinner, softer, and more asymmetrically bent than those of normal mites, losing their barrier-like function against acaricide penetration. As a result, CN may enhance the penetration ability of this complex on T. urticae [38,60].

Conclusions
In summary, the significant synergistic effects were observed when CN was combined with MT and OMT as pesticidal agents: against P. xylostella, the CTC of MT/OMT (8/2)/CN was 213; against T. urticae, the CTC of MT/OMT (3/7)/CN was 252. Furthermore, these mixtures displayed good control efficiency against P. xylostella and T. urticae in the greenhouse. Importantly, the enzymes activity changes over time of CarE and GST in P. xylostella treated with CN, MT/OMT (8/2) and MT/OMT (8/2)/CN were explored. Due to the accumulation of toxicant leading to a decrease in the detoxification ability, the time for reaching the lowest point of the CarE activity in the treated groups was different, whereas the time for reaching the lowest point of the GST activity in the treated groups was the same (at 36 h). Notably, by SEM analysis, the toxicology study suggested that the destruction of the cuticle layer crest of T. urticae by MT/OMT (3/7)/CN may be the main cause of their death. These results will pave the way for the future study of different combinations of MT-OMT-CN and the application of CN as a synergist with other bioactive natural products as pesticidal candidates in crop protection.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/toxins15040240/s1, Biological assay; Pictures of control effects of different complexes against P. xylostella; Pictures of control effects of different complexes against T. urticae. Figure S1. Pictures of control efficiency of CK after 1st day (a), 3rd day (b) and 5th day (c) against P. xylostella in the greenhouse; Figure S2. Pictures of control efficiency of MT/OMT (8/2, 0.2 mg/mL) after 1st day (a), 3rd day (b) and 5th day (c) against P. xylostella in the greenhouse; Figure S3. Pictures of control efficiency of MT/OMT (8/2, 0.2 mg/mL)/CN after 1st day (a), 3rd day (b) and 5th day (c) against P. xylostella in the greenhouse; Figure S4. Pictures of control efficiency of CK after 1st day (a), 3rd day (b) and 5th day (c) against T. urticae in the greenhouse; Figure S5. Pictures of control efficiency of MT/OMT (3/7, 0.2 mg/mL) after 1st day (a), 3rd day (b) and 5th day (c) against T. urticae in the greenhouse; Figure S6. Pictures of control efficiency of MT/OMT (3/7, 0.2 mg/mL)/CN after 1st day (a), 3rd day (b) and 5th day (c) against T. urticae in the greenhouse.
Author Contributions: J.X., S.F., Y.W., H.W. and S.Z. who have made substantial contributions to the present work are named in the manuscript. M.L. and H.X. designed the work, analyzed the data, and wrote and revised the paper. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China (Project No. 32272592).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The dataset utilized in this study is available upon request.