The Insecticidal Efficacy and Physiological Action Mechanism of a Novel Agent GC16 against Tetranychus pueraricola (Acari: Tetranychidae)

Simple Summary Spider mite is major pest in agriculture and have developed resistance to commonly used pesticides. Therefore, it is urgent to discover new pesticides to control the pest. In order to provide alternatives for its management, we evaluated the effectiveness of a new agent GC16 against the spider mite Tetranychus pueraricola. Then, we preliminarily revealed the its acaricidal mechanism of action based on the damage of cuticle and organelles of mites. We confirmed that GC16 has a good controlling effect on T. pueraricola and it is not harmful to Picromerus lewisi and Harmonia axyridis. Our research provides not only an alternative pesticide for the management of spider mites, but also guidance for the application of GC16 in sustainable agriculture. Abstract Chemical control plays a crucial role in pest management but has to face challenges due to insect resistance. It is important to discover alternatives to traditional pesticides. The spider mite Tetranychus pueraricola (Ehara & Gotoh) (Acari: Tetranychidae) is a major agricultural pest that causes severe damage to many crops. GC16 is a new agent that consists of a mixture of Calcium chloride (CaCl2) and lecithin. To explore the acaricidal effects and mode of action of GC16 against T. pueraricola, bioassays, cryogenic scanning electron microscopy (cryo-SEM) and transmission electron microscopy (TEM) were performed. GC16 had lethal effects on the eggs, larvae, nymphs, and adults of T. pueraricola, caused the mites to dehydrate and inactivate, and inhibited the development of eggs. GC16 displayed contact toxicity rather than stomach toxicity through the synergistic effects of CaCl2 with lecithin. Cryo-SEM analysis revealed that GC16 damaged T. pueraricola by disordering the array of the cuticle layer crest. Mitochondrial abnormalities were detected by TEM in mites treated by GC16. Overall, GC16 had the controlling efficacy on T. pueraricola by cuticle penetration and mitochondria dysfunction and had no effects on Picromerus lewisi and Harmonia axyridis, indicating that GC16 is likely a new eco-friendly acaricide.


Introduction
Sustainable agriculture focuses on producing long-term crops with minimal effects on the environment and is being watched around the world [1][2][3]. Biodiversity is an important part of sustainable agriculture [3,4]. Considering the damage to biodiversity caused by conventional pesticides, there is a need to develop new alternative and eco-friendly pesticides to protect biodiversity [5].

Bioassay of GC16 against Different Stages of T. pueraricola Egg Bioassay
The ovicidal toxicity of GC16 to T. pueraricola eggs was assessed by the petri dishspraying method according to a previous study with some modifications [33][34][35][36][37]. In total, 15 adult females were placed on a 20 mm diameter bean leaf disc placed on 0.3% agar in a petri dish for egg deposition. After 12 h, the females were removed, and the leaf discs were evenly and fully sprayed with a power sprayer. After the discs had dried, the eggs were counted, and the petri dishes with the treated leaf discs were placed in an incubator at 25 ± 2 • C with 65 ± 10% RH on a 16:8 (L/D) photoperiod. Five concentrations of GC16 were used to generate a regression equation (0, 1.25, 5, 10, 20 g/L). Each treatment was represented by three replicate leaf discs. The number of emerged larvae was counted daily until all the eggs for control had hatched except died eggs with physiological causes.

Larva and Nymph Bioassay
For the bioassays with larvae and nymphs, 20 adult female T. pueraricola were placed on leaf discs on 0.3% agar to lay eggs for 12 h [14,35]. Once the larvae/nymphs had emerged, their numbers were counted using a microscope, and the dead larvae/nymphs and other instars were removed. Then, the discs were sprayed with GC16 solutions (six concentrations of GC16; 0, 0.83, 1.67, 3.33, 6.67, 13.33 g/L) according to the same method described above. The leaf discs with larvae and nymphs were dried and stored in the incubator described previously. After 24 h, the living larvae and nymphs were counted.

Adult Bioassay
According to Ay (2005) [34], mortality tests were performed before each experiment to determine the range of concentrations that would produce 10~95% mortality. Adult female mites (30~50) were transferred to each bean leaf disc (2 cm diameter) placed in a petri dish, and each dish was covered with a lid containing small holes to avoid condensation of water vapor [14]. All experiments were conducted using three replicates of each concentration of GC16 (0, 0.83, 1.67, 3.33, 6.67, 13.33 g/L). The leaf discs with mites were examined under a microscope to remove the dead and inactive mites and then the discs were sprayed with a power sprayer as described above. After air-drying, the leaf discs were placed back into the plastic petri dishes (3.5 cm diameter) with 0.3% agar to preserve moisture, a method modified according to Xu et al. (2018) [14]. The petri dishes with the treated mites were placed in an incubator described earlier. After 24 h, the numbers of live and dead mites were counted. Mites were recorded as dead if they failed to move when touched with a soft brush. The results were not used if the mortality in the control exceeded 15%.

Bioassays of GC16 by Different Methods Slide-Dip Assay
As recommended by the FAO, 2-3 cm long pieces of double-sided tape were adhered to glass slides [35,38]. The backs (rather than feet or mouthparts) of uniform adult females were gently stuck to the tape using a small brush. There were 45 mites on each slide, and the experiment was repeated three times. The slides were kept in an incubator for 4 h, and the mites were examined with a microscope (SZ51, Olympus, Tokyo, Japan). Dead and inactive individuals were removed. Slides containing mites were dipped into 6.67 g/L GC16 and shaken gently for 5 s. The excess liquid was quickly removed with absorbent paper. The control group was treated in the same way with water. The slides were then placed in a glass petri dish with soaked gauze on the bottom and plastic wrap with a small hole on the top and placed in an incubator as mentioned earlier. After 24 h, the dead mites and live mites were counted. A mite was considered dead if it did not respond to light touch with a small brush. If the mortality for the control exceeded 15%, the trial was repeated.

Leaf-Dip Assay
Following the methods of Xu et al. (2018) [14] and Wang et al. (2015Wang et al. ( , 2016 [13,39], bean leaf discs (2 cm in diameter) were dipped into a 6.67 g/L GC16 solution for 10 s, while leaf discs dipped into water were set as control. The leaf discs were then placed on filter paper to dry. After drying, the leaf discs were backed up and attached to 0.3% agar in a 3.5 cm diameter plastic petri dish. Approximately 25~30 adult female mites were transferred to each disc, and each petri dish was covered by a cover with small holes to avoid water vapor condensation. The experiment was repeated three times. Petri dishes containing the mites being tested were kept in incubators under the same conditions as those described previously. After 24 h, the numbers of live and dead mites were counted in each petri dish. Mites that did not move after being touched by a soft brush were recorded as dead. The results were not used if the mortality rate for the control treatment exceeded 15%.

Spraying Assay
The spraying assay was conducted in a similar manner as the assay in the section Adult Bioassay [2]. Approximately 35 healthy adult female mites were transferred to bean leaves, and three replicates were performed. Dead and inactive mites were removed under a microscope. Then, a solution containing 6.67 g/L GC16 was sprayed equally on the bean leaves with the mites with a power sprayer. Then, the plants with the mites were kept in incubators at the conditions described previously. Twenty-four hours later, the numbers of live and dead mites were counted. Mites that did not move after being touched by a soft brush were recorded as dead. Sprayed water was used as a control under the same conditions. If the mortality rate for the control exceeded 15%, the experiments were repeated.

Bioassays for the Different Components of GC16
Bioassays for the different components of GC16 in adult female T. pueraricola were performed by the spraying method as described in the previous paragraph with a GC16 concentration of 6.67 g/L. Water was used as a blank control, and the CaCl 2 and lecithin treatments were administered at concentrations of 3.03 g/L and 3.64 g/L, respectively.

Observation of Poisoning Symptoms for T. pueraricola
To observe poisoning symptoms [40][41][42], 25 mites were carefully transferred to bean leaves. T. pueraricola were treated with GC16 at concentrations of 6.67 g/L and 2.00 g/L by the spraying method, and water was used as the control. Observations were made under a microscope (SZ51, Olympus, Tokyo, Japan) at 20 min, 40 min, and then every 2 h from 2 to 24 h; each observation was made with three replicates.

Effects of GC16 on the Morphology of Female Adult T. pueraricola
By the slide-dip method (details described above), 6.67 g/L GC16 was used to evaluate the effects on the morphology of female adult T. pueraricola. Treatment with water under the same condition was set as the control. After treatment for 24 h and 48 h, mites were photographed by microscopy (LEICA M205 FA, Wetzlar, Germany), and the body lengths and widths of mites were measured [43]. The relative shrinkage rate (Rst) of length = (body length of control for n h-body length of GC16 for n h)/body length of control for n h (n is the number of hours after treatment; h means hour); the Rst of width was calculated the same.

Effects of GC16 on the Egg Hatching Rate and Developmental Duration of T. pueraricola
Fresh, healthy, and uniform female adult mites were selected and placed on common bean leaves. After 12 h of laying eggs, the adult mites were removed with a brush, and approximately 20~40 eggs were kept on each leaf [13,35]. The mite eggs were treated with 10 g/L GC16 by the petri dish-spraying method described above, with water as a control, and each treatment consisted of three biological replicates. The eggs were observed and photographed under a microscope (LEICA M205 FA, Wetzlar, Germany) every day, and the hatching rate and development time (time after egg lay) were calculated.

Cryo-SEM (Scanning Electron Microscopy)
The tested mites were treated with water (control), GC16, CaCl 2 , or lecithin. The concentration of GC16 was set to its LC 50 (2.00 g/L), and the concentrations of CaCl 2 (0.90 g/L) or lecithin (1.10 g/L) corresponded to the ratio in GC16. After treatment for 24 h, live mites were used for scanning/transmission electron microscopy (SEM/TEM) analysis.
According to Walther (2001) [46], to avoid chemical fixation and drying artifacts and obtain the most direct and real images of mites in a defined physiological state, a fast frozen technique was used. For cryo-SEM, fresh mites were directly and gently glued to the sample table and frozen in supercooled liquid nitrogen for 2 min. The samples of each replicate were then transferred to a preparation chamber at −140 • C. Next, sublimation was performed at −90 • C for 10 min, followed by coating twice for 60 s each time. The samples were observed and photographed with a ZEISS Sigma 300 scanning electron microscope.

TEM (Transmission Electron Microscopy)
Sample preparation was the same as that described above. A previous method was modified as appropriate [47][48][49][50]. Samples were fixed overnight at 4 • C using 2.5% glutaraldehyde in 0.1 M PB (pH 7.4). Samples were then washed with 0.1 M PB (pH 7.4) three times for 15 min each time. Afterward, the samples were post-fixed with 1% OsO 4 for 2 h at 4 • C, washed with 0.1 M PB (pH 7.4) three times for 15 min each time, followed by serial ethanol dehydration and acetone transition for 5 min, embedded in Epon 812 resin, and polymerized at 60 • C for 48 h. Serial ultrathin sections with a uniform thickness (60 nm) were made using a Leica EM UC7 ultramicrotome. The ultrathin sections were then loaded onto 50-mesh Cu grids and double-stained with 2% uranyl acetate and lead citrate before observation with a JEM 1400 Plus transmission electron microscope at 120 kV.

The Effects of GC16 on Non-Target Organisms
The target pest mite T. pueraricola and non-target organisms P. lewisi and H. axyridis were treated with 6.67 g/L GC16 using the spraying method mentioned above in the section Spraying Assay. The P. lewisi and H. axyridis were placed in insect-rearing cages (120 mesh, 30 cm × 30 cm × 30 cm) that provided yellow mealworm and aphids, respectively. In total, 20~40 insects were tested each repeat (three repeats each treatment), and insects treated with water in the same way were set as control. Twenty-four hours later, the dead insects were counted and the mortality rate was calculated.

Statistical Analysis
For the developmental stage bioassay data, the slope ± SE, LC 50 values, 95% fiducial limits, chi-square values, and degrees of freedom (df) were calculated by probit analysis using Polo Plus 2.0 software (LeOra software, Berkeley, CA, USA).
The other data were analyzed using SPSS software, v. 25.0 (SPSS Inc., Chicago, IL, USA). The graphs were created by Sigmaplot 14.0 (Systat Software Inc., San Jose, CA, USA) and grouped by Adobe Illustrator 2021 (Adobe Systems Inc., San Jose, CA, USA). After normality test, the data were in accordance with normal distribution or approximate normal distribution. Differences between the effects of different bioassay methods or different components were analyzed using one-way analysis of variance (ANOVA). Significant differences between treatments were based on Tukey's honestly significant difference (HSD) test. Differences in body length/width among the different treatments were analyzed by one-way ANOVA with Tukey's honestly significant difference (HSD) test. Differences in the egg hatching rate and development duration between the two groups were compared using independent sample t tests. One-way ANOVA with Tukey's honestly significant difference (HSD) test was used to analyze the differences of effects of GC16 on non-target organisms across three organisms, and an independent sample t test was used to compare differences of pesticides treatments (GC16 vs. Control) for the same organism. Statistical significance was set at p < 0.05.

Bioassays of GC16 against Different Stages of T. pueraricola
To evaluate the effects of GC16 against T. pueraricola, the LC 50 values were determined by spraying method ( Table 1). The LC 50 values of GC16 against eggs, larvae, nymphs, and adults of T. pueraricola were in the range of 1.266~2.239 g/L, and the LC 90 values ranged from 5.951 to 26.888 g/L. Results indicated that GC16 had clear insecticidal effects on all instars and stages of T. pueraricola, and their mortality increased with increasing concentrations of GC16; however, the same concentration of GC16 had different lethal effects on mites in different stages. At a concentration of 6.67 g/L, the mortality of female adults reached over 80% (Table S1).

Bioassays of GC16 by Different Bioassay Method
The spray method and slide-dip method are commonly used techniques to determine the contact toxicity of pesticides, while the leaf-dip method can better test the stomach toxicity. To determine the mode of action of GC16, we compared its toxicity by different bioassay methods. The results showed that the mortality rate of T. pueraricola treated with 6.67 g/L GC16 by the leaf-dip method was 4.94%, while the mortality rates of the spraying method and slide-dip method were both greater than 80%, which were significantly higher than that of the leaf-dipping method (Figure 1). There was no significant difference between the spray method and the slide-dip method. The results of these experiments indicated that GC16 mainly acted on mites through contact.
toxicity. To determine the mode of action of GC16, we compared its toxicity by different bioassay methods. The results showed that the mortality rate of T. pueraricola treated with 6.67 g/L GC16 by the leaf-dip method was 4.94%, while the mortality rates of the spraying method and slide-dip method were both greater than 80%, which were significantly higher than that of the leaf-dipping method (Figure 1). There was no significant difference between the spray method and the slide-dip method. The results of these experiments indicated that GC16 mainly acted on mites through contact.

Bioassays for the Different Components of GC16
GC16 is composed of a mixture of CaCl2 and lecithin. To determine which component is the main active component and/or how the two components synergize, the lethal efficacy of each component was tested and the results were compared. The mortality rates of mites were less than 10% when CaCl2 or lecithin acted alone, and there were no significant differences between control and the CaCl2/lecithin alone groups. However, the mortality of mites treated for 24 h with GC16 reached 80%, and the coapplication of lecithin + CaCl2 significantly increased mortality compared with mites treated with lecithin or CaCl2 alone ( Figure 2). These data indicated that CaCl2 or lecithin alone were not lethal to mites, but the combination of lecithin + CaCl2 (GC16) produced very different results.

Bioassays for the Different Components of GC16
GC16 is composed of a mixture of CaCl 2 and lecithin. To determine which component is the main active component and/or how the two components synergize, the lethal efficacy of each component was tested and the results were compared. The mortality rates of mites were less than 10% when CaCl 2 or lecithin acted alone, and there were no significant differences between control and the CaCl 2 /lecithin alone groups. However, the mortality of mites treated for 24 h with GC16 reached 80%, and the coapplication of lecithin + CaCl 2 significantly increased mortality compared with mites treated with lecithin or CaCl 2 alone ( Figure 2). These data indicated that CaCl 2 or lecithin alone were not lethal to mites, but the combination of lecithin + CaCl 2 (GC16) produced very different results.

Observation of Poisoning Symptoms
To investigate the mechanism by which GC16 influenced T. pueraricola, the poisoning and death symptoms of T. pueraricola were observed. After GC16 treatment, the mites first entered the quiescent stage (Table 2). Then, they died and later shriveled at high concentrations. At a low concentration, some mites gradually resumed movement, while the others moved slightly and then became sluggish until death. Control (0) feed and ovi-feed and ovi-feed and ovi-feed and ovi-feed and ovi-feed and ovi-feed and ovi-

Observation of Poisoning Symptoms
To investigate the mechanism by which GC16 influenced T. pueraricola, the poisoning and death symptoms of T. pueraricola were observed. After GC16 treatment, the mites first entered the quiescent stage (Table 2). Then, they died and later shriveled at high concentrations. At a low concentration, some mites gradually resumed movement, while the others moved slightly and then became sluggish until death.

Effects of GC16 on the Morphology of Female Adult T. pueraricola
After GC16 treatment, most of the tested mites died 24 h later, but the control mites treated with water were still alive and active enough to walk, forage, and oviposit. Under a microscope, it was clearly seen that the mite bodies became small, crumpled, and shriveled and the legs became bent and curled up after treatment with GC16 ( Figure 3). Compared with the water control, the body lengths of the mites treated with GC16 were significantly shortened at 24 h and 48 h, and the relative shrinkage rates were 14% and 25%, respectively (Table 3). In addition, the body widths of the mites treated with GC16 for 48 h were significantly smaller than those of the control, and the relative shrinkage rate reached 14%.

Effects of GC16 on the Hatching Rate and Developmental Duration of T. pueraricola Egg
After treatment with GC16, it was found that the mite eggs gradually shriveled, became withered and deformed, and could not hatch successfully. However, under the same conditions, the eggs treated with water could molt and hatch normally (Figure 4). The hatching rates of eggs treated with GC16 and control were 14.30% and 94.38%, respectively, and there was a significant difference between them (t = 4.576, df = 4, p < 0.001 Table  S2). Moreover, the development duration of the control eggs in water was 4.07 days, while their development duration after GC16 treatment was 5.10 days, a significant difference of 1.03 days longer than that in water (t = 1.480, df = 4, p = 0.005), indicating that GC16 could significantly reduce the hatching rate of eggs and prolong the egg development duration.

Effects of GC16 on the Hatching Rate and Developmental Duration of T. pueraricola Egg
After treatment with GC16, it was found that the mite eggs gradually shriveled, became withered and deformed, and could not hatch successfully. However, under the same conditions, the eggs treated with water could molt and hatch normally (Figure 4). The hatching rates of eggs treated with GC16 and control were 14.30% and 94.38%, respectively, and there was a significant difference between them (t = 4.576, df = 4, p < 0.001 Table S2). Moreover, the development duration of the control eggs in water was 4.07 days, while their development duration after GC16 treatment was 5.10 days, a significant difference of 1.03 days longer than that in water (t = 1.480, df = 4, p = 0.005), indicating that GC16 could significantly reduce the hatching rate of eggs and prolong the egg development duration.

Cryo-SEM Analysis
According to the above results, the application of GC16 caused the insect bodies to dehydrate and atrophy; however, is this related to cuticle damage? Considering that cuticle penetration plays an important role as an insecticide mechanism, to determine the cuticle integrity of mites treated with GC16, cryo-SEM was performed. After GC16 treatment, the crest lines on the dorsal surfaces of T. pueraricola showed disordered and irregular arrangements, and the cuticle ridges snuggled close each other ( Figure 5). In contrast, the dorsal dermatoglyphs of control (water-treated) T. pueraricola were arranged in an orderly and regular manner, and the cuticle ridge was evenly and regularly distributed. Additionally, there were no obvious abnormalities in the dorsal crest with CaCl 2 or lecithin treatment alone. Furthermore, no obvious differences were seen in the forelegs, hind legs, abdomens, or peritremes between the mites receiving different treatments. The treatments include GC16, Control (water), CaCl 2 , and lecithin. The parts that were photographed included the back, dorsal crest, abdomen, ventral texture, foreleg, hind leg, and peritreme.

TEM Analysis
From previous poisoning symptom observations, mites displayed inactive and motionless states after GC16 treatment, and it is unclear whether this state is related to energy metabolism. To determine the cause behind these symptoms, the ultramicrostructure of the inner tissue was observed. TEM observations showed that the endoplasmic reticulum, mitochondria, and nuclear membrane system were not damaged, and the cuticle was compact and had regular protuberances in the control (water group) ( Figure 6). The submicroscopic structure of the GC16 group was also clearly visible, with no abnormalities in the nucleus or endoplasmic reticulum but obvious abnormalities in the mitochondria were found. Specifically, the mitochondria were swollen and malformed, the intercristae matrix was vacuolated, and a flocculent amorphous substance appeared in the mitochondrial lumen. Compared with the water control, the cuticles of the mites treated with GC16 were dissolved, protrusion was seriously damaged, and the arrangement of the crest was irregular. Notably, there was no clear cuticle damage or organelle damage in the CaCl 2 treatment group. In the lecithin treatment group, except for mitochondrial abnormalities (the mitochondria swelled irregularly and their cristae became fractured and fuzzy), additional changes were not noted.

The Effects of GC16 on the Non-Target Organisms
Safety assessment for non-target organisms is an important part of pesticide environmental toxicology, which guides agricultural production. Here, the mortality rate for mites treated with GC16 was more than 80%, while the mortality rates for non-target organisms P. lewisi and H. axyridis under GC16 treatment were less than 10% and there were no significant differences on mortality rates for them between GC16 treatment and the blank control, respectively (Table 4). This indicates that GC16 has a good control efficacy on the spider mite T. pueraricola, while it has no lethal effects on non-target organisms, and GC16 may have the potential to be developed as an eco-friendly acaricide. Note: Values are mean ± SE. The same uppercase letter in a column expresses no significant difference among three organisms according to the one-way ANOVA and Tukey's honestly significant difference (HSD) test, and the same lowercase letter in a row indicates no significant difference between GC16 treatment (6.67 g/L) and control (water) based on independent sample t test at p < 0.05. The F and p values for HSD test of GC16 and Control across the three organisms are F(2,6) = 925.992, p < 0.001 and F(2,6) = 3.973, p = 0.080, respectively.

Discussion
Exploring novel pesticides has been an important part of integrated pest management (IPM) in the current situation of agricultural development. In this study, to evaluate the performance of a new agent, GC16, on T. pueraricola, we carried out bioassays with mites at different developmental stages with different treatment methods; we also examined the bioassays for different components of GC16 in addition to their combination. The results showed that GC16 had effects on the eggs, larvae, nymphs, and adults of T. pueraricola by contact with the synergistic reaction mechanism of lecithin and CaCl 2 . Subsequently, ultrastructures of the mites were observed. The combined results demonstrated that GC16 killed mites by damaging their cuticles to first dehydrate and then destroying the mitochondria to disrupt metabolism, making the mites inactive.
In general, the median lethal concentration (LC 50 ) is the elemental parameter to analyze the acaricidal activity of acaricides. The LC 50 value is affected by the pest species, type of pesticide, bioassay method, bioassay time, and treatment environment [51][52][53]. Previous studies have reported that the LC 50 values of avermectin, bifenazate, etoxazole, and spirodiclofen against T. cinnabarinus over 24 h by the slide-dip method were 3.2 × 10 −6 , 14.932 × 10 −3 , 4.4 × 10 −4 , and 0.356 g/L, respectively [52]. Additionally, the LC 50 values of the botanical pesticide scoparone against T. cinnabarinus and T. urticae by the slide-dip method were found to be 0.279 and 0.906 g/L, respectively [53]. The LC 50 value at 24 h of osthole to T. urticae was 0.332 g/L by the spraying method [2]. The LC 50 of the crude acetone extract from Aloe vera L. against female adult T. cinnabarinus was 6.165 g/L by the slide-dip method after 24 h [51]. Herein, we found that the LC 50 value of GC16 against female adult T. pueraricola was 2.00 g/L. In contrast to commercial pesticides, the LC 50 value of GC16 was greater but at an intermediate level when compared with the plant-derived extract. Combined with the above studies, different pesticides have different acaricidal activities against different spider mite species, and GC16 has the potential to become a new acaricide compared with the plant-derived extracts.
Poison symptom investigation is the first step in understanding the mechanism of action of pesticides. To investigate the mechanism of GC16, we observed poisoning and death symptoms of T. pueraricola. The findings revealed that mites became stationary after GC16 treatment and then died with curly legs and shrunken and wizened bodies, which is somewhat similar to the paralyzing effects of nerve agents but without the excitement [54]. In addition, after treatment with abamectin, pyridazin, curcumin, and scopolamine, T. cinnabarinus showed symptoms of excitement, coma, stasis, and death [43]. There have also been other previous studies on insect poisoning symptoms. Essential oils and monoterpenes had knockdown effects on Musca domestica [40]. Distinct poisoning symptoms, such as extended proboscis, expanded wings, unhooked wings, extended legs, and twisted bodies, were also observed in Apis mellifera mellifera [42].
Naturally, insecticidal agents control insects through a variety of mechanisms, including contact, stomach, repellent, fumigant, and systemic methods or through food intake prevention or oviposition inhibition, etc. [40,49,51]. Zhang et al. (2013) found that the A. vera L. leaf acetone extract had contact acaricidal, repellent, fumigant, and oviposition inhibitory activities against T. cinnabarinus [51]. Ma et al. (2021a) reported that 1,3,4-oxadiazoles possessed excellent contact activity and weak systemic activity against E. lanigerum [49]. Here, a study of the mode of action demonstrated that GC16 had contact activity against T. pueraricola, similar to the mite contact activity of botanical extracts from A. vera and Artemisia annua [51,55]. The egg hatching inhibition and ovicidal activity of GC16 against T. pueraricola is consistent with azadirachtins against the maize stem borer Chilo partellus [56]. Moreover, egg hatching inhibition and the delay in egg hatching of C. partellus were supposedly due to the overall detrimental effects of azadirachtins on the reproductive systems of C. partellus [56]. Furthermore, scoparone was found to bind to the Vg protein and lower Vg gene expression to inhibit egg development in T. cinnabarinus [57]. Whether the decrease in egg hatching rate and prolongation of the developmental duration observed in this work are related to the reproductive system needs further exploration.
Undoubtedly, investigating the action mechanisms of pesticides against pests is an important strategy to develop new prospective pesticides. To explore the action mechanism, SEM, TEM, cuticle permeability, enzyme activity, gene expression profile, and RNAi are generally analyzed [49,52,53,58,59].
Cryo-SEM is an important method to study the surfaces of biological samples rich in water. Compared with traditional SEM, there is no sample pretreatment processes required for cryo-SEM, which would inevitably be related to sample distortion, shrinkage, or a loss of the inner cellular soluble components; therefore, we can obtain the most realistic images of sample shape and structure [46,60]. In this work, through cryo-SEM, we found that the cuticle layer of T. pueraricola was destroyed and its arrangement was disordered by GC16, similar to another finding, i.e., graphene oxide can absorb and impair the structure of the cuticle layer of mites [52]. The insect cuticle is its primary protective barrier against the penetration of pesticides. Previous studies have shown that pesticides more easily penetrate weaker and damaged cuticles, and cuticle damage is positively correlated with insecticide permeability and insect mortality [61,62]. Moreover, cuticle permeability is regarded to be associated with insecticide sensitivity, and damaged cuticles are often accompanied by dehydration and shriveling [52,58]. Therefore, the reason for death of T. pueraricola in this study might be that the impaired cuticle layer lost its protective function against the penetration of GC16.
In terms of TEM, ultrastructural changes were detected in T. pueraricola, indicating that GC16 might exert its acaricidal activity by destroying the mitochondria and perturbing the cuticle layer array. It has been previously reported that the steroid PSNW targets the midgut cells of Mythimnazus separata Walker by destroying the cell membrane and mitochondria [63]. In addition, the target site of the steroid 1,3,4-oxadiazole was demonstrated to be the mitochondria and nucleus in the midgut tissues of Eriosoma lanigerum [49]. Mitochondria are the powerhouses of the cell and mitochondrial dysfunction is related to oxidative homeostasis and lipid and energy metabolism [50,64]. For example, zebrafish exposed to triazoles had impaired mitochondrial oxidative phosphorylation and oxidative stress as well as dysregulation of lipid metabolism, which resulted in developmental disorders and movement disorders [64]. In this study, we found mitochondrial dysfunction in mites treated with GC16 accompanied by motionless poisoning symptoms, which were inferred to be related to lipid or energy dysmetabolism. In addition, the mitochondrial dysfunction phenomenon of mites after GC16 treatment was similar to treatment with cyflumetofen, which was demonstrated to be an inhibitor of complex II in the mitochondrial electron transport chain [65]. Mitochondrial abnormalities were also seen in mites in the lecithin group; however, the corresponding mortality rate of the mites after this treatment was low, which might be because lecithin acted alone with a sublethal effect rather than a lethal effect. It was also previously reported that lecithin could induce mitochondrial membrane alterations in mammals but lecithin effectively protected certain sperm quality characteristics against freezing-induced damage [66]. Lecithin is structurally similar to the cell membrane (both contain phospholipids). Moreover, the epicuticle is the outermost layer of the insect integument and mainly composed of lipids and proteins. According to the principle of "like dissolves like", lecithin can dissolve cell membranes and the cuticles of insects in theory. In addition, calcium chloride has the property of water absorption and may influence Ca 2+ balance of the mite. Therefore, we speculate that it is the symmetrical structure of GC16 [inorganic ions + organic substance (dissolve membrane)] that caused the cuticle of mite to be adsorbed, dissolved, and lose water and to lead to ionic imbalance.
Overviewing the action mechanism of pesticides, the Ca 2+ homeostasis disruption and cuticle permeability increase hypotheses were mentioned. For example, Zhou et al. (2021a) reported that curcumin might activate and overexpress the CaM gene and disrupt Ca 2+ homeostasis in T. cinnabarinus to achieve the control effect [67]. Scopoletin acts by regulating the calcium signaling pathway and disrupting intracellular Ca 2+ homeostasis [68]. Further results showed that the acaricidal mechanism of scopoletin on T. cinnabarinus may be related to the calcium channel gene TcT-VDCC [69]. In addition, the mechanism of action of scoparone against T. cinnabarinus is by targeting the interface between CaM1 and L-VGCC to activate the CaM binding site located in the IQ motif at the L-VGCC Cterminus [53]. Moreover, scopoletin could act on mites by inhibiting chitinase (CHIT) gene expression [70], and graphene oxide could inhibit the expression of the cuticle protein (CPR) gene to disturb the construction of the cuticle layer and increase cuticle permeability and acaricide sensibility [52]. Additionally, in Blattella germanica, low expression of CYP4G19 disordered the array of the lipid layer, enhanced cuticle permeability, and compromised insecticide tolerance [58]. Because CaCl 2 is an important component of GC16, whether the application of GC16 affects the calcium homeostasis in mites needs further exploration. However, the destruction of the cuticles of the mites in this study supports the cuticle penetration hypothesis.
Putting the above together, GC16 exhibited a stronger lethal effect on T. pueraricola than lecithin or CaCl 2 alone. In addition, GC16 destroyed and disordered the cuticles of the mites, while those treated with lecithin or CaCl 2 alone had intact and regular cuticles. In contrast to the normal mitochondria of the control and CaCl 2 group, there were mitochondrial abnormalities, such as inner ridge fracture and degradation, in the GC16 group and lecithin group. The combined results suggest that GC16 broke the cuticles of the mites by the coapplication of lecithin + CaCl 2 and lecithin was the main source of T. pueraricola mitochondria damage, indicating that cuticle damage was more important than mitochondrial dysfunction for the lethal effects of GC16 against mites.

Conclusions
In conclusion, we found that GC16 caused insecticidal efficacy against T. pueraricola through contact by disordering the arrangement of the crest in the cuticular layer and destroying the mitochondria. Considering that 6.67 g/L GC16 (recommended concentration for the control of T. pueraricola) has no lethal effects on natural enemy insects P. lewisi and H. axyridis, it may possess the potential to be developed as an ecological agent. However, the effects of GC16 on the overall ecosystem need to be further evaluated in field applications. Our findings may accelerate the development of novel ecological pesticides to control destructive spider mites worldwide. Furthermore, this work will provide alternative pesticide support for pest control for sustainable agriculture; for example, planting of