Behavioral response of Panonychus citri (McGregor) (Acari: Tetranychidae) to synthetic chemicals and oils

Mite culture

Mite culture was regularly maintained since 2019, on lemon leaves with the water-saturated sponge. The culture was reared in the growth chamber with a 16:8h (Light:Dark) photoperiod and 26 ± 1 °C temperature. One to three-day-old adult females (He et al., 2011; Alves, Casarin & Omoto, 2018) were used for said experimentation reared in the laboratory for several generations (more than 50 generations). The 1–3-day-old adult females were used due to fully developed adultery and ready for egg-laying after 4–5 days. The mite culture was shifted to the open-air environment 1 month before the experiment to acclimatize.

Plants

Citrus plants (Citrus reticulata) approximately 1–2 months old were used after shifting to the pots. The plants with 7–8 leaves were used by leaving six leaves (3 on the right and left side) and cutting them. All plants were washed three times with water to be sure not to have any arthropods on them. The bottom of each plant stem was wrapped with wet tissue paper and maintained wet to keep mites on the plant. All plants were manured and watered accordingly under reasonable conditions during January.

Chemicals

SYP-9625 30% EC and Abamectin 5% EC, EnSpray 99% EC (EnSpray 99), and vegetable oil 99% were used in this research. Chemicals and EnSpray were bought from the local market. The degummed vegetable oil was extracted from household daily kitchen vegetable waste that was provided by the Institute of Zoology, Guangdong Academy of Sciences.

Each chemical toxicity was calculated using a modified leaf dip bioassay (Wang et al., 1971; Nauen, 2005) previously in the laboratory. The selection concentrations of each chemical were made with 10–90% corrected mortality after 24 h. Lethal and sublethal concentrations of each chemical were calculated by probit analysis using SPSS version 22.0 software (Weinberg & Abramowitz, 2016). In this experiment, we used LC₃₀ (0.065%, 0.049%, 0.024% and 0.08%) and LC₅₀ (0.196%, 0.110%, 0.051% and 0.024%) for SYP-9625, Abamectin, Vegetable oil and EnSpray 99, respectively.

Experimental methodology

The method adopted by Iftner & Hall (1983) was followed for the current experiment. Letters were assigned to leaves surfaces as; adaxial right (ADR), adaxial left (ADL), abaxial right (ABR), and abaxial left (ABL). We used a free choice and no choice method by dividing it into nine small experiments, as shown in Fig. 1. Chemicals were applied to the treated leaf surface with a hand sprayer. Untreated part of leaflet or surfaces was protected from chemicals spraying by cardboard shield and plastic bags. Each plant’s ground surface was covered with plastic with double side sticky tape on the edge. The right adaxial surface was selected for easy to release mites (20 mites × 3 surfaces) and identified mites location from the inoculated surface after 30 min of chemicals application. Mites were captured 24 h by location as per the experimental layout. The mites on the leaf surface, wet tissue paper (chemically treated), and plastic cover (chemical sprayed) were considered as dead. The mites not found as live or dead were considered missing mites. The experiments were used with three replications.

The treated, un-treated, and half-treated data were combined for leaf surfaces (ADR, ADL, ABAR, and ABL) further analysis. All experiments were performed in open-air environmental conditions.

Statistical analysis

The mean number of mites (LC₃₀ vs LC₅₀, Treated vs Un-treated, Treated vs Half-treated, and Adaxial vs Abaxial) were analyzed using an independent sample t-test. The difference between control and treatments captured mites means were analyzed using the general linear model (GLM) for ANOVA with Tukey’s HSD test (P < 0.05). All statistical analysis procedures were calculated with Minitab^® 17.3.1 version (Minitab, 2016). Graphical representation was done using GraphPad prism^®(Motulsky, 2007) and OriginPro (Edwards, 2002).

A correlation analysis was conducted by comparing toxicity (% mortality) and % mites present on treated, un-treated, and half-treated surfaces to better understand the relationship between the behavioral responses of P. citri. Pearson correlation (r) and calculating t distribution value formulas were used in R.

$$r={\displaystyle \frac{\sum (x-mx)(y-my)}{\sqrt{\sum {\left(x-mx\right)}^2\sum {\left(y-my\right)}^2}}}$$

$$t={\displaystyle \frac{r}{\sqrt{1-r^2}}\sqrt{n-2}}$$

n is the length of factor (df = n − 2) in two vectors (x (toxicity) and y (mites observed on treated or untreated or half treated surfaces)) while mx and my are the means of vectors. The significant level can be determined by the t-value.

Toxicity

Compared to control, acute toxicity of treatments was found significantly different within each dose in all experiments except in exp. no. 8 at LC₃₀. There was significant difference between doses within abamectin (For exp. no. 3; t_−5.56 = −5.00; P = 0.007), SYP-9625 (For exp. no. 4; t_−7.78 = −3.50; P = 0.025) and EnSpray (For exp. no. 4; t_−8.89 = −8; P = 0.001, For exp. no. 6; t_−6.67 = −3.464; P = 0.026) and vegetable oil (For exp. no. 8; t_−4.44 = −2.828; P = 0.047) than others. Differences in toxicity (from LC₃₀ to LC₅₀) of chemicals to adult (female) P. citri occurred among experiments depending on application methods, with ranges in SYP-9625, abamectin, vegetable oil, and EnSpray of 1.156–2.399-fold, 1.33–5.556 fold, 1.249–5.005 fold and 0–8.889 fold, respectively. Maximum toxicity (%) was observed in the no-choice experiment (the whole plant treated—exp. no. 9), and SYP-9625 more toxic (except in exp. no. 2) than others in all experiments (Table 1).

Re-captured Panonychus citri

According to Fig. 1, the experimental layout is further divided into three parts; Treated vs Untreated (Experiments 1–6), Treated vs Half-treated (Experiments 7–8), and the whole plant treated (Adaxial vs Abaxial) (Experiment 9).

Mites dispersal within the ADR surface were observed 40–82.24% (LC₃₀) and 53.7–94.067% (LC₅₀) from treated to untreated. The difference between treated and untreated was significantly recognizable. A significant difference was observed in all treatments between the mean number of mites captured on the treated and un-treated on the ADR: SYP-9625 (t_−9.22 = −5.56; P = 0.000), Abamectin (t_−7.667 = −8.37; P = 0.000), vegetable oil (t_−10.78 = −9.17; P = 0.000) and EnSpray 99 (t_−8.11 = −5.78; P = 0.000) except control (t_−8.11 = −5.78; P = 0.097), at LC₃₀ while similar results found by applying the LC₅₀ doses. The number of mites captured on the treated ADR surface was lower than the number of mites captured on the untreated surface. A maximum number of mites were observed under the un-treated ADR surface at LC₃₀ dose of vegetable oil than in the others (Fig. 2).

On the Adaxial surface of left side (ADL), a significant difference was observed within all treatments between the mites captured on the treated and un-treated ADL surfaces: control (t_−2.778 = −2.94; P = 0.015 and t_−2.778 = −2.94; P = 0.015), SYP-9625 (t₋₆ = −3.45; P = 0.006 and t_−10.22 = −8.72; P = 0.000), Abamectin (t_−5.778 = −6.28; P = 0.000 and t_−7.78 = −5.29; P = 0.001), vegetable oil (t_−4.22 = −2.37; P = 0.042 and t_−4.67 = −3.78; P = 0.004) and EnSpray 99 (t_−4.33 = −3.99; P = 0.002 and t_−9.11 = −4.77; P = 0.001) on the LC₃₀ and LC₅₀ doses respectively. A higher number of mites captured on the un-treated surface at LC₅₀ of SYP-9625 than others (Fig. 3).

The Panonychus citri less visited the abaxial surface than the adaxial surface, so a small number of mites (Mean ± SE) were captured but enough for the difference between treated and untreated surfaces. At LC₃₀ doses, the data collected from abaxial surface of right side (ABR) was significantly different on treated and untreated surfaces: Abamectin (t_−1.889 = −6.8; P = 0.000), EnSpray 99 (t_−1.889 = −3.3; P = 0.005) and control (t_−1.444 = −2.25; P = 0.041). By treating ABR with lethal concentrations (LC₅₀) was significantly different on treated and untreated surfaces by treated with vegetable oil (t_−3.222 = −4.24; P = 0.002) while SYP-9625, abamectin, and EnSpray 99 were similar between the replication within treated or untreated. The number of mites found maximum on the un-treated ABR surface treated with LC₅₀ of abamectin (6.78 ± 0.813) (Fig. 4).

The difference between treated and un-treated was observed significant within all treatments: SYP-9625 (t₋₂ = −4.1; P = 0.003), abamectin (t_−3.11 = −4.37; P = 0.002), vegetable oil (t₋₂ = −4.94; P = 0.001) and EnSpray 99 (t_−2.33 = −3.61; P = 0.006) except control at LC₃₀ doses on the abaxial surface of left side (ABL). No mites were observed after treatment with LC₅₀ doses on ABL except vegetable oil (t_−3.33 = −4.87; P = 0.001) and control (non-significant). Maximum number of mites found on un-treated surfaces depending on the concentration of chemicals (Fig. 5).

On the adaxial surfaces, difference between treated and half-treated surfaces were found similar (non-significant) at LC₃₀ except on vegetable oil application (t_4.33 = 2.8, P = 0.038) while at LC₅₀, all treatments found significant different (For SYP-9625: t_8.5 = 8.77, P = 0.000; abamectin: t_9.167 = 10.51, P = 0.000; vegetable oil: t_6.167 = 9.43, P = 0.000; EnSpray: t_8.5 = 7.54, P = 0.001) (Fig. 6).

On the abaxial surfaces between treated and half-treated number of mites was significantly different at LC₃₀: abamectin (t_2.167 = 2.89; P = 0.023), vegetable oil (t_2.67 = 2.42; P = 0.038) and EnSpray 99 (t_3.17 = 3.03; P = 0.014) except SYP-9625 and control. At LC₅₀, a significant difference was observed between treated and half-treated surfaces with all mites repelled from treated surfaces (SYP-9625, abamectin and EnSpray 99) (Fig. 7).

In no choice teste (whole plant treated), a significant difference was observed within all treatments (between adaxial and abaxial surfaces): SYP-9625 (t₋₄ = −6.71; P = 0.001), Abamectin (t_−3.17 = −2.53; P = 0.035), vegetable oil (t₋₈ = −5.37; P = 0.003) and EnSpray 99 (t_−7.67 = −3.04; P = 0.029) except control (t_−1.33 = −1.15; P = 0.285) at LC₃₀ doses while all treatments found no difference between adaxial and abaxial surfaces at LC₅₀ doses (Fig. 8).

Correlation analysis

The correlation between toxicity vs treated and toxicity vs un-treated on both surfaces, either right or left, were found negatively correlated except EnSpray and abamectin (Toxicity vs Treated) at LC₃₀ and LC₅₀, respectively (Table S1).

The relationship between toxicity and treated surfaces was positively correlated at LC₃₀ on adaxial (SYP-9625, abamectin, and EnSpray) and abaxial surfaces (abamectin and EnSpray). There was a significant correlation between toxicity and sublethal half-treated abaxial surfaces of SYP-9625, abamectin, and EnSpray 99. There was a positive correlation between toxicity and lethal half-treated adaxial surface for vegetable oil and EnSpray 99. In contrast, on the abaxial surface, only SYP-9625 was found positively correlated (Table S2). In a no-choice experiment (the whole plant treated), a positive correlation was observed by treatment with vegetable oil (toxicity vs adaxial) at both concentrations (Table S3).