Lethal and Sublethal Effects of Afidopyropen and Flonicamid on Life Parameters and Physiological Responses of the Tobacco Whitefly, Bemisia tabaci MEAM1

Abstract

The tobacco whitefly, Bemisia tabaci MEAM1, is a destructive pest that damages plants by sucking plant juice and transmitting viruses. B. tabaci insecticide resistance contributes to population resurgence, and new insecticides are continually needed. Flonicamid and afidopyropen are selective pesticides with high insecticidal activity against piercing–sucking pests and safety to non-target species. We determined the toxicity of flonicamid and afidopyropen to B. tabaci, investigated the sublethal effects on life parameters, and studied physiological responses to them. Flonicamid and afidopyropen were highly toxic to B. tabaci, with LC₅₀ values of 12.795 mg/L (afidopyropen) and 25.359 mg/L (flonicamid) to nymphs and 4.711 mg/L (afidopyropen) and 11.050 mg/L (flonicamid) to adults. Sublethal concentrations (LC₁₀ and LC₂₀) reduced the longevity and fecundity of the B. tabaci F0 generation. Transgenerational effects were caused by exposure to sublethal concentrations of flonicamid and afidopyropen. Nymph mortality increased, development was delayed, fecundity decreased, and adult longevity was shortened. Population parameters such as the intrinsic rate of growth (r), net reproductive rate (R₀), and finite rate of growth (λ) were significantly decreased compared to the control. The activity of detoxifying enzymes, such as GSTs and P450, were induced by flonicamid and afidopyropen at 72 h, while CarE was inhibited. The expression levels of eleven P450 genes and four GST genes were significantly higher than in the control. In conclusion, flonicamid and afidopyropen have excellent acute toxicity and continuous control effects on B. tabaci. Higher GST and P450 activities and gene expression levels may play important roles in the detoxification metabolic process.

1. Introduction

The tobacco whitefly, Bemisia tabaci Middle East-Asia Minor 1 (MEAM1), is a serious plant pest [1,2]. The pest directly damages plants by feeding on the phloem and secretes honeydew on plant surfaces which serves as a substrate for sooty mold, resulting in black blight [3,4]. Furthermore, B. tabaci also transmits viruses such as cotton leaf curl virus (CLCV), tomato yellow leaf curl virus (TYLCV), tomato chlorosis virus (ToCV), and tobacco mosaic virus (TMV) [5,6]. In some cases, the damage resulting from the plant viruses transmitted by B. tabaci exceeds the direct harm from feeding. Whitefly control mainly relies on the use of chemical insecticides such as organophosphates, carbamates, pyrethroids, and neonicotinoids [7,8]. However, the massive and repetitive application of synthetic insecticides for the management of pests has resulted in insecticide resistance, reduced populations of natural enemies, environmental pollution, and threats to human health [9,10,11]. The tobacco whitefly is a rapidly evolving complex species, including various biotypes, and different biotypes possess different sensitivity to insecticides. It is reported that B-type and Q-type B. tabaci have developed resistance to most insecticides including organophosphates, carbamates, pyrethroids, and neonicotinoids [2,12]. New effective chemical insecticides are the key approach to solve the resistance issues of whiteflies.

Flonicamid and afidopyropen are systemic insecticides with selective activity against piercing–sucking insects, in which they typically lead to motion incoordination and feeding disorders [13]. Afidopyropen could interfere with the insect stringer and cause the insect to lose coordination and sense of direction, which results in starvation, desiccation, and death [13,14]. Flonicamid is a feeding blocker that inhibits insect stylet penetration into plant tissues, resulting in starvation and death [15,16]. Flonicamid and afidopyropen are highly effective against many important sucking pests, including species in Liviidae, Aleyrodidae, and Aphididae, and they have limited negative effects on non-targets when used according to the label [13,16,17]. This advantage supports the integration of biological and chemical pest management [13,17]. Flonicamid and afidopyropen use seldom develops cross-resistance to other insecticides [17,18]; therefore, their use provides alternative chemical control options. Both molecules are good candidates for use in insecticide resistance management programs.

Following the field application of insecticides, biodegradation, soil adsorption, natural decomposition, and rainwater leaching reduce the initial dose to lower concentrations. These sublethal concentrations typically do not kill the target organism but they may affect its behavior, development, and reproduction. Insects that contact the sublethal deposits can show sublethal effects [19,20]. In some cases, sublethal doses have strong effects on arthropod population fitness such as extending development time and reducing fecundity [21,22]. The life parameters of the F1 generation of B. tabaci exposed to LC₂₅ doses of dinotefuran, cycloxaprid, and spirotetramat were significantly inhibited and they had increased developmental time, decreased survival, and reduced fecundity [7,8,23]. However, sublethal effects sometimes have a positive impact on insects [24]. Sublethal concentrations of imidacloprid had significant adverse effects on the life parameters (survival, longevity, and fecundity) of the F0 generation of B. tabaci, but they did not decrease biological parameters, population increase rate, or the sex ratio of the F1 generation [23,25].

In some cases, sublethal concentrations of insecticides may accelerate the development and stimulate reproduction of target pests and contribute to the recovery of pest populations [26,27,28]. Upon exposure to sublethal concentrations of insecticides, insects undergo complex physiological changes to eliminate the toxicity of insecticides, such as the activities and gene expression levels of detoxification enzymes [29]. P450 activity in B. tabaci, after treatment with LC₅₀ of chlorogenic acid, was enhanced, and the expression levels of six P450 genes significantly increased. These results suggested P450 was involved in chlorogenic acid detoxification [30]. Exposure to sublethal concentration stress can reduce detoxification metabolism ability. Detoxification ability is usually related to increased insecticide resistance and field control failures [31]. Therefore, determining the sublethal effects of insecticides on pest life parameters and physiological responses is significant for anticipating the field control effects and assessment of insecticide resistance risks.

Here, the toxicity of afidopyropen and flonicamid against B. tabaci MEAM1 and their sublethal effects on the life parameters of F0 and F1 generations were studied. The activities of detoxifying enzymes were also assayed. Our results would be useful to provide a sound basis for the chemical control of B. tabaci and optimize rational field application of afidopyropen and flonicamid.

2. Materials and Methods

2.1. Insect Materials

The B. tabaci MEAM1 strain maintained under laboratory conditions was originally obtained from greenhouse tomato plants (Solanum lycopersicum L.) in Liaocheng, China, in October 2020. The colony was established on cucumber plants (Cucumis sativus L.) planted in flowerpots (15 cm diameter × 20 cm height) and maintained in the psychrometric room of Shandong Agricultural University for more than 15 generations. The environmental conditions were 25 ± 1 °C, 60 ± 5% relative humidity, and a 12:12 h (L:D) photoperiod. From 2020, the colony was maintained without any exposure to insecticides.

2.2. Insecticides

Technical afidopyropen (92.5%) was provided by Badische Anilin und Sodafabrik (Shanghai, China); 95% imidacloprid was provided by Hailir Pesticides and Chemicals Group (Qingdao, China); 99.1% dinotefuran, 98% pymetrozine, and 98.5% flonicamid were provided by Shandong United Pesticide Industry Co., Ltd. (Jinan, China); 95.9% sulfoxaflor was provided Kedihua Agricultural Technology Co., Ltd. (Shanghai, China).

2.3. Bioassay

2.3.1. Insecticide Susceptibility

Stock solutions (5000 mg·L⁻¹) of insecticides were made in analytical grade acetone or in dimethyl sulfoxide (BASF) and then diluted with deionized water containing 0.05% (v/v) Tween 80 to generate five serial dilutions. Toxicity to B. tabaci adults was detected according to the method reported by Shafi et al. [32], with minor modifications. Cucumber plants were grown in plastic pots (10 × 15 × 10 cm), and plants that were about 20 d old with 4–5 leaves were selected and placed in mesh cages (20 × 20 × 30 cm) in the laboratory. The cucumber leaves were immersed for 10 s in different concentrations of the insecticides. After air drying, approximately 20 healthy B. tabaci adults were transferred to the cage. After 2 h, 20 whiteflies were retained in each cage, and all the cages were maintained in the psychrometric room with normal conditions. For the control, the cucumber leaves were treated by distilled water containing 0.05% (v/v) Tween 80. Every concentration treatment contained three repetitions, and each bioassay consisted of five concentrations. Whitefly mortality was recorded every 24 h until 72 h. The tested adults that were unable to move after slight touch were considered dead. For the nymph toxicity bioassay, fresh cucumber plants were inoculated with B. tabaci adults. After 24 h, the plants were transferred to a new cage without any adults. Ten days later, cucumber plants with B. tabaci nymphs were immersed in tested insecticide solution for 10 s, and 50 whitefly nymphs were retained on each plant. The other steps were similar to those described above. Nymphs that remained unhatched after 5 d were considered dead, and the surviving nymphs emerged and left behind exuviae, while the dead nymphs dried up.

2.3.2. Sublethal Effect Bioassay

The sublethal bioassay was carried out according to the description by Tang et al. [33], with minor modifications.

F0 generation: according to the bioassay results, LC₁₀ and LC₂₀ solutions of afidopyropen and flonicamid were prepared. Three-week-old plants with 4–5 leaves were selected and immersed into the insecticide solution. During the period, plastic film was used to cover the pots to prevent the soil from falling. B. tabaci adults were treated according to the methods described in Section 2.3.1. After 72 h, the surviving whiteflies (ten males and ten females) were transferred to an environment with normal, untreated conditions and fed on fresh cucumber plants. The survival rate, longevity, and fecundity were recorded until all whiteflies died. Every treatment contained three replicates, and each replicate contained 20 pairs of adults.

F1 generation: the B. tabaci adults were treated according to the method described for the sublethal effect bioassay on the F0 generation. On the 2nd day after oviposition of the parental adults, fresh cucumber plants with eggs were gathered, and 10 eggs were retained on each leaf under an anatomical microscope. A total of 150 eggs were chosen for the life table analyses. The eggs were observed daily to record their hatch rate. After hatching, nymph survival was recorded daily. After emergence, the adults were paired (one male and one female), transferred to a new cage, and fed on fresh cucumber plants. All cages were maintained under normal conditions, and the survival rate, nymph developmental time, oviposition period, adult longevity, and fecundity were recorded daily until the death of all whiteflies. The raw data of all individuals in the life table were analyzed according to the age–stage two-sex life table theory. The developmental time, longevity, fecundity, and life table parameters of B. tabaci were calculated using the bootstrap method included in the computer program TWOSEX-MS Chart (Version 2023.02.20) [34]. Differences in B. tabaci life parameters of the F1 generation among the different treatments were compared using the paired test included in the software.

The age-specific survival rate (l_x) was calculated as follows:

$$l_x={\sum }_{j=1}^{\beta \beta }{s}_{xj}$$

(1)

where β = the number of stages.

The age-specific fecundity (m_x) is calculated as follows:

$$m_x={\displaystyle \frac{\sum _{j=1}^{\beta }{s}_{xj}{f}_{xj}}{\sum _{j=1}^{\beta }{s}_{xj}}}$$

(2)

The net reproductive rate (R₀) is calculated as follows:

$$R_0={\sum }_{x=0}^{\mathrm{\infty }}{l}_x{m}_x$$

(3)

The intrinsic rate of increase (r) is calculated as follows:

$${\sum }_{x=0}^{\mathrm{\infty }}{e^{-r(x+1)}l}_x{m}_x=1$$

(4)

The finite rate (λ) is calculated as follows:

$$\mathsf{\lambda }=e^r$$

(5)

The mean generation time (T) is defined as the length time that is needed by a population to increase to R₀-fold its current size, which is calculated as follows:

$$\mathrm{T}={\displaystyle \frac{{InR}_0}{r}}$$

(6)

The relative fitness (R_f) is defined as the ratio the value of R₀ of every treatment compared with that of the Control.

2.4. Detoxifying Enzyme Activities Assay

2.4.1. Sample Preparation

Approximately 1000 B. tabaci adults were treated with LC₁₀ and LC₂₀ concentrations of afidopyropen and flonicamid. After 24, 48, and 72 h, the surviving adults were chosen as the tested sample. The treated adults were homogenized using an automatic grinding machine in cold 0.05 M phosphate buffer (pH 7.8) that contained 0.1 mM ethylenediamine tetraacetic acid and 1% polyvinylpyrrolidone. After centrifuging at 10,000× g for 10 min at 4 °C, the supernatants were collected as the enzyme samples [35]. The protein concentration of every sample was detected using the Bradford assay. Each treatment had five replicates with 50 individuals per replicate.

2.4.2. Enzyme Activity Assay

Carboxylesterase (CarE): The 2.3 mL reaction mixtures, containing a 50 μL enzyme sample, 1.8 mL 0.03 M α-naphthyl acetate (as substrate), and 0.45 mL phosphate buffer (0.04 mol/L, pH 7.0), were incubated at 37 °C for 10 min. Then, 0.9 mL stopping solution (0.2 g fast blue-B salt in 20 mL distilled water plus 50 mL 5% sodium dodecyl sulfate, SDS) was added to terminate the reactions. The absorbance was read at 600 nm after 3 min.

Glutathione-S-transferase (GST): The OD value of 275 μL reaction mixtures containing 75 μL 0.6 mM 1-chloro-2,4-dinitrobenzene (CDNB) (as the substrate), 150 μL 6 mM reduced glutathione (GSH), and 50 μL enzyme solution was monitored at 340 nm and 27 °C for 5 min.

Cytochrome P450: The reaction mixtures contained 180 μL of ethoxy coumarin solution as a substrate (100 μmol/L) and 20 μL of enzyme solution and were incubated at 37 °C for 30 min, at which point the reaction was stopped with 20 μL 15% trichloroacetic acid (m/v). Then, the fluorescence intensity was detected using a SpectraMax Gemini XPS (Thermo Fisher Scientific, Waltham, MA, USA). The amount of ethoxycoumarin converted to hydroxycoumarin per mg protein per min was used as the enzyme activity.

2.5. Gene Expression Assay

B. tabaci adults were exposed to the LC₁₀ and LC₂₀ of afidopyropen and flonicamid, and the surviving individuals were gathered at 72 h as the test insects. Total RNA was extracted using an RNApure Tissue Kit (DNase I) (ComWin Biotech, Taizhou, China). cDNA was synthesized using a SYBR1 PrimeScript RT–qPCR Kit II (Takara Biotechnology, Beijing, China). The mRNA levels of eleven CYP450 genes and four GST genes were measured using reverse transcriptase–quantitative polymerase chain reaction (RT–qPCR). The average threshold cycle (Ct) was calculated per sample. The relative level of each gene was defined as the increase (in folds) compared with the amount of β-actin. Each gene was analyzed in triplicate in each of three biologically independent treatments, and each replicate contained 40 surviving B. tabaci adults. All detailed gene information is listed in Table S1.

2.6. Statistical Analyses

In the bioassays, the number of surviving B. tabaci adults at 72 h or number of nymphs at 120 h was used for the regression analyses. The regression equations were followed by Probit analyses in PASW Statistics 18.0.0 (2009, SPSS Inc., Quarry Bay, Hong Kong), in which LC₅₀ values and 95%CL were calculated. The differences in the life parameters of F0 generation enzyme activities and gene expression levels were analyzed by one-way analysis of variance followed by Tukey’s HSD multiple comparisons at the 0.05 level.

3. Results

3.1. Toxicity of Six Insecticides to B. tabaci

The results indicated that sulfoxaflor had excellent toxicity against B. tabaci nymphs, with an LC₅₀ value of 6.645 mg/L, followed by dinotefuran. Afidopyropen and flonicamid were also highly toxic to nymphs, with LC₅₀ values of 12.795 and 25.359 mg/L, respectively (

Table 1. Bioassay results of six insecticides administered to B. tabaci nymphs and adults.

Insecticides	Stages	Regression Equation (y = ax + b)	LC10 (95%CL) (mg/L)	LC50 (95%CL) (mg/L)	LC90 (95%CL) (mg/L)	Chi-Square	df	p
Imidacloprid	Nymph	y = −3.648x + 7.494	28.323 (20.665–35.805)	47.256 (37.638–56.437)	113.372 (98.662–130.622)	9.267	18	0.953
Dinotefuran	Nymph	y = −2.665x + 3.750	3.825 (2.375–5.334)	7.709 (5.568–9.786)	25.544 (21.462–30.741)	9.925	18	0.934
Sulfoxaflor	Nymph	y = −3.652x + 4.390	3.985 (2.837–5.120)	6.645 (5.182–8.041)	15.926 (13.797–18.294)	7.933	18	0.980
Afidopyropen	Nymph	y = −3.744x + 4.922	7.341 (4.916–8.732)	12.795 (9.017–17.490)	26.631 (19.987–32.679)	11.986	18	0.848
Flonicamid	Nymph	y = −3.696x + 5.772	12.268 (8.706–19.783)	25.359 (17.129–33.438)	49.425 (39.672–61.811)	11.688	18	0.863
Pymetrozine	Nymph	y = −3.056x + 6.517	35.910 (22.624–54.202)	67.729 (46.517–88.505)	165.625 (145.891–190.348)	8.009	18	0.979
Imidacloprid	Adult	y = −3.231x + 7.068	32.158 (22.627–41.528)	57.314 (42.889–69.222)	153.923 (132.188–187.608)	8.111	18	0.977
Dinotefuran	Adult	y = −3.680x + 5.896	10.116 (7.053–14.822)	16.802 (12.326–21.118)	40.012 (30.806–51.969)	8.682	18	0.967
Sulfoxaflor	Adult	y = −2.567x + 3.650	3.680 (2.028–5.205)	7.616 (5.123–9.748)	26.405 (20.071–34.028)	9.027	18	0.959
Afidopyropen	Adult	y = −3.346x + 3.638	2.696 (1.677–3.935)	4.711 (3.068–5.908)	12.231 (8.386–16.202)	6.915	18	0.991
Flonicamid	Adult	y = −3.646x + 5.190	6.621 (4.318–8.814)	11.050 (8.473–14.506)	26.521 (20.850–33.494)	9.743	18	0.940
Pymetrozine	Adult	y = −3.063x + 5.992	17.329 (11.410–23.392)	31.881 (23.660–39.828)	90.388 (76.978–108.790)	8.371	18	0.973

The number of surviving adults at 72 h or nymphs at 120 h was used for the regression analyses.

). These values were lower than that of pymetrozine. Afidopyropen had the highest toxicity against adults with an LC₅₀ value of 4.711 mg/L, followed by dinotefuran (7.616 mg/L) and flonicamid (11.050 mg/L). Nymphs were more susceptible to neonicotinoids than adults, while adults were more susceptible to afidopyropen and flonicamid than nymphs.

3.2. Sublethal Effects of Flonicamid and Afidopyropen on B. tabaci

3.2.1. Sublethal Effects on the F0 Generation

LC₁₀ and LC₂₀ of afidopyropen and flonicamid produced significant adverse effects on the F0 generation of B. tabaci (

Table 2. Sublethal effects of flonicamid and afidopyropen on survival rate, fecundity, longevity, and hatchability of B. tabaci adults in the F0 generation.

Treatments	Survival Rate (%)	Male Longevity (d)	Female Longevity (d)	Fecundity (Eggs/Female)	Hatchability (%)
Afidopyropen—LC10	85.50 ± 2.75 b	6.64 ± 0.12 c	4.96 ± 0.12 c	51.16 ± 1.24 b	88.27 ± 0.61 b
Afidopyropen—LC20	73.00 ± 2.89 c	4.99 ± 0.13 e	3.83 ± 0.11 d	36.43 ± 1.10 d	81.99 ± 0.98 c
Flonicamid—LC10	89.00 ± 2.65 ab	7.16 ± 0.13 b	5.89 ± 0.09 b	43.67 ± 1.05 c	84.08 ± 0.86 c
Flonicamid—LC20	81.50 ± 2.75 b	5.97 ± 0.10 d	4.92 ± 0.11 c	35.01 ± 1.06 d	75.59 ± 1.35 d
CK	95.50 ± 0.96 a	8.23 ± 0.16 a	6.33 ± 0.13 a	68.33 ± 1.58 a	95.50 ± 0.47 a

The values (mean ± SE) followed by different letters in the same row have a significant difference.

). At 72 h point after treatment, the survival rates were 85.50% (LC₁₀) and 73.00% (LC₂₀) in the afidopyropen treatments, while they were 89.00% (LC₁₀) and 81.50% (LC₁₀) for flonicamid treatments, decreasing by 10%, 22.5%, 6.5%, and 14% compared to the control (95.50%). Longevity reduction, fecundity reduction, and decreased hatching rate were also observed after treatment. Female longevity after treatment with LC₂₀ doses of afidopyropen and flonicamid was 3.83 d and 4.92 d, respectively, shortened by 2.50 d and 1.41 d, respectively, compared to the control (6.33 d). The fecundity was 36.43 eggs/adult (afidopyropen) and 35.01 eggs/adult (flonicamid), respectively, decreasing by 46.69% and 48.76%, compared to the control (68.33 eggs/adult). For the same pesticide, an increase in treatment concentration increased the magnitude of the adverse effect.

3.2.2. Transgenerational Effects on F1 Generation

Exposed to sublethal concentrations of afidopyropen and flonicamid, the development of B. tabaci nymphs became faster (

Table 3. Transgenerational effects of flonicamid and afidopyropen on B. tabaci in the F1 generation.

Stages		CK	Afidopyropen—LC10	Afidopyropen—LC20	Flonicamid—LC10	Flonicamid—LC20
Developmental time (d)	Egg	5.06 ± 0.06 a	4.81 ± 0.07 c	4.54 ± 0.07 b	5.20 ± 0.05 a	4.75 ± 0.06 b
	Larva I	3.65 ± 0.05 c	3.99 ± 0.06 b	4.24 ± 0.07 a	3.97 ± 0.06 b	3.92 ± 0.07 a
	Larva II	3.17 ± 0.06 b	3.36 ± 0.05 ab	3.59 ± 0.07 a	3.20 ± 0.06 ab	3.09 ± 0.06 b
	Larva III	2.78 ± 0.05 b	3.06 ± 0.07 ab	3.35 ± 0.09 a	3.11 ± 0.07 ab	3.17 ± 0.07 ab
	Larva IV	5.66 ± 0.07 c	6.03 ± 0.08 ab	6.35 ± 0.07 a	5.72 ± 0.07 bc	5.65 ± 0.08 c
	Total larvae	15.24 ± 0.13 c	16.47 ± 0.14 b	17.54 ± 0.19 a	16.02 ± 0.11 bc	15.89 ± 0.13 bc
Longevity (d)	Female	12.74 ± 0.26 a	10.92 ± 0.22 c	9.62 ± 0.17 d	11.48 ± 0.19 b	10.32 ± 0.19 c
	Male	10.66 ± 0.25 a	8.73 ± 0.20 b c	8.02 ± 0.19 c	9.85 ± 0.18 ab	9.16 ± 0.19 b
APOP	(d)	1.71 ± 0.08 b	1.87 ± 0.08 ab	1.84 ± 0.06 ab	1.93 ± 0.09 a	1.98 ± 0.06 a
TPOP	(d)	22.03 ± 0.20 b	23.11 ± 0.23 ab	23.44 ± 0.23 a	23.17 ± 0.19 ab	22.61 ± 0.21 b
Mortality (%)	Egg	2.66 ± 1.32 d	10.65 ± 2.51 c	18.66 ± 3.18 b	18.67 ± 3.18 b	24.11 ± 3.28 a
	Larva I	0.67 ± 0.67 c	4.66 + 1.72 b	7.33 ± 2.14 ab	7.99 + 2.21 ab	9.32 ± 2.36 a
	Larva II	2.01 ± 1.15 a	2.67 ± 1.31 a	3.98 ± 1.59 a	4.68 ± 1.72 a	3.33 ± 1.47 a
	Larva III	2.00 ± 1.14 a	3.99 ± 1.65 a	3.33 ± 1.46 a	2.67 ± 1.32 a	5.33 ± 1.18 a
	Larva IV	2.00 ± 1.13 a	3.99 ± 1.59 a	5.37 ± 1.83 a	0.67 ± 0.65 a	2.01 ± 1.11 a
	Total larvae	6.68 ± 1.37 b	15.31 ± 2.17 ab	20.01 ± 2.54 a	16.01 ± 2.63 a	19.99 ± 2.69 a
	Female	51.99 ± 4.07 a	41.34 ± 4.04 b	33.34 ± 3.84 cd	35.99 ± 3.93 bc	31.35 ± 3.79 d
	Male	38.67 ± 3.97 a	32.67 ± 3.82 b	28.01 ± 3.66 c	29.33 ± 3.72 bc	24.65 ± 3.52 d
Fecundity	(Eggs)	73.79 ± 1.47 a	52.47 ± 1.45 b	38.81 ± 1.03 d	67.39 ± 1.44 a	46.45 ± 1.36 c

APOP, adult preovipositional period; TPOP, total preovipositional period (from egg to first oviposition). The values (mean ± SE) followed by different letters in the same column are significantly different between treatments at the 5% significance level using the paired bootstrap test.

). The total nymph development times of LC₂₀ and LC₁₀ of afidopyropen treatment were 17.54 and 16.47 d, respectively, shortened by 1.23 and 2.30 d, respectively, compared to the control (15.24 d), while they were 16.02 and 15.89 d for the LC₂₀ and LC₁₀ flonicamid treatment, respectively, and not significantly different than the control. Egg development was shortened by sublethal treatments. The LC₂₀ and LC₁₀ afidopyropen treatment values were 4.81 d and 4.54 d, respectively, and these were shortened by 0.25 d and 0.52 d, respectively.

The adult longevity of B. tabaci treated with LC₂₀ and LC₁₀ doses of afidopyropen and flonicamid was shortened significantly compared to the control. Female longevity was 10.92 and 9.62 d for the LC₂₀ and LC₁₀ afidopyropen treatments, respectively, compared to the control (12.74 d). The reduction of adult longevity after treatment with flonicamid was less than that of afidopyropen treatment. The values were 11.48 d and 10.32 d for the LC₂₀ and LC₁₀ flonicamid treatments, respectively. These values were prolonged by 0.56 d and 0.70 d, respectively, compared to the same sublethal concentrations of afidopyropen. For males, similar results were observed.

High mortality of B. tabaci nymphs occurred. The afidopyropen treatment values were 15.31% (LC₁₀) and 20.01% (LC₂₀), increasing by 8.63% and 13.33%, respectively, compared to the control (6.68%). For the flonicamid treatments, the nymph mortality was 16.01 (LC₁₀) and 19.99 (LC₂₀), increasing by 9.33% and 13.31%, respectively, compared to the control. No significant differences were observed between afidopyropen and flonicamid treatments.

The fecundity of B. tabaci adults treated by afidopyropen and flonicamid was less than the control. The values were 52.47 eggs/female (LC₁₀) and 38.81 eggs/female (LC2₀) for afidopyropen, significantly decreasing by 28.89% and 47.40%, respectively, compared to the control (73.79 eggs/female). For flonicamid treatments, the fecundity values were 46.45 eggs/female (LC₂₀) and 67.39 eggs/female (LC₁₀). The LC₂₀ value was significantly decreased by 35% compared to the control. However, there was no significant difference between the LC10 value and the control. Furthermore, oviposition of adults was delayed in the LC₂₀ afidopyropen treatment, and the TPOP (23.44 d) was longer than the control (22.03 d).

The results of age–stage-specific survival rate (S_xj) also indicated that the survival rate decreased, development was delayed, and longevity was shortened (Figure 1). For LC₂₀ and LC1₀ afidopyropen treatments, the peak period of adult emergence was at the 26th to 27th d and 24th to 25th d, respectively, while it was the 22nd to 23rd d in the control. For flonicamid treatments, the peak period of adult emergence was at the 22nd to 23rd d and 23rd to 24th d, respectively. The survival time of female adults was from the 20th to 36th d and the 20th to 39th d for LC₂₀ and LC₁₀ afidopyropen treatments, respectively. Survival ranged from the 19th to 38th d and the 17th to 34th d for the LC₂₀ and LC₁₀ flonicamid treatments, respectively, which are shorter periods than those of the control (17th to 37th d).

The female age-specific fecundity (f_xj), age-specific fecundity of the total population (m_x), and age-specific maternity (l_xm_x) of the B. tabaci F1 generation after afidopyropen and flonicamid treatment also indicated that fecundity declined compared to the control. The area of the curve (f_xj, m_x, and l_xm_x) and the coordinate axis represent the reproductive potential of the population. From Figure 2, the highest reproductive potential was observed in the control, followed by the LC₁₀ flonicamid and LC₁₀ afidopyropen treatments. The lowest reproductive potential was observed in the LC₂₀ afidopyropen treatment.

For the afidopyropen treatment, the r, λ, and R₀ of B. tabaci treated by LC₂₀ were 0.092 d⁻¹, 1.096 d⁻¹, and 12.94 eggs, while they were 0.110 d⁻¹, 1.117 d⁻¹, and 21.69 eggs for the LC₁₀ treatment (

Table 4. Sublethal effects of flonicamid and afidopyropen on F1 generation population parameters of B. tabaci.

Parameters	CK	Afidopyropen—LC10	Afidopyropen—LC20	Flonicamid—LC10	Flonicamid—LC20
r	0.134 ± 0.003 a	0.110 ± 0.004 bc	0.092 ± 0.004 d	0.113 ± 0.004 b	0.101 ± 0.005 c
λ	1.143 ± 0.004 a	1.117 ± 0.004 b	1.096 ± 0.005 c	1.119 ± 0.005 b	1.106 ± 0.005 bc
Ro	38.37 ± 4.86 a	21.69 ± 2.20 b	12.94 ± 1.52 c	24.25 ± 2.69 b	15.19 ± 1.88 c
T	27.23 ± 0.23 bc	27.87 ± 0.22 ab	27.75 ± 0.23 ab	28.21 ± 0.21 a	26.92 ± 0.22 b
Rf	1.000	0.711	0.526	0.913	0.629

r, intrinsic rate of increase; λ, finite rate of increase; Ro, net reproductive rate; T, mean generation time; Rf, relative fitness. The values (mean ± SE) followed by different letters in the same column have significant differences between treatments at 5%.

). The minimum value was observed in the LC₂₀ treatment, intermediate value in the LC₁₀ treatment, and maximum value in the control treatment. For the flonicamid treatment, the values of r, λ, and R₀ were 0.101 d⁻¹, 1.106 d⁻¹, and 15.19 eggs, respectively, which were lower than that of flonicamid LC₁₀ treatment (0.113 d⁻¹, 1.119 d⁻¹, and 24.25 eggs) and the control. Comparing the flonicamid and afidopyropen treatments, the adverse effects of the latter were slightly greater than that of the former at the same sublethal concentration. The relative fitness R_f obtained for LC₂₀ and LC₁₀ afidopyropen treatments was 0.526 and 0.711, respectively, which is lower than the control, and the values for the LC₂₀ and LC₁₀ flonicamid treatments were 0.629 and 0.913, respectively.

3.3. Sublethal Effects of Flonicamid and Afidopyropen on Detoxifying Enzymes

The increase in the activities of B. tabaci detoxification metabolism enzyme (GST and P450) was observed after treatment with LC₁₀ and LC₂₀ of flonicamid and afidopyropen, while the CarE activity decreased (Figure 3).

Afidopyropen treatment first decreased GST and then it was increased. In the LC₂₀ afidopyropen treatment, the activity was lower than that of the control at 24 and 48 h, decreasing by 29.21% and 24.51%, respectively, and increased by 54.00% at 72 h. Meanwhile, the GST activity in the flonicamid treatment increased gradually after treatment, and at 48 and 72 h, the GST activity in the LC₂₀ flonicamid treatment was increased by 49.72% and 66.18%, respectively.

P450 activity increased after treatment with afidopyropen and flonicamid. The LC₂₀ afidopyropen treatment increased P450 activity by 32.32%, 112.22%, and 158.60%, compared to the control, at 24 h, 48 h, and 72 h, respectively. Slight dose effects were observed, and the activity in the afidopyropen treatment was slightly higher than the P450 activity in the flonicamid treatment at the same concentrations.

The CarE activity in B. tabaci treated with afidopyropen and flonicamid decreased when compared to the control. As the treatment concentration increased, the enzyme activity decreased more significantly. In the LC₂₀ afidopyropen treatment the CarE activity decreased by 51.60%, 49.27%, and 28.59% compared to the control at 24 h, 48 h, and 72 h, respectively. The CarE activity decreased by 47.55%, 56.30%, and 66.04% at 24 h, 48 h, and 72 h, respectively, when treated with LC₂₀ flonicamid.

3.4. Sublethal Effects on Expression of Genes Related to P450 and GSTs

Exposure to afidopyropen and flonicamid induced high expression levels of P450 and GST genes. For P450 genes, the expression level of CYP4CS6, CYP6DZ8, and CYP6EN1 increased significantly, which was more than 10 times higher than the control when treated by LC₁₀ and LC₂₀ of afidopyropen (Figure 4). Considerably higher expression levels of CYP6JM1, CYP6DZ6, and CYP6DW were also observed than the control, and the expression of CYP3133A1, CYP315A1, CYP4CS5, and CYP6EM1 increased slightly. The gene expression levels of CYP6CX4 and CYP6JG1 were not significant.

For GST genes, a higher concentration (LC₂₀) of afidopyropen and flonicamid induced higher expression levels of the four GST genes (Figure 4). The expression levels of GSTd11 and GSTd8 were upregulated more significantly, and the expression level of GSTd11 after treatment with LC₂₀ flonicamid and afidopyropen was 14.85 times and 5.39 times higher than that of the control, while the expression level of GSTd8 after treatment by LC₂₀ flonicamid and afidopyropen was 4.65 times and 9.09 times higher than the control. No significant differences in GSTs3 and GSTd7 among different sublethal concentration treatments were observed.

4. Discussion

New insecticides with high efficiency, low non-target toxicity, and environmental safety are important for achieving effective control of whitefly populations [36]. We found that flonicamid and afidopyropen are highly toxic to B. tabaci nymphs and adults, with toxicity levels exceeding imidacloprid and comparable to dinotefuran and sulfaxaflor [2,12], commonly used insecticides for controlling piercing–sucking pests. Compared to the environmentally friendly insecticide pymetrozine, which has a similar mode of action, flonicamid and afidopyropen demonstrated considerably higher insecticidal activity [37]. Given the use restrictions on sulfoxaflor and dinotefuran due to their toxicity to beneficial organisms and rising pest resistance, flonicamid and afidopyropen present effective alternatives, offering high selective toxicity and good environmental safety.

In addition to the direct mortality caused by high doses of pesticides, sublethal doses may cause behavioral and physiological effects on insects that survive initial pesticide exposure [20]. We documented a significant decrease in the life parameters of B. tabaci adults directly exposed to LC₁₀ and LC₂₀ treatments of flonicamid and afidopyropen. The hatching rate of the offspring from treated adults also decreased significantly. Zhou et al. [38] reported that when B. tabaci was treated with an LC₂₅ dose of afidopyropen, the longevity, fecundity, and hatching rate of the offspring also decreased significantly. Similarly, sublethal (LC₁₀) concentrations of afidopyropen also reduced the longevity, fecundity, and oviposition duration of Aphis gossypii Glover adults [33]. This may be because afidopyropen and flonicamid interfere with insect feeding and behavior. Since insects expend considerable energy in the physiological resistance to chemical insecticides, they survive at the expense of population expansion and there is a trade-off in the allocation of energy resources [39,40].

Exposure to sublethal concentrations of insecticides not only affects the contemporary generation (F0) of target pests but can also have transgenerational effects on the F1 progeny [41]. Here, sublethal concentrations of afidopyropen and flonicamid also resulted in negative impacts on the B. tabaci F1 population. As the sublethal concentration increased, the adverse effects were more significant. For afidopyropen, the LC₂₀ concentration significantly slowed down the development of B. tabaci nymphs, reduced adult longevity, and reduced the survival and fecundity. Unlike afidopyropen, the LC₂₀ concentration of flonicamid did not prolong the developmental duration. Interestingly, no stimulatory effects of low concentrations were found in either afidopyropen or flonicamid. Our B. tabaci results are consistent with previous studies where B. tabaci treated with the sublethal concentrations of afidopyropen suffered decreased fecundity and reduced adult longevity [32]. When B. tabaci were treated with sublethal concentrations of flonicamid, feeding was inhibited, egg hatching declined, and nymph mortality increased [42]. The population parameters can reflect the relative adaptability of the tested insect population, which helps predict environmental adaptability, proliferation, and attenuation [32,35]. Our results indicated that the life parameters (r, λ, and R₀) of the B. tabaci F1 population pretreated with afidopyropen and flonicamid were lower than those of the control. This indicated that afidopyropen and flonicamid significantly inhibited the propagation of the progeny population. Ding et al. [43] reported that key population parameters of A. gossypii, including R₀, r, λ, and F, were decreased significantly, while T was increased after treatment with sublethal concentration of afidopyropen. However, in contrast to our results, Abbas et al. [42] reported that B. tabaci pretreatment with an LC₂₅ concentration of flonicamid increased the net reproductive rate (R₀) of the F1 population compared to the untreated control. As determined in previous reports, sublethal effects on the life parameters of the target pests are related to the type of insecticides and pest species. Our study indicated that LC₁₀ and LC₂₀ of afidopyropen and flonicamid inhibited the propagation of the progeny population of B. tabaci, and this had a continuous control effect on the F1 offspring population.

When pests ingest pesticides, physiological responses occur to detoxify and eliminate the toxins [2,12]. Determining the source of pesticide resistance can be facilitated through the assessment of detoxification enzymes, which act to convert and destroy the pesticide chemistry, thereby diminishing their toxicity or safeguarding the target sites via blocking action [44]. In the present study, LC₂₀ and LC₁₀ of afidopyropen and flonicamid increased P450 activity in B. tabaci at 48 and 72 h after treatment. For GST, the activity decreased at 24 and 48 h after afidopyropen treatment and increased at 72 h when treated with flonicamid while the GST activity increased at 48 and 72 h. Similarly, the detoxifying enzyme (CarE, GSTs, and P450s) activities of B. tabaci in diafenthiuron-resistant strains were higher than those in diafenthiuron-susceptible strains [45]. After continuous sublethal pymetrozine treatments, CarE, GST, and P450 activities in F11 and F12 B. tabaci generations were higher than in the pymetrozine-susceptible strains [46]. However, the research performed by Zhou et al. [38] indicated that only the GST of B. tabaci was induced by a sublethal concentration (LC₂₅) of afidopyropen, with no significant differences between treatments in the activities of P450 or CarE. Given all this, we believe that the detoxification enzymes (GSTs and P450) play key roles in the metabolic detoxification of afidopyropen and flonicamid in B. tabaci.

The high expression level of P450 and GST genes plays an important role in the development of insecticide resistance in insects and enhances the adaptability of insects to insecticide stress [47,48]. In the present study, we found that three of the twelve P450 genes were significantly upregulated after treatment with a sublethal concentration of afidopyropen and flonicamid. These genes were CYP4CS6, CYP6DZ8, and CYP6EN1, followed by CYP6JM1, CYP6DZ6, and CYP6DW4. Additionally, the upregulation of four GST genes (GSTs3, GSTd7, GSTd8, and GSTd11) was detected. He et al. [35] reported that a higher expression level of GSTd7 in B. tabaci MED may be an important reason for stronger insecticide tolerance than that of B. tabaci MEAM1. At 48 h after exposure to a sublethal concentration (LC₂₅) of imidacloprid, nine P450 genes (CYP4CS2, CYP4CS5, CYP4CS6, CYP4CS8, CYP6DW4, CYP6DW5, CYP6DW6, CYP6DZ8, and CYP6EM1) in B. tabaci were upregulated significantly. These genes may be positively correlated with imidacloprid resistance [49]. Furthermore, overexpression of CYP6CX4 or GSTs2 of whiteflies enhanced the resistance to flupyradifurone significantly, and silencing these two genes increased the susceptibility [50]. Thus, higher expression levels of P450 and GST genes were closely associated with the B. tabaci detoxification of afidopyropen and flonicamid.

5. Conclusions

This study confirmed higher toxicity of flonicamid and afidopyropen to B. tabacii than imidacloprid. These molecules cause rapid death (in 72 h) at moderate concentrations. At sublethal concentrations they had continuous adverse effects on surviving individuals and caused transgenerational effects on the progeny. Sublethal concentrations inhibited the life parameters and expansion of the progeny population. The activities of GSTs and P450s were significantly increased after insecticide exposure, accompanied by upregulation in the expression levels of six P450 genes and four GST genes. These genes may play significant roles in the detoxification process. Therefore, afidopyropen and flonicamid can be used as efficient alternative pesticides.