Introduction
Aphis gossypii Glover (Hemiptera: Aphididae) is a globally significant agricultural pest. While it primarily infests malvaceous plants, such as cotton, it also severely affects a wide range of other crops including species from Solanaceae, Cucurbitaceae, and Rutaceae families (Ebert and Cartwright, Reference Ebert and Cartwright1997). The piercing–sucking feeding behaviour of A. gossypii leads to leaf curling and malformation, which stunts plant development (Dong et al., Reference Dong, Shang, Guo, Wang, Zhu, Liang and Shi2025). Moreover, A. gossypii acts as a vector for various plant viruses, and the losses caused by virus transmission often far exceed those resulting from its direct feeding damage (Zeyen et al., Reference Zeyen, Stromberg and Kuehnast1987).
The control of A. gossypii continues to rely predominantly on chemical strategies, which remain the most effective approach to date (Shrestha and Parajulee, Reference Shrestha and Parajulee2013). Since Bayer introduced the first-generation neonicotinoid insecticide, imidacloprid (IMI), in 1991, this class of insecticides has played a major role in managing A. gossypii. However, the repeated and excessive use of IMI has resulted in the development of resistance and cross-resistance to multiple pesticides in many countries (Chen et al., Reference Chen, Li, Chen, Ma, Liang, Liu, Song and Gao2017). Sulfoxaflor (SUL), a sulfoximine insecticide, is also widely used for the management of A. gossypii. IMI has been extensively employed since its introduction due to its high efficacy, relatively low toxicity, and broad-spectrum activity against various sucking pests (Shi et al., Reference Shi, Jiang, Wang, Qiao, Wang and Wang2011). Although SUL was introduced later, its overreliance and improper use have already led to varying levels of resistance in A. gossypii. Currently, multiple regions have reported differing degrees of resistance of A. gossypii to both IMI and SUL (Ma et al., Reference Ma, Tang, Liang, Li and Gao2021; Wang et al., Reference Wang, Zhu, Cui, Wang, Huang, Ji, Yang and Rui2021a; Ullah et al., Reference Ullah, Xu, Gul, Güncan, Hafeez, Gao and Song2022).
Toxicity of IMI and SUL to A. gossypii
Table 1 Long description
Toxicity results are listed for two treatments, SUL and IMI, using columns for R2, LC30 in milligrams per liter with 95 percent confidence limits, LC50 in milligrams per liter with 95 percent confidence limits, LC95 in milligrams per liter with 95 percent confidence limits, chi square, and degrees of freedom. For SUL, R2 is 0.97, LC30 is 2.73 with 95 percent confidence limits from 1.64 to 3.94, LC50 is 7.13 with limits from 5.14 to 9.30, and LC95 is 145.08 with limits shown as 93.20 to 273.98 slash 0. Chi square for SUL is 4.37 and degrees of freedom is 5. For IMI, R2 is 0.96, LC30 is 7.66 with 95 percent confidence limits from 5.44 to 10.01, LC50 is 18.18 with limits from 14.37 to 22.65, and LC95 is 273.34 with limits from 176.94 to 501.98. Chi square for IMI is 5.31 and degrees of freedom is 5.
a 95% Confidence limits; bchi-square value (χ 2); cdegrees of freedom (df).
Low concentrations of insecticides lead to sublethal effects on insects, which may not cause direct mortality but can still impact multiple biological traits of the insects (Guedes et al., Reference Guedes, Smagghe, Stark and Desneux2016). The manifestation of sublethal effects depends on both the type of pesticide used and the target pest species. Some studies indicate that under certain conditions, sublethal exposure may lead to pest resurgence (Zeng et al., Reference Zeng, He, Wu, Tang, Gu, Ding and Zhang2016). Furthermore, the expression of reproductive-related genes are also influenced by these sublethal effects. Vitellogenin (Vg) is a crucial nutrient source for insect embryos and directly affects reproduction by regulating ovarian development. Its expression level is frequently employed as a biomarker to assess reproductive fitness in insects (Zhao et al., Reference Zhao, Sun, Xiao, Tan, Jiang and Bai2018). Vitellogenin receptors (VgR) are the basis of vitellogenesis (Rabhi et al., Reference Rabhi, Esancy, Voisin, Crespin, Le Corre, Tricoire-Leignel, Anton and Gadenne2014) and are critical for yolk protein absorption and ovarian maturation (Cong et al., Reference Cong, Yang, Jiang, Niu, Shen, Ran and Wang2015). They also play a broader role in oocyte development (Xu et al., Reference Xu, Shen, Wu, Mao, Jia, Zhang, Xia and Lin2023). The endocytosis mediated by VgR ensures efficient uptake of Vg into oocytes (Tufail and Takeda, Reference Tufail and Takeda2009). Current research on insect Vg primarily examines how the sublethal effects of insecticides alter their expression (Yao et al., Reference Yao, Xu, Dong, Que, Quan and Chen2018). Existing evidence shows that different insecticides can exert distinct sublethal impacts on the expression of Vg and VgR within the same pest species. For example, sublethal exposure to thiamethoxam leads to downregulation of Vg and VgR expression in the F1 generation of A. gossypii, thereby reducing their fecundity. In contrast, sublethal doses of acetamiprid can upregulate Vg and VgR expression, ultimately facilitating population growth (Ullah et al., Reference Ullah, Gul, Desneux, Tariq, Ali, Gao and Song2019b, Reference Ullah, Gul, Desneux, Gao and Song2019c).
Mepiquat chloride (DPC) is a widely utilised plant growth regulator (PGR) that modifies cotton canopy architecture (Wang et al., Reference Wang, Mu, Du, Chen, Tian, Zhang and Li2014). It promotes a compact canopy structure and induces synchronised flowering and boll setting. This regulator has been shown to effectively control growth, improve cotton quality, and increase yield (Siebert and Stewart, Reference Siebert and Stewart2006). Since the 1980s, it has been extensively employed in major cotton-growing regions worldwide. Existing studies indicate that spraying DPC at different concentrations can influence the growth and development of A. gossypii (Zhou et al., Reference Zhou, Wang, Ma and He2011). However, it remains unclear whether the combination of insecticides with DPC affects the toxicity to A. gossypii.
Previous studies have shown that DPC reduces the reproductive capacity of A. gossypii and directly modulates aphid insulin signalling and juvenile hormone (JH) biosynthesis pathways via downregulating key genes (such as Ag4E-1B-like and AgJHAMT-like) (Zhou et al., Reference Zhou, Wang, Ma and He2011; Qiu et al., Reference Qiu, Ma, Chen, Song, Hu, Wu, Shan, Wang, Ren, Ma and Luo2025). Furthermore, studies indicate that neonicotinoids (e.g. IMI) and sulfoximines (e.g. SUL) often exert sublethal effects on A. gossypii, leading to altered reproduction and pest resurgence (Ullah et al., Reference Ullah, Gul, Desneux, Gao and Song2019c; Shang et al., Reference Shang, Yao, Zhu, Wang, Li, Zhang, Gao, Wu, Niu, Ji, Luo and Cui2021). Given DPC’s capacity to interfere with aphid reproductive pathways, we hypothesise that combinations of DPC with insecticides may modify sublethal effects of these insecticides. Therefore, this study aims to evaluate the bioactivity of DPC in combination with IMI or SUL against A. gossypii and to investigate the sublethal effects of these mixtures on the development and reproduction of A. gossypii and their progeny through population growth parameters and qPCR analysis. The findings will help to clarify the influence of DPC on the sublethal effects of insecticides on A. gossypii and provide a scientific basis for the rational use of pesticides in the field.
Effect of DPC on the toxicity of IMI and SUL, DPC1: 50 mg/L DPC; DPC2: 100 mg/L DPC; IMI: LC30 value of IMI; SUL: LC30 value of SUL. The notation ns represent non-significant differences (P > 0.05). Same letters (a and a) denote no statistically significant differences (P > 0.05). Results are shown as the mean ± standard error (SE).
Materials and methods
Experimental materials
Aphis gossypii was sourced from cotton plants at the Institute of Cotton Research of CAAS in Anyang, China, and was maintained on cotton leaves (CCRI 49) for more than 10 generations. The rearing took place in a lab-controlled setting, where the temperature was kept steady at 26 ± 1℃ and the relative humidity at 65 ± 5%, following a 16 h light and 8 h dark cycle, without any insecticide exposure.
Technical grade of DPC (98%) was procured from Zhong Mian Xiao Kang Biotechnology Co., Ltd (Anyang), while technical grades of IMI (97%) and SUL (95%) were purchased from Anyang Quanfeng Biological Technology Co., Ltd (Anyang).
Bioassay
The toxicity of IMI and SUL to A. gossypii was determined using the insect dip bioassay recommended by the Food and Agriculture Organization (FAO, 1979). The technical grades of IMI and SUL were completely dissolved in acetone and subsequently diluted with a 0.1% Triton X-100 aqueous solution to create six concentration gradients (active ingredient concentration: 160, 80, 40, 20, 10, and 5 mg/L for SUL and 200, 100, 50, 25, 12.5, and 6.25 mg/L for IMI). These concentrations constituted the treatment groups. The wingless adults of A. gossypii were chosen and dipped in the solution for 5 s, then taken out and dried on the surface. A control solution with the same acetone concentration and 0.01% Triton X-100 served as a control. Cotton leaves (CCRI 49), which were washed and air-dried at room temperature prior to use, were placed upside down on 1.8% agar Petri dishes, and 30 treated adult A. gossypii were transferred onto each leaf. Mortality was recorded after 48 h; A. gossypii that showed no movement when gently touched with a soft brush was considered dead. Each treatment was replicated three times.
Effects of IMI combined with DPC on adult longevity (A) and fecundity (B) in the F0 generation of A. gossypii, as well as total longevity (C) and fecundity (D) in the F1 generation. The letters (a, b, and c) represent significant differences (P < 0.05). Results are shown as the mean ± standard error (SE).
Figure 2 Long description
Four bar graphs labeled A, B, C and D. Graph A shows adult longevity in days for the F0 generation, with categories Control, IMI-LC30, IMI plus DPC1 and IMI plus DPC2. Control has the highest longevity at approximately 12 days, while IMI plus DPC2 has the lowest at around 7 days. Graph B displays fecundity in offspring per individual for the F0 generation, with Control showing the highest fecundity at about 40 offspring and IMI plus DPC2 showing the lowest at approximately 23 offspring. Graph C illustrates total longevity in days for the F1 generation, with all treatments showing similar values around 15 days. Graph D presents fecundity in offspring per individual for the F1 generation, with Control having the highest fecundity at about 50 offspring and IMI plus DPC2 having the lowest at approximately 30 offspring. The letters a, b and c above the bars indicate statistical differences, with a representing the highest value group and b or c indicating lower value groups. The graphs show that IMI and DPC treatments generally reduce both longevity and fecundity compared to the Control group. Bar colors differentiate treatments but do not add meaning beyond the labels.
Previous research indicates that DPC exhibits no significant insecticidal activity against A. gossypii (Qiu et al., Reference Qiu, Ma, Chen, Song, Hu, Wu, Shan, Wang, Ren, Ma and Luo2025). Based on the recommended field doses of DPC provided by the China Pesticide Information Network, concentrations of 50 mg/L (DPC1) and 100 mg/L (DPC2) were selected to be combined with the LC30 of IMI (IMI-LC30) and SUL (SUL-LC30); the mixed groups were termed IMI + DPC1, IMI + DPC2, SUL + DPC1, and SUL + DPC2. The same methods and statistical analysis of mortality were applied as described earlier. Briefly, A. gossypii adults were immersed in solutions of DPC1, DPC2, IMI-LC30, IMI + DPC1, IMI + DPC2, SUL-LC30, SUL + DPC1, SUL + DPC2, or a control (which consisted of the same concentration of acetone and 0.1% Triton X-100) for a duration of 5 s. Subsequently, the individuals were dried, and each adult was placed on a fresh cotton leaf. Mortality was recorded after 48 h. Each treatment included 30 adults, and these assays were repeated three times.
Effects of sublethal doses of IMI combined with DPC on the population parameters of A. gossypii across the F0 and F1 generations
Table 2 Long description
Population parameters r per day, lambda per day, R0 offspring per individual, and GRR offspring per individual are reported for F0 and F1 generations under Control, IMI LC30, IMI plus DPC1, and IMI plus DPC2, as mean plus or minus standard error with letter markers. For F0, r is 0.4323 a in Control, 0.3717 b in IMI LC30, 0.3821 b in IMI plus DPC1, and 0.3811 b in IMI plus DPC2. For F0, lambda is 1.5408 a in Control, 1.4503 b in IMI LC30, 1.4654 b in IMI plus DPC1, and 1.4640 b in IMI plus DPC2. For F0, R0 is 40.61 a in Control, 27.70 b in IMI LC30, 23.91 b in IMI plus DPC1, and 22.73 b in IMI plus DPC2; GRR is 45.30 a in Control, 41.62 a in IMI LC30, 32.74 b in IMI plus DPC1, and 33.29 b in IMI plus DPC2. For F1, r is 0.4314 ab in Control, 0.4539 a in IMI LC30, 0.4240 b in IMI plus DPC1, and 0.4281 b in IMI plus DPC2; lambda is 1.5394 ab in Control, 1.5745 a in IMI LC30, 1.5282 b in IMI plus DPC1, and 1.5343 b in IMI plus DPC2. For F1, R0 is 36.35 c in Control, 49.55 a in IMI LC30, 43.19 b in IMI plus DPC1, and 41.26 bc in IMI plus DPC2; GRR is 40.06 c in Control, 58.20 a in IMI LC30, 50.43 b in IMI plus DPC1, and 47.63 b in IMI plus DPC2. Letter markers differ within each row across treatments.
Note: Data are mean ± standard error (SE). Different lowercase letters in the same row indicate significant difference between the treatment and the control groups at P < 0.05.
Effects of DPC on the sublethal effects of IMI and SUL
Approximately 1400 apterous adult A. gossypii were transferred onto 1.8% agar Petri dishes containing fresh cotton leaves. After 24 h, the adults were removed and the newborn nymphs were maintained on the leaves until they reached adulthood (designated as the F0 generation). These newly emerged F0 adults were divided into seven groups, each group of adults (200 aphids) was dipped into solutions of IMI-LC30, IMI + DPC1, IMI + DPC2, SUL-LC30, SUL + DPC1, SUL + DPC2, and control for 5 s. After being dried, each adult was transferred onto a fresh cotton leaf.
After 48 h, the aphids (approximately 20 adults per sample, collected three samples) of each treatment group were collected for RNA extraction. Concurrently, 60 surviving F0 adults (from each treatment) were selected to observe the development and reproduction of A. gossypii. Subsequently, 60 newly emerged nymphs produced by the F0 generation (designated as the F1 generation, which were used to construct the life tables) were selected and placed on a fresh cotton leaf. Their development was monitored daily, and the number of offspring produced was recorded. All aphids were monitored until death. Fresh leaves and plates were replaced every 2–3 days. Furthermore, adult aphids of the F1 generation were collected from the treatments for RNA extraction. Samples (approximately 20 adults each) were collected within 48 h after moulting, with three replicates per treatment.
RNA extraction and reverse transcription
The quality of total RNA extracted from samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was assessed with a spectrophotometer (Nanodrop2000c, Thermo Scientific, Waltham, MA, USA). According to the manufacturer’s instructions, cDNA synthesis was carried out using the TransScript ® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech Co., Ltd, China), with extracted RNA as the template. The cDNA produced was stored at −20℃.
Quantitative real-time PCR
The StepOnePlus™ Real-Time PCR System (CFX Opus 96; Bio-Rad, Singapore) was employed to perform quantitative real-time PCR (RT-qPCR), with the reaction mixture prepared in line with the manufacturer’s instructions for TransStart ® Top Green qPCR SuperMix (+Dye I/ + Dye II) (TransGen Biotech Co., Ltd, China). Each reaction comprised 10 µL of qPCR Mix, 1 µL of cDNA, 0.4 µL of forward and reverse primers, with nuclease-free water added to a final volume of 20 µL. The thermal cycling conditions were set as follows: 95℃ for 30 s, followed by 40 cycles of 95℃ for 10 s and 60℃ for 30 s. Using the β-actin gene (F: 5′-TGGACTCTGGTGACGGTGTCTC-3′ and R: 5′-ATTTCTCTTTCAGCGGTGGTGG-3′) from A. gossypii served as an internal reference gene, and primers for Vg (F: 5′-ACCACTGCACACTCGGATAA-3′ and R: 5′-CGGCTTGCATGAACCAGTAG-3′) and VgR (F: 5′-CAGGGCGAACGTCCATACAAC-3′ and R: 5′-GTATCCACTGCAATCAGATTGC-3′) were designed and synthesised based on the literature (Ma et al., Reference Ma, Tang, Xia, Lv and Gao2019; Wang et al., Reference Wang, Zhu, Wang, Ji, Wang, Huang, Rui and Cui2023a). The relative expression levels of Vg and VgR were calculated by 2−∆∆Ct method (Livak and Schmittgen, Reference Livak and Schmittgen2001).
Effects of IMI combined with DPC on relative expression level of Vg and VgR in the F0 (A and B) and F1 (C and D) generation of A. gossypii. The letters (a, b, and c) represent significant differences (P < 0.05). The results are shown as the mean ± standard error (SE).
Figure 3 Long description
Four bar graphs labeled A, B, C and D. Graph A shows the relative expression of Vg in the F0 generation, with bars labeled Control, IMI-LC30, IMI+DPC1 and IMI+DPC2. The Control group has the highest expression, followed by IMI-LC30, IMI+DPC1 and IMI+DPC2. Graph B displays the relative expression of VgR in the F0 generation, with the Control group having the highest expression, followed by IMI-LC30 and lower expressions in IMI+DPC1 and IMI+DPC2. Graph C shows the relative expression of Vg in the F1 generation, with the Control group having a lower expression than IMI-LC30, while IMI+DPC1 and IMI+DPC2 have similar lower expressions. Graph D presents the relative expression of VgR in the F1 generation, with the Control group having the highest expression, followed by IMI-LC30 and lower expressions in IMI+DPC1 and IMI+DPC2. Each graph includes annotations with letters a, b and c indicating significant differences.
Effects of SUL combined with DPC on adult longevity (A) and fecundity (B) in the F0 generation of A. gossypii, as well as total longevity (C) and fecundity (D) in the F1 generation. The letters (a, b, and c) represent significant differences (P < 0.05). Results are shown as the mean ± standard error (SE).
Figure 4 Long description
A bar chart with the y axis labeled, Adult longevity days, ranging from 0 to 15. The x axis categories are Control, SUL LC30, SUL plus DPC1 and SUL plus DPC2. Letters above bars are a for Control, b for SUL LC30, b for SUL plus DPC1 and b for SUL plus DPC2. Bar heights are approximately 12 for Control, approximately 9 for SUL LC30, approximately 8 for SUL plus DPC1 and approximately 7 for SUL plus DPC2. The image B showing a bar chart with the y axis labeled, Fecundity offspring per individual, ranging from 0 to 50. The x axis categories are Control, SUL LC30, SUL plus DPC1 and SUL plus DPC2. Letters above bars are a for Control, b for SUL LC30, b for SUL plus DPC1 and b for SUL plus DPC2. Bar heights are approximately 40 for Control, approximately 30 for SUL LC30, approximately 28 for SUL plus DPC1 and approximately 25 for SUL plus DPC2. The image C showing a bar chart with the y axis labeled, Total longevity days, ranging from 0 to 20. The x axis categories are Control, SUL LC30, SUL plus DPC1 and SUL plus DPC2. Letters above bars are a for Control, a for SUL LC30, a for SUL plus DPC1 and a for SUL plus DPC2. Bar heights are approximately 16 for Control, approximately 16 for SUL LC30, approximately 15 for SUL plus DPC1 and approximately 15 for SUL plus DPC2. The image D showing a bar chart with the y axis labeled, Fecundity offspring per individual, ranging from 0 to 50. The x axis categories are Control, SUL LC30, SUL plus DPC1 and SUL plus DPC2. Letters above bars are c for Control, a for SUL LC30, b for SUL plus DPC1 and bc for SUL plus DPC2. Bar heights are approximately 35 for Control, approximately 45 for SUL LC30, approximately 40 for SUL plus DPC1 and approximately 38 for SUL plus DPC2.
Effects of sublethal doses of SUL combined with DPC on the population parameters of A. gossypii across the F0 and F1 generations
Table 3 Long description
Population parameters are listed for F0 and F1 generations across four treatments: Control, SUL LC30, SUL plus DPC1, and SUL plus DPC2. For F0, r per day is 0.4323 plus or minus 0.0052 for Control, 0.4120 plus or minus 0.0051 for SUL LC30, 0.3959 plus or minus 0.0068 for SUL plus DPC1, and 0.4007 plus or minus 0.0071 for SUL plus DPC2. For F0, lambda per day is 1.5408 plus or minus 0.0081 for Control, 1.5100 plus or minus 0.0077 for SUL LC30, 1.4858 plus or minus 0.0102 for SUL plus DPC1, and 1.4928 plus or minus 0.0106 for SUL plus DPC2. For F0, R0 offspring per individual is 40.61 plus or minus 2.01 for Control, 30.43 plus or minus 2.10 for SUL LC30, 27.67 plus or minus 2.21 for SUL plus DPC1, and 24.96 plus or minus 2.23 for SUL plus DPC2; GRR offspring per individual is 45.30 plus or minus 1.45, 38.96 plus or minus 2.50, 36.20 plus or minus 1.64, and 35.30 plus or minus 3.95 in the same treatment order. For F1, r per day is 0.4314 plus or minus 0.0887 for Control, 0.4566 plus or minus 0.0082 for SUL LC30, 0.4507 plus or minus 0.0586 for SUL plus DPC1, and 0.4281 plus or minus 0.0095 for SUL plus DPC2. For F1, lambda per day is 1.5394 plus or minus 0.0134 for Control, 1.5787 plus or minus 0.0129 for SUL LC30, 1.5695 plus or minus 0.0149 for SUL plus DPC1, and 1.5548 plus or minus 0.0103 for SUL plus DPC2. For F1, R0 offspring per individual is 36.35 plus or minus 1.98 for Control, 45.66 plus or minus 0.94 for SUL LC30, 41.30 plus or minus 1.36 for SUL plus DPC1, and 38.57 plus or minus 1.71 for SUL plus DPC2; GRR offspring per individual is 40.06 plus or minus 2.73, 48.43 plus or minus 0.91, 45.73 plus or minus 1.13, and 42.50 plus or minus 0.91 in the same treatment order. Each value includes a lowercase letter marker shown in the table for within row comparisons.
Note: Data are mean ± standard error (SE). Different lowercase letters in the same row indicate significant difference between the treatment and the control groups at P < 0.05.
Data analysis
Bioassay data were analysed using IBM SPSS Statistics 26.0.0 to determine the LC30, LC50, and LC95 values. The longevity, fecundity, intrinsic rate of increase (r), finite rate of increase (λ), net reproductive rate (R 0), and gross reproductive rate (GRR) were calculated based on the formula reported in literature (Chi and Liu, Reference Chi and Liu1984; Chi, Reference Chi1988). The TWOSEX-MSChart computer program was used to calculate the life table parameters (http://140.120.197.173/Ecology/, Ver. 12/08/2022). The mean and SE of the life table parameters were determined through 100,000 bootstrap replicates. Furthermore, the significant differences in life table parameters among different groups were evaluated using the paired bootstrap test within TWOSEX-MSChart (Wei et al., Reference Wei, Chi, Guo, Li, Zhao and Ma2020). Statistical analysis of the toxicity of DPC to A. gossypii was performed using independent sample t-tests in SPSS. Bioassay and qPCR data were analysed using one-way analysis of variance (ANOVA) followed by least significant difference multiple comparisons in SPSS to evaluate significant differences. The ANOVA is presented with the degrees of freedom (df), F-value, and P-value. Bar graphs were generated using GraphPad Prism (version 9.0.0).
Results
Influence of DPC on the susceptibility of A. gossypii to IMI and SUL
The A. gossypii exhibited differing susceptibility to IMI and SUL (table 1). The A. gossypii was more susceptible to SUL than to IMI, as indicated by lower LC30 (2.73 mg/L) and LC50 (7.13 mg/L) values for SUL compared to IMI (LC30 = 7.66 mg/L, LC50 = 18.18 mg/L).
Treatment with DPC1 resulted in a mortality of 7.98% for A. gossypii, while treatment with DPC2 led to a mortality of 10.00%. No significant difference was observed between the two concentrations (fig. 1). The corrected mortalities of IMI + DPC1 (33.34%) and IMI + DPC2 (31.76%) did not show significant differences when compared to the IMI-LC30 group (30.83%). Similarly, SUL + DPC1 and SUL + DPC2 showed mortalities of 34.35% and 31.61%, respectively, without statistically significant differences compared to SUL-LC30 (29.13%). These findings suggest that the addition of DPC did not significantly alter the LC30 values of IMI and SUL against A. gossypii.
Effect of DPC on the sublethal effects of IMI on A. gossypii
Compared to the control, treatment with IMI-LC30 resulted in a significant reduction in the adult longevity of F0 A. gossypii, decreasing from 11.90 to 9.22 days (paired bootstrap test: P = 0.025). When combined with DPC (IMI + DPC1 and IMI + DPC2), adult longevity further decreased to 8.60 days and 7.41 days (paired bootstrap test: P = 0.002, P < 0.001), respectively. Although no significant differences were observed among the three IMI-treated groups (IMI-LC30, IMI + DPC1, and IMI + DPC2), all showed statistically significant reductions compared to the control (fig. 2A). Fecundity in the IMI-LC30 group decreased from 40.61 to 27.70 offspring per individual, while the IMI + DPC1 and IMI + DPC2 groups declined to 23.91 and 22.73 offspring per individual, respectively. All treatments resulted in significantly lower fecundity than the control (paired bootstrap test: all P < 0.001) (fig. 2B). Furthermore, the parameters including r, λ, and R 0 were significantly reduced across all treatment groups compared to the control (paired bootstrap test: all P < 0.001) (table 2).
In the F1 generation, no significant differences in total longevity were observed among the three IMI-treated groups and the control (fig. 2C). However, fecundity increased significantly in the IMI-LC30 and IMI + DPC1 (paired bootstrap test: P < 0.001, P = 0.007), with the IMI-LC30 treatment rising to 49.55 offspring per individual (36.35 for control). Although both IMI + DPC1 and IMI + DPC2 significantly reduced fecundity compared to IMI-LC30 (paired bootstrap test: P = 0.001 P < 0.001), the fecundity in the of IMI + DPC1 remained remarkably higher than that in the control group (paired bootstrap test: P = 0.007), whereas the value of IMI + DPC2 group did not surpass that of the control (fig. 2D). Both the r and λ were not significantly higher in IMI-LC30 than in the control; nevertheless, an upward trend was observed. No significant differences were detected among the combination treatments (IMI + DPC1 and IMI + DPC2), nor between any combination and the control. The IMI-LC30 treatment showed significantly higher R 0 and GRR values than the control, while IMI + DPC1 and IMI + DPC2 exhibited lower values than IMI-LC30 (paired bootstrap test: R 0: P = 0.001, P < 0.001; GRR: P = 0.006, P = 0.001), yet they remained elevated compared to the control (paired bootstrap test: R 0: P = 0.007, P = 0.073; GRR: P < 0.001, P = 0.028) (table 2).
Impact of DPC on the expression of Vg and VgR in A. gossypii treated with a sublethal dosage of IMI
In the F0 generation of A. gossypii, the relative expression levels of Vg and VgR were significantly lowered by IMI-LC30 treatment (one-way ANOVA: Vg: P = 0.003; df = 3, F = 20.724 and VgR: P = 0.036; df = 3, F = 21.220), with the expression levels of these two genes downregulated to 0.57 and 0.77 compared to the control, respectively (fig. 3A, B). When IMI was combined with DPC, no significant differences in Vg expression were observed among the IMI-treated groups. However, compared to the IMI-LC30 group, the expression of VgR in the IMI + DPC1 and IMI + DPC2 groups was further significantly downregulated to 0.44 and 0.35, respectively (one-way ANOVA: P = 0.008, P = 0.002; df = 3, F = 21.220).
In the F1 generation, Vg expression in the IMI-LC30 group increased significantly to 1.26 compared to the control (one-way ANOVA: P = 0.006; df = 3, F = 16.056), while in the IMI + DPC1 and IMI + DPC2 groups, it returned to levels that were not significantly different from the control (fig. 3C). Although VgR expression did not differ significantly between the IMI-LC30 and the control, it decreased markedly when IMI was applied in combination with DPC, with the IMI + DPC1 and IMI + DPC2 further significantly downregulated to 0.52 and 0.41, respectively (one-way ANOVA: P < 0.001, P < 0.001; df = 3, F = 69.430) (fig. 3D).
Effect of DPC on the sublethal effects of SUL on A. gossypii
Aphis gossypii treated with SUL-LC30, SUL + DPC1, and SUL + DPC2 resulted in a significant reduction in adult longevity, fecundity, r, λ, R 0, and GRR in the F0 generation compared to the control (paired bootstrap test: adult longevity: P = 0.003, P < 0.001, P < 0.001; fecundity: all P < 0.001; r: P = 0.005, P < 0.001, P < 0.001; λ: P = 0.005, P < 0.001, P < 0.001; R 0: all P < 0.001; GRR: P = 0.027, P < 0.001, P = 0.012) (fig. 4 and table 3). This trend was consistent across the IMI-treated groups. Specifically, adult longevity decreased from 11.89 days in the control to 8.73 days for SUL-LC30, 8.26 days for SUL + DPC1, and 7.55 days for SUL + DPC2 (fig. 4A). Furthermore, fecundity significantly declined from 40.61 offspring per individual in the control to 30.43 for SUL-LC30, 27.67 for SUL + DPC1, and 24.69 for SUL + DPC2, respectively (fig. 4B). However, no significant differences in adult longevity and fecundity were observed among the SUL-treated groups (SUL-LC30, SUL + DPC1, and SUL + DPC2).
In the F1 generation, no significant differences in total longevity were observed between the SUL-treated groups and the control (fig. 4C). Consistent with the findings related to IMI, exposure to SUL-LC30 significantly increased F1 fecundity, with the number of offspring per individual rising from 36.35 in the control to 45.66 in the SUL-LC30 group (paired bootstrap test: P < 0.001). However, the SUL + DPC1 group showed a significant decrease in fecundity, dropping to 41.30 compared to the SUL-LC30 group (45.66) (paired bootstrap test: P = 0.039). Furthermore, the SUL + DPC2 group (38.57) demonstrated a continued declining trend in fecundity compared to SUL + DPC1, although this difference was not statistically significant. Nevertheless, fecundity in the DPC combination treatments (SUL + DPC1 and SUL + DPC2) remained higher than that of the control (paired bootstrap test: P = 0.039, P = 0.395) (fig. 4D). Both the r and λ were significantly higher in all SUL-treated groups than in the control. While no statistically significant differences were observed in r or λ between the DPC combination treatments and the SUL-LC30 group (paired bootstrap test: both P = 0.035), both parameters exhibited a consistent decreasing trend. Similarly, although the R₀ and GRR were reduced in the combination groups compared to the SUL-LC30 group, these parameters still did not fall below the levels of control group.
Effects of SUL combined with DPC on relative expression level of Vg and VgR in the F0 (A and B) and F1 (C and D) generation of A. gossypii. The letters (a, b, and c) represent significant differences (P < 0.05). Results are shown as the mean ± standard error (SE).
Figure 5 Long description
A bar chart with the vertical axis label, Relative expression of Vg, ranging from 0.0 to 1.2. The horizontal axis categories are Control, SUL dash LC30, SUL dash DPC1, SUL dash DPC2. The Control bar is the tallest, near 1.0, with the letter a above it. The SUL dash LC30 bar is lower, near 0.4, with the letter b above it. The SUL dash DPC1 bar is lower, near 0.4, with the letter b above it. The SUL dash DPC2 bar is higher than SUL dash LC30 and SUL dash DPC1, near 0.5, with the letter b above it. Each bar has an error bar. The image B showing a bar chart with the vertical axis label, Relative expression of VgR, ranging from 0.0 to 1.2. The horizontal axis categories are Control, SUL dash LC30, SUL dash DPC1, SUL dash DPC2. The Control bar is the tallest, near 1.0, with the letter a above it. The SUL dash LC30 bar is lower, near 0.5, with the letter b above it. The SUL dash DPC1 bar is near 0.5, with the letter b above it. The SUL dash DPC2 bar is lower than SUL dash LC30 and SUL dash DPC1, near 0.4, with the letter b above it. Each bar has an error bar. The image C showing a bar chart with the vertical axis label, Relative expression of Vg, ranging from 0.0 to 1.5. The horizontal axis categories are Control, SUL dash LC30, SUL dash DPC1, SUL dash DPC2. The SUL dash LC30 bar is the tallest, near 1.2, with the letter a above it. The Control bar is lower, near 1.0, with the letter b above it. The SUL dash DPC1 bar is near 0.9, with the letter b above it. The SUL dash DPC2 bar is near 1.0, with the letter b above it. Each bar has an error bar. The image D showing a bar chart with the vertical axis label, Relative expression of VgR, ranging from 0.0 to 1.5. The horizontal axis categories are Control, SUL dash LC30, SUL dash DPC1, SUL dash DPC2. The Control bar is near 1.0 with the letter a above it. The SUL dash LC30 bar is near 1.0 with the letter a above it. The SUL dash DPC1 bar is lower, near 0.7, with the letter a above it. The SUL dash DPC2 bar is lower, near 0.6, with the letter a above it. Each bar has an error bar. Across the four bar charts, the same four treatment categories are shown in each plot and each plot includes letters above bars using a and b and error bars for each category.
Impact of DPC on the expression of Vg and VgR in A. gossypii treated with a sublethal dosage of SUL
In A. gossypii’s F0 generation, the SUL-treated groups significantly suppressed the relative expression of the Vg and VgR genes (fig. 5). Specifically, the expression of Vg in the SUL-LC30, SUL + DPC1, and SUL + DPC2 groups decreased to 0.37, 0.35, and 0.47 (one-way ANOVA: P < 0.001, P < 0.001, P < 0.001; df = 3, F = 63.564), respectively, while the relative expression of VgR decreased to 0.51, 0.51, and 0.35 (one-way ANOVA: P = 0.002, P = 0.002, P < 0.001; df = 3, F = 12.806), respectively (fig. 5A, B).
In the F1 generation, Vg expression increased to 1.14 in the SUL-LC30 group (one-way ANOVA: P = 0.038; df = 3, F = 11.317), while it decreased to 0.82 in the SUL + DPC1 and to 0.84 in the SUL + DPC2. In contrast, none of the treatments induced significant changes in VgR expression compared to the control (fig. 5C, D). Therefore, the SUL-LC30 treatment significantly upregulated Vg expression, while SUL + DPC1 and SUL + DPC2 resulted in a notable downregulation of Vg expression.
Discussion
IMI and SUL are currently widely used insecticides for controlling A. gossypii. These two insecticides, though from different classes, both act on nicotinic acetylcholine receptors (Wang et al., Reference Wang, Zhu, Cui, Wang, Huang, Yang, Ji and Rui2021b). Previous studies have shown that laboratory-selected SUL-resistant strains of A. gossypii exhibit varying levels of cross-resistance to neonicotinoids such as IMI and acetamiprid (Wang et al., Reference Wang, Zhang, Liu, Ding, Huang, Yao and Liang2024). Furthermore, sublethal exposure to insecticides can significantly affect pest development and reproduction (Liang et al., Reference Liang, Ma, Chen, Tang, Xia, Chi and Gao2019), with effects that may be either negative or positive (Wang et al., Reference Wang, Huang, Liu and Liang2022). Notably, the sublethal effects of many insecticides can lead to pest resurgence. Several researches have demonstrated that sublethal exposures to lambda-cyhalothrin and IMI significantly enhance the fecundity of the F1 generation of A. gossypii (Ullah et al., Reference Ullah, Gul, Desneux, Qu, Xiao, Khattak, Gao and Song2019a; Qiu and Chen, Reference Qiu and Chen2024). These findings align with our results, indicating that exposure to sublethal doses of IMI and SUL significantly inhibited reproduction in the F0 generation but led to a pronounced resurgence in the F1 generation. These findings were further supported by corresponding changes in the expression levels of Vg and VgR. Furthermore, several studies have reported that sublethal effects can also suppress pest population growth. For instance, treatments with four different sublethal concentrations of insecticides were shown to inhibit population increase in Bemisia tabaci (Hemiptera: Aleyrodidae) (He et al., Reference He, Zhao, Zheng, Weng, Biondi, Desneux and Wu2013). The manifestation of these divergent responses may be influenced by multiple factors, including pest species, insecticide type, and applied concentration (Qu et al., Reference Qu, Xiao, Li, Chen, Biondi, Desneux, Gao and Song2015).
During field applications of PGRs, A. gossypii and other pests inevitably come into direct contact with these compounds. PGRs can induce the production of secondary metabolites in plants, which may exhibit repellent or toxic effects on insects (Liu et al., Reference Liu, Hong, Li, Bing, Chen, Jing, Gershenzon, Lou, Baldwin and Li2023). Some PGRs have been reported to inhibit pest growth and development. For instance, low concentrations of indoleacetic acid affect the development of Galleria mellonella (Lepidoptera: Pyralidae), while higher concentrations exhibit insecticidal activity (Abo Elsoud et al., Reference Abo Elsoud, Ghoneim, Hamadah and El-Hela2021). However, certain PGRs may also exacerbate pest damage (Gupta et al., Reference Gupta, Yadav and Bhattacharya2009). Increased concentrations of DPC elevate gossypol levels in cotton plants, thereby inhibiting pests such as Helicoverpa armigera (Lepidoptera: Noctuidae) (Qu et al., Reference Qu, Zhang and Ma2008). Additionally, DPC reduces the fecundity of Tetranychus cinnabarinus (Acari: Tetranychidae), likely due to morphological changes in cotton leaves, including thicker mesophyll and higher trichome density, which impede feeding (Bai et al., Reference Bai, Sun, Shu, Sun, Zhu and Xu1997). Moreover, DPC could interfere with hormone pathways in insects. Direct exposure to DPC inhibits development and reduces the reproductive capacity of A. gossypii, significantly downregulating the expression of AgJHAMT-like (Qiu et al., Reference Qiu, Ma, Chen, Song, Hu, Wu, Shan, Wang, Ren, Ma and Luo2025), which is associated with JH synthesis. Additionally, RNA interference targeting JHAMT in Nilaparvata lugens Stål (Hemiptera: Delphacidae) reduces both Vg expression and fecundity (Lu et al., Reference Lu, Chen, Liu, Zhang, Chen and Zhou2016). This study confirms that DPC alone exhibits no significant contact toxicity against A. gossypii. However, when combined with sublethal doses of insecticides, DPC did not significantly enhance toxicity but did suppress population resurgence. Therefore, the combination of DPC with IMI and SUL inhibits the population growth of A. gossypii. These findings offer new insights into the management of sublethal effects and the optimisation of insecticide combinations.
Population parameters in pests induced by the sublethal effects of insecticides are often accompanied by alterations in the expression of reproduction-related genes. Sublethal doses of SUL and flonicamid markedly enhance the expression of the Vg gene in the F1 generation of A. gossypii (Shi et al., Reference Shi, Luo, Lv, Zhang, Desneux, You, Li, Ullah and Ma2022; Wang et al., Reference Wang, Huang, Yuan, Zhang and Liang2023b). These findings are consistent with the results of the present study, which showed that treatment of A. gossypii with LC30 of IMI and SUL led to the upregulation of Vg in the F1 generation. Previous studies have reported that sublethal doses of both SUL and lambda-cyhalothrin upregulated the expression of VgR in the offspring of A. gossypii (Wang et al., Reference Wang, Zhu, Wang, Ji, Wang, Huang, Rui and Cui2023a; Qiu and Chen, Reference Qiu and Chen2024). In contrast, the present study demonstrated that IMI and SUL at LC30 concentrations reduced VgR expression in the F0 generation and did not alter the gene expression in the F1 generation. These discrepancies may be attributed to differences in aphid susceptibility to insecticides or variations in experimental methodologies. Furthermore, this study demonstrates that the combination of DPC with sublethal doses of IMI or SUL reduces the fecundity of A. gossypii and suppresses population resurgence in the F1 generation, suggesting that this suppression may be associated with the downregulation of Vg and VgR expression.
In summary, the results of this study indicate that the combination of DPC with IMI or SUL did not enhance toxicity or produce a significant increase in mortality. However, the combined application of DPC with IMI or SUL significantly affected the sublethal effects on A. gossypii. Specifically, while combinations of sublethal doses of the insecticides with DPC did not significantly affect the longevity, fecundity, and population growth of the F0 generation compared to insecticide-only treatments, they reduced the fecundity, r, λ, R 0, and GRR of the F1 generation. This demonstrates that the combination DPC with IMI or SUL effectively suppresses population resurgence. This study provides rational support for the application of DPC and insecticides in the field.
Acknowledgements
This work was supported by the Agricultural Science and Technology Innovation Program (ASTIP) (CAAS-ZDRW202412), the earmarked fund for CARS (CARS-15-21), the Key Research and Development Program in Xinjiang Uygur Autonomous Region (2024B02003), and Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences.
Author contributions
Y.M., X.R., and H.H. conceived the study. X.S., C.W., and Y.M. designed the experiments. K.W. and Y.Q. performed the experiments and analysed data. K.W. wrote the manuscript. X.R., Y.S., and D.W. contributed with and revised the manuscript. All authors read and approved the final manuscript.
Competing interests
The authors declare no conflict of interest.
