Enhancing insecticide efficacy: synergistic and sublethal impacts of a pirimicarb-imidacloprid mixture on Myzus persicae Sulzer (Hemiptera: Aphididae)

Abstract

Aphid resistance to insecticides necessitates management strategies that improve efficacy while minimizing chemical input, including combinations of active ingredients with distinct modes of action. This study evaluated the individual and combined toxicity of pymetrozine, pirimicarb, and imidacloprid against third-instar nymphs and adults of the green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae). Using a 48-h leaf-dip bioassay, insecticides were tested individually and in 1:1 binary combinations, and interactions were quantified using the combination index (CI). Sublethal effects of the most effective mixture were further assessed at the LC₃₀ level (16.424 mg l⁻¹) using age-stage, 2-sex life table analysis and biochemical assays. Among the tested combinations, the imidacloprid-pirimicarb mixture produced the strongest combined effect (CI < 0.5) in both developmental stages, whereas the other mixtures showed antagonistic interactions (CI > 1). This mixture resulted in LC₅₀ values of 39.269 mg l⁻¹ for nymphs and 122.553 mg l⁻¹ for adults, corresponding to substantial reductions in effective concentration and active ingredient requirements compared with single-insecticide treatments. Sublethal exposure significantly (P < 0.05) reduced demographic parameters, including the intrinsic rate of increase (r), net reproductive rate (R₀), fecundity, and longevity. Biochemical assays also revealed developmental stage-dependent alterations in acetylcholinesterase, esterase, and total protein levels following exposure to the mixture. Overall, the findings demonstrate that the imidacloprid-pirimicarb combination enhances toxicity against M. persicae under laboratory conditions and may contribute to reduced insecticide input, although additional studies are required to clarify the nature of the interaction and evaluate its applicability under field conditions.

Graphical abstract

Introduction

The green peach aphid, Myzus persicae Sulzer (1776) (Hemiptera: Aphididae), is one of the most important polyphagous insect pests in agricultural systems worldwide. This species exhibits a remarkably broad host range and has been reported to infest more than 400 plant species across approximately 50 botanical families, including field crops, vegetables, fruit trees, and industrial crops such as tobacco. In addition to causing direct damage through phloem feeding, which leads to reduced plant growth and yield quality, M. persicae is of particular concern due to its efficiency as a vector of numerous plant viruses, thereby posing a serious threat to crop productivity (Eigenbrode et al. 2018, Sharma et al. 2022).

Among its many host plants, chili pepper (Capsicum annuum L.), a member of the family Solanaceae, is especially vulnerable to infestation by M. persicae. Chili pepper is a globally important horticultural crop with high economic value, widely cultivated for both fresh consumption and processing. Owing to its significant market demand and economic importance, C. annuum plays a key role in agricultural production systems worldwide (Mandal et al. 2023). Continuous aphid activity throughout the year, particularly under greenhouse conditions, can result in severe infestations and if left unmanaged, yield losses in pepper crops may reach up to 50 t ha⁻¹ (Pathipati et al. 2018).

Chemical control remains one of the most widely used and cost-effective approaches for aphid management due to its rapid efficacy in improving crop quantity and quality (Khan and Riyaz 2018, Sharma et al. 2019). Repeated and excessive use of insecticides has not only threatened pollinator populations but has also promoted the development of resistance in M. persicae (Lin et al. 2021). The emergence of resistance mechanisms and their adverse effects on non-target organisms underscore the urgent need for innovative pest management strategies. One promising approach is the use of insecticide mixtures with different modes of action that may enhance toxicity and reduce application rates. Such mixtures can reduce application doses, delay resistance development, and enhance the sustainability of IPM programs (Vidau et al. 2011, Yi et al. 2012, Taillebois and Thany 2016). Synergism is commonly defined as the enhancement of an insecticide’s efficacy by compounds that exhibit little or no toxicity when applied alone. Such interactions are frequently associated with altered detoxification processes that increase insect susceptibility to insecticides (Metcalf 1967, Casida 1970). Consequently, the assessment of detoxification enzyme activities provides valuable insight into the physiological basis of enhanced toxicity observed in insecticide combinations. Among these enzymes, esterases and acetylcholinesterase (AChE) play pivotal roles in the detoxification and target-site sensitivity of several classes of insecticides. Alterations in β-esterase and AChE activities have frequently been associated with metabolic resistance and changes in insect susceptibility (Li et al. 2007, Hazarika et al. 2025).

Exposure of pest populations to insecticide mixtures with different modes of action can reduce the survival of resistant phenotypes and may delay the accumulation of resistance alleles more effectively than rotational applications alone (Madgwick and Kanitz 2023, Sparks 2025). However, the biological and physiological consequences of such mixtures require careful evaluation to ensure their sustainable use within IPM programs.

Despite the potential advantages of insecticide mixtures, information on both lethal and sublethal effects of insecticide mixtures on aphid pests remains limited, emphasizing the need for comprehensive life table analyses and enzymatic assessments. Accordingly, the present study was conducted to investigate the lethal and sublethal effects of binary mixtures of imidacloprid, pymetrozine, and pirimicarb on third-instar nymphs and adults of M. persicae. Population parameters, including age-specific survival rate (l_x), age-specific fecundity (m_x), intrinsic rate of increase (r), net reproductive rate (R₀), mean generation time (T), age-stage life expectancy (e_xj), gross reproductive rate (GRR), age-stage survival (S_xj), and finite rate of increase (λ), were assessed. In addition, biochemical activities such as protein, esterase content, and acetylcholinesterase activity were evaluated to determine whether insecticide mixtures could provide maximum efficacy at minimal technical concentrations.

Materials and Methods

Host Plant and Insect Rearing

For the establishment and maintenance of green peach aphid (M. persicae) colonies, hot pepper plants (Capsicum annuum L. cv. Meenakshi, F1) were used as host plants. A total of 50 pepper plants were maintained continuously for colony propagation. To ensure a constant supply of suitable host plants, 10 new plants were cultivated every 2 wk, and host plants were replaced when necessary; plant replacement occurred three times during the experimental period.

The initial aphid population consisted of approximately 100 apterous adult individuals obtained from a single greenhouse colony at the University of Mohaghegh Ardabili (Iran), which had no history of insecticide application. After collection, aphids were transferred to untreated pepper plants and reared under laboratory conditions without exposure to insecticides to reduce handling stress and ensure uniform susceptibility prior to bioassays.

All pepper plants used for colony maintenance and experiments were free from insecticide treatments and were grown in pesticide-free soil to prevent any potential contamination. Aphid colonies were maintained under controlled environmental conditions (25 ± 5 °C, 16:8 h light: dark photoperiod).

For insecticidal bioassays, leaves were excised from pepper plants at the six-leaf growth stage and used to assess aphid mortality. Experimental plants were maintained under the same controlled conditions throughout the study (Zeng et al. 2016).

Chemical Compounds

Technical grade insecticides including imidacloprid (97%), pymetrozine (95%), and pirimicarb (97%) were obtained from Ariashimi Co. (Tehran, Iran).

Acute Toxicity

To evaluate the potential for reducing application rates of technical-grade insecticides, the toxic effects of imidacloprid, pymetrozine, and pirimicarb were evaluated individually and in binary mixtures at a 1:1 ratio. Preliminary range-finding bioassays were conducted to determine appropriate concentration ranges for each insecticide and life stage. Based on these preliminary tests, 5 concentrations were selected for each treatment for probit analysis, prepared using a serial dilution factor of approximately 2 between successive concentrations, in accordance with standard arthropod bioassay procedures (Robertson et al. 2007).

All insecticide solutions were prepared in distilled water containing 1.5% dimethyl sulfoxide (DMSO). An untreated control (distilled water only) and a solvent control (distilled water with 1.5% DMSO) were included to account for any effects of the solvent on aphid mortality (Robertson et al. 2007). Mortality in the control treatments remained below 10% throughout the experiments; therefore, observed mortality data were corrected using Abbott’s formula prior to probit analysis (Abbott 1925). Each concentration was tested in four independent replicates, with 20 aphids per replicate. All bioassays, including controls, were conducted simultaneously under identical environmental conditions to ensure reproducibility and comparability of LC₅₀ estimates.

Bioassays were performed using the leaf-dip method. Detached pepper leaves were immersed in insecticide solutions for 20 s, air-dried under shaded conditions at room temperature for approximately 1 h, and placed individually into 9 cm glass Petri dishes. For each replicate, 20 third-instar nymphs or 20 adults of the M. persicae were transferred onto treated leaves. Mortality was assessed after 48 h, and aphids were considered dead if they failed to respond to gentle probing with a fine brush (Zeng et al. 2016).

The concentration ranges of the technical-grade insecticides tested against third-instar nymphs and adults of M. persicae were as follows: imidacloprid (40–1,000 mg l⁻¹ for nymphs and 55–1,300 mg l⁻¹ for adults), pymetrozine (3,162.27–7,000 mg l⁻¹ for nymphs and 5,000–8,000 mg l⁻¹ for adults), pirimicarb (1,000–3,000 mg l⁻¹ for nymphs and 2,000–4,500 mg l⁻¹ for adults). For the binary mixtures (1:1), the concentration ranges were 20–200 mg l⁻¹ (nymphs) and 30–600 mg l⁻¹ (adults) for pirimicarb-imidacloprid, 200–2,500 mg l⁻¹ (nymphs) and 1,000–3,700 mg l⁻¹ (adults) for pymetrozine-imidacloprid, 1,000–3,000 mg l⁻¹ (nymphs) and 1,500–4,200 mg l⁻¹ (adults) for pymetrozine-pirimicarb.

The potential combined effects of the two pesticide compounds were evaluated using the method described by Chou and Talalay (1984). In this approach, the two pesticides were mixed in various ratios, and their combined effect on target mortality was evaluated. The degree of interaction was quantified using the Combination Index (CI). The CI was calculated using Equation (1).

$$CI=\frac{{LD}_x^{2m}}{{LD}_x^2}+\frac{{LD}_x^{1m}}{{LD}_x^1}$$ (1)

where ${LD}_x^{1m}$ and ${LD}_x^{2m}$ represent the concentrations of the first and second pesticides in the mixture that produce ${LD}_x$ mortality, and ${LD}_x^1$ and ${LD}_x^2$ represent the concentrations of each pesticide applied individually that produce the same ${LD}_x$ mortality. CI values were interpreted as follows: CI < 1 indicates a synergistic effect, CI = 1 indicates an additive effect, and CI > 1 indicates an antagonistic effect. This method allows precise determination of the nature of interaction between two pesticides at different concentration ratios.

To facilitate comparison between single insecticides and binary mixtures, the percentage reduction in LC₅₀ values was calculated according to the following Equation (2):

$$\mathrm{\%}\mathrm{ }\mathit{Reduction}=\frac{LC50\mathrm{ }\mathit{single}-\mathrm{ }LC50\mathrm{ }\mathit{mixture}}{LC50\mathrm{ }\mathit{single}}\times 100$$ (2)

This approach has been widely used to quantify relative increases in toxicity and synergistic effects in insecticide mixture studies (Cedergreen 2014).

Sublethal Toxicity and Life Table Bioassays

To evaluate the sublethal effects of the pirimicarb-imidacloprid mixture on the life table parameters of the M. persicae, a cohort of 100 adult females was released onto pepper leaves inside plastic clip cages with mesh ventilation. After 24 h, only the newly produced, same-aged nymphs were retained, and the adult females were removed. First-instar nymphs were used for age synchronization.

Pepper leaves were dipped in LC₃₀ solutions (16.424 mg l⁻¹; each compound at 8.212 mg l⁻¹), which were chosen to simulate realistic field exposure levels while producing measurable sublethal effects. Preliminary bioassays confirmed that this concentration produced sublethal effects with limited mortality, validating its use for life table studies.

From the synchronized cohort, 50 third-instar nymphs per treatment were randomly selected and individually placed on treated leaves inside clip cages. Aphids were exposed to the treated leaves for 48 h, during which daily survival and developmental data were recorded. After the 2-d exposure period, all aphids were transferred to fresh, untreated pepper leaves, which were replaced every 3 d to maintain leaf quality and allow continued monitoring of development and reproduction (Campbell 1989).

Daily observations continued until all individuals reached the third instar of the next generation. From the adults that produced nymphs, 50 first-instar nymphs per treatment were individually transferred onto untreated leaves to continue monitoring development. Life table parameters, including the intrinsic rate of increase (r), net reproductive rate (R₀), finite rate of increase (λ), and mean generation time (T), were calculated using the age-stage, two-sex life table method (Chi et al. 2023). All parameters were estimated using the TWOSEX-MSChart program.

Enzymatic Assays

For enzymatic assays, third-instar nymphs and adults of M. persicae exposed to the LC₃₀ concentration of the 1:1 mixture of pirimicarb-imidacloprid and untreated controls were used. For each treatment, four biological replicates were prepared, with 25 nymphs and 15 adults per replicate. Aphids were collected in equal numbers for each replicate. Samples were homogenized in 70 μl sodium phosphate buffer (0.04 M, pH 7) using a VIZION homogenizer at 200 rpm for 10 min on ice. Homogenates were centrifuged at 15,000 rpm for 15 min at 4 °C using a SIGMA 1-14K refrigerated centrifuge, and the supernatant was collected as the enzyme extract. All biochemical assays were performed in triplicate for each replicate (Piri et al. 2020).

β-Naphthyl acetate, RR-blue salt, 5,5-dithiobis-2-nitrobenzoic acid (DTNB), acetylthiocholine iodide, Bradford reagent, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Total Protein Quantification

Total protein content was determined using the Bradford method (Bradford 1976). Protein concentration, as an indicator of energy storage, was measured in third-instar nymphs and adults of the green peach aphid. For each concentration, 10 μl of protein solution was mixed with 290 μl of Bradford reagent. The same procedure was applied to the enzyme extracts of the pesticide treatments. Absorbance was recorded at 595 nm using a microplate reader (Biochrom Anthos, UK). All treatments were tested in triplicate.

Esterase Activity

Esterase activity was determined following the method of Han et al. (1998). A reaction mixture consisting of 20 μl enzyme extract and 70 μl phosphate buffer (40 mM, pH 7.0) was combined with 90 μl of substrate solution containing β-naphthyl acetate (10 mM). The mixture was incubated at 30 °C for 10 min in a W350B water bath. Subsequently, 90 μl of RR-blue salt was added to the micro tubes. Absorbance was measured at 540 nm using a microplate reader. Substrate without enzyme extract served as the blank.

Acetylcholinesterase (AchE) Activity

Acetylcholinesterase activity was assayed according to Ellman et al. (1961). A reaction mixture containing 80 μl phosphate buffer (0.1 M, pH 7.0), 50 μL 5,5-dithiobis-2-nitrobenzoic acid (DTNB, 10 mM), and 40 μl enzyme extract was incubated at 25 to 27 °C in a W350B water bath for 10 min. Following incubation, 50 μl of acetylthiocholine iodide (10 mM) was added. Absorbance was recorded at 405 nm using a microplate reader. A blank containing substrate without enzyme extract was included. All assays were performed in triplicate.

Statistical Analysis

Data distribution was evaluated for normality prior to analysis using the Kolmogorov–Smirnov test. For toxicity assays, mortality data were expressed as the proportion of dead individuals per experimental unit and analyzed using Probit regression with a binomial error structure to estimate lethal concentrations (LC₃₀ and LC₅₀) and their corresponding confidence intervals. Separate models were fitted for each life stage and treatment. Model adequacy was assessed using goodness of fit statistics. Biochemical assay results were analyzed based on three biological replicates per treatment in each of two independent experimental runs, and data from both runs were pooled for statistical analysis. Following confirmation of normality, enzyme activity and total protein content were compared between treated and control groups using one-way analysis of variance (ANOVA). When only two groups were involved, differences between means were evaluated using independent-samples t-tests at a significance level of 0.05. Population growth parameters were analyzed according to the age-stage, 2-sex life table theory (Chi and Liu 1985, Chi 1988, Chi and Su 2006) using TWOSEX-MS Chart software (version 2023). Variances and standard errors were estimated by bootstrapping with 100,000 resampling’s, and treatment effects were assessed using paired bootstrap tests at the 5% significance level. All statistical analyses were conducted using SPSS software (version 26), except for life table analyses, which were performed using TWOSEX-MS Chart.

Results

Toxicity of Insecticides and Their Mixtures

Mortality data for imidacloprid and pirimicarb generally met the assumption of normality for both developmental stages. However, mortality data for adult aphids exposed to pirimicarb and pymetrozine deviated from normality. In these cases, data were log-transformed [ln(x + 1)] to satisfy the assumptions of normality before further analyses.

The probit-log concentration regression lines for imidacloprid, pirimicarb, and pymetrozine against third-instar nymphs and adult females of M. persicae are presented in Fig. 1. For all insecticides and both developmental stages, probit mortality increased consistently with increasing log concentration, indicating a clear dose-dependent response. The observed probit values closely matched the expected values estimated by the probit model, demonstrating an adequate goodness of fit of the regression lines. Differences in susceptibility were evident between developmental stages. Third-instar nymphs showed steeper regression slopes than adults for imidacloprid (1.07 ± 0.138 vs. 0.84 ± 0.136), indicating greater probit responses at lower log concentrations. However, for pirimicarb and pymetrozine, steeper slopes were observed in adults (4.22 ± 0.567 and 4.99 ± 0.949, respectively) compared with nymphs (2.78 ± 0.404 and 3.34 ± 0.571, respectively) (Table 1). Overall, the probit-log concentration regressions indicate differential toxicity among the tested insecticides and developmental stages, providing a quantitative basis for subsequent comparisons of lethal concentrations.

Fig. 1.

Probit-log concentration regression lines for imidacloprid, pirimicarb, and pymetrozine against third-instar nymphs and adult females of M. persicae after 48 h exposure. Panels A and B show nymph and adult mortality responses to imidacloprid, respectively; panels C and D show responses to pirimicarb; and panels E and F show responses to pymetrozine.

Table 1.

Toxicity (mg l^−¹) of pirimicarb, imidacloprid, pymetrozine against third-instar nymphs and adults of M. persicae

Pesticides	Life stages	N  a	Lethal concentration (95% confidence limits) (mg a.i. l-1)			Slope ± SE	χ  2  ${df=3}^{\mathrm{b}}$	Sig.c
			LC30	LC50	LC90
Imidacloprid	Nymph	480	61.652 (36.883–87.481)	193.176 (144.241–257.899)	3,148.578 (1,696.123–8,628.787)	1.07 ± 0.138	1.661	0.646c
	Adult	480	80.855 (39.596–124.626)	344.768 (242.337–516.637)	11,931.818 (4,541.842–74,536.559)	0.84 ± 0.136	1.189	0.0756c
Pirimicarb	Nymph	480	1,121.085 (902.314–1,286.839)	1,731.068 (1,548.211–1,930.577)	5,005.042 (3,896.738–7,733.055)	2.78 ± 0.404	0.596	0.897c
	Adult	480	2,610.808 (2,358.840–2,814.905)	3,465.066 (3,228.668–3,775.092)	6,920.681 (5,804.413–9,265.254)	4.22 ± 0.567	4.169	0.244c
Pymetrozine	Nymph	480	4,105.926 (3,567.037–4,506.404)	5,859.751 (5,356.490–6,665.681)	13,975.735 (10,710.127–23,438.730)	3.34 ± 0.571	6.995	0.072c
	Adult	480	6,216.265 (5,784.037–6,604.795)	7,902.670 (7,322.108–9,098.867)	14,207.918 (1,147.345–22,735.635)	4.99 ± 0.949	1.473	0.688c

^aNumber of subjects.

^b df = degrees of freedom.

^cSig.= significance level (P-value).

P > 0.05 indicates an acceptable fit of the probit model. respectively. Since the significance level is greater than 0.05, no heterogeneity factor is used in the calculation of confidence limits.

The probit-log concentration regression lines for the binary mixtures of pirimicarb- imidacloprid, pymetrozine-imidacloprid, and pirimicarb-pymetrozine (1:1) against third-instar nymphs and adult females of M. persicae are presented in Fig. 2. For all mixtures and both developmental stages, probit mortality increased with increasing log concentration, indicating a clear concentration dependent response. The observed probit values generally showed good agreement with the expected values estimated by the probit model, although slight deviations were evident at the lower and higher log concentration ranges for some mixtures. Differences in susceptibility between developmental stages were apparent, with third-instar nymphs exhibiting higher probit responses at lower log concentrations than adult females in most binary combinations. Among the tested mixtures, combinations containing imidacloprid tended to produce higher probit responses across the tested concentration range, particularly in third-instar nymphs, whereas mixtures involving pymetrozine showed comparatively moderate responses. These probit-log concentration regressions demonstrate that the toxicity of insecticide mixtures varied depending on both the chemical combination and the developmental stage of the aphids.

Fig. 2.

Probit-log concentration regressions for binary mixtures (1:1) of insecticides against third-instar nymphs and adult females of M. persicae after 48 h exposure. Panels A and B represent pirimicarb + imidacloprid; panels C and D represent pymetrozine + imidacloprid; and panels E and F represent pirimicarb + pymetrozine.

Table 1 summarizes the estimated lethal concentrations of pirimicarb, imidacloprid, and pymetrozine for third-instar nymphs and adult females of M. persicae. Probit analysis yielded adequate fits for all treatments, as indicated by non-significant values (P > 0.05). Sensitivity differed between life stages, with third-instar nymphs generally more responsive than adult females across all insecticides. Among the tested compounds, imidacloprid exhibited the highest toxicity, pymetrozine the lowest, and pirimicarb intermediate effects. Variation in the slopes of the regression lines suggests differences in the homogeneity of responses within the aphid populations. These results provide a quantitative basis for comparison with insecticide mixtures in subsequent analyses.

The toxicity and interaction effects of binary mixtures of pirimicarb, imidacloprid, and pymetrozine against M. persicae are summarized in Table 2. Probit regression provided acceptable fits for all mixtures, as indicated by non-significant χ² values (P > 0.05).

Table 2.

Toxicity (mg l^–¹) and Combination Index (CI) of the binary mixtures of pirimicarb, imidacloprid and pymetrozine against third-instar nymphs and adults of M. persicae

Pesticides	Life stages	Lethal concentration (95% confidence limits) (mg a.i. l−1)			Slope ± SE	χ   2  ${\mathit{df}=3}^{\mathbf{b}}$	Sig.	CI	Effect
		LC30	LC50	LC90
Pirimicarb + imidacloprid	Nymph	16.42 (9.53–22.98)	39.26 (29.45–49.01)	330.55 (217.52–665.05)	1.38 ± 0.194c	4.513	0.211c	0.22	Synergism
	Adult	27.94 (11.131–46.324)	122.553 (83.04–177.29)	4,542.91 (1,747.63–30,405.25)	0.83 ± 0.143c	3.863	0.277c	0.39	Synergism
Pirimicarb + Pymetrozine	Nymph	1,103.18 (856.89–1,279.42)	1,755.75 (1,559.84–1,975.22)	2,794.35 (2,411.51–3,553.23)	2.61 ± 0.401c	2.570	0.463c	1.31	Antagonism
	Adult	1,717.61 (1,446.86–1,926.69)	2,503.12 (2,275.47–2,755.30)	6,283.25 (5,088.12–8,931.43)	3.22 ± 0.435c	1.012	0.798c	1.03	Antagonism
Pymetrozine + imidacloprid	Nymph	165.38 (61.36–271.35)	674.42 (465.40–963.75)	20,932.22 (7,839.15–179,053.9)	0.86 ± 0.168c	0.976	0.807c	3.60	Antagonism
	Adult	860.09 (603.07–1,060.80)	1,472.385 (1,232.94–1,684.52)	5,477.74 (4,164.763–,864.35)	2.24 ± 0.338c	0.540	0.910c	4.45	Antagonism

^a95% confidence limits.

^bDegrees of freedom.

^cSince the significance level is greater than 0.050, no heterogeneity factor is used in the calculation of confidence limits.

Based on CI (Equation (1)), pirimicarb-imidacloprid exhibited synergistic effects (CI < 1), while pirimicarb-pymetrozine and pymetrozine-imidacloprid showed antagonistic interactions (CI > 1) across the tested concentrations (Table 2).

The binary mixture of pirimicarb and imidacloprid markedly reduced the LC₅₀ values of both compounds in third-instar nymphs and adults of M. persicae (95% confidence intervals are provided in Tables 1 and 2). For third-instar nymphs, the LC₅₀ of the mixture was 39.26 mg l⁻¹ (corresponding to 19.63 mg l⁻¹ of each insecticide), representing reductions of 89.83% for imidacloprid and 98.86% for pirimicarb relative to individual treatments. In adults, LC₅₀ values decreased to 122.553 mg l⁻¹ for both compounds, corresponding to reductions of 82.22% for imidacloprid and 98.31% for pirimicarb. These results clearly indicate a strong synergistic effect of the pirimicarb-imidacloprid combination, allowing substantial enhancement of toxicity at lower concentrations.

Sublethal Toxicity and Life Table Bioassays

The intrinsic rate of increase (r) of M. persicae was significantly higher in the control treatment (0.29 ± 0.0075 d⁻¹) than in the pirimicarb + imidacloprid treatment (0.18 ± 0.01 d⁻¹), indicating substantial suppression of population growth (Table 3). Similarly, the net reproductive rate (R₀) and finite rate of increase (λ) were significantly reduced from 44.28 ± 3.18 offspring individual⁻¹ and 1.33 ± 0.01 d⁻¹ in the control to 8.45 ± 1.41 offspring individual⁻¹ and 1.20 ± 0.015 d⁻¹, respectively, under the combined treatment (P < 0.05).

Table 3.

Reproductive and life-table parameters (mean ± SE) of M. persicae under control treatments and the pirimicarb-imidacloprid mixture

Parameters	Control	Pirimicarb + imidacloprid	CI of difference 95%
APOP (d)	0.94 ± 0.07 a	1.19 ± 0.17 a	−0.117–0.617
TPOP (d)	6.72 ± 0.15 a	6.54 ± 0.21 a	−0.333–0.701
Ovipositional period (d)	18 ± 0.93 a	8 ± 0.96 b	6.957–12.192
Fecundity (nymphs/female)	45.03 ± 3.15 a	10.52 ± 1.64 b	27.541–41.492
Longevity (d)	24.4 ± 1.009 a	12.63 ± 0.92 b	9.085–14.449
Preadult survival rate	0.98 ± 0.0166 a	0.80 ± 0.0492 b	0.078–0.282
R  0 (offspring/individual)	44.28 ± 3.18 a	8.45 ± 1.41 b	28.999–42.674
T (d)	13.03 ± 0.29 a	11.61 ± 0.40 b	0.475–2.421
r (d-1)	0.29 ± 0.0075 a	0.18 ± 0.01 b	0.079–0.138
λ (d-1)	1.33 ± 0.01 a	1.20 ± 0.015 b	0.097–0.173
GRR (offspring/individual)	62.18 ± 3.95 a	25.27 ± 4.57 b	25.072–48.751
DT (d)	3.77 ± 0.2850 a	2.3833 ± 0.06224 b	0.853–1.995

SE was estimated by bootstrapping (100,000 resamples). APOP, TPOP, T, r, λ, DT, and R。 represent the adult pre-ovipositional period, total pre-ovipositional period, mean generation time, intrinsic rate of increase, finite rate of increase, doubling time, and net reproductive rate, respectively. Means within each row followed by the same letter are not significantly different at P >0. 05. CI = confidence interval.

Parameters associated with population turnover and reproductive performance were also markedly affected by the pirimicarb + imidacloprid mixture. Gross reproductive rate (GRR), fecundity, longevity, and doubling time (DT) decreased significantly from 62.18 ± 3.95 offspring individual⁻¹, 45.03 ± 3.15 offspring, 24.4 ± 1.009 d, and 3.77 ± 0.2850 d in the control to 25.27 ± 4.57 offspring individual⁻¹, 10.52 ± 1.64 offspring, 12.63 ± 0.92 d, and 2.3833 ± 0.06224 d, respectively, in the combined treatment (Table 3). In contrast, neither the adult pre-ovipositional period (APOP) nor the total pre-ovipositional period (TPOP) differed significantly between treatments, with values ranging from 0.94 ± 0.07 to 1.19 ± 0.17 d for APOP and 6.54 ± 0.21 to 6.72 ± 0.15 d for TPOP.

The age-specific survival rate (l_x), age-specific fecundity (m_x) of the offspring generation, and the age-stage-specific fecundity (f_xj)-defined as the number of nymphs produced by each female at age x and developmental stage j-peaked on day 14 in the control with an average of 3.61 nymphs, and on day 26 in the pirimicarb-imidacloprid combination treatment with an average of 2 nymphs (Fig. 3).

Fig. 3.

Age-specific survival rate (l_x), age-specific fecundity (m_x), and age-stage female fecundity (f_x) of M. persicae under control conditions and a pirimicarb + imidacloprid mixture. Panel A represents the control treatment, and panel B represents the pirimicarb + imidacloprid treatment.

Age-stage life expectancy (e_xj) declined with increasing age in both treatments. The maximum life expectancy of adult M. persicae was 37 d in the control and 29 d in the combined treatment (Fig. 4).

Fig. 4.

Age- and life-stage–specific life expectancy (E_xj) of M. persicae under control conditions A) and the pirimicarb + imidacloprid mixture B).

The age-stage-specific survival and reproduction rate (S_xj) results are shown in Fig. 5 the highest probability of surviving from newly hatched first-instar nymph to adult was 0.90 in the control and 0.62 in the pirimicarb-imidacloprid treatment. The age-stage-specific reproductive value (V_xj) reached its maximum for female M. persicae at 11.84 nymphs on day 11 in the control and 5.62 nymphs on day 12 in the combined treatment (Fig. 6).

Fig. 5.

Age-stage–specific survival rate (S_xj) of M. persicae under control conditions A) and the pirimicarb + imidacloprid mixture B).

Fig. 6.

Age-stage–specific reproductive value (V_xj) of M. persicae under control conditions A) and the pirimicarb + imidacloprid mixture B).

Physiological Disruptions

Total protein content differed significantly between treatments in both third-instar nymphs (F = 98.36; df = 1,4; P < 0.05) and adult M. persicae (F = 191.57; df = 1,4; P < 0.05). In third-instar nymphs, the highest mean protein content was observed in the control, whereas in adults the pirimicarb-imidacloprid treatment resulted in higher protein levels (Fig. 7).

Fig. 7.

Comparison of mean total protein content in nymphs and adults of M. persicae under control and pirimicarb + imidacloprid treatments. Panel A represents total protein content in nymphs, and panel B represents total protein content in adults. Bars represent mean ± SEM. Different letters above bars indicate significant differences according to Student’s t-test (P < 0.05).

Esterase activity differed significantly between treatments at both developmental stages, with a significant increase in third-instar nymphs and a significant decrease in adults under the combined treatment (Fig. 8). In third-instar nymphs, esterase activity was significantly higher in the pirimicarb-imidacloprid treatment compared with the control (F = 9.81; df = 1,4; P < 0.05). In contrast, adult aphids exhibited significantly higher esterase activity in the control treatment than in the combined treatment (F = 98.36; df = 1,4; P < 0.05).

Fig. 8.

Comparison of mean specific activity of β-esterase in third-instar nymphs and adult M. persicae under control and pirimicarb + imidacloprid treatments. Panel A represents β-esterase activity in nymphs, and panel B represents β-esterase activity in adults. Bars represent mean ± SEM. Different letters above bars indicate significant differences according to Student’s t-test (P < 0.05).

The results showed that the specific activity of acetylcholinesterase differed significantly between the control and the pirimicarb-imidacloprid mixture treatments at both developmental stages, ie, third-instar nymphs (F = 21.46; df = 1,4; P < 0.05) and adults (F = 240.32; df = 1,4; P < 0.05), at the 5% probability level. In third-instar nymphs, AChE activity was significantly elevated under the combined treatment compared with the control, whereas in adults the opposite pattern was observed, with higher enzyme activity recorded in the control treatment (Fig. 9).

Fig. 9.

Comparison of mean acetylcholinesterase (AChE) activity in third-instar nymphs and adult M. persicae under control and pirimicarb + imidacloprid treatments. Panel A represents AChE activity in nymphs, and panel B represents AChE activity in adults. Bars represent mean ± SEM. Different letters above bars indicate significant differences according to Student’s t-test (P < 0.05).

Discussion

The overuse of chemical insecticides poses several ecological and agronomic risks, including toxicity to non-target organisms such as natural enemies and pollinators, accumulation of residues in the environment, and acceleration of resistance development in pest populations (Ahmad 2008, Muniz-Junior et al. 2023). Reducing insecticide doses is therefore a key goal of sustainable pest management, helping to mitigate these negative impacts while maintaining control efficacy. In this study, the observed reductions in LC₅₀ provide mechanistic indicate that insecticide mixtures may lower the effective toxic concentration under controlled conditions, potentially reducing overall pesticide inputs without compromising efficacy (IRAC 2023, Muniz-Junior et al. 2023).

The present study demonstrates that the pirimicarb-imidacloprid mixture exerts significant synergistic effects on M. persicae under laboratory conditions. This pattern is biologically plausible, as imidacloprid stimulates nicotinic acetylcholine receptors, while pirimicarb inhibits acetylcholinesterase, leading to intensified neural stress and overwhelmed detoxification pathways, particularly in metabolically active nymphs (Ahmad 2008, Taillebois and Thany 2022, Fukuto 1990). In contrast, mixtures containing pymetrozine showed antagonistic interactions under the experimental conditions. This response may be related to the feeding inhibitory activity of pymetrozine, which could reduce insect exposure to the companion insecticide and consequently limit overall toxicity during the 48-h bioassay period (Silva et al. 2024).

Age-stage life table analysis demonstrated that sublethal exposure to the pirimicarb-imidacloprid mixture negatively affected the survival and reproductive performance of M. persicae. Treated aphids showed delayed and reduced fecundity, lower survival probability, and significant declines in reproductive and population growth parameters, including reproductive value (V_xj), intrinsic rate of increase (r), net reproductive rate (R₀), finite rate of increase (λ), life expectancy (e_xj), and gross reproductive rate (GRR). In contrast, pre-ovipositional periods were not significantly affected. These findings suggest that sublethal exposure primarily influences survival and reproductive capacity rather than developmental timing, which is consistent with previous studies on neonicotinoid and carbamate insecticides (Choi et al. 2005, Desneux et al. 2007, Amini Jam et al. 2014, Zeng et al. 2016).

The results showed that the effects of treatments on total protein content were dependent on developmental stage. Total protein content is often used as an indicator of overall metabolic status and physiological stress in insects exposed to insecticides (Vontas et al. 2001). The significant differences in protein content observed between treatments and developmental stages in the present study further support the notion that the pirimicarb-imidacloprid mixture imposes substantial metabolic demands on M. persicae. The lower protein content recorded in treated nymphs may indicate protein degradation or reduced protein synthesis under insecticide-induced stress, whereas the increased protein levels observed in treated adults could be associated with the induction of detoxification enzymes or stress-related proteins (Vontas et al. 2001, Heidel-Fischer and Vogel 2015, Jin et al. 2025).

The results showed a developmental stage-dependent response of general esterase activity to the pirimicarb-imidacloprid mixture. General esterases are among the most important metabolic enzymes involved in the detoxification of xenobiotic, including carbamate and neonicotinoid insecticides (Li et al. 2007, Montella et al. 2012). Enhanced esterase activity has frequently been associated with metabolic resistance in aphids and other hemipteran pests. In this study, the pirimicarb-imidacloprid mixture significantly altered general esterase activity compared with the control at both developmental stages. The differential esterase responses observed between nymphs and adults may indicate that susceptibility to the pirimicarb-imidacloprid mixture is strongly influenced by developmental physiology. Because immature stages undergo rapid growth and intense metabolic activity, insecticide exposure may alter enzymatic regulation differently from adults, potentially leading to temporary activation of detoxification pathways in nymphs. In contrast, the reduced esterase activity detected in adults may reflect cumulative physiological stress or disruption of metabolic homeostasis caused by simultaneous exposure to multiple insecticidal modes of action. Similar stage-specific biochemical responses have been documented in other aphid species exposed to neurotoxic insecticides (Bass et al. 2014, Bass and Nauen 2023).

Overall, these findings suggest that the enhanced toxicity observed in the mixture may be related to the complementary action of the two compounds and stage-dependent physiological responses, with stage-dependent physiological sensitivity likely influencing the overall response. In line with this pattern, the pirimicarb-imidacloprid mixture induced pronounced biochemical alterations in M. persicae, affecting both enzymatic systems and overall protein status. The contrasting pattern observed in adults-suppressed detoxification-related enzyme activities alongside elevated protein content-suggests a shift in metabolic allocation toward stress adaptation rather than efficient detoxification. Such responses are consistent with stress-induced synthesis of protective and regulatory proteins under xenobiotic pressure (Hemingway 2000, Kültz 2005, Li et al. 2007).

Previous studies have reported that mixtures containing neonicotinoids may enhance toxicity in insect pests by simultaneously affecting multiple physiological pathways (Taillebois and Thany 2022). In the present study, the pirimicarb-imidacloprid mixture produced a strong combined effect against M. persicae under laboratory conditions, as reflected by reduced LC₅₀ values and CI estimates. This combined effect may be associated with complementary modes of action between the two insecticides, where imidacloprid targets nicotinic acetylcholine receptors and pirimicarb inhibits acetylcholinesterase, leading to disruption of neural signaling (Ahmad 2008, Sparks et al. 2020, Blumel et al. 2001). The Insecticide Resistance Action Committee (IRAC) highlights that mixtures of insecticides with dissimilar modes of action may delay resistance development, although careful ecological evaluation is required (Sparks & Nauen 2015). Integration of biochemical and life-table data further supports that this combined effect reflects combined disruption of multiple neural pathways (Casida and Durkin 2013, IRAC 2023).

However, it should be noted that these findings do not directly indicate field-level efficacy or improvements in integrated pest management. Considering these points, additional studies should examine whether the observed combined effects remain consistent, as well as under semi-field and field conditions and with a broader range of mixture ratios (eg, 3:1 and 1:3). Integrating molecular and enzymatic approaches would help clarify the mechanisms underlying stage-dependent responses and provide a stronger basis for evaluating the ecological relevance and practical implications of insecticide mixtures in sustainable pest management.

Conclusion

Sublethal exposure to the pirimicarb-imidacloprid mixture significantly affected the survival, fecundity, adult longevity, and population growth of M. persicae. Integration of biochemical and life-table analyses showed that the combined treatment altered detoxification- and neurophysiology-related responses and increased toxicity at lower concentrations under laboratory conditions. The reduced LC₅₀ values and CI estimates suggest a strong combined effect of the mixture in both nymphs and adults, although additional studies are required to determine whether the observed interaction reflects true synergism or a strong additive response. In addition, because pymetrozine primarily acts as a feeding inhibitor with relatively slower toxic action, longer exposure periods may be necessary to fully evaluate its interaction patterns in binary mixtures.

Despite these findings, the pirimicarb-imidacloprid mixture should be considered a complementary component of IPM rather than a standalone control strategy. Further investigations are needed to evaluate field efficacy, environmental persistence, effects on natural enemies and other non-target organisms, and the potential role of these mixtures in resistance management. Future studies could include isobologram or response surface analyses, resistant aphid populations, semi-field and field experiments, a wider range of mixture ratios, and molecular assessments of detoxification and acetylcholinesterase-related genes to further clarify the mechanisms and practical applicability of these insecticide combinations in sustainable aphid management.

Author Contributions

 Mehri Zargani (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Investigation [equal], Methodology [equal], Resources [equal], Software [equal], Supervision [equal], Validation [equal], Visualization [equal], Writing-original draft [equal]), Hooshang Rafiee Dastjerdi (Conceptualization [supporting], Data curation [supporting], Formal analysis [supporting], Funding acquisition [supporting], Investigation [supporting], Methodology [supporting], Project administration [lead], Resources [supporting], Software [supporting], Supervision [supporting], Validation [supporting], Visualization [supporting], Writing-review & editing [supporting]), Bahram Naseri (Conceptualization [supporting], Methodology [supporting]), Asgar Ebadollahi (Conceptualization [supporting], Investigation [supporting], Methodology [supporting], Resources [supporting], Software [supporting], Supervision [supporting], Validation [supporting], Visualization [supporting], Writing-review & editing [supporting]), Vahid Mahdavi (Conceptualization [supporting], Formal analysis [supporting], Methodology [supporting], Writing-review & editing [supporting])

Funding

This work is based upon research funded by Iran National Science Foundation (INSF) under project No. 4047297 and carried out at the University of Mohaghegh Ardabili.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability

Data will be made available on request.