Introduction

The cowpea weevil beetle, Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae), emerges as a particularly devastating postharvest pest on a global scale1,2. The larvae of this species feed and develop exclusively on different stored grains and legumes such as cowpea grains, mung, and adzuki beans2. Significant damages related to feeding inside the seeds by larvae cause direct damage to the grains3,4. Adult insects focus their short lifespan on mating and laying eggs on beans2. Following oviposition on the seeds, hatched first-instar larvae bore into the beans and thus, these larvae progress by consuming the grains internally, transforming into pupae, and ultimately completing their life cycle as adult beetles2,3.

The management of C. maculatus involves the utilization of insecticides, which have been identified as more cost-effective and efficient than alternative practices5. Nevertheless, applying chemical controls has been associated with many detrimental consequences, jeopardizing both human health and the environment6. It also exacerbates pest control due to resistance development7,8,9,10. The search for alternative management has become a significant challenge to avoid these chemical side effects11,12and safeguarding cowpeas in storage systems could be performed utilizing eco-friendly methods such as using essential oils (EO)13,14.

The aromatic plants that release volatile compounds are rich in essential oils that generally exhibit lower toxicity than synthetic insecticides to mammals and the environment14,15. Furthermore, essential oils are generally found in aromatic plants. One economically, environmentally, and locally available plant is peppermint (Mentha piperita L.)16. Peppermint essential oil has been shown to have antibacterial, antifungal, and insecticidal activity17,18,19.

While research on this plant’s insecticidal properties has primarily focused on mosquito species and houseflies17,20scant literature exists regarding its efficacy against stored product insects16,21,22. For example, an experiment21 showed essential oil derived from M. piperita exhibits promising insecticidal potential against various stored product insect pests, including Sitophilus oryzae, Rhyzopertha dominica, and Tribolium castaneum. They reported a correlation between the mortality percentage increase and peppermint concentration oil that makes M. piperita a functional alternative to synthetic insecticides in some pest population management21.

While peppermint (M. piperita) essential oil is not currently a commercially registered insecticide, it represents a strong candidate for development due to its broad-spectrum insecticidal properties, rapid biodegradability, and low mammalian toxicity23. Given increasing concerns over resistance to synthetic insecticides and their environmental impact, there is growing interest in identifying natural alternatives that are both effective and sustainable. Life table analysis provides a powerful framework for understanding the long-term population-level impacts of such compounds, even before commercial formulation. Thus, this study provides critical pre-commercial data on the sublethal and transgenerational effects of peppermint oil on C. maculatus, helping to establish its potential for future use in integrated pest management (IPM) systems.

Insect populations can modulate the efficacy of pesticides by activating or inhibiting their internal enzyme system of which antioxidant enzymes systems like superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST), function to maintain low levels of free radicals within insect cells, thereby mitigating cellular damage24,25,26. Furthermore, the enzyme activities serve as a valuable biomarker for identifying organisms exposed to sublethal pesticide concentrations27. Studies on C. maculatus have shown an inverse relationship between the concentration of Acalypha wilkesiana oil extract and the activity of detoxification enzymes like SOD, CAT, and GST28.

Insect responses to toxicants, including essential oils, can vary between males and females due to sex-specific differences in metabolism, immune defense, and reproductive roles29. Understanding these differences is crucial for evaluating the ecological impact of botanical insecticides. Moreover, recent evidence indicates that sublethal exposure to plant-derived compounds may induce physiological or epigenetic changes that persist across generations30,31. However, studies assessing peppermint EO’s sex-specific and transgenerational impacts on stored product pests are scarce. Our study addresses this gap by investigating both gender-based susceptibility in F₀ and potential effects on life table parameters and antioxidant enzyme activity in F₁ and F₂ generations.

While the insecticidal effects of peppermint (M. piperita) essential oil (EO) have been studied against stored grain pests23,32,33most research to date has focused on immediate toxicity endpoints in a single generation. However, little is known about how sublethal EO exposure in parental insects influences offspring performance and physiological stress markers in subsequent generations. Given the growing interest in transgenerational effects of botanical insecticides34,35our study explores whether exposure of the F₀ generation to LC₃₀ peppermint EO concentration modulates life table parameters and antioxidant enzyme activities in F₁ and F₂ generations. Furthermore, integrating demographic toxicology with biochemical assays provides novel insights into how EO-induced stress may be biochemically inherited and expressed across generations. Thus, this study, first of all, aims to evaluate the constituents of the peppermint oils using Gas Chromatography–Mass Spectrometry (GC-MS), secondly to explore the efficiency of the oil on C. maculatus females and males, and thirdly, to assess the sublethal impact of the oil on the following two generations (F1 and F2) of the insects when F0 generation exposed to the oil using a two-sex life table analysis. Also, we aimed to investigate the activity of antioxidant enzymes such as Superoxide dismutase (SOD), Catalase (CAT), and Glutathione S-transferase (GST) of F1 and F2 generations following exposure of F0 generation to sublethal concentrations of EO.

Materials and methods

Insect colony

The population of C. maculatus originated from a laboratory stock reared on cowpea grains at the Department of Plant Protection, the University of Tehran, College of Agriculture & Natural Resources, Karaj, Alborz, Iran (35°48’25.3"N 50°59’38.3” E). The cowpeas were kept at − 20 °C for 24 h before use, following standard laboratory practice to reduce the risk of microbial or hidden pest contamination in stored legumes. However, it is acknowledged that this method may not be fully effective against C. maculatus eggs, as previous studies have demonstrated their tolerance to − 20 °C conditions36. Therefore, this step was intended primarily as a precautionary measure rather than a definitive disinfection method. Then, C. maculatus colonies were reared on cowpeas for at least three generations before they were used in the experiments. The cowpea weevils were reared on cowpea (Vigna unguiculata) at 28 ± 2 ºC, relative humidity of 60 ± 5%, and photoperiod of 16:8 (L: D) h. The cowpeas containing eggs (10 grains to each Petri dish) were transferred to small Petri dishes (6 cm diameter, 1 cm depth). They were carefully checked daily until the emergence of adults.

Plant collection and extraction of essential oil

Peppermint (M. piperita) leaves were collected during the whole flowering stage in mid-summer from the Botanical Garden of the University of Tehran, College of Agriculture and Natural Resources, Karaj, Iran (35°48’25.3"N 50°59’38.3” E). This season was selected as it corresponds to the peak period of essential oil accumulation and bioactive compound concentration in peppermint leaves37. The leaves were stored in the dark at the laboratory temperature for one week and then crushed into powder. For essential oil extraction, the obtained peppermint powder was used by hydrodistillation38. Briefly, 100 g of powdered dried M. piperita leaves were subjected to hydrodistillation in distilled water (1:10 w/v) for three hours using a Clevenger-type apparatus. Following desiccation with anhydrous sodium sulfate, the essential oil was stored at 4 °C for further analysis38.

Gas Chromatography-Mass (GC-MS) spectrometry analysis

The essential oil constituents of M. piperita were analyzed using gas chromatography-mass spectrometry (GC-MS) on a Polaris Q GC-MS system (Thermo Electron Corporation, Germany) equipped with a DB-5 capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm)38.

The analysis was performed based on the following conditions: The oven temperature was programmed as isothermal at 40 °C for 1 min, then raised to 250 °C at 6 °C/min and held at this temperature for 4 min. Helium was used as carrier gas at a 1.0 ml/min rate. The injection volume was 1 µL, and the injector temperature was maintained at 250 °C in splitless mode. The GC column effluent was introduced directly into the mass spectrometer. The ionization was performed by electron impact (EI) at 70 eV. The essential oils components were identified by matching their mass spectral peaks available with Wiley, NIST, and NBS mass spectral libraries, and by comparing fragmentation patterns and retention times with published data in the literature and authentic reference compounds, where available39.

Bioassay procedures

The concentration-response bioassay was carried out using Whatman filter paper number 1 to determine the effect of essential oil toxicity by respiratory exposure (the mortality covering the range of 10–90%)40. Thus, five concentrations of peppermint oil were determined based on performed pre-tests, which included 3, 3.4, 3.8, 4.3, and 5 ppm for females and 1.5, 1.8, 2.2, 2.8, and 3.5 ppm for males. Separate concentration ranges were used for males and females based on preliminary trials indicating sex-specific differences in susceptibility to peppermint essential oil. This approach is supported by literature reporting that females in many insect species, including C. maculatus, often exhibit higher tolerance to chemical and botanical insecticides due to larger body mass, greater energy reserves, and enhanced detoxification enzyme activity, particularly related to reproduction and longevity29. Therefore, distinct ranges were required to determine LC₅₀ values for each sex accurately.

The Petri dish (6 cm diameter, 1 cm depth) was covered with treated filter paper on its top, and 10 adults of the same age (one day old of both sexes separately) were released into the Petri dish, and then the Petri dish was sealed with parafilm. All treatments were replicated four times, and 10 adult insects were used for each replication. All assays were conducted under controlled environmental conditions of 28 ± 2 °C, 60 ± 5% relative humidity (RH), and a photoperiod of 16:8 h (light: dark). Mortality was recorded 24 h after exposure. To confirm the mortality of adult C. maculatus, the immovable ones touched with a fine bristle brush were considered dead.

Sublethal effects of peppermint essential oil on developmental and reproductive performance in two generations of C. maculatus

We aim to explore whether the treatment of adult males and females (F0 generation) with the sublethal concentrations of peppermint leaves affects the following (subsequent) two generations of the beetle. To test it, the newly adult males and females were exposed to two sublethal concentrations of LC10 (2.527 ppm for females and 1.211 ppm for males) and LC30 (2.984 ppm for females and 1.567 ppm for males) of essential oil as described in the bioassay procedures. Adult males and females of C. maculatus were distinguished based on established morphological characteristics. Specifically, sex was identified under a stereomicroscope by examining the posterior abdominal segments. Males possess a more pointed abdomen with a narrow pygidium and lack females’ dark, thickened patches. Females, in contrast, have a broader and more rounded abdomen, and the elytra often cover the pygidium. Previous studies have widely used this morphological differentiation for accurate sex determination41.

We also set up one control group without treatment. After 24 h, 100 males and females were randomly selected from each sublethal concentration treatment. Each couple (male and female) was transferred into a new Petri dish (6 cm diameter, 1 cm depth) and used for life table studies42. The oviposition container had new cowpea seeds (10 seeds in each Petri dish) as an oviposition substrate for weevils to lay eggs. After 6 h of adult oviposition, all laid eggs on the surface of cowpea seeds were removed except one egg. Every grain bearing one egg (100 replicates for each treatment) was transferred into a new container (a Petri dish). The Petri dishes were placed in a growth chamber set at the experimental conditions mentioned above and checked daily for the development of insects. The developmental time of immature stages (egg, larvae, and pupa) and their viability were measured. Besides, the adult emergence was checked daily until the death of the last individual. In another experiment, upon adult weevil emergence of the new generation (F2 generation), male and female individuals cohabitated within Petri dishes (6 cm diameter, 1 cm depth), and each Petri dish contained five cowpea grains as oviposition substrates. After 24 h, the number of eggs was counted, and 300 eggs, including 100 eggs from each treatment and the control group, were put in a growth chamber. Their development, TPOP (Total pre-ovipositional period, oviposition period, fecundity, and mortality were recorded until the death of the last individual.

Life table parameters of C. maculatus

The population growth parameters of C. maculatus under the effects of peppermint essential oil were estimated using standard life table formulas. The net reproduction rate (R₀) was calculated as the sum of the products of age-specific survival (lx) and age-specific fecundity (mx), i.e., R₀ = Σlxmx. The cohort generation time (Tk) was computed as Tk = Σx·lxmx / Σlxmx, where x represents the age in days43,44. The intrinsic rate of increase (rm) was estimated iteratively using the Euler–Lotka equation: Σe^(–rm·x)·lxmx = 143,44. The finite rate of increase (λ) was derived as λ = e^rm, while the mean generation time (T) was calculated using T = ln(R₀)/rm43,44. The innate capacity for increase (rc) was also estimated as rc = ln(R₀)/Tk43,44. The gross reproduction rate (GRR), representing the total number of offspring a female would produce over her lifetime regardless of survival, was calculated as GRR = Σmₓ43,44.

Enzyme assays

The activity of three key antioxidant enzymes, including glutathione S-transferase (GST), catalase (CAT), and superoxide dismutase (SOD) was evaluated in C. maculatus populations following exposure to peppermint essential oil. Enzyme activity was measured in adults that survived 24 h of exposure to the oil’s sublethal concentrations (LC₁₀ and LC₃₀). Samples were taken from two generations: (i) first-generation (F₁) survivors that were less than 24 h old at the time of analysis, and (ii) newly emerged adults (< 24 h old) of the second generation (F₂), whose parents (F₀) had been exposed to the sublethal treatments45.

For each experimental group (treatment × generation × sex), three biological replicates were prepared. In each replicate, 10 individuals (either all males or all females, < 24 h old) were pooled and homogenized together in a 1:10 (w/v) ratio using Sorensen’s buffer (0.05 M, pH 7.4). The homogenate was centrifuged at 10,000 rpm for 10 min at 4 °C. The resulting supernatants were transferred into new tubes and stored at − 20 °C until enzymatic assays were performed.

Determination of glutathione S transferase (GST)

Glutathione S transferase (GST) activity was assayed by monitoring the increase in the concentration of the conjugation product of GSH and 1-chloro-2, 4-dinitrobenzene (CDNB) according to the alteration in the absorbance of 340 nm light by CDNB (GST substrate) over time46. One unit (U) of GST activity was defined as the amount of enzyme that catalyzes the conjugation of 1 micromole (µmol) of reduced glutathione (GSH) with 1-chloro-2,4-dinitrobenzene (CDNB) per minute at 25 °C and pH 7.0, as monitored by the increase in absorbance at 340 nm (ε = 9.6 mM⁻¹ cm⁻¹). Due to sexual dimorphism in C. maculatus, different homogenate volumes were used for males (20 µL) and females (10 µL) to equalize the total protein content introduced into the GST reaction mixture. Specifically, 10 µL of female homogenate and 20 µL of male homogenate were added to the reaction mixture containing 50 mM phosphate buffer (pH 7.0), 0.05 M Tris-HCl buffer, 15 mM CDNB (1-chloro-2,4-dinitrobenzene), and 10 mM GSH (reduced glutathione), as per previously established methods.

Determination of catalase (CAT) activity

CAT activity was evaluated47. One unit (U) of CAT activity was defined as the amount of enzyme that decomposes 1 micromole (µmol) of hydrogen peroxide (H₂O₂) per minute at 25 °C and pH 7.0, based on the decrease in absorbance at 240 nm (ε = 40 mM⁻¹ cm⁻¹). Tissue homogenates were prepared in 50 mM phosphate buffer (pH 7.0). The assay principle relies on monitoring the decrease in absorbance of the reaction mixture at λ = 240 nm over time. This mixture contains a sample extract and hydrogen peroxide (H2O2) at a concentration of 30 mM, serving as the CAT substrate.

Determination of superoxide dismutase (SOD) activity

SOD activity was measured48,49. One unit (U) of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the reduction of nitro blue tetrazolium (NBT) by superoxide radicals under the assay conditions at 25 °C and pH 7.8, measured at 560 nm. The reaction mixture comprised 1.17 µM riboflavin, 0.13 M methionine, 0.1 µM EDTA, and 0.75 µM NBT, prepared in a total volume of 90 mL using 50 mM sodium phosphate buffer (pH 7.8). Control samples (blanks) identical in composition to the treatments, except for the enzyme source in the treatments, were incubated in the dark alongside the reaction mixtures. All samples were incubated at 25 °C for 20 min under fluorescent light and dark conditions. The change in absorbance at 560 nm was measured against the blanks using a spectrophotometer. SOD activity was expressed in units per milligram of protein (U mg⁻¹ protein).

Protein determination

Protein concentrations were determined before enzyme assays using the Bradford method, and all results were expressed as specific activities (U/mg protein).

Data analysis

Lethal concentration (LC₅₀ and LC₃₀) values were estimated using Probit analysis in POLO-Plus 1.0, which is designed explicitly for bioassay data and provides goodness-of-fit statistics and confidence intervals. The effects of essential oil concentration, sex, and generation, as well as their interactions, on development time, reproductive period, fecundity, and antioxidant enzyme activities (GST, CAT, and SOD) were analyzed using multifactorial analysis of variance (ANOVA). Before analysis, the data were tested for normality and homogeneity of variances using the Shapiro–Wilk and Levene’s test, respectively. When ANOVA revealed significant main or interaction effects (p < 0.05), post hoc multiple comparison tests were conducted using both Tukey’s Honestly Significant Difference (HSD) test and Bonferroni correction to determine specific differences among treatment groups. All statistical analyses were performed using IBM SPSS Statistics version 27, and results were expressed as mean ± standard error (SE).

In addition, TWOSEX-MSChart (version 2022) was used to analyze life table parameters following the age-stage, two-sex life table theory based on Chi’s method38. The bootstrap technique50 was employed to estimate means and standard errors for these growth parameters. Additionally, paired bootstrap tests were conducted to identify statistical differences between treatment groups regarding development, fecundity, reproduction periods, and population growth51,52.

Results

Chemical composition of M. piperita essential oil identified by GC-MS

The chemical constituents of M. piperita essential oil were identified using gas chromatography-mass spectrometry (GC-MS), and a total of 52 compounds were detected (Table 1). The most abundant constituents included isopulegol (Cyclohexanol, 5-methyl-2-(1-methylethyl)-, (1.alpha.,2.beta.,5.alpha.)-(.+/-.) (26.613%), L-menthone (20.284%), cis-menthone (cyclohexanone, 5-methyl-2-(1-methylethyl)-, cis-) (8.528%), eucalyptol (5.855%), camphene (Bicyclo[4.1.0]heptane, 3,7,7-trimethyl-, [1 S-(1.alpha.,3.beta.,6.alpha.)]-) (5.754%), camphor (4.882%), and D-carvone (3.984%). These major components are primarily oxygenated monoterpenes and are known for their biological activities, such as insecticidal, antioxidant, and repellent effects. Other notable compounds included caryophyllene oxide (1.213%), endo-borneol (1.662%), linalool (0.415%), and thymol (0.549%), which contribute to the essential oil’s bioactivity. The identification confidence was rated as “High” for all constituents based on library match scores (NIST database), and molecular formulas and CAS numbers were provided to ensure compound verification (Table 1).

Table 1 Chemical constituents of the essential oil from M. piperita.
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Supplementary Fig. 1 shows the total ion chromatogram (TIC) of M. piperita essential oil. The chromatogram illustrates the oil’s chemical complexity, with over 50 resolved peaks across a retention time range of 4 to 40 min. The highest-intensity peaks were observed between 6 and 15 min, indicating that most volatile components are relatively low-boiling compounds, consistent with monoterpenes and oxygenated monoterpenes. Significant peaks were detected at retention times of 10.069 min, 10.606 min, 10.312 min, 12.270 min, and 13.679 min, corresponding to the most abundant constituents identified by GC-MS, including 1-menthone, iso-menthone, camphor, D-carvone, and α-pinene oxide. The chromatographic profile demonstrates high resolution and sharp peak separation, supporting the reliability of qualitative and semi-quantitative analyses. This GC-MS fingerprint provides a foundational chemical basis for interpreting the biological effects of the essential oil on C. maculatus observed in subsequent assays.

Toxicity of M. piperita essential oil against C. maculatus

The concentration–mortality response of C. maculatus to peppermint essential oil was assessed using Probit analysis for both adult males and females. The lethal concentration (LC) values, including LC₁₀, LC₃₀, LC₅₀, and LC₉₀, indicated that males were more susceptible to the essential oil than females, as evidenced by lower LC values across all mortality thresholds. For instance, the LC₅₀ for males was 1.894 ppm (95% CI: 1.769–1.968), while that for females was significantly higher at 3.349 ppm (95% CI: 3.216–3.462). The slopes of the concentration-response curves, 10.484 ± 1.081 for females and 6.778 ± 0.660 for males, reflect the rate of increase in mortality with concentration, suggesting a steeper response in females. No mortality was recorded for the control. In addition, the LC10 values were 1.211 ppm for males and 2.527 ppm for females, while the LC30 values were 1.567 ppm for males and 2.984 ppm for females (Supplementary Table 1). The chi-square (χ²) values for both sexes (female: 11.463, male: 5.497) with 18 degrees of freedom were statistically non-significant (p > 0.05), indicating a good fit of the data to the Probit model and no significant deviation between observed and expected mortalities. This suggests the reliability of the concentration–response relationship observed.

Concentration-dependent effects of essential oil on developmental parameters

The impact of adult exposure (F0) to two sublethal concentrations of M. piperita essential oil (LC10 and LC30) on the egg, larva, pupa, adult longevity, and total life span of their offspring (two generations) is presented in Table 2. For egg duration, both generation and the interaction between EO and sex were statistically significant. This suggests that the generational lineage strongly influenced egg developmental time and was differentially affected by essential oil treatments depending on sex (Table 2; Supplementary Table 2). In contrast, larval plus pupal duration was significantly influenced only by the interaction between generation and EO, while main effects of generation, EO concentration, and sex were not statistically significant. This implies that responses to the EO varied across generations but not consistently across sexes or EO concentrations alone.

Table 2 Developmental traits of C. maculatus across two generations following exposure to M. piperita essential oil.
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Regarding adult longevity, generation, sex, and generation × sex interaction had significant effects. Notably, females exhibited higher longevity than males across all treatments, and this sex-dependent pattern varied between generations (Table 2; Supplementary Table 2).

For total life span, significant main effects were observed for generation and sex, along with a significant generation × sex interaction. These findings reveal a sex- and generation-dependent pattern in lifespan, although EO concentration itself did not significantly affect total life span (Table 2; Supplementary Table 2).

Essential oil-induced changes in reproductive performance

A multifactorial ANOVA was conducted to evaluate the effects of M. piperita essential oil, generation, and their interaction on three key reproductive traits of C. maculatus females: total pre-oviposition period (TPOP), oviposition period, and total fecundity (Table 3; Supplementary Table 3). Adult females initiated oviposition immediately upon emerging. For TPOP, the analysis revealed that the main effect of generation was highly significant, indicating that reproductive timing varied strongly between the two generations. The interaction effect between generation and EO concentration was also significant, suggesting that the impact of EO on TPOP depended on the generational context. However, the main effect of EO concentration alone was not statistically significant, indicating no universal concentration-dependent effect.

Table 3 Reproductive traits and fecundity of C. maculatus females across two generations following exposure to M. piperita essential oil.
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A significant interaction was found between generation and EO for the oviposition period, though neither factor alone was significant. This suggests that changes in oviposition timing were driven by the combined influence of EO exposure and generational lineage rather than by either factor in isolation. In contrast, fecundity (total egg output) was influenced both by EO concentration and the interaction between generation and EO. Although generation alone did not significantly influence fecundity, the combination of generational effects and EO exposure strongly influenced female reproductive output (Table 3; Supplementary Table 3).

These findings suggest that M. piperita essential oil alters reproductive performance in C. maculatus in a concentration- and generation-dependent manner, particularly affecting fecundity and reproductive timing when both factors interact.

Essential oil-induced changes in population parameters

The results of the population parameters in C. maculatus revealed that exposure to sublethal concentrations of M. piperita essential oil had significant effects, particularly at the LC₃₀ level. In the F₁ generation, GRR and R₀ were both significantly lower (p < 0.05) in the LC₃₀ treatment compared to both the LC₁₀ and control groups, indicating reduced reproductive capacity. The intrinsic rate of increase (r) and the finite rate of increase (λ) also showed significant reductions in the LC₃₀ group (p < 0.05) compared to the control. At the same time, the mean generation time (T) remained statistically unchanged among treatments (Table 4).

Table 4 The effects of different treatments of M. piperita essential oil on the population parameters (Mean ± SE) of C. maculatus.
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In the F₂ generation, the GRR and R₀ values were significantly higher in the LC₁₀ treatment than in the control (p < 0.05), suggesting a potential stimulatory or hormetic effect at low concentrations. The LC₃₀ treatment also produced a GRR value statistically different from the control and LC₁₀. Additionally, the intrinsic rate of increase (r) and λ were significantly increased (p < 0.05) in both LC₁₀ and LC₃₀ treatments compared to the control. At the same time, the LC₃₀ group exhibited the shortest mean generation time (27.95 ± 0.11 days), which was significantly different from all other treatments (p < 0.05). These findings highlight that essential oil can significantly influence population growth parameters in a concentration- and generation-dependent manner, with more pronounced effects emerging in the second generation (Table 4).

Modulation of GST activity following essential oil exposure

The effect of M. piperita essential oil on Glutathione S-Transferase (GST) activity in male and female C. maculatus adults across two generations is presented in Fig. 1. Glutathione S-transferase activity significantly varied among essential oil concentrations, sexes, and generations. GST activity increased concentration-dependently in both generations, with the highest activity observed in LC₃₀-treated males. In Generation I, GST activity in LC₃₀-treated males increased by 61.5% compared to the control group (7.780 ± 0.078 U/mg protein vs. 4.880 ± 0.122 U/mg protein U/mg protein), while the increase in LC₁₀ males was approximately 30.1%. In females, the GST activity increase was also notable but less pronounced—33.2% in LC₃₀ and 14.0% in LC₁₀ compared to controls (Fig. 1; Supplementary Table 4).

Fig. 1
figure 1

Effect of two concentrations (LC10 and LC30) of M. piperita essential oil on the activity of the Glutathione S-transferase (GST) enzyme. Values are expressed as mean ± standard error (SE). Different lowercase letters (a, b, c) indicate significant differences among treatments (Control, LC₁₀, LC₃₀) within the same sex. Different uppercase letters (A, B) indicate significant differences between sexes (male vs. female) within each treatment. Statistical analysis was performed using Tukey’s HSD test at the 5% significance level (p < 0.05).

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This pattern was also observed in Generation II. GST activity in LC₃₀ males increased by 56.5% and in LC₁₀ males by 42.8% relative to the control group. Females again showed increases: 61.1% (LC₃₀) and 38.2% (LC₁₀) relative to the control (Fig. 1; Supplementary Table 4). A multifactorial ANOVA (Supplementary Table 4) revealed that the main effects of generation, essential oil concentration, and sex were all statistically significant (p < 0.001), as were their two-way and three-way interactions, including generation × EO, generation × sex, EO × sex, and generation × EO × sex (all p < 0.05). These findings suggest that M. piperita essential oil induces GST activity in a concentration-dependent manner, with males exhibiting consistently higher enzymatic response than females.

Alterations in catalase (CAT) activity across generations

The impact of M. piperita essential oil on catalase (CAT) activity in male and female C. maculatus across two generations is illustrated in Fig. 2. In Generation I, CAT activity did not show a statistically significant difference among essential oil treatments, ranging from approximately 3.31 ± 0.06 to 3.56 ± 0.08 U/mg protein. However, a notable concentration-dependent increase in CAT activity was observed in Generation II. Specifically, CAT activity significantly increased in both sexes at LC₁₀ and LC₃₀ treatments compared to the control, with females exhibiting slightly higher activity than males at each concentration level (Fig. 2).

Fig. 2
figure 2

Effect of two concentrations (LC10 and LC30) of M. piperita essential oil on the activity of the catalase (CAT) enzyme. Values are expressed as mean ± standard error (SE). Different lowercase letters (a, b, c) indicate significant differences among treatments (Control, LC₁₀, LC₃₀) within the same sex. Different uppercase letters (A, B) indicate significant differences between sexes (male vs. female) within each treatment. Statistical analysis was performed using Tukey’s HSD test at the 5% significance level (p < 0.05).

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The multifactorial ANOVA results (Supplementary Table 5) confirmed that the main effects of generation, essential oil concentration (EO), and sex were all highly significant (p < 0.001), along with all interaction terms: generation × EO, generation × sex, EO × sex, and generation × EO × sex. The large partial eta-squared values (η² > 0.6 for most terms) suggest strong effect sizes for both main and interaction effects. The observed pattern implies that M. piperita essential oil upregulates CAT activity in a concentration-dependent manner, only in the second generation, potentially indicating a transgenerational antioxidant response.

Generation-specific effects on superoxide dismutase (SOD) activity

The activity of superoxide dismutase (SOD) was significantly affected by essential oil concentration (EO), sex, and generation (p < 0.001 for all main effects). A strong interaction was observed between EO × sex, indicating differential antioxidant responses between males and females under essential oil exposure. The three-way interaction (generation × EO × sex) was also significant, reflecting complex modulation of SOD activity across generations, concentrations, and sexes (Fig. 3; Supplementary Table 6).

Fig. 3
figure 3

Effect of two concentrations (LC10 and LC30) of M. piperita essential oil on the activity of the Superoxide dismutase (SOD) enzyme. Values are expressed as mean ± standard error (SE). Different lowercase letters (a, b, c) indicate significant differences among treatments (Control, LC₁₀, LC₃₀) within the same sex. Different uppercase letters (A, B) indicate significant differences between sexes (male vs. female) within each treatment. Statistical analysis was performed using Tukey’s HSD test at the 5% significance level (p < 0.05).

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As shown in Fig. 3, in both generations, SOD activity decreased significantly with increasing concentrations of the essential oil (LC10 and LC30) in both sexes. In Generation I, the highest activity was recorded in control males (13.27 ± 0.06 U/mg protein) and females (10.26 ± 0.11 U/mg protein), while the lowest activity was observed at LC30 (3.26 ± 0.03 U/mg protein in males; 1.66 ± 0.09 U/mg protein in females). A similar trend was noted in Generation II, with more SOD activity compared to Generation I (Fig. 3; Supplementary Table 6). These findings underscore the concentration-dependent oxidative stress imposed by the essential oil and reveal a generational compounding effect, with females generally exhibiting lower enzymatic activity than males (Fig. 3; Supplementary Table 6).

Discussion

Plant essential oils (EOs) have been gaining interest because these natural insecticides effectively control many insect pests. These oils (Eos) are complex mixtures of biologically active compounds consisting mainly of monoterpenes, sesquiterpenes, and phenylpropanoids that have already proven effective insecticides53. They are active against arthropod pests, are not toxic to mammals, and are more environmentally friendly than conventional chemical insecticides54,55. Since EOs are especially effective against stored-product insect pests, including eggs, larvae, and adults54some formulations based on essential oils are already available in the market for insect pest control in indoor conditions56.

This study, found that the chemical profile of M. piperita essential oils mainly are isopulegol (26.613%), L-menthone (20.284%), cis-menthone (8.528%), eucalyptol (5.855%), camphene (5.754%), camphor (4.882%), and D-carvone (3.984%), whilst another study reported different major component for essential oil of M. piperita, including menthone (30.63%), menthol (25.16%), menthofuran (6.47%), β-phellandrene (5.59%), isomenthone (4.74%), menthol acetate (4.61%), and pulegone (4.39%)57. Also, other studies reported different major components for M. piperita58,59. These fluctuations might be ascribed to a range of factors, such as climatic conditions, genetic characteristics, geographical location, timing of collection, methods of extraction, plant’s stage of development, age, and interactions with environmental factors59,60.

Research suggests a correlation between elevated monoterpenoid content and insecticidal efficacy. Peppermint oil, rich in monoterpenoids, demonstrates effectiveness as both an adulticide and a larvicide against C. maculatus61. Studies indicate that peppermint oil achieves a mortality rate of up to 90% in adults when applied at a concentration of 1.0 ml after 48 h, showcasing its potent insecticidal properties61.

The present study also demonstrated that C. maculatus is susceptible to peppermint oil. Similar phenomena have been reported for other insects as well. For example, studies indicate that peppermint oil can achieve up to 100% mortality in pests such as the red flour beetle (T. castaneum) and the lesser grain borer (R. dominica) at effective concentrations, with notable results showing 94% adult mortality and 82% larval mortality after treatment with 12% peppermint oil62,63.

Our findings revealed that peppermint essential oil exhibited sex-specific toxicity, with male C. maculatus adults being more susceptible than females. This pattern is consistent with earlier studies, which report that females in many insect species, including bruchids, often show higher tolerance to botanical and synthetic insecticides. Such differences are generally attributed to greater body mass, thicker cuticles, and enhanced detoxification enzyme activity in females, which are often linked to reproductive physiology and longevity29,64,65. Additionally, females demonstrate elevated activities of detoxification enzymes, such as general esterase, which are crucial for metabolizing harmful compounds65. This increased enzymatic activity is often linked to reproductive physiology, as females allocate resources to reproduction and detoxification processes, potentially leading to a trade-off between fecundity and longevity66.

Moreover, we observed that sublethal exposure of the F₀ generation significantly influenced key life table parameters and antioxidant enzyme activities in F₁ and F₂ generations, suggesting a transgenerational stress effect. Similar transgenerational impacts of essential oil and insecticide exposure have been reported in C. maculatus, C. chinensis, Spodoptera exigua, Spodoptera frugiperda, and aphids, where sublethal treatments affected survival, development, egg number, hatchability, and enzyme activity in subsequent generations67,68,69,70,71. Similarly, sublethal concentrations of Artemisia annua and Rosmarinus officinalis essential oils decreased fecundity and longevity in Tetranychus urticae, affecting the intrinsic rate of increase in the population72. These results support the growing evidence that epigenetic inheritance or stress-induced physiological programming may mediate the long-term effects of botanical insecticides.

The present study also demonstrated the effect of peppermint EO on the reduction of C. maculatus fecundity, which is in congruence with other studies that report lemongrass, rosemary, Vanillosmopsis arborea, Eucalyptus camaldulensis, and Heracleum persicum essential oils caused egg number reduction in C. maculatus11,73,74. The fecundity of Trogoderma granarium females was reduced after larval exposure to the LC30 concentration of Piper nigrum and Artemisia khorassanica essential oils75. This reduction in fecundity can be viewed as an adaptive response, as it may enhance the species’ survival by promoting reproductive strategies that mitigate the harmful effects of environmental stressors, such as essential oil exposure74. The implications for pest control are substantial; by integrating peppermint EO into management strategies, it can effectively disrupt the reproductive cycle of C. maculatus, thereby reducing population growth and damage to stored crops76,77. This approach offers a natural alternative to synthetic insecticides and aligns with sustainable agricultural practices, potentially leading to long-term pest management solutions.

The age-stage, two-sex life table offers distinct advantages for insect population ecology research, enabling the prediction of population growth course over both short- and long-term perspectives78. Based on our results, the r, GRR, R0, and λ were significantly reduced in LC30, especially in the first generation, compared to control groups. A reduction in these parameters could decrease the speed of C. maculatus population growth79. Also, LC30 concentrations of M. piperita essential oil reduced the life table parameters of T. urticae80. Correspondingly, it has been reported75 that fumigation with P. nigrum and A. khorassanica essential oils significantly reduced the life table parameters (R₀, rm, λ, and T) of T. granarium. This detrimental effect is primarily attributed to extended immature development times, decreased insect survival rates, and diminished fecundity.

Interestingly, in the F₂ generation, the gross reproduction rate (GRR) was significantly higher in the LC₁₀ treatment compared to the control group. This may reflect a hormetic effect, wherein low-concentration exposure to a stressor can stimulate physiological responses that enhance reproductive performance81. In insects, low concentrations of specific essential oils have been reported to upregulate stress-related signaling pathways or alter endocrine responses that may temporarily favor fecundity82. However, this stimulatory effect was not observed at the higher concentration (LC₃₀), suggesting a concentration-dependent trade-off between adaptive response and toxicity. Further investigation into the molecular mechanisms underlying such responses is warranted.

The effect of EO on the antioxidant enzyme was shown: all the enzymes measured were affected by the toxicant’s sublethal effect, reflecting an organism’s biochemical/metabolic disturbances in subsequent generations83,84.

In our experiments, GST activity increased significantly in both male and female C. maculatus following exposure to sublethal concentrations of M. piperita essential oil, with the most pronounced elevation observed at LC₃₀. This enhanced GST response likely reflects a detoxification mechanism, as glutathione S-transferases play a key role in conjugating reactive oxygen species (ROS) and xenobiotic compounds85. The greater upregulation of GST at LC₃₀ suggests that higher EO concentrations induce stronger oxidative or chemical stress, prompting the insect’s biochemical defense system to respond more robustly. This pattern aligns with findings in other insect species where elevated GST activity is associated with enhanced resistance or adaptation to environmental stressors86,87. Therefore, the GST increase indicates metabolic stress and may reflect an adaptive physiological response to mitigate EO-induced cellular damage, particularly in a concentration-dependent manner. The elevated GST activity at LC₃₀ in both generations also implies that the oxidative challenge imposed by essential oils can persist or even intensify in progeny, underscoring the transgenerational biochemical impact of botanical insecticides. Some studies88,89 have shown that when T. castaneum was subjected to Callistemon citrinus and Artemisia dracunculus essential oils, its GST activity was affected by the plants’ EOs.

Insects primarily rely on ascorbate peroxidase and SOD-CAT systems to mitigate oxygen toxicity90,91. The activity of these systems can be influenced (increased or decreased) depending on the severity, duration, and nature of the stress encountered92. In our study, the parental essential oil exposure resulted in a significant increase in CAT activity in generation II and reduction in SOD activity in both generations of C. maculatus. The sex-specific differences observed in CAT activity in generation II, point to possible underlying physiological or metabolic differences in oxidative stress responses between males and females. Another study on C. maculatus93 investigated the acute effects of R. officinalis EO, observing significant increases in CAT activity following short-term exposure. Similarly, elevated CAT activity in Ephestia kuehniella larvae was observed after dietary exposure to EO constituents like α-pinene and thymol94.

When the effect of Carum carvi essential oil were tested on two stored product pests, Tenebrio molitor and Tribolium confusum they showed a contradictory effect on the insect i.e. a reduction of SOD in T. confusum and an increase of SOD in T. molitor95. Also, the essential oil of Lippia turbinata caused an increase in CAT and SOD activity of the stored product pest Rhipibruchus picturatus96. Another study97 has reported elevation of Drosophila melanogaster CAT and GST activities and a null effect on SOD activity following treatment by Psidium guajava EO.

While several studies have documented the alteration of GST, CAT, and SOD activity in insects directly exposed to essential oils88,89,94,95,98few have addressed whether these effects extend to subsequent generations. However, these studies primarily address acute responses and do not delve into the potential heritable or transgenerational effects of EO-induced oxidative stress. This gap in the literature underscores the novelty of our findings, which suggest that sublethal EO exposure can lead to persistent oxidative stress responses, as evidenced by altered CAT and SOD activity, across multiple generations.

These results suggest that sublethal exposure to M. piperita essential oil does not merely cause short-term physiological stress but can also trigger inherited biochemical changes, such as altered antioxidant enzyme activity. Since these effects were observed across generations and were accompanied by changes in reproductive and developmental traits, they may have implications for long-term population fitness. Over time, such stress responses could influence ecological dynamics (e.g., pest persistence, interaction with other species) and potentially shape adaptive responses at the evolutionary level99,100. Numerous studies have demonstrated that more resistant species exhibit higher SOD and CAT activity, as well as a more significant induction of activity and/or a higher CAT-to-SOD ratio95,101. Essential oil-based pesticides offer a potential advantage in pest management due to their complex composition, which slows the development of resistance in insect populations and environmental contamination102.

In conclusion, this study demonstrates that M. piperita essential oil caused C. maculatus mortality, and its sublethal concentrations affected life history traits and antioxidant enzyme activity of subsequent generations. Notably, GST, CAT, and SOD enzyme activities were modulated in a concentration- and generation-dependent manner, indicating that oxidative stress is a likely mechanism of deleterious effect observed in the transgenerational study. The reproductive capacity of the beetles was also significantly reduced following sublethal EO exposure. These findings provide novel insight into the transgenerational stress effects of plant-derived compounds and support the potential of M. piperita essential oil as a botanical insecticide in sustainable pest management strategies. Future studies may further explore formulation optimization and long-term ecological impacts of such biopesticides.