Comparison of the chemical composition and toxicity of lavender and spearmint plants’ essential oils on cowpea weevil, Callosobruchus maculatus (F.) (Col.: Chrysomelidae)

Introduction

The cowpea weevil, Callosobruchus maculatus (F.) (Col: Chrysomelidae), is a well-known global pest of leguminous crops. The insect host range includes cowpeas, peas, chickpeas, mung beans, lentils, soybeans, and other pulses (Kébé et al., 2017; Tuda et al., 2014). These legume crops provide approximately 20-25% protein in food, about 50-60% carbohydrates, and 1-2% fat, thereby serving as a staple source for about a billion people worldwide (Beck, 2014; França et al., 2021). Therefore, high tropical protein contents in grain seeds and their rich carbohydrate content make them highly susceptible to consumption by pests, especially the cowpea weevil.

In cowpea seed infestation, female weevils lay eggs on the seed surface. Following hatching, the larvae enter the seed to feed on the endosperm until they complete their larval and pupal stages, emerging as adults (Amiri & Bandani, 2023). The feeding behavior not only decreases seed weight but also creates holes in the surface that serve as entry points for opportunistic microorganisms, especially fungi (Farrel et al., 2002; Rajendran, 2020). The damage caused by the larvae results in both qualitative and quantitative losses, and in severe situations, entire crop yields can be destroyed due to the weevil's rapid development, high reproductive capacity, short life cycle, and adaptability to varying climates (Lima et al., 2004; Kpoviessi et al., 2019; Masoumi et al., 2021).

Control of C. maculatus has historically been achieved through treatment with chemical insecticides, such as aluminum phosphide (Phostoxin), which is a viable economic option. However, the overuse of such chemicals has led to problems, including the development of insecticide resistance, environmental contamination, and adverse health effects among consumers (Talukder, 2009). Therefore, immediate and alternative eco-friendly methods of pest management should be developed that minimize their impacts on the environment and human health. Bio-insecticides derived from natural sources are promising and provide a safer, sustainable alternative for pest control (Aimad et al., 2021).

Plant essential oils (EOs) have been gaining interest because these natural insecticides are very effective in controlling many insect pests in nature. These oils are complex mixtures of bioactive compounds, primarily consisting of monoterpenes, sesquiterpenes, and phenylpropanoids, which have already proven effective as insecticides (Gupta et al., 2025). They account for a diverse spectrum of arthropod pests, are less toxic to mammals, and are more environmentally friendly than conventional chemical insecticides (Campolo et al., 2018; Zimmermann et al., 2022). There are various methods for applying essential oils, including fumigation, topical application, and residual assays; all three have demonstrated effectiveness across the life stages of stored-product pests, such as eggs, larvae, and adults (Campolo et al., 2018). Some formulations based on essential oils are already available in the market for insect pest control (Dwivedy et al., 2016).

Among these important sources of essential oils, we find some members of the Lamiaceae family, such as lavender (Lavandula angustifolia Miller) and spearmint (Mentha spicata L.), well-known for their antioxidant, biological, and even pharmaceutical properties (Chauhan et al., 2008; de Sousa Barros et al., 2015; Smigielski et al., 2009; Smigielski et al., 2015). Essential oils from these plants have shown insecticidal activity against various insect species. Additionally, the chemical composition of these oils is influenced by factors such as climate, genotype, and soil conditions (Smigielski et al., 2009; Betlej et al., 2024). Lavender oil has been shown to have both contact and ingestion toxicity against pea leaf weevils, Sitona lineatus (Fadil et al., 2023), granary weevils, Sitophilus granarius (Germinara et al., 2017), and stored grain borers, Rhyzopertha dominica (Tine et al., 2019). Similarly, spearmint oil has exhibited insecticidal activity against pests such as the diamondback moth (Plutella xylostella) (Yi et al., 2016) and Spodoptera littoralis (Ferrati et al., 2023).

This study aimed to investigate the chemical composition of L. angustifolia essential oils in comparison to M. spicata using gas chromatography-mass spectrometry (GC-MS) analysis. Additionally, we aimed to explore (1) the insecticidal activity of these essential oils against male and female C. maculatus and (2) the sublethal effects of these oils on biological parameters such as egg-laying rate, egg hatchability, larval and pupal development durations, adult emergence, and lifespan of the individual male and female weevils. While the insecticidal potential of essential oils has been studied to some extent so far, this study presents several novel contributions and implications. First, it presents a comparative analysis of two pharmacologically important essential oils — lavender (L. angustifolia) and spearmint (M. spicata) — whose detailed chemical profiles and toxic effects on C. maculatus have not been studied simultaneously before. Second, the study extends beyond acute toxicity by examining sublethal (LC₃₀) impacts on insect biological traits, including oviposition, larval development time, hatching success, and adult longevity- traits that are crucial for understanding population dynamics. Third, the research highlights sex-based sensitivity differences, a topic seldom explored in botanical pesticide studies, offering insights into potential physiological or hormonal mechanisms behind differential EO susceptibility. These features demonstrate the innovative integration of chemistry, toxicology, and reproductive biology to enhance sustainable pest control strategies.

Materials and methods

Insect rearing and maintenance

The cowpea weevil, C. maculatus, was obtained from a population maintained at the Physiology Laboratory of the Plant Protection Department in the Faculty of Agriculture and Natural Resources of Tehran University. The insects were reared on cowpea seeds (Vigna unguiculata) in a germinator set at 30 ± 1°C with a relative humidity of 60 ± 5% and complete darkness. Only newly emerged adults (<24 hours old) were used for the experiments (Amiri & Bandani, 2023).

Essential oil (EO) extraction and chemical composition assessment

Spearmint leaves (M. spicata) and lavender flowers (L. angustifolia) were collected from Alborz Province, Karaj, Iran (35°48'25.3"N, 50°59'38.3"E, elevation: 1340 m) to prepare the essential oils (EOs). The material was allowed to dry under shade conditions for 3 days (Mohtashami et al., 2012), after which it was ground by an electric mill (Moulinex, LM2 model) with a sample mass of 100 grams during each extraction. The essential oils were extracted via water distillation using a Clevenger apparatus (Glass Making Unit, Iran Scientific and Industrial Research Organization). The extraction process lasted 3 hours. The oils obtained were dehydrated with sodium sulfate, stored in glass containers covered with aluminum foil, and refrigerated at 4°C until further analysis (Golestani Kelat et al., 2011).

The chemical analysis of essential oils was performed using a gas chromatography-mass spectrometry (GC-MS) system based on the TRACE GC and TRACE MS from Agilent Technologies, USA. A 30-m-long HP-5 column was used (0.25 mm outer diameter and 0.25 µm inner diameter). The temperature program was conducted in such a way that it rose from 60°C to an ending temperature of 250°C at a rate of 5°C/min. The injection temperature was 260°C with helium as the carrier gas. The volatile components were determined using Xcalibur v2 software (Wiley and NIST libraries). The relative percentages of each component were determined based on GC peak areas.

Bioassays of essential oils

Bioassays were conducted according to the methods described by Amiri and Bandani (2023). Preliminary tests were performed to determine concentrations that would lead to mortality of 20-80% among adults. Then, five concentrations were selected at logarithmically spaced intervals and tested against both males and females. Regarding the determination of concentrations of spearmint oil on the male insect, the following concentrations were utilized: 0.0 (control), 4.6, 8.17, 16.05, 32.95, and 65 µL/L air, and for the female adults: 0.0 (control), 10.36, 20.33, 40.69, 80.7, and 126.47 µL/L air. For lavender oil, concentrations for the male adults were: 0.0 (control), 2.3, 4.6, 8.5, 16.05, and 32.65 µL/L air, and for the female adults: 0.0 (control), 5.75, 10.17, 21.5, 46.23, and 83 µL/L air. Therefore, five concentrations with a control were utilized for each essential oil, and each treatment was replicated five times, and in each replicate, 10 insects were used. Thus, in total, we used 300 adults in each assay (N = 300).

Acetone was the solvent diluent for the essential oils; therefore, the control adults were treated with only acetone. The fumigation chambers were all made from 250 mL glass bottles for each treatment. Ten adults (either male or female) were placed in each bottle. A measured volume of the essential oil was applied to a Whatman No. 1 filter paper (1.5 cm diameter) using a micropipette. As described by Wang et al. (2006), the filter paper was air-dried for 5 minutes to remove the potential lethality of acetone. The filter paper was suspended in the center of the bottle cap with a thread, and the bottle was sealed with parafilm to prevent volatile loss. The number of dead and living insects was recorded after 24 hours. An insect was counted as dead if there was no apparent reaction to touch stimulation using a fine brush on its antennae or legs.

Sublethal effects of EOs

Sublethal effects were assessed by exposing male and female adults separately to the sublethal concentration (30%) of either lavender or spearmint essential oils for 24 hours. Sublethal concentrations were defined as the concentration causing 30% mortality (LC₃₀). They were selected because LC₃₀ provides a practical balance between producing measurable biological effects and maintaining a sufficient number of survivors for subsequent life-history measurements (fecundity, hatchability, development time, longevity). Use of LC₃₀ (and nearby low-percentile LC values such as LC₁₀–LC₃₀) is well established in insect toxicology and studies of essential-oil sublethal effects, allowing the detection of sublethal physiological and behavioral responses without the strong demographic truncation that would result from higher lethal concentrations (Izakmehri et al., 2013).

Data analysis

The normality of the data was confirmed using the Shapiro-Wilk and Kolmogorov-Smirnov tests. LC values (LC₃₀, LC₅₀, and LC₉₀) were computed using Poloplus 2 software (Finney, 1971). Essential oils for toxicity and relative sensitivity between male and female adults were compared using the method of Robertson et al. (2007). The sublethal effects on biological parameters were analyzed using SPSS v26 software. The means were examined using Tukey's test at 5%. Data plots were created using Microsoft Excel (Office 2016).

Results

Chemical composition of essential oils

Gas chromatography-mass spectrometry (GC-MS) was used to identify the components of lavender and spearmint essential oils. Lavender essential oil consisted of 29 components, whereas spearmint essential oil consisted of 30 components (Table 1). The major constituents of lavender essential oil were linalool (38.01%), linalyl acetate (21.27%), eucalyptol (9.6%), camphor (8.07%), lavandulol (5.11%), trans-caryophyllene (2.12%), and caryophyllene oxide (1.6%). The predominant constituents of spearmint essential oil included carvone (76.42%), menthol (3.29%), eucalyptol (3.27%), D-limonene (3.12%), cis-dihydrocarveol (2.47%), borneol (2.09%), and pulegone (1.04%).

Toxic effects of essential oils 

Table 2 summarizes the mortality rates of cowpea weevils exposed to different concentrations of lavender and spearmint essential oils. Both types of essential oils exhibited concentration-dependent toxicity, leading to increased mortality of the adults at higher concentrations of the essential oils. In the control, there was no mortality. The lowest concentration (2.3 µL /L air) of lavender essential oil resulted in 10.2% male weevil mortality, and the highest concentration (14.95 µL /L air) caused 77.5% mortality in males. The female weevil mortality rate ranged from 15% at the lowest concentration (5.75 µL /L air) to 87.5% at the highest concentration (23 µL /L air). On the other hand, spearmint essential oil at similar concentrations caused significantly lower mortality compared to lavender oil.

Lethal concentrations of both essential oils are summarized in Table 2. Lavender essential oil was more toxic than spearmint essential oil. For L. angustifolia, the LC₅₀ for males was 5.27 µL/L (95% CI: 4.65–5.98) and for females was 10.12 µL/L (95% CI: 9.10–11.16). These intervals do not overlap, indicating a statistically significant difference in susceptibility between sexes. For M. spicata, the LC₅₀ for males was 9.45 µL/L (95% CI: 8.62–10.48) and for females was 17.36 µL/L (95% CI: 16.24–18.62). Again, the non-overlapping confidence intervals indicate a significant difference between males and females.

Male weevils were generally more susceptible to both oils than females, as shown in Table 3. The LC₅₀ ratio was highest for lavender, with a value of 1.92, compared to 1.84 for spearmint (Table 3). The LC₅₀ ratio of lavender to spearmint was 1.79 for females and 1.72 for males. For LC₉₀ ratios, the 95% confidence intervals (Cis) for male: female comparisons overlapped for both lavender (1.08–2.26) and spearmint (1.09–2.04), indicating that these differences were not statistically significant at the 5% level. In contrast, LC₅₀ ratios showed non-overlapping CIs between sexes, consistent with significant differences in susceptibility at the median lethal concentration (Table 3). Statistical analysis showed no significant difference in the toxicity between lavender and spearmint essential oils. The hypotheses of equality and parallelism of the regression lines were not rejected (Tables 2 and 3).

Effects of essential oils on biological parameters of the cowpea weevil

The summaries of the effects of sublethal concentrations (LC₃₀) of lavender and spearmint essential oils on the biological parameters of the cowpea weevil, such as egg production, egg hatchability, development time, eclosion rate, and adult longevity, are given in Table 4. Egg production under both oils was significantly less than the number of eggs laid by the control insects. However, there was no significant difference in the number of eggs laid between the two essential oils. The total eggs laid by weevils exposed to lavender were 62.09 ± 1.40 eggs, while those exposed to spearmint essential oil laid 64.78 ± 1.23 eggs. Control insects laid 80.55 ± 1.33 eggs (P ≤ 0.05).

Both essential oils also significantly reduced the rate of egg hatchability. Hatchability for eggs deposited by females exposed to lavender essential oil was 52.94 ± 1.35%, and eggs deposited by females exposed to spearmint essential oil had a hatchability of 58.32 ± 1.45%. The control group had a hatchability rate of 73.88 ± 1.24% (P ≤ 0.05). Immature development time was significantly prolonged in both essential oil treatments compared to the control group. The developmental time in the lavender treatment was 25.96 ± 0.15 days, and in the spearmint treatment, it was 23.84 ± 0.19 days. In the control group, the immature development period was 20.99 ± 0.19 days (P ≤ 0.05). Both essential oils, lavender and spearmint, contributed to diminishing the percentage of insect emergence. Eclosion rates were as follows: lavender eclosion was 61.07±1.27%, spearmint eclosion was 62.91± 1.91%, and control eclosion was 71.75± 1.03% (P ≤ 0.05).

There was a significant decrease in the lifespans of both male and female adult weevils when exposed to both essential oils. Male weevils treated with lavender oil had a lifespan of 5.18±0.34 days, and those treated with spearmint oil had a lifespan of 6.11 ± 0.27 days. Meanwhile, female weevils had a lifespan of 7.7 ± 0.42 days under lavender treatment and 7.58 ± 0.52 days under spearmint treatment. In the control group, males lived for 9.01 ± 0.67 days, and females lived for 11.94 ± 0.37 days (P ≤ 0.05) (Table 4).

Discussion

The use of botanical-derived insecticides in agriculture spans hundreds of years in countries such as China, Egypt, Greece, and India (Germinara et al., 2017). Interestingly, the primary benefits of botanical-based insecticides include rapid biodegradability, minimal environmental pollution, no toxicity toward non-target organisms, and reduced potential for resistance formation (Isman, 2006; Ebadollahi, 2011). However, the most widely used botanical insecticides are essential oils (EOs), which are commonly used for pest control, especially indoors. EOs are primarily comprised of terpenoid compounds that exert insecticidal effects by interfering with various metabolic, biochemical, physiological, and behavioral processes in insects (Tripathi et al., 2009).

Factors such as plant genotype, climate, location, soil conditions, and morphology contribute to variability in the chemical constituents of essential oils (Germinara et al., 2017). The study revealed that the major components of lavender EO were linalool (40.23%), linalyl acetate (21.47%), eucalyptol (10.6%), camphor (8.43%), and lavandulol (5.25%). These results align with previous research, such as that by Ebadollahi et al. (2014), which reported the most abundant constituents of lavender EO to be linalool (28.63%), eucalyptol (18.65%), and 1-borneol (15.94%). Other authors, such as Tine et al. (2021) and Kozuharova et al. (2023), have isolated linalool and linalyl acetate as the key components. For example, the major components of lavender reported were linalool and linalyl acetate in amounts of 20.42% and 13.24%, respectively (Tine et al., 2021). Germinara et al. (2017) studied the oil's chemical composition and reported a variety of compounds comprising linalool (23.8%), eucalyptol (12.0%), borneol (10.7%), and camphor (2.8%). Chemical profiles of lavender EO vary between studies due to climatic and soil conditions and genetic differences (Kaya et al., 2018).

Our spearmint (M. spicata) essential oil study shows the presence of carvone (78.71%), menthol (3.58%), eucalyptol (3.47%), and limonene (2.36%). These results confirm the finding of Mogosan et al. (2017), who identified carvone (41.22%) and menthol (12.78%) as the major components in spearmint EO. Carvone (48.5%) and limonene (20.7%) were also identified as the principal components by Brahmi et al. (2016); however, the relative proportions of the different constituents may vary across different studies. According to Fitsiou et al. (2016), spearmint EO had a very high percentage of carvone (85.4%). These variations are a consequence of soil conditions, genetic varieties, and climatic factors, all of which influence the chemical composition of essential oils (Facundo et al., 2008; Ebadollahi et al., 2014).

Our experiment demonstrated that both lavender and spearmint essential oils resulted in significant mortality in cowpea weevils (C. maculatus). Insect mortality is directly proportional to the concentration of the applied oil, indicating a clear dose dependence of the EOs on insect mortality. This finding was corroborated by research published in the past regarding lavender EO, which demonstrated toxicity effects on many insect pests (Shaaya et al., 1997; Rozman et al., 2007; Abdelgaleil et al., 2009; Pugazhvendan et al., 2012; Germinara et al., 2017). Similarly, spearmint EO has also been affirmed to be very toxic against numerous pests, such as the Mediterranean moth (Ephestia kuehniella) and Indian meal moth (Plodia interpunctella) (Eliopoulos et al., 2015). Regarding spearmint efficacy, evidence has also been provided against other storage insects, such as the lesser grain borer (Rhyzopertha dominica) (Souza et al., 2016). Apart from that, spearmint oil was found to have fumigation toxicity against mosquitoes (Culex pipiens) and houseflies (Musca domestica), with LC50 values of around 43 and 65 microliters per liter of air, respectively (Mohafrash et al., 2020).

The study found that the lethal concentration (LC₅₀) values for spearmint EO were 9.45 microliters per liter for males and 17.36 microliters per liter for females, while the LC₅₀ values for lavender EO were 5.27 microliters per liter of air for males and 10.12 microliters per liter for females. This indicates that male insects are more sensitive to both essential oils than female insects, with males being nearly twice as vulnerable.

The greater susceptibility of male C. maculatus to lavender and spearmint essential oils compared to females may be influenced by physiological and biochemical differences between the sexes, as has been reported in other beetle species. For example, previous studies have documented that females often have larger body size and thicker cuticles, which can reduce the penetration of fumigants and lipophilic compounds such as essential oils. Moreover, the large body size of the female insects results in a lower surface area-to-volume ratio, potentially reducing the rate of essential oil absorption through the cuticle (George et al., 2015; Balabanidou et al., 2018). Other work has shown that higher metabolic rates and respiratory activity in males can increase the uptake of volatile compounds (Guedes et al., 2003). In addition, lipid reserves in females, which support reproduction, might act as temporary sinks for lipophilic constituents of essential oils, potentially moderating acute toxicity (Leyria et al., 2024). Additionally, males often exhibit higher metabolic rates and increased respiratory activity, resulting in faster absorption of volatile compounds through their spiracles, which leads to greater toxicity. (Guedes et al., 2003). Another feature of the insect fat body is that it functions as a significant site for protein, enzyme, and peptide production (analogues to the human liver), functioning as a detoxification site (Wronska et al., 2023; Huang et al., 2022). Another critical factor is enzymatic detoxification; males may possess lower baseline levels or different isoforms of detoxifying enzymes such as cytochrome P450 monooxygenases, esterases, and glutathione S-transferases, which are crucial for neutralizing toxic compounds (Adesanya et al., 2018; Pavela & Benelli, 2016; Navarro-Roldán et al., 2020; Liu et al., 2021). Together, these physiological differences likely contribute to females' higher tolerance to EO exposure. These findings align with research indicating that insect susceptibility to plant-derived pesticides varies by gender (Souza et al., 2016; Lee et al., 2002). While these mechanisms were not directly examined in the present study, they are consistent with published findings in related systems and may help explain the observed sex-based differences in susceptibility.

The measured LC₅₀ values for spearmint EO are noteworthy because they resemble those for the lesser grain borer published by Souza et al. (2016), demonstrating the broader applicability of spearmint as a fumigant for insect pest management. Interestingly, there was no significant difference in the toxicity of lavender and spearmint essential oils against cowpea weevils, which coincides with the findings of Amiri and Bagheri (2020), who also did not detect any significant difference in the toxicity of spearmint and rosemary oils against the same pest. This, however, contrasts with Heydarzadeh and Moravvej (2012), who reported different toxicity levels of distinct essential oils (e.g., fennel, summer savory, and species of Teucrium) against C. maculatus. Their study found that males were more sensitive than females, in line with our findings.

The insecticidal properties of essential oils are often attributed to the activity of individual components within these oils (Isman et al., 2007). Spearmint essential oil, for instance, is very high in carvone, which has been associated with its toxic effects on several insect pests, such as the lesser grain borer (Khalfi et al., 2006). Similarly, linalool, a major component in lavender essential oil, has been associated with highly toxic effects on several insect species, including the lesser grain borer and red flour beetle (Rozman et al., 2007). Yang et al. (2021) also confirmed the insecticidal potential of M. spicata EO components such as carvone and limonene against termites, thereby adding more weight to the role of essential oils in integrated pest management.

Furthermore, the sublethal effects of lavender and spearmint essential oils on the biology of the cowpea weevil demonstrated that sublethal concentration treatments reduced the lifespan of adults, the number of eggs laid per female, and the hatching rate, and increased the duration of the larval and pupal stages before adulthood. These results further confirm those of Izakmehri et al. (2013), which showed that the use of Eucalyptus camaldulensis and Heracleum persicum essential oils helps reduce the egg-laying and survival rates of C. maculatus. Studies on terpenes, such as limonene and eugenol, have also shown similar effects on reproductive and developmental parameters in insect pests (Barbosa et al., 2013). The sublethal effects demonstrated in this study suggest the potential application of lavender and spearmint essential oils in integrated pest management, as they affect the pest's reproductive capacity and developmental stages.

Conclusion

The results of this study provide strong evidence that essential oils from lavender (L. angustifolia) and spearmint (M. spicata) have significant insecticidal effects on the cowpea weevil (C. maculatus), with lavender essential oil demonstrating higher toxicity. Both oils also exert a sublethal impact on the pest's biological parameters, including lifespan, egg-laying rates, hatching rates, and developmental time. These findings suggest that lavender and spearmint essential oils could serve as effective alternatives to chemical pesticides in pest management strategies, offering a more environmentally friendly and safer approach to controlling C. maculatus populations. Future research should further investigate the synergistic effects of these oils in combination with other botanical insecticides and assess their long-term efficacy under field conditions.