Stored-grain pests pose a serious threat to both the quantity and quality of grains world-wide. The multivoltine cowpea weevil, Callosobruchus maculatus Fab. (Coleoptera: Bruchidae), causes significant losses in stored pulses. These losses may range from 5-10% in temperate regions to 20-30% in tropical countries (Saranya et al., 2019). The infestations not only lead to direct losses due to grain consumption but also increase temperature and moisture levels, promoting the rapid growth of molds, including toxic species. The pulse beetle is classified as a primary Insect-pest because it bores into the grain, consumes the kernel and completes its development inside. Most insects, including the pulse beetle, undergo four developmental stages: egg, larva, pupa and adult (Anu et al., 2019). Traditionally, the control of stored-grain pests has depended largely on synthetic chemicals, many of which pose significant risks to human health and the environment. Due to its ozone-depleting properties, growers are gradually reducing their use of methyl bromide as a post-harvest fumigant. Similarly, the repeated use of phosphine has led to the disruption of biological systems and the development of insect-pest resistance (Rani et al., 2012).
       
Plant-based products, including powders, extracts, essential oils and pure secondary metabolites, serve as active ingredients in botanical insecticides, which are an important category of biopesticides gaining traction in stored product pest management (Shunmugadevi and Radhika, 2020; Verma et al., 2020). Monoterpenes, which are the main constituents of essential oils isolated from aromatic plants through hydro-distillation or solvent extraction (Abdelgaleil, 2024), are lipophilic liquids with high volatility. This affect insects in various ways, including toxicity, repellency, antifeedant properties, fumigant action, growth inhibition, suppression of reproductive behavior and reductions in fecundity and fertility. Monoterpenes are considered promising alternatives to conventional synthetic insecticides for several reasons: these are safe for mammals, biodegradable and exhibit low persistence in the environment, which minimizes risks to non-target organisms and reduced the likelihood of accumulation in the food chain (Akbar et al., 2022).
       
Considering the intriguing biological and beneficial properties of monoterpenes, along with their potential for controlling stored-product insect-pests, this study was conducted to evaluate the insecticidal potential (both contact and fumigant toxicity) of six monoterpenes against C. maculatus. Additionally, the study explored the effects of the most potent monoterpene on the AChE activity of the target Insect-pests to elucidate its possible mode of action.

Test Insect-pest culture
 
The present investigation was conducted during 2023–2025 at the Department of Entomology, Tamil Nadu Agricultural University (TNAU), Coimbatore, Tamil Nadu, India. The C. maculatus beetles were reared under laboratory conditions (30±5°C and 70±5% RH) using fresh cowpea seeds in one-liter glass jars containing 250 g of seeds, which were covered with fine mesh cloth for ventilation (Wahba et al., 2018). One- to three-day-old adult beetles were used in the toxicity tests and all experimental procedures were conducted under the same conditions as the insect culture protocol.
 
Monoterpenes tested
 
Citral, β-citronellol, eucalyptol, geraniol, β-myrcene and thymol were purchased from Sigma-Aldrich Chemical Company, Mumbai, India. The purity of the components varied between 95 and 99%.
 
Contact toxicity bioassay
 
The contact toxicity of monoterpenes against C. maculatus adults was tested by following direct application method (Broussalis et al., 1999). The five different concentrations of each compound, ranging from 80 to 400 µL /cm² was prepared using acetone as a solvent. 500 µl of each concentration was sprayed separately onto 9 cm-diameter petri plates. After allowing 15 minutes for the solvent to evaporate, 20 adult insects of C. maculatus were introduced in each plate and covered with a lid. Every chemical and concentration in the experiment was repeated four times. The control plates were treated with acetone alone. Vaseline was applied to the Petri plate lids to prevent the settling of insects. The mortality was assessed at 24, 48 and 72 hours after treatment. Insects were considered dead if their appendages remained immobile or moribund when gently touched with a camel hair brush.
 
Fumigation toxicity bioassay
 
The fumigant toxicity of the monoterpenes was assessed by the method proposed by Rani and Rajasekharreddy (2010). The monoterpenes were dissolved in acetone and diluted to working concentrations of 100, 80, 60, 40 and 20 µL/ml of air. A plastic jar with a capacity of 250 ml functioned as a fumigation chamber. The filter paper that was affixed to under surface of the plastic jar lid was treated individually with varying amounts of the monoterpenes. The small diet cups (3.0 cm × 1.5 cm), each containing 20 adult insects and sealed with nylon (60-mesh) cloth, were placed inside each plastic jar. The jars were then closed with lids, ensuring that the test insects did not come into direct contact with the monoterpene compounds. Acetone was used as the control under the same conditions. Each plastic jar was then closed with a lid. The experiment was replicated four times. The number of dead insects was recorded after 24, 48 and 72 hours of exposure and the percentage mortality (%) for each concentration was computed.
 
AChE inhibition assay
 
Acetylcholine iodide (ATChI) and 5, 5’- dithio-bis (2-nitrobenzoic) acid (DTNB) used in the study was procured from Sigma-Aldrich Chemicals company. AChE inhibition was assessed using the colorimetric method described by Ellman et al. (1961), with ATChI as substrate. Adults of C. maculatus (0.5 g) were individually homogenized in 5 mL of 0.1 M ice-cold phosphate buffer (pH 7.0) using a Teflon glass tissue homogenizer. Homogenates were centrifuged (5,000 rpm for 20 min at 0°C) and supernatants were transferred into a new microcentrifuge and utilized as the enzyme source. Enzyme aliquots (50 μl) and DTNB (100 μl of 0.01 M) were combined with 2.8 ml of 0.1 M phosphate buffer (pH 8.0). Monoterpene test solutions (20 μl), prepared in acetone, were added to the mixture. Control treatments were added with acetone (20 μl) instead of monoterpene. The mixtures were incubated at 37°C for 15 min. The reactions were initiated by adding ATChI (30 μl) followed by incubation at 37°C for 10 min. Absorbance was measured at 412 nm using of Unico 1200-Spectrophotometer. The monoterpenes were examined at three concentrations 0.0025, 0.05 and 0.25 M. Four replicates were carried out for each concentration and control. Each test and control were corrected by blanks for nonenzymic hydrolysis. Percentage of AChE activity inhibition was computed as follows:

 
Where,
OD_B = Optical density of the blank enzyme.
OD_T = Optical density of the treatment.
 
Statistical analysis
 
All experiments were carried out in a Completely Randomized Design (CRD). The data collected from each dose-response bioassay were subjected to probit analysis (Finney, 1971) to determine the LC50, LT50 and 95% fiducial limits using the SPSS 28.0 software program (Statistical Package for Social Sciences, Armonk, NY). The percentage inhibition of AChE activity was analyzed and significant differences among treatment means were compared at 0.01 and 0.05 probability levels using Tukey’s HSD test.

Contact toxicity
 
The contact toxicity assay showed that toxicity increased with both concentration and exposure time. At the lowest concentration of 80 µL/cm², thymol exhibited 31.25% mortality after 24 hours, which increased to 65.00% after 72 hours. At 400 µL/cm², thymol recorded the highest mortality rates of 78.75%, 90.00% and 96.25% after 24, 48 and 72 hours of exposure, respectively. Geraniol also exhibited relatively strong contact toxicity, with mortalities of 78.75%, 88.75% and 95.00% at 400 µL/cm² after 24, 48 and 72 hours, respectively. In contrast, β-citronellol and citral elicited moderate contact toxicity, with 91.25% and 87.50% mortalities, respectively, after 72 hours at 400 µL/cm². β-Myrcene and eucalyptol showed the least activity, with mortalities of 32.50%, 40.00% and 63.75%, 73.75% at 80 and 400 µL/cm² after 72 hours of exposure to C. maculatus (df = 21; F = 73.84; P<0.05; Table 1).


       
Thymol and geraniol were found to have strong contact toxicity, with the lowest LC₅₀  (Lethal concentrations) values of 54.37 µL/cm² and 62.49 µL/cm², respectively, after 72 hours of exposure (Table 2), followed by β-citronellol (77.50 µL/cm²) and citral (94.31 µL/cm²). In contrast, eucalyptol (166.98 µL/cm²) and β-myrcene (243.86 µL/cm²) demonstrated the least efficacy, with the highest LC₅₀  values. Based on these LC₅₀ values, thymol was identified as the most potent monoterpene for managing C. maculatus.


       
The time required for monoterpenes to cause 50% mortality (LT₅₀) was determined for the highest concentration of 400 µL/cm². The results indicated the lowest LT₅₀ value of 7.97 hours for thymol, followed closely by geraniol with an LT₅₀  of 8.72 hours. The LT₅₀  values for β-citronellol and citral were 10.19 hours and 14.08 hours, respectively. In contrast, higher LT₅₀ values were observed for eucalyptol (23.75 hours) and β-myrcene (41.37 hours) (Table 3).


       
Monoterpenes are key bioactive compounds in essential oils, widely used in fragrances, flavorings and medicinal formulations such as expectorants and antiseptics. These are considered promising for environmentally friendly insect pest control due to their selectivity, toxicity to insects and biodegradability. Research has shown that these are effective against various insect species of medical, veterinary and agricultural importance, serving as ovicides, larvicides and adulticides. The intrinsic properties of monoterpenes interfere with the basic metabolic, biochemical and physiological functions of insect pests. In addition to metabolic and physiological alterations, the ingestion of essential oils or monoterpenes may also produce histological modifications, particularly affecting the regenerative cells in the midgut. This leads to disruption of the epithelium and impairs the replacement of functional epithelial cells (Campolo et al., 2018).
       
When insects come into direct contact with thymol, it causes disorganization of the cell membrane, leading to a loss of permeability (Park et al., 2017). This could explain its effectiveness as a contact insecticide in this study, where thymol recorded the highest mortality of 78.75%, 90.00% and 96.25% after 24, 48 and 72 hours of exposure, respectively, at 400 µL/cm². Similarly, geraniol also exhibited relatively strong contact toxicity, with mortalities of 78.75%, 88.75% and 95.00% at 400 µL/cm² after 24, 48 and 72 hours of exposure, respectively. Geraniol, an alcohol, can penetrate insects’ respiratory systems due to its viscosity, leading to their death by suffocation and dehydration through its effect on the cuticle (chitin). At lower doses, geraniol alters the olfactory signals of the host, stimulating insect receptors and acting as a repellent (Brari and Thakur, 2015). The present findings regarding the contact toxicity of geraniol and β-myrcene against pulse bruchids align with those of Abdelgaleil et al. (2009), who reported the high toxicity of geraniol and the inactive nature of myrcene against Sitophilus oryzae (L.). This investigation also revealed that β-citronellol caused 91.25% mortality and citral caused 87.50% mortality after 72 hours of exposure at 400 µL/cm². β-Citronellol, a key active component in citronella oil, has irritant properties that can damage the integuments of insects (Vasconcelos et al., 2015 and Sharma et al., 2016). Insects exposed to lemongrass essential oil or citral exhibited lower respiration rates, decreased locomotor activity and avoided treated surfaces (de Souza Alves et al., 2019). Soe et al., (2020) reported that essential oils of clove, cinnamon, lengukas, citronellol and kaffir lime showed contact toxicity against C. maculatus.           

Fumigation toxicity
 
At a concentration of 100 µL/mL of air, citral demonstrated a mortality rate of 90.00% after 72 hours of exposure, followed by 83.33%, 78.33%, 65.00% and 53.33% mortality at concentrations of 80, 60, 40 and 20 µL/mL of air, respectively. The data clearly indicated moderate toxicity for thymol (78.33%), β-citronellol (76.67%), geraniol (73.33%) and β-myrcene (68.33%). However, eucalyptol, even at the highest concentration of 100 µL/mL, resulted in the lowest mortality rates of 16.67%, 28.33% and 36.67% at 24, 48 and 72 hours, respectively (df = 21; F = 34.45; P<0.05; Table 4).


       
The concentration-mortality response assessed after 72 hours of exposure revealed the lowest LC₅₀  value of 21.35 µL/mL for citral, followed by thymol, geraniol, β-citronellol and β-myrcene, with LC₅₀  values of 26.79, 33.82, 35.20 and 50.09 µL/mL of air, respectively. In contrast, eucalyptol exhibited the lowest fumigant activity, with an LC₅₀ of 215.51 µL/mL of air (Table 5).


       
Lethal times (LT₅₀) for C. maculatus adults exposed to 100 µL/mL of air for the tested monoterpenes exhibited considerable overlap in their effectiveness against the target insect. Notably, citral had the shortest LT₅₀ at 26.84 hours, followed closely by β-citronellol with an LT₅₀  of 32.10 hours. In contrast, geraniol, thymol, β-myrcene and eucalyptol had LT₅₀  values of 37.40 hours, 38.90 hours, 53.55 hours and 114.94 hours, respectively (Table 6).


       
Fumigant toxicity is a major focus of monoterpene research due to the distinctive toxic effects of the vapors from these natural compounds on stored product insect-pests. Monoterpenes can volatilize, allowing them to disperse in the air and penetrate storage areas, effectively reaching insects in cracks and crevices. When inhaled by insect-pests, monoterpenes can induce toxic effects, leading to mortality (Changbunjong, 2022). The present study highlighted citral, β-citronellol, geraniol, myrcene and thymol as effective fumigants. Similar findings were reported by Ajayi et al., (2014) and Reis et al., (2016), who identified 1,8-cineole, menthone, geraniol, geranial, citronellol and citronellal as the most potent fumigant compounds against C. maculatus (F.). Additionally, a study by Oyedeji et al., (2020) found that citral was a more effective fumigant than other tested essential oils, with an LC₅₀ value of 0.19 µL/L air after 24 hours of exposure. Wahba et al. (2018) reported that the fumigant toxicity of eugenol and carvacrol exhibited the highest toxic effects, with LC₅₀ values of 34.58 and 37.34 mg/L air, respectively, after 72 hours of exposure, while thymol demonstrated moderate toxicity with an LC₅₀ value of 45.32 mg/L air.
 
Inhibitory effects of monoterpenes on AChE activity
 
The in vitro inhibitory effect of potent monoterpenes-β-citronellol, thymol and geraniol-on AChE from C. maculatus adults was examined at concentrations of 0.0025, 0.05 and 0.25 M. β-Citronellol was the most effective inhibitor of C. maculatus AChE activity, with 7.84%, 30.76% and 56.66% inhibition (Fig 1A; df = 12; F = 142.48; P<0.01), followed by thymol with 4.01%, 24.48% and 51.81% inhibition at 0.0025, 0.05 and 0.25 M, respectively (Fig 1B; df = 12; F = 124.31; P<0.01). In contrast, geraniol was the least effective inhibitor, with 1.82%, 8.83% and 26.84% inhibition at 0.0025, 0.05 and 0.25 M, respectively (Fig 1C; df = 12; F = 305.79; P<0.01).


       
Essential oils function as neurotoxins, paralyzing insects and leading to their death. One of the mechanisms by which these oils operate is the inhibition of acetylcholinesterase (AChE), making this enzyme one of the most studied targets in stored-product insects (Campolo et al., 2018; Kavallieratos et al., 2021). AChE is a key enzyme involved in neuronal and neuromuscular communication in insects, differing from its mammalian counterpart by just one residue. This distinction makes AChE an attractive target for newly developed insecticides. Essential oils have been identified as a potential source of insecticides due to their ability to modify AChE activity in certain stored-product pests.
       
Previous studies examining the mode of action of monoterpenes against various pests have demonstrated that the symptoms observed in these pests resemble those caused by organophosphate and carbamate insecticides, suggesting that AChE may be the primary target for monoterpenes. Inhibition of acetylcholinesterase (AChE) activity can cause an accumulation of acetylcholine at neuromuscular junctions, leading to the disruption of nerve transmission and, ultimately, insect mortality (Rajashekar et al., 2014). A comparison of the inhibitory effects of monoterpenes on AChE in C. maculatus revealed the highest inhibition of AChE by β-citronellol and thymol. In contrast, geraniol demonstrated strong insecticidal activity but was a weak AChE inhibitor. These findings suggest that while AChE may be a target for monoterpenes, other targets may also exist. Previous research by Abdelgaleil (2009) showed that when comparing monoterpene toxicity against Sitophilus oryzae (L.) with their effects on AChE activity, certain monoterpenes, including cuminaldehyde, 1,8-cineole, (-)-limonene and (L)-fenchone, exhibited strong insecticidal and potent AChE inhibitory activities. However, compounds like (-)-carvone and geraniol, while having strong insecticidal properties, were weak AChE inhibitors. Saad et al., (2018) reported that monoterpenes such as (-)-citronellal, p-cymene, (-)-menthone, α-pinene, α-terpinene and (-)-terpinene-4-ol had a significant inhibitory effect on the AChE activity of Tribolium castaneum Herbst larvae. According to them, (-)-citronellal was the most effective inhibitor among the six monoterpenes tested, with an IC₅₀ value of 9.82 mM. The inhibition percentages were 38.8%, 41.7%, 35.0% and 27.2% after treatment with essential oils from Cymbopogon citratus, Lantana camara, Antrodia camphorata and Imperata cylindrica against S. oryzae, respectively, relative to the control (Tawfeek et al., 2021). In an in vitro inhibition study conducted by Shahriari et al., (2018), α-pinene, trans-anethol and thymol were found to inhibit the AChE activity of Ephesitia kuniella Zeller, with IC₅₀ values of 0.864, 0.49 and 0.137 mg/mL, respectively. López et al. (2018) reported that carvacrol and thymol inhibited the AChE activity of Anisakis simplex Rudolphi, with IC₅₀ values of 0.113 and 0.625 mg/mL, respectively. Similarly, Kumrungsee et al., (2014) found that thymol, 1,8-cineole and essential oil from Psidium guajava inhibited AChE activity in Plutella xylostella (L.) larvae. However, some reports concluded that there is little or no correlation between the in vitro inhibition of AChE by monoterpenes and their contact or fumigant toxicities in insects (Isman and Tak, 2017).

The present investigation identified geraniol, β-citronellol, thymol and citral as the most effective monoterpenes, demonstrating both contact and fumigant toxicity against C. maculatus. These compounds could be utilized to mitigate infestations of stored-product insects. Additionally, they target various mechanisms, including the inhibition of the AChE enzyme, which may help slow down resistance development in these insects. However, large-scale validation of the most effective monoterpenes could lead to the development of natural insecticides based on these compounds, offering an eco-friendly solution for controlling stored-product pests.

The authors thank the Department of Entomology, Tamil Nadu Agricultural University for furnishing all the necessary resources for the research.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All experimental procedures involving insects were conducted in accordance with institutional guidelines for laboratory handling and care. No vertebrate animals or human participants were involved in this study.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.