Post-harvest management of food grains and products is one of the major challenges owing to several factors that can aggravate the post-harvest losses. The efforts are being made to reduce these losses by following several tactics based on the prevailing factors causing losses to ensure the food security of the ever-growing global population. Besides increasing food production and productivity, Food and Agricultural Organisation (FAO) suggested an alternative approach for food security by focussing on the postharvest losses of agricultural production, which is approximately, 10% in developed countries and exceed 20.5% in developing countries¹. There are several biotic and abiotic factors which are responsible for post-harvest losses of grains². Among the biotic factors storage insect pests are one of the critical factors which can cause damage to the food grains in storage either directly or indirectly. The direct damage is mainly due to feeding on grains (either whole or broken) whereas indirect damage pertains to contamination of food grains due to presence of faeces, webbings and body parts³.

The storage insect pests such as red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae) and pulse beetle Callosobruchus spp (Coleoptera: Bruchidae) are most important coleopteron stored grain pests generally found infesting several commodities. Red flour beetle is a cosmopolitan polyphagous pest whose adults and larvae are responsible for severe economic damage in stored products, feeding on several dried foods including flours, fruits and grains³. Moreover, Tribolium spp. are also known to produce carcinogenic compounds called quinones, which leads to allergies, dermatitis and other health disorders⁵. Pulse beetle Callosobruchus spp. is also a cosmopolitan field-to-store pest ranked as important post-harvest insect pest⁶. Approximately, 10–40% of the total annual production of pulses is lost annually due to damage by pulse beetles in tropical countries⁷.

Traditionally fumigant like phosphine and few contact insecticides such as malathion, deltamethrin are used for management of storage pests in India. Several efforts are being made to develop alternative approaches to reduce the failure of traditionally used insecticides. The development of plant essential oil (EO) based products for storage pest management is one among these approaches. The plant essentials, basically a mixture of several volatile compounds have proved to possess several properties such as antimicrobial, antioxidant, anti-inflammatory, anticancer, antiparasitic etc. Besides, several plant essential oils are found to possess very promising insecticidal activities^8,9. Apart for their insecticidal properties, EOs are considered to be important tool in pest management due their broad spectrum of activity and mode of action due to presence of several active compounds⁹ making them ideal candidate for resistance management¹¹.

Himalayan pencil cedar Juniperus macropoda (Family: Cupressaceae) and Himalayan cedar, Cedrus deodara (Family: Pinaceae) are two woody conifer plants known for their diverse biological activity. There are about 75 species of Juniperus found in diverse geographical region right from sea level to above timberline¹². In Himalayas, especially Northern India there is around six species of genus Juniperus, of which Juniperus communis is widespread¹³. J. macropoda is one of the species which is mainly found in northern western Himalayas and is already characterised for its biological activity^14,15. C. deodara is a species of cedar, native to western Himalayas, widely used in Indian system of medicine due to its nutritional and pharmaceutical effects. It is known to have diverse biological activities such as an anti-inflammatory, anti-hyperglycaemic, analgesic, antiulcer, antispasmodic, antibacterial, insecticidal, molluscicidal, anticancer etc.¹⁶. To our knowledge there are very few reports on insecticidal potential of EO of J. macropoda, although insecticidal properties have been studies for other species such as Juniperus formosana¹⁷, Juniperus oxycedrus spp oxycedrus¹⁸, Juniperus phoenisea¹⁹, Juniperus recurve and Juniperus communis²⁰ EO of C. deodara have been reported to possess insecticidal activity against storage pests such as Tenebrio molitor²¹, Callosobruchus chinensis²², Sitosphilus oryzae²³.

In present study the EOs of these two Himalayan plants were evaluated for their fumigation toxicity and repellent activity against T. castaneum and C. maculatus.

The test insects, T. castaneum and C. maculatus, used for the bioassay were obtained from the Storage Laboratory, Division of Entomology, ICAR-IARI, New Delhi. The mass multiplication of insects was carried out in insect culture room maintained at 28–30 ± 2 ^o C temperature and 75 ± 5% relative humidity. T. castaneum was reared on wheat flour mixed with yeast (10:1 w/w) at 12% moisture content in a glass jar (covered with muslin cloth) whereas C. maculatus were reared on mung bean, Vigna radiata seeds. To obtain all individual of same generation, 40–50 adults were released in rearing container and allowed to lay eggs for two days and then removed to obtain uniform age eggs.

To know the real time volatiles emitted from essential oils, the dynamic headspace collection methodology²⁴ was followed (Fig. 2). 500 µl of essential oil was pipetted on the cotton and kept at the base of Borosil glass reagent bottle (1L). Pre-filtered air was pushed into the glass bottle at 0.5 l/min and air laden with volatiles was pulled out with vacuum pump (0.5 l/min) which goes through volatile trap (Porapak Q (150 mg, 80/100 mesh: Supelco). All connections were made with Teflon tape and silicon tubes to avoid any contamination. Collection of volatiles was done for 3 hours for each oil. Elution of the entrained volatiles was done using 300 µl dichloromethane DCM (≥ 99.7% purity; CDH (P) Ltd). The eluted samples were collected in 2ml capacity HPLC (screw-cap) vials (Borosil Glass Works Ltd, Mumbai) and stored at -20°C till further use.

The qualitative and quantitative analysis of headspace extracts was carried out by using Shimadzu QP2010 Ultra gas chromatography-mass spectroscopy (GC-MS) equipped with Rtx-5 MS capillary column of 30 m length, 0.250 mm Diameter and 0.25 µm film thickness. 1µl (injection volume) samples were introduced using an autosampler with split ratio of 5:1 with an injector temperature of 230°C. Helium gas served as carrier at a constant flow rate of 1 ml/min. Initially column temperature was 40°C held for 4 minutes and then ramped at 10°C/min to 220°C and held for 1 min and finally increased at 15°C/min to 260 and held for 1 min. The GC column was then calibrated using an n-alkanes series (C₈H₁₈-C₂₁H₄₄), and retention indices (RIs) of the components were determined under identical operating conditions. Compound identification was performed by comparing the obtained RIs with published literature values and by matching the mass spectra with those from inbuilt NIST 14 Mass Spectral Library (2023).

Fumigation activity of EOs was evaluated against larvae and adult of T. castaneum and adult of C maculatus. Round bottom glass bottles (250 ml volume) with airtight lid (stopcock) were used as fumigation chambers to assay the fumigation toxicity²⁵. Based on preliminary dose-range finding bioassay (to determine the dose range giving 20–80% mortality), larvae and adult of T. castaneum were exposed to five different concentrations of J. macropoda EOs (300 µl/l to 400 µl/l and 500 µl/l to 900 µl/l respectively, for larvae and adult). Similarly, for EO of C. deodara, T. castaneum larvae and adult of were exposed five different concentrations ranging between 60 µl/l to 140 µl/l and 40 µl/l to 200 µl/l, respectively. For bioassay against adult of C maculatus, the concentrations were ranged between 8 µl/l to 40 µl/l and 4 µl/l to 24 µl/l, respectively, for J. macropoda and C. deodara. The required quantity of EO in each concentration was measured with micropipette and loaded on the cotton balls, which were hung inside the fumigation bottle from stopcock. A cotton ball without essential oil (EO) treatment was used as the control in the experiment. The fumigations bottles were closed immediately to prevent the escape of insects and EO vapours. For each concentration and the control, 30 individuals of the test insect stages of T. castaneum and C. maculatus were released separately into bottles for the fumigation bioassay. Each treatment was replicated three times. After 48 hours of exposure, insect mortality was recorded by gently touching each individual with a camel-hair brush to confirm any movement, if present. The moribund insects were considered dead.

The amino acid sequences of target proteins, acetylcholinesterase (AChE), GABA receptors (GABArs), and NADH: ubiquinone oxidoreductase for T. castaneum and C. maculatus were retrieved from the UniProt database (www.uniprot.org) using their respective UniProt Ids (Table 6). The amino acid sequences of species-specific OR proteins were also retrieved from the same database in order to evaluate repellent activity. Further, three-dimensional protein structures were generated through homology modelling using SWISS-MODEL server (swissmodel.expasy.org). The resulting structural models were validated for stereochemical quality and structural reliability before proceeding to molecular docking studies.

The modelled protein structure underwent systematic preparation for molecular docking analysis. In this preparation, polar hydrogen atoms were added to accurately reflect electrostatic interactions, water molecules were removed to avoid potential interference, and Kollman charges were assigned to each atom to account for partial atomic charges. The prepared protein structure was then further converted and saved in PDBQT format, which is compatible with AutoDock Vina software generated by Trott and Olson (2010)³¹.

The three-dimensional molecular structures of major essential components from each EO were retrieved from PUBCHEM database (pubchem.ncbi.nlm.nig.gov.) in SDF format. These ligand structures were processed through energy minimization and conformational optimization procedures by using Discovery Studio Visualizer (BIOVIA, Dassault Systèmes, San Diego, CA, USA; version 21.1). The optimized ligand structure was subsequently converted to PDBQT format using AutoDockTools (ADT) to ensure compatibility with molecular docking software.

The chemical composition analysis of plant essential oils is primarily undertaken to elucidate the bioactive compounds present, which may function as effective agents in various applications including biomedicine, biopesticides development, and other environmentally sustainable solutions. Extensive efforts have been carried out for exploration of J. macropoda and C. deodara bio-actives. Despite their ethnobotanical significance, the bioactive potential of these EOs remains largely underexplored, warranting comprehensive phytochemical investigations. In practical situation where fumigation is involved for insecticidal action, the volatiles emitted from essential oils brings mortality in insects, hence we analysed the headspace sample of EOs to identify such volatiles. The chemo-profiling of the volatiles from J. macropoda EO were characterized by a high abundance of monoterpenes and monoterpenoids wherein 4-terpineol, limonene, and γ-terpinolene, β-thujene, 4-carene, α-pinene, and β-myrcene constituted the major part. Essential oils from plant species belonging to Juniperus have previously been reported to contain monoterpenes and monoterpenoids, although considerable compositional variation has been observed among different species and geographical regions^32–35. The compositional variation was also evident from the studies by Stappen et al.¹⁵ from Western Himalaya and Dahmane et al.³⁶ from Algeria, who reported sabinene, cedrol, α-pinene and 4-terpineol as the major constituents of J. macropoda oil from Western Himalaya. Similar to present study, the sesquiterpenes and sesquiterpenoids in minor quantity have also been reported by Krutca et al.³³. The contrasting chemical profiles observed across Juniperus species underscore the significant influence of geographical and environmental factors on terpenoid biosynthesis, with important implications for their biological activities and industrial applications. Converse, the C. deodara EO was found rich in sesquiterpenes (41.92 ± 0.051%), predominantly characterized by himachalane-type compounds (α-cuprenene, α-himachalene, γ-himachalene), consistent with previous investigations^20,37. Kumar et al. reported comparable sesquiterpene profiles, including α-himachalene (13.83%), γ-himachalene (12.00%), β-himachalene (37.34%), deodaron (0.43%), α-atlantone (4.53%), γ-(Z)-atlantone (2.77%), γ-(E)-atlantone (3.34%) and, α-(E)-atlantone (10.63%)³⁷. This sesquiterpene predominance represents a characteristic chemotaxonomic marker of Cedar species and correlates strongly with their documented bioactivities^21,39,40,41. The monoterpene fraction in C. deodara was substantially lower (12.36 ± 0.041%) than that observed in J. macropoda oil, reflecting species-specific biosynthetic pathways and distinct ecological adaptations. Notably, the presence of aromatic hydrocarbons (22.77 ± 0.016%), including benzaldehyde (19.40%), p-cymene, and benzoic acid, corroborates earlier findings by Saab et al.⁴². The marked differences in essential oils from these two Himalayan plants highlight the need for detailed bioactivity studies to assess their potential.

There are very few reports on fumigation toxicity of EOs of J. macropda and C. deodara against storage insect pests. The present study revealed C. deodara oil as most effective fumigant against both test insects. The C. deodara EO have also been reported to possess very good fumigation toxicity against a storage pest, rice weevil Sitophilus oryzae with 53.33% percent mortality after 30 days of exposure²³. The toxicity of Cedar wood oil alone was found very effective than its combination with neem oil and neem oil alone²¹. Contrast to fumigation toxicity potential of C. deodara oil in present study, Gupta et al. did not find very encouraging toxicity against C. maculatus (LC₅₀ = 1487.29 µl/l) and C. chinensis (LC₅₀ = 1716.80 µl/l)²⁰.

Our studies revealed that J. macropoda oil was not much effective (T. castaneum larvae LC₅₀ = 357.332 µl/l, T. castaneum adult LC₅₀ = 724.656 µl/l, and C. maculatus adult LC₅₀ = 25.679 µl/l) as compared to C. deodara oil (T. castaneum larvae LC₅₀ = 103.906 µl/l, T. castaneum adult LC₅₀ = 123.097 µl/l, and C. maculatus adult LC₅₀ = 16.125 µl/l). But, toxicity of J. macropoda EO in present study is more promising than those reported by Gupta et al.²⁰ for toxicities of EOs from J. communis (LC₅₀ = 1945.44 µl/l) and J. recurva (LC₅₀ = 2369.76 µl/l) against C. maculatus, this may be attributed to differential composition of EOs. The EOs of J communis and J recurva were dominated by camphene which is a monoterpene compound.

Apart from fumigation toxicity, EO from Juniperus species have been studied for its repellent and contact activity against insect pests^15,17,19,43. The repellent effect is an important characteristic in the choice of essential oil for the management of stored grain pests because high repellency can lower the infestation and the consequent reduction or absence of oviposition⁴⁴. In the present study essential oil of C. deodara shows significantly higher repellent activity against T. castaneum adults at all doses (10ng, 50 ng, and 100ng). The antifeedant/repellent activity of this plant EO have also been reported by Buneri et al. against mealworm beetle (Tenebrio molitor) larvae²¹. Koc et al. also reported potential repellent effect of another species C. libani on brown dog tick species (Rhipicephalus sanguineus)⁴⁵. The EOs of J. macropoda was found to be most effective repellent at concentration of 50 ng and 100 ng against T. castaneum but not against C. maculatus. Similar to this, Guo, et al. reported that the essential oil of J. formosana showed strong repellent activity against T. castaneum with the percentage repellency (PR) over 80% at 2 h¹⁷. The other species of Juniperus and C. deodara essential oil have been reported as promising repellents²⁰. The variation in the repellent activity could be due to differential volatile profiles.

Molecular docking serves as a useful tool in pest management by providing predictive insights onto ligand-protein interactions, facilitating the identification of potential molecular target, enabling structure-based identification of ecofriendly insecticidal or repellent agents⁴⁶. Literature shows that, acetylcholinesterase enzyme is reported to be most potent target for fumigation toxicity of EOs²⁷. Besides this key enzyme, EOs are also reported to act on other biological targets of insects such as ion channels or respiratory system^47–49. Further odorant receptors (ORs) on insect antennae play a crucial role in insect behaviour, sensitization of them by EOs may cause repellent effect in insects. Considering this, we performed in silico molecular docking of major components of both EOs against acetylcholinesterase (AchE), GABA receptors (GABArs), and NADH: ubiquinone oxidoreductase and Odorant receptors of both test insect species. In silico studies revealed that C. deodara sesquiterpenoids including γ-himachalene, α-cuprenene, α-himachalene, and α-(E)-atlantone showed consistently strong binding affinities to all four target proteins in both insect species. The toxicity and repellency observed in the present study is may be due to this binding suggesting the potential target site. The other EOs with these sesquiterpenoids as major components has also been proved good fumigants against storage pests ^39,50. A strong binding of δ-cadinene to AChE in both insects may be responsible for fumigation and repellent property of J. macropoda EO. This compound has also been proved to possess larvicidal against malaria, dengue and filariasis causing mosquitoes⁵¹. In comparison to compounds from J. macropoda, C. deodara compounds often showed stronger and more stable binding affinities, consistent with bioassay results indicating the superior insecticidal activity of C. deodara essential oil. The most often targeted protein with highest binding values was AChE, which is a vital enzyme in the insect nervous system that hydrolyses acetylcholine, a key neurotransmitter in nerve impulse transmission. Inhibition of AChE by essential Oils disrupts synaptic signalling, causing neurological dysfunction, paralysis, and ultimately insect mortality. This mechanism underlies the insecticidal (fumigation) potential of these essential oils. The present study concludes that EOs of C. deodara and J. macropoda can be a promising candidate for further in-depth studies and development of suitable formulations for management of storage pests.

The present investigation provides comprehensive insights into the chemical composition, bio-efficacy, and molecular interaction of Cedrus deodara and Juniperus macropoda essential oils against major storage insect pests. Distinct chemotypes were identified, with C. deodara oil characterized by sesquiterpene-rich derivatives and J. macropoda dominated by monoterpene and monoterpenoids. These compositional differences translated into markedly higher fumigation toxicity and repellency of C. deodara oil. Molecular docking analyses substantiated these finding, revealing strong and stable binding of key sesquiterpenoids (e.g., γ-himachalene, α-himachalene, α-cuprenene) to vital insect target proteins, particularly acetylcholinesterase, indicating a plausible neurotoxic mode of action. Collectively, the integrated chemical, biological and computational evidence underscores the potential of C. deodara essential oil as a potent and eco-compatible fumigant and repellent for sustainable post-harvest pest management.