Exploring the Efficacy of Four Apiaceae Essential Oils against Nine Stored-Product Pests in Wheat Protection

The Apiaceae family, known for aromatic plants producing bioactive essential oils (EOs), holds significance across sectors, including agrochemicals. This study evaluated the insecticidal potential of four Apiaceae EOs from Crithmum maritimum L., Trachyspermum ammi (L.) Sprague ex Turrill, Smyrnium olusatrum L., and Elwendia persica (Boiss.) Pimenov and Kljuykov against various significant storage pests (Sitophilus oryzae (L.), Trogoderma granarium Everts, Rhyzopertha dominica (F.), Tribolium castaneum (Herbst), T. confusum Jacquelin du Val, Oryzaephilus surinamensis (L.), Alphitobius diaperinus (Panzer), Acarus siro L., and Tenebrio molitor L.) on wheat. Insect mortality rates were monitored at intervals of 1, 2, 3, 4, 5, 6, and 7 days. Smyrnium olusatrum EO exhibited the highest efficacy, followed by T. ammi, C. maritimum, and E. persica EOs, although efficacy varied by species, developmental stage, and concentration. Notably, complete mortality occurred for several pests at 1000 ppm of S. olusatrum and T. ammi EOs. Gas chromatography–mass spectrometry (GC–MS) analysis revealed key compounds in these EOs, including myrcene, germacrone, and curzerene in S. olusatrum EO, and thymol, γ-terpinene, and p-cymene in T. ammi EO. These findings emphasize their potential as botanical insecticides. Smyrnium olusatrum and T. ammi EOs emerge as promising eco-friendly pest management options due to their efficacy, highlighted compound composition, and availability of biomass from both wild and cultivated sources.


Introduction
The substantial dependence on synthetic pesticides for managing arthropod pests, particularly within food supplies, presents notable environmental as well as human health hazards [1,2].These hazards encompass mammalian toxicity, environmental pollution, and bioaccumulation.Furthermore, the frequent emergence of resistance among insect species to prevalent compounds significantly constrains their efficacy [3].Amidst this concern, the European Union advocates for a considerable decrease in chemical pesticide usage, integrating innovative concepts and advanced technologies aligned with Integrated Pest Management (IPM) tenets [4,5].
In recent years, multiple investigations have been carried out to assess the efficacy of natural pesticidal formulations, such as diatomaceous earth, entomopathogenic nematodes and fungi, nanoemulsions (NEs), microemulsions, and essential oils (EOs), as grain protectants against different pests of storages, as well as other pests, exhibiting promising results [31][32][33][34][35][36][37][38][39].Extracted from various plant parts like leaves, seeds, roots, and flowers, EOs represent secondary plant metabolites with substantial applications in aromatherapy, perfumery, and cosmetics industry [40].These EOs and their constituents hold valuable potential in diverse fields such as agriculture, environment, and human health.Their significance extends further as they have attracted considerable attention for the development of insecticides and acaricides [41][42][43].Indeed, research efforts have extensively concentrated on assessing the contact effects of EOs on insect species by application onto filter paper, or by topical administration onto the dorsal region of the insect thorax, while also investigating their efficacy as botanical repellents [44][45][46][47][48]. Various EOs and their physiologically active constituents have garnered significant recommendations as promising contact toxicants and fumigants because of their volatile characteristics, leaving negligible residual impact [49].Among several botanical sources of EOs, the family of Apiaceae is one of the most important worldwide for the high economic value and cultivation systems developed in temperate and Mediterranean regions [50].Apiaceae EOs find applications in food and beverages (alcoholic and non-alcoholic drinks), pharmaceutics, cosmetics, and perfumery.Recently, the agrochemical industry has been investigating these products as potential candidates within the scenario of replacing synthetic pesticides with safer and eco-friendly alternatives (e.g., the farm-to-fork strategy of the EU).
Crithmum maritimum L., found extensively along Western Europe's Mediterranean coastlines, is a facultative halophyte renowned for its diverse culinary and practical applications [51].The leaves boast significant antioxidant and nutritional properties, while the seeds contain valuable edible oil and secondary compounds with promising industrial uses [52].A biennial plant, Smyrnium olusatrum L., found across the Mediterranean region from North Africa to the British Isles, is distinguished by the plentiful presence of sesquiterpenes equipped with a furan ring [53].Among these compounds, isofuranodiene predominates.This plant potentially exhibits neuritogenic, anti-inflammatory, and anti-cancer effects, along with insecticidal and acaricidal properties [54].The Persian Trachyspermum ammi (L.) Sprague ex Turrill, referred to as ajwain, is cultivated for its fruits.This species contains around 2-4.4% brown EO rich in thymol, widely used in treating gastrointestinal issues, bronchial problems, and lack of appetite.This EO demonstrates anti-aggregatory, antimicrobial, and fungicidal effects.Ajwain serves as traditional remedy for various human and animal ailments, possessing stimulant, antispasmodic, and carminative properties [55][56][57].Elwendia persica (Boiss.)Pimenov and Kljuykov (syn.Bunium persicum (Boiss.)B.Fedtsch.),found in dry temperate regions of Central Asia like India, Kazakhstan, Afghanistan, Pakistan, Egypt, and Iran, yields fruits serving as spice, antiseptic, and carminative agents.Studies revealed that its EO primarily comprises γ-terpinene, cuminaldehyde, and p-cymene [58,59].Additionally, major antioxidants such as p-coumaric acid, caffeic acid, and kaempferol are isolated from the fruit's polar fraction [60].The plant nomenclature follows POWO [61].
The insecticidal efficacy of EOs derived from C. maritimum, S. olusatrum, T. ammi, and E. persica has been previously assessed against various pests of the storage environment using different application methods such as fumigation, NEs, and direct aqueous solution application [62][63][64][65][66][67][68][69].Despite the potential displayed by these EOs, their efficacy is often limited to a single developmental stage of the targeted pest, such as adults or larvae, and never as grain protectants.Hence, there remains a pressing need for research into novel insecticidal formulations capable of eradicating multiple developmental stages of either one or multiple storage pests when applied directly to grains.Thus, the objective of the current investigation was to assess the pesticidal impact of EOs derived from C. maritimum, S. olusatrum, T. ammi, and E. persica against different developmental stages of nine storage pests known for their detrimental impact, including the understudied species A. diaperinus, A. siro, T. granarium, and T. molitor, over multiple time intervals.

Effectiveness against Alphitobius Diaperinus Larvae and Adults
Between and within exposures, all main effects and respective interactions were significant for both adults and larvae of A. diaperinus (Table 2).After one day of exposure, S. olusatrum resulted in 28.9 and 45.6% mortality of A. diaperinus larvae at 500 and 1000 ppm, respectively, while pirimiphos-methyl only reached 8.9% mortality.At 500 ppm, S. olusatrum caused more than 68% mortality after seven days of exposure, while at 1000 ppm, complete mortality (100%) was observed after six days of exposure, significantly outperforming pirimiphos-methyl at both concentrations.Following S. olusatrum, C. maritimum EO caused 13.3 and 44.4% mortality to A. diaperinus larvae, while the remaining EOs had no effect on mortality.Concerning A. diaperinus adults, there was no effect on mortality observed for any EO at 500 ppm, while at 1000 ppm, C. maritimum EO caused 13.3% mortality, followed by T. ammi, exhibiting 6.7% mortality after 7 days of exposure (Table 3).

Effectiveness against Tribolium Castaneum Larvae and Adults
For T. castaneum, between and within exposures, all main effects and the respective interactions were significant for both adults and larvae (Table 2).At 500 ppm, S. olusatrum, T. ammi, and E. persica EOs caused T. castaneum larvae 17.8, 10.0, and 3.3% mortality after one day of exposure, respectively, while C. maritimum had no effect on mortality.After seven days of exposure, however, the aforementioned EOs caused 77.8, 61.1, 48.9, and 67.8% mortality, respectively, with mortality caused by S. olusatrum (77.8%) significantly higher than that of pirimiphos-methyl (56.7%).After four days of exposure, S. olusatrum EO resulted in complete mortality of T. castaneum larvae, followed by T. ammi (93.3%),E. persica (65.7%), and C. maritimum (61.1%), all significantly outperforming pirimiphos-methyl (34.4%).Complete mortality was observed for T. ammi EO after five days of exposure, while at the last exposure interval, C. maritimum caused 91.1% mortality, followed by E. persica at 90.0% mortality.Adults of T. castaneum were only affected by S. olusatrum EO three days post-exposure at 500 ppm.Mortality reached 13.3% at the final exposure interval, while pirimiphos-methyl failed to affect mortality at all exposure intervals.At 1000 ppm, S. olusatrum EO caused T. castaneum adults 26.7% mortality after two days of exposure, reaching 81.1% mortality after seven days, followed by E. persica (13.3%), T. ammi (5.6%), and C. maritimum (5.6%) (Table 4).For each EO, within each row, means followed by the same uppercase letter are not significantly different (df = 6, 62; Tukey HSD test at p = 0.05).For each concentration, within each column, means followed by the same lowercase letter are not significantly different (df = 4, 44; Tukey HSD test at p = 0.05).No significant differences were recorded where no letters exist.
No statistical analysis was performed where dashes exist.Due to zero values, mortality data for adults exposed to C. maritimum, T. ammi, and E. persica EOs at 500 ppm are not shown.

Effectiveness against Tenebrio Molitor Larvae and Adults
For T. molitor, between exposures, all main effects and respective interactions were significant for both larvae and adults.Within exposures, exposure × concentration was not significant for larvae and adults, and exposure × concentration × EO type was not significant for larvae (Table 2).Regarding T. molitor larvae, only S. olusatrum EO affected mortality at 500 ppm, which ranged from 3.3 to 11.1% from four to seven days, respectively.At 1000 ppm, S. olusatrum EO caused 2.2-18.9%larval mortality from two to seven days of exposure, followed by C. maritimum reaching 4.4% mortality after seven days, and E. persica (2.2%), while T. ammi did not affect mortality at any exposure interval.Adults of T. molitor were mostly affected by T. ammi EO, which resulted in 13.3-100% mortality from one to six days of exposure at 500 ppm, significantly outperforming pirimiphos-methyl (7.8-51.1%).Following T. ammi EO, S. olusatrum and E. persica exhibited 75.6 and 7.8% mortality after seven days of exposure, respectively, while C. maritimum did not affect the mortality of T. molitor adults at all exposure intervals.At 1000 ppm, T. ammi EO remained the most efficient, exhibiting 27.8-100% after one to four days, respectively, significantly outperforming pirimiphos-methyl at every exposure interval (10.0-35.6%,respectively).Smyrnium olusatrum EO caused 96.7% mortality after seven days of exposure, followed by E. persica (26.7%), while C. maritimum only caused 1.1% mortality (Table 6).

Effectiveness against Trogoderma Granarium Larvae and Adults
All main effects and respective interactions were significant for both adults and larvae between exposures.Within exposures, only exposure × concentration was not significant for larvae of T. granarium (Table 2).Larvae of T. granarium were only affected by S. olusatrum and T. ammi, exhibiting 3.3% mortality for both EOs after one day of exposure at 500 ppm, which reached 50.0 and 41.1% for each EO, respectively, after seven days of exposure, significantly higher than pirimiphos-methyl (26.7%).The remaining EOs did not affect mortality at any exposure interval.At 1000 ppm, S. olusatrum EO exhibited the highest efficacy against larvae of T. granarium, exhibiting 13.3 to 93.3% mortality after one to seven days of exposure, remaining significantly higher than pirimiphos-methyl (0.0-25.6%, respectively).Following this EO, T. ammi exhibited 68.9% mortality after seven days of exposure, while C. maritimum and E. persica only reached 17.8 and 4.4%, respectively.Concerning the adults of T. granarium, mortalities between one day (18.9%) and two days (40.0%) post-exposure were significantly higher when treated with T. ammi at 500 ppm, reaching 78.9% at seven days post-exposure.A significant increase in mortalities was also observed for S. olusatrum EO, exhibiting 27.8-70.0%mortalities after two to four days after exposure, respectively, ultimately reaching 81.1% mortality after seven days of exposure, outperforming T. ammi.The remaining EOs, C. maritimum, and E. persica, reached 48.9 and 42.2% mortality at the last exposure interval, respectively.At 1000 ppm, complete mortality was achieved by S. olusatrum EO after four days of exposure, followed by T. ammi (88.9%), both significantly higher than pirimiphos-methyl (42.2%).After seven days of exposure, all EOs exhibited significantly higher mortality rates than pirimiphos-methyl (70.0%), with T. ammi resulting in complete mortality, followed by C. maritimum (91.1%) and E. persica (87.8%) (Table 7).

Effectiveness against Oryzaephilus Surinamensis Larvae and Adults
For O. surinamensis, all main effects and respective interactions were significant for both adults and larvae between exposures.Within exposures, only exposure × concentration and exposure × concentration × EO type were not significant for adults (Table 2).Larvae of O. surinamensis exhibited the highest susceptibility to S. olusatrum, resulting in mortalities ranging from 23.3 to 57.8% after one to seven days of exposure at 500 ppm.Following S. olusatrum EO, T. ammi caused up to 53.3% mortality after seven days of exposure, with C. maritimum (26.7%) and E. persica (6.7%) following.Mortalities caused by S. olusatrum and T. ammi EOs were significantly higher than pirimiphos-methyl after two days of exposure, exhibiting 72.2 and 47.8%, respectively, compared to the latter (8.9%) at 1000 ppm.Complete mortality was achieved by S. olusatrum EO after five days of exposure, while T. ammi resulted in 100% mortality after six days of exposure.At the final exposure interval, C. maritimum and E. persica EOs resulted in 61.1% and 53.3% mortalities, respectively.O. surinamensis adults suffered higher mortalities to S. olusatrum (21.1%), T. ammi (12.2%), and C. maritimum (10.0%) than E. persica EO (8.9%) after two days at 500 ppm.All EOs exhibited significantly higher mortalities than pirimiphos-methyl from three to seven days of exposure at 500 ppm.Mortalities reached 48.9, 43.3, 41.1, and 33.3% for T. ammi, C. maritimum, S. olusatrum, and E. persica EOs, respectively, at seven days post-exposure.A similar trend was observed between the EOs at 1000 ppm.Elwendia persica EO caused low (10.0%)mortality after one day of exposure, while T. ammi did not affect mortality.However, after two days of exposure, S. olusatrum, T. ammi, and C. maritimum EOs outperformed E. persica, exhibiting 36.7,26.7, and 25.6% mortalities, respectively, compared to the later (22.2%).All, nevertheless, demonstrated significantly higher mortalities than pirimiphosmethyl (2.2%) at the same exposure interval.At the final exposure interval, C. maritimum EO resulted in the highest mortality (77.8%), followed by T. ammi (76.7%), S. olusatrum (75.6%), and E. persica (65.6%), although the only significant difference among treatments was observed between all EOs and pirimiphos-methyl (11.1%) (Table 8).For each EO, within each row, means followed by the same uppercase letter are not significantly different (df = 6, 62; Tukey HSD test at p = 0.05).For each concentration, within each column, means followed by the same lowercase letter are not significantly different (df = 4, 44; Tukey HSD test at p = 0.05).No significant differences were recorded where no letters exist.

Effectiveness against Sitophilus Oryzae Adults
For S. oryzae adults, between exposures, all main effects and respective interactions were significant.Within exposures, only exposure × concentration was not significant (Table 2).At 500 ppm, S. oryzae adults exhibited 20.0 and 3.3% mortality when treated with S. olusatrum and C. maritimum, respectively, after one day of exposure, while the remaining EOs did not affect mortality.At seven days post-exposure, S. olusatrum and C. maritimum EOs caused 90.0 and 33.3% mortalities, respectively, followed by E. persica (6.7%).No effect on mortality was observed for T. ammi EO at any exposure interval at 500 ppm.At 1000 ppm, C. maritimum caused 94.4% after the last exposure interval.Smyrnium olusatrum EO resulted in complete mortality after three days of exposure, significantly higher than pirimiphos-methyl (38.9%).Elwendia persica EO affected mortality at four days of exposure (3.3%), ultimately reaching 11.1% mortality at the last exposure interval.Mortality caused by T. ammi remained low from one to seven days of exposure, exhibiting 3.3 to 8.9% mortality, respectively (Table 10).

Effectiveness against Acarus Siro Nymphs and Adults
Between exposures, all main effects were significant for A. siro nymphs and adults, apart from concentration in the case of adults.The respective interaction was insignificant.Within exposures, exposure × concentration and exposure × concentration × EO type were not significant for nymphs and adults (Table 2).At 500 ppm, A. siro nymphs were affected only by S. olusatrum and T. ammi EOs after one day of exposure, exhibiting low mortalities (1.1% for both EOs).After seven days of exposure, S. olusatrum EO resulted in the highest mortality rates, demonstrating 55.6%, followed by T. ammi (47.8%), C. maritimum (17.8%), and E. persica (6.7%), with mortalities gradually increasing over time.At 1000 ppm, a similar trend was observed after seven days of exposure, with S. olusatrum EO remaining the most efficient (63.3%), followed by T. ammi (56.7%), C. maritimum (32.2%), and E. persica (15.6%).Regarding A. siro adults, all S. olusatrum, T. ammi, and C. maritimum EOs exhibited 2.2% mortality at 500 ppm after one day of exposure, while E. persica did not affect mortality.At the final exposure interval, S. olusatrum EO resulted in the highest mortality rate (67.8%), followed by T. ammi (55.6%), C. maritimum (26.7%), and E. persica (15.6%).At 1000 ppm, S. olusatrum, T. ammi, and C. maritimum EOs exhibited 4.4, 3.3, and 3.3% mortalities, respectively, after one day of exposure, whereas E. persica did not affect mortality.After seven days of exposure, the aforementioned EOs caused 76.7, 65.6, and 45.6%, respectively, with E. persica remaining less effective, causing 23.3% mortality (Table 11).For each EO, within each row, means followed by the same uppercase letter are not significantly different (df = 6, 62; Tukey HSD test at p = 0.05).For each concentration, within each column, means followed by the same lowercase letter are not significantly different (df = 4, 44; Tukey HSD test at p = 0.05).Due to zero values, mortality data for adults exposed to T. ammi EO at 500 ppm are not shown.For each EO, within each row, means followed by the same uppercase letter are not significantly different (df = 6, 62; Tukey HSD test at p = 0.05).For each concentration, within each column, means followed by the same lowercase letter are not significantly different (df = 4, 44; Tukey HSD test at p = 0.05).No significant differences were recorded where no letters exist.Due to zero values, mortality data for adults exposed to T. ammi EO at 500 ppm are not shown.For each EO, within each row, means followed by the same uppercase letter are not significantly different (df = 6, 62; Tukey HSD test at p = 0.05).For each concentration, within each column, means followed by the same lowercase letter are not significantly different (df = 4, 44; Tukey HSD test at p = 0.05).No significant differences were recorded where no letters exist.

Discussion
Generally, EO chemical composition is influenced by diverse factors, such as climate, altitude, growth conditions, soil type, agricultural practices, and harvesting time [71].The chemical composition identified in the E. persica EO aligns with previously reported compositions of EOs derived from wild Iranian plants.These typically showcase a prevalence of γ-terpinene, γ-terpinen-7-al, and cumin aldehyde [72][73][74][75].On the contrary, the identified chemical composition differs from the one reported for cultivated Iranian E. persica, in which cumin aldehyde, α-terpinen-7-al, and γ-terpinen-7-al levels are usually higher than those of γ-terpinene [76].Regarding S. olusatrum EO, its chemical composition differs from that reported by Quassinti et al. [77].A lower percentage of isofuranodiene was detected, together with a higher one of the monoterpenes myrcene and β-phellandrene.Isofuranodiene is a thermosensitive compound and undergoes Cope rearrangement under conventional gas chromatographic runs, producing curzerene [78].For this reason, the values derived by GC-MS are usually underestimated.Moreover, the chemical composition of the EO could be influenced by the geographic origin of the plant used in this study, which differs from the one used by Quassinti et al. [77].Regarding C. maritimum EO, its chemical composition aligned with that reported by Piatti et al. [79] concerning major EO constituents.However, our study highlighted a lower percentage of γ-terpinene and a higher content of dillapiole.The chemical composition of C. maritimum EO is influenced by diverse factors, and many chemotypes have already been reported [80].On the contrary, T. ammi EO was mainly dominated by thymol, γ-terpinene, and p-cymene, and this result is in line with the chemical compositions already reported in the literature [81].
Amidst botanical pesticides, EOs have garnered escalating interest due to their notable effectiveness coupled with their characteristic of low persistence in the environment [82][83][84].Moreover, EOs often present low toxicity to non-target organisms and mammals [35,85,86].In addition, these plant-derived products are frequently distinguished by possessing diverse modes of action, thereby diminishing the likelihood of resistance development, a noteworthy characteristic in the context of IPM [86,87].In the current research, multiple EOs have demonstrated their insecticidal potential against diverse pests exhibiting tolerance to conventional insecticides.Among all EOs, S. olusatrum was the most efficient, followed by T. ammi, C. maritimum, and E. persica, although the effectiveness of each EO varied depending on the species, developmental stage, and concentration applied.Past studies indicate that C. maritimum exhibited insecticidal effects against S. oryzae, T. confusum, T. castaneum, R. dominica, and O. surinamensis when applied on filter paper, or for the evaluation of fumigant and contact toxicity [65][66][67].The effectiveness of S. olusatrumderived isofuranodiene and S. olusatrum NE against T. granarium, T. molitor, T. confusum, and T. castaneum has been previously investigated [68,88].Trachyspermum ammi EOs have been tested against A. siro, T. granarium, T. molitor, T. confusum, T. castaneum, O. surinamensis, R. dominica, and S. oryzae as fumigants, repellants, or NEs on wheat [63,64,69], except for A. diaperinus.As for E. persica EO, its impact has only been tested for its repellent effect and contact toxicity against T. castaneum [62].
In general, most EOs tested here had little to no effect against A. diaperinus adults; nevertheless, S. olusatrum EO had a remarkable effect on larvae, causing complete mortality after six days of exposure at 1000 ppm, significantly outperforming pirimiphos-methyl.However, EOs tested here had little to no effect against adults of this pest.Interestingly, the mortality rates of A. diaperinus larvae after five and seven days of exposure to S. olusatrum EO at 1000 ppm were identical to the resulting mortality rates exhibited by Kavallieratos et al. [89] when treating larvae with the concentration of deltamethrin indicated on the formulation label.The difference in mortality rates between larvae and adults may be partially attributed to differences in structural and physiological characteristics of the cuticle across different insect life stages [90,91].
A noteworthy discovery of the current investigation is the efficiency of several tested EOs in controlling Tribolium spp., since both T. confusum and T. castaneum have been recognized for decades as highly tolerant species to numerous insecticides [34,[92][93][94].Both S. olusatrum and T. ammi EO demonstrated complete mortality of T. castaneum larvae after five and four days, respectively, at 1000 ppm, with S. olusatrum EO also exhibiting high mortality rates for adults, whereas pirimiphos-methyl could not control the species in this developmental stage.The elevated mortality in adults of T. castaneum holds profound significance since adults are more tolerant than larvae [95][96][97].Regarding T. confusum, larval mortality was high for all EOs tested at both concentrations, with T. ammi EO resulting in complete larval mortality after four days of exposure at the high concentrations applied.In line with T. castaneum, T. confusum adults also exhibit higher tolerance compared to larvae [95].This has become evident in the present investigation, since the EOs applied had little effect on adult mortality, whereas no effect was observed for pirimiphos-methyl.Nevertheless, previous investigations on the effectiveness of EO derived from Carlina acaulis L. against T. confusum exhibited remarkable results after two days of exposure in both larvae and adults of this species [98], demonstrating that the various modes of action of different EOs are reflected in the observed mortality rates.
In general, EOs demonstrate insufficient efficacy in controlling T. molitor larvae.For example, the application of Tanacetum vulgare L. EO on wheat caused 8.9% mortality of T. molitor larvae at seven days post-exposure [99].The low effectiveness of EOs against larvae of this pest has become evident in the current investigation as well.Nevertheless, remarkable adult mortality rates were observed in T. molitor adults when treated with T. ammi and S. olusatrum EOs.Complete mortality was induced by the former at both concentrations, while the latter achieved a 96.7% mortality rate at 1000 ppm.These specific EOs have exhibited exceptional efficacy against T. molitor adults compared to other botanical formulations.For instance, after seven days of initial exposure, the following EOs resulted in varying mortality rates: Santalum album L. EO exhibited a rate of 60 [69,99,100].
Various insecticidal formulations based on β-cyfluthrin, deltamethrin, or the bacteriocin microcin extracted from Citrobacter spp.have highlighted a higher tolerance among larvae of T. granarium compared to adults [27,101,102].In the present study, S. olusatrum and T. ammi EOs exhibited remarkable efficacy for the management of both larvae and adults of this species, with S. olusatrum EO demonstrating exceptionally high to complete suppression of both developmental stages at 1000 ppm, significantly outperforming pirimiphos-methyl.Hence, these EOs might serve as viable alternatives to synthetic organophosphorus insecticides such as pirimiphos-methyl.
In the current study, all EOs exhibited exceptional efficiency against O. surinamensis, with S. olusatrum and T. ammi EOs successfully suppressing larvae of this species, and all EOs demonstrated high mortality rates for adults (65.6-77.8%), in both cases significantly outperforming pirimiphos-methyl.Alternative insecticides yielded notably lower efficacy.For instance, T. vulgare EO resulted in the mortality of 93.3% of larvae and 13.3% of adults [99].On the other hand, Mentha longifolia (L.) Huds.EO-based NE caused the death of 86.7% of larvae and 63.3% of adults of O. surinamensis at 1000 ppm, following a seven-day exposure to treated wheat [103].When subjected to single and combined treatments with two concentrations of the entomopathogenic nematode Steinernema carpocapsae (Weiser) (50 IJs/cm 2 , 100 IJs/cm 2 ) and the entomopathogenic fungus Beauveria bassiana (Bals.-Criv.)Vuill.(1 × 10 6 conidia/mL), mortalities of O. surinamensis adults ranged from 9.38 to 48.64% at the same exposure interval [38].
Concerning the effectiveness of the tested EOs against adults of R. dominica, all treatments resulted in elevated mortalities at 1000 ppm and seven days post-exposure, ranging from 45.6 to 95.6%.These are rather elevated mortality rates considering that S. carpocapsae (50 IJs/cm 2 , 100 IJs/cm 2 ) and B. bassiana (1 × 10 6 conidia/mL) alone and combined re-sulted in 25.43-71.22%mortality of this pest after seven days of exposure [38].Nevertheless, C. acaulis EO at 1000 ppm resulted in complete mortality of R. dominica adults at four days post-exposure [98].The aforementioned research showcased the remarkable efficacy of the EO derived from C. acaulis for the control of R. dominica compared to the EOs tested in the current study [98].However, in this study, the efficacy of S. olusatrum EO significantly surpassed that of C. acaulis against S. oryzae.In the present investigation, S. olusatrum EO treatment completely suppressed S. oryzae adults at three days post-exposure at 1000 ppm, as opposed to C. acaulis, which suppressed this species after six days of exposure [98].Nonetheless, achieving rapid and complete suppression of S. oryzae adults stands as a significant accomplishment, considering the aforementioned challenges in its control.For instance, in a recent study, combined applications of spinosad with hexaflumuron, lufenuron, and chlorfluazuron on wheat, as well as individual insecticide treatments, resulted in 2.7-56.3%mortalities after seven days of exposure [104].Additionally, it has been documented that the application of 25 µL/mL of nerolidol or linalool on rice after seven days of exposure resulted in 58.22 and 79.72% S. oryzae adult mortality, respectively [105].
Concerning A. siro, the investigation into the effectiveness of environmentally friendly pesticides when applied to commodities is scarce.Only a limited number of natural pesticides, such as zeolites, have undergone assessment for A. siro control.However, EOs have previously showcased their effectiveness against this pest.As per Kavallieratos et al. [69], when 1000 ppm of Pimpinella anisum L. EO-based NE and T. ammi EO-based NE were assessed as single and combined treatments on wheat, they resulted in mortality rates ranging from 29.8 to 38.1% for adults and 21.1 to 86.6% for nymphs, observed 7 days after exposure.The findings of this study align with previously observed mortalities of this pest when subjected to green pesticides.Nonetheless, the efficacy of C. acaulis EO stood out significantly, achieving mortality rates of 91.1% for adults and 95.6% for nymphs of A. siro [98].
Examining various EOs against different pests is imperative due to the diverse outcomes observed, contributing to a more comprehensive pest control strategy.For instance, Kavallieratos et al. [98] demonstrated complete mortality in T. molitor larvae using C. acaulis EO at 1000 ppm over four days, while the EO showed lower effectiveness for adults.Conversely, in our research, T. ammi EO achieved complete mortality in T. molitor adults at the same concentration and exposure interval, with no impact on larvae.This combination suggests potential benefits in integrating these EOs for pest management, underscoring the necessity for thorough, multifaceted investigations.This study expands our understanding of the efficacy of C. maritimum, S. olusatrum, T. ammi, and E. persica EOs against various developmental stages of nine storage pests.Smyrnium olusatrum and T. ammi EOs displayed notable effectiveness against a diverse range of arthropod pests, although the performance of each EO varies concerning the targeted species.The mechanism by which EOs affect insect pests is not completely clear.However, their mode of action always relies on the chemical nature and interactions of their major components.Regarding the pesticidal action of S. olusatrum EO, its bioactivity could be ascribed to the presence of furanosesquiterpenes, mainly represented by isofuranodiene.This compound has already been reported for its marked pesticidal action on T. granarium, P. truncatus, Tetranychus urticae Koch [68,106], but also on T. molitor, T. castaneum, and T. confusum [88].Like other molecules containing the furan moiety, isofuranodiene is susceptible to lactonization [107,108], producing five-membered reactive compounds.The latter, under photo-oxidation, can produce toxic radicals lethal to numerous insects and pests [108,109].In the case of isofuranodiene, its phototoxins could bind, through Michael addition, to the thiol groups of proteins and enzymes such as those involved in the detoxicative functions (e.g., glutathione) [110], with the consequent production of oxidative stress and damage to the tissues.The action of isofuranodiene could be reinforced by that of germacrone and myrcene (20.5 and 29.4% of the EO composition, respectively).Germacrone has been effective against T. urticae, Lasioderma serricorne (F.), and T. castaneum [106,111,112]; while myrcene demonstrated contact toxicity and repellent effects on T. castaneum and Liposcelis bostrychophila Badonnel [113].
Concerning T. ammi EO, its pesticidal potential is strongly correlated with the presence of thymol as a major compound (38.9% of the total composition).Indeed, it was effective against insect vectors such as Culex pipiens L. [114] and Musca domestica L. [115].Thymol is reported as a potent inhibitor of acetylcholinesterase (AChE), but also as a molecule interacting with GABA-A and octopamine receptors [114].In addition to this compound, pcymene (28.0%) and γ-terpinene (28.8%) could contribute to the effect registered for T. ammi EO and synergize the activity of thymol, as reported in previous research [116,117].
Alternatively, γ-terpinene represents one of the major compounds found in C. maritimum EO, accounting for 36.1% of the total EO composition.This molecule has exhibited larvicidal activity against various insects, such as Anopheles anthropophagus (Xu and Feng), Aedes aegypti L., and Aedes albopictus Skuse (Diptera: Culicidae) [118][119][120][121][122][123].It also displayed the ability to suppress the growth and development of Zeugodacus cucurbitae (Coquillett) (Diptera, Tephritidae) larvae, compromising the immune system of the insect [124].As with other monoterpenoids, γ-terpinene has been reported to be a competitive inhibitor of acetylcholinesterase (AChE) [125], but the exact mechanism of action is yet to be defined.The pesticidal action of C. maritimum EO could be ascribed to the action of γ-terpinene, as well as to the synergistic activity of this compound with β-phellandrene, dillapiole, thymol methyl ether, and p-cymene (14.7, 12.0, 11.2, and 10.1% of the total composition, respectively), all of which have demonstrated insecticidal and pesticidal properties [126][127][128].
Concerning E. persica EO, its chemical composition was mainly dominated by γ-terpinene (35.8%) and cumin aldehyde (16.6%).In earlier investigations, γ-terpinene demonstrated insecticidal activity, paralleled by cumin aldehyde, which exhibited toxicity against larvae of both C. pipiens and A. albopictus [129,130].Therefore, the pesticidal activity of E. persica EO could be ascribed to the action of both γ-terpinene and cumin aldehyde, but also to the synergism of these compounds with other minor constituents such as α-terpinen-7-al and γterpinen-7-al.
The wide availability of biomass from S. olusatrum and T. ammi from spontaneous populations and cultivations, respectively, underscores the potential scalability of these EOs within agrochemical applications.Following the outcomes of the current study, further comprehensive assessments of these EOs are advised.These evaluations should encompass individual and combined analyses to determine their effectiveness across diverse insects, mites, commodities, and concentrations.This approach may facilitate the creation of eco-friendly multi-pesticide formulations suitable for storage environments.

Plant Materials
Crithmum maritimum was harvested in 2020 from a cultivation located in the Municipality of Camerano (AN), Italy (N 43 • 32 ′ 08 ′′ ; E 13 • 33 ′ 03 ′′ , 135 m a.s.l., North exposure, with an average incline of 5%).The texture of the soil was silty, with very little presence of sand.The climate at the station is sub-Mediterranean-low-mesotemperate.The cultivation process commenced in 2019 using indigenous seeds.Germination took place in January within a greenhouse, facilitated by a nursery plant tray.Subsequently, transplantation occurred in agricultural land in late March.The cultivation was organic, with no fertilization or irrigation.The plants were not provided with any growing operations or plant protection.Full irradiation was ensured, avoiding the presence of natural weeds through manual operation until the plant reached full-grown habitus to cover its surroundings.Manual seed harvesting occurred in October 2020, coinciding with the full maturation of the seed phase, characterized by the desiccation of the floral structures (umbellifers).Upon collection, the seeds were stored in the farm owner's warehouse, ensuring strict adherence to hygienic, food-related, and GACP (Good Agricultural and Collection Practices) standards.The ripened seeds (schizocarps) of wild T. ammi were collected from Ardabil Province (Iran, N 38  06 ′ , 2053 m a.s.l.) in the province of Khorasan Razavi, at the full ripening stage in June 2020.The plant locations were specified at the flowering stage and, after identification, voucher specimens (codex no 4517 and 4512, respectively) were deposited in the Herbarium of the Department of Horticultural Science, University of Maragheh, Iran.Smyrnium olusatrum flowers were collected in April 2020 during its blooming period from San Severino Marche, central Italy (N 43 • 13 ′ 44.9 ′′ ; E 13 • 10 ′ 29.1 ′′ , 236 m a.s.l.); a voucher specimen (codex: CAME #29339) was stored in the Herbarium Camerinensis, c/o School of Bioscience and Veterinary Medicine, University of Camerino, Italy.

Isolation of EOs
The EOs of the four plant species tested here were obtained through hydro-distillation using a Cleavenger-type apparatus placed on top of a 10 L round flask.A mantle system Falc MA (Falc Instruments, Treviglio, Italy) was employed for heating.Yields of 4.45 and 1.79% (w/w) were obtained from the distillation of 0.6 kg of T. ammi and C. marithimum schizocarps, respectively, in 6 L of deionized water.Concerning E. persica, 0.820 kg of plant seeds were distilled using 7 L of distilled water, and the EO was isolated with a yield of 3.72% (w/w).Regarding S. olusatrum, 2.2 kg of fresh material were placed in 6 L of deionized water, and a yield of 0.90% w/dry weight (dw) was achieved.The dw of the plant material was estimated by determining the water content (81.80%) by drying the fresh flowers in an oven at 110 • C for 16 h.

Chemical Analysis of EOs
The chemical analysis of the EOs was performed employing an Agilent 6890 N gas chromatograph furnished with a single quadrupole 5973N mass spectrometer and an autosampler 7863 (Agilent, Wilmington, DE, USA).The separation of the EOs' constituents was achieved through an HP-5MS capillary column (30 m length, 0.25 mm i.d., 0.1 µm film thickness; 5% phenylmethylpolysiloxane) supplied by Agilent (Folsom, CA, USA).The EOs were diluted 1:100 in n-hexane before the analysis.The analytical conditions and the interpretation of the chromatograms were in line with those previously reported [131].

Insect and Mite Species
The colonies were maintained in complete darkness at the Laboratory of Agricultural Zoology and Entomology, affiliated with the Agricultural University of Athens.Oryzaephilus surinamensis was reared using a blend composed of rolled oats, fragmented brewer's yeast, and wheat in a ratio of 5 parts oats: 1 part yeast: 5 parts wheat.Alphitobius diaperinus were sustained on a diet consisting of yeast and wheat bran in a 1:3 ratio, supplemented with apple cubes for added moisture.Rhyzopertha dominica, T. granarium, and S. oryzae were exclusively maintained on intact wheat grains.Tribolium species were cultivated on a diet composed of wheat flour supplemented with an extra 5% of brewer's yeast.Tenebrio molitor received oat bran and slices of potato to enhance moisture levels.Acarus siro were maintained on a diet comprising wheat germ, brewer's yeast, and oat flakes in a 10:1:10 ratio.For the insect species, the temperature was set at 30 • C with a relative humidity (RH) of 65%, while for the mite species, the conditions were 25 • C at 80% RH.The involved insect participants were of indeterminate sex and under 14 days old for adult T. molitor, Tribolium spp., S. oryzae, R. dominica, and O. surinamensis, younger than 24 h for T. granarium adults, or less than one week old for A. diaperinus adults.Regarding the larvae used in the experiments, they ranged from 3rd to 4th instar for the Tribolium species and O. surinamensis, measured between 1 to 1.4 cm in length for T. molitor, were of medium size (2-4 mm) for T. granarium, or were smaller than 0.7 cm in length for A. diaperinus.The A. siro specimens were of unidentified sex.They were acquired from colonies ranging in age between 1 and 21 days, while identification of nymphs and adults was based on external morphology, particularly discerning shorter body setae in the former [132].

Bioassays
The bioassays commenced consequent to the conduction of preliminary trials that evaluated two concentrations: 500 and 1000 ppm, representing 500 and 1000 µL EO of each EO per kg of wheat, respectively.The test solutions of EOs were formulated by first dissolving 125 and 250 µL of each EO in pure ethanol at a ratio of 1:1 (v/v) for 500 and 1000 ppm, respectively.Subsequently, the mixture was adjusted to reach a total volume of 750 µL using Tween 80 in a 0.3% (v/v) aqueous solution [98].The application of EO solutions was conducted by evenly spraying 0.25 kg of wheat separately on trays via the BD-134 K airbrush (Fengda ® , London, UK), with each tray representing a treatment replicate.Supplementary solutions, comprising (i) water, (ii) 99.8% ethanol, (iii) carrier control (Tween 80 in combination with ethanol and water), and (iv) a positive control, i.e., Actellic EC containing the active component pirimiphos-methyl (50%) at the concentration appearing on the pesticide label (5 ppm = 5 µL/kg wheat), were administered to additional lots for control purposes.Each of these solutions was applied using distinct airbrushes.The treated amounts of wheat were transferred into glass containers with a capacity of 1 L, where a manual agitation lasting 10 min was meticulously performed.This was undertaken to guarantee the uniform dispersion of both the EO solutions and controls throughout the wheat kernels.Following this, three sub-samples of either 10 g or 1 g of wheat, designated for insects and mites, respectively, were extracted from every treated 1 L container of wheat (EO or control) using individual spoons.These wheat sub-samples were precisely weighed on filter paper using the Precisa XB3200D electronic balance (Alpha Analytical Instruments, Athens, Greece).Distinct filter paper was utilized between each weighing.Subsequently, the 10 g and 1 g samples were conveyed into glass vials selected specifically for the insect species (125 mm in height and 75 mm in diameter) or the mite species (60 mm in height and 27 mm in diameter).To facilitate air circulation within the larger vials, their caps were equipped with a 15 mm in diameter opening sealed with fabric, whereas the smaller vials were fitted with caps featuring perforations [99,132].
The interior walls near the vial caps were coated with a 60 wt% by weight dispersion of polytetrafluoroethylene (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) in water to secure the arthropods inside.Arthropod species were individually introduced in groups of 10 adults, larvae, or nymphs into the designated vials.The vials were then placed in incubators set at 30 • C and 65% RH for S. oryzae, R. dominica, O. surinamensis, T. molitor, A. diaperinus, and the Tribolium species, and at 25 • C and 80% RH specifically for A. siro throughout the experimental duration [131].To prevent cannibalism among A. diaperinus individuals, every sub-replication comprised 10 vials, each housing a single arthropod [132].After exposure intervals from 1 to 7 days, mortality assessments were conducted using the Olympus SZX9 stereomicroscope (Bacacos S.A., Athens, Greece).Dead specimens were carefully examined with individual fine brushes for each concentration of controls or EOs, ensuring that any movement from the arthropods was detected.This entire process was repeated three times, comprising three replications and three sub-replications, incorporating new sets of wheat, arthropods, and glass vials for each iteration.

Data Analysis
Control mortality, staying below 5%, required no adjustment.To standardize variance, the dataset was log (x + 1) transformed before analysis [133,134].Data on each arthropod species underwent analysis using a repeated measures model, with exposure as the repeated factor and mortality as the response variable.The main effects considered were EO type and concentration [135].All analyses were executed using JMP 16.2 software [136].For mean separation, the Tukey-Kramer HSD test at a 0.05 significance level was applied [137].
a Compounds are reported depending on the order of their elution from an HP-5MS capillary column.bLinearretention index experimentally obtained through the analysis of a homologous series of C 7 -C 30 alkanes on HP-5MS capillary column.cLinearretention index reported inAdams (2007)[Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry; Allured Publishing Corp., Carol Stream, IL, USA].d Relative area percentage values are means of two independent analyses, showing RSD% values < 20% in all cases.e Identification methods: Std, derived from the comparison with pure standards; MS, derived from comparison with WILEY, ADAMS, and NIST 08 MS libraries; RI, based on comparison of RI with those found in ADAMS, FFNSC2, and NIST 08 libraries.f tr, % < 0.05.g Results of the quantitative analysis are altered due to thermal degradation phenomena.

Table 2 .
MANOVA parameters depicting the main effects and their interactions leading to the observed mortalities of Alphitobius diaperinus, Tribolium castaneum, T. confusum, Tenebrio molitor, Trogoderma granarium, and Oryzaephilus surinamensis larvae and adults, Rhyzopertha dominica and Sitophilus oryzae adults, and Acarus siro nymphs and adults, between and within exposures (error df = 80 for all species and developmental stages).

Table 3 .
Mean (%) mortality ± standard errors (SEs) of Alphitobius diaperinus larvae and adults after 1-7 days in wheat treated with Crithmum maritimum, Smyrnium olusatrum, Trachyspermum ammi, and Elwendia persica essential oils (EOs) at two concentrations, with positive control, pirimiphos-methyl.No statistical analysis was performed where dashes exist.Due to zero values, mortality data for adults exposed to any EO at 500 ppm are not shown.Due to zero values, mortality data for adults exposed to S. lustratum and T. ammi EOs at 1000 ppm are not shown.
For each EO, within each row, means followed by the same uppercase letter are not significantly different (df = 6, 62; Tukey HSD test at p = 0.05).For each concentration, within each column, means followed by the same lowercase letter are not significantly different (df = 4, 44; Tukey HSD test at p = 0.05).No significant differences were recorded where no letters exist.No statistical analysis was performed where dashes exist.Due to zero values, mortality data for adults exposed to C. maritimum, S. olustratum, and T. ammi EOs at 500 ppm are not shown.

Table 7 .
Mean (%) mortality ± standard errors (SE) of Trogoderma granarium larvae and adults after 1-7 days in wheat treated with Crithmum maritimum, Smyrnium olusatrum, Trachyspermum ammi, and Elwendia persica essential oils (EOs) at two concentrations, with positive control, pirimiphos-methyl.For each EO, within each row, means followed by the same uppercase letter are not significantly different (df = 6, 62; Tukey HSD test at p = 0.05).For each concentration, within each column, means followed by the same lowercase letter are not significantly different (df = 4, 44; Tukey HSD test at p = 0.05).No significant differences were recorded where no letters exist.Due to zero values, mortality data for larvae exposed to C. maritimum and E. persica EOs at 500 ppm are not shown.