Identification of Volatile Compounds and Insecticidal Activity of Essential Oils from Origanum compactum Benth. and Rosmarinus officinalis L. against Callosobruchus maculatus (Fab.)

This work was undertaken to investigate the volatile compounds and insecticidal activity of essential oils (EOs) from Origanum compactum Benth. and Rosmarinus oﬃcinalis L. against the crop pest Callosobruchus maculatus (Fab.). Essential oils of Origanum compactum (EOC) and Rosmarinus oﬃcinalis L . (EOR) were extracted by use of hydrodistillation, and their volatile compounds were proﬁled by gas chromatography-mass spectrometry (GC-MS). The insecticidal activity of extracted EOC and EOR was evaluated against C. maculatus . GC-MS analysis revealed that carvacrol (70.88%) and 1,8-cineole (62.35%) were the major constituents of EOC and EOR, respectively. EOC exhibited a potent insecticidal activity with calculated LC 50 values of 6.77 and 3.57 μ L/L air, 24 and 48h posttreatment, respectively. Comparable LC 50 values were obtained for EOR recording 6.25 and 3.82 μ L/ L, 48h posttreatment. The eﬀects of fumigation by the tested EOs on fertility (egg hatching) and the emergence of adult C. maculatus were also investigated. Notably, EOC completely abolished egg fertility judged by the abrogation of emergence of adults, regardless of the tested dose. By contrast, EOR completely inhibited the fertility and the emergence of C. maculatus adults at the dose of 16 μ L/10g. The outcome of the present study highlights the utility of the EOs from O. compactum Benth. and R. oﬃcinalis L. as natural sources of eﬀective and ecofriendly pest-control agents.


Introduction
Nowadays, many environmental challenges influence agriculture worldwide. In addition to poor soil quality and cultivation techniques, there are problems related to insect pests. Pests attacking stored food legumes result in significant damage and loss of both quality and quantity. While losses attributed to pests are estimated to be around 40% in Africa, they do not exceed 3% in developed countries [1].
Despite the magnitude of losses caused by insect pests, a limited number of studies have shed light on pests in Africa. In Morocco, C. maculatus causes serious damage to stored legume foods, where C. maculatus larvae develop and feed on the cotyledons of legumes, particularly when no measures are taken. Pests are capable of destroying a crop within 4-5 months according to the Food and Agriculture Organization (FAO). Losses due to insect pests reached 35% of global agricultural production [2].
C. maculatus can be considered as one of the most growing challenges throughout the tropical and subtropical regions. It is a worrying pest of several pulses including Vigna unguiculata, Cicer arietinum, Glycine max, and Phaseolus vulgaris.
ese pulses are an important food source for millions of people based in tropical and subtropical areas. Cowpea seeds are most attacked pulses by C. maculatus and cause maximum damage, which could reach 2-5 kg seeds within 45-90 days when stored under optimal temperature (30 ± 10 C) and moisture conditions (75 ± 3%) [3].
Insecticides represent one of the most used control methods to manage insect pests. However, the resistance of insects to modern insecticides is still a great challenge facing chemical insecticides. In addition, these chemicals can possess risks to consumers and cause even harmful effects in the long term [4]. e plant kingdom represents a potentially effective alternative as a source of natural pest-control agents. Aromatic plants contain essential oils (EOs) that possess natural insecticidal activities. Hence, the insectcontrolling potential of plant-derived EOs has been widely tested against pests attacking stored grains through their insecticidal potencies [5][6][7][8]. In this context, several researchers have reported lethal effects of EOs against plant and human pests [9,10].
is work aimed to investigate the profile of volatile compounds and fumigant activity of EOs from Origanum compactum Benth. and Rosmarinus officinalis L. against Callosobruchus maculatus (Fab).

Materials and Methods
2.1. Insect Breeding. C. maculatus was obtained from a local warehouse and subsequently bred in glass jars with 500 g of Vigna unguiculata seeds. Jars were maintained at a temperature of 27 ± 1°C, relative humidity of 70 ± 5%, and a photoperiod of 14 h (light)/10 h (dark).

Plant
Material. O. compactum was harvested from the region of Taounate from Morocco, whereas R. officinalis was harvested from the region of Taza, Morocco. ereafter, the studied plants were identified by a botanist before being deposited at the Herbarium of Sidi Mohamed Ben Abdellah University. Next, the leaves were cleaned and dried in the shade at room temperature for 15 days.

Extraction of Essential Oils.
One hundred grams of O. compactum and R. officinalis leaves were soaked in 750 mL of distilled water before being extracted at 100°C by use of a Clevenger apparatus for 4 h. e obtained EOs were dehydrated with anhydrous sodium sulfate before being stored in a refrigerator at 4°C until further use [11].

Gas Chromatography-Mass Spectrometry Analysis.
e volatile compounds of the studied plants were determined by using GC-MS. Briefly, 0.1 μL of the sample was injected for analysis using a gas chromatograph coupled to a mass spectrophotometer (Agilent Technologies 5973 with an Agilent 19091S-433 HP-5MS column, 30 m long, 0.25 mm inner diameter, and 0.25 μm film thickness of the stationary phase) in positive mode. Helium was used as the carrier gas, with a typical pressure range (psi) of 0.9 mL/sec. e oven temperature program was set between 60 and 300°C for 10 min and then held at 300°C for 20 min. e detector temperature was set at 250°C, whilst the injector temperature was set at 260°C. Identification of compounds was performed by comparing retention times with standards of the database [12].

Insecticidal Activity Test.
e insecticidal activity test was carried out to evaluate the activity of the essential oils in a vapor phase as reported in earlier work [11]. To achieve this goal, Whatman paper discs (3 × 3 cm) impregnated with different concentrations of the tested EOs (4,8,12,16, and 20 μL/L of air) were attached to the inner surface of the stoppers of each jar to avoid their direct contact with the insects. Next, 10 g of cowpea seed and five pairs of C. maculatus aged from 0 to 48 h were separately introduced into each jar. Total mortality of insect individuals by each dose was recorded daily for 5 days. Egg-laying capacity of the females of C. maculatus was calculated by use of a magnifying binocular. Jars were subsequently maintained at a temperature of 27 ± 1°C, relative humidity of 70 ± 5%, and a photoperiod of 14 h (light)/10 h (dark) until the emergence phase of adults.

Statistical Analysis.
e results were expressed as means (±SD). A two-way analysis of variance (ANOVA) was used to analyze the effect of varying doses and exposure periods on mortality and fecundity of females and the emergence of adult C. maculatus. Significant differences between treatments were calculated by using Tukey's multiple range tests (p < 0.05). e lethal concentration LC 50 , LC 90 , chi-square, and 95% confidence intervals for each regression coefficient were calculated by use of probit analysis [13]. A significant difference was considered when p < 0.05.

Journal of Chemistry
As given in Table 4, both EOC and EOR showed dose and exposure time-dependent insecticidal activities, leading to100% of adult mortality 72 h postexposure. At the highest dose, EOC induced 90.0% of adult mortality (20 μL/L air/ 10 g) 24 and 48-hour posttreatment, whereas at the highest dose, EOR exhibited 100% of adult mortality 24-hour posttreatment. No mortalities were recorded in control groups.

Effect on Fecundity.
e fecundity of C. maculatus females was strongly affected by the insecticidal effects of EOs tested. e obtained results showed a significant decrease in the number of eggs laid by females after being exposed to the vapor of EOC and EOR relative to the control ( Figure 3 and Table 5). At the highest dose used for testing (20 μL/10 g seeds), the two EOs completely inhibited the fecundity of females relative to the control value of 196.66 ± 11.54. ANOVA analysis indicated that the EOmediated toxicity against C. maculatus fecundity was highly significant as a function of increasing concentrations (F � 1123.48; df � 5, 24; p < 0.0001). Moreover, there is no significant difference between EOC and EOR towards the fecundity of females (F � 6.31; df � 1, 24; P � 0.0191). is can be explained by the fact that C. maculatus has sensitivity towards EOs of the tested aromatic plants.

Effect on Fertility.
e obtained results showed that both EOC and EOR significantly reduced the egg hatchability when compared to the control in a dose and time-dependent manner ( Figure 4 and Table 5). For all tested EOC doses, egg hatching was not recorded compared to the control fecundity rate of 94.02 ± 4.08.
Similarly, EOR exhibited a potent egg hatching inhibitory effect, wherein the dose of 16 μL/10 g, resulted in complete abrogation of egg hatchability (Figure 4; Table 5). EOC possessed a toxic effect on the fertility of C. maculatus eggs irrespective of the tested dose, whereas EOR inhibited the fertility at the highest tested dose. erefore, EOC exhibited a far more potent inhibitory effect on egg hatchability than EOR.   Figure 3: Effects of essential oil of EOC and EOC on the fecundity of C. maculatus (mean ± SD).

Effect on Adult Emergence.
e obtained results showed no C. maculatus adults' emergence in Vigna unguiculata. Seeds provisory treated with EOC regardless of the used dose; meanwhile, the total inhibition of C. maculatus adults' emergence by EOR was recorded for the hastiest dose used for testing 16 μL/10 g ( Figure 5 and Table 5). erefore, EOC exhibited higher inhibitory activity on the emergence rate of C. maculatus adults as compared to that of EOR.

Discussion
e obtained results showed that EOC was higher in carvacrol (70.88%), which is in agreement with a previously published study [14], reporting that carvacrol was the major constituent of EO extracted from Origanum compactum Benth. (43.97%). Additionally, other studies reported variable concentrations of carvacrol present in EO of O. compactum of 47.85% and 31.22% [15]. e current study, the result of which are reported here, found that 1,8-cineole (62.35%) was the major compound of EOR. Similarly, these findings were conforming to those reported by Ait-Ouazzou and co-workers [16], who showed that 1,8-cineole was the main constituent of the EO extracted from R. officinalis (43.99%).
Considering the insecticidal activity of the tested EOs against C. maculatus, the obtained results clearly indicated that EOC efficiently controlled C. maculatus, which is in agreement with previous reports [17], which revealed that EO of O. compactum possessed high insecticidal activity against Spodoptera littoralis larvae, with an LD 50 of 0.05 mL/ larva. Similarly, O. compactum possessed the insecticidal effect against adults of Musca domestica and Mayetiola destructor [17−19]. In this study, EOR was shown to be effective against C. maculatus, which is corroborated by findings reported by another previous work [20]. In that study, authors reported that EO of R. officinalis was bioactive against C. maculatus. Accordingly, Douiri and co-workers reported the insecticidal effect for EO of Rosmarinus species on C. maculatus males and females with LC 50 varying from 5.51 to 2.43 μL/L air and 6.80 to 3.04 μL/L air, respectively [21].
In the present work, the insecticidal effect of the studied oils resulted in a significant reduction in the number of eggs laid per female. It is thus fitting to conclude that our results were comparable with those reported by Douiri and co-workers [22], who showed that EOs from Asteraceae species efficiently controlled C. maculatus potently impacting their fecundity, longevity, fertility (89.03-93.40%), and success rate (80-90%). In addition, LC 50 was determined to be 2.5 and 23.3 μL/L of air for females and 2.56 and 46.07 μL/L for males. In this context, Bounechada et al. stated that the leaf powder of Ocimum basilicum completely abrogated the emergence of Trogoderma granarium, which  16 2.66 ± 0.57   Journal of Chemistry indicated that Ocimum basilicum might serve as an ecofriendly control agent specifically for this pest species [2]. Furthermore, it was reported that at a dose of 33.3 μL/L, the essential oils of Melaleuca quinquenervia and Ocimum gratissimum significantly reduced the oviposition of the C. maculatus females by 98.78% ± 0.87 and 99.94 ± 0.35%, respectively [23,24]. In this study, the obtained results showed that EOs efficiently controlled the fertility of C. maculatus (hatching eggs). In this case, EOC completely inhibited the hatching of eggs laid by the female C. maculatus, regardless of the used dose. Meanwhile, the total inhibition of hatching eggs by EOR was obtained by the highest dose used. Specifically, at the dose of 400 μL, EOs extracted from O. basilicum and O. gratissimum inhibited the hatching of eggs of C. maculatus [25]. Similarly, Ketoh et al. stated that C. schoenanthus EO inhibited hatching egg and development of neonate C. maculatus larvae at the dose of 33.3 μL/mL [26]. In addition, EO of Z. multiflora has been previously reported to exhibit a strong insecticidal effect against eggs, larvae, and adults of C. maculatus [27].
Our results also showed that the tested EOs efficiently controlled the emergence of C. maculatus adults. EOC has completely prevented the total emergence of adults irrespective of the concentration used, whereas EOR prevented the emergence of C. maculatus when applied at the highest dose. Moussa Kéïta et al. [25] with a drop in the emergence of C. maculatus adults to 0 and 4% follow exposure to EOs of Ocimum basilicum and Ocimum gratissimum. It was also reported that the emergence of C. maculatus F 1 adults was significantly inhibited by EO of Alpinia calcarata at concentrations of 0.80 g/L using fumigant toxicity [28]. Similarly, the emergence of C. maculatus has been previously reported to be also controlled by Allium sativum [21].
EOs from aromatic plants exhibit a potent insecticidal effect by fumigation, contact, and repulsion assays [5,8,29]. EOs are known for their ovicidal, repellent, and insecticidal activities against various insects attacking stored products [29]. e mechanism of action (MOA) of EOs against insects was investigated by Renoz and co-workers who reported that EOs resulted in Sitophilus granarius death by altering a variety of key biological processes and activities, namely, muscular and neurological systems, cellular respiration, protein synthesis, development, reproduction, and insects' behavior [30]. Rajendran et al. reported that terpenoids have gained particular attention among other constituents of EOs because of their potent fumigant effect against stored grain insects [31]. In this context, it has been postulated that C. maculatus could absorb EOs along with their components, for example, terpenoids. Consequently, the toxicity of the tested EOs in the current study is hypothesized to be attributed to the presence of carvacrol [32,33].
In the present work, the insecticidal activity of both EOC and EOR could be due to bioactive compounds identified in the oils, particularly carvacrol, which is known for its bioactivity including the insecticidal effect [34]. In the current study, carvacrol was found to be the major component in EOC with 70.88%, whereas 1,8cineole was reported to be the dominant constituent in EOR with 62.35%. Taken together, these monoterpene compounds could be responsible for the biological activity of these EOs. Additionally, 1,8-cineole, borneol, and thymol have been previously reported to exert adverse toxicities against S. oryzae adults at the lowest dose (0.1 μL/720 mL volume), 24 h posttreatment by fumigation [35]. Camphor and linalool caused 100% mortality for R. dominica adults, and this has been attributed to monoterpenoids contained in EOs accounting for the observed insecticidal activity [4]. It was also reported that carvacrol, linalool, thymol, terpineol, and eugenol inhibited the emergence of A. obtectus adults [36,37]. Citral and 1,8-cineole contained in EO were found to be ovicides and strong inhibitors of the emergence of adult houseflies. Eugenol and (−)-menthone powerfully inhibited adult emergence C. maculatus adults [38]. e MOA by which terpenes can exert this insecticidal effect have been reported in an earlier work [28]. EO constituents can operate synergistically or individually depending on which insect pest is being targeted. For example, the two components D-limonene and α-terpineol showed a synergistic toxicity against Trichoplusia ni, whereas no correlation was found with toxicity against Spodoptera frugiperda.
e MOA underlying the synergistic interaction of 1,8-cineole and camphor, the major constituent of the EO of R. officinalis against Trichoplusia ni, has already been reported by Tak et al. [39], who showed that 1,8-cineole enhances the penetration of camphor into the blood circulation through the insect's body wall referred to as integument.
e MOA of the reported insecticidal activity of EOs has been thoroughly investigated by Rattan and co-workers [40], who reported that EOs and their components, particularly thymol, result in insect death through the inhibition of acetylcholinesterase, thereby leading to its accumulation eventually causing hyperstimulation of nicotinic and muscarinic receptors and disrupted neurotransmission. Moreover, it might act by blocking the octopamine receptors through tyramine receptors cascade or by disruption of the octopaminergic system [40].

Conclusion
e obtained results revealed that the studied EOs efficiently controlled the insect life cycle, which could be attributed to its richness in specific bioactive monoterpenoid alcohols such as carvacrol. Taken together, the outcome of the present study highlights the benefits of the EOs extracted from Origanum compactum Benth. and Rosmarinus officinalis L. as effective ecofriendly pestcontrol agents. Further investigation is therefore warranted to evaluate the safety of these EOs and their nontarget toxicities against mammals and humans.

Data Availability
e data used to support the findings are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.