Botanical Oils Isolated from Simmondsia chinensis and Rosmarinus officinalis Cultivated in Northern Egypt: Chemical Composition and Insecticidal Activity against Sitophilus oryzae (L.) and Tribolium castaneum (Herbst)

The rice weevil, Sitophilus oryzae (L.), and the red flour beetle, Tribolium castaneum (Herbst), are key stored-product pests in Egypt and worldwide. The extensive use of synthetic insecticides has led to adverse effects on the environment, human health, and pest resistance. As a result, environmentally friendly pest management alternatives are desperately required. The botanical oils of jojoba, Simmondsia chinensis (L.), and rosemary, Rosmarinus officinalis L. plants growing in Egypt were extracted, identified by gas chromatography/mass spectrometry (GC–MS), and evaluated for their insecticidal activity against S. oryzae and T. castaneum. The main constituents identified in BOs were carvyl acetate (20.73%) and retinol (16.75%) for S. chinensis and camphor (15.57%), coumarin (15.19%), verbenone (14.82%), and 1,8-cineole (6.76%) for R. officinalis. The S. chinensis and R. officinalis BOs caused significant contact toxicities against S. oryzae and T. castaneum adults, providing LC50 values of 24.37, 68.47, and 11.58, 141.8 ppm at 3 days after treatment (DAT), respectively. S. chinensis oil exhibited significant fumigation toxicity against both insects; however, it was more effective against S. oryzae (LC50 = 29.52 ppm/L air) than against T. castaneum (LC50 = 113.47 ppm/L air) at 3 DAT. Although the essential oil (EO) of R. officinalis significantly showed fumigation toxicity for S. oryzae (LC50 = 256.1 and 0.028 ppm/L air at 1 and 3 DAT, respectively), it was not effective against T. castaneum. These BOs could be beneficial for establishing IPM programs for suppressing S. oryzae and T. castaneum.

Rosmarinus officinalis L. (Lamiales: Lamiaceae) is a medicinal plant native to Europe, but it has been cultivated in different areas around the world [31][32][33]. The EO of R. officinalis has been widely used in medicine due to its powerful antibacterial, cytotoxic, antimutagenic, antioxidant, antiphlogistic, and chemopreventive characteristics [34,35]. In a trial conducted in Argentina, the EO of R. officinalis was found to be highly effective on Tribolium castaneum [36]. The jojoba plant, Simmondsia chinensis (L.) (Caryophyllales: Simmondsiaceae), is a monotypic species native to the Sonoran Desert in North America [37]. The oil of jojoba seeds is a mixture of long-chain esters (97-98%) of fatty acids and fatty alcohols. It has been reported that the oil has significant analgesic, antipyretic, anti-inflammatory, antioxidant, anti-bacterial, and anti-parasitic properties [38]. Furthermore, it has a lethal effect on the adults of S. oryzae (L.) [39].
The bioactivity of these BOs is affected by their chemical profiles [40][41][42][43][44]. Furthermore, the environmental conditions of different regions impact the chemical constituents of these plants [35,40]. It has been reported that BOs collected from different growing areas at different periods have different chemical compositions and therefore may exhibit different biological activities [35,41]. However, while many studies have been conducted in different countries to assess the insecticidal performance of various BOs against stored-product insects, the available data on BOs extracted from plants growing in Egypt and their bioactivity against common stored-product pests are scarce. Therefore, in the present study, we extracted S. chinensis and R. officinalis BOs from plants cultivated in Northern Egypt and identified their chemical composition by GC-MS. In addition, the toxicity of those isolated BOs against the rice weevil, S. oryzae, and the red flour beetle, T. castaneum, was studied under laboratory conditions.

Contact Bioassay
Mortality of S. oryzae and T. castaneum adults caused by the contact bioassay of different concentrations of S. chinensis BO at 1, 2, and 3 DAT is shown in Table 2. All the evaluated concentrations of S. chinensis oil significantly reduced the number of treated S. oryzae adults at 1, 2, and 3 DAT in comparison with controls. The most effective activity was caused by using a concentration of 200 ppm of S. chinensis oil, showing 90, 93.3, and 96.7% mortality at 1, 2, and 3 DAT, respectively. The same trend was repeated against T. castaneum adults, except for concentrations of 10 and 20 ppm, which were not significantly different compared to control at 1 and 2 DAT. The concentration of 200 ppm showed the best activity at 1, 2, and 3 DAT. It is observed that mortality of both insects treated with S. chinensis BO was increased when oil concentration and/or exposure time were increased. The estimated contact LC 50 values were 54.35, 36.94, and 24.37 ppm/cm 2 for S. oryzae, whereas in the case of T. castaneum they were 181.3, 101.5, and 68.47 ppm/cm 2 , respectively, at 1, 2, and 3 DAT (Table 3). Table 2. Mortality (±SD) of S. oryzae and T. castaneum adults following contact bioassay of S. chinensis oil.

Mortality of S. oryzae
Mortality of T. castaneum 0.00 ± 0.00 f 0.00 ± 0.00 e 0.00 ± 0.00 f 0.00 ± 0.00 e 0.00 ± 0.00 e 0.00 ± 0.00 f The treatments of high concentrations (≥50 ppm/cm 2 ) of R. officinalis EO showed significant contact toxicities of S. oryzae adults higher than control at 1 DAT (Table 4). Moreover, all the evaluated concentrations significantly decreased the number of S. oryzae adults at 2 and 3 DAT in comparison with control. The lowest two concentrations of R. officinalis EO (10 and 20 ppm/cm 2 ) were not able to cause a reduction in the number of adults T. castaneum at 1, 2, or 3 DAT, showing no mortality. The highest concentration (200 ppm/cm 2 ) of R. officinalis EO showed significant activity against T. castaneum adults, providing 40, 53.3, and 67% mortality, respectively, at 1, 2, and 3 DAT. The LC 50 values of R. officinalis EO recorded on S. oryzae were 115.8, 40.54, and 11.58 ppm/cm 2 at 1, 2, and 3 DAT, respectively ( Table 5). The values decreased by about a third every day of exposure time. At 1, 2, and 3 DAT, the LC 50 values against T. castaneum were 281.9, 213.7, and 141.8 ppm/cm 2 , respectively. The LC 50 values recorded against T. castaneum were higher than those against S. oryzae. 0.00 ± 0.00 e 0.00 ± 0.00 e 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 d 0.00 ± 0.00 d

Discussion
Different previous studies have documented the insecticidal characteristics of many plant-derived substances that would enable them to play an effective role in the integrated pest management (IPM) of stored-product pests. These substances may be advantageous because they are selective for pests and have no or little negative impact on non-target organisms or the environment [1,27]. Furthermore, many of these products are biodegradable by soil microorganisms and are low in toxicity to mammals [42]. BOs and their major constituents are complementary tools for pest control, in particular of stored-product insects [23]. They can be used in IPM of stored-product insects for pest prevention, early pest detection, or pest control [15,43,44]. In the present study, the BOs of S. chinensis and R. officinalis were extracted from local plants growing in Egypt, identified by GC-MS, and evaluated against S. oryzae and T. castaneum by contact and fumigation bioassays under laboratory conditions. The chemical compositions of the extracts from R. officinalis and S. chinensis BOs were similar to those previously reported for the oils isolated from plants cultivated in Egypt [44] and other countries around the world [31,42,[45][46][47][48][49][50]. However, the percentages of constituents differed. This can be attributed to many factors, including the difference in geographical site, collection period, environmental and climate conditions, and the nutritional status of the plants [44,51]. In 2016, Abdelgaleil et. al. extracted the EO of R. officinalis growing in the same area of plant collection as the present study (Northern Egypt) [44]. The main common constituents identified in both studies were 1,8-cineole, camphor, a-pinene, verbenone, and L-linalool. Moreover, in the current study, we report the presence of coumarin (15.19%) and hymecromone (5.56%) in the R. officinalis EO. However, additional research and information are ideally required to support our findings regarding the presence of those compounds. These two compounds have previously been found in plant extracts such as Orysan sativa [52]. The variation of the active ingredients within a plant extract would improve its mechanism of action and thus its biological effectiveness [53]. It is reported that linalool has an acetylcholinesterase inhibition and repellence effect against some insects [54,55]. Furthermore, the presence of terpenes and aromatic volatile compounds in the BOs had anti-bacterial and anti-fungal effects and protected food products for a long time without affecting their quality [54,56]. The 1.8-cineole was found to be highly effective against adults of S. oryzae when it was applied at a concentration of 0.1 mL/720 mL volume at 24 h of exposure, whereas camphor was found to be more effective towards Rhyzopertha dominica, with mortality of 100% [57,58]. The GC-MS analysis revealed that the major component found in S. chinensis BO was carvyl acetate (20.73%). It is a natural product identified in different plant sources such as citrus, Mentha pulegium, and Santolina chamaecyparissus [59].
In this study, both oils of S. chinensis and R. officinalis exhibited different degrees of toxicity against S. oryzae and T. castaneum. These results are in harmony with those previously found by Abdel-Rahman and Mahmoud [60], who observed high contact activity of S. chinensis oil against S. oryzae adults. The estimated LC 50 and LC 90 values of S. chinensis against S. oryzae were 1.17 and 2.76% (v/v), respectively, at 4 DAT. In a trial conducted in Egypt, the repellent and toxicant effects of eight BOs on T. castaneum adults were studied [54]. The oil of S. chinensis provided 73.33% adult repellency at 180 min after exposure at a concentration of 15% (v/v). The contact toxicity of the S. chinensis BO at the same concentration showed a relatively low LC 50 value (10.73% v/v) at 1 DAT [54].
However, the degree of toxicity depended on the bioassay method [44] and the targeted insect. For example, S. chinensis BO was effectively excellent against S. oryzae (90% mortality) and moderate against T. castaneum (70% mortality) when it was used in the contact bioassay at 1 DAT. In addition, the S. chinensis and R. officinalis BOs were significantly effective as contact toxicants against both S. oryzae and T. castaneum adults. However, both were more effective against S. oryzae than on T. castaneum adults. While S. chinensis oil exhibited significant fumigation toxicity against S. oryzae (LC 50 = 29.52 ppm/L air), higher than that on T. castaneum (LC 50 = 1134.7 ppm/L air), the R. officinalis EO had a moderate effect on S. oryzae (LC 50 = 256.1 ppm/L air at 1 DAT) and was ineffective against T. castaneum. Those results are in harmony with those obtained by Saroukolai et al. (2010), who found that the extracted Thymus persicus EO was 70 times more effective against S. oryzae than T. castaneum adults [1]. In different studies, R. officinalis EO showed strong fumigation toxicity against S. oryzae. The estimated LC 50 values varied from 26.71 mg/L air to 53.6 286 µL/L air [29,60]. Similarly, Origanum vulgare EO was found to be an efficacious fumigant rather than a contact against S. oryzae [44]. Furthermore, A. monosperma and P. graveolens had high contact toxicity against S. oryzae but were ineffective fumigants [44]. On the other hand, the oil of Cupressus sempervirens caused strong fumigant toxicity for S. oryzae but was not effective in the contact method [61]. In summary, the results of the biological performance of these botanical oils is affected by application methods [62,63], and this should be considered when developing IPM programs for such insects.

Insect Colonies
S. oryzae and T. castaneum insects used in the current experiment originated from a susceptible strain that was previously reared in the laboratory of the Plant Protection Department, Faculty of Agriculture (Saba-Basha), Alexandria University, Egypt. For S. oryzae rearing, four hundred adults (mixed sexes and ages) were placed into 2 L glass jars containing 500 g of sterilized wheat grains (var. Sakha 68) [5,6,64]. To avoid the escape of insects, about 7-10 cm from the inside upper part of the used jars were painted with Vaseline. For well ventilation, jars were covered with pieces of muslin fixed to the neck of the jars by two rubber bands [65]. After one week, all the added adults were removed using 8-mesh sieves, and the culture was kept in a hygrothermal conditioned cabinet (28 ± 2 • C and 70 ± 5% R.H.). The culture was thereafter investigated daily, and the emerged adults were used in bioassays. These procedures were performed to obtain groups of adults of the same and known age. All the same procedures and conditions used with S. oryzae were followed in T. castaneum rearing except the used medium (wheat flour, bran, and dry yeast at a rate of 17:5:1, respectively). Adult insects used in the current experiment were 2-3 weeks old [43].

Extraction of Botanical Oils
The seeds of jojoba, Simmondsia chinensis (L.), were collected from a farm located in Al-Adl village, Gharb Al-Nubaria region, Nubaria city, Al-Behieira Governorate, Egypt (30 • 39 51.9 N, 30 • 07 28.0 E). Healthy jojoba seeds were selected and transferred to the laboratory of the Plant Protection Department, Faculty of Agriculture (Saba Basha), Alexandria University in plastic bags. The BO from seeds was extracted by the pressing method [66]. The seeds were well dried for 14 days. The oil extraction process was made at the room temperature, using a manual hydraulic press (SPECAC, London, UK) with a load configuration of 0-5 tones. About 50 g of seeds was added to the press cylinder and subjected to the pressing process. The yielded oil (v/w%) was determined and then saved for further chemical analysis and bioassays. For the extraction of R. officinalis EO, fresh leaves were collected from a known nursery located in the Abees region, Alexandria governorate, Egypt, and transferred in plastic bags to the laboratory. Leaves were dried at room temperature (26 ± 1 • C) for five days and were subjected to hydro-distillation using a Clevenger apparatus for 6 h [67][68][69][70][71]. The resulting oil was filtered, dried over anhydrous sodium sulfate, expressed as v/w% of the dry matter [44], and stored in the laboratory refrigerator at 4 • C until usage.

Chemical Composition of Botanical Oils
The chemical composition of BOs was performed using a Thermo-Logical Gas Chromatography (GC Follow 1300)/Mass Spectrometer (ISQ7000 show; Thermo Logical) apparatus (Agilent Technologies, Santa Clara, CA, USA) [47,62]. A Thermo TR-50MS capillary column (30 m in length × 250 µm in breadth × 0.25 µm in thickness of film) was used as a GC column. The spectroscopic location in GC-MS included an electron ionization framework that used high-energy electrons (70 eV) and a 300 • C MS exchange line temperature. Unadulterated helium gas (99.995%) was used as the carrier gas with a flow rate of 1 mL/min. The column temperature was programmed (60 • C for 2 min, 100 • C at 10 • C/min for 5 min, 150 • C at 10 • C/min for 5 min, 200 • C at 10 • C/min for 5 min, and 250 • C at 10 • C/min for 20 min). One microliter of the arranged extricates was infused in a partless mode.

Contact Bioassay
The contact activity of six serial concentrations (10,20,50,100,150, and 200 ppm) of both S. chinensis and R. officinalis BOs was evaluated against the adults of S. Oryzae and T. castaneum with the method previously described [29]. The BOs were diluted in acetone (Al-Nasr Pharmaceutical Chemicals Co., Obour, Egypt). One mL of each concentration was placed in a 9 cm Petri dish by a micropipette and spread uniformly on the whole surface of the dish. Acetone was allowed to evaporate, leaving a thin film of the oils on the surface of dishes [41]. Twenty adults (same age and weight) of each insect were separately added to the Petri dish. Dishes without BOs acted as controls. All treatments were replicated five times. At 1, 2, and 3 days after treatment (DAT), the percentages of adult mortality were recorded and corrected using Abbott's formula [1,72].

Fumigation Bioassay
To evaluate the fumigation toxicity of BOs against S. oryzae and T. castaneum adults, six oil concentrations (10, 20, 50, 100, 150, and 200 ppm/L air) were evaluated following the previously described bioassay [29,74]. One-liter glass jars were used as fumigation chambers. Each oil concentration was evenly added to a filter paper piece (2 × 3 cm) fixed in the subsurface of the screw caps of jars. The inner side of the jar's neck was painted with Vaseline to prevent direct contact of insects with the treated filter paper. Caps were directly screwed tightly onto the jars, each containing 20 adults (same age and weight). The filter papers in the controls were treated with acetone only. Each treatment was replicated five times. The adult mortality was calculated at 1, 2, and 3 DAT, and the LC 50 values (ppm/L air) were considered [73].

Statistical Analysis
The generalized linear model (GLM) was used to perform a one-way analysis of variance on the insect mortality data [32]. Means were then compared by the Duncan's least significant difference (LSD) test [75] using SAS software V. 8.2 (SAS Institute Inc., Cary, NC, USA) [76]. Differences were considered significant at α = 0.05. The LdP line computerized software program was used to calculate the probit analyses of LC 50 values and their fiducial limits (confidence intervals) for botanical oils according to Finney (1971) [77].

Conclusions
Botanical oils have been widely investigated for their biological activity against a wide range of agricultural pests, including stored-product pests. Many studies have confirmed the potency of these products; however, their toxicity is dependent on a number of factors (e.g., the chemical composition of the BO, the targeted pest, and the bioassay method). The data of the current study suggest the high contact and fumigation effects of S. chinensis and R. officinalis BOs against S. oryzae and T. castaneum. Thus, it can be concluded that these BOs are a promising approach in terms of decreasing chemical pesticide use, and they should be considered for an effective IPM strategy for S. oryzae and T. castaneum.

Conflicts of Interest:
The authors declare no conflict of interest.

BO
Botanical oil EO Essential oil LC 50 Lethal concentration for 50% of tested insects IPM Integrated pest management GC-MS Gas chromatography/mass spectrometry DAT Days after treatment RT Retention time