Ocimum basilicum Essential Oil

AWad, Mona, Nancy, Amer ·, Hassan, N. & M., Moataz A., 2024, Towards Sustainable Pest Management: Toxicity, Biochemical Effects, and Molecular Docking Analysis of Ocimum basilicum (Lamiaceae) Essential Oil on Agrotis ipsilon and Spodoptera littoralis (Lepidoptera: Noctuidae), Neotropical Entomology 53 (3), pp. 669-681 : 672-676

Chemical Composition of Ocimum basilicum Essential Oil

The chemical compounds of basil EO were identified using GC–MS as shown in Table 1 and Fig. 1 . The main bioactive compounds included linalool (29.34%), 3,7-dimethyl-2,6-octadienal (13.16%), 2,6-octadienal, 3,7-dimethyl-, (Z)- (8.82%), and 3-cyclohexen-1-ol,4-methyl-1-(1-methylethyl)- (7.20%).

Toxicity of O. basilicum EO to A. ipsilon and S. littoralis Larvae

LC 15 and LC 50 values of O. basilicum EO to the second instar larvae of both insects are shown in Table 2 . The LC 15 values were 706.29 and 784.93 mg /L while the LC 50 values were 2748.04 and 2665.70 mg /L to A. ipsilon and S. littoralis , respectively ( Table 2 ).

Effect of O. basilicum EO on the Development of A. ipsilon and S. littoralis

As shown in Table 3 , when the 2nd instar larvae of both insects were treated with the LC 15 and LC 50 of O. basilicum , a highly significant elongation in the larval duration (F = 25.63; df = 3, 378; P <0.0001) was recorded. Nevertheless, no significant difference (P> 0.05) was observed in the pupal duration except for the case of LC 50 with A. ipsilon . In addition, no significant difference was found in pupation (F = 0.39; df = 2, 17; P = 0.682), emergence (F = 0.14; df = 2, 17; P = 0.874), or female pupal weight (F = 6.12; df= 2,169; P = 0.003). Instead, the male pupal weight (F = 0.33; df = 2, 173; P = 0.721) of S. littoralis decreased after treating the larvae with LC 15 and LC 50 values. The proportion of emerged females of A. ipsilon slightly decreased (by 0.58-fold) after the treatment of the second instar larvae with LC 15 (LC 15: Χ 2 = 4.26; P =0.039) while it slightly increased (by 1.22-fold) after the treatment with LC 50 (Χ 2 = 4.17; P = 0.041). As shown in Fig. 2 , the same pattern was recorded for S. littoralis (LC 15: Χ 2 = 0.22; P = 0.642 andLC 50: Χ 2 = 0.06; P = 0.814).

Effect of O. basilicum EO on Detoxifying Enzymes

CarE (a -esterase and β -esterase), cytochrome P-450, and GST activities were assessed after 24, 48, 72, and 96 h of treating the 2nd instar larvae of A. ipsilon and S. littoralis with O. basilicum EO. As shown in Table 4 , the CarE activities increased with all treatments in A. ipsilon , while were decreased in S. littoralis ( Table 5 ). In contrast, O. basilicum EO significantly induced the MFO activity in both insects ( Table 4 and 5 ) after 24, 48, and 72 h from treatments. Interestingly, GST activity significantly increased in A. ipsilon ( Table 4 ) and decreased in S. littoralis ( Table 5 ).

Docking Investigation

Docking on the receptor of GST (PDB ID: 1PN9) The docking procedure ( Fig. 3 ) was initially validated by re-docking with the co-crystallized s-Hexylglutathione ligand (GTX) in the enzyme binding pocket with an energy score (S) of − 4.558 kcal/mol. As shown in Table 6 , the docking energy score of the docked compound (linalool) with the enzyme receptor (PDB ID: 1PN9) was − 4.7748 kcal/mol, which is higher than that of the co-crystallized ligand. In addition, linalool bonded with serine (SER 9) residue ( Table 6 ). The overall bonding connections by OH-bonds of the relevant amino acid residue against the docked molecule are depicted in Fig. 3 .

Means that do not share a letter in row are significantly different

Discussion

Insecticide resistance is a critical problem in insect management. Resistance develops through such mechanisms as resistance to penetration, target-site alteration, and enhanced activity of detoxification enzymes ( Tangtrakulwanich and Reddy 2014). Accordingly, essential oils have been used as insecticides due to their ability to act on multiple targets. They can enhance the insecticidal effect and are promising as an alternative to traditional insecticides ( Isman 2020; Duque et al. 2023). In general, the Ocimum genus is well known for its insecticidal effect against diverse insect pests ( Rodríguez-González et al. 2019). The basic chemical composition of Ocimum plants is highly variable and may rely on the genetic properties of the plant and the cultivation conditions ( Vieira and Simon 2000). Herein, we analyzed and identified the chemical composition of basil ( O. basilicum ) EO using GC–MS and the analysis revealed that the major constituents were linalool (29.34%), the most abundant compound, 3,7-dimethyl-2,6-octadienal (13.16%), 2,6-octadienal, 3,7-dimethyl-, (Z)- (8.82%), and 3-cyclohexen-1-ol,4-methyl-1-(1-methylethyl)- (7.20%). The results also revealed that this plant may belong to linalool chemotype, which could have a repellent and toxic activities against insects ( Rozman et al. 2007; Chaaban et al. 2019).

Regarding the toxicity of O. basilicum EO , no significant difference in its LC 50 values between A. ipsilon and S. littoralis (about 1.03-fold). Beside toxicity, the sublethal effects on the behavioral and physiological parameters may play a key role in insect pests management (de Araújo et al. 2017). Our experiment showed that the LC 15 and LC 50 values of O. basilicum EO significantly prolonged the larval duration in both insects, in comparison with the control. Similarly, the pupal durations of A. ipsilon were significantly prolonged after administering the 2nd instar larvae with LC 50 of O. basilicum EO. However , no significant differences in the pupal duration of S. littoralis were recorded after treating the 2nd instar larvae with LC 15 and LC 50, compared to the control.

Means that do not share a letter in column are significantly different

Means that do not share a letter in column are significantly different

As to pupation, emergence percentage, and female pupal weight, no significant difference was observed after the treatment of the second instar larvae of both insects with LC 15 and LC 50 of O. basilicum EO. Nevertheless , the male pupal weight of S. littoralis was significantly decreased after treating the larvae with LC 15 and LC 50 values. It has been reported that poor nutrition before pupation affected pupa development and prolonged the pupa duration ( Aqueel et al. 2015). Earlier studies also confirmed the sublethal effects of chemical or bio-insecticides in a number of lepidopteran pests including A. ipsilon ( Moustafa et al. 2021a and 2022), S. littoralis ( Moustafa et al. 2021b and 2023a), Plutella xylostella (Linnaeus) ( Lepidoptera : Plutellidae ) ( Wang et al. 2023), Mamestra brassicae (Linnaeus) ( Lepidoptera : Noctuidae ) ( Moustafa et al. 2016 and 2023b), and Tuta absoluta ( Kandil et al. 2020) . According to Santos et al. (2017) and Huisamen et al. (2023), the sublethal effect on individuals that is followed by physiological impairment can negatively affect the insect activities and population growth.

Detoxification enzymes are key players in insecticide metabolism in insects ( Fouad et al. 2022; You et al. 2022; Aioub et al. 2023; Moustafa et al. 2023c; Prasannakumar et al. 2023). Insect resistance is usually accompanied with enhanced activity of these enzymes ( David et al. 2013). In fact, the EOs mode of action needs further understanding. The EOs elicit such distinct neurotoxic symptoms as hyperactivity, agitation, paralysis, and knockdown ( Ahmadi et al. 2022). Besides, some studies reported that EOs inhibit detoxifying enzymes (P450s, CarEs, and GSTs) in insects ( Tak et al. 2016; Huang et al. 2020). As a target for insecticides, GST is crucial for pesticide detoxification. It converts lipid metabolites of insecticides or combines with toxic molecules via chelation, to protect tissues from oxidative stress ( Korkina 2016; Liao et al. 2017). As revealed by our results, GST activities increased significantly by 4.4-, 4.2-, 2.4-, and 2.1-fold after 24, 48, 72, and 96 h of treating A. ipsilon larvae with LC 50 of O. basilicum EO. However , a different pattern was recorded for S. littoralis larvae , where theLC 50 of O. basilicum EO caused a noticeable inhibition of GST activity 3.8-, 2.5-, 3.6-, and 6.1-fold after 24, 48, 72, and 96 h after treatment, as compared with control. The inhibition of GST can be attributed to the presence of various ingredients in basil EO that act through various modes of action ( Liao et al. 2017) while the GST enhanced activity could be an indicator of the adaptation of insects to xenobiotics ( Koirala et al. 2022). The cytochrome P450 system protects insects from poisons ( Liao et al. 2017). Our study indicated that O. basilicum EO significantly induced the cytochrome P450 activity by about 2.3-fold in A. ipsilon larvae after 24 h of treatment and by about 1.5-, 1.7-, and twofold in S. littoralis larvae after 24, 48, and 72 h of treatment with the LC 50.

Interestingly, the esterase family of enzymes hydrolyzes ester bonds in insecticides and changes their activities that would result in chemical stress in insects ( Gong et al. 2021). Our results showed that the treatment of the 2nd instar larvae withLC 15 and LC 50 of O. basilicum EO caused a significant increase in β -esterase activity in A. ipsilon whereas a significant decline was observed in S. littoralis , after 48 and 72 h of treatment.

Being more sensitive to essential oils than P450s and CarEs, GST may serve as the primary target of essential oils. Therefore, the decrease in GST activity may be one major cause of insect mortality. In this context, the molecular docking study enables us to specify the most optimal ligands for the GST enzyme. The docking energy score of linalool, the main constituent of basil EO, with the enzyme receptor (PDB ID: 1PN9) was higher than that of the co-crystallized ligand, which confirmed the strong binding between the compound and the receptor. In docking simulations, a lower energy score indicates stronger binding or greater engagement ( Shahbaaz et al. 2017). Our current findings are consistent with the experimental findings of in vitro assay. Interaction with the detoxification enzymes is recognized to be the most critical element influencing the biological activity of the compounds against enzymes. For instance, the interaction between citral in Cymbopogon citratus EO and cytochrome P-450 enzyme of S. littoralis was reported by Moustafa et al. (2023a). Overall, the results could provide better understanding of the mode of action of O. basilicum EO at the molecular level, particularly linalool binding affinity with GST receptor.

Figures

Fig.1 Chemical structure of the main bioactive compounds of basil EO, O. basilicum

Fig. 2 Sex ratio of the emerged adults of Agrotis ipsilon and Spodoptera littoralis after treating the 2nd instar larvae with LC15 and LC50 of Ocimum basilicum essential oil

Fig. 3 2D and 3D molecular docking simulation of the interactions between the s-Hexylglutathione ligand (GTX) in the enzyme binding pocket and linalool with the active site of the receptor of GST (PDB ID: 1PN9)