Toxicological Activity of Some Plant Essential Oils Against Tribolium castaneum and Culex pipiens Larvae

In the present work, essential oils (EOs) from Schinus terebinthifolius (ripe and unripe fruits and leaves), Origanum majorana (air-dried aerial parts), and Psidium guajava (leaves) were assayed for their insecticidal activity against red flour beetle (Tribolium castaneum) and Culex mosquito larvae (Culex pipiens). Several components were identified in the EOs using Gas chromatography–mass spectrometry (GC/MS), of which ∆-3-carene (25.9%), γ-terpinene (19.4), and γ-elemene (7.1%) were the major ones in S. terebinthifolius ripe fruits, α-pinene (48.9%), germacrene D (12.9%), and α-thujene (7.7%) in S. terebinthifolius unripe fruits, γ-elemene (11.7%), spathulenol (10.1%), β-elemene (9.2%), and p-cymene (9.1%) in S. terebinthifolius leaves, α-pinene (25.5%), (E)-caryophyllene (15.7%), (E)-nerolidol (16.7%), and cedran-8-ol (8.8%) in P. guajava leaves, and terpinen-4-ol (21.7%), γ-terpinene (16.5%), and sabinene (10.1%) in O. majorana air-dried aerial parts. The lethal concentration (LC50) was calculated for tested EOs at different time periods (after 6, 12, 24, 48, and 72 h). After 6 h of treatment, the LC50 was 33.3 and 6.8 μg/L air for S. terebinthifolius ripe and unripe fruits, respectively, and >40 μg/L air for EOs of S. terebinthifolius leaves, O. majoranaair-dried aerial parts, and P. guajava leaves. After 24 h of treatment, the LC50 was 4.2, <2, 5, >40, and 6.1 μg/L air for EOs of S. terebinthifolius ripe fruits and leaves, O. majorana leaves, and P. guajava leaves, respectively. On the other hand, the LC50 values decreased when the exposed period was increased to 72 h, and were <2 μg/L air for EOs of S. terebinthifolius ripe fruits, unripe fruits, and leaves along with P. guajava leaves, respectively, and 37.912 for EO of O. majorana leaves. The LC50 value after 24 h of exposure of S. terebinthifolius unripe fruit EO was under 2 μg/L air, which means that the EO of S. terebinthifolius ripe fruit had a strong effect on adult T. castaneum adults compared to other tested EOs using the fumigation method. The present data confirm that the EOs of O. majorana leaves and S. terebinthifolius unripe fruits and leaves were more effective as larvicide than the EOs of S. terebinthifolius ripe fruits and P. guajava leaves on C. pipiens at a higher concentration (100 mg/L) when applied by the dipping method. EOs from S. terebinthifolius unripe or ripe fruits and leaves and P. guajava leaves were more effective as adulticide than EO of O. majorana leaves against T. castaneum when applied by the fumigant method.


Introduction
Botanicals are basically secondary metabolites that serve as a defence mechanism for plants to withstand the continuous selection pressure from herbivore predators and other environmental factors.

Fumigant Assay on Red Flour Beetle
The fumigation experiment was carried out at 26 ± 1 • C and 65 ± 5% RH. Newly emerged adults (1-15 days old) were used in fumigant studies. The fumigant method for the 5 EOs was tested against T. castaneum adults. Glass jars (1 L) were used as fumigation chambers (replicates) and filter paper pieces (3 × 3 cm) were joined to the undersurface of the screw caps of the jars. The 5 EOs were applied to the filter paper pieces by 2, 5, 10, 20, and 40 µL/L air. Every jar as a replicate containing 20 insects as treatment and control were repeated 3 times. Filter paper pieces were treated with acetone (Loba Chemie Pvt. Ltd., laboratory reagents & fine chemicals, Mumbai, India) alone as a control. Control insects were kept under the same conditions with acetone. The insect mortality percentage was observed after 6, 24, 48, and 72 h of treatment and the lethal concentration causing 50% mortality (LC 50 ) expressed as mg/L air was calculated from log-concentration mortality regression lines. Insects were considered dead when no leg or antenna movements were recorded. The fumigant method assay was performed as described by Finney [49], El-Bakry et al. [50], and Huang et al. [51].

Bioassay Toxicity of Mosquitos
The tested EOs of S. terebinthifolius ripe and unripe fruits and leaves, O. majorana air-dried aerial parts, and P. guajava leaves were examined for bioassays [30] on newly second instar larvae of C. pipiens. This experiment was conducted by the dipping method using four concentrations of each oil (10,25,50, and 100 mg/L). Three replicates for each concentration were prepared. Each replicate including 50 C. pipiens larvae was separately put into a 200-mL plastic cup containing 100 mL of distilled water. The tested EO solutions were added to the cups and suspended with 0.05 mL of Tween-20.
The C. pipiens larvae were exposed to 10, 25, 50, and 100 mg/L of tested EOs in 100 mL of distilled water. In the control cups, only solvent (absolute acetone) was dissolved in the water. Treated and control larvae were held in the same conditions used for colony rearing. Larval mortality was recorded 24 and 48 h after treatment and continued to the end of the larval stage. Larvae were considered dead when they did not rise to the surface of the solution or when they did not respond to a stimulus. Additionally, pupal and adult mortality was calculated. The longevity parameter was calculated for each development stage of C. pipiens.

Red Flour Beetle Experiment
Fumigant Toxicity of Tested Essential Oils Figure 1 shows the statistical significance of the main effects (oil source, oil concentration, and time of exposure). Oil of S. terebinthifolius unripe fruits showed the highest mortality of T. castaneum ( Figure 1A). With increased oil concentration and exposure time, mortality increased significantly ( Figure 1B,C). Additionally, the interaction between two factors ( Figure 1D-F) showed significant effects on the mortality percentage of T. castaneum.

4).
The fumigant experiment applied to adult T. castaneum with different times and concentrations showed that adult mortality increased gradually with increased concentrations from 2 to 40 µg/L air and time from 6 h to 72 h of exposure. Figure 2 illustrates the effects of tested EOs on adult T. castaneum, with dead insects (shown in black) due to accumulation of CO2 in the tracheas of insects treated with the fumigation method, when compared to normal T. castaneum (in brown).   The mortality values were 31.66% and 75% after 24 h of exposure to EO of O. majorana leaves and P. guajava leaves, respectively, and were 56.66% and 93.33% after 48 h. The mortality values were 68.33% and 100% after 72 h with EO O. majorana leaves and P. guajava leaves at 40 µg/L air, respectively ( Table 4). The great effect of EO of S. terebinthifolius unripe fruits on adult T. castaneum after 6 h of exposure at 40 µg/L air was shown by adult mortality of 76.66%. After 6 h at 40 µg/L air with EO of S. terebinthifolius leaves, O. majorana leaves, and P. guajava leaves, the adult mortality was under 50%, while it was 61.66% and 76.66% with EO of S. terebinthifolius ripe and unripe fruits, respectively. After 72 h at 5 µg/L air with EOs of S. terebinthifolius ripe fruits, unripe fruits, and leaves, O. majorana leaves, and P. guajava leaves, adult mortality was 86.66%, 100%, 90%, 28.33%, and 88.33%, respectively, while the control was recorded as a standard reference. After 12 and 24 h of exposure with acetone as the control, mortality was 1.66%, but was 3.33% after 48 h and 8.33% after 72 h ( Table 4).
The fumigant experiment applied to adult T. castaneum with different times and concentrations showed that adult mortality increased gradually with increased concentrations from 2 to 40 µg/L air and time from 6 h to 72 h of exposure. Figure 2 illustrates the effects of tested EOs on adult T. castaneum, with dead insects (shown in black) due to accumulation of CO 2 in the tracheas of insects treated with the fumigation method, when compared to normal T. castaneum (in brown).     Table 3 presents the mortality percentages of T. castaneum as affected by the three factors, oil source, oil concentration, and time period, with fumigant application. After 48 h of treatment, the mortality ranged from 58.3% to 100% with EO of S. terebinthifolius ripe fruits, was 100% with EO of S. terebinthifolius unripe fruits, ranged from 55% to 100% with EO of S. terebinthifolius leaves, and ranged from 16.6% to 56.6% with EO of O. majorana leaves, and 61.6% to 93.3% with EO of P. guajava leaves. By comparison, mortality was 8.3% with the control ( Table 2).
The lethal concentration causing 50% mortality (LC 50 ) of T. castaneum was calculated for tested EOs at different time periods (6,12,24,48, and 72 h). After 6 h of treatment, the LC 50 was 33.3, 6.8, >40, >40, and >40 µg/L air for EOs of S. terebinthifolius ripe fruits, S. terebinthifolius unripe fruits, S. terebinthifolius leaves, O. majorana leaves, and P. guajava leaves, respectively. After 24 h of treatment, the LC 50 was 4.2, <2, 5.1, >40, and 6.1 µg/L air for EOs of S. terebinthifolius ripe fruits, S. terebinthifolius leaves, O. majorana leaves, and P. guajava leaves, respectively. After 24 h of treatment, the LC 50 of S. terebinthifolius unripe fruit oil was under 2 µg/L air, which means that the EO of S. terebinthifolius ripe fruits had a stronger effect on T. castaneum adults than other tested EOs using the fumigation method (Table 5).  Table 6 shows the significant effects of oil concentrations and oil sources and their interaction with mortality and longevity of C. pipiens at different stages (larval, pupal, and adult). All treatments showed highly significant effects on mortality and longevity except the interaction between the EO source and the EO concentration for longevity at the larval stage. The EOs were tested for their toxicity against the second instar larvae of C. pipiens. The five EOs showed pronounced insecticidal activity on immature stages (larva and pupa). After 24 h of treatment with EO of S. terebinthifolius ripe fruits, unripe fruits, and leaves, O. majorana leaves, and P. guajava leaves, the larval mortality was 15.3%, 34.6%, 30.6%, 36.6%, and 16.6% at 100 mg/L, respectively ( Table 7). The larval mortality recorded after 48 h of treatment with the tested EOs was 17.3%, 36.6%, 32.6%, 38.6%, and 18.6% at 100 mg/L, respectively.
The total larval mortality was recorded during the larval stage for each concentration to examine the larvicidal activity of the tested EOs against C. pipiens. Table 7 shows that total larval mortality ranged from 26% to 33.3% with EO of S. terebinthifolius ripe fruits, 40.6% to 68% with EO of S. terebinthifolius unripe fruits, 30% to 50% with EO of S. terebinthifolius leaves, 42.6% to 78% with EO of O. majorana leaves, and 24% to 36.6% with EO of P. guajava leaves at 10 to 100 mg/L, and was 3.3% as a control. Mortality increased with growing concentration and time of exposure.
The present data confirm that the EOs of O. majorana leaves and S. terebinthifolius unripe fruits and leaves were more effective as larvicide than EOs of S. terebinthifolius ripe fruits and P. guajava leaves on C. pipiens at a higher concentration (100 mg/L). Figures 3 and 4 show the destroyed digestive system (rupture) in larvae of C. pipiens, which results in increased larval mortality within a short time   The present data confirm that the EOs of O. majorana leaves and S. terebinthifolius unripe fruits and leaves were more effective as larvicide than EOs of S. terebinthifolius ripe fruits and P. guajava leaves on C. pipiens at a higher concentration (100 mg/L). Figures 3 and 4 show the destroyed digestive system (rupture) in larvae of C. pipiens, which results in increased larval mortality within a short time (24-48 h) with treatment by EO of S. terebinthifolius unripe fruits, while EO of O. majorana leaves led to a 78% mortality at 100 mg/L.
The effects of the tested EOs on immature stages were recorded as mortality percentages. As shown in Table 3, the mortality percentages increased gradually with increased oil concentration (from 10 to 100 mg/L). Pupal mortality ranged from 26% to 40% with EO of S. terebinthifolius ripe fruits, 42

Adult Stage
Adult mortality ranged from 36% to 58% with EO of S. terebinthifolius ripe fruits, 58.6% to 94% with EO of S. terebinthifolius unripe fruits, 46% to 78% with EO of S. terebinthifolius leaves, 62.6% to 100% with EO of O. majorana leaves, and 34.6% to 63.3% with EO of P. guajava leaves at 100 mg/L, and was 5.3% in the control. Mortality increased with a growing concentration and time of exposure (Table 7).
Adult longevity reached 27.3, 15.4, 20.5, 11.8, and 24.9 days with 100 mg/L of EO of S. terebinthifolius ripe fruits, unripe fruits, and leaves, O. majorana leaves, and P. guajava leaves, respectively, and was 44.3 days with the control. EO from O. majorana leaves and S. terebinthifolius unripe fruits strongly reduced adult longevity by approximately 65% to 73% when compared with the control, which means that both EOs had insecticidal activity on the adult stage, which is an important vector for severe and highly infectious diseases in humans.

Lethal Concentrations of LC50
The results were obtained using probit regression line parameters of C. pipiens with five essential oils at five interval concentrations, and the lethal concentration causing 50% mortality (LC50) was calculated for the tested EOs on larval and adult stages at different time periods (after 6, 12, 24, 48, and 72 h) to examine the larvicidal and insecticidal activity.
The LC50 values of total larval mortality were >100, 31.2, >100, 24.1, and >100 mg/L for EOs of S. terebinthifolius ripe fruits, unripe fruits, and leaves, O. majorana leaves, and P. guajava leaves (Table  8), respectively. This means that the oils of O. majorana leaves and S. terebinthifolius unripe fruits had stronger larvicidal activity against C. pipiens larvae than the other tested EOs applied by the dipping method.
In addition, Table 8 shows that the LC50 of adults was >50, 10.9, 20.1, 9.7, and >50 mg/L for EOs of S. terebinthifolius ripe fruits, unripe fruits, and leaves, O. majorana leaves, and P. guajava leaves, The effects of the tested EOs on immature stages were recorded as mortality percentages. As shown in Table 3, the mortality percentages increased gradually with increased oil concentration (from 10 to 100 mg/L). Pupal mortality ranged from 26% to 40% with EO of S. terebinthifolius ripe fruits, 42.6% to 72% with EO of S. terebinthifolius unripe fruits, 30% to 58% with EO of S. terebinthifolius leaves, 44.6% to 82% with EO of O. majorana leaves, and 24% to 42.6% with EO of P. guajava leaves at 10 to 100 mg/L. The tested EO of S. terebinthifolius ripe fruits, S. terebinthifolius unripe fruits, S. terebinthifolius leaves, O. majorana leaves, and P. guajava leaves affected larval and pupal longevity of C. pipiens. Larval longevity at 100 mg/L was 20.6, 16.3, 18.2, 14.8, and 19.9 days, respectively, while it was 8.3 days in the control (Table 7).
On the other hand, pupal longevity was affected by treatment with 100 mg/L of EO of S. terebinthifolius ripe or unripe fruits and leaves, O. majorana leaves, and P. guajava leaves, with values at 63.4, 37.1, 43.1, and 46.4 h, respectively, while it was 32.2 h in the control (Table 4).

Adult Stage
Adult mortality ranged from 36% to 58% with EO of S. terebinthifolius ripe fruits, 58.6% to 94% with EO of S. terebinthifolius unripe fruits, 46% to 78% with EO of S. terebinthifolius leaves, 62.6% to 100% with EO of O. majorana leaves, and 34.6% to 63.3% with EO of P. guajava leaves at 100 mg/L, and was 5.3% in the control. Mortality increased with a growing concentration and time of exposure (Table 7).
Adult longevity reached 27.3, 15.4, 20.5, 11.8, and 24.9 days with 100 mg/L of EO of S. terebinthifolius ripe fruits, unripe fruits, and leaves, O. majorana leaves, and P. guajava leaves, respectively, and was 44.3 days with the control. EO from O. majorana leaves and S. terebinthifolius unripe fruits strongly reduced adult longevity by approximately 65% to 73% when compared with the control, which means that both EOs had insecticidal activity on the adult stage, which is an important vector for severe and highly infectious diseases in humans.

Lethal Concentrations of LC 50
The results were obtained using probit regression line parameters of C. pipiens with five essential oils at five interval concentrations, and the lethal concentration causing 50% mortality (LC 50 ) was calculated for the tested EOs on larval and adult stages at different time periods (after 6, 12, 24, 48, and 72 h) to examine the larvicidal and insecticidal activity.
The LC 50 values of total larval mortality were >100, 31.2, >100, 24.1, and >100 mg/L for EOs of S. terebinthifolius ripe fruits, unripe fruits, and leaves, O. majorana leaves, and P. guajava leaves (Table 8), respectively. This means that the oils of O. majorana leaves and S. terebinthifolius unripe fruits had stronger larvicidal activity against C. pipiens larvae than the other tested EOs applied by the dipping method. In addition, Table 8 shows that the LC 50 of adults was >50, 10.9, 20.1, 9.7, and >50 mg/L for EOs of S. terebinthifolius ripe fruits, unripe fruits, and leaves, O. majorana leaves, and P. guajava leaves, respectively. Therefore, the essential oils of O. majorana leaves and S. terebinthifolius unripe fruits had strong insecticidal activity against C. pipiens.

Chemical Constituents of the Essential Oils
Several compounds have been identified in the studied plant materials. α-Pinene was identified with a high percentage in EO from unripe fruits of S. terebinthifolius, which agreed with Ennigrou et al. [21], who reported that α-pinene was found in amounts of 26.3% (immature fruits) and 13.9% (mature fruits). α-Cadinol, elemol, germacrene-D, and ∆-3-carene are the most common compounds identified in the EO of leaves and fruits of S. terebinthifolius [53]. ∆-3-carene (25.9%) was the most abundant compound in EO of S. terebinthifolius ripe fruits. Previously it was reported that the main chemical compounds of EO from S. terebinthifolius ripe fruits from Brazil were myrcene, limonene, and germacrene-D [54], while, in another report, ∆-3-carene, and α-pinene dominated in fruit EO [23].

Fumigant Toxicity on T. Castaneum
The LC 50 ranged from <2 to 33.3 µg/L air for EO of S. terebinthifolius ripe fruits, <2 to 6.8 µg/L air for EO of S. terebinthifolius unripe fruits, <2 to 65.1 µg/L air for EO of S. terebinthifolius leaves, 37.9 to >40 µg/L air for EO of O. majorana leaves, and <2 to 60.2 µg/L air for EO of P. guajava leaves, which means that the EO of S. terebinthifolius unripe fruits had a stronger effect on T. castaneum adults than the other tested EOs using the fumigation method. Our results agree with those of Abdelgaleil et al. [68] who reported that the EO of O. vulgare (LC 50 = 1.6 µg/L air) was the most potent toxicant against S. oryzae adults. At the same time, EO of S. terebinthifolius possessed strong fumigant toxicity (LC 50 <30 mg/L air).
Savory and marjoram EOs had 72.5% and 67.5% mortality, respectively, on T. castaneum adults when exposed to 150 µL/L air for 24 h [69]. The insecticidal activity of oil of Origanum leaves in a vapor-phase toxicity bioassay against T. castaneum adults reached LC 50 = 73.7 µL/L air [70], while the EOs obtained from leaves and flowers showed insecticidal activity against T. castaneum adults [71]. Thymol and other compounds of O. majorana EO, showed insecticidal activity against S. oryzae and R. dominica adults [72].
The P. guajava treatments caused significantly higher mortality at 21 days of exposure when compared to the control. None of the treatments of P. guajava achieved 100% mortality throughout the experimental period. Since mortality was found to be directly proportional to exposure time and concentration, increased mortality might be attained by increasing either or both [73].
For the mode of toxic action, some monoterpenes had an inhibitory effect on acetylcholinesterase activity [74,75], bound with octopamine receptors [76] and GABA-gated chloride ion channels [77].

Mosquitocide Activity of Tested Essential Oils
In this study, five EOs belonging to several classes was examined to compare their relative toxicity against C. pipiens larvae. The EOs of O. majorana leaves and S. terebinthifolius leaves and unripe fruits showed larvicidal toxicity. The tested EOs had LC 50 values for the larval and adult stages under 100 mg/L (9.7-90.9 mg/L), except for the EOs of S. terebinthifolius ripe fruits and leaves as well as P. guajava leaves, which had LC 50 of total larval mortality of 18,475.3, 115.6, and 1719.1 mg/L, respectively. Therefore, the EOs of O. majorana leaves, S. terebinthifolius leaves, and unripe fruits EOs have potential as effective mosquitocides. In addition, the bioactivity of most monoterpenes against C. pipiens was evaluated in the present experiment. The leaves of the Origanum herb are rich in EO, which confers its characteristic and fragrance. The larval toxicity of some plant extracts, EOs, and phytochemicals against C. pipiens has been reported [78][79][80][81].
With the present results, total larval mortality at 10 to 100 mg/L ranged from 40.6% to 68% with EO of S. terebinthifolius unripe fruits, while it was 42.6% to 78% with EO of O. majorana leaves. Mortality increased with a growing concentration and time of exposure. The present data confirms that the EOs of O. majorana leaves as well as S. terebinthifolius unripe fruits and leaves had a more larvicidal effect than EOs of S. terebinthifolius ripe fruits and P. guajava leaves on C. pipiens at the higher concentration (100 mg/L).
The tested EOs of S. terebinthifolius ripe/unripe fruits and leaves, O. majorana leaves, and P. guajava leaves affected larval and pupal longevity of C. pipiens due to prolonged larval longevity. Larval longevity at 100 mg/L was 20.6, 16.3, 18.2, 14.8, and 19.9 days, respectively. Similar to Abd El Meguid et al. [82], the toxicological activity of four plant oils including O. majorana had prominent mosquitocidal activity against A. caspius and C. pipiens, along with toxic effects against larvae and pupae.
The most abundant identified compound of EOs of S. terebinthifolia fruits and seeds was ∆-3-carene and the least abundant identified compound was γ-elemene. The EOs were observed to have mosquitocidal activity against An. gambiae, An. Arabiensis, and C. quinquefasciatus. The mortality of C. quinquefasciatus ranged from 0.5% to 96.7%, and of An. gambiae from 13.7% to 97.9% [23].
From the present results, the adult mortality ranged from 36% to 58% with EO of S. terebinthifolius ripe fruits, 58.6% to 94% with EO of S. terebinthifolius unripe fruits, 46% to 78% with EO of S. terebinthifolius leaves, 62.6% to 100% with EO of O. majorana leaves, and 34.6% to 63.3% with EO of P. guajava leaves at 100 mg/L. The LC 50  From the previously identified chemical components in the tested EOs, it can be considered that they have insecticidal properties against immature stages of C. pipiens and the adult stage of T. castaneum.

Conclusions
The present data confirm that the essential oils of O. majorana leaves and S. terebinthifolius unripe fruits and leaves have more larvicidal effect than those of S. terebinthifolius ripe fruits and P. guajava leaves on C. pipiens at a higher concentration (100 mg/L) when applied by the dipping method. Additionally, EOs of S. terebinthifolius unripe and ripe fruits, P. guajava leaves, and S. terebinthifolius leaves have more adulticidal effect than O. majorana leaf oil against T. castaneum when applied by the fumigant method.