Evaluation of the Toxicity of Satureja intermedia C. A. Mey Essential Oil to Storage and Greenhouse Insect Pests and a Predator Ladybird

The use of chemical insecticides has had several side-effects, such as environmental contamination, foodborne residues, and human health threats. The utilization of plant-derived essential oils as efficient bio-rational agents has been acknowledged in pest management strategies. In the present study, the fumigant toxicity of essential oil isolated from Satureja intermedia was assessed against cosmopolitan stored-product insect pests: Trogoderma granarium Everts (khapra beetle), Rhyzopertha dominica (Fabricius) (lesser grain borer), Tribolium castaneum (Herbst) (red flour beetle), and Oryzaephilus surinamensis (L.) (saw-toothed grain beetle). The essential oil had significant fumigant toxicity against tested insects, which positively depended on essential oil concentrations and the exposure times. Comparative contact toxicity of S. intermedia essential oil was measured against Aphis nerii Boyer de Fonscolombe (oleander aphid) and its predator Coccinella septempunctata L. (seven-spot ladybird). Adult females of A. nerii were more susceptible to the contact toxicity than the C. septempunctata adults. The dominant compounds in the essential oil of S. intermedia were thymol (48.1%), carvacrol (11.8%), p-cymene (8.1%), and γ-terpinene (8.1%). The high fumigant toxicity against four major stored-product insect pests, the significant aphidicidal effect on A. nerii, and relative safety to the general predator C. septempunctata make terpene-rich S. intermedia essential oil a potential candidate for use as a plant-based alternative to the detrimental synthetic insecticides.

Oleander aphid {Aphis nerii Boyer de Fonscolombe (Hemiptera: Aphididae)}, as a cosmopolitan obligate parthenogenetic aphid, is a common insect pest of many ornamental plants comprising several species of Asclepiadaceae, Apocynaceae, Asteraceae, Convolvulaceae, and Euphorbiaceae, especially in greenhouse conditions. Along with direct damage, A. nerii is able to transmit pathogenic (hold for 1 min), increase to 100 • C at 8 • /min, increase to 185 • C at 5 • /min, increase to 280 • C at 15 • /min, and hold at 280 • C for 2 min. The carrier gas was helium (99.999%) with flow rate of 1 mL/min. Essential oil was diluted in methanol, and 1 µL solution was injected (split 1:10 at 0.75 min). The identification of components was performed by comparing mass spectral fragmentation patterns and retention indices with those reported in the databases [28][29][30].

Insects
The required colonies of Oryzaephilus surinamensis and Rhyzopertha dominica were reared on wheat grains for several generations at the Department of Plant Production, Moghan College of Agriculture and Natural Resources, University of Mohaghegh Ardabili (Ardabil province, Iran). Tribolium castaneum and Trogoderma granarium adults were collected from infested stored wheat grains in Moghan region (Ardabil province, Iran). Insects were identified by Asgar Ebadollahi. Fifty unsexed pairs of adult insects were separately released onto wheat grains and removed from breeding container after 48 h. Wheat grains contaminated with insect eggs were separately kept in an incubator at 25 ± 2 • C, 65 ± 5% relative humidity and a photoperiod of 14:10 (L:D) h. Finally, one to fourteen-day-old adults of O. surinamensis, R. dominica, T. castaneum and T. granarium were designated for fumigant bio-assays.
Aphis nerii and its natural predator Coccinella septempunctata were used to evaluate the contact toxicity of the S. intermedia essential oil. Cohorts of apterous adult females of A. nerii and unsexed adults of C. septempunctata were taken directly from homegrown oleander (Nerium oleander L.) and a chemically untreated alfalfa (Medicago sativa L.) field (Moghan region, Ardabil province, Iran), respectively.

Fumigant Toxicity
The fumigant toxicity of S. intermedia essential oil was tested on adults of O. surinamensis, R. dominica, T. castaneum, and T. granarium. To determine the fumigant toxicity of the essential oil, filter papers (Whatman No. 1, 2 × 2 cm) were impregnated with essential oil concentrations and were attached to the under surface of the screw cap of glass containers (340-mL) as fumigant chambers. A series of concentrations (4.71-14.71, 7.06-20.88, 20.59-58.82, and 8.82-35.29 µL/L for O. surinamensis, R. dominica, T. castaneum, and T. granarium, respectively) was organized to assess the toxicity of S. intermedia essential oil after an initial concentration setting experiment for each insect species. Twenty unsexed adults (1-14 days old) of each insect species were separately put into glass containers and their caps were tightly affixed. The same conditions without any essential oil concentration were used for control groups and each treatment was replicated five times. Insects mortality was documented 24, 48 and 72 h after initial exposure to the essential oil. Insects were considered dead when no leg or antennal movements were observed [31].

Contact Toxicity
The contact toxicity of S. intermedia essential oil against the apterous adult females of A. nerii and unsexed adults of C. septempunctata was tested through filter paper discs (Whatman No. 1), 9 cm diameter, positioned in glass petri dishes (90 × 10 mm). Range-finding experiments were established to find the proper concentrations for each insect. Concentrations ranging from 200 to 750 µg/mL for A. nerii and from 500 to 1400 µg/mL for C. septempunctata were prepared via 1.00% aqueous Tween-80 as an emulsifying agent. Each solution (200 µL) was applied to the surface of the filter paper. Ten insects were separately released onto each treated disc, the dishes sealed with Parafilm ® and kept at 25 ± 2 • C, 65 ± 5% relative humidity and a photoperiod of 16:8 h (light:dark). Except for the addition of essential oil concentrations, all other procedures were unchanged for the control groups. Four replications were made for each treatment and mortality was documented after 24 h. Aphids and ladybirds were considered dead if no leg or antennal movements were detected when softly prodded [32,33].

Data Analysis
The mortality percentage was corrected using Abbott's formula: Pt = [(Po − Pc)/(100 − Pc)] × 100, in which Pt is the corrected mortality percentage, Po is the mortality (%) caused by essential oil concentrations and Pc is the mortality (%) in the control groups [34].
Analysis of variance (ANOVA) and Tukey's test at p = 0.05 were used to statistically identify the effects of independent factors (essential oil concentration and exposure time) on insect mortality and the differences among mean mortality percentage of insects, respectively. Probit analysis was used to estimate LC 50 and LC 95 values with 95% fiducial limits, the data heterogeneity and linear regression information using SPSS 24.0 software package (Chicago, IL, USA).

Fumigant Toxicity
Analysis of variance (ANOVA) revealed that the tested concentrations of S. intermedia essential oil (F = 239.462 and p < 0.0001 for O. surinamensis, F = 223.629 and p < 0.0001 for R. dominica, F = 169.615 and p < 0.0001 for T. castaneum, and F = 89.032 and p < 0.0001 for T. granarium with df = 4, 45) and the Foods 2020, 9, 712 5 of 12 considered exposure times (F = 212.855 and p < 0.0001 for O. surinamensis, F = 281.180 and p < 0.0001 for R. dominica, F = 84.705 and p < 0.0001 for T. castaneum, and F = 84.501 and p < 0.0001 for T. granarium with df = 2, 45) had significant effects on the mortality of all insect pests. According to Figure 1 and relatively high R 2 values, there is a positive correlation between the fumigation of essential oil concentrations and the mortality of four storage insect pests at all exposure times. Furthermore, the steep slopes indicate a homogenous toxic response among beetles to the essential oil.

Fumigant Toxicity
Analysis of variance (ANOVA) revealed that the tested concentrations of S. intermedia essential oil (F = 239.462 and p < 0.0001 for O. surinamensis, F = 223.629 and p < 0.0001 for R. dominica, F = 169.615 and p < 0.0001 for T. castaneum, and F = 89.032 and p < 0.0001 for T. granarium with df = 4, 45) and the considered exposure times (F = 212.855 and p < 0.0001 for O. surinamensis, F = 281.180 and p < 0.0001 for R. dominica, F = 84.705 and p < 0.0001 for T. castaneum, and F = 84.501 and p < 0.0001 for T. granarium with df = 2, 45) had significant effects on the mortality of all insect pests. According to Figure 1 and relatively high r 2 values, there is a positive correlation between the fumigation of essential oil concentrations and the mortality of four storage insect pests at all exposure times. Furthermore, the steep slopes indicate a homogenous toxic response among beetles to the essential oil. According to Table 2, an obvious difference in the mean mortality percentage of all tested storage insect pests was detected, as essential oil concentration and exposure time were increased. For According to Table 2, an obvious difference in the mean mortality percentage of all tested storage insect pests was detected, as essential oil concentration and exposure time were increased. For example, 25.00% mortality of O. surinamensis adults was observed at 4.71 µL/L and 24-h exposure time, which had increased to 80.00% and 100% at 14.71 µL/L after 24 and 72 h, respectively. It is apparent that the essential oil of S. intermedia gave at least 90% mortality against all tested stored-product insect pests at 58.82 µL/L after 72 h ( Table 2). Based on lower LC 50 values of those stored-product insect pests tested, O. surinamensis was significantly the most susceptible insect to the essential oil of S. intermedia at all time intervals. In contrast, the adults of T. castaneum with highest LC 50 and LC 95 values were the most tolerant to fumigation with S. intermedia essential oil. Furthermore, the susceptibility of insect pests to the fumigation of S. intermedia essential oil followed in the order: O. surinamensis > R. dominica > T. granarium > T. castaneum (Table 3).

Contact Toxicity
The tested concentrations of S. intermedia essential oil demonstrated significant contact toxicity on both A. nerii (F = 27.682, df = 4, 15; p < 0.0001) and C. septempunctata (F = 35.607, df = 4, 15; p < 0.0001). A positive correlation between essential oil concentrations and the mortality of A. nerii and C. septempunctata in the contact assay is also apparent, based on the high R 2 values (Figure 1). Comparisons of the mean mortality percentage of A. nerii and its predator C. septempunctata caused by  Table 4. The mortality percentages of both insects increased with increasing essential oil concentrations, but their susceptibility to the essential oil was noticeably different. For example, 62.50% mortality was documented for A. nerii at 500 µg/mL essential oil concentration while its predator C. septempunctata was more tolerant and exhibited only 17.50% mortality at this concentration (Table 4). Table 4. Mean mortality ± SE of the adults of Aphis nerii and Coccinella septempunctata exposed to the different concentration of Satureja intermedia essential oil after 24 h.

Insect
Concentration ( The results of the probit analysis for the contact toxicity of S. intermedia essential oil against A. nerii and C. septempunctata adults are shown in Table 5. According to low LC 50 and LC 95 values, the adult females of A. nerii were more susceptible to contact toxicity of S. intermedia essential oil than the adults of C. septempunctata.

Discussion
The susceptibility of O. surinamensis, R. dominica, T. castaneum and T. granarium adults to the essential oil of S. intermedia with 24-h LC 50  Artemisia sieberi Besser (24-h LC 50 = 33.80 µL/L) also had notable fumigant toxicity against the adults of T. granarium [42,43]. The toxicity of all the above-mentioned essential oils was augmented when the exposure time was prolonged. These findings support the results regarding the time-dependent susceptibility of O. surinamensis, R. dominica, T. castaneum and T. granarium to plant essential oils. The differences in observed LC 50 values are likely due to the differences in the essential oil compositions from the different plant species and possibly to differences in the experimental conditions. Furthermore, the S. intermedia essential oil with low 24-h LC 50 value was more toxic on O. surinamensis than A. foeniculum, A. filipendulina, and A. millefolium essential oils, on R. dominica than A. graveolens essential oil, on T. castaneum than Lippia citriodora essential oil, and on T. granarium than S. molle essential oil. The terpenes, especially thymol, carvacrol, p-cymene and γ-terpinene, were recognized as the main components of S. intermedia essential oil in the present study. In the study of Sefidkon and Jamzad, thymol (32.3%), γ-terpinene (29.3%), p-cymene (14.7%), elemicin (4.8%), limonene (3.3%), and α-terpinene (3.3%) were the main components of S. intermedia essential oil [20]. In another study, thymol (34.5%), γ-terpinene (18.2%), p-cymene (10.5%), limonene (7.3%), α-terpinene (7.1%), carvacrol (6.9%), and elemicin (5.3%) were found to be major components in the essential oil of S. intermedia [23]. In the present study, however, limonene was a minor component (0.5%), and neither elemicin nor α-terpinene were detected. Ghorbanpour et al. reported the terpenes thymol (32.3%), p-cymene (14.7%), γ-terpinene (3.3%), and carvacrol (1.0%), and the phenylpropanoid elemicin (4.8%) as the main components in the essential oil of S. intermedia [22], while the concentrations of γ-terpinene and carvacrol were much lower compared to the present findings. The differences in the chemical profile of the plant essential oils are likely due to the internal and external factors such as seasonal variation, geographical features, plant growth stage, and different extraction conditions [19,44,45]. The insecticidal properties of several terpenes, especially monoterpene hydrocarbons and monoterpenoids, which accounted for 88.9% of the S. intermedia essential oil in the present study, have been documented in recent investigations. For example, insecticidal activities of p-cymene, α-pinene, γ-terpinene, 1,8-cineole, and limonene have been demonstrated against several detrimental insect pests [46][47][48][49][50]. Previous studies have also indicated that the monoterpenoids thymol and carvacrol had significant toxicity against insect pests [46,51,52]. Accordingly, the insecticidal efficiency of S. intermedia essential oil can be attributed to such components.
The contact toxicity of the essential oil of Eucalyptus globulus Labill. against A. nerii has been reported by Russo et al. [53]. Although this is the only previous study to investigate the susceptibility of A. nerii to a plant essential oil, its findings confirm the results of the present study about the possibility of A. nerii management through plant essential oils. Indeed, the toxicity of S. intermedia essential oil was evaluated for the first time in the present study against A. nerii and its natural enemy C. septempunctata. The essential oil of S. intermedia was more toxic on A. nerii (LC 50 : 418 µg/mL) than the predator ladybird C. septempunctata (LC 50 : 914 µg/mL), suggesting that the predator was more tolerant than the aphid to S. intermedia essential oil, which is very valuable in terms of predator protection. Similar results were obtained for controlling aphids [54,55] and some other insect pests [56][57][58] using plant-derived essential oils along with protecting their predators. However, the destructive side-effects of some essential oils on parasitoids have been reported [59][60][61]. Therefore, it is important to select efficient pesticides with lower side effects on natural enemies at operative concentrations to the pests, which has been achieved in the current study.

Conclusions
In conclusion, the terpene-rich essential oil of S. intermedia has significant fumigant toxicity against the adults of O. surinamensis, R. dominica, T. castaneum, and T. granarium, and may be considered as a natural effective fumigant on stored products. This bio-rational agent also has significant contact toxicity on the adult females of A. nerii, one of the cosmopolitan insect pests of ornamental plants. Furthermore, the predator ladybird C. septempunctata was more tolerant to the essential oil than the aphid. Accordingly, S. intermedia essential oil can be nominated as an eco-friendly efficient insecticide by decreasing the risks associated with the application of synthetic chemicals. However, the exploration of any side-effects of the essential oil on other useful insects such as parasitoids and pollinators, its phytotoxicity on the treated plants and crops, any adverse tastes or odors on stored products, and the preparation of novel formulations to increase its stability in the environment for practical utilization are needed.