Acute Toxicity and Sublethal Effects of Lemongrass Essential Oil and Their Components against the Granary Weevil, Sitophilus granarius

In the present work, we evaluate the toxic and repellent properties of lemongrass (Cymbopogon citratus (DC. ex Nees) Stapf.) essential oil and its components against Sitophilus granarius Linnaeus as an alternative to insecticide use. The lethal dose (LD50 and LD90), survivorship, respiration rate, and repellency on adults of S. granarius exposed to different doses of lemongrass oil and some of its components were evaluated. The chemical composition of the essential oil was found to have the major components of neral (24.6%), citral (18.7%), geranyl acetate (12.4%), geranial (12.3%), and limonene (7.55%). Lemongrass essential oil (LD50 = 4.03 µg·insect–1), citral (LD50 = 6.92 µg·insect–1), and geranyl acetate (LD50 = 3.93 µg·insect–1) were toxic to S. granarius adults. Survivorship was 99.9% in insects not exposed to lemongrass essential oil, decreasing to 57.6%, 43.1%, and 25.9% in insects exposed to LD50 of essential oil, citral, and geranyl acetate, respectively. The insects had low respiratory rates and locomotion after exposure to the essential oil, geranyl acetate, and citral. Our data show that lemongrass essential oils and their components have insecticidal and repellent activity against S. granarius and, therefore, have the potential for application in stored grain pest management schemes.


Introduction
Chemical synthetic insecticides are used to control insects in stored grain facilities. Phosphine is commonly used in noxious gas form to control stored product pests worldwide [1]. Other alternative chemical methods to fumigants consist of protectants with long residual efficacies that target a broad spectrum of species [2]. Insecticides such as pirimiphos-methyl, spinetoram, and spinosad are grain protectants and provide a rapid, lethal effect in stored product pests [3][4][5]. However, these insecticides cause environmental pollution [6], atmosphere ozone-depletion [7], toxic waste [8], have a long residual period of toxicity [9], and have documented insecticide resistance [1]. Among the alternative strategies to insecticides, the use of plant essential oils has been proposed for insect control in stored grains [10,11].
Plant essential oils have different properties such as biodegradability, selectivity to target pests, and can reduce the use of conventional insecticides [12,13]. Plant essential oils are volatile substances

Gas Chromatography(GC) Analysis
Quantitative analysis of the lemongrass essential oil was performed in triplicate on a Shimadzu gas chromatograph model GC-17A equipped with a flame ionization detector (FID; Shimadzu Corporation, Kyoto, Kansai, Japan), using chromatographic conditions: a fused silica capillary column (30 m × 0.22 mm) with a DB-5 bound phase (0.25 µm film thickness); column pressure 110 kPa; helium carrier gas at a flow rate of 1.8 mL min −1 ; injector temperature 205 • C; detector temperature of 260 • C; column temperature programmed to start at 40 • C (isothermal remaining for 2 min) and increased from 3 • C min −1 to 260 • C (isotherm remaining at 260 • C, for 10 min). A sample of 1 µL (1% w/v in dichloromethane) was injected, using split mode (split ratio 1:10).

Gas Chromatography/Mass Spectrometry (GC/MS) Analysis
The identification of lemongrass essential oil components was performed with GC/MS mass-coupled gas chromatograph (CGMS-QP 5050A; Shimadzu Corporation, Kyoto, Kansai, Japan). One µL of essential oil containing 1% dichloromethane was injected in the splitless mode (1:10 ratio). The gas carrier used was helium, with 1.8 mL −1 constant flow rate on an Rtx ® -5MS fused silica capillary column (30 m, 0.25 × 0.25 mm; Restek Corporation, Bellefonte, PA, USA), using the Crossbond ® stationary phase (35% diphenyl and 65% dimethyl polysiloxane). The initial temperature of the injector and detector was 40 • C, for 3 min, with a temperature increase from 3 • C/min to 300 • C and held for 25 min. For mass spectrometer detection, an electron ionization mode with ionization energy of 70 eV was programmed to detect masses in the range of 29-600 Da. Lemongrass oil components were identified using their Kovats indexes from original literature [39][40][41], by comparisons of their mass spectra and retention times with those of (C 3 −C 24 ) n-alkanes and mass spectral data deposited in the Wiley 07 Spectroteca and National Institute of Standards and Technology (NIST08 and NIST11) databases.

Toxicity Test of Lemongrass Essential Oil and Components
Terpenoids of lemongrass essential oil, including citral and geranyl acetate, were purchased from Merck KGaA (Darmstadt, Germany). Lemongrass essential oil, citral, and geranyl acetate were diluted in 10 mL of acetone to obtain six doses (1.56, 3.12, 6.25, 12.5, 25, and 50 µg·insect −1 ). Serial doses and a control (acetone) were used to determine the dose-response relationship and estimate lethal doses. Each dose solution (1 µL) was applied on the bodies of 50 newly-emerged (24-hour-old) S. granarius adults using a Hamilton microsyringe (model 7001, KH Hamilton Storage GmbH, Domat/Ems, Switzerland). The insects were placed individually in glass vials (20 × 100 mm), covered with a piece of organza, and fed on wheat grains. Three replicates of 50 weevils were used for each dose. The experimental design was completely randomized. The number of dead insects was recorded after 24 h of exposure. Insects were considered dead if unable to walk when prodded with a fine hair brush.

Time-Mortality Test
Adults of S. granarius were individualized in glass vials (20 × 100 mm) and exposed to the lethal doses (LD 50 and LD 90 ) of lemongrass essential oil and components determined in the dose-response relationship. Exposure procedures and conditions were the same as described in Section 2.5. The number of alive insects was recorded every 6 h for 2 d. Three completely randomized replicates were used with all essential oil and component doses. Acetone was used as a control.

Respiration Rate
Respiration rate of S. granarius adults was evaluated for 3 h after exposure to LD 50 and LD 90 essential oil and its components. The granary weevils treated with acetone were used as the control group. The respirometry measurement was detected with a TR3C CO 2 analyzer (Sable System International, Las Vegas, NV, USA) and recorded by a data acquisition system (ExpeData, Sable System International) using the methodology adapted from previous studies [42,43]. For CO 2 quantification, a S. granarius adult was placed in a respirometry chamber (25 mL) and the chamber was connected to a closed air system. Then, the gas in the respiratory chamber was pumped to the O 2 and CO 2 analyzers, and compared with those from the control. To quantify the CO 2 produced inside each chamber, an airstream scrubbed compressed O 2 via drietite/acarite column was pumped through the chamber at a flow of 100 mL min −1 for 2 min. Sitophilus granarius adults were weighed on a Shimadzu analytical balance model AY220 (Shimadzu Corporation, Kyoto, Japan) before and after the test. Sixteen completely randomized replicates were used to evaluate essential oil, components, and control.

Behavioral Repellency Response
Adults of S. granarius were placed in Petri dishes (90 mm diameter), with filter paper discs (Whatman TM , Fisher Scientific, Leicestershire, LE, UK) at the bottom of the plate used as arenas. Half of the arena was treated with 250 µL of lemongrass essential oil and their components at the LD 50 or LD 90 , and the other half with acetone and air-dried for five min [44]. An S. granarius was released in the center of the arena and monitored for 10 min. Twenty (Males/females, 1 ratio) insects were used and the experimental design was completely randomized. Behavioral repellency was recorded using a Canon digital camcorder model XL1 3CCD NTSC (Canon, Tokyo, Japan) with a 16X video lens (ZoomXL 5.5-88 mm, Canon, Tokyo, Japan). The measurement of the distance walked and time spent on each half-arena were obtained with the aid of a video tracking system (ViewPoint Life Sciences, Montreal, Canada). Weevils that spent <1 min or 50% of the time in the half-arena treated with components were considered repelled or irritated, respectively [44,45].

Statistical Analysis
The toxicity data were submitted to Probit analysis to obtain a dose-mortality curve [46]. The time-mortality data were analyzed for survival analysis (Kaplan-Meier estimators, log-rank test) with the Origin Pro 9.1 software (OriginLab Corporation, Northampton, MA, USA). Respiration rate data were submitted to two-way ANOVA and Tukey's HSD test (p < 0.05). Behavioral repellency response (walked distance and resting time) data were submitted to one-way ANOVA, and a Tukey's HSD (p < 0.05) test was also used for comparison of means. Respiration rate and behavioral repellency response were arcsine-transformed to meet assumptions of normality and homoscedasticity. Statistical procedures were analyzed by SAS 9.0 software (SAS Institute, Campus Drive Cary, NC, USA).

Toxicity Test
The dose-response model provided a good fit to the data (p > 0.05), allowing the determination of toxicological endpoints and confirming the toxicity of lemongrass essential oil and its components to S. granarius ( Table 2). The LD 50 of the essential oil was 4.03 µg·insect −1 (3.29-4.94 µg·insect −1 ). The bioassay indicated that geranyl acetate was the most toxic component, with an LD 50 of 3.93 µg·insect −1 (3.25-4.77) µg·insect −1 , followed by citral (LD 50 = 6.92 µg·insect −1 ; range of 5.63-8.58 µg·insect −1 ). Both components were used in subsequent tests. Mortality was less than 1% in the control.

Toxicity Test
The dose-response model provided a good fit to the data (p > 0.05), allowing the determination of toxicological endpoints and confirming the toxicity of lemongrass essential oil and its components to S. granarius ( Table 2). The LD50 of the essential oil was 4.03 µg insect −1 (3.29-4.94 µg insect −1 ). The bioassay indicated that geranyl acetate was the most toxic component, with an LD50 of 3.93 µg insect −1 (3.25-4.77) µg insect −1 , followed by citral (LD50 = 6.92 µg insect −1 ; range of 5.63-8.58 µg insect −1 ). Both components were used in subsequent tests. Mortality was less than 1% in the control.

Respiration Rate
The respiration rate of S. granarius was influenced by exposure to lemongrass essential oil and its components at LD 50 and LD 90 (Figure 4). For LD 50 , respiration rates differed after 3 h of exposure (F 3,59 = 8.83; p < 0.001). The highest mean respiration rate was observed in control insects (1.84 µL of CO 2 h −1 ), followed by insects exposed to lemongrass essential oil (1.52 µL of CO 2 h −1 ), citral (1.39 µL of CO 2 h −1 ), and geranyl acetate (1.32 µL of CO 2 h −1 ). Similar results were obtained with treatments at LD 90 ; respiration rates differed after 3 h of exposure (F 3,59 = 7.47; p < 0.001), with mean rates of 1.73 µL CO 2 h −1 in the control, 1.46 µL CO 2 h −1 in insects exposed to essential oil, 1.16 µL CO 2 h −1 in insects exposed to citral, and 1.12 µL CO 2 h −1 in insects exposed to geranyl acetate.

Discussion
This study investigated the chemical composition of lemongrass essential oil and assessed the insecticidal and repellent activities of the essential oil and its terpenoids citral and geranyl acetate against S. granarius under laboratory conditions. The chemical quantitative and qualitative analyses revealed 13 components of lemongrass essential oil. The components present in larger quantities in the lemongrass are neral, citral, geranyl acetate, geranial, limonene, and camphene, which have been reported for this essential oil [39][40][41]. Terpenoids are secondary metabolites with several functions in plant physiology, cell membranes [47,48], and defense of plants against insects and pathogens, as demonstrated for more complex components [48,49]. In lemongrass essential oil, citral and geranyl acetate may have a neurotoxic effect on S. granaries with rapid lethality, as reported for other insects [49][50][51]. Although the mode of action of this essential oil and its components has not been fully elucidated, their toxic effects suggest a viable alternative for the management of stored product pests.
Insecticidal and repellent action of lemongrass essential oil and its terpenoids against S. granarius were found in bioassays under laboratory conditions. Lemongrass topically applied was toxic against S. granarius adults (LD50 = 4.03 µg insect −1 ) and mortality increased in a dose-dependent manner, as also reported in other pests [52][53][54]. Sitophilus granarius adults exposed to high doses of lemongrass essential oil (LD50 and LD90) showed muscle contractions and changes in locomotion, and when exposed to LD90, paralysis without recovery. In this case, symptoms were consistent in S. granarius, confirming neurotoxicity. There is a set of results that point to effects on the nervous system of insect pests such as Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) [55], Callosobruchus maculatus

Discussion
This study investigated the chemical composition of lemongrass essential oil and assessed the insecticidal and repellent activities of the essential oil and its terpenoids citral and geranyl acetate against S. granarius under laboratory conditions. The chemical quantitative and qualitative analyses revealed 13 components of lemongrass essential oil. The components present in larger quantities in the lemongrass are neral, citral, geranyl acetate, geranial, limonene, and camphene, which have been reported for this essential oil [39][40][41]. Terpenoids are secondary metabolites with several functions in plant physiology, cell membranes [47,48], and defense of plants against insects and pathogens, as demonstrated for more complex components [48,49]. In lemongrass essential oil, citral and geranyl acetate may have a neurotoxic effect on S. granaries with rapid lethality, as reported for other insects [49][50][51]. Although the mode of action of this essential oil and its components has not been fully elucidated, their toxic effects suggest a viable alternative for the management of stored product pests.
Insecticidal and repellent action of lemongrass essential oil and its terpenoids against S. granarius were found in bioassays under laboratory conditions. Lemongrass topically applied was toxic against S. granarius adults (LD 50 = 4.03 µg·insect −1 ) and mortality increased in a dose-dependent manner, as also reported in other pests [52][53][54]. Sitophilus granarius adults exposed to high doses of lemongrass essential oil (LD 50 and LD 90 ) showed muscle contractions and changes in locomotion, and when exposed to LD 90 , paralysis without recovery. In this case, symptoms were consistent in S. granarius, confirming neurotoxicity. There is a set of results that point to effects on the nervous system of insect pests such as Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) [55], Callosobruchus maculatus Fabricius (Coleoptera: Chrysomelidae) [56], and Trichoplusia ni Hübner (Lepidoptera: Noctuidae) [50] after lemongrass essential oil exposure (by contact or fumigation). These data show that the topical application of different doses of lemongrass essential oil in small volumes is toxic against S. granarius.
In this study, a high variation in S. granarius survival is mediated by the interaction of the lemongrass essential oil, citral, and geranyl acetate with target sites in the nervous system [51]. Time periods to induce mortality in S. granarius by this essential oil and components were from 24 to 48 h. The low survivorship of this insect seems to be due to the rapid action of lemongrass essential oil, citral, and geranyl acetate, as observed in other coleopteran pests of grains such as Oryzaephilus surinamensis Linnaeus (Coleoptera: Silvanidae), Rhyzopertha dominica Fabricius (Coleoptera: Bostrichidae), and Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) after plant terpenoid exposure [29,61,62]. In this study, the compared effects of the lemongrass essential oil and its components on S. granarius occurred at various time periods. The lower time-mortality of insects exposed to LD 50 of lemongrass essential oil in comparison with its compounds citral and geranyl acetate may be due to the lower amount of the components in the essential oil blend, ranging, i.e., citral 18.5% and geranyl acetate 12.5%. The rapid insecticidal activity against S. granarius suggests that lemongrass essential oil and its components can be effective against this stored product pest. Thus, they may be a valuable alternative to synthetic chemical insecticides, especially in the management of pest populations that have developed resistance to chemical insecticides.
The lemongrass essential oil, geranyl acetate, and citral negatively affect the respiration rate of S. granarius up to 3 h after exposure, which indicates the physiological stress caused by the components. The respiration of insects is affected by the energy necessary for their metabolism to produce physiological defense against essential oils [27,43]. Different respiratory responses have been reported for other insects exposed to essential oils and components in Podisus nigrispinus Dallas (Heteroptera: Pentatomidae) [51], Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) [63], and T. molitor [21]. Low respiratory rates result in high physical conditioning damage because the energy is reallocated at the expense of physiological processes [42,43] with the potential to affect muscle activity, causing permanent paralysis [43,45]. Inhalation of fumigant essential oils is associated with insect respiration rate [45,51]. Our results show that S. granarius exposed to lemongrass essential oil, geranyl acetate, and citral had a decrease in the respiration rates, suggesting a possible fitness cost and energy reallocation.
The behavioral response tests show that lemongrass essential oil, geranyl acetate, and citral affect S. granarius. Some insect pests alter their locomotion when exposed to lemongrass essential oil and its components and avoided the toxic environments after the detection of the chemical components [21,22,42]. Plant essential oils have been claimed to disrupt the recognition of the substrate, which impairs the orientation and locomotor activity of insects [29,44,64]. According to the results here obtained, the odor of essential oil and its components is repulsive to S. granarius. Terpenoids cross the insect body barrier through the spiracles and trachea [22,44] and could lead to important consequences in the control of insect pests of stored grains [65]. Our results show that S. granarius adults are repelled by lemongrass essential oil, geranyl acetate, and citral, suggesting that the use of lemongrass essential oil and its components may introduce an innovative approach to control this pest through manipulation of its foraging and avoidance behavior.

Conclusions
This study shows the potential of lemongrass essential oil, citral, and geranyl acetate as an insecticide or repellent IPM approach to manage S. granarius. These compounds caused significant effects on the mortality, respiration depletion, and repellency in this pest of stored grains. Additionally, the insecticide effects of lemongrass essential oil can be due to the synergism of components and their ability to penetrate the insect body or through the respiratory system. Lemongrass essential oil, citral, and geranyl acetate have toxic and sublethal effects on S. granarius and can be an alternative to synthetic chemical insecticides.