Abstract
Oils extracted from Cymbopogon citratus, Lantana camara, Artemisia camphorata, and Imperata cylindrica plants were used as potential insecticides against the rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae). The phytochemical composition of the isolated oils was identified by gas chromatograph-mass spectrometry (GC-MS). Oil contact toxicities were evaluated against the adults of S. oryzae. The activities of acetylcholinesterase (AChE), alkaline phosphatase (ALP), and transaminases enzymes (AST) were measured. L. camara oil (LC50 = 9.81 mg/cm2) demonstrated the highest effect, followed by C. citratus oil (LC50 = 10.89 mg/cm2), A. camphorata EO (LC50 = 16.12 mg/cm2), and I. cylindrica oil (LC50= 36.85 mg/cm2) against the adults of S. oryzae. The inhibition percentages of AChE were 38.8, 41.7, 35.0, and 27.2%; ALP were 42.4, 49.3, 28.1, and 18.7%; AST were 33.9, 38.7, 20.8, and 11.8%; and ALT were 22.7, 30.5, 14.6, and 9.6% after treated S. oryzae with oils from C. citratus, L. camara, A. camphorata and I. cylindrica, respectively. The highest abundant compounds in C. citratus were geranial (25.95%), nerylacetal (8.85%), and neral (8.45%), in L. camara were caryophyllene (12.2%), and 3-elemene (8.89%), in A. camphorata were germacrene D-4-ol (20.83%), and borneol (19.47%), and in I. cylindrica were 5-phenylundecane (10.68%), and 6-phenyldodecane (8.70%).
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Potential Insecticidal Activity of Four Essential Oils against the Rice Weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae)
Mohamed E. Tawfeek,a Hayssam M. Ali,b Mohammad Akrami,c and Mohamed Z. M. Salem d,*
Oils extracted from Cymbopogon citratus, Lantana camara, Artemisia camphorata, and Imperata cylindrica plants were used as potential insecticides against the rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae). The phytochemical composition of the isolated oils was identified by gas chromatograph-mass spectrometry (GC-MS). Oil contact toxicities were evaluated against the adults of S. oryzae. The activities of acetylcholinesterase (AChE), alkaline phosphatase (ALP), and transaminases enzymes (AST) were measured. L. camara oil (LC50 = 9.81 mg/cm2) demonstrated the highest effect, followed by C. citratus oil (LC50 = 10.89 mg/cm2), A. camphorata EO (LC50 = 16.12 mg/cm2), and I. cylindrica oil (LC50= 36.85 mg/cm2) against the adults of S. oryzae. The inhibition percentages of AChE were 38.8, 41.7, 35.0, and 27.2%; ALP were 42.4, 49.3, 28.1, and 18.7%; AST were 33.9, 38.7, 20.8, and 11.8%; and ALT were 22.7, 30.5, 14.6, and 9.6% after treated S. oryzae with oils from C. citratus, L. camara, A. camphorata and I. cylindrica, respectively. The highest abundant compounds in C. citratus were geranial (25.95%), nerylacetal (8.85%), and neral (8.45%), in L. camara were caryophyllene (12.2%), and 3-elemene (8.89%), in A. camphorata were germacrene D-4-ol (20.83%), and borneol (19.47%), and in I. cylindrica were 5-phenylundecane (10.68%), and 6-phenyldodecane (8.70%).
Keywords: Oily extracts; Chemical composition; Contact toxicity; Sitophilus oryzae; Acetylcholinesterase; Aspartate transaminase; Alanine transaminase
Contact information: a: Department of Applied Entomology and Zoology, Faculty of Agriculture, 21545 El-Shatby, Alexandria University, Alexandria, Egypt; b: Botany and Microbiology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia c: Department of Engineering, University of Exeter, Exeter EX4 4QF, UK; d: Forestry and Wood Technology Department, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria 21545, Egypt;
* Corresponding Author: zidan_forest@yahoo.com
INTRODUCTION
Stored-product pests cause critical misfortunes in weight and quality of the stored grains and cereal products (Fields and Korunic 2000; Neethirajan et al. 2007). The grain weevils (Curculionidae) are major pests of stored grains such as wheat, maize, and rice. The rice weevil, Sitophilus oryzae L. (Coleoptera: Curculionidae), is one of the most hazardous stored grain pests throughout the world (Pugazhvendan et al. 2009). Females deposit eggs into grain; larvae are legless and remain in the grain kernel for their entire duration. Feeding of S. oryzae larvae and adults can bring down grain weight by up to 75%, diminishing the dietary benefit and germination of the grains resulting in lower prices for seed grain (Dal Bello et al. 2000).
The protection of stored grain using synthetic pesticides is still the method of choice (Rattan 2010). However, many problems are associated with these chemicals, such as insect resistance, toxicity to mammals and other living organisms, toxic residues in stored products, increasing costs of application, and environmental contamination (Arthur et al. 2014). Therefore, there is interest in finding alternative ways for stored products protection. Natural pesticides with low mammalian toxicity are recommended for suppressing insect pests especially in storage (Parugrug and Roxas 2008).
Botanicals are plant-inferred materials that can be utilized as an important component in integrated pest management (IPM) for prevailing insect pests (Abdelsalam et al. 2019; Salem et al. 2020; Mosa et al. 2021; Salem et al. 2021; Moustafa et al. 2021). Botanical pesticides are biodegradable, have little or no deleterious impact on the environment and non-target living beings, cheap, easily produced, and may impede the advancement of resistance (Isman 2005; Rajendran and Sriranjini 2008). Essential oils (EOs) from different botanical parts have shown promising insecticidal activities. EOs from Cymbopogon citratus, C. nardus, and Eucalyptus citriodora have repellent effects against Anopheles arabiensis mosquitoes (Solomon et al. 2012). The EO composition of C. citratus EO showed the presence of neral, geranial, and β-pinene as main compounds, while in C. nardus they were citronellal, nerol, and citronellol; both oils showed low contact toxicity against Dinoderus porcellus L. (Coleoptera: Bostrichidae) (Loko et al. 2021). When tested against three stored grain insects, Sitophilus oryzae, Rhyzopertha dominica, and Tribolium castaneum, plant EOs could play an important role in control of stored-grain insects especially S. oryzae and could be recommended for use as a part of IPM program in stored grains (Tawfeek et al. 2017).
Acetylcholinesterase (AChE, EC 3.1.1.7) is a key enzyme that terminates nerve impulses by catalyzing the hydrolysis of neurotransmitter, acetylcholine, in the nervous system of various organisms. Its inhibition leads to paralysis and death (Zibaee 2011). Alkaline phosphatase (ALP, EC 3.1.3.1) is a set of hydrolytic enzymes, which hydrolyzes phosphomonoesters under alkaline conditions causing cytolysis of tissues during the insect development (Miao 2002). Aspartate aminotransferase (AST, EC 2.6.1.1) and alanine aminotransferase (ALT, EC 2.6.1.2) are also known as glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT), respectively. The aminotransferases are important enzymes catalyzing amino acid catabolism and are critical in carbohydrate and protein metabolism (Zibaee et al. 2008). They are altered during various pathological and physiological activities (Etebari et al. 2005).
This work evaluated the insecticidal activities of the extracted oils from four plant species (Cymbopogon citratus, Lantana camara, Artemisia camphorata, and Imperata cylindrica) against Sitophilus oryzae (L.) adults. Their inhibitory effects on the enzyme activities of AChE, ALP, AST, and ALT were estimated.
EXPERIMENTAL
Insect Rearing
The rice weevil Sitophilus oryzae (L.) adults were gathered from infested wheat grains (Triticum aestivum L.). Insect rearing was performed at the laboratory of Applied Entomology and Zoology Department, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria, Egypt. The culture was maintained in plastic containers (26 × 30 × 20 cm) at 28 ± 2 °C and 65 ± 5 % relative humidity without exposure to any insecticide.
Extraction of Plant Oils
Fresh leaves collected from four plants and authenticated at the Department of Forestry and Wood Technology, Faculty of Agriculture, Alexandria University, Egypt, were used for the oil extraction. Oils from Cymbopogon citratus, Lantana camara, and Artemisia camphorata were extracted by the Clevenger method, where 100 g fresh weight from each material were cut to small pieces and put in 2-L flask containing 1000 mL of distilled water and subjected to hydrodistillation for 3 h to extract the essential oil (EO) (Salem et al. 2014, 2020). For Imperata cylindrica, 100 g were cut into small pieces, extracted by soaking in n-hexane solvent (200 mL) for 6 h under shaking, and filtered with Whatman No. 1 filter paper under suction pressure (Hamada et al. 2018). The n-hexane oily liquid extract (HOE) was separated and concentrated by evaporating the n-hexane solvent under vacuum using a rotary evaporator at 45 °C. The HOE was stored in brown tubes (Salem et al. 2019; Mohamed et al. 2020).
Contact Toxicity
The insecticidal activity of the four oils was tested against S. oryzae adults by the residual film technique (Qi and Burkholder 1981), under laboratory conditions at 12:12 h (light: dark photoperiod). Serial dilutions of tested oils from C. citratus, L. camara, A. camphorata, and I. cylindrica at 2, 5, 10, 20, 30, and 60 mg/cm2 were prepared in acetone as a solvent. There were four replicates for each treatment in addition to controls. One mL of each concentration was placed on the bottom of each Petri dish (9 cm diameter). After the acetone was evaporated, 10 adult of rice weevils were placed into each dish. The same procedure was used for the control treated with acetone. Mortality percentages were recorded after 72 h of treatment. The LC50 and LC90 values as well as the slope of lines were calculated according to Finney (1952). It should be noted that, according to the residual film technique used in the bioassay test, the concentration is attributed to the area Unit. A unit of area means a spread rate at which one milligram of a substance is spread over the area of one square centimeter, which used as a dose calculation unit.
Biochemical Assays
Homogenate preparation
After treatment with LC50 values of the tested oils, S. oryzae adults were separately weighted and homogenized in 10 volumes (w/v) of ice cold 0.1 M phosphate buffer (pH 7.2) using a Teflon glass tissue homogenizer. In the ALP assay, the samples were homogenized in 10 volumes (w/v) of ice cold 0.1 M phosphate buffer (pH 9.8). The homogenates were centrifuged at 5000 rpm for 30 min at 4 °C. The obtained supernatants were divided into small portions and stored at -20 °C. Three replicates were used for each treatment.
Total soluble protein
The total protein content was determined calorimetrically according to the method of Lowry et al. (1951) by using bovine serum albumin (BSA) as a standard.
AChE activity
Acetylcholinesterase (AChE) activity was determined according to Ellman et al. (1961). The reaction mixture contained 2.78 mL of 0.1 M phosphate buffer (pH 8.0); 0.1 mL of ten times diluted DTNB reagent solution (39.5 mg of 5, 5-dithiobis-[2-nitro-benzoic acid] and 15 mg of sodium bicarbonate dissolved in 10 mL of 0.1 M phosphate buffer, pH 7.2); 0.1 mL of the supernatant; and 0.02 mL of 0.075 M acetylthiocholine iodide. The yellow color formed was measured after 10 min at 412 nm. Specific activity was expressed as µmol acetylthiocholine hydrolyzed/min/mg protein.
Alkaline phosphatase activity
Alkaline phosphatase (ALP) activity (Klin 1972) was determined using a diagnostic kit (Diamond Co., Cairo, Egypt). In this method, 20 μL of the enzyme source was added to 1000 μL of 1.0 M diethanolamine buffer (pH 9.8) containing 0.6 mM magnesium ions and 1 mM p-nitrophenyl phosphate, mixed in the cuvette, incubated for 30 sec. With the stopwatch started simultaneously, the output was read again after exactly 1, 2, and 3 min at 405 nm using a spectrophotometer (Sequoia-Turner Model 340; Texas City, TX, USA). Specific activity was expressed as IU/mg protein/hr.
Transaminases activity
Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined as previously described (Reitman and Frankel 1957). In this method, 100 μL of enzyme source was added to 500 μL of 0.1 M phosphate buffer (pH 7.2) containing 80 mM L-aspartate as a substrate for AST or 80 mM D-L-alanine as a substrate for ALT, and 4 mM ά-ketoglutarate.
After incubating the mixture for 30 min at 37 °C, 500 μL of developing color reagent (4 mM 2, 4-dinitrophenylhydrazine) was added, and the solution was incubated for 20 min at room temperature. Lastly, 5 mL of 0.4 N NaOH was added, and the mixture was left at room temperature for 5 min. An assay mixture without enzyme source was used as the blank, and the absorption was measured at 546 nm. AST and ALT specific activities were expressed as IU/mg protein/hr.
GC–MS Analysis of the Oils
The chemical composition of the extracted oils was analyzed with a Trace GC Ultraho-ISQ mass spectrometer (Thermo Scientific, Waltham, MA, USA) with a direct capillary column TG–5MS (30 m × 0.25 mm × 0.25 µm film thickness). Oils were diluted in n-hexane solvent (3 n-hexane: 1 oil) before being injected to the GC–MS. The used carrier gas was He (flow rate of 1 mL/min).
The program and oven temperatures conditions can be found in previous work (Moustafa et al. 2021). The Xcalibur 3.0 data system (Thermo Fisher Scientific) with the Match factor from the GC–MS literature is a very intelligent tool to identify chemical constituents, where the value ≥650 is acceptable to confirm the compounds (El-Sabrout et al. 2019; Behiry et al. 2020; Salem et al. 2020; Abd-Elkader et al. 2021; Ali et al. 2021; Moustafa et al. 2021).
Statistical Analysis
Means were compared for significance using one-way analysis of variance (ANOVA) test with Least Significant Difference (LSD0.05) (2005). Mortality rates were corrected according to Abbott’s formula (Abbott 1925) and plotted against concentrations as log/probit regression lines. LC50, LC90 values, and the toxicity index, as well as the slope of the lines were calculated according to Finney (1952).
RESULTS AND DISCUSSION
Insecticidal Activity of the Extracted Oils
The effects of four natural plant oils from Cymbopogon citratus, Lantana camara, Artemisia camphorata, and Imperata cylindrica on the rice weevil Sitophilus oryzae adults were tested after 72 h from treatment by residual film technique. As shown in Table 1, among the four natural plant oils used, the L. camara EO exhibited the highest effect against the adults of S. oryzae (LC50 = 9.81 mg/cm2), followed by C. citratus EO (LC50 = 10.89 mg/cm2), A. camphorata EO (LC50 = 16.12 mg/cm2), and I. cylindrica HOE (LC50= 36.85 mg/cm2).
Table 1. Contact Toxicity of the Oils against S. oryzae Adults after 72 h from Treatment by Residual Film Technique
In vivo Effect of Oils on AChE and ALP Activities in S. oryzae Adults
The changes in AChE and ALP enzymes activities in S. oryzae are displayed in Table 2. There was no significant difference in the AChE activity of S. oryzae adults after being treated with LC50 of four EOs. The inhibition percentages were 38.8, 41.7, 35.0, and 27.2% after treatment with Eos from C. citratus, L. camara, A. camphorata, and I. cylindrica, respectively, relative to the control. The inhibition percentages in ALP activity were 42.4, 49.3, 28.1, and 18.7% after treatment with C. citratus, L. camara, A. camphorata, and I. cylindrica oils, respectively, relative to the control.
Table 2. Changes in Acetylcholinesterase (AChE) and Alkaline Phosphatase (ALP) Activities of S. oryzae Adults as Affected by LC50 of Extracted Oils
In vivo Effect of Oils on AST and ALT Activities in S. oryzae Adults
Table 3 shows the inhibitory effects in the activities of both AST and ALT in S. oryzae adults. The inhibition percentages of AST activity were 33.9, 38.7, 20.8, and 11.8% after being treated S. oryzae adults with C. citratus, L. camara, A. camphorata, and I. cylindrica oils, respectively, compared with the control. Meanwhile, the inhibition percentages of ALT activity were 22.7, 30.5, 14.6, and 9.6% after treating S. oryzae adults with the same previous extracts compared with the control.
Table 3. Changes in Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) Activities of S. oryzae Adults as Affected by LC50 of the Extracted Oils
Chemical Composition of the Oils
Table 4 presents the chemical components of the EO from C. citratus, where the highest abundant compounds were geranial (25.95%), nerylacetal (8.85%), neral (8.45%), linalool oxide (6.36%), 2-furanmethanol (5.4%), cis-linalool oxide (4.29%), 10-hydroxygeraniol (3.92%), cis-verbenol (3.85%), cis-linalool oxide (2.38%), 4-methyl-valeric acid (2.25%), and sobrerol 8-acetate (2.19%).
Table 5 shows the chemical composition of the EO from L. camara corresponding to the highest abundant compounds caryophyllene (12.2%), 3-elemene (8.89%), Germacrene D (5.46%),1,2-cyclopentanedione (3.73%), 5-hydroxymethylfurfural (3.57%), 2,3-dihydro-benzofuran (coumaran) (3.53%), geranyl vinyl ether (2.98%), 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one (2.97%), adipic acid dimethyl ester (2.73%), 3,7-dimethyl-2,3-epoxy-6-octenol (2.71%), hydroxy-dimethyl ester butanedioic acid (2.69%), 2-furylmethanol (2.47%), 2(5H)-furanone (2.11%), and 1-ethylpentyl acetate (2.02%).
The chemical composition of the EO from A. camphorata is shown in Table 6, where the highest abundant compounds were germacrene D-4-ol (20.83%), borneol (19.47%), 1,8-cineole (7.71%), longiverbenone (5.15%), ascaridol (4.42%), camphor (4.16%), cyperotundone (2.86%), cedran-8-ol (2.53%), 7-epi-silphiperfol-5-ene (2.08%), (E)-isovalencenol (2.04%), β-dihydroagarofuran (2.03%), (Z)-piperitol (1.90%), and α-bisabolol oxide A (1.76%).
Table 4. EO Composition of Cymbopogon citratus leaves by GC-MS
Table 5. EO Composition from Lantana camara Leaves
Table 6. EO Composition from Artemisia camphorata Leaves