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RESEARCH ARTICLE
Year : 2022 | Volume
: 59
| Issue : 4 | Page : 320-326
Biological toxicity of Ruta graveolens essential oil against three species of diptera Drosophila melanogaster, Culex pipiens and Culiseta longiareolata
Hayette Bouabida, Djemaa Dris
Water and Environment Laboratory, Echahid Cheikh Larbi Tebessi University Tebessa, Algeria
Correspondence Address:
Hayette Bouabida
Water and Environment Laboratory, Echahid Cheikh Larbi Tebessi University, Tebessa
Algeria
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/0972-9062.353272
Background & objectives: Recently, the use of biodegradable and environment friendly plant-based bioinsecticides has received a great deal of attention from researchers to control insect disease vectors. The aim of this research is to determine the larvicidal efficacy of Ruta graveolens essential oil against third instar larvae of two species of mosquito (Culex pipiens and Culiseta longiareolata) and a biological model Drosophila melanogaster.
Methods: Culiseta longiareolata and Culex pipiens larvae were collected from untreated areas located in Tebessa and Drosophila melanogaster, the wild strain collected from rotten apples in the Tebessa region. Ruta graveolens essential oil has been tested at different concentrations between 2.5μ/mL and 140μL/mL against third instar larvae of the three species under standard laboratory conditions according to the recommendations from the Word Health Organization. The effects were examined on mortality, growth and the main components (proteins, carbohydrates, lipids).
Results: The essential oil showed larvicidal activity with LC50 and LC90 values (10.85μL/mL, 70.95μL/mL and 39.4μL/mL), (26μL/mL, 144.5μL/mL and 89.57μL/mL) against third instar larvae of Drosophila melanogaster, Culex pipiens and Culiseta longiareolata respectively. In addition, it disrupted the growth and several morphological malformations were observed. It also affected growth and the main components (proteins, carbohydrates, lipids).
Interpretation & conclusion: The essential oil affected growth and energy reserves for all three species. The results indicated that the essential oil of Ruta graveolens has good potential as a source of natural larvicides.
Keywords: larvicidal activity; mosquitoes; essential oil; Drosophila melanogaster; growth; energy reserves
Diptera are dangerous medicinal and veterinary insect pests in the world[1]. In particular, mosquitoes (Culicidae), which transmit many viruses, parasites and nematodes, that cause dangerous diseases[2]. Cx. pipiens is one of the most abundant mosquito species in the Mediterranean region[3] and mates in open spaces and requires a blood meal for laying[4]. Culex pipiens is a vector of West Nile Virus (WNV), periodic filariasis and deadly encephalitis[5]. WNV has caused epidemics such as invasive neurological diseases around the world over the past decades[6]. The mosquito Culiseta longiareolata is multivoltin[7] and hibernated as larvae and adult females[8] with abundance in many countries such as Europe, Africa and Asia[9]. Culiseta longiareolata is the main vector of avian malaria and various arboviruses. It can also be the vector of many human diseases (brucellosis, avian influenza, West Nile Encephalitis)[10]. Thomas Hunt Morgan (in 1910) was the first to introduce Drosophila melanogaster (Drosophilidae) as a research model. Recently, the fruit fly is considered as a model organism in toxicology studies[11],[12],[13]. Control of mosquito larvae depends on the use of synthetic insecticides[14], but unfortunately chemicals are expensive, in addition to their negative impact on human and animal health[15]. Therefore, there is a need to develop insecticides that are safe for the environment and humans. In this context, vegetable essential oils have been shown to be environmentally safe insecticides and have little or no effect on non-target organisms[16],[17]. The aim of the present investigation was to determine the insecticidal properties of a Mediterranean plant Ruta graveolens from the south-eastern region of Algeria, against two species of mosquitoes (Culex pipiens and Culiseta longiareolata) and a biological model Drosophila melanogaster and its effect on growth and main biochemical components (proteins, carbohydrates and lipids).
Material & MethodsTest insects
The two mosquito species (Cx. pipiens and Cs. longiareolata) were collected from sites in the Tebessa region (south-eastern Algeria). Each of the 30 larvae was reared in vessels containing 150 mL of tap water and kept at 25°C. The larvae were fed a mixture of dry biscuit and yeast. The water was changed every 2 days as described previously[18].
Drosophila melanogaster (Meigen, 1830), the wild strain was harvested from rotten apples in the Tebessa region. The flies were reared in 250 mL glass vials containing about 5 mL of standard medium (corn flour, agar and a preservative). They were maintained at a temperature of 25±2°C and a relative humidity of 65% as previously described[19].
Plant material, extraction and yield of essential oil
The leaves of the Ruta graveolens plant (Rutaceae) were harvested in 2020 in a semi-arid region in Algeria (Tebessa), the plant material was dried in the open air and in the shade; 100 g of the dry matter of the plant with 1000 mL of distilled water are introduced into a Clevenger type hydrodistillator for 3 h for extraction. The essential oil has been dried with anhydrous sodium sulfate, then recovered and stored at 4°C in the dark in a glass flask. The yield of essential oil is the ratio between the weight of the oil extracted and the weight of the dry matter of the plant, evaluated from 6 extractions.
Larvicidal tests
Larvicidal activity against Culex pipiens and Culiseta longiareolata
The larvicidal activity of Ruta graveolens EO was evaluated as described previously[20]. The preparations of increasing concentrations from 6 μL/mL up to 140 μL/ mL, each concentration dissolved in 1 ml of ethanol. For each concentration carried out, four replicates each contain 30 larvae of the third instar of Culex pipiens and Culiseta longiareolata. The treatment was applied in jars each containing 150 mL of dechlorinated water and food for 24 h according to WHO recommendations. In parallel, the positive control series received 1 mL of ethanol while the negative control series contains only water. Mortality was recorded 24 h after treatment.
Toxicability of Ruta graveolens essential oil against third instar Drosophila melanogaster larvae
The effect of essential oil of Ruta graveolens on the mortality of fruit fly larvae was studied according to the method by (Miyazawa et al, 2004)[21] with some rectifications. In this experiment, the third instar larvae of fruit flies were used to determine the toxicity potential of Ruta graveolens EO. The composition of the feed medium was supplemented by different concentrations (2.5, 5, 10, 20 and 30 μL/mL). The test compounds were dissolved in 1 mL of acetone and mixed in 50 mL of artificial diet. A control diet was treated with 1 mL of acetone.
The artificial diet was poured into glass vials; 30 third instar larvae were selected to be placed in each vial, for each concentration 4 replicates. Mortalities were recorded after exposure to EO at 24 h, 48 h, 72 h and then corrected according to Abbott’s formula (1925), lethal and sublethal concentrations (LC25, LC50 and LC90).
Growth determination
Newly excavated third instar larvae of the three species (Culex pipiens, Culiseta longiareolata and Drosophila melanogaster) were exposed to the LC50 of Ruta graveolens EO (70.95 μL/mL, 39.41 μL/mL and 10.85 μL/mL) respectively for 24 h. The body weight of the surviving larvae was measured using an analytical balance. Weight data were expressed in mg/larva.
Estimation of biochemical composition
Body biochemical analysis of third instar larvae of three species were extracted and quantified as previously described[1]. The newly moulted Drosophila melanogaster, Culex pipiens and Culiseta longiareolata larvae (L3) were collected (4 replicate seach containing ten individuals) were weighed and extracted into 1m L of trichloroacetic acid (20%). In short, the quantification of proteins was carried out according to the method of Bradford[22], carbohydrates according to the method of Duchateau and Florkin[23] and lipids by the method of Goldsworthy[24] with the following standards: serum albumin bovine, glucose and sunflower oil. Regarding reagents using Coomassie Brilliant Blue, anthrone and vanillin for protein, carbohydrate and lipids respectively. The data were expressed in μg/larva.
Statistical analysis
Statistical analysis was performed using MINITAB software (version 16, Penn State College, PA, USA) and GRAPH PAD PRISM 7. The results obtained were expressed as the mean ± the standard deviation (SD).
Ethical statement: Not applicable
ResultsLarvicidal activity against Culex pipiens and Culiseta longiareolata
The application of essential oil of Ruta graveolens with different concentrations (20, 40, 60, 90, 120 and 140 μL/mL), (6, 16, 33, 50, 70, 90 μL/mL) on larvae (L3) of Culex pipiens and Culiseta longiareolata showed larvicidal activity with a dose-response relationship [Figure 1]. Corrected mortality was noted at 24 h after treatment. Positive controls (ethanol) had no significant effect against the two mosquito species. The values of the sublethal and lethal concentrations (LC25, LC50 and LC90) are mentioned in the [Table 1].
Table 1: Toxicity of R. graveolens EO applied on larvae (L3) of Cx. pipiens and Cs longiareolata: Determination of lethal and sublethal concentrations (μL/mL)
Figure 1: Effects of R. graveolens EO applied on larvae (L3) of Cx. pipiens (A) and Cs. longiareolata (B): Corrected mortality(%)Toxi ability of Ruta graveolens essential oil against third instar Drosophila melanogaster
Larvae
[Figure 2] shows the corrected mortality of Drosophila melanogaster after exposure to different concentrations of the tested EO. The highest mortality was observed at a concentration of 30μL/mL of Ruta graveolens at 24, 48 and 72 h after treatment. All the differences between the concentrations used were significant at 24 h, 48 h and 72 h (P<0.001), the sublethal and lethal concentrations (LC25, LC50 and LC90) of EO with their respective confidence intervals (95%) are calculated and given in [Table 2]. After treatment, physical deformities of pupae and adults were recorded in Drosophila melanogaster as the appearance of wrinkled wings make flight impossible, reduction in size and incomplete pupation [Figure 3].
Table 2: Toxicity of R. graveolens EO applied on larvae (L3) of Drosophila melanogaster: Determination of lethal and sublethal concentrations (μL/mL)
Figure 2: Effects of R. graveolens EO applied on larvae (L3) of Drosophila melanogaster: Corrected mortality (%).
Figure 3: (A) pupa no treated (B) size reduction (C) incomplete pupation (D) Adults no treated (E) wrinkled wings Make (F) wrinkled wings MakeGrowth determination
Whole body weights were estimated for the third instar larvae of three dipterus species at different times after treatment with R. graveolens EO [Table 3]. In this study, the treatment with the lethal concentrations CL50 (70.95, 39.41 and 10.85 μL/mL) of R. graveolens EO for the Cx. pipiens, Cs. longiareolata and D. melanogaster species respectively. The treatment induces a significant reduction in weight larvae of Cx. pipiens and Cs. ongiareolata at 24 h (p=0.026, p=0.021) and at 48 h (p =0.024, p=0.016). While this decrease is significant at 72 h (p=0.044) for Cs. longiareolata and not significant for Cx. pipiens. Concerning the D. melanogaster larvae, their weight also affected with the treatment at 24 h (p=0.006), 48 h (p=0.011) and at 72 h (p =0.026) as compared to control.
Table 3: Effects of R. graveolens EO (LC50) on weight (mg) against larvae of Cx. pipiens, Cs. longiareolata and D. melanogasterEstimation of biochemical composition
The amounts of the protein, carbohydrate and lipid components were determined in the whole bodies of third instar larvae of Cx. pipiens, Cs. longiareolata [Table 4] and D. melanogaster [Table 5] at different times (24 h, 48 h and 72 h). Our study showed a decrease in the protein content of larvae of Cx. pipiens, Cs. longiareolata and D. melanogaster treated with R. graveolens EO at 24 h (p=0.030, p=0.012 and p=0.025), at 48 h (p=0.020, p=0.003 and p=0.013) and at 72 h (p= 0.032, p= 0.022 and p=0.037) respectively. Carbohydrate levels showed significant reduction at 24 h (p=0.028) for Cs. longiareolata larvae and at 72 h (p=0.025) in D. melanogaster larvae.
Table 4: Effects of R. graveolens EO (LC50) on the proteins, carbohydrates and lipids (μg/ individual) on the larvae of Cx. pipiens and Cs. longiareolata
Table 5: Effects of R. graveolens EO (LC50) on the proteins, carbohydrates and lipids (μg/ individual) on the larvae of D. melanogasterFinally, R. graveolens also reduced lipid levels in third instar larvae of Cx pipiens and D. melanogaster to 24 h (p=0.031 and p=0.023) respectively and a decrease for Cs. longiareolata larvae during the periods tested (24 h p=0.018, 48 h p=0.022 and 72 h p =0.031) as compared to control.
DiscussionRuta graveolens EO has a larvicidal power against mosquitoes and fruitflies at LC50 after 24 h with the following values: 70.95, 39.41 and 10.85 μL/mL for Culex pipiens, Culiseta longiareolata and Drosophila melanogaster respectively. The Rutaceae family is known as an excellent source of essential oils with insecticidal properties. The essential oil of Zanthoxylum limonella has larvicidal activity against Ae. aegypti and Cx. quinquefasciatus with the following concentrations: 1%, 5% and 10% with the values of LT50 = 9.78, 5.61, and 0.24 h for Ae. aegypti and Cx. quinquefasciatus had LT50 = 28.46, 20.25 and 1.01 h[25]. The essential oils of citrus limon peels demonstrated significant larvicidal properties against third instar larvae of Aedes aegypti, with an LC50 value = 15.48 mg·L-126. The work of Sheng et al.,[21] showed that the application of 100ppm of essential oils from 8 plants Citrus aurantium (leaf and flower) Citrus bergamia (skin) Citrus paradisi (fruit) Citrus limon (fruit) Citrus vulgaris (skin) Citrus medica (skin) Citrus reticulata var. tangerine (skin) Citrus reticulata (skin) belong to the Rutaceae family showed larvicidal activity against Aedes albopictus with a significant mortality of 97.5 ± 2.9, 82.5 ± 2.9, 50.0 ± 4.1 and 47.5 ± 2.9% with Citrus medica, Citrus paradise, Citrus reticulata and Citrus vulgari respectively. In addition, research from Marin et al.,[28] indicates that leaves of Citrus sinensis and Murraya koenigiiwere (Rutaceae) collected in India show activity on third instar larvae of Aedes aegypti and Culex quinquefasciatus with lethal concentrations LC50 (Citrus sinensisegalle) of 0.69 and 0.61 and for M. koenigii it was 1.05% and 0.54% respectively for the two mosquito species. The essential oil of fruits of Poncirus trifoliata had a significant toxic effect on fruit flies and shows an efficacy of up to 90% depending on the dose and time; doses 100 and 200 μg/ml revealed a mortality of 76.2 and 93.2%, respectively after 36 hours of exposure[29]. In the study by De Lacerda Neto et al.,[30], the Dysphania ambrosioides essential oil presented significant action on D. melanogaster mortality. This effect depends on the exposure time (1 h–8 h) and the oil concentration (1, 2 and 4 mg/mL). Similar results have also been reported by Sousa Silveira et al.,[31], who demonstrated that the toxicity of thymol and carvacrol against D. melanogaster were demonstrated with EC50 values of 17.96 and 16.97 μg/ mL, respectively, with 48 h of exposure. The essential oil of a plant native to Mexico (Hyptis mociniana) has shown larvicidal activity against the larvae of Drosophila melanogaster[32]. The results of Tolou et al.[33] indicated that the following plants (Achillea millefolium, Lavandula angustifolia, and Mentha piperita) of spearmint had toxicity against Bracon hebetor bee in fumigation. Fertility is influenced by the body size of mosquitoes and therefore determines their potential for disease transmission[34]. In the present study, the treatment with the EO of Ruta graveolens (LC50) induced a significant reduction in the weight larvae of Culex pipiens, Culiseta longiareolata and Drosophila melanogaster compared to controls in all periods tested. Several studies have reported similar observations using other plant EOs such as Lavandula dentata[35], Ocimum basilicum21 and Rosmarinus officinalis[16]. In the present study, biochemical analysis revealed a decrease in the protein, carbohydrate and fat content in the whole body of the third instar larvae of Culex pipiens, Culiseta longiareolata and Drosophila melanogaster after exposure of Ruta graveolens essential oil compared to the control series. Our results are in agreement with those of Bouguerra & Boukoucha[17] in which a decrease in proteins, carbohydrates and lipids were recorded after application of essential oils of Origanum glandulosum against Culex pipiens larvae.
ConclusionThe present study confirmed the toxic properties of essential oil of Ruta graveolens and showed larvicidal activity against three species of Diptera Drosophila melanogaster, Culex pipiens and Culiseta longiareolata. Additionally, the essential oil of Ruta graveolens affected larval growth for all three species, this caused a change in the biochemical composition of the larvae. Based on these results we can suggest that this essential oil can be used as a natural insecticide against insects.
Conflict of interest: None
AcknowledgementsThe authors thank University of Echahid Cheikh Larbi Tebessi, Tebessa, Algeria and the Ministry of High Education and Scientific Research of Algeria.
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