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Larvicidal potential of carvacrol and terpinen-4-ol from the essential oil of Origanum vulgare (Lamiaceae) against Anopheles stephensi, Anopheles subpictus, Culex quinquefasciatus and Culex tritaeniorhynchus (Diptera: Culicidae)
Research in Veterinary Science 104 (2016) 77–82
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Larvicidal potential of carvacrol and terpinen-4-ol from the essential oil of Origanum vulgare (Lamiaceae) against Anopheles stephensi, Anopheles subpictus, Culex quinquefasciatus and Culex tritaeniorhynchus (Diptera: Culicidae) Marimuthu Govindarajan a,⁎, Mohan Rajeswary a, S.L. Hoti b, Giovanni Benelli c,⁎ a b c
Unit of Vector Control, Phytochemistry and Nanotechnology, Department of Zoology, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India Regional Medical Research Centre, Nehru Nagar, Belgaum 590010, Karnataka, India Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80, 56124 Pisa, Italy
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Article history: Received 15 September 2015 Received in revised form 16 November 2015 Accepted 29 November 2015 Available online xxxx Keywords: Arbovirus Filariasis Malaria Eco-friendly larvicides Mosquito vectors Plant-borne mosquitocides
a b s t r a c t Mosquito-borne diseases represent a deadly threat for millions of people worldwide. However, the use of synthetic insecticides to control Culicidae may lead to resistance, high operational costs and adverse non-target effects. Nowadays, plant-borne mosquitocides may serve as suitable alternative in the fight against mosquito vectors. In this study, the mosquito larvicidal activity of Origanum vulgare (Lamiaceae) leaf essential oil (EO) and its major chemical constituents was evaluated against the malaria vectors Anopheles stephensi and An. subpictus, the filariasis vector Culex quinquefasciatus and the Japanese encephalitis vector Cx. tritaeniorhynchus. The chemical composition of the EO was analyzed by gas chromatography–mass spectroscopy. GC–MS revealed that the essential oil of O. vulgare contained 17 compounds. The major chemical components were carvacrol (38.30%) and terpinen-4-ol (28.70%). EO had a significant toxic effect against early third-stage larvae of An. stephensi, An. subpictus, Cx. quinquefasciatus and Cx. tritaeniorhynchus, with LC50 values of 67.00, 74.14, 80.35 and 84.93 μg/ml. The two major constituents extracted from the O. vulgare EO were tested individually for acute toxicity against larvae of the four mosquito vectors. Carvacrol and terpinen-4-ol appeared to be most effective against An. stephensi (LC50 = 21.15 and 43.27 μg/ml, respectively) followed by An. subpictus (LC50 = 24.06 and 47.73 μg/ml), Cx. quinquefasciatus (LC50 = 26.08 and 52.19 μg/ml) and Cx. tritaeniorhynchus (LC50 = 27.95 and 54.87 μg/ml). Overall, this research adds knowledge to develop newer and safer natural larvicides against malaria, filariasis and Japanese encephalitis mosquito vectors. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Arthropods are dangerous vectors of deadly pathogens and parasites, which may hit as epidemics or pandemics in the increasing world population of humans and animals (Mehlhorn, 2008; Mehlhorn et al., 2012). Mosquitoes (Diptera: Culicidae) represent a key threat for millions of people worldwide, since they act as vectors for devastating pathogens and parasites, including malaria, yellow fever, dengue, West Nile, chikungunya, and filariasis. The approach to combat these diseases largely relied on interruption of the disease transmission cycle by either targeting the mosquito eggs, larvae or pupae through spraying of stagnant water breeding sites or by killing the adult mosquitoes using insecticides (Joseph et al., 2004; Benelli, 2015a). Larvicidal tools are a successful way of reducing mosquito densities in their ⁎ Corresponding authors. E-mail addresses: [email protected] (M. Govindarajan), [email protected] (G. Benelli).
http://dx.doi.org/10.1016/j.rvsc.2015.11.011 0034-5288/© 2015 Elsevier Ltd. All rights reserved.
breeding places before they emerge into adults. However, the larvicidal action mainly rely to the use of synthetic chemical insecticides, mostly organophosphates and pyrethroids), insect growth regulators (e.g. diflubenzuron, methoprene), and/or microbial control agents. Although effective, the repeated use of chemical pesticides damage natural biological control systems and may lead to widespread development of resistance. These problems have warranted the need for developing alternative strategies using eco-friendly products (see Benelli, 2015b; Pavela, 2015a for recent reviews). Plants offer an alternative source of insect-control agents because they contain a range of bioactive chemicals (Govindarajan et al., 2011a,b, 2013; Benelli et al., 2015a,b,c), many of which are selective and have little or no harmful effect on non-target organisms and the environment (Benelli et al., 2015a). In this scenario, huge efforts recently focused on plant extracts or other phytochemicals as potential sources of mosquitocidal or mosquito-repellent tools (Sukumar et al., 1991; Govindarajan, 2010a; Govindarajan et al., 2011a; Benelli, 2016). In particular, essential oils (EO) have received much attention as potentially
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useful bioactive compounds against insects (Cheng et al., 2003; Pavela, 2015b) showing a broad spectrum of activity against insect pests, low mammalian toxicity and degrading rapidly in the environment. For instance, cheap EO obtained from Cymbopogon citratus (Sukumar et al., 1991), Tagetus minuta (Perich et al., 1995), Mentha piperita (Ansari et al., 2000a), Dalbergia sisoo (Ansari et al., 2000b), Lippia sidoides (Carvalho et al., 2003), Hyptis martiusii (Araujo et al., 2003), Clausena anisata (Govindarajan, 2010b) and other plant species (Cheng et al., 2003; Traboulsi et al., 2005; Pavela et al., 2014) recently showed promising larvicidal activity against mosquito vectors. Origanum vulgare L. is a spice herb from the family Lamiaceae. It grows from 20 to 80 cm in height, with opposite leaves and purple flowers produced in erect spikes. It is native to western and southwestern Eurasia and the Mediterranean region. The main components of its EO are phenolic compounds, carvacrol and thymol. However, the chemical compositions vary depending on geographical region and session of collecting (Faleiro et al., 2003). Dried O. vulgare leaves are currently used in many processed foods such as beverages, cured meat products, snack foods, and milk products. Some species belonging to the genus Origanum are used as fragrance components in soaps, detergents, perfumes, cosmetics, flavorings, and pharmaceuticals (Bernath and Padulosi, 1996). O. vulgare EO has antibacterial, antifungal, antiparasitic, antimicrobial and antioxidant properties. Even though the EO and the constituents of many Origanum species have been studied, (Halim et al., 1991; Shafaghat, 2011) only few reports on the antimicrobial and antioxidant activities of the Origanum EO are available (Daferera et al., 2000; Esen et al., 2007; Busatta et al., 2008) and limited information is available about its larvicidal potential against mosquito vectors of medical and veterinary importance (Pavela, 2015a). On this basis, here we report the larvicidal activity of the O. vulgare leaf essential oil grown in southern India and its major chemical constituents against four important mosquito species, the malaria vectors Anopheles stephensi and An. subpictus, the filariasis vector Culex quinquefasciatus and the Japanese encephalitis vector Culex tritaeniorhynchus.
of 4 °C min−1 and held at this temperature for 5 min. The inlet and interface temperatures were 250 and 280 °C, respectively. The carrier gas was helium at a flow rate of 1.0 mL min−1 (constant flow). The sample (0.2 μL) was injected with a split of 20:1. Electron impact mass spectrometry was carried out at 70 eV. Ion source and quadrupole temperatures were maintained at 230 and 150 °C, respectively. The identification of compounds was based on the comparison of their retention indices and mass spectra with those in commercial libraries NIST 98.1 and Mass Finder 3.1. The concentration of each essential oil component was calculated from the integration area of the chromatographer.
2. Materials and methods
Larvicidal activity of the O. vulgare EO and its major compounds, carvacrol and terpinen-4-ol, were evaluated following WHO (2005). Carvacrol and terpinen-4-ol were purchased from Sigma-Aldrich (Germany). EO was tested at 30, 60, 90, 120, and 150 μg/ml. Furthermore, each compound was tested at various concentrations (ranging from 10 to 100 μg/ ml). EO or/and individual compounds were dissolved in 1 ml DMSO, then diluted in 249 ml of filtered tap water to obtain each of the desired
2.1. Plant material and extraction of essential oil O. vulgare was collected from Nilgiris, Western Ghats, Tamil Nadu, India. It was authenticated at the Department of Botany, Annamalai University. Vouchers specimens are deposited at the herbarium of Plant Phytochemistry Division, Department of Zoology, Annamalai University. EO was obtained by the hydro-distillation of 3 kg of fresh leaves in a Clevenger apparatus for 8 h. The oil layer was separated from the aqueous phase using a separating funnel. The resulting essential oil was dried over anhydrous sodium sulfate. The essential oil was stored in dark at 4 °C until the testing phase. 2.2. Gas chromatography Gas chromatography (GC) was carried on a Varian gas chromatograph equipped with a flame ionization detector and a BPI (100% dimethyl polysiloxane) capillary column. Helium at a flow rate of 1.0 mL min−1 and 8 psi inlet pressure was employed as a carrier gas. Temperature was programmed from 60 to 220 °C at 5 °C min−1 with a final hold time of 6 min. The injector and detector temperatures were maintained at 250 and 300 °C, respectively. The sample (0.2 μL) was injected with 1:20 split ratio. 2.3. Gas chromatography–mass spectrometry Gas chromatography–mass spectrometry (GC–MS) was performed using an Agilent 6890 GC equipped with 5973 N mass selective detector and an HP-5(5% phynyl methyl polysiloxane) capillary column. The oven temperature was programmed from 50 to 280 °C at the rate
2.4. Mosquitoes Laboratory-bred pathogen-free strains of mosquitoes were reared in the vector control laboratory, Department of Zoology, Annamalai University. The larvae were fed on dog biscuits and yeast powder in the 3:1 ratio. At the time of adult feeding, these mosquitoes were 3–4 days old after emergences (maintained on raisins and water) and were starved for 12 h before feeding. Each time, 500 mosquitoes per cage were fed on blood using a feeding unit fitted with Parafilm as membrane for 4 h. Anopheles stephensi, Anopheles subpictus, Cx. quinquefasciatus and Cx. tritaeniorhynchus were fed during 6:00 to 10:00 p.m. A membrane feeder with the bottom end fitted with Parafilm was placed with 2.0 ml of the blood sample (obtained from a slaughter house by collecting in a heparinized vial and stored at 4 °C) and kept over a netted cage of mosquitoes (Govindarajan and Sivakumar, 2014). The blood was stirred continuously using an automated stirring device, and a constant temperature of 37 °C was maintained using a water jacket circulating system. After feeding, the fully engorged females were separated and maintained on raisins. Mosquitoes were held at 28 ± 2 °C, 70–85% relative humidity, with a photoperiod of 12-h light and 12-h dark. 2.5. Larvicidal activity
Table 1 Chemical composition of Origanum vulgare essential oil; RI = retention index; MS = mass spectra. Peak
Component
Retention time (Kovats index)
Composition (%)
Mode of identification
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
α-thujene Sabinene p-cimene γ-terpinene cis-p-menth-2-en-1-ol trans-p-menth-2-en-1-ol Borneol Terpinen-4-ol α-terpineol Carvacrol methyl ether Thymol Carvacrol Trans-caryophillene Germacrene D β-bisabolene Espatulenol Caryophillene oxide Total
932 974 1025 1056 1121 1141 1169 1177 1188 1241 1292 1298 1422 1483 1508 1582 1586
1.92 2.18 1.46 2.12 1.49 1.20 2.39 28.70 4.28 1.46 2.80 38.30 2.62 1.37 1.25 1.82 1.87 97.23
RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS
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3. Results The yield of O. vulgare leaf essential oil was 13.6 ml/kg fresh weight. Table 1 shows the constituents of the essential oil, their percentage composition and their Kovats Index (KI) values listed in order of elution. A total of 17 compounds representing 97.23% of the essential oil were identified. The major constituents of this oil were carvacrol (38.30%), and terpinen-4-ol (28.70%). Chemical structures of two major compounds were shown in Fig. 1. The percentage compositions of remaining 15 compounds ranged from 1.20% to 4.28%. The 24 h larvicidal results of essential oil and its two major compounds against mosquito vectors An. stephensi, An. subpictus, Cx. quinquefasciatus and Cx. tritaeniorhynchus are presented in Tables 2–4, respectively. The essential oil from the leaves of O. vulgare exhibited significant larvicidal activity, with the LC50 values of 67.00, 74.14, 80.35 and 84.93 μg/ml, respectively. The two major pure constituents extracted from the O. vulgare EO were tested individually against the four mosquito vector larval populations. Carvacrol and terpinen-4-ol appeared to be most effective against An. stephensi (LC 50 = 21.15 and 43.27 μg/ml) followed by An. subpictus (LC50 = 24.06 and 47.73 μg/ml), Cx. quinquefasciatus (LC50 = 26.08 and 52.19 μg/ml) and Cx. tritaeniorhynchus (LC50 = 27.95 and 54.87 μg/ml). 4. Discussion
Fig. 1. Chemical structure of the two major constituents of Origanum vulgare essential oil: (a) carvacrol, (b) terpinen-4-ol.
concentrations. The control was prepared using 1 ml of DMSO in 249 ml of water. Twenty early third instar larvae were introduced into each solution. For each concentration, five replicates were performed, for a total of 100 tested larvae. Larval mortality was recorded at 24 h after exposure, during which no food was given to the larvae. The lethal concentrations (LC50 and LC90) were calculated by probit analysis (Finney, 1971). The Statistical Package of Social Sciences 12.0 software was used for all the analyses. Results with P b 0.05 were considered to be statistically significant.
Plant essential oils often show a broad spectrum of bioactivity against arthropod pests, due to the presence of several active ingredients that act through several mechanisms. Their lipophilic nature facilitates them to interfere with basic metabolic, biochemical, physiological and behavioral functions of insects (Benelli, 2015b; Pavela, 2015a). Our results shed light on the promising potential of O. vulgare essential EO and its major constituents carvacrol and terpinen-4-ol as larvicides against for mosquito vectors, An. stephensi, An. subpictus, Cx. quinquefasciatus and Cx. tritaeniorhynchus. Notably, the EO pure constituents carvacrol and terpinen-4-ol were more than two fold more active than the whole O. vulgare EO in larvicidal assays against early third instar larvae of An. stephensi, An. subpictus, Cx. quinquefasciatus and Cx. tritaeniorhynchus. In our experiments, mosquito larval mortality was dosage dependent. Previous studies on other Lamiaceae species are in agreement
Table 2 Larvicidal activity of Origanum vulgare essential oil against the mosquito vectors Anopheles stephensi, Anopheles subpictus, Culex quinquefasciatus and Culex tritaeniorhynchus. Mosquito species
Concentration (μg/ml)
24 h mortality (%)±SDa
LC50 (μg/ml) (LCL-UCL)
LC90 (μg/ml) (LCL-UCL)
χ2
An. stephensi
30 60 90 120 150 30 60 90 120 150 30 60 90 120 150 30 60 90 120 150
26.4 ± 1.0 42.1 ± 0.6 64.5 ± 0.2 82.7 ± 0.8 100.0 ± 0.0 23.6 ± 0.7 36.4 ± 0.4 59.3 ± 2.0 76.7 ± 0.8 98.2 ± 1.0 20.3 ± 0.3 32.6 ± 0.6 55.8 ± 1.0 71.2 ± 2.0 96.4 ± 0.4 17.8 ± 0.6 29.4 ± 0.8 52.6 ± 1.2 68.3 ± 1.6 95.2 ± 0.8
67.00 (60.14–73.21)
128.56 (119.30–140.75)
7.607 n.s.
74.14 (67.34–80.47)
139.03 (128.94–152.42)
7.486 n.s.
80.35 (73.66–86.76)
146.80 (136.12–161.04)
7.330 n.s.
84.93 (78.42–91.32)
150.88 (140.04–165.33)
7.110 n.s.
An. subpictus
Cx. quinquefasciatus
Cx. tritaeniorhynchus
No mortality was observed in the control. SD = standard deviation. LCL = 95% lower confidence limit. UCL = 95% upper confidence limit. χ2 = chi-square. n.s. = not significant (α = 0.05) a Values are mean ± SD of five replicates.
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Table 3 Larvicidal activity of carvacrol against the mosquito vectors Anopheles stephensi, Anopheles subpictus, Culex quinquefasciatus and Culex tritaeniorhynchus. Mosquito species
Concentration (μg/ml)
24 h mortality (%)±SDa
LC50 (μg/ml) (LCL-UCL)
LC90 (μg/ml) (LCL-UCL)
χ2
An. stephensi
10 20 30 40 50 10 20 30 40 50 10 20 30 40 50 10 20 30 40 50
28.4 ± 0.6 45.3 ± 0.8 67.6 ± 2.0 84.2 ± 1.0 100.0 ± 0.0 25.7 ± 0.6 36.2 ± 0.8 62.4 ± 0.2 77.6 ± 0.8 97.3 ± 1.0 23.4 ± 0.8 32.6 ± 1.2 58.9 ± 2.0 71.3 ± 0.6 95.2 ± 0.8 19.2 ± 0.6 29.4 ± 1.4 55.3 ± 1.2 68.6 ± 0.8 93.8 ± 0.6
21.15 (18.76–23.27)
41.89 (38.82–45.96
6.511 n.s.
24.06 (21.69–26.24)
46.36 (42.92–50.96)
6.243 n.s.
26.08 (23.71–28.33)
49.62 (45.83–54.74
7.020 n.s.
27.95 (25.70–30.15)
50.84 (47.06–55.89)
5.875 n.s.
An. subpictus
Cx. quinquefasciatus
Cx. tritaeniorhynchus
No mortality was observed in the control SD = standard deviation. LCL = 95% lower confidence limit. UCL = 95% upper confidence limit. χ2 = chi-square. n.s. = not significant (α = 0.05). a Values are mean ± SD of five replicates.
with our results (Murugan et al., 2015; Pavela, 2015a; Govindarajan et al., 2016a,b). For instance, Govindarajan et al. (2012) evaluated that larvicidal activity of EO from Mentha spicata against Aedes aegypti, An. stephensi and Cx. quinquefasciatus (LC50 of 49.71 ppm for An. stephensi, 56.08 ppm for Ae. aegypti and 62.62 ppm for Cx. quinquefasciatus). In bioassays conducted with pure EO constituents, carvacrol and terpinen-4-ol showed great potential as plant-borne mosquito larvicides. Similarly, Kweka et al. (2012) tested the leaf essential oil of Plectranthus amboinicus against An. gambiae larvae, showing that carvacrol and thymol was potent compounds with LC50 and LC90 values were 55 and 99 ppm, respectively. Pavela et al. (2014) found that L-(−)-menthol from Mentha piperita EO was effective against Cx. quinquefasciatus with LC50 and LC90 of 54 and 88 ppm. Cavalcanti et al. (2004) found that eugenol and 1,8-cineole from Ocimum gratissimum had LC50 values were 60 ppm, when tested against Ae. aegypti larvae at 24 h. Further research showed that the monoterpenes β-asarone, p-cymene, (+)-limonene, linalyl acetate, myrcene, α- phellandrene, (+)-β-pinene, (−)-βpinene, α-terpinene, γ- terpinene and terpinolene (and perhaps
thymol), phenyl propenes safrole and eugenol, and diallyl disulfide have potent larvicidal action on one or more species of mosquitoes of medical and veterinary relevance (Pohilt et al., 2011). Cheng et al. (2004) also demonstrated that cinnamaldehyde, cinnamyl acetate, and eugenol had an excellent larvicidal activity against Ae. aegypti larvae in 24 h with an LC50 value of 29, 33, and 33 μg/mL, respectively. In other investigation, Araujo et al. (2003) found that 1,8-cineole induced 100% larval mortality of Ae. aegypti after 1 day with a dosage of 100 mg/L. Cheng et al. (2003) reported the larvicidal activity of linalool against Ae. aegyti larvae (LC50 = 50 ppm). Tiwary et al. (2007) observed the larvicidal activity of linalool rich EO of Zanthoxylum armatum against different mosquito species including Cx. quinquefasciatus (LC50 = 49 ppm), Ae. aegypti (LC50 = 54 ppm) and An. stephensi (LC50 = 58 ppm). Rahuman et al. (2000) reported that n-hexadecanoic acid in Feronia limonia dried leaves was effective against fourth-instar larvae of Cx. quinquefasciatus, An. stephensi and Ae. aegypti with LC50 values of 129.24, 79.58, and 57.23 μg/ml, respectively. Kiran et al. (2006) showed that pregeijerene, geijerene, and germacrene D isolated from
Table 4 Larvicidal activity of terpinen-4-ol against Anopheles stephensi, Anopheles subpictus, Culex quinquefasciatus and Culex tritaeniorhynchus. Mosquito species
Concentration (μg/ml)
24 h mortality (%)±SDa
LC50 (μg/ml) (LCL-UCL)
An. stephensi
20 40 60 80 100 20 40 60 80 100 20 40 60 80 100 20 40 60 80 100
27.3 ± 0.6 44.6 ± 0.2 65.2 ± 0.8 84.7 ± 0.6 100.0 ± 1.0 23.8 ± 0.2 42.3 ± 1.0 60.1 ± 0.6 76.5 ± 1.8 98.2 ± 1.0 20.8 ± 1.6 38.5 ± 0.8 54.7 ± 1.0 71.2 ± 0.4 96.4 ± 0.2 18.3 ± 1.2 35.6 ± 0.6 51.9 ± 1.4 69.2 ± 0.6 95.6 ± 0.8
43.27 (38.62–47.43)
84.13 (78.03–92.16)
6.723 n.s.
47.73 (42.94–52.09)
92.46 (85.56–101.70)
7.088 n.s.
52.19 (47.51–56.62)
98.41 (90.98–108.42)
7.532 n.s.
54.87 (50.36–59.23)
100.26 (92.84–110.22)
7.296 n.s.
An. subpictus
Cx. quinquefasciatus
Cx. tritaeniorhynchus
LC90 (μg/ml) (LCL–UCL)
χ2
No mortality was observed in the control. SD = standard deviation. LCL = 95% lower confidence limit. UCL = 95% upper confidence limit. χ2 = chi-square. n.s. = not significant (α = 0.05). a Values are mean ± SD of five replicates.
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Chloroxylon swietenia DC had LC50 values of 28.3, 43.4, and 63.6 lg/ml when tested against fourth-instar Ae. aegypti larvae and LC50 values of 25.8, 41.2, and 59.5 lg/ml when tested against fourth-instar An. stephensi larvae at 24 h. 5. Conclusions Overall, this research adds knowledge to develop newer and safer natural larvicides against malaria, filariasis and Japanese encephalitis mosquito vectors. The plant tested in the study is available in large quantities in India. The cost involved in the preparation of this essential oil is minimal. Most importantly, essential oils are generally more biodegradable and environmental-friendly, if compared synthetic insecticides currently marketed. Conflicts of interest The authors declare no conflicts of interest. Compliance with ethical standards All applicable international and national guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. Acknowledgments The authors would like to thank Professor and Head, Department of Zoology, Annamalai University for the laboratory facilities provided. We also acknowledge the cooperation of staff members of the VCRC (ICMR), Pondicherry. References Ansari, M.A., Vasudevan, P., Tandon, M., Razdan, R.K., 2000a. Larvicidal and mosquito repellent action of peppermint (Mentha piperita) oil. Bioresour. Technol. 71, 267–271. Ansari, M.A., Razdan, R.K., Tandon, M., Vasudevan, P., 2000b. Larvicidal and repellent actions of Dalbergia sisoo Roxb. (F. Leguminosae) oil against mosquitoes. Bioresour. Technol. 73 (3), 207–211. Araujo, E.C.C., Silveira, E.R., Lima, M.A.S., Neto, M.A., Andrade, I., Lima, M.A.A., 2003. Insecticidal activity and chemical composition of volatile oils from Hyptis martiusii benth. J. Agric. Food Chem. 51, 3760–3762. Benelli, G., 2015a. Research in mosquito control: current challenges for a brighter future. Parasitol. Res. 114, 2801–2805. Benelli, G., 2015b. Plant-borne ovicides in the fight against mosquito vectors of medical and veterinary importance: a systematic review. Parasitol. Res. 114 (9), 3201–3212. Benelli, G., 2016. Plant-mediated biosynthesis of nanoparticles as an emerging tool against mosquitoes of medical and veterinary importance: a review. Parasitol. Res. http://dx.doi.org/10.1007/s00436-015-4800-9. Benelli, G., Bedini, S., Cosci, F., Toniolo, C., Conti, B., Nicoletti, M., 2015a. Larvicidal and ovideterrent properties of neem oil and fractions against the filariasis vector Aedes albopictus (Diptera: Culicidae): a bioactivity survey across production sites. Parasitol. Res. 114, 227–236. Benelli, G., Bedini, S., Flamini, G., Cosci, F., Cioni, P.L., Amira, S., Benchikh, F., Laouer, H., Di Giuseppe, G., Conti, B., 2015b. Mediterranean essential oils as effective weapons against the West Nile vector Culex pipiens and the Echinostoma intermediate host Physella acuta: what happens around? An acute toxicity survey on non-target mayflies. Parasitol. Res. 114 (3), 1011–1021. Benelli, G., Murugan, K., Panneerselvam, C., Madhiyazhagan, P., Conti, B., Nicoletti, M., 2015c. Old ingredients for a new recipe? Neem cake, a low-cost botanical byproduct in the fight against mosquito-borne diseases. Parasitol. Res. 114, 391–397. Bernath, J., Padulosi, S., 1996. Origanum dictamnus L. and Origanum vulgare L. ssp. hirtum (Link) Letswaart: traditional uses and production in Greece. Proceedings of the IPGRI International Workshop on Oregano. CIHEAM, Valenzano, Bari, Italy, pp. 8–12. Busatta, C., Vidal, R.S., Popiolski, A.S., Mossi, A.J., Dariva, C., Rodrigues, M.R.A., Corazza, F.C., Corazza, M.I., Oliveira, J.V., Cansian, R.I., 2008. Application of Origanum majorana L. essential oil as an antimicrobial agent in sausage. Food Microbiol. 25, 207–211. Carvalho, A.F.U., Melo, V.M.M., Craveiro, A.A., Machado, M.I.L., Bantim, M.B., Rabelo, E.F., 2003. Larvicidal activity of the essential oil from Lippia sidoides Cham. against Aedes aegepti L. Mem. Inst. Oswaldo Cruz 98, 569–571. Cavalcanti, E.S.B., Morais, S.M., Lima, M.A.A., Santana, E.W.P., 2004. Larvicidal activity of essential oils from Brazilian plants against Aedes aegypti L. Mem. Inst. Oswaldo Cruz 99, 541–544.
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Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Larvicidal potential of carvacrol and terpinen-4-ol from the essential oil of Origanum vulgare (Lamiaceae) against Anopheles stephensi, Anopheles subpictus, Culex quinquefasciatus and Culex tritaeniorhynchus (Diptera: Culicidae) Marimuthu Govindarajan a,⁎, Mohan Rajeswary a, S.L. Hoti b, Giovanni Benelli c,⁎ a b c
Unit of Vector Control, Phytochemistry and Nanotechnology, Department of Zoology, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India Regional Medical Research Centre, Nehru Nagar, Belgaum 590010, Karnataka, India Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80, 56124 Pisa, Italy
a r t i c l e
i n f o
Article history: Received 15 September 2015 Received in revised form 16 November 2015 Accepted 29 November 2015 Available online xxxx Keywords: Arbovirus Filariasis Malaria Eco-friendly larvicides Mosquito vectors Plant-borne mosquitocides
a b s t r a c t Mosquito-borne diseases represent a deadly threat for millions of people worldwide. However, the use of synthetic insecticides to control Culicidae may lead to resistance, high operational costs and adverse non-target effects. Nowadays, plant-borne mosquitocides may serve as suitable alternative in the fight against mosquito vectors. In this study, the mosquito larvicidal activity of Origanum vulgare (Lamiaceae) leaf essential oil (EO) and its major chemical constituents was evaluated against the malaria vectors Anopheles stephensi and An. subpictus, the filariasis vector Culex quinquefasciatus and the Japanese encephalitis vector Cx. tritaeniorhynchus. The chemical composition of the EO was analyzed by gas chromatography–mass spectroscopy. GC–MS revealed that the essential oil of O. vulgare contained 17 compounds. The major chemical components were carvacrol (38.30%) and terpinen-4-ol (28.70%). EO had a significant toxic effect against early third-stage larvae of An. stephensi, An. subpictus, Cx. quinquefasciatus and Cx. tritaeniorhynchus, with LC50 values of 67.00, 74.14, 80.35 and 84.93 μg/ml. The two major constituents extracted from the O. vulgare EO were tested individually for acute toxicity against larvae of the four mosquito vectors. Carvacrol and terpinen-4-ol appeared to be most effective against An. stephensi (LC50 = 21.15 and 43.27 μg/ml, respectively) followed by An. subpictus (LC50 = 24.06 and 47.73 μg/ml), Cx. quinquefasciatus (LC50 = 26.08 and 52.19 μg/ml) and Cx. tritaeniorhynchus (LC50 = 27.95 and 54.87 μg/ml). Overall, this research adds knowledge to develop newer and safer natural larvicides against malaria, filariasis and Japanese encephalitis mosquito vectors. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Arthropods are dangerous vectors of deadly pathogens and parasites, which may hit as epidemics or pandemics in the increasing world population of humans and animals (Mehlhorn, 2008; Mehlhorn et al., 2012). Mosquitoes (Diptera: Culicidae) represent a key threat for millions of people worldwide, since they act as vectors for devastating pathogens and parasites, including malaria, yellow fever, dengue, West Nile, chikungunya, and filariasis. The approach to combat these diseases largely relied on interruption of the disease transmission cycle by either targeting the mosquito eggs, larvae or pupae through spraying of stagnant water breeding sites or by killing the adult mosquitoes using insecticides (Joseph et al., 2004; Benelli, 2015a). Larvicidal tools are a successful way of reducing mosquito densities in their ⁎ Corresponding authors. E-mail addresses: [email protected] (M. Govindarajan), [email protected] (G. Benelli).
http://dx.doi.org/10.1016/j.rvsc.2015.11.011 0034-5288/© 2015 Elsevier Ltd. All rights reserved.
breeding places before they emerge into adults. However, the larvicidal action mainly rely to the use of synthetic chemical insecticides, mostly organophosphates and pyrethroids), insect growth regulators (e.g. diflubenzuron, methoprene), and/or microbial control agents. Although effective, the repeated use of chemical pesticides damage natural biological control systems and may lead to widespread development of resistance. These problems have warranted the need for developing alternative strategies using eco-friendly products (see Benelli, 2015b; Pavela, 2015a for recent reviews). Plants offer an alternative source of insect-control agents because they contain a range of bioactive chemicals (Govindarajan et al., 2011a,b, 2013; Benelli et al., 2015a,b,c), many of which are selective and have little or no harmful effect on non-target organisms and the environment (Benelli et al., 2015a). In this scenario, huge efforts recently focused on plant extracts or other phytochemicals as potential sources of mosquitocidal or mosquito-repellent tools (Sukumar et al., 1991; Govindarajan, 2010a; Govindarajan et al., 2011a; Benelli, 2016). In particular, essential oils (EO) have received much attention as potentially
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useful bioactive compounds against insects (Cheng et al., 2003; Pavela, 2015b) showing a broad spectrum of activity against insect pests, low mammalian toxicity and degrading rapidly in the environment. For instance, cheap EO obtained from Cymbopogon citratus (Sukumar et al., 1991), Tagetus minuta (Perich et al., 1995), Mentha piperita (Ansari et al., 2000a), Dalbergia sisoo (Ansari et al., 2000b), Lippia sidoides (Carvalho et al., 2003), Hyptis martiusii (Araujo et al., 2003), Clausena anisata (Govindarajan, 2010b) and other plant species (Cheng et al., 2003; Traboulsi et al., 2005; Pavela et al., 2014) recently showed promising larvicidal activity against mosquito vectors. Origanum vulgare L. is a spice herb from the family Lamiaceae. It grows from 20 to 80 cm in height, with opposite leaves and purple flowers produced in erect spikes. It is native to western and southwestern Eurasia and the Mediterranean region. The main components of its EO are phenolic compounds, carvacrol and thymol. However, the chemical compositions vary depending on geographical region and session of collecting (Faleiro et al., 2003). Dried O. vulgare leaves are currently used in many processed foods such as beverages, cured meat products, snack foods, and milk products. Some species belonging to the genus Origanum are used as fragrance components in soaps, detergents, perfumes, cosmetics, flavorings, and pharmaceuticals (Bernath and Padulosi, 1996). O. vulgare EO has antibacterial, antifungal, antiparasitic, antimicrobial and antioxidant properties. Even though the EO and the constituents of many Origanum species have been studied, (Halim et al., 1991; Shafaghat, 2011) only few reports on the antimicrobial and antioxidant activities of the Origanum EO are available (Daferera et al., 2000; Esen et al., 2007; Busatta et al., 2008) and limited information is available about its larvicidal potential against mosquito vectors of medical and veterinary importance (Pavela, 2015a). On this basis, here we report the larvicidal activity of the O. vulgare leaf essential oil grown in southern India and its major chemical constituents against four important mosquito species, the malaria vectors Anopheles stephensi and An. subpictus, the filariasis vector Culex quinquefasciatus and the Japanese encephalitis vector Culex tritaeniorhynchus.
of 4 °C min−1 and held at this temperature for 5 min. The inlet and interface temperatures were 250 and 280 °C, respectively. The carrier gas was helium at a flow rate of 1.0 mL min−1 (constant flow). The sample (0.2 μL) was injected with a split of 20:1. Electron impact mass spectrometry was carried out at 70 eV. Ion source and quadrupole temperatures were maintained at 230 and 150 °C, respectively. The identification of compounds was based on the comparison of their retention indices and mass spectra with those in commercial libraries NIST 98.1 and Mass Finder 3.1. The concentration of each essential oil component was calculated from the integration area of the chromatographer.
2. Materials and methods
Larvicidal activity of the O. vulgare EO and its major compounds, carvacrol and terpinen-4-ol, were evaluated following WHO (2005). Carvacrol and terpinen-4-ol were purchased from Sigma-Aldrich (Germany). EO was tested at 30, 60, 90, 120, and 150 μg/ml. Furthermore, each compound was tested at various concentrations (ranging from 10 to 100 μg/ ml). EO or/and individual compounds were dissolved in 1 ml DMSO, then diluted in 249 ml of filtered tap water to obtain each of the desired
2.1. Plant material and extraction of essential oil O. vulgare was collected from Nilgiris, Western Ghats, Tamil Nadu, India. It was authenticated at the Department of Botany, Annamalai University. Vouchers specimens are deposited at the herbarium of Plant Phytochemistry Division, Department of Zoology, Annamalai University. EO was obtained by the hydro-distillation of 3 kg of fresh leaves in a Clevenger apparatus for 8 h. The oil layer was separated from the aqueous phase using a separating funnel. The resulting essential oil was dried over anhydrous sodium sulfate. The essential oil was stored in dark at 4 °C until the testing phase. 2.2. Gas chromatography Gas chromatography (GC) was carried on a Varian gas chromatograph equipped with a flame ionization detector and a BPI (100% dimethyl polysiloxane) capillary column. Helium at a flow rate of 1.0 mL min−1 and 8 psi inlet pressure was employed as a carrier gas. Temperature was programmed from 60 to 220 °C at 5 °C min−1 with a final hold time of 6 min. The injector and detector temperatures were maintained at 250 and 300 °C, respectively. The sample (0.2 μL) was injected with 1:20 split ratio. 2.3. Gas chromatography–mass spectrometry Gas chromatography–mass spectrometry (GC–MS) was performed using an Agilent 6890 GC equipped with 5973 N mass selective detector and an HP-5(5% phynyl methyl polysiloxane) capillary column. The oven temperature was programmed from 50 to 280 °C at the rate
2.4. Mosquitoes Laboratory-bred pathogen-free strains of mosquitoes were reared in the vector control laboratory, Department of Zoology, Annamalai University. The larvae were fed on dog biscuits and yeast powder in the 3:1 ratio. At the time of adult feeding, these mosquitoes were 3–4 days old after emergences (maintained on raisins and water) and were starved for 12 h before feeding. Each time, 500 mosquitoes per cage were fed on blood using a feeding unit fitted with Parafilm as membrane for 4 h. Anopheles stephensi, Anopheles subpictus, Cx. quinquefasciatus and Cx. tritaeniorhynchus were fed during 6:00 to 10:00 p.m. A membrane feeder with the bottom end fitted with Parafilm was placed with 2.0 ml of the blood sample (obtained from a slaughter house by collecting in a heparinized vial and stored at 4 °C) and kept over a netted cage of mosquitoes (Govindarajan and Sivakumar, 2014). The blood was stirred continuously using an automated stirring device, and a constant temperature of 37 °C was maintained using a water jacket circulating system. After feeding, the fully engorged females were separated and maintained on raisins. Mosquitoes were held at 28 ± 2 °C, 70–85% relative humidity, with a photoperiod of 12-h light and 12-h dark. 2.5. Larvicidal activity
Table 1 Chemical composition of Origanum vulgare essential oil; RI = retention index; MS = mass spectra. Peak
Component
Retention time (Kovats index)
Composition (%)
Mode of identification
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
α-thujene Sabinene p-cimene γ-terpinene cis-p-menth-2-en-1-ol trans-p-menth-2-en-1-ol Borneol Terpinen-4-ol α-terpineol Carvacrol methyl ether Thymol Carvacrol Trans-caryophillene Germacrene D β-bisabolene Espatulenol Caryophillene oxide Total
932 974 1025 1056 1121 1141 1169 1177 1188 1241 1292 1298 1422 1483 1508 1582 1586
1.92 2.18 1.46 2.12 1.49 1.20 2.39 28.70 4.28 1.46 2.80 38.30 2.62 1.37 1.25 1.82 1.87 97.23
RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS
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3. Results The yield of O. vulgare leaf essential oil was 13.6 ml/kg fresh weight. Table 1 shows the constituents of the essential oil, their percentage composition and their Kovats Index (KI) values listed in order of elution. A total of 17 compounds representing 97.23% of the essential oil were identified. The major constituents of this oil were carvacrol (38.30%), and terpinen-4-ol (28.70%). Chemical structures of two major compounds were shown in Fig. 1. The percentage compositions of remaining 15 compounds ranged from 1.20% to 4.28%. The 24 h larvicidal results of essential oil and its two major compounds against mosquito vectors An. stephensi, An. subpictus, Cx. quinquefasciatus and Cx. tritaeniorhynchus are presented in Tables 2–4, respectively. The essential oil from the leaves of O. vulgare exhibited significant larvicidal activity, with the LC50 values of 67.00, 74.14, 80.35 and 84.93 μg/ml, respectively. The two major pure constituents extracted from the O. vulgare EO were tested individually against the four mosquito vector larval populations. Carvacrol and terpinen-4-ol appeared to be most effective against An. stephensi (LC 50 = 21.15 and 43.27 μg/ml) followed by An. subpictus (LC50 = 24.06 and 47.73 μg/ml), Cx. quinquefasciatus (LC50 = 26.08 and 52.19 μg/ml) and Cx. tritaeniorhynchus (LC50 = 27.95 and 54.87 μg/ml). 4. Discussion
Fig. 1. Chemical structure of the two major constituents of Origanum vulgare essential oil: (a) carvacrol, (b) terpinen-4-ol.
concentrations. The control was prepared using 1 ml of DMSO in 249 ml of water. Twenty early third instar larvae were introduced into each solution. For each concentration, five replicates were performed, for a total of 100 tested larvae. Larval mortality was recorded at 24 h after exposure, during which no food was given to the larvae. The lethal concentrations (LC50 and LC90) were calculated by probit analysis (Finney, 1971). The Statistical Package of Social Sciences 12.0 software was used for all the analyses. Results with P b 0.05 were considered to be statistically significant.
Plant essential oils often show a broad spectrum of bioactivity against arthropod pests, due to the presence of several active ingredients that act through several mechanisms. Their lipophilic nature facilitates them to interfere with basic metabolic, biochemical, physiological and behavioral functions of insects (Benelli, 2015b; Pavela, 2015a). Our results shed light on the promising potential of O. vulgare essential EO and its major constituents carvacrol and terpinen-4-ol as larvicides against for mosquito vectors, An. stephensi, An. subpictus, Cx. quinquefasciatus and Cx. tritaeniorhynchus. Notably, the EO pure constituents carvacrol and terpinen-4-ol were more than two fold more active than the whole O. vulgare EO in larvicidal assays against early third instar larvae of An. stephensi, An. subpictus, Cx. quinquefasciatus and Cx. tritaeniorhynchus. In our experiments, mosquito larval mortality was dosage dependent. Previous studies on other Lamiaceae species are in agreement
Table 2 Larvicidal activity of Origanum vulgare essential oil against the mosquito vectors Anopheles stephensi, Anopheles subpictus, Culex quinquefasciatus and Culex tritaeniorhynchus. Mosquito species
Concentration (μg/ml)
24 h mortality (%)±SDa
LC50 (μg/ml) (LCL-UCL)
LC90 (μg/ml) (LCL-UCL)
χ2
An. stephensi
30 60 90 120 150 30 60 90 120 150 30 60 90 120 150 30 60 90 120 150
26.4 ± 1.0 42.1 ± 0.6 64.5 ± 0.2 82.7 ± 0.8 100.0 ± 0.0 23.6 ± 0.7 36.4 ± 0.4 59.3 ± 2.0 76.7 ± 0.8 98.2 ± 1.0 20.3 ± 0.3 32.6 ± 0.6 55.8 ± 1.0 71.2 ± 2.0 96.4 ± 0.4 17.8 ± 0.6 29.4 ± 0.8 52.6 ± 1.2 68.3 ± 1.6 95.2 ± 0.8
67.00 (60.14–73.21)
128.56 (119.30–140.75)
7.607 n.s.
74.14 (67.34–80.47)
139.03 (128.94–152.42)
7.486 n.s.
80.35 (73.66–86.76)
146.80 (136.12–161.04)
7.330 n.s.
84.93 (78.42–91.32)
150.88 (140.04–165.33)
7.110 n.s.
An. subpictus
Cx. quinquefasciatus
Cx. tritaeniorhynchus
No mortality was observed in the control. SD = standard deviation. LCL = 95% lower confidence limit. UCL = 95% upper confidence limit. χ2 = chi-square. n.s. = not significant (α = 0.05) a Values are mean ± SD of five replicates.
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Table 3 Larvicidal activity of carvacrol against the mosquito vectors Anopheles stephensi, Anopheles subpictus, Culex quinquefasciatus and Culex tritaeniorhynchus. Mosquito species
Concentration (μg/ml)
24 h mortality (%)±SDa
LC50 (μg/ml) (LCL-UCL)
LC90 (μg/ml) (LCL-UCL)
χ2
An. stephensi
10 20 30 40 50 10 20 30 40 50 10 20 30 40 50 10 20 30 40 50
28.4 ± 0.6 45.3 ± 0.8 67.6 ± 2.0 84.2 ± 1.0 100.0 ± 0.0 25.7 ± 0.6 36.2 ± 0.8 62.4 ± 0.2 77.6 ± 0.8 97.3 ± 1.0 23.4 ± 0.8 32.6 ± 1.2 58.9 ± 2.0 71.3 ± 0.6 95.2 ± 0.8 19.2 ± 0.6 29.4 ± 1.4 55.3 ± 1.2 68.6 ± 0.8 93.8 ± 0.6
21.15 (18.76–23.27)
41.89 (38.82–45.96
6.511 n.s.
24.06 (21.69–26.24)
46.36 (42.92–50.96)
6.243 n.s.
26.08 (23.71–28.33)
49.62 (45.83–54.74
7.020 n.s.
27.95 (25.70–30.15)
50.84 (47.06–55.89)
5.875 n.s.
An. subpictus
Cx. quinquefasciatus
Cx. tritaeniorhynchus
No mortality was observed in the control SD = standard deviation. LCL = 95% lower confidence limit. UCL = 95% upper confidence limit. χ2 = chi-square. n.s. = not significant (α = 0.05). a Values are mean ± SD of five replicates.
with our results (Murugan et al., 2015; Pavela, 2015a; Govindarajan et al., 2016a,b). For instance, Govindarajan et al. (2012) evaluated that larvicidal activity of EO from Mentha spicata against Aedes aegypti, An. stephensi and Cx. quinquefasciatus (LC50 of 49.71 ppm for An. stephensi, 56.08 ppm for Ae. aegypti and 62.62 ppm for Cx. quinquefasciatus). In bioassays conducted with pure EO constituents, carvacrol and terpinen-4-ol showed great potential as plant-borne mosquito larvicides. Similarly, Kweka et al. (2012) tested the leaf essential oil of Plectranthus amboinicus against An. gambiae larvae, showing that carvacrol and thymol was potent compounds with LC50 and LC90 values were 55 and 99 ppm, respectively. Pavela et al. (2014) found that L-(−)-menthol from Mentha piperita EO was effective against Cx. quinquefasciatus with LC50 and LC90 of 54 and 88 ppm. Cavalcanti et al. (2004) found that eugenol and 1,8-cineole from Ocimum gratissimum had LC50 values were 60 ppm, when tested against Ae. aegypti larvae at 24 h. Further research showed that the monoterpenes β-asarone, p-cymene, (+)-limonene, linalyl acetate, myrcene, α- phellandrene, (+)-β-pinene, (−)-βpinene, α-terpinene, γ- terpinene and terpinolene (and perhaps
thymol), phenyl propenes safrole and eugenol, and diallyl disulfide have potent larvicidal action on one or more species of mosquitoes of medical and veterinary relevance (Pohilt et al., 2011). Cheng et al. (2004) also demonstrated that cinnamaldehyde, cinnamyl acetate, and eugenol had an excellent larvicidal activity against Ae. aegypti larvae in 24 h with an LC50 value of 29, 33, and 33 μg/mL, respectively. In other investigation, Araujo et al. (2003) found that 1,8-cineole induced 100% larval mortality of Ae. aegypti after 1 day with a dosage of 100 mg/L. Cheng et al. (2003) reported the larvicidal activity of linalool against Ae. aegyti larvae (LC50 = 50 ppm). Tiwary et al. (2007) observed the larvicidal activity of linalool rich EO of Zanthoxylum armatum against different mosquito species including Cx. quinquefasciatus (LC50 = 49 ppm), Ae. aegypti (LC50 = 54 ppm) and An. stephensi (LC50 = 58 ppm). Rahuman et al. (2000) reported that n-hexadecanoic acid in Feronia limonia dried leaves was effective against fourth-instar larvae of Cx. quinquefasciatus, An. stephensi and Ae. aegypti with LC50 values of 129.24, 79.58, and 57.23 μg/ml, respectively. Kiran et al. (2006) showed that pregeijerene, geijerene, and germacrene D isolated from
Table 4 Larvicidal activity of terpinen-4-ol against Anopheles stephensi, Anopheles subpictus, Culex quinquefasciatus and Culex tritaeniorhynchus. Mosquito species
Concentration (μg/ml)
24 h mortality (%)±SDa
LC50 (μg/ml) (LCL-UCL)
An. stephensi
20 40 60 80 100 20 40 60 80 100 20 40 60 80 100 20 40 60 80 100
27.3 ± 0.6 44.6 ± 0.2 65.2 ± 0.8 84.7 ± 0.6 100.0 ± 1.0 23.8 ± 0.2 42.3 ± 1.0 60.1 ± 0.6 76.5 ± 1.8 98.2 ± 1.0 20.8 ± 1.6 38.5 ± 0.8 54.7 ± 1.0 71.2 ± 0.4 96.4 ± 0.2 18.3 ± 1.2 35.6 ± 0.6 51.9 ± 1.4 69.2 ± 0.6 95.6 ± 0.8
43.27 (38.62–47.43)
84.13 (78.03–92.16)
6.723 n.s.
47.73 (42.94–52.09)
92.46 (85.56–101.70)
7.088 n.s.
52.19 (47.51–56.62)
98.41 (90.98–108.42)
7.532 n.s.
54.87 (50.36–59.23)
100.26 (92.84–110.22)
7.296 n.s.
An. subpictus
Cx. quinquefasciatus
Cx. tritaeniorhynchus
LC90 (μg/ml) (LCL–UCL)
χ2
No mortality was observed in the control. SD = standard deviation. LCL = 95% lower confidence limit. UCL = 95% upper confidence limit. χ2 = chi-square. n.s. = not significant (α = 0.05). a Values are mean ± SD of five replicates.
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Chloroxylon swietenia DC had LC50 values of 28.3, 43.4, and 63.6 lg/ml when tested against fourth-instar Ae. aegypti larvae and LC50 values of 25.8, 41.2, and 59.5 lg/ml when tested against fourth-instar An. stephensi larvae at 24 h. 5. Conclusions Overall, this research adds knowledge to develop newer and safer natural larvicides against malaria, filariasis and Japanese encephalitis mosquito vectors. The plant tested in the study is available in large quantities in India. The cost involved in the preparation of this essential oil is minimal. Most importantly, essential oils are generally more biodegradable and environmental-friendly, if compared synthetic insecticides currently marketed. Conflicts of interest The authors declare no conflicts of interest. Compliance with ethical standards All applicable international and national guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. Acknowledgments The authors would like to thank Professor and Head, Department of Zoology, Annamalai University for the laboratory facilities provided. We also acknowledge the cooperation of staff members of the VCRC (ICMR), Pondicherry. References Ansari, M.A., Vasudevan, P., Tandon, M., Razdan, R.K., 2000a. Larvicidal and mosquito repellent action of peppermint (Mentha piperita) oil. Bioresour. Technol. 71, 267–271. Ansari, M.A., Razdan, R.K., Tandon, M., Vasudevan, P., 2000b. Larvicidal and repellent actions of Dalbergia sisoo Roxb. (F. Leguminosae) oil against mosquitoes. Bioresour. Technol. 73 (3), 207–211. Araujo, E.C.C., Silveira, E.R., Lima, M.A.S., Neto, M.A., Andrade, I., Lima, M.A.A., 2003. Insecticidal activity and chemical composition of volatile oils from Hyptis martiusii benth. J. Agric. Food Chem. 51, 3760–3762. Benelli, G., 2015a. Research in mosquito control: current challenges for a brighter future. Parasitol. Res. 114, 2801–2805. Benelli, G., 2015b. Plant-borne ovicides in the fight against mosquito vectors of medical and veterinary importance: a systematic review. Parasitol. Res. 114 (9), 3201–3212. Benelli, G., 2016. Plant-mediated biosynthesis of nanoparticles as an emerging tool against mosquitoes of medical and veterinary importance: a review. Parasitol. Res. http://dx.doi.org/10.1007/s00436-015-4800-9. Benelli, G., Bedini, S., Cosci, F., Toniolo, C., Conti, B., Nicoletti, M., 2015a. Larvicidal and ovideterrent properties of neem oil and fractions against the filariasis vector Aedes albopictus (Diptera: Culicidae): a bioactivity survey across production sites. Parasitol. Res. 114, 227–236. Benelli, G., Bedini, S., Flamini, G., Cosci, F., Cioni, P.L., Amira, S., Benchikh, F., Laouer, H., Di Giuseppe, G., Conti, B., 2015b. Mediterranean essential oils as effective weapons against the West Nile vector Culex pipiens and the Echinostoma intermediate host Physella acuta: what happens around? An acute toxicity survey on non-target mayflies. Parasitol. Res. 114 (3), 1011–1021. Benelli, G., Murugan, K., Panneerselvam, C., Madhiyazhagan, P., Conti, B., Nicoletti, M., 2015c. Old ingredients for a new recipe? Neem cake, a low-cost botanical byproduct in the fight against mosquito-borne diseases. Parasitol. Res. 114, 391–397. Bernath, J., Padulosi, S., 1996. Origanum dictamnus L. and Origanum vulgare L. ssp. hirtum (Link) Letswaart: traditional uses and production in Greece. Proceedings of the IPGRI International Workshop on Oregano. CIHEAM, Valenzano, Bari, Italy, pp. 8–12. Busatta, C., Vidal, R.S., Popiolski, A.S., Mossi, A.J., Dariva, C., Rodrigues, M.R.A., Corazza, F.C., Corazza, M.I., Oliveira, J.V., Cansian, R.I., 2008. Application of Origanum majorana L. essential oil as an antimicrobial agent in sausage. Food Microbiol. 25, 207–211. Carvalho, A.F.U., Melo, V.M.M., Craveiro, A.A., Machado, M.I.L., Bantim, M.B., Rabelo, E.F., 2003. Larvicidal activity of the essential oil from Lippia sidoides Cham. against Aedes aegepti L. Mem. Inst. Oswaldo Cruz 98, 569–571. Cavalcanti, E.S.B., Morais, S.M., Lima, M.A.A., Santana, E.W.P., 2004. Larvicidal activity of essential oils from Brazilian plants against Aedes aegypti L. Mem. Inst. Oswaldo Cruz 99, 541–544.
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