Laboratory bioassays on insecticidal activity of essential oils (EOs) extracted from six Mediterranean plants (Achillea millefolium, Lavandula angustifolia, Helichrysum italicum, Foeniculum vulgare, Myrtus communis, and Rosmarinus officinalis) were carried out against the larvae of the Culicidae mosquito Aedes albopictus. The chemical composition of the six EOs was also investigated. Results from applications showed that all tested oils had insecticidal activity, with differences in mortality rates as a function of both oil and dosage. At the highest dosage (300 ppm), EOs from H. italicum, A. millefolium, and F. vulgare caused higher mortality than the other three oils, with mortality rates ranging from 98.3% to 100%. M. communis EO induced only 36.7% larval mortality at the highest dosage (300 ppm), a similar value to those recorded at the same dosage by using R. officinalis and L. angustifolia (51.7% and 55%, respectively). Identified compounds ranged from 91% to 99%. The analyzed EOs had higher content of monoterpenoids (80–99%) than sesquiterpenes (1–15%), and they can be categorized into three groups on the basis of their composition. Few EOs showed the hydrocarbon sesquiterpenes, and these volatile compounds were generally predominant in comparison with the oxygenated forms, which were detected in lower quantities only in H. italicum (1.80%) and in M. communis (1%).
Mosquitoes are mainly significant as human pest in Europe, since their bite causes a local skin reaction but also, in some cases, serious allergic and systemic reactions such as angioderma and urticaria (Peng et al. 1999). Their importance is more relevant in the tropical countries as vectors of very dangerous diseases such as malaria, filariasis, yellow fever, dengue, and other viral infections, which contribute significantly to poverty and social debility (James 1992). Mosquitoes are ecologically important components of the aquatic and terrestrial food chain, then they are the most important group of insects in terms of public health importance, and thus, appropriate control programs are justified. Until a few years ago, only the adults were sprayed, but now, it is well known that a more efficient way to reduce mosquito populations is to target the larvae. In Italy, as well as in other countries (Yang et al. 2002), larvae control usually relies on continued application of organophosphates and insect growth regulators, such as diflubenzuron and methoprene. With the introduction in Italy of Aedes albopictus in 1990 and the continuous increase of its population, synthetic insecticides applications were intensified. Although effective, their repeated use has had undesirable effects on nontarget organisms, on human health, and on the development of resistance (Severini et al. 1993).
All over the world, there is a need to find alternatives to synthetic insecticides. From this point of view, botanical pesticides are promising since they are effective, environmentally friendly, easily biodegradable, and often inexpensive. Essential oils (EOs) are well known for their antibacterial, antifungal, antimite, and overall insecticidal activities (Cheng et al. 2003). It is acknowledged that many compounds isolated and identified from various plant extracts can exert toxic activity against mosquito species. Feronia limonia (Rutaceae) acetone extract from dried leaves provided a potent mosquito larvicide, identified as n-hexadecanoic acid (Rahuman et al. 2000). Thymol, one of the major components of Lippia sidoides (Verbenaceae) EO, was identified as an active component for the larvicidal action against Aedes aegypti, causing 100% larval mortality (Carvalho et al. 2003). 1,8-Cineole has showed remarkable insecticidal effect against A. aegypti larvae (Araujo et al. 2003). Chantraine et al. (1998) reported that the monoterpenes (E)-anethole and E-nerolidol were found to be the active ingredients of the most toxic EOs extracted from various Bolivian plant species. E-anethole was one of the most effective larvicidal constituents from leaves of Cinnamomum osmophloeum (Lauraceae) used against A. aegypti (Cheng et al. 2004). Recently, many other plant extracts were evaluated for their insecticidal properties against mosquito larvae (Michaelakis et al. 2009; Koliopoulos et al. 2010; Evergetis et al. 2009; Mathew et al. 2009; Pavela et al. 2009; Kamaraj et al. 2009; Kannathasan et al. 2008; Rahuman et al. 2008; Senthilkumar et al. 2008; Anees 2008).
Since further studies are important to increase the knowledge of new plant substances to be used against mosquito species, in the present study, the chemical composition of six EOs extracted from Mediterranean plants—Achillea millefolium, Lavandula angustifolia (Lavandula spica), Helichrysum italicum, Foeniculum vulgare, Myrtus communis and Rosmarinus officinalis—and the larvicidal activity against the Culicidae mosquito A. albopictus were investigated.
Materials and methods
EO sampling and GC analysis
A. millefolium, M. communis, R. officinalis, H. italicum, F. vulgare, and L. angustifolia leaves were collected on Elba Island in 2006 and hydrodistilled in the farm directly (Hines & Teo) using Clevenger apparatus after the drying process (Italian Pharmacopoeia 2008). An amount of each EO (A, H, F, L, M, R) was diluted in n-hexane (HPLC grade) and analyzed by GC–FID and GC–MS to define its aromatic profile.
All EOs were diluted in n-hexane (HPLC solvent grade, 10%) and injected in GC–FID (injection volume, 1 μl, HP-WAX and HP-5 capillary columns) and GC–MS (injection volume, 0.1 μl, DB-5 capillary column). The GC analyses were carried out with HP-5890 Series II instrument, equipped with HP-WAX and HP-DB-5 capillary columns (30 m × 0.25 μm, 0.25-μm film thickness), working with the following temperature program: 60°C for 10 min, ramp of 5°C/min up to 220°C; injector and detector temperatures 250°C; carrier gas nitrogen (2 ml/min); detector dual FID; split ratio 1:30; injection volume of 1 μl; 10% n-hexane solution. Identification of the EO constituents, for both columns, was achieved by comparing their retention times with those of pure authentic samples and by mean of their linear retention indices (LRIs), relative to a series of n-hydrocarbons (C9–C23) on the two different columns.
The GC/EIMS analyses were performed by a Varian CP-3800 gas chromatograph, equipped with an HP DB-5 capillary column (30 m × 0.25 mm; coating thickness, 0.25 μm) and a Varian Saturn 2000 ion trap mass detector. Analytical conditions are as follows: injector and transfer line temperatures, 220 and 240°C, respectively; oven temperature programmed from 60 to 240°C at 3°C/min; carrier gas helium at 1 ml/min; injection volume, 0.1 μl (10% n-hexane solution); split ratio, 1:30. The identification of the constituents was based on the comparison of retention times with those of authentic samples by comparing their LRIs relative to a series of n-hydrocarbons (C9–C23) and by computer matching by two commercial databases (NIST 2000 and ADAMS), as well as a homemade library mass spectra built up from pure substances or known oils and MS literature data. Furthermore, molecular weights of all identified substances were confirmed by GC/CIMS using MeOH as CI ionizing gas.
Insects cultures and rearing conditions
Mosquito larvae of A. albopictus utilized in bioassays were obtained from field-collected eggs deposited by wild females on a bar of masonite placed outside in a dark vase containing water. The eggs batches, daily collected, were kept wet for 24 h and then placed in mineral water in laboratory at 25 ± 1°C and natural summer photoperiod for hatching. The newly emerged larvae were then isolated in groups of ten specimens in 100 cc tubes with mineral water and a small amount of dog or cat food. Larvae were daily controlled until they reached the fourth instar, when they were utilized for bioassays (within 12 h).
Bioassays and statistical analysis
Three groups of 20 fourth-instar larvae were isolated in 250-ml beakers and exposed to test dosage of 50, 100, 150, 200, 250, and 300 ppm of the oil in mineral water with 0.1% of Tween 80 for 24 h, according to standard World Health Organization (1981) procedure; 250 ml beakers with the same number of larvae and mineral water with 0.1% of Tween 80 were used as the control. The mortality was recorded after 24 h, at the end of the test, during which no food was given to the larvae. The mortality of the larvae was reported as an average from three replicates, and mortality percentage rates were corrected using the Abbott’s formula (Abbott 1925). Original data were transformed into arcsine square root percentage values before statistical analysis. Data were processed by JMP of SAS (1999) using a linear model with two factors with interactions, oil and dosage:
where y ij is the observation, μ is the overall mean, O i the oil (i = 1–6), D j the dosage (j = 1–6), O i * D j the interaction between oil and dose, and e ij the residual error. Averages were separated by Tukey–Kramer HSD test (Sokal and Rohlf 1981). For simplicity sake, only one level of probability (P < .05) was used for the significance of differences between the means. Lethal concentrations (LC50 and LC90) were calculated by using SigmaPlot© software (Systat Software Inc., California).
EOs quality control was performed by gas chromatography to define their volatile composition before carrying out each specific biological assay. Identified compounds ranged from 91% to 99% (Tables 1 and 2), and many typical volatile constituents of the analyzed EOs have been reported in the literature for the selected species. Analyzed EOs had higher content of monoterpenoids (80–99%) than sesquiterpenes (1–15%), and they can be categorized into three different groups on the basis of their chemical composition:
EOs with large amounts of hydrocarbons monoterpenes from M. communis (M, 63%) and R. officinalis (R, 58%).
EOs with large amounts of oxygenated monoterpenes from A. millefolium (A, 52%), F. vulgare (F, 66%), and L. angustifolia (L, 66%).
EOs with both significant amounts of oxygenated monoterpenes (49%) and the highest level of sesquiterpenes (15%) from H. italicum (E).
Few species showed the hydrocarbon sesquiterpenes in their EOs, and these volatile compounds were generally present in larger quantity, in comparison with the oxygenated forms, which were detected in lower quantity only in H. italicum (E, 1.80%) and in M. communis (M, 1%). The GC–MS analysis of the EOs identified the typical constituents reported in the literature for the selected species.
Results from EOs applications indicated that all tested oils had insecticidal activity, with relevant differences in mortality rates, as a function of the oil (F = 154.21, df = 5, P < .0001) and dosage (F = 269.97, df = 5, P < .0001) (Table 3). There was also a significant interaction, dosage × oil (F = 8.51, df = 25, P < .0001). For all tested oils, it was evident that larval mortality was dosage dependent. At the highest dosage (300 ppm), EOs from H. italicum, A. millefolium, and F. vulgare showed a higher mortality than the other three oils, with mortality percentage rates ranging from 98.3 to 100 (Table 4). Bioassays showed that F. vulgare was the most toxic oil against A. albopictus larvae (LC50 = 142.9 and LC90 = 239.2) followed by H. italicum (LC50 = 178.1 and LC90 = 288.6) and by A. millefolium (LC50 = 211.3 and LC90 = 328.0) (Table 5). For this latter EO, a larger dosage (350 ppm) was tested with respect to the other two oils to obtain a mortality of 100% in order to calculate the LC values. A statistically not different toxicity was showed by F. vulgare EO at the dosage rate of 300, 250, and 200 ppm (100%, 98.3%, and 96.7%, respectively) (Table 4). Mortality rates caused by A. millefolium and H. italicum EOs at 250 ppm dose (63.3% and 81.7%, respectively) were not statistically different from each other. At a dosage of 300 ppm, M. communis, R. officinalis, and L. angustifolia showed a significantly lower larval mortality than other oils, with percentage rates ranging from 36.7% to 55%. In fact, M. communis EO induced only a 36.7% larval mortality at higher dosage, a similar rate to those recorded with the same dosage by using R. officinalis and L. angustifolia (51.7% and 55%, respectively). Lastly, for all the tested oils, no significant differences were recorded in larval mortality at the lowest dosage of 50 ppm.
Discussion and conclusions
All the selected Mediterranean plant EOs showed insecticidal activity against A. albopictus larvae, thus widening the spectrum of action of these oils already reported in the literature as effective against several other insect and mite species. It was proved that different mortality responses are a function of both oil type and dosage rate. EOs from H. italicum and A. millefolium showed high toxicity at the highest dosage of 300 ppm, with mortality rates rising to 100%, while F. vulgare EO showed a significant mortality starting from the lower dosage of 200 ppm, therefore suggesting their relatively higher toxicity at lower doses. To date, no data are available on larvicidal mosquito properties of H. italicum EO, while ethanolic extract of A. millefolium showed repelling properties against A. aegypti, with the most active components identified as stachydrine and a phenolic compound pyrocatechol (Tunòn et al. 1994). Jaenson et al. (2006) evidenced that extracts of A. millefolium leaves significantly reduced Aedes mosquito bites in the field in southern Sweden, and, according to our results, (-)-germacrene, β-pinene, sabinene, and α-pinene were identified as the more abundant volatile compounds known to have insecticidal and repellent properties. Our bioassays performed by using fennel extracts substantially confirmed the F. vulgare insecticidal activity already reported in literature against different mites and insects, even though the composition of this EO showed some differences in comparison to the previous studies. As a matter of fact, Mimica-Dukić et al. (2003) reported (E)-anethole (72.27–74.18%), fenchone (11.32–16.35%), and methyl chavicol (3.78–5.29%) as main constituents of the F. vulgare EO, while our analysis showed a higher amount of methyl chavicol (43.5%), a similar content of fenchone (11.8%) and a much lower quantity of (E)-anethole (9.8%). However, this latter component is known to have effective larvicidal activity against mosquito species (Chantraine et al. 1998; Cheng et al. 2004). Traboulsi et al. (2005) showed that extracts of F. vulgare leaves were toxic against Culex pipiens larvae, and terpineol and 1,8-cineole (both not found in our EOs) were the most effective components in repellency tests. Saad et al. (2006) reported a strong miticidal effect of fennel EO, where anethole (47.6%), pinene (11.7%), and 1,8-cineole (10.3%) were the major constituents. It can be noted that α-pinene was present in our fennel EO, while anethole and 1,8-cineole were not detected. Research performed by Lee et al. (2006) suggested that the acaricidal activity of F. vulgare seed EO oil was mainly due to their constituents carvone and naphthalene, but only the former was found at very low level (0.3%) in our oil.
Results from R. officinalis, L. angustifolia, and M. communis at the highest dosage showed a significantly lower A. albopictus larval mortality than other oils, with percentage values which reached only 55%. Many published works suggest a prevalent repellent mosquitoes effect of these EOs. Choi et al. (2002) tested the repellent activity of Lavandula officinalis and R. officinalis EOs against C. pipiens pallens, showing an effective repellent effect mainly to adult mosquitoes due to α-terpinene, carvacrol, and thymol. In our study, we found very low levels of thymol and only in R. officinalis, while α-terpinene, carvacrol, and thymol were never detected in the other tested EOs. EO from R. officinalis leaves was found to be ovicidal and repellent against three different mosquito species (Prajapati et al. 2005) and ovicidal against two stored-product insects (Tunç et al. 2000). Microcapsules containing R. officinalis EO were tested against Limantria dispar larvae and evidenced interesting larvicidal effects. A chemical composition showed as its major constituents α-pinene (38.9%), 1,8-cineole (8.6%), bornyl-acetate (8.3%), and camphene (8.0%) (Moretti et al. 2002). Regarding larvicidal bioassays using M. communis EO, it was evident that this oil was less toxic than all the other tested oils. This result was at variance with previous data reported in the literature. As a matter of fact, extracts from leaves and flowers of M. communis were found to be toxic against fourth-instar larvae of C. pipiens molestus, and thymol, carvacrol, (1R)-(+)-α-pinene, and (1S)-(-)-α-pinene were the most toxic pure components (Traboulsi et al. 2002). In our study, both thymol and carvacrol were absent in M. communis EO, while α-pinene was the most abundant component. Recently, Amer and Mehlhorn (2006a,b) tested the larvicidal effect of EOs extracted from 41 plants against third instar larvae of three different mosquitoes, showing that M. communis EO caused 100% larval mortality at low dosage (50 ppm) against A. aegypti, while—according to our results—L. angustifolia and R. officinalis showed a lower mortality at the same dosage (63.3% and 16.7%, respectively).
In conclusion, the present article improves the knowledge on the composition and the mosquito larvicidal properties of EOs extracted from six Mediterranean plants. Overall, the present investigation revealed that EOs from F. vulgare, H. italicum, and A. millefolium have remarkable toxic effects against A. albopictus larvae. However, further investigations are needed to elucidate this activity against other mosquito species, and also the active ingredient(s) of the extracts responsible for larvicidal activity should be identified and utilized, if possible, in preparing a commercial formulation/product to be used as a mosquitocidal. A plant origin insecticide may be well defined and harmless to humans and other nontarget organisms. For this purpose, use of botanical derivatives in mosquito control, instead of synthetic insecticides, could reduce the costs and environmental effects.
Abbott WS (1925) A method of computing the effectiveness of an insecticide. J Econ Entomol 18:265–267
Amer A, Mehlhorn H (2006a) Persistency of larvicidal effect of plant oil extracted under different storage conditions. Parasitol Res 99:473–477
Amer A, Mehlhorn H (2006b) Larvicidal effect of various essential oils against Aedes, Anopheles and Culex larvae (Diptera, Culicidae). Parasitol Res 99:466–472
Anees AM (2008) Larvicidal activity of Ocimum sanctum Linn. (Labiatae) against Aedes aegypti (L.) and Culex quinquefasciatus (Say). Parasitol Res 103(6):1451–1453
Araujo ECC, Silveira ER, Lima MAS, Andrade Neto M, De Andrade IL, Lima MAA, Santiago GMP, Mesquita ALM (2003) Insecticidal activity and chemical composition of volatile oils from Hyptis martiusii Benth. J Agric Food Chem 51(13):3760–3762
Carvalho AFU, Maciel Melo VM, Craveiro AA, Machado MIL, Bantim MB, Rabelo E (2003) Larvicidal activity of the essential oil from Lippia sidoides Cham. against Aedes aegypti L. Mem Inst Oswaldo Cruz Rio de Janiero 98(4):569–571
Chantraine JM, Laurent D, Ballivian C, Saavedra G, Ibanez R, Vilaseca LA (1998) Insecticidal activity of essential oils on Aedes aegypti larvae. Phytother Res 12(5):350–354
Cheng S, Liu J, Tsai K, Chen W, Chang S (2004) Chemical composition and mosquito larvicidal activity of essential oils from leaves of different Cinnamomum osmophloeum provenances. J Agric Food Chem 52:4395–4400
Cheng SS, Chang HT, Chang ST, Tsai KH, Chen WJ (2003) Bioactivity of selected plant essential oils against the yellow fever mosquito Aedes aegypti larvae. Bioresour Technol 89:99–102
Choi W, Park B, Ku S, Lee S (2002) Repellent activity of essential oils and monoterpenes against Culex pipiens pallens. J Am Mosq Control Assoc 18(4):348–351
Evergetis E, Michaelakis A, Kioulos E, Koliopoulos G, Haroutounian SA (2009) Chemical composition and larvicidal activity of essential oils from six Apiaceae family taxa against the West Nile virus vector Culex pipiens. Parasitol Res 105(1):117–124
Italian Pharmacopoeia (2008) Farmacopea Ufficiale Italiana, XII edizione, Roma.
Jaenson TGT, Palsson K, Borg-Karlson A (2006) Evaluation of extracts and oils of mosquito repellent plants from Sweden and Guinea-Bissau. J Med Entomol 43(1):113–119
James AA (1992) Mosquito molecular genetics: the hands that feed bite back. Science 257(5066):37–38
Kamaraj C, Bagavan A, Rahuman AA, Zahir AA, Elango G, Pandiyan G (2009) Larvicidal potential of medicinal plant extracts against Anopheles subpictus Grassi and Culex tritaeniorhynchus Giles (Diptera: Culicidae). Parasitol Res 104(5):1163–1171
Kannathasan K, Senthilkumar A, Venkatesalu V, Chandrasekaran M (2008) Larvicidal activity of fatty acid methyl esters of Vitex species against Culex quinquefasciatus. Parasitol Res 103(4):999–1001
Koliopoulos G, Pitarokili D, Kioulos E, Michaelakis A, Tzakou O (2010) Chemical composition and larvicidal evaluation of Mentha, Salvia, and Melissa essential oils against the West Nile virus mosquito Culex pipiens. Parasitol Res 107(2):327–335
Lee C, Sung B, Lee H (2006) Acaricidal activity of fennel seed oils and their main components against Tyrophagus putrescentiae, a stored-food mite. J Stored Prod Res 42:8–14
Mathew N, Anitha MG, Bala TSL, Sivakumar SM, Narmadha R, Kalyanasundaram M (2009) Larvicidal activity of Saraca indica, Nyctanthes arbor-tristis and Clitoria ternatea extracts against three mosquito vector species. Parasitol Res 104(5):1017–1025
Michaelakis A, Papachristos D, Kimbaris A, Koliopoulos G, Giatropoulos A, Polissiou MG (2009) Citrus essential oils and four enantiomeric pinenes against Culex pipiens (Diptera: Culicidae). Parasitol Res 105(3):769–773
Mimica-Dukić N, Kujundžić S, Soković M, Couladis M (2003) Essential oils composition and antifungal activity of Foeniculum vulgare Mill. obtained by different distillation conditions. Phytother Res 17(4):368–371
Moretti MD, Sanna-Passino G, Demontis S, Bazzoni E (2002) Essential oil formulation useful as a new tool for insect pest control. AAPs PharmSciTech, 3 (2) article 13, http://www.aapspharmscitech.org.
Pavela R, Vrchotová N, Tříska J (2009) Mosquitocidal activities of thyme oils (Thymus vulgaris L.) against Culex quinquefasciatus (Diptera: Culicidae). Parasitol Res 105(5):1365–1370
Peng Z, Yang J, Wang H, Simons FER (1999) Production and characterisation of monoclonal antibodies to two new mosquito Aedes aegypti salivary protein. Insect Biochem Mol Biol 29(10):909–914
Prajapati V, Tripathi AK, Aggrawal KK, Khanuja SPS (2005) Insecticidal, repellent and oviposition-deterrent activity of selected essential oils against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. Bioresour Technol 96:1749–1757
Rahuman AA, Venkatesan P, Gopalakrishnan G (2008) Mosquito larvicidal activity of oleic and linoleic acids isolated from Citrullus colocynthis (Linn.) Schrad. Parasitol Res 103(6):1383–1390
Rahuman AA, Gopalakrishnan G, Ghouse BS, Arumugam S, Himalayan B (2000) Effect of Feronia limonia on mosquito larvae. Fitoterapia 71:553–555
Saad E, Hussien R, Saher F, Ahmed Z (2006) Acaricidal activity of some essential oils and their monoterpenoidal constituents against house dust mite, Dermatophagoides pteronyssinus (Acari: Pyroglyphidae). J Zhejiang Univ 7(12):957–962
Senthilkumar A, Kannathasan K, Venkatesalu V (2008) Chemical constituents and larvicidal property of the essential oil of Blumea mollis (D. Don) Merr. against Culex quinquefasciatus. Parasitol Res 103(3):959–962
Severini C, Romi R, Marinucci M, Rajmond M (1993) Mechanism of insecticide resistance in field populations of Culex pipiens from Italy. J Am Mosq Control Assoc 9:164–168
Sokal RR, Rohlf FJ (1981) Biometry. Freeman and Company, New York
Traboulsi AF, El-Haj S, Tueni M, Taoubi K, Abi Nader N, Mrad A (2005) Repellency and toxicity of aromatic plant extracts against the mosquito Culex pipiens molestus (Diptera: Culicidae). Pest Manag Sci 61(6):597–604
Traboulsi AF, Taoubi K, El-Haj S, Bessiere JM, Rammal S (2002) Insecticidal properties of essential plant oils against the mosquito Culex pipiens molestus (Diptera: Culicidae). Pest Manag Sci 58(5):491–495
Tunç I, Berger BM, Erler F, Dağc F (2000) Ovicidal activity of essential oils from five plants against two stored-product insects. J Stored Prod Res 36:161–168
Tunòn H, Horsell W, Bohlin L (1994) Mosquito repellent activity of compounds occurring in Achillea millefolium L. (Asteraceae). Econ Bot 48(2):111–120
World Health Organization (1981) Instruction for determining the susceptibility or resistance of mosquito larvae to insecticide. WHO/VBC/81.807
Yang YC, Lee SG, Lee HK, Kim MK, Lee SH, Lee HS (2002) A piperidine amide extracted from Piper longum L. fruit shows activity against Aedes aegypti mosquito larvae. J Agric Food Chem 50:3765–3767
About this article
Cite this article
Conti, B., Canale, A., Bertoli, A. et al. Essential oil composition and larvicidal activity of six Mediterranean aromatic plants against the mosquito Aedes albopictus (Diptera: Culicidae). Parasitol Res 107, 1455–1461 (2010). https://doi.org/10.1007/s00436-010-2018-4
- Insecticidal Activity
- Mosquito Species
- Larval Mortality