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Idesia (Arica)

versión On-line ISSN 0718-3429

Idesia vol.35 no.2 Arica jun. 2017  Epub 13-Mayo-2017 

Larvicide activity of essential oils on Aedes aegypti L. (Díptera: Culicidae)


Actividad larvicida de aceites esenciales en Aedes aegypti L. (Diptera: Culicidae)


Toshik Iarley da Silva1; Antonio Carlos Leite Alves1; Francisco Roberto de Azevedo1* ; Cláudia Araújo Marco1; Hernandes Rufino dos Santos1; Raul Azevedo1

1 Universidade Federal do Cariri, Centro de Ciencias Agrárias e da Biodiversidade, Crato-CE, Brazil. * Autor por correspondencia:


Aedes aegypti L. is the most important mosquito in terms of public health in the world. It transmits a virus that causes several diseases, among them, dengue fever. Traditionally, the intensive use of insecticides has been the main strategy to control A. aegypti and to eliminate adult mosquitos or their larvae; however, this may lead to resistance by the mosquitos and damages to human health and the environment. Essential oils from medicinal plants have shown high efficiency as alternative larvicides. Therefore, the objective of this paper was to evaluate the larvicide potential of Vanillosmopsis arborea and Hyptis suaveolens on Aedes aegypti larvae. The essential oils were extracted by the hydrodistillation method using a Clevenger equipment. The completely randomized design (CRD) was used with a 2x5 factorial, with two essential oils and five concentration levels (200; 150; 100; 50; and 0 ppm). Each one of the ten treatments was replicated four times with ten larvae each, totaling 40 plots. Analyses were made regarding LC10, LC50, LC90, test of means, and regression and efficiency analysss. The V. arborea oil showed values of LCW = 39.69 ppm, LC50 = 48.98 ppm, and LC90 = 60.45 ppm, while the H. suaveolens oil showed values of LCW = 78.46 ppm, LC50 = 139.7 ppm and LC90 = 246.48 ppm. The main chemical component of V. arborea was a-bisabolol (94.17%), while for H. suaveolens, it was 1,8-cineol (72.23%). The essential oils showed a larvicide effect on A. aegypti larvae.

Key words: Mosquito, Dengue fever, Biolarvicide, Medicinal plants.


Aedes aegypti L. es el mosquito de salud pública más importante del mundo. Esto es debido a que es un transmisor del virus que causa muchas enfermedades, específicamente el dengue. Tradicionalmente, el uso intensivo de insecticidas ha sido la estrategia principal para eliminar los mosquitos adultos o sus larvas; Sin embargo el uso intensivo de insecticidas puede estimular una resistencia en los mosquitos y daños a la salud o al medio ambiente. Los aceites esenciales de plantas medicinales se habían mostrado de alta eficiencia como larvicidas alternativos. Por lo tanto, evaluamos la eficiencia de los aceites esenciales de Vanillosmopsis arborea y Hyptis suaveolens en larvas de Aedes aegypti. Los aceites esenciales se extrajeron mediante el método de hidrodestila-ción utilizando un equipo Clevenger. Se utilizó un diseño completamente al azar (CRD) utilizando un factorial 2x5 que el aceite esencial de V. arborea y H. suaveolens a concentraciones de cinco concentraciones (200, 150, 100, 50 y 0 ppm) respectivamente. Cada uno de los diez tratamientos se replicó cuatro veces utilizando diez larvas cada uno, totalizando 40 muestras. CLW CL50, CL90, ensayo promedio, análisis de regresión y eficiencia. El aceite de V. arborea mostró valores de CLW = 39,69 ppm, CL50 = 48,98ppm y CL90 = 60,45 ppm, mientras que el aceite de H. suaveolens mostró valores de CLW = 78,46ppm, CL50 = 139,7 ppm y CL90 246,48 ppm. El principal componente químico de V. arborea fue a-bisabolol (94,17%), mientras que para H. suaveolens fue 1,8-cineol (72,23%). Ambos aceites esenciales probados mostraron efecto larvicida sobre las larvas de A. aegypti.

Palabras clave: Mosquito, Dengue, Biolarvicida, Plantas medicinales.


Despite the continuous efforts to eliminate diseases transmitted by vectors, dengue fever still is the most important disease transmitted by mosquitos in Brazil, where many outbreaks have been reported since the 1980's (Souza et al, 2015). Dengue fever epidemics oftentimes create chaos in the communities where they occur, causing great social agitation and economic damages.

The process of growing urbanization provided favorable conditions for A. aegypti to propagate, specially due to the intensive use of disposable materials, thus increasing the number of potential breeding sites for the vector mosquito of Flavivirus, responsible for causing the dengue fever. It is disseminated according to the expansion of A. aegypti, which currently occupies almost all of the cosmo-tropical areas of the world (Porto et al., 2008).

The recent new estimation of the global incidence of dengue fever on developed and developing regions of the world, including Latin America and the Caribbean, indicated that approximately 40% of the global population were susceptible to dengue infection (Chadee and Martínez, 2016). The incidence of dengue increased 30 times over the last decade worldwide (Dias and Moraes, 2014). In 2012, it was considered as the most important viral disease transmitted by a mosquito in the world, native in over 100 countries of the tropical and subtropical regions, where over 2.5 billion people live. It is estimated that, among this population, 50-100 million are annually infected, with 500,000 severe cases. Approximately 2.5% of the infected people die, and most of them are children from Asian and Latin American countries (Dias and Moraes, 2014).

The prevention and control of the virus that causes dengue currently depend on controlling the vector mosquito. Different methods have been suggested to control the vector (as Mechanical, biological and chemical); however, several of these methods are limited due to its microevolution (Louise et al, 2016). The components of the integrated control of vectors include surveillance, reduction of sources (environmental management), biological and chemical control using insecticides and repellents, traps, and management of the resistance to insecticides, as well as public information campaigns which aim to reduce the incidence of mosquitoes (Braga and Valle, 2007).

For the A. aegypti larvae control, the main larvicide used in Brazil is organophosphate temephos (Costa et al, 2010). The indiscriminate use of insecticide substances may cause mosquito resistance against these compounds, compromising its control by this strategy. In fact, deficiencies to control A. aegypti determined by operational failures in campaigns, as well as by the existence of temephos-resistance population has been observed (Lima et al, 2006).

Considering the vector resistance, it is fundamental to look for alternatives that may make it feasible to control it. Among them, a highlight is the use of essential oils extracted from medicinal and/or aromatic plants. Plant essential oils are strong candidates, since, in some cases, they are highly active, readily available on tropical countries and economically feasible (Silva et al, 2008).

Around 27% of the plants studied due to their larvicide activity against A. aegypti were collected in Brazil, from which, 77% were collected in the Northeast region of Brazil (Dias and Moraes, 2014).

The aim of this research was to evaluate, under laboratory conditions, the larvicide potential of the essential oils of Vanillosmpsis arborea and Hyptis suaveolens on Aedes aegypti larvae.

Material and Methods

The essential oil of wild alfazema-brava [Hyptis suaveolens (L.) Poit] was extracted from plant leaves collected in the city of Lavras da Mangabeira, state of Ceará, Brazil. The essential oil of candeeiro [Vanillosmopsis arborea Baker] was extracted from stems collected in the municipality of Crato, state of Ceará.

The essential oils were extracted using the hydrodistillation method on a Clevenger equipment. Thus, 300 g of each material were weighted and submerged into 2,000 mL of distilled water in a flat-bottom flask with capacity for 5,000 mL, establishing an extraction period of 120 minutes. After the extraction period, the essential oil was removed from the equipment with the aid of a Pasteur pipette and stored on eppendorfs covered with tin foil in a domestic refrigerator.

In order to obtain the eggs of the vector insect, ovitraps were used, installed on the regions of Lameiro, Seminário, Vila Alta and the Downtown area of the city of Crato, Ceará. These traps were constituted by a black polypropylene plant pot, with capacity for 400 mL, containing water and a pressed wood pallet (Eucatextype), 3x11 cm, inserted vertically in relation to the wall of the pot.

In order to obtain the larvae, the pallets containing the collected eggs were placed on beakers with capacity for 2,000 mL. Then, water was added and the beakers were taken to a Biochemical Oxygen Demand (B.O.D.) climatized chamber under controlled temperature conditions of 25±1°C, relative humidity of the air of 70±1% and photoperiod of 12 hours. After the hatching of the larvae, the pallets were removed and the larvae were mainted uder the same conditions and fed with organic matter until they reached the third instar.

The essential oils were sent to the Research Institute on Drugs and Medicines (IPeFarM) of the Federal University of Paraíba for chromatographic analysis. Each essential oil was diluted for a concentration of 200 ppm. For such, 50 mg of each essential oil was weighted on an analytical scale (0.0001), adding 245 mL of distilled water and 5 mL of Dimethylsulfoxide (DMSO). The solution was stored on a volumetric flask (500 mL) and manually stirred until a homogeneous solution was obtained. After preparing the solution at 200 ppm, fractions were obtained from it, and they were diluted with distilled water in order to obtain the 150, 100 and 50 ppm solutions, such concentrations were suggested by the authors of this work.

For each treatment, ten larvae were used between the third and fourth instar. The larvae were removed from the beaker where they were stored using a Pasteur pipette, removing the water excess, and they were placed in polyethylene cups with capacity for 50 mL. Then, with the help of a volumetric pipette (25 mL), 25 mL of the solution was added to each cup containing the larvae. After 24, 48 and 72 hours of exposition of the larvae to the treatments, the number of dead larvae was recorded; the larvae that did not move or that did not respond to stimulations with the Pasteur pipette were considered as dead.

The completely randomized design (CRD) was used with a 2x5 factorial, with two essential oils (H. suaveolens and V. arborea) and five concentration levels (200; 150; 100; 50 and 0 ppm). Each one of the 10 treatments was replicated four times with ten larvae each, totaling 40 plots. From the obtained data, the means were evaluated, and they were then submitted to regression analysis using the SISVAR-UFLA program. LC10 (Lethal Concentration enough to cause 10% of mortality), LC50 (Lethal Concentration enough to cause 50% of mortality) and LC90 (Lethal Concentration enough to cause 90% of mortality) were analyzed using the Probit analysis, using the StatPlus v5 program (AnalystSoft Inc.), with a reliability interval of 5% of significance. The larvae mortality efficiency was determinate as a percentage using Abbott's formula (1925):


E = Efficiency
Nc = Number of alive individuals on the control treatment
Nt = Number of alive individuals treated

Results and Discussion

The chemical composition of the H. suaveolens and V. arborea essential oils is shown on table 1.

Table 1. Chemical composition of the H. suaveolens and V. arborea essential oils.

*Retention time.

The GC/MS analysis identified 34 chemical compounds representing 100% of the H. suaveolens essential oil. The compounds with the highest concentration on this essential oil were 1,8-cineole (72.23%), β-pinene (6.41%), sabinene (4.24%), bicyclogermacrene (2.46%) and δ-3-carene (2.12%), as shown on Table 1.

The main compounds from on fresh and dry leaves of H. suaveolens from Guinea Bissau were sabinene, limonene and terpinolene, while in Laos, the main compounds found were mainly sabinene, α-phellandrene, 1,8-cineole, β-phellandrene and limonene (Ashitani et al, 2015). According to these authors, the highest concentration of 1,8-cineole (46.6%) was found in plants cultivated and distilled in Laos.

Similar results were found by Oliveira et al (2013) when testing concentrations of the Piper aduncum essential oil (which showed 53.9%of 1,8 cineole), whose main compound was the isolated 1,8 cineole. The above-mentioned authors also observed that this main compound did not show any larvicide action regarding A. aegypti.

The studies mentioned above state that, possibly, the isolated substance does not show the same action when compared to essential oils. This may be related to the synergism of the substances found on essential oils.

The H. suaveolens species belongs to the Lamiaceae family and it is commonly known in Brazil as bamburral, sambacoité, mentrasto-do-grande, cheirosa, alfavacao, alfavaca-de-caboclo, alfazema-de-caboclo, alfazema-brava, etc., and it is used in popular medicine as antitussive, sudorific, antispasmodic and useful to treat gouty. It is an annual plant and it occurs spontaneously on pastures and annual and perennial cultures (Maia et al, 2008).

With the GC/MS analysis of the V. arborea essential oil, 100% of its chemical composition could be identified, corresponding to 13 compounds (Table 1), whose main compound was a-bisabolol (94.17%). The other compounds with the highest concentrations were: methyl eugenol (2.39%), a -bisabolol oxide (1.03%) and vanilosmine (0.51%).

On studies conducted by Marco et al (2015) on the content, yield and quality of the V. arborea essential oil, they highlight that the main compounds were: a-bisabolol (93.83%), methyl eugenol (1.54%) and a-bisabolol oxide (0.72%). Similar values were found in this research regarding the chemical constitution of V. arborea.

The V. arborea species belongs to the Asteraceae family, and it has an acknowledged economic value due to its anti-inflammatory properties, due to the presence of sesquiterpene a-bisalobol, present on the essential oil extracted from its wood. It has a good-quality wood, which is highly resistant to the weather, and it has a high amount of essential oil. This characteristic promotes its burn, causing an intense flame, which justifies its traditional name, candeeiro (Cavalcanti and Nunes, 2002).

The efficiency of the concentrations of V. arborea essential oil on A. aegypti larvae are shown on Table 2.

Table 2. Efficiency of the concentrations of V. arborea essential oil on A. aegypti larvae.

It is possible to observe that when the concentration of the essential oil increased, the number of dead larvae was higher. Concentrations over 100 ppm during a larvae exposure period of 12 hours caused 100% of larvae mortality. This shows that, under the evaluated conditions, a concentration of 100 ppm is enough to cause the maximum effect of the test. For the 50 ppm concentration, a mortality index is observed across all observed times.

The efficiency reaches 100% at concentrations above 100 ppm, while concentrations of 50 ppm show an efficiency of 53.58% (Table 2). When the total efficiency was compared with the larvae exposure periods at a concentration of 50 ppm, it was observed that the 12-hour period represented 66.66% of mortality, while the 24, 36 and 48-hour periods represented 4.64%, 9.47% and 4.64%, respectively.

The essential oils with the highest larvicide action against A. aegypti mentioned in the literature, according to Dias and Morais (2014), were collected from Callitris glaucophylla Joy (LC50=0.69 ppm), Juniperus virginiana L. (LC50=1 ppm), Thymus serpyllum L. (LC50=1 ppm), and Amyris balsamifera L. (LC50=1 ppm).

On studies conducted by Autran et al (2009) with the essential oil extracted from various parts of Piper marginatum Jacq., these authors conclude that the oil extracted from the inflorescences showed higher potential on A. aegypti larvae with LC10 values of 13.8 and LC50 values of 20.0 ppm; the authors did not calculate LC90.

Table 3 shows the efficiency of the H. suaveolens essential oil concentrations on A. aegypti larvae.

Table 3. Efficiency of the H. suaveolens essential oil concentrations on A. aegypti larvae.

Similarly to what happens to the V. arborea essential oil, for the H. suaveolens oil, it is observed that the highest number of dead larvae is observed when the concentration increases. At the exposure period of 12 hours, the highest mortality number occurs for concentrations above 100 ppm. The 100 ppm concentration is the one that promotes mortality across all observed periods, followed by the 200 ppm concentration, which promotes mortality at the 24 and 36-hour period of exposure. The 50 ppm concentration shows no mortality, and it is noteworthy that it has no toxic effect.

Regarding efficiency, it is observed that the 200 ppm concentration showed 65% of efficiency after 12 hours of exposure, showing total efficiency of 82.5%. When the comparison of the total efficiency to the exposure period of larvae at the 100 ppm concentration was conducted, it was observed that the 12-hour period represented 50% of mortality, while the 24, 36 and 48-hour periods represented 8.33%, 16.66% and 25.0%, respectively. For the 150 ppm concentration, the 12-hour exposure period was the most representative one, representing 89.47% of the efficiency, while the 24-hour period showed 10.53%. At the 200 ppm concentration, the mortality efficiency was also higher at the 12-hour period, showing a value of 78.78%. The 24, 36 and 48-hour periods showed 18.18%, 3.03% and 0.0%, respectivety.

Similar results were found by Oliveira et al (2013), when analyzing the larvicide action of Piper aduncum L. on A. aegypti, where the authors point out that the 100% mortality was reached after 24 hours of exposure with treatments at 500 and 1,000 ppm concentrations. After 48 hours of exposure, the mortality rates were 80 and 50% for 250 and 100 ppm concentrations, respectively.

The total means of the concentrations of V. arborea and H. suaveolens essential oils when submitted to regression analysis (Figure 1 and Figure 2) characterizes the efficiency of the tested treatments. It is observed that, as the concentrations increase, the number of dead larvae increases polynomially, highlighting the toxic activity of these oils on A. aegypti.

Figure 1. Total mortality of A. aegypti larvae after exposure to the V. arborea and H. suaveolens essential oil submitted to regression analysis.

Figure 2. Total mortality of A. aegypti larvae after exposure to the V. arborea and H. suaveolens essential oil submitted to regression analysis.

On studies conducted by Leite et al (2009) on the larvicide activity of some medicinal plants on A. aegypti, an inhibition of the larvae development was observed between 27 and 72 hours at an assay with Rosmarinus officinalis L. and Mentha piperita L. essential oils. When applied at a concentration of 20 ppm, the R. officinalis oil inhibited 80% of the larvae feasibility, while the M. piperita essential oil at the same concentration showed a 40% inhibition. These oils caused 100% of larvae mortality at concentrations of 40 and 80 ppm, respectively. Similar results were obtained on our research when comparing the V. arborea results to M. piperita.

Kiran et al (2006), when evaluating the larvicide activity of the essential oil extracted from the leaves and stems of Chlororylon swietenia DC. On A. aegypti and Anopheles stephensi, pointed out that, as it may be expected, with the increase on the concentration, an increase also occurs on the number of dead individuals. On their study, they concluded that the LC50 values for A. aegypti and A. stephensi were 16.5 and 14.9 ppm and 20.2 and 19.0 ppm for the oil extracted from the leaves and from the stem, respectively. Such values were higher, in terms of efficiency, to the oils tested on this research. The same occurs with the results obtained by Cavalca et al (2010) when they studied the Eucalyptus cinerea essential oil, which showed a high larvicide power with LC50 and LC90 equal to 38 and 27 ppm, respectively.

Table 4 shows the minimal (LC10), medium (LC50) and maximal (LC90) concentrations of V. arborea and H. suaveolens essential oils able to cause mortality on A. aegypti larvae.

Table 4. Minimal (LC10), medium (LC50) and maximal (LC90) concentrations of V. arborea and H. suaveolens essential oils able to cause mortality on A. aegypti larvae.

The V. arborea essential oil showed LC10 of 39.69 ppm (mg/L), LC50 of 48.98 ppm and LC90 of 60.45 ppm. Furtado et al (2005) report that the V. arborea essential oil induced the highest larvicide action evaluated on their experiment, with LC50 of 15.9 p.m. and LC90 of 28.5 ppm, while the Ocimum gratissimum L. essential oil showed the lowest activity, with LC50 of 95.80 ppm and LC90 of 102.86 ppm.

The H. suaveolens essential oil showed LC10 of 78.46 ppm, LC50 of 139.7 ppm, and LC90 of 246.48 ppm. Cavalcanti et al (2004), when researching on the larvicide activity of some Brazilian plants on A. aegypti, pointed out that LC50 for H. suaveolens was 261 ppm; this value was higher than the one found on this study. However, the value found by the authors is still higher than LC90 (246.48 ppm) found on this study.

On studies conducted by Silva et al (2008) with H. fruticosa and H. pectinata essential oils, they highlight that these plants showed LC50 of 502 and 366 ppm, respectively. On this study, it was observed that H. suaveolens has a larvicide effect that is higher than other plants from the same botanic genus.

The Citrus aurantium L. essential oil showed the highest efficiency, with respective values of LC50 = 22.64 ppm and LC90 = 83.77 ppm, while the Citrus sinensis L. essential oil was the least effective one, with LC50 = 77.55 ppm and LC90 = 351.36 ppm (El-Akhal et al, 2015). Similar values than the ones found by these authors were found on this research, considering the analysis and oscillations of LC50 and LC90 for both studied species.

Komalamisra et al (2005) consider potential larvicides based on natural products with LC50<50 ppm as active, 50 ppm<LC50<100 ppm as moderately active, 100 ppm<LC50<750 ppm as effective, and LC50>750 ppm as inactive. Kiran et al (2006) consider the compounds with LC50<100 ppm as the ones with a significant larvicide effect. If the criterion from Komalamisra et al (2005) is used, it is observed that the V. arborea oil is characterized as active, while H. suaveolens is considered as effective.

The main components of essential oils are terpenes, which are substances with larvicide action with low impact on human and animal health and on the environment, favoring their application on field studies. The oil with the highest larvicide action was the onde from Piper marginatum, with LC50 of 8.29 ppm followed by P. aduncum, with LC50 = 30.19 ppm. The P. nigrum oil showed LC50 = 75.85 ppm (Costa et al, 2010).

The oil extracted from the Anacardium humile Saint Hill leaves caused a toxic effect on A. aegypti larvae, defining LC50 at a concentration of 20.9 ppm, which makes the product a potential larvicide, despite its high toxicity. The minimal concentration able to cause mortality (LC10) was 4.15 ppm, and the maximum toxicity (LC90) was 39.8 ppm (Porto et al, 2008).

Although synthetic products are still advantageous and widely used, natural products have the potential to offer efficient and safer repellence to humans and the environment (Correa and Salgado, 2011).

In fact, insecticides derived from essential oils have several important benefits. Due to their volatile nature, there is a much lower risk level for the environment when compared to the current synthetic insecticides. Populations of predators, parasitoids and pollinator insects are less impacted due to the minimal residual activity, making essential-oil based insecticides compatible with integrated pest control and management programs (Koul et al, 2008).


The essential oils of Hyptis suaveolens and Vanillosmospsis arborea, offer great potential as agents of control against larvae of Aedes aegypti that is considered one of the biggest public health problems in the world. The use of essential oils as insecticides has great potential for use in the integrated management of larval control programs. Since they are biodegradable and non-toxic compounds and it is an alternative method of minimizing the harmful effects of some pesticidal compounds on the environment.

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Fecha de Recepción: 24 Enero, 2017. Fecha de Aceptación: 4 Abril, 2017.

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