Dissuasive and Biocidal Activity of Salvia Officinalis (Lamiaceae) With Induction Of Malformations In Aedes Aegypti (Diptera: Culicidae)

Abstract

Spanish

Different studies have evaluated the biocidal activity of essential oils (EO) in mosquito larvae of medical importance. However, limited research focused on analyzing the EO effect at all the stages of the life cycle of Aedes aegypti has been done. This study evaluates the biological activity of the EO Salvia officinalis against Ae. aegypti. The ovicidal activity was evaluated at 1, 5, 37 and 50 mg.L⁻¹ concentrations in eggs from 0-12 h and eggs from 0-72 h. Larvicidal, pupicidal and adulticidal activities were assessed at exploratory concentrations (EC) and multiple concentrations. We employed an EC of 1,000 mg.L⁻¹ for repellent activity and used an exposure time of 0-2 min and 2-15 min on the forearms of volunteers. The deterrent action was estimated at EC of 5, 50 and 200 mg.L⁻¹. The EO caused malformations in embryos as well as larvae alteration. The highest larvicidal activity was at 63 and 76 mg.L⁻¹ (27 ± 13.4 and 37 ± 18.6%) with 24 h exposure. The greatest pupicidal mortality was at 310 and 390 mg.L⁻¹ (89 ± 1.53 and 100 ± 0%) with 48 h exposure. Adulticidal mortality at 300 mg.L⁻¹ was 57.5 ± 0% and the percentage of repellency was 42 ± 4.7%. The dissuasive action at 200 mg.L⁻¹ was 97 ± 4.81%, with an oviposition activity rate of -0.94. S. officinalis showed a biocidal effect on embryos and mortality of pupae and adults, indicating that its potential use in control programs should be focused on these stages of development.

Keywords

Botanical insecticide oviposition dissuasive activity essential oil ovicidal activity 1-8 cineol

Introduction

Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) is the main vector of pathologies of major impact in urban and peri-urban areas of tropical and subtropical zones, namely Dengue, Chikungunya and Zika virus (Rodríguez-Morales 2015a, 2015b). This mosquito is distributed under 2,200 masl although in Colombia, it is reported at altitudes of up to 2,302 masl, (Ruiz-López et al. 2016). Ae. aegypti is present in 80 % of the national territory, where a large part of the population lives and is susceptible to the three pathologies mentioned above (Singh et al. 2012; Soonwera and Phasomkusolsil 2014; Castrillón et al. 2015).

The efforts to stop the circulation of diseases transmitted by Ae. aegypti are focused on vector control through the use of chemical insecticides and the elimination of their breeding sites (Licastro et al. 2010; Rodríguez 2002). For juvenile phases, temephos and growth regulators are used, and for the control of adults, pyrethroid insecticides are applied (Álvarez González et al. 2014; Ardila-Roldán et al. 2013; Maestre-Serrano and Gómez-Camargo 2013). However, the vector has not shown a population decline due to the inappropriate application of insecticides. This has led to an increase in the number of cases of diseases caused by arboviruses in the neotropical regions (Castrillón et al. 2015; Rodríguez-Morales 2015a, 2015b). Additionally, the indiscriminate use of chemicals induces environmental contamination and generates resistance to the active principles in populations of Ae. aegypti (Leyva et al. 2012; Maheswaran and Ignacimuthu 2012; Bhatt et al. 2013).

As an alternative to insecticides of a synthetic origin (organophosphates-OP and pyrethroids-PI), the use of essential oils (EOs) and extracts obtained from plants have been proposed. These botanical products are synthesized naturally by the secondary metabolism of plants when exposed to sudden changes in temperature or as a chemical defense mechanism against the harmful action of pathogens and phytophagous insects (Ávalos and Pérez-Urria 2009; Koul et al. 2008; Leyva et al. 2012; Arango 2013). The EOs offer multiple applications in vector control programs that are not found in traditional insecticides. For example, biocidal action can be achieved throughout the insect's life cycle (egg, larva, pupa and adult), as well as benefits such as repellent and dissuasive action (Maheswaran and Ignacimuthu 2012; Zoubiri and Baaliouamer 2011).

Different studies have evaluated the biocidal activity of EOs in mosquito larvae of medical importance (Aciole et al. 2011; El-Gendy and Shaalan 2012; Elango et al. 2012; Gokulakrishnan et al. 2013; Leyva et al. 2012; Mathivanan et al. 2010; Ponnusamy et al. 2010; Suman et al. 2013). However, the majority of researches have not focused on analyzing the effects of EO at all of Ae. aegypti life cycle stages and as such have not detected other significant biocidal effects. In this sense, the present study sought to evaluate whether Salvia officinalis L. (Lamiales: Lamiaceae) EO has ovicidal, larvicidal, pupicidal and adulticidal activity together with repellent, attractive and dissuasive action in Ae. aegypti.

Materials and methods

Abbreviations

EO, Essential oil; LC, Lethal concentration; DMSO, Dimethyl Sulfoxide; OAI, oviposition activity index; MWHD, Microwave-assisted hydrodistillation; LD, Lethal doses; KT, knockdown time; RO, olfactory receptor; EDO, odour degrading enzymes.

Essential oil extraction

S. officinalis EO was provided by the “Centro Nacional de Investigación para la Agroindustrialización de Plantas Aromáticas y Medicinales Tropicales - CENIVAM” of the Universidad Industrial de Santander. The oil extraction methodology, as well as its chemical characterization, was carried out in accordance with the methodology described by Ríos et al. (2017).

Aedes aegypti colony

Bioassays were performed with a colony of Ae. aegypti, Rockefeller strain, maintained in an insectary at 25 ± 5 °C, with a humidity of 70 ± 5% and photoperiod (12:12). Male adults were fed on a permanent basis with a sugar solution of 10 % honey (75 % carbohydrates). Females were blood-fed on an albino Wistar rat (WI IOPS AF/Han strain) provided by Universidad Industrial de Santander bioterium. This research was approved by the Ethics Committee (CEINCI) (Minutes No. 11, 8 May 2015).

To obtain the larvae in the experiments, eggs were submerged in mineral water obtained through osmosis (Pure Pro EC106M-P; Pure Pro Water Corporation, Bogotá, Colombia) plus TetraMin Tropical Flakes® fish concentrate (Manufactured by Tetra GMBH Henenteich, Melle, Germany). After emergence, the larvae were kept in plastic trays and fed every three days until they reached the appropriate instar for each assay.

The ovicidal activity

We used the Rezende et al. (2008) and Suman et al. (2013) methodology to establish the ovicidal activity. We separated 20 gravid females of Ae. aegypti in a security cage with dimensions of 40 x 40 x 40 cm. Inside were six plastic cups with 50 µL of EO dissolved in dimethyl sulfoxide (DMSO) at different concentrations plus a glass as a control treatment (49 mL of water + 1 mL of 0.5% DMSO). Initially, we used a screening selection with exploratory concentrations (5, 50 and 200 mg.L⁻¹). Then, we evaluated multiple concentrations of 1, 5, 37 and 50 mg.L⁻¹. The inside of each glass was coated with Whatman® #1 filter paper cut in half and folded into a cone shape, in order to collect and count the eggs. The oviposition lasted for eight consecutive days, changing the filter paper daily. Three replicates were used by concentration and the experiment was repeated on three different days.

When the females oviposited, in order to obtain embryonated eggs and non-embryonated eggs, the time of oviposition was considered: from 0 to 12 h for non-embryonated eggs; and more than 72 h embryonated eggs. Between 50 and 100 eggs per glass were taken at random. In each case, the eggs were transferred to individual containers according to the evaluated concentration, where they were subsequently counted and examined under a stereoscope in order to verify their integrity. The hatching percentage of the eggs was evaluated up to 120 h after oviposition, submerging the eggs obtained for each concentration in mineral water. The first instar larvae that emerged were counted under a microscope, and eggs that did not hatch after seven days were considered unviable. The hatching value of the eggs was estimated as the percentage of eggs that went on to the larval stage in a normal manner.

To determine whether the EO caused alteration in embryonic development and hatching of Ae. aegypti, a random sample of non-embryonated and embryonated eggs from glass treated at different concentrations (1, 5, 37 and 50 mg.L⁻¹) was selected. The eggs selected from each concentration and exposure time were immersed in a solution of 80 % sodium hypochlorite for 10 min until the eggshell lost its pigmentation and became transparent. These eggs were examined under a microscope to detect morphological abnormalities caused by exposure to the EO (malformed embryos, malformations in the head and abdomen and merged corporal tagmas). The morphological changes were recorded to calculate the percentage of abnormalities in relation to the embryo's maturation times.

Larvicidal activity

Larvae at the final L₃ and initial L₄ stages were used. These were transferred individually to 200 mL plastic cups with 99 mL of mineral water. 1 mL of the EO of S. officinalis was added to each plastic cup for a final volume of 100 mL where the concentration-response bioassays were performed. Firstly, we conducted exploratory concentration of 30, 300, and 1,000 mg.L⁻¹ (Vera et al. 2014). Subsequently, multiple concentrations were evaluated (30, 47, 51, 63 and 76 mg.L⁻¹) in order to establish mortalities between 2 and 98 %. In each case, the negative control treatment was DMSO at 0.5% and positive control with the insecticide propoxur (1.50 mg.L⁻¹) were used in this assay. Quadruplicate tests (40 larvae) were performed for each concentration evaluated by repeating the experiment three times on different days until a total of 120 larvae were used by each treatment. The larvae were not fed throughout the treatment. Mortality at 24 and 48 h was recorded in each assay. The results of mortality and survival were subjected to Probit analysis (Wiegand 1972).

Pupicidal activity

Twenty pupae, between 12 and 24 h of age, were removed with a Pasteur pipette and placed in four plastic cups containing 48 mL of mineral water and 2 mL of EO of S. officinalis (final volume: 50 mL). The selective screening was carried out with exploratory concentrations of 250, 310, and 390 mg.L⁻¹ of EO. Pupae in the control group were placed in water with 0.5 % dimethyl sulfoxide (DMSO) as this was the solvent used in the EO, with no toxicity to Ae. aegypti. Pupicidal activity was determined by the percentage of dead pupae at 24 and 48 h; the pupae that showed no movement under external stimuli were considered dead. The replicas and experimental design followed the same criteria of larvicidal activity.

Adulticidal activity

The adulticidal activity of EO was assessed, using the CDC bottle bioassay method (Brogdon and McAllister 1998). The interiors of Wheaton 250 mL glass bottles were treated with 1 mL of EO of S. officinalis at exploratory concentrations of 30, 300 y 1,000 mg.L⁻¹. In the present study, the use of acetone (1 mL) as a negative control is based on the fact that this substance is used as a solvent for EOs and is highly volatile. This characteristic is extremely important, since when applying the treatments, it is necessary that the solvent evaporates, avoiding its interference in the results obtained (Debboun et al. 2007, World Health Organization 2009).

In each bottle, 10 individuals (males and females) were introduced, and mortality was recorded every 15 min for three hours. Triplicate tests (30 adults) were performed for each concentration evaluated by repeating the experiment on three times on different days. The mortality percentage was calculated after 2 min of exposure, followed by every 15 min thereafter for up to 2 h, and a final recorded at 24 h of exposure.

Repellent activity

Twenty females of Ae. aegypti between 5 and 10 days emerged, copulated, fed only of carbohydrates and were maintained nulliparous. They were placed in rectangular acrylic chambers (18 cm x 5 cm x 4 cm) (Castillo et al. 2017). Each device had an opening of 29 mm in diameter at the lower end so that only a section of the forearm of the volunteer is exposed to mosquitoes and treatment. These tests followed the methodology of the American Society for Testing and Materials (ASTM) and ASTM-E951-94 for repellency bioassays (Debboun et al. 2007).

Volunteers were used after signing the informed consent approved by the “Comite de Ética en Investigación Científica” (CEINCI), Minutes No. 3/2013. A 4 cmβ area was initially marked on the forearm with removable ink for application of the treatments. The right arm was used to evaluate 1 mL of S. officinalis EO, at a single concentration of 1,000 mg.L⁻¹ and the left arm was used for the control treatment (1 mL of acetone at 99 %). After the application of the treatments, we waited 20 min (drying time) for the ubication of the acrylic chamber in each arm with the females of Ae. aegypti. During the test, the number of bites and landings were recorded at 0 min and 2 min, and subsequently, in the interval of 2 to 15 min. DEET was used as a positive control. Four replicates of each experiment were performed on different days (40 adults for treatment), which always consisted of four different volunteers for each test, with a maximum exposure time of 1 hour. In all the experiments, the protection percentage was estimated according to the formula proposed by Phasomkusolsil and Soonwera (2011) in time intervals of 0-2 min, from 3 to 15 min and 15 min onwards, as follows:

% Protection = \frac{N c - N t}{N c}

Nc = Number of bites received by control arm; Nt = Number of bites received by the treated arm.

Additionally, the percentage of bites was calculated according to the authors Tawatsin et al. (2001) and Thavara et al. (2001):

Biting % = \frac{B}{60} x 100

Where: B = Total numbers of bites by the end of the test.

Oviposition dissuasive activity

Ten females that were nulliparous, gravid and with a visibly swollen abdomen due to the blood supply were placed in four aluminium cages of 70 x 70 x 70 cm. Four 30 mL plastic cups were introduced in each of the cages, each with 14.25 mL mineral water with 75 µL of S. officinalis EO (final volume: 15 mL). We used a screening selection with exploratory concentrations (5, 50 and 200 mg.L⁻¹). The control treatment was 14.25 mL of water + 75 µL of 0.5 % DMSO. They were subsequently marked and covered with Whatman® #1 filter paper cut in half manually and folded into a cone shape, to allow the oviposition of Ae. aegypti females.

The dissuasive oviposition activity was evaluated for 6 days. Every 24 h the plastic cups were rotating inside the cages counterclockwise and maintaining the same concentrations mentioned above. Each experiment was performed by triplicate and repeated for four different days, counting the number of eggs laid per day, over 6 days. The experiment was evaluated by the Oviposition Activity Index (OAI) between + 1 to – 1 as follows:

Oviposition Activity Index (OAI) = \frac{N t - N s}{N t + N s}

Nt = Total number of eggs in the treatment; Ns = Total number of eggs in the control.

Values greater than or equal to + 0.3 can be considered as attractants, and less than or equal to - 0.3, are considered as repellents (Kramer and Mulla 1979).

Additionally, we calculated the Inhibition Effect (IE) of the different treatments on the oviposition:

Inhibition Effect (IE) % = \frac{N c - N t}{N c}

Nc = Total number of eggs in the control; Nt = Total number of eggs in the treatment (El-Gendy and Shaalan 2012; Phasomkusolsil and Soonwera 2012).

Statistical analyses

The data was tabulated and descriptive statistics, normality test of Kolmogorov-Smirnorv and, Shapiro Wilk were employed. When normal distribution was found, ANOVA and Tukey or Newman-Keuls test were applied. When distribution was not normal, non-parametric tests were employed (Kruskal-Wallis test), and when it was significant, multiple comparison tests were used. According to the experiment, the Spearman correlation test was applied with P ≤ 0.05 considered as statistically significant. The results were analyzed with Statistics V11.

Results

Chemical composition of essential oil

The EO of S. officinalis showed a yield of 0.4 %. The main components of the EO were the monoterpenes 1,8-cineol (26.6 %), α-tujone (18.1 %), the sesquiterpene trans-β-caryophyllene (7.3 %) and the hydrocarbon α-humulene (5.4 %) (Ríos et al. 2017).

Ovicidal activity

We obtained 580 non-embryonated eggs (0-12 h) and 1,054 embryonated eggs (0-72 h). When comparing hatching percentages in eggs from 0-12 hours, a decrease in hatching percentage is observed as the concentration of EO increases, going from 74.7 % for 1 mg.L⁻¹ to 0 % for 50 mg.L⁻¹ (Fig. 1). In the case of eggs from 0-72 hours, an increase in hatching percentage is observed at 5 mg.L⁻¹ (97.6 %), 37 mg.L⁻¹ (100 %) and 50 mg.L⁻¹ (77.6 %) when compared to the other concentrations evaluated. Statistically significant differences were found between the treatments of eggs from 0-12 hours to 37 mg.L⁻¹ and 50 mg.L⁻¹, and the control group (ANOVA test P ≤ 0.05). No statistically significant differences were found between the treatments of the 0-72 hour eggs and the control group [Kruskal-Wallis test: H (4, N = 10) = 7.228125, P = 0.1243]. However, when observing the hatching percentages obtained, it seems that the AE can reduce the hatching percentage of eggs with a few hours of exposure, even at low concentrations (Fig. 1).

Figure 1.

Egg hatching percentage of Aedes aegypti from 0-12 h and 0-72 h (± SD), in different concentrations of Salvia officinalis EO (1, 5, 37 and 50 mg.L⁻¹). ^a,b,c Different letters indicate significant statistical differences between concentrations between eggs from 0-12 h versus the control group (ANOVA test P ≤ 0.05).

In relation to embryonic development, eggs from 0-12 h showed damage in the cephalic capsule. At the highest concentration (50 mg.L⁻¹) 3.4 % of eggs presented malformations. The formation of structures, such as eyes and mouthparts, were not observed. At concentrations of 37 and 50 mg.L⁻¹, the thorax was not adequately formed, and the embryo was completely deformed or nonexistent (Fig. 2). There was a positive correlation between the increase in the concentration of the EO and malformations (R = 0.66, P ≤ 0.05).

Figure 2.

Types of malformations presented in the embryos of Aedes aegypti for each Salvia officinalis EO concentrations in eggs and percentages of malformations from 0-12 h: 1 mg.L⁻¹: malformed embryos, indistinguishable cephalic capsules; 5 mg.L⁻¹: malformations in the head, segments of the abdomen with malformations; 37 mg. L⁻¹: malformed and fused with the thorax; 50 mg.L⁻¹: weak chorion, the embryos are not clearly distinguishable. Eggs without distinguishable embryo, not viable to go on to the larval stage. Eggs from 0-72 h: 1 mg.L⁻¹: malformations in the head, an abnormal increase in egg size, thorax and abdomen with malformations; 5 mg. L⁻¹: malformations in the head, or cephalic capsule indistinguishable, thorax of small size; 37 mg.L⁻¹: embryos with scarcely distinguishable thorax, large head and abdominal segments with malformations; 50 mg.L⁻¹: indistinguishable sections of the embryo, malformations in the head and abdominal segments.

Regarding eggs from 0-72 h, the percentage of malformations was lower than in eggs of 1-12 h, in 20.7 % at a concentration of 37 mg.L⁻¹. The Spearman correlation coefficient showed a negative relationship (R = -0.265, P ≤ 0.05) suggesting that malformations in embryos from 0-72 h do not have a relationship directly proportional to the increase in EO concentration. The malformations caused by the EO were present in the head (non-existent mouthparts, large cephalic capsule), abnormal formation of the thorax and abdomen (scarcely differentiated, incomplete and with malformations at the end of the last segments) (Fig. 2).

The abnormal hatching of the eggs was significantly higher in eggs from 0-12 h at 50 mg.L⁻¹ when compared to the control group (ANOVA test P ≤ 0.05), where the percentage of abnormalities in the hatching was 36.6 %. Regarding eggs from 0-72 h, significant statistical differences were found between the concentration of 37 mg.L⁻¹ and the rest, except at 50 mg.L⁻¹ (ANOVA test P ≤ 0.05). The highest percentage of abnormal hatching was recorded at 37 mg.L⁻¹ at 3.7 %. When comparing hatching for each concentration, statistically significant differences were found at 50 mg.L⁻¹ between eggs from 0-12 h and from 0-72 h (NK test P = 0.0034) (Fig. 3).

Figure 3.

Percentage of eggs of Aedes aegypti with abnormal hatching obtained from 0-12 h and 0-72 h, in different concentrations of Salvia officinalis EO (1, 5, 37 and 50 mg.L⁻¹). Different letters indicate significant statistical differences. ^{a, b, c} between the eggs from 0-12 h versus the control group (Tukey test P≤ 0.05), ^{d, e, f} between eggs from 0-72 h versus the control group (Tukey test P < 0.05). ^{c, f} and statistically significant differences at 50 mg. L⁻¹ between eggs from 0-12 h and from 0-72 h (NK test P = 0.0034).

In relation to the larvae obtained from the eggs in each of the treatments and concentrations evaluated, a higher percentage of abnormalities was observed between 37 and 5 mg.L⁻¹ for eggs from 0-12 h (15.6 %). No significant statistical differences were found in eggs from 0-12 h [KW test H (4, N = 10) = 5.951613; P = 0.2028]. In eggs from 0-72 h, a higher percentage of abnormalities was observed at 5 mg.L⁻¹ (7.5 %) and 50 mg.L⁻¹ (3.4 %). No significant statistical differences were found in eggs from 0-72 h [KW test H (4, N = 10) = 5.834483; P = 0.2119] when compared to the control group. No statistically significant differences were found when comparing larval abnormalities from 0 -12 h and from 0-72 h at each concentration; 1 mg.L⁻¹ (KW Test P = 0.5353), 5 mg.L⁻¹ (Tukey Test P ≥ 0.05), 37 mg.L⁻¹ [KW test H (2, N = 6) = 4, 80; P = 0.090], and 50 mg.L⁻¹ [KW test H (2, N = 6) = 1.30; P = 0.522] (Fig. 4). Regarding the abnormalities observed, morphological damages were detected, mainly represented by the decrease in the amount of chitin in the head of the larvae and in the head-thorax junction. In addition, it was observed that the larvae showed slow or abnormal movements, and in some cases, death.

Figure 4.

Percentage of abnormal larvae hatching of Aedes aegypti observed in eggs from 0-12 h and 0-72 h obtained at different concentrations of Salvia officinalis EO (1, 5, 37 and 50 mg.L⁻¹).

Larvicidal activity

The highest mortality value was observed at 76 mg.L⁻¹ at 24 h (37 ± 18.6) and 48 h (37 ± 19.2). No significant differences were found between the treatments evaluated at 24 h and 48 h [KW test: H (5, N = 18) F = 8.072327 P = 0.1523; KW test: H (5, N = 18) F = 7.970630 P = 0.1579]. The lethal concentrations of S. officinalis EO at 24 h were LC₅₀ = 76.43 mg.L⁻¹ (71.84 - 83.79), LC₉₅ = 123.92 mg.L⁻¹ (106.98 - 136.75). At 48 h the lethal concentrations were LC₅₀ = 123.92 mg.L⁻¹ (106.98 - 136.75), LC₉₅ = 198.20 mg.L⁻¹ (149.17 - 322.5) (Table 1).

Table 1.

Larvicidal activity (in mortality percent ± SD) of the essential oil of Salvia officinalis against Aedes aegypti at 24 and 48 h. LC₅₀ is the lethal concentration causing mortality of 50% of organisms exposed to the treatment. LC₉₅ is the lethal concentration causing mortality of 95% of organisms exposed to the treatment. The confidence interval is given in parentheses. The statistical analysis was well adjusted to the Probit model (Finney 1949).

Concentration (mg. L⁻¹)	Mortality (% ± SD)
Concentration (mg. L⁻¹)	24 h ± SD	48 h ± SD
0	0	0
30	3 ± 2.3	5 ± 1.7
47	6 ± 3.3	16 ± 6.8
63	27 ± 13.4	34 ± 14.8
76	37 ± 18.6	37 ± 19.2
Temephos	100 ± 0	100 ± 0
LC₅₀	76.4 (71.8 - 83.8)	123.9 (106.9 - 136.7)
LC₉₅	123.9 (106.9 - 136.7)	198.2 (149.2 - 322.5)
X ²	5.46	1.37

SD: Standard deviation; LC: Lethal Concentration; X²: Chi squared.

Pupicidal activity

The highest pupicidal action was found in the concentration of 390 mg.L⁻¹, at 24 h and 48 h of exposure (69 ± 3.1 and 100) (Table 2). It was observed that the pupicidal action increased significantly from 24 to 48 h of EO exposure. Statistically significant differences were observed with respect to the negative control at 24 h [KW test: H (3, N = 12) F = 7.533333 P = 0.0567], and 48 h [KW test: H (3, N = 12) F = 8.569604 P = 0.0356].

Table 2.

Pupicidal activity (in mortality percent ± SD) of the essential oil of Salvia officinalis against Aedes aegypti at 24 and 48 h. * Statistically significant differences between negative control and mortality percent.

Concentration (mg. L⁻¹)	Mortality (% ± SD)
Concentration (mg. L⁻¹)	24 h ± SD	48 h ± SD
0	0	0
250	16 ± 1.5	73 ± 3.5 *
310	44 ± 5.8	89 ± 1.5 *
390	69 ± 3.1	100 *

SD: Standard deviation of mortality percent at 24 and 48 h.

Adulticidal activity

It was observed that Ae. aegypti mortality increased significantly with higher concentrations of the EO and greater exposure time [60 min KW test H (3, N = 16) = 9.548033; P = 0.022; 120 min KW test H (3, N = 16) = 9.118421; P = 0.028; 24 h KW test H (3, N = 16) = 11.72727; P = 0.0084]. The highest percentage of mortality was reached at concentrations of 30 and 1,000 mg.L⁻¹ (100 %) at 24 h of exposure. From 0 to 2 min of exposure, no effect was observed in the exposed individuals (Table 3).

Table 3.

Adulticidal activity (in mortality percent ± SD) of the essential oil of Salvia officinalis against Aedes aegypti at 0-2 min, 60 min, 120 min and 24 h. ^a,b,c Equal letters indicate significant statistical differences between control and mortality percentage observed during test intervals.

Concentration (mg.L⁻¹)	Mortality (% ± SD)
Concentration (mg.L⁻¹)	0-2 min	60 min	120 min	24 h
0	0	0	0	0
30	0	55 ± 3ª	62 ± 2^b	100^c
300	0	40 ± 2.4ª	50 ± 3^b	100^c
1,000	0	65 ± 2.6ª	70 ± 2.2^b	100^c

Mosquito repellency activity

The highest percentage of protection was observed at the time interval from 0 to 2 min (67 %), with a bite percentage of 3.3%. No statistically significant differences were found between the time intervals and the negative and positive controls (DEET) (ANOVA P = 0.667563). However, significant statistical differences were found between the time intervals (NK test P = 0.041) (Table 4).

Table 4.

Protection percentage (% ± SD) and bite percentage of the essential oil of Salvia officinalis against Aedes aegypti evaluated during test intervals 0-2, 0-15 min. ^a,b Different letters indicate significant statistical differences between time intervals of protection percentage (Newman-Keuls Test P = 0.041).

Time	Protection percentage (% ± SD)	Control ± SD	DEET ± SD	% Bite
2 min	67ª ± 1	6 ± 2	0 ± 2	3.3
15 min	26^b ± 4.7	19 ± 3.5	0 ± 0	23.3

SD: Standard deviation of protection percentage in negative control and positive control with DEET.

Oviposition deterrent activity

In relation to the deterrence of oviposition, the three concentrations evaluated (5, 50 and 200 mg.L⁻¹) of S. officinalis showed significant inhibition percentages > 80 % [KW test H (3, N = 32) = 10, 39201; P = 0.015]. The concentration of 200 mg.L⁻¹ presented the highest value of oviposition inhibition, directly related to the value obtained in the Oviposition Activity Index (-0.94). Accordingly, female Ae. aegypti avoid laying their eggs in all the treatments evaluated (Table 5).

Table 5.

Oviposition inhibition activity of the essential oil of Salvia officinalis against Aedes aegypti. OAI: Oviposition activity index (negative values indicate a greater number of eggs in the controls, positive values indicate a greater number of eggs in the treatments). IE: Inhibition effect (% ± SD). * Significant statistical differences between control and Inhibition effect (IE) [KW test H (3, N = 32) = 10, 39201; P = 0.015].

Concentration (mg.L⁻¹)	IE % (% ± SD)	OAI
0	0 ± 103.2	0
5	92 ± 12.7 *	- 0.853
50	91 ± 13 *	- 0.831
200	97 ± 4.8 *	- 0.941

Discussion

According to Abou-Elnaga (2014), five chemotypes of the essential oil of S. officinalis have been characterized according to major components (1. Camphor > α-tujone > 1,8-cineol > β-tujone, 2. Camphor > α-tujone > β-tujone > 1,8-cineol; 3. β-tujone > camphor > 1,8-cineol > α-tujone; 4. 8-cineol > camphor > α-tujone > β-tujone; and 5. α-tujone > camphor > β-tujone > 1,8-cineol). 1,8-cineole constitutes the major component (26.6 %) of the essential oil of S. officinalis used in this study, corresponding to chemotype 4. This variation of chemotypes is explained by the inter and intra-specific factors of the genotype, plant cultivation conditions (climate and geographical location), the vegetative organ used for its extraction, time of year in which it was collected and extraction conditions (Bernotiené et al. 2007; Santana et al. 2012).

The chemical characterization described coincides with that reported by Castañeda et al. (2007), who also performed extraction by microwave-assisted hydrodistillation (MWHD) of plants obtained from the same department (Santander, municipality of Sucre) with the major compounds 1,8-cineol (26.6 %) and a- tujona (18.3 %).

Bioassays of ovicidal activity

According to the larvae hatching results, it was observed that Ae. aegypti were susceptible to biocidal action by penetration of S. officinalis EO. This indicates that, although the embryos are protected by a hard shell (Phasomkusolsil and Soonwera 2011), they were permeable to EO. The exposure of eggs from 0-12 h with EO was more effective in decreasing hatching percentage. Accordingly, it can be inferred that exposure time, the concentration used, and embryo age directly affects ovicidal activity (Valarmathy et al. 2011). In relation to the 0-72 eggs where the embryo is already formed, apart from causing morphological damage to the embryo, larvae hatching is also affected. This explains the percentage of emerged larvae that presented abnormalities with the concentrations used.

Arivoli and Tennyson (2011) mention that some substances of vegetable origin can induce deformities and degenerative effects during the development of mosquito eggs, as observed in embryos exposed to EO. According to Valarmathy et al. (2011), longer exposure time to the toxic agent increases the entry capacity of compounds through the shell and affects embryogenesis. In this study, high percentages of abnormalities were observed in eggs from 0-12 h, which are related to the passage of the compounds of S. officinalis through the egg chorion. In general, the chorion is more resistant to drying and the action of external compounds up to two hours after oviposition. During this period, structural modifications occur in its constituent proteins, which lead to its insolubilization (Devillers et al. 2014; Rezende et al. 2008). It is precisely in this time that the components of the S. officinalis EO can penetrate the egg and exert its ovicidal action, or cause malformations. After these two hours, the entry of the essential oil into the egg occurs in smaller quantities through the small pores in the surface of the shell.

With respect to the hatching patterns, the 0-72 h eggs presented significant percentages of larvae malformations. These are directly related to embryo affectation, or damage in the structural conformation of the shell, which would prevent normal eclosion. In relation to the larvae, the type of damage that predominated was the little cephalic chitinization, observed mostly in the eggs of this same period. Even when the larva changed state, this type of damage continued until the pupal stage. Thus, the exposure time of the EO components causes the possible inhibition of chitin synthesis and generates a weaker integument (Arivoli and Tennyson 2011). Accordingly, any type of damage caused at the embryonic stage will affect the following stages of insect development. In this regard, Arivoli and Tennyson (2011) mention that the metamorphic abnormalities caused in early stages of development, also cause abnormalities in pupae and adults, which adds up as an important effect that justifies the utility of EO in mosquito control.

Although high inhibition of hatching could be expected in eggs from 0-72 h, it is important to note that during this time, the embryo was fully developed; therefore, inhibition was not significant. Accordingly, the effect of the essential oil possibly caused physiological alterations of the hormonal type. This phenomenon is similar to that observed with pyriproxyfen (juvenile hormone analogue) and azadirachtin (ecdysone agonist or molting hormone) (Suman et al. 2013). Here the effects are observable in the later development of larval stages, represented in scarce cephalic chitinization, malformations at the digestive tract level and abnormal behaviour, which was consistent with this study.

Larvicidal activity

S. officinalis presents larvicidal action against mosquitoes and the LC₅₀ and LC₉₀ presented in the study are lower than those reported by Pavela (2008). Pavela (2008) studied the larvicidal activity of 56 species of plants from the Euro-Asian region, including S. officinalis, with a mortality of 84.6% at an LD₅₀ of 159 mg.L⁻¹ and an LD₉₀ > 500 mg.L⁻¹ in Culex quinquefasciatus larvae. According to this result, Pavela did not consider the plant S. officinalis as an important larvicidal agent. The same was indicated by Arivoli and Tennyson (2011) who mentioned that some species of Salvia have moderate larvicidal activity, with LC₅₀ values between 100 and 250 mg.L⁻¹. However, more recent studies than those mentioned, show that this plant is considered an important agent for the production of insecticidal molecules. For instance, Ali et al. (2015) presented different species of Salvia with larvicidal activity similar to the results of our study for larvae of Ae. aegypti, with reports of LC₅₀ of 56.9 mg.L⁻¹ and LC₉₀ of 92.4 mg.L⁻¹; and Abou-Elnaga (2014) with LC₅₀ of 25.1 mg.L⁻¹ and LC₉₀ of 120.5 mg.L⁻¹ at 24 h of exposure, and an LC₅₀ of 18.4 mg.L⁻¹ and LC‰ of 106, 8 mg.L⁻¹ at 48 h. The lethal concentrations obtained by these authors are lower than those reported in our study. This might be explained by the major compounds reported by these authors, which where are different from those detected in this study. For example, viridiflorol (20.4 %) and a-tujone (25.8 %) in Ali et al. (2015), and miristicin (20.58 %), sabinene (15.37 %) and terpinen-4-ol (14.92 %) in Abou-Elnaga (2014).

The content of monoterpenoids in the essential oil of S. officinalis (thujone, camphor and 1,8-cineol) has a strong larvicidal effect against other mosquito species, such as Anopheles and Culex (Abou-Elnaga 2014). The major component of S. officinalis in this work, 1,8-cineol, has been reported with insecticidal action against various insect species, even being extracted from other plant species (Leyva et al. 2009). Kishore et al. (2011) mention that 1,8-cineole isolated from leaves of Hyptis martiusii Benth (Lamiaceae) showed a larvicidal effect on Ae. aegypti at concentrations of 25 mg.mL⁻¹ (10 % mortality), 50 mg.mL⁻¹ (53 % mortality) and 100 mg.mL⁻¹ (100 % mortality). Lucia et al. (2007), evaluated the larvicidal activity of Eucaliptus grandis W. Hill ex Maid (Myrtaceae) and its major components, and report that 1,8-cineol showed low larvicidal activity, with an LC₅₀ of 57.2 mg.L⁻¹.

Pupicidal activity

The pupae of Ae. aegypti have strong cuticle sclerotization as the hatching time of the adult individual approaches, going from a thin, smooth and colorless layer, to a darker and thicker cuticle before emergence (Popa et al. 2011). This degree of cuticle sclerotization contributes to a lower susceptibility of this stage to the action of external agents (Ramar et al. 2013), whether from synthetic or natural origin.

Since the pupa stage is more resistant, there are few studies that report the mortality of essential oils in mosquitoes, and the high concentrations used to achieve 100 % mortality. No studies were found in the literature involving the effect of S. officinalis on Ae. aegypti. However, when examining the concentrations used in studies evaluating the action of different plant species, it is evident that the concentrations used are between 160 and 1,000 mg.L⁻¹ (Gleiser et al. 2007; Ramar et al. 2013) and between 100 and 1,000 µL / mL (Sivagnaname and Kalyanasundaram 2004; Souza et al. 2011; Candido et al. 2013; Phasomkusolsil and Soonwera 2013).

The concentrations used in this paper to observe the pupicidal effect showed mortality percentages higher than 50 % at 48 h of exposure in concentrations that did not exceed 400 mg.L⁻¹. Since the pupae used for the bioassay were less than 24 h of emergence, that is, they had a less sclerotic cuticle, it is possible that the observed mortality is related to the action by contact with the components of the AE present in the liquid medium. Since the cuticle of insects is made up of different regions that differ markedly with respect to their chemical composition and properties, some studies consider that there is an ideal polarity value for the entry of xenobiotics in the organism (Lucia et al. 2013).

Adulticidal activity

The observed adulticidal effect is possibly due to the presence of monoterpenes in the EA. Studies indicate that they are the cause of a neurotoxic effect similar to that of organophosphorus compounds and carbamates in the inhibition of the enzyme acetylcholinesterase (Lucia et al. 2013). In this study, the major compounds, 1,8-cineol and α-tujone, play an important adulticidal role, due to the fact that both have neurotoxic effect in mosquitoes, reflected in a knockdown effect (Lucia et al. 2007; Lucia et al. 2009a). Being volatile monoterpenes, these compounds enter the body through the tracheae and tracheoles, entering in circulation through these ducts until they reach the hemolymph, where they are absorbed by proteins and spread throughout the body of the insect (Lucia et al. 2013). Although there are few studies evaluating the adulticidal effect of S. officinalis, the action times reported in this study coincide with those published by Lucia et al. (2009b), who obtained a fumigant effect of 1,8-cineol with a knockdown time (KT_50min) between 4.95-5.73, attributable to a neurotoxic effect of this compound. Subsequently, Lucia et al. (2011) evaluated the fumigant action of 15 plants of the genus Eucalyptus and found a KT₅₀ between 10.65-0.076 min for species with a high content of 1,8-cineol. The minor adulticidal activity obtained with 300 mg.L⁻¹ is possibly related to the volatility of the compounds of the EO. Lucia et al. (2009b) reported that species with a high content of 1,8-cineole in their essential oils have a greater fumigant activity against Ae. aegypti, and this activity is related with the vapor pressure of this compound.

Repellency activity

The first line of defense against mosquito bites is the application of repellents. For this reason, to understand that there are substances as EOs that have this effect on mosquitoes can be a powerful tool to prevent its bites (Nerio et al. 2010; Jaramillo Ramirez et al. 2012). The protection and repellency percentages obtained from 0 to 2 min indicate that S. officinalis EO constitutes a repellent and a deterrent element (Phasomkusolsil and Soonwera 2011) against bites in this period, with a subsequent decrease in its effectiveness in the period from 2 to 15 min. This effect may be due to the fact that repellents work by creating a vapor barrier that prevents the insect from coming into contact with the surface of the skin (Nerio et al. 2010), but this same vapor phase of the EA makes them effective only during a relatively short period of time due to the high volatility and the type of components involved (Leyva et al. 2012; Phasomkusolsil and Soonwera 2011).

Some authors suggest that monoterpenes such as a-pinene, 1,8-cineol, eugenol, limonene, terpinolene, citronellol, citronellal, camphor and thymol present insecticidal activity in adult mosquitoes as natural repellents (Leyva et al. 2009; Nerio et al. 2010; Abou-Elnaga 2014). Individual compounds of the oil, such as 1,8-cineol, are detected by a specific olfactory receptor (RO) of the antenna of the mosquitoes (located in the sensilla's), which, in combination with odor degrading enzymes (EDO) recognizes the stimulus (Jaramillo Ramirez et al. 2012). If the EO compound acts by inhibiting the activity or production of the EDO enzymes, the specific olfactory receptor (RO) continues to function even though there would be no enzymes to break it down, which would cause confusion and disorientation in the insects, making them unable to locate their target (Jaramillo Ramirez et al. 2012).

However, this effect cannot only be attributed to these components, since bioactivity is associated with the joint presence of monoterpenes and sesquiterpenes in the EO (Nerio et al. 2010; Leyva et al. 2012) as is the case with S. officinalis. Therefore, in the case of the S. officinalis EO, a formulation should be considered to help stabilize their volatile components and improve protection times.

Deterrent activity

The high percentages of effectiveness in the inhibition of oviposition indicate that the females are sensitive to the smell of EO. The egg oviposition restriction action occurs because the sensory system of the insects is able to detect the volatile compounds of S. officinalis EO, which, when dissipated in the oviposition medium, produce secondary metabolites that act together or independently to avoid laying eggs (Navarro-Silva et al. 2009; El-Gendy and Shaalan 2012).

Davis and Bowen (1994) mention that mosquitoes use physical and chemical signals to detect potential oviposition sites through sensory receptors in the antennae, which respond to specific stimuli depending on the stage of development. Gravid females will be more likely to look for suitable oviposition sites, with adequate color and optical density of the water, texture, moisture and temperature of the oviposition substrate (Guha et al. 2012).

Only a few studies are available on the deterrent potential of EO from different plants. It has been reported that flavonoid compounds and formulations based on neem reduce oviposition and affect Ae. aegypti fertility (Guha et al. 2012). However, the EO of S. officinalis has monoterpenes and sesquiterpenes, which due to their volatility can cause an evasive action on the part of the females to these compounds, acting as semiochemicals (Navarro-Silva et al. 2009). These data support the values obtained at the three concentrations for the oviposition activity index (OAI), indicating high deterrent action potential against Ae. aegypti.

In conclusion, S. officinalis EOs presented a biocidal effect in all stages of development of the Ae. aegypti mosquito. However, this effect was more pronounced in the egg, pupa and adult stages (mortality and detergent activity). The results of the present study helped to understand the form like the S. officinalis EO has an insecticide effect over the different developmental stages of Ae. aegypti. According to this, the information contributes presented here can be useful to propose the components of EO of S. officinalis as possible replacement candidates for conventional chemical substances like temephos.

Origin and funding

This study was part of master´s dissertation of Ruth Castillo and carried thanks to the financing of COLCIENCIAS, “Autonomous Patrimony, National Fund for Financing for Science, Francisco José de Caldas”, through contract RC-0572-2012- Bio-Network - CENIVAM.

Author contribution

The first author, Ruth M. Castillo-Morales, performed the experiments, analyzed the information, and wrote the paper. The second author, Jonny E. Duque, worked on designing experiments, analyses, and writing the paper.

Footnotes

Acknowledgements

To Dr Elena E. Stashenko for supplying the essential oil. To the members of the Research Group on Tropical Diseases (CINTROP), especially to Gustavo Rincón and Juliana Cuadros. This study was conducted thanks to the financing of COLCIENCIAS, “Autonomous Patrimony, National Fund for Financing for Science, Francisco José de Caldas”, through contract RC-0572-2012- Bio-Network -CENIVAM. Thank the referees who have reviewed the work.

References

ABOU-ELNAGA

Z. S.

2014. Insecticidal bioactivity of eco-friendly plant origin chemicals against Culex pipiens and Aedes aegypti (Diptera: Culicidae). Journal of Entomology and Zoology Studies 2(6): 340–347. https://www.entomoljournal.com/archives/2014/vol2issue6/PartG/75-258.pdf

ACIOLE

S. D. G.

; PICCOLI

C. F.

; DUQUE

L. J. E.

; COSTA

E. V.

; NAVARRO-SILVA

M. A.

; MARQUES

F. A.

; SALES MAIA

B. H. L. N.

; PINHEIRO

M. L. B.

; REBELO

M. T.

2011. Insecticidal activity of three species of Guatteria (Annonaceae) against Aedes aegypti (Diptera: Culicidae). Revista Colombiana de Entomología 37(2): 262–268. http://www.scielo.org.co/scielo.php?script=sci_arttext&pid=S0120-04882011000200018

ALI

; TABANCA

; DEMIRCI

; BLYTHE

E. K.

; ALI

; CAN BASER

K. H.

; KHAN

I. A.

2015. Chemical composition and biological activity of four Salvia essential oils and individual compounds against two species of mosquitoes. Journal of Agricultural and Food Chemistry 63(2): 447–456. 10.1021/jf504976f

ÁLVAREZ GONZÁLEZ

; PONCE GARCÍA

; OVIEDO

; BRICEÑO

; FLORES SUÁREZ

A. E.

2014. Mecanismos asociados a la resistencia al derribo “kdr” a la deltametrina en Aedes aegypti del occidente de Venezuela. Boletín de Malariologia y Salud Ambiental 54(1): 58–67. http://ve.scielo.org/pdf/bmsa/v54n1/art07.pdf

ARANGO

M. C.

2013. Intervención de los compuestos secundarios en las interacciones biológicas. pp. 191–256. In: Ringuelet

; Viña

(Eds.). Productos naturales vegetales Editorial de la Universidad de La Plata. lera edición. Universidad Nacional de La Plata. La Plata, Buenos Aires, Argentina. 261 p. http://sedici.unlp.edu.ar/handle/10915/27885 [Review date: 27 April 2018].

ARDILA-ROLDÁN

; SANTACOLOMA

; BROCHERO

2013. Estado de la sensibilidad a los insecticidas de uso en salud pública en poblaciones naturales de Aedes aegypti (Diptera: Culicidae) del departamento de Casanare, Colombia. Revista Biomédica 33(3): 446–458. 10.7705/biomedica.v33i3.1534

ARIVOLI

; TENNYSON

2011. Studies on the mosquitocidal activity of Murraya koenigii (L.) Spreng (Rutaceae) leaf extracts against Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus (Diptera: Culicidae). Asian Journal of Experimental Biology Sciences 2(4): 721–730.

ÁVALOS

G. A.

; PÉREZ-URRIA

C. E.

2009. Metabolismo secundario de plantas. Reduca (Biología) 2(3): 119–145. http://www.revistareduca.es/index.php/biologia/article/view/798/814

BERNOTIENĖ

; NIVINSKIENĖ

; BUTKIENĖ

; MOCKUTÉ

2007. Essential oil composition variability in sage (Salvia officinalis L.). Chemija 18(4): 38–43. http://mokslozurnalai.lmaleidykla.lt/publ/0235-7216/2007/4/38-43.pdf

10.

BHATT

; GETHING

; BRADY

O. J.

; MESSINA

J. P.

; FARLOW

A. W.

; MOYES

C. L.

; DRAKE

J. M

; BROWNSTEIN

J. S.

; HOEN

A. G.

; SANKOH

; MYERS

M. F.

; GEORGE

D. B.

; JAENISCH

; WINT

G. R. W.

; SIMMONS

C. P.

; SCOTT

T. W.

; FARRAR

J. J.

; HAY

S. I.

2013. The global distribution and burden of dengue. Nature 496: 504–507. 10.1038/nature12060

11.

BROGDON

W. G

; McALLISTER

J. C.

1998. Simplification of adult mosquito bioassays through use of time-mortality determinations in glass bottles. Journal of the American Mosquito Control Association 14(2): 159–164. https://www.biodiversitylibrary.org/content/part/JAMCA/JAMCA_V14_N2_P159-164.pdf

12.

CANDIDO

L. P.

; CAVALCANTI

M. T.

; BESERRA

E. B.

2013. Bioactivity of plant extracts on the larval and pupal stages of Aedes aegypti (Diptera, Culicidae). Revista da Sociedade Brasileira de Medicina Tropical 46(4): 420–425. 10.1590/0037-8682-0118-2013

13.

CASTAÑEDA

M. L.

; MUÑOZ

; MARTÍNEZ

J. R.

; STASHENKO

E. E.

2007. Estudio de la composición química y la actividad biológica de los aceites esenciales de diez plantas aromáticas colombianas. Scientia et Technica 13(33): 165–166. https://www.redalyc.org/pdf/849/84903338.pdf

14.

CASTILLO

R. M.

; STASHENKO

; DUQUE

J. E.

2017. Insecticidal and repellent activity of several plant-derived essential oils against Aedes aegypti. Journal of the American Mosquito Control Association 33(1): 25–35. 10.2987/16-6585.1

15.

CASTRILLÓN

J. C.

; CASTAÑO

J. C.

; URCUQUI

2015. Dengue en Colombia, diez años de evolución. Revista Chilena de Infectología 32(2): 22–29. 10.4067/S0716-10182015000300002

16.

DAVIS

E. E.

; BOWEN

1994. Sensory physiological basis for attraction mosquitoes. Journal of the American Mosquito Control Association 10(2): 316–325. https://www.biodiversitylibrary.org/content/part/JAMCA/JAMCA_V10N2_P316-325.pdf

17.

DEBBOUN

; FRANCES

S. P.

; STRICKMAN

2007. Standard methods for testing mosquito repellents. pp 103–110. In: Debboun

; Frances

; Strickman

(Ed.). Insect repellents: principles, methods, and uses. Taylor and Francis Group. CRC Press. Boca Ratón, Florida, EE. UU. 464 p.

18.

DEVILLERS

; LAGNEAU

A. C.

; LATTES

; GARRIGUES

J. C.

; CLÉMENTÉ

M. M.

; YÉBAKIMA

2014. In silico models for predicting vector control chemicals targeting Aedes aegypti. SAR and QSAR in Environmental Research 25(10): 805–835. 10.1080/1062936X.2014.958291

19.

EL-GENDY

N. A.

; SHAALAN

E. A.

2012. Oviposition deterrent activity of some volatile oils against the filaria mosquito vector Culex pipiens. Journal of Entomology 6(6): 435–441. 10.3923/je.2012.435.441

20.

ELANGO

; RAHUMAN

A. A.

; KAMARAJ

; BAGAVAN

; ZAHIR

A. A.

2012. Adult emergence inhibition and adulticidal activity of leaf crude extracts against Japanese encephalitis vector, Culex tritaeniorhynchus. Journal of King Saud University – Science 24(1): 73–80. 10.1016/j.jksus.2010.08.012

21.

FINNEY

D. J.

1949. The adjustment for a natural response rate in Probit Analysis. Annals of Applied Biology 36(2): 187–195. 10.1111/j.1744-7348.1949.tb06408.x

22.

GLEISER

R. M.

; BONINO

M. A.

; ZYGADLO

J. A.

2007. Bioactividad de aceites esenciales de Minthostachys mollis contra mosquitos. Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas 6(6): 350–351. https://www.redalyc.org/pdf/856/85617472019.pdf

23.

GOKULAKRISHNAN

; KUPPUSAMY

; SHANMUGAM

; APPAVU

; KALIYAMOORTHI

2013. Pupicidal and repellent activities of Pogostemon cablin essential oil chemical compounds against medically important human vector mosquitoes. Asian Pacific Journal of Tropical Disease 3(1): 26–31. 10.1016/S2222-1808(13)60006-7

24.

GUHA

; SEENIVASAGAN

; BANDYOPADHYAY

; IQBAL

S. T.

; SATHE

; SHARMA

; PARASHAR

B. D.

; KAUSHIK

M. P.

; THANVIR

; SATHE

; SHARMA

; PARASHAR

B. D.

; KAUSHIK

M. P.

2012. Oviposition and flight orientation response of Aedes aegypti to certain aromatic aryl hydrazone esters. Parasitology Research 111(3): 975–982. 10.1007/s00436-012-2921-y

25.

JARAMILLO RAMIREZ

G. I.

; LOGAN

J. G.

; LOZA-REYES

; STASHENKO

; MOORES

G. D.

2012. Repellents inhibit P450 enzymes in Stegomyia (Aedes) aegypti. PLoS ONE 7(11): 1–8. 10.1371/journal.pone.0048698

26.

KISHORE

; MISHRA

B. B.

; TIWARI

V. K.

; TRIPATHI

2011. A review on natural products with mosquitocidal potentials. Research Signpost: 335–365.

27.

KOUL

; WALIA

; DHALIWAL

G. S.

2008. Essential oils as green pesticides: Potential and constraints. Biopesticides International 4(1): 63–84.

28.

KRAMER

W. L.

; MULLA

M. S.

1979. Oviposition attractants and repellents of mosquitoes: oviposition responses of Culex mosquito to organic infusions. Environmental Entomology 8(6): 1111–1117. 10.1093/ee/8.6.1111

29.

LEYVA

; MARQUETTI

M. C.

; TACORONTE

J. E.

; SCULL

; TIOMNO

; MESA

; MONTADA

2009. Actividad larvicida de aceites esenciales de plantas contra Aedes aegypti (L.) (Diptera: Culicidae). Revista Biomédica 20(1): 5–13. https://www.medigraphic.com/pdfs/revbio/bio-2009/bio091b.pdf

30.

LEYVA

; TIOMNO

; TACORONTE

J. E.

; MARQUETTI

M. C.

; MONTADA

2012. Essential plant oils and insecticidal activity in Culex quinquefasciatus. pp. 221–238. In: Perveen

(Ed.). Insecticides - Pest Engineering. Ed. Intech, 538 p. ISBN 978-953-307-895-3. 10.5772/27818

31.

LICASTRO

; HÉCTOR

; SECCACINI

; HARBURGUER

; LUCIA

; ZERBA

2010. Innovación en herramientas de control del mosquito Aedes aegypti (Diptera: Culicidae), transmisor del dengue en Argentina. Ciencia e Investigación 60(4): 49–57. http://aargentinapciencias.org/wp-content/uploads/2017/11/revista_cei60-4.pdf

32.

LUCIA

; GONZALEZ-AUDINO

; SECCACINI

; LICASTRO

; ZERBA

; MASUH

2007. Larvicidal effect of Eucalyptus grandis essential oil and turpentine and their major components on Aedes aegypti larvae. Journal of the American Mosquito Control Association 23(3): 299–303. 10.2987/8756-971X(2007)23[299:LEOEGE]2.0.CO;2

33.

LUCIA

; HARBURGUER

; LICASTRO

; ZERBA

, MASUH

2009a. Efficacy of a new combined larvicidal-adulticidal ultralow volume formulation against Aedes aegypti (Diptera: Culicidae), vector of dengue. Parasitology Research 104(5): 1101–1107. 10.1007/s00436-008-1294-8

34.

LUCIA

; LICASTRO

; ZERBA

; AUDINO

P. G.

; MASUH

2009b. Sensitivity of Aedes aegypti adults (Diptera: Culicidae) to the vapors of Eucalyptus essential oils. Bioresource Technology 100(23): 6083–6087. 10.1016/j.biortech.2009.02.075

35.

LUCIA

; JUAN

L. W.

; ZERBA

E. N.

; HARRAND

; MARCÓ

; MASUH

H. M.

2011. Validation of models to estimate the fumigant and larvicidal activity of Eucalyptus essential oils against Aedes aegypti (Diptera: Culicidae). Parasitology Research 110: 1675–1686. 10.1007/s00436-011-2685-9

36.

LUCIA

; ZERBA

; MASUH

2013. Knockdown and larvicidal activity of six monoterpenes against Aedes aegypti (Diptera: Culicidae) and their structure-activity relationships. Parasitology Research 112(12): 4267–4272. 10.1007/s00436-013-3618-6

37.

MAESTRE-SERRANO

; GÓMEZ-CAMARGO

2013. Dengue: Epidemiología, políticas públicas y resistencia de vectores a insecticidas. Revista Ciencias Biomédicas 4(2): 302–317.

38.

MAHESWARAN

; IGNACIMUTHU

2012. A novel herbal formulation against dengue vector mosquitoes Aedes aegypti and Aedes albopictus. Parasitology Research 110: 1801–1813. 10.1007/s00436-011-2702-z

39.

MATHIVANAN

; GOVINDARAJAN

; ELUMALAI

; KRISHNAPPA

; ANANTHAN

2010. Mosquito larvicidal and phytochemical properties of Ervatamia coronaria Stapf. (Family: Apocynaceae). Journal of Vector Borne Diseases 47(3): 178–180. http://www.mrcindia.org/journal/issues/473178.pdf

40.

NAVARRO-SILVA

M. A.

; MARQUES

F. A.

; DUQUE

L. J. E.

2009. Review of semiochemicals that mediate the oviposition of mosquitoes: a possible sustainable tool for the control and monitoring of Culicidae. Revista Brasileira de Entomologia 53(1): 1–6. 10.1590/S0085-56262009000100002

41.

NERIO

L. S.

; OLIVERO-VERBEL

; STASHENKO

2010. Repellent activity of essential oils: A review. Bioresource Technology 101(1): 372–378. 10.1016/j.biortech.2009.07.048

42.

PAVELA

2008. Larvicidal effects of various Euro-Asiatic plants against Culex quinquefasciatus Say larvae (Diptera: Culicidae). Parasitology Research 102(3): 555–559. 10.1007/s00436-007-0821-3

43.

PHASOMKUSOLSIL

; SOONWERA

2011. Comparative mosquito repellency of essential oils against Aedes aegypti (Linn.), Anopheles dirus (Peyton and Harrison) and Culex quinquefasciatus (Say). Asian Pacific Journal of Tropical Biomedicine 1(1): 113–118. 10.1016/S2221-1691(11)60136-6

44.

PHASOMKUSOLSIL

; SOONWERA

2012. The effects of herbal essential oils on the oviposition deterrent and ovicidal activities of Aedes aegypti (Linn.), Anopheles dirus (Peyton and Harrison) and Culex quinquefasciatus (Say). Tropical Biomedicine 29(1): 138–150. 10.1016/S2221-1691(11)60136-6

45.

PHASOMKUSOLSIL

; SOONWERA

2013. Efficacy of Thai herbal essential oils against three immature stages of Aedes aegypti (Linn), Anopheles dirus (Peyton and Harrison) and Culex quinquefasciatus (Say). Journal of Herbal Medicine 2(2): 25–35.

46.

PONNUSAMY

; XU

; BÖRÖCZKY

; WESSON

D. M.

; AYYASH

L. A.

; SCHAL

; APPERSON

C. S.

2010. Oviposition responses of the mosquitoes Aedes aegypti and Aedes albopictus to experimental plant infusions in laboratory bioassays. Journal of Chemical Ecology 36(7): 709–719. 10.1007/s10886-010-9806-2

47.

POPA

J. C.

; CASTILLO

R. M.

; PÉREZ

M. G.

; FIGUEREDO

; MONTADA

2011. Metamorfosis y emergencia de Aedes aegypti fuera del medio acuático y nuevo reporte de importancia entomológica y epidemiológica en Santiago de Cuba. Revista Cubana de Higiene y Epidemiologia 49(2): 173–182. http://scielo.sld.cu/pdf/hie/v49n2/hie04211.pdf

48.

RAMAR

; PAULRAJ

M. G.

; IGNACIMUTHU

2013. Screening of pupicidal activity of some essential oils against Culex quinquefasciatus Say. Journal of Medicinal Plant Research 1(2): 9–12. https://www.academia.edu/4708450/Screening_of_pupicidal_activity_of_some_essential_oils_against_Culex_quinquefasciatus_Say

49.

REZENDE

G. L.

; MARTINS

A. J.

; GENTILE

; FARNESI

L. C.

; PELAJO-MACHADO

; PEIXOTO

A. A.

; VALLE

2008. Embryonic desiccation resistance in Aedes aegypti: presumptive role of the chitinized serosal cuticle. BMC Developmental Biology 8(1): 82. 10.1186/1471-213X-8-82

50.

RÍOS

; STASHENKO

E. E.

; DUQUE

J. E.

2017. Evaluation of the insecticidal activity of essential oils and their mixtures against Aedes aegypti (Diptera: Culicidae). Revista Brasileira de Entomologia 61(4): 307–311. 10.1016/j.rbe.2017.08.005

51.

RODRÍGUEZ-MORALES

A. J.

2015a. Aedes: un eficiente vector de viejos y nuevos arbovirus (dengue, chikungunya y zika) en las Américas. Revista Cuerpo Medicina 8(2): 50–52. http://cmhnaaa.org.pe/ojs/index.php/rcmhnaaa/article/view/192/162

52.

RODRÍGUEZ-MORALES

A. J.

2015b. No era suficiente con dengue y chikungunya: llegó también Zika. IMedPub Journals 11(2-3): 1–4. https://www.archivosdemedicina.com/medicina-de-familia/no-era-suficiente-con-denguey-chikungunya-lleg-tambinzika.pdf

53.

RODRÍGUEZ

2002. Estrategias para el control del dengue y del Aedes aegypti en las Américas. Revista Cubana de Medicina Tropical 54(3): 189–201. http://scielo.sld.cu/pdf/mtr/v54n3/mtr04302.pdf

54.

RUIZ-LÓPEZ

; GONZÁLEZ-MAZO

; VÉLEZ-MIRA

; GÓMEZ

G. F.

; ZULETA

; URIBE

; VÉLEZ-BERNAL

I. D.

2016. Presencia de Aedes (Stegomyia) aegypti (Linnaeus, 1762) y su infección natural con el virus dengue en alturas no registradas para Colombia. Revista Biomédica 36(2): 303–308. 10.7705/biomedica.v36i2.3301

55.

SANTANA

; CABRERA

; GIMÉNEZ

; GONZÁLEZ-COLOMA

; SÁNCHEZ-VIOQUE

; DE LOS MOZOS-PASCUAL

; RODRÍGUEZ-CONDE

M. F.

; LASERNA-RUIZ

; USANO-ALEMANY

; HERRAIZ

2012. Perfil químico y biológico de aceites esenciales de plantas aromáticas de interés agro-industrial en Castilla-La Mancha (España). Grasas y Aceites 63(2): 214–222. 10.3989/gya.129611

56.

SINGH

; SINGH

P. R.

; MOHANTY

M. K.

2012. Toxicity of a plantbased mosquito repellent/killer. Interdisciplinary Toxicology 5(4): 184–191. 10.2478/v10102-012-0031-4

57.

SIVAGNANAME

; KALYANASUNDARAM

2004. Laboratory evaluation of methanolic extract of Atlantia monophylla (Family: Rutaceae) against immature stages of mosquitoes and non-target organisms. Memorias do Instituto Oswaldo Cruz 99(1): 115–118. 10.1590/S0074-02762004000100021

58.

SOONWERA

; PHASOMKUSOLSIL

2014. Mosquito repellent from Thai essential oils against dengue fever mosquito (Aedes aegypti (L.)) and filarial mosquito vector (Culex quinquefasciatus (Say)). African Journal of Microbiology Research 8(17): 1819–1824. 10.5897/AJMR2014.6737

59.

SOUZA

T. M.

; FARIAS

D. F.

; SOARES

B. M.

; VIANA

M. P.

; LIMA

G. P. G.

; MACHADO

L. K. A.

; MORAIS

S. M.

; CARVALHO

A. F. U.

2011. Toxicity of Brazilian plant seed extracts to two strains of Aedes aegypti (Diptera: Culicidae) and nontarget animals. Journal of Medical Entomology 48(4): 846–851. 10.1603/ME10205

60.

SUMAN

D. S.

; WANG

Y. W.

; BILGRAMI

A. L.

; GAUGLER

2013. Ovicidal activity of three insect growth regulators against Aedes and Culex mosquitoes. Acta Tropica 128(1): 103–109. 10.1016/j.actatropica.2013.06.025

61.

TAWATSIN

; WRATTEN

S. D.

; SCOTT

R. R.

; THAVARA

; TECHADAMRONGSIN

2001. Repellency of volatile oils from plants against three mosquito vectors. Journal of Vector Ecology 26(1): 76–82. https://pubmed.ncbi.nlm.nih.gov/11469188/

62.

THAVARA

; TAWATSIN

; CHOMPOOSRI

; SUWONKERD

; CHANSANG

U. R.

; ASAVADACHANUKORN

2001. Laboratory and field evaluations of the insect repellent 3535 (ethyl butylacetylaminopropionate) and deet against mosquito vectors in Thailand. Journal of the American Mosquito Control Association 17(3): 190–195. https://pubmed.ncbi.nlm.nih.gov/14529087/

63.

VALARMATHY

; GOVINDARAJU

; ELUMALAI

2011. Studies on ovicidal activity of plant essential oil formulation against the eggs of important vector mosquitoes, Anopheles stephensi (Liston), Culex quinquefasciatus (Say) and Aedes aegypti (L.) at laboratory condition. International Journal of Current Research 3(6): 378–381. https://www.recentscientific.com/sites/default/files/Download_107.pdf

64.

VERA

S. S.

; ZAMBRANO

D. F.

; MÉNDEZ-SANCHEZ

S. C.

; RODRÍGUEZ-SANABRIA

; STASHENKO

E. E.

; DUQUE-LUNA

J. E.

2014. Essential oils with insecticidal activity against larvae of Aedes aegypti (Diptera: Culicidae). Parasitology Research 113(7): 2647–2654. 10.1007/s00436-014-3917-6

65.

WIEGAND

1972. Finney, D. J.: Probit analysis. 3. Aufl. Cambridge University Press, Cambridge 1971. XV, 333 S., 41 Rechenbeispiele, 20 Diagr., 8 Tab., 231 Lit., L 5.80. Biometrische Zeitschrift 14(1): 72–72. 10.1002/bimj.19720140111

66.

WORLD HEALTH ORGANIZATION. 2009. Guidelines for efficacy testing of spatial repellents for human skin. World Health Organization. 1–30. https://apps.who.int/iris/handle/10665/70072

67.

ZOUBIRI

; BAALIOUAMER

2011. Potentiality of plants as source of insecticide principles. Journal of Saudi Chemical Society 18(6): 925–938. 10.1016/j.jscs.2011.11.015