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Abstract
This study investigated the toxic effects of essential oils isolated from Siparuna guianensis against Aedes aegypti, Culex quinquefasciatus (eggs, larvae, pupae, and adult) and Aedes albopictus (C6/36) cells. The oviposition-deterring activity, egg viability, and repellence activity in the presence of different essential oils concentrations were determined. The essential oils showed high toxicity to all developmental stages of A. aegypti and C. quinquefasciatus. Furthermore, the oils also showed high repellent activity towards the adult stage of mosquitoes (0.025 to 0.550 μg/cm2 skin conferred 100% repellence up to 120 min) and in contact with cultured insect cells (C6/36) induced death possibly by necrosis. The results presented in this work show the potential of S. guianensis essential oils for the development of an alternative and effective method for the natural control of mosquitoes in homes and urban areas.
Citation: Aguiar RWS, dos Santos SF, da Silva Morgado F, Ascencio SD, de Mendonça Lopes M, Viana KF, et al. (2015) Insecticidal and Repellent Activity of Siparuna guianensis Aubl. (Negramina) against Aedes aegypti and Culex quinquefasciatus. PLoS ONE 10(2): e0116765. https://doi.org/10.1371/journal.pone.0116765
Academic Editor: Immo A. Hansen, New Mexico State University, UNITED STATES
Received: July 7, 2014; Accepted: December 13, 2014; Published: February 3, 2015
Copyright: © 2015 Aguiar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), www.cnpq.br, SECT (Secretária de Ciência Tecnologia do estado do Tocantins), http://www.sedecti.to.gov.br/sedecti/, and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), www.capes.gov.br. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Aedes aegypti L. (Diptera: Culicidae) and Culex quinquefasciatus Say (Diptera: Culicidae) are insect vectors responsible for the transmission of several human diseases caused by protozoans (malaria), nematodes (lymphatic filariasis) and viruses (West Nile encephalitis, dengue fever, yellow fever and chikungunya fever). In 2010, there were more than 1.6 million cases of dengue virus infection in Latin America, and in 2013, 204,650 cases only in Brazil [1]. Dengue fever is a debilitating disease caused by a flavivirus and transmitted mainly by female A. aegypti mosquitoes. Because there are no vaccines available, the most effective way to prevent this disease is to control the insect vector. Filariasis is caused by the roundworm Wuchereria bancrofti (Cobb.), which is transmitted by C. quinquefasciatus. This parasite is endemic to many tropical regions, with an estimated 120 million individuals infected in 81 countries and more than 1 billion people are at risk of contracting the infection. In Brazil, the Metropolitan Region of Recife, located in the State of Pernambuco, has endemic areas of lymphatic filariasis, which constitutes a major challenge to public health [2].
In Brazil, chemical insecticides play an important role in the control of insect vectors [3]. However, the extended use of organochlorines, organophosphates, carbamates, and pyrethroids without any resistance management strategy has led to the emergence of several insecticide resistant populations. Therefore, there has been a resurgence of mosquito-borne diseases [4]. Chemicals derived from plants have been shown to function as general toxicants, growth and reproductive inhibitors, oviposition deterrents, fumigants for adult insects, and repellents. They can therefore provide an alternative for chemical repellent formulations used in mosquito control. [5, 6, 7].
Siparuna guianensis Aubl. (Family: Siparunaceae), also known as capitiú and negramina, is present in almost all of Brazil, but with greater frequency in Northern Brazil [8]. Chemical and ethnobiological studies on S. guianensis are limited, with only a few reports of traditional medicine in Central and South America, including the use of juice from leaves against fevers, and as a postpartum antibiotic [9]. In Brazil, S. guianensis is used in folk medicine and the leaves and flowers are considered carminative, aromatic, anti-dyspeptic, diuretic, and have stimulant and febrifuge properties [10]. However, no insecticidal activity has been reported for S. guianensis plants. Some plants (e.g., Melia azedarach L., Sida acuta Burm. f., Hyptis suaveolens (L.) Poit., Acorus calamus L., Feronia limonia L., Andrographis paniculata Burm.f.) have a natural toxicity against insect pests [11, 12, 13, 14, 15, 16]. In addition, essential oils from Eucalyptus saligna Sm. [17], Pinus caribaea Mor., Pinus tropicalis Mor. [18], and Chenopodium ambrosioides L. [19] have been shown to be toxic to mosquitoes. However, there are no thorough investigations into the insecticidal effects of the essential oils and their constituents from the leaf, stem, and fruit of S. guianensis. A recent study has shown that S. guianensis essential oil is also toxic to Rhipicephalus microplus also known as cattle-tick, an important tick that parasitize a variety of livestock species [20].
In this paper, we analyzed the chemical constituents of essential oils from the leaf, stem, and fruit of S. guianensis and tested their effects against A. aegypti and C. quinquefasciatus. We investigated the larvicidal, ovicidal, adulticidal, oviposition-deterring, and repellent effects of S. guianensis essential oil against A. aegypti and C. quinquefasciatus in vivo and its toxicity against C6/36 cells (Aedes albopictus Skuse).
Materials and Methods
Plant material and hydrodistillation
Siparuna guianensis was collected in Gurupi (11°43'45" latitude S, 49°04'07" longitude W) and Formoso do Araguaia (11°47'48" latitude S, 49°31'44" longitude W), Tocantins state, Brazil (The collections were approved by Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq, nº 010580/2013–1). Taxonomic identification was confirmed by experts at the herbarium of the Federal University of Tocantins (Campus Porto Nacional), where samples were deposited with reference number 10.496. Essential oils of S. guianensis were extracted from the leaves, stem, and fruit by hydrodistillation in a Clevenger apparatus, as described by [21]. The present study did not involve endangered or protected species.
Cells and insects
C6/36 Aedes albopictus cells were grown in Leibovitz L-15 medium (Gibco) supplemented with 5% fetal bovine serum (FBS) in standard adherent cell culture plates (Techno Plastic Products, Trasadingen, Switzerland), at 28°C. The colonies of A. aegypti and C. quinquefasciatus were originally established from insects collected from the field in the state of Tocantins, Brazil, (11°40'55.7" latitude S, 49°04'3.9" longitude W), where no insecticides have been used for the control of mosquitoes. Insects were maintained in the laboratory for at least five generations before being used in bioassays (The collections were approved by Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq, nº 010580/2013–1).. All bioassays were conducted at 26 ± 1°C, 60.0 ± 5% RH, and a 12-h light and 12-h dark photoperiod. Fish food pellets (larvae), 10% honey solution (male adult) and rabbit blood (female adult) were used as food sources.
C6/36 cell viability
In order to analyze the effect of essential oils of S. guianensis on the viability of mosquito cells in culture C6/36 cells from A. albopictus were incubated with serial dilutions of the essential oils of S. guianensis and cell viability was determined by the trypan blue exclusion method in the Countess Automated Cell Counter by Invitrogen (Carlsbad, CA, USA), using the manufacturer specified protocol. Adherent cells were plated at concentrations of 2 × 105 cells/mL and incubated for 2 h at 28°C. The medium supernatant was replaced with new medium containing different concentrations of essential oil (0.86 to 0.086 µg/mL) mixed with dimethyl sulfoxide (DMSO). A negative control medium containing only DMSO was also prepared. The cells were then incubated at 28°C for 24 h and each treatment was performed in triplicate. After incubation, the cells were aspirated and an aliquot was mixed with equal amounts of trypan blue stain (0.4%). Cell viability was determined by the Countess Cell Counter using the following parameters: minimum size 7 µm, maximum size 30 µm, and circularity 80%. Statistical analysis to determine LC50 concentration was done with GraphPad Prism v6.04 (San Diego, CA, USA). Light microscopy evaluation (Axiovert 100; Zeiss, Oberkochen, Germany) was also carried out to observe the cytopathic effects induced by the essential oil.
Chemical composition analysis of essential oils
The GC analysis of S. guianensis essential oils was performed using a Chemito 8510 GC (Chemito Technologies Pvt. Ltd, Mumbai, India) instrument equipped with a data processor. A BP-5 wide-bore capillary column (30 m × 0.53 mm i.d., 1.0-mm film thickness) was used for separation of the sample components (sample size 0.03 mL, measured using a Hamilton GC syringe with a 1.0-mL cap.). Hydrogen was used as the carrier gas at a flow rate of 5 mL/min and 20 psi inlet pressure. The GC column oven temperature went from 70°C to 210°C at a rate of 2.5°C/min, with a final hold time of 5 min. Both injector and detector (FID) temperatures were maintained at 230°C. GC-MS analysis was carried out on a Trace DSQ MS (Thermo Electron Corporation, Waltham, MA, USA), using a BP-5 capillary column (30 m × 0.25 mm × 0.25 μm), with helium as the carrier gas at a flow rate of 1 mL/min; split ratio 1:20. The column temperature went from 65°C to 210°C (10-min hold) at 3°C/min. Mass spectra were recorded in the range of 40–650 amu, operating at 70 eV, and the ion source temperature was maintained at 200°C. The constituents of the oil were identified using standard reference compounds and by matching the mass spectra fragmentation pattern with NIST Mass Spectra Library stored in the GC-MS database.
Ovicidal test analysis
Fifty A. aegypti eggs were exposed (for 24h) to three concentrations (0.5, 1.0, and 2.0 µg/mL in 500 µL of DMSO and 29.5 mL of distilled water) of S. guianensis essential oils, that were chosen from serial dilutions that had minimum and maximum effects on egg hatching. Each test was replicated ten times. Distilled water mixed with 500 µL DMSO served as the control. In the case of C. quinquefasciatus, one egg raft was used for each replication. The percentage of egg viability was calculated by the following formula: %V = (T—I)/T × 100, where %V is the percentage egg viability, T is the number of viable eggs in control treatment without application of essential oil, and I is the number of viable eggs after treatment with essential oil.
Mosquito ovipositor-deterrence test analysis
The effect of S. guianensis essential oils on egg-laying by A. aegypti and C. quinquefasciatus was carried out according to the methodology previously described [12]. Twenty-five gravid females fed on rabbit blood (1–7 d old) and fifty males were simultaneously exposed, in an entomological cage (35 cm width x 23 cm depth x 47 cm length), to ovitraps containing different extract concentrations or controls and incubated at 28 ± 2°C. The cages contained six replicates in plastic cups (100 mL) containing 0.5, 1.0, 1.5, and 2.0 µg of essential oil in 500 μL of DMSO and a control with tap water and 500 µL of DMSO. The test was repeated four times. The numbers of eggs and egg rafts were counted on the seventh day after treatment. The percentage of oviposition inhibition was calculated as previously described [22].
Larvicidal and pupicidal activity assay
Initially, we tested the toxicity of the essential oils obtained from the stem, fruit, and leaf of S. guianensis against the fourth instar larvae of A. aegypti and C. quinquefasciatus, according to the methodology previously described [23]. Because the highest yield and toxicity was obtained from the leaves, we chose S. guianensis leaf essential oil for further assays. Toxicity tests for essential oil were conducted against first, second, third, and fourth instar larvae and pupae. Twenty-five insects were placed in 100-mL cups with 29.5 mL of degassed distilled water, and 500 µL of DMSO solution containing the essential oil of S. guianensis. For first and second larvae, we used 0.10–0.95 µg/mL of essential oil and 0.50–2.50 µg/mL for the third and fourth instar larvae and pupae. Each cup was left at room temperature. The control was treated as above but without essential oil. Each treatment was done with three replicates. Toxicity was reported as LC50 and LC95, representing the concentrations in µg/mL that killed 50 and 95% of mosquitoes.
Adult toxicity assay
The essential oil of S. guianensis was dissolved in acetone to reach the desired concentrations (0.20–0.80 µg/mL). An aliquot (0.50 mL) of the test solution was dispensed in glass bottles of 50 mL, with an internal area of 60 cm2. The solvent was allowed to evaporate at room temperature and the essential oil of S. guianensis stayed in the inner wall of the glass bottle [11]. Fifty female A. aegypti and C. quinquefasciatus adults were then introduced into the glass bottle and insect mortality (LC50 and LC95) was recorded 1 h after the treatment. The experiment was repeated three times.
Time response assay
For each developmental phase of A. aegypti and C. quinquefasciatus, insect mortality was initially estimated by performing concentration response curves. Mortality was recorded after 0.5–55 min of exposure to essential oil of S. guianensis and the response time (LT) activity was reported as LT50 and LT95, in min.
Repellent activity assay
The repellent activity and RD50 of the essential oil of S. guianensis was performed according to the methodology described by [24, 25]. One hundred blood-starved A. aegypti and C. quinquefasciatus female insects, 4–5 days old, were kept in a net cage (24 × 24 × 24 cm3). The arms of the person conducting the test were cleaned with ethanol. After air-drying, 25 cm2 of the dorsal side of the skin on each arm was exposed, and the remaining area was covered by rubber gloves. S. guianensis essential oil was dissolved in ethanol, while ethanol alone served as a negative control and DEET (14.55%), present in a commercial product, as a positive control. The essential oil of S. guianensis at a concentration of 0.025–0.550 µg/cm2 was applied to one arm and the control and treated arms were introduced simultaneously into the cage. The numbers of bites were counted over 10 min every 120 min, from 4 p.m. to 6 p.m. The experiment was repeated five times. The percentage of protection was calculated by using the following formula, as previously described [26]:
Where P (%) is the percentage of protection, Nc is the number of bites received by the control arm, and Nt is the number of bites received by the treated arm.
Statistical analysis
LC and LT were determined by probit analysis [27]. Mortality data were corrected using the Abbott’s formula [28]. Significant differences between values were determined by using analysis of variance followed by Tukey’s test (P<0.01 and P<0.05). Statistical analysis was performed using SISVAR 4.6 [29], and graphs were produced using SIGMA PLOT 11.0 (Systat Software, Inc. San Jose, USA).
Results and Discussion
Essential oils purified from the leaves, stem, and fruit of S. guianensis were analyzed by GC and GC-MS and their qualitative and quantitative compositions were determined (Table 1). The main components of oils from the leaves were β-myrcene (79.71%) and 2-undecanone (14.58%). From the stem, the major components were β-myrcene (26.91%), δ-elemene (20.92%), germacrene D (9.42%), α-limonene (7.91%), and bicyclo-germacrene (7.79%). The main components in the fruit were 2-tridecanone (38.75%), 2-undecanone (26.5%) and β-myrcene (16.42%).
The observed concentration of the main constituents in the essential oils of S. guianensis was different from that reported by [20, 30, 31], suggesting a considerable variability in the oil samples studied. Essential oils from S. guianensis samples collected in different Amazon regions and Minas Gerais Estate were shown to have variable amounts of major compounds [32, 20]. These changes in the composition of essential oils might arise from several environmental (climatic, seasonal, or geographic) and genetic differences, since the specimen analyzed was collected from a region of the Brazilian cerrado (a savannah-like area), which is a completely different biome when compared with the Amazon.
Our analysis identified mainly sesquiterpene hydrocarbons, oxygenated sesquiterpenes and monoterpene hydrocarbons in the essential oils of the fresh leaves, stems and fruits of S. guianensis.
Essential oils purified from S. guianensis dramatically influenced the viability of A. aegypti and C. quinquefasciatus eggs (Fig. 1). At a concentration of 0.5 µg/mL, less than 20% of egg hatching was observed for both insects (Fig. 1). For concentrations of 1.0 and 2.0 µg/mL, only 5% of egg hatching was observed for both insects (Fig. 1). Other plant essential oils and/or leaf extracts have also shown toxicity to mosquito eggs. Pinus caribaea and Pinus tropicalis essential oils and the leaf extract from Chenopodium ambrosioides have been shown to be toxic to to A. aegypti eggs [18] and C. quinquefasciatus egg rafts [19], respectively.
Each datum represents the mean of four replicates. Means followed by different letters are significantly different (P<0.01) by Tukey’s test.
We found that the essential oils also proved to be deterrents to oviposition in both species of mosquitoes. The amount of A. aegypti and C. quinquefasciatus eggs lain in ovitraps treated with different concentrations of essential oil of S. guianensis was lower than that in controls (Fig. 2A and 2B). Oviposition-inhibiting effects against A. aegypti and C. quinquefasciatus have also been observed for other plant essential oils [11, 12, 33].
Control: Number of laid eggs ovitraps treated with water + DMSO. Different letters between columns indicate significant differences. Means followed by different letters are significantly different (P<0.01). Analysis of variance followed by Tukey’s test.
Stem, leaf, and fruit essential oils of S. guianensis showed high toxicity against the fourth instar A. aegypti and C. quinquefasciatus larvae (Table 2). The LC50 values obtained for stem, leaf, and fruit essential oils were 1.76, 0.98, and 2.46 µg/mL against A. aegypti and 1.36, 0.89, and 2.45 µg/mL against C. quinquefasciatus, respectively. The leaf essential oil showed the lowest LC50 towards fourth instar larvae of A. aegypti and C. quinquefasciatus, so we chose this extract to perform the toxicity tests for all insect stages (egg, larval, pupal, and adult).
Toxicity of the S. guianensis essential oil against the fourth instar A. aegypti and C. quinquefasciatus larvae were higher than those from essential oils derived from the fruit of Juniperus macropoda and seeds of Pimpinella anisum [11]. The LD95 values of both species were 171.3 and 115.7 µg/mL against A. aegypti and 204.8 and 149.7 µg/mL against C. quinquefasciatus, respectively. Leaf and stem essential oils of Chloroxylon swietenia against A. aegypti [34] showed LD50 values of 16.5 and 20.4 µg/mL, respectively. The larvicidal effect of S. guianensis essential oil was also superior to that found by [23], who used essential oils of Eucalyptus camaldulensis and Eucalyptus urophylla against A. aegypti and A. albopictus. Their LC50 values were 95.5 and 31.0 µg/mL against A. aegypti and 295.8 and 55.3 µg/mL against A. albopictus for E. camaldulensis and E. urophylla, respectively.
The LC50 and LC95 values of S. guianensis essential oil differ depending on the stage of development of mosquitoes (Table 3). We found that the LC50 of S. guianensis essential oil against the pupae of A. aegypti (LC50: 1.69 µg/mL) and C quinquefasciatus (LC50: 1.31 µg/mL) was higher than that of all larval stages (Table 3). The essential oil was more toxic to younger larvae of A. aegypti and C. quinquefasciatus. The LC50 values for the first instar A. aegypti and C. quinquefasciatus larvae were 0.21 µg/mL and 0.31 µg/mL, respectively (Table 3). This is higher than the LC95 value of a synthetic insecticide (temephos) (0.072 mg/L-1) against C. quinquefasciatus third instar larvae [3]. A previous study showed that temephos has an LC50 value of 0.0023 µg/mL towards third instar susceptible A. aegypti larvae, which is lower than the LC50 of the S. guianensis essential oil (0.75 µg/mL) [35]. Nevertheless, isolation and identification of larvicidal components in S. guianensis essential oil could lead to the development of alternative compounds to replace synthetic insecticide [36]. Besides insects, S. guianensis essential oils were recently shown to be toxic to the tick R. Microplus [20].
The essential oil of S. guianensis LT50 and LT95 were obtained using the LC95 concentrations determined for each stage of A. aegypti and C. quinquefasciatus (Table 4). The time required to achieve 95% mortality (LT95) was 40.97 min for the pupae of C. quinquefasciatus and 45.79 min for pupae of A. aegypti (Table 4). However, for the larval and adult stages, it took less than 33 min to kill 95% of both stages. These results could be associated with the amount of essential oil that can penetrate the pupae when compared with the larval and adult stages or to differences in metabolic detoxification [36].
Besides its toxicity, the essential oil of S. guianensis shows promise as a repellent (Table 5). The doses to repel 50% of the adult mosquito population (RD50) were 0.438 and 0.662 µg/cm2 for A. aegypti and C. quinquefasciatus, respectively. Repellent values of 0.450 to 0.550 µg/cm2 obtained 100% repellency for A. aegypti and C. quinquefasciatus for up to 120 min (Table 6). From these results, the essential oil of S. guianensis has the potential to be an excellent candidate for a mosquito-repellent formulation. Furthermore, the repellent activity obtained by the essential oil concentration above 0.450 µg/cm2 was higher than that of a commercial product usually sold in Brazil as an insect repellent (with N,N-diethyl-m-toluamide [DEET] 14.55% as the active ingredient), which was used as a positive control, and had a maximum protection of 30 min (Table 6).
The compounds 2-undecanone and 2-tridecanone, which are major components of the essential oil of S. guianensis, have been shown to be involved in the resistance of tomatoes to herbivorous pests [37] and in repellence to blood-feeding insects [38]. In this context, repellent properties of the essential oil of S. guianensis can be associated with oxygenated compounds, as shown in Table 1.
We also analyzed the in vitro toxicity of essential oil of S. guianensis on C6/36 cells derived from A. albopictus. As observed by light microscopy (Fig. 3), the cytopathic effect of the essential oil of S. guianensis can be associated with the loss of plasma membrane integrity, leading to cell death by necrosis in a dose-dependent manner. As observed in Fig. 3, a concentration 0.86 µg/mL S. guianensis essential oil lowered cell viability of the C6/36 cells to less than 20%, compared with 90% in the control (S1 Fig.). The LC50 determined to be 0.15 µg/mL.
B, C, and D: C6/36 cells treated with essential oil of S. guianensis at concentrations 0.2, 0.4, and 0.8 µg/mL, respectively.
This study showed that S. guianensis essential oil has significant ovicidal, larvicidal, pupicidal, and adulticidal effects as well as repellent activity against A. aegypti and C. quinquefasciatus. Furthermore, we showed a high cytotoxic effect on A. albopictus C6/36 cells. Considering the wide distribution of S. guianensis throughout neotropical areas of the world and the growing resistance of A. aegypti and C. quinquefasciatus populations to synthetic organic pesticides [3, 36], S. guianensis could be further studied as a source of potential alternative strategies for controlling and repelling insect vectors.
Conclusion
The essential oil of S. guianensis is highly toxic to all developmental stages of A. aegypti and C. quinquefasciatus. Furthermore, it has a high mosquito repellent activity. These properties of the S. guianensis essential oil demonstrate its potential for use as a natural insecticide against mosquitoes.
Supporting Information
S1 Fig. Viability of C6/36 cells from A. albopictus treated with different concentrations of essential oils of S. guianensis.
https://doi.org/10.1371/journal.pone.0116765.s001
(TIF)
Author Contributions
Conceived and designed the experiments: RWSA JD BMR. Performed the experiments: SFS FSM KFV. Analyzed the data: SDA JD RWSA FSM. Contributed reagents/materials/analysis tools: MML BMR RWSA SDA. Wrote the paper: BMR RWSA.
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