Insecticidal and Repellent Activity of Siparuna guianensis Aubl. (Negramina) against Aedes aegypti and Culex quinquefasciatus

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.


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 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 × 10 5 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 LC 50 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 LC 50 and LC 95 , 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 cm 2 . 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 (LC 50 and LC 95 ) 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 LT 50 and LT 95 , in min.

Repellent activity assay
The repellent activity and RD 50 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 cm 3 ). The arms of the person conducting the test were cleaned with ethanol. After air-drying, 25 cm 2 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/cm 2 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).
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.
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].
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 LC 50 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 LC 50 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 LD 95 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 LD 50 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 LC 50 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 LC 50 and LC 95 values of S. guianensis essential oil differ depending on the stage of development of mosquitoes (Table 3). We found that the LC 50 of S. guianensis essential oil against the pupae of A. aegypti (LC 50 : 1.69 µg/mL) and C quinquefasciatus (LC 50 : 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 LC 50 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 LC 95 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 LC 50 value of 0.0023 µg/mL towards third instar susceptible A. aegypti larvae, which is lower than the LC 50 of the S. guianensis essential oil (0.75 µg/mL) [35]. Nevertheless, isolation and  Entomocidal and Repellent Activities of S. guianensis Essential Oils 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 LT 50 and LT 95 were obtained using the LC 95 concentrations determined for each stage of A. aegypti and C. quinquefasciatus ( Table 4). The time required to achieve 95% mortality (LT 95 ) 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 (RD 50 ) were 0.438 and 0.662 µg/cm 2 for A. aegypti and C. quinquefasciatus, respectively. Repellent values of 0.450 to 0.550 µg/cm 2 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/cm 2 was higher than that of a commercial 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 LC 50 determined to be 0.15 µg/mL. 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