Introduction
Filariasis is a disease that is caused by nematodes. These nematodes are transmitted to humans via mosquitoes during a blood meal (CDC, Global Health, 2010). Mosquitoes are a serious threat to public health as they act as vectors that help in transmission of diseases that can be morbid and fatal (Becker et al., Reference Becker, Petriae, Zgomba, Boase, Dahl, Lane and Kaiser2003). Owing to lack of proper medication and vaccines for treating mosquito-borne diseases, an alternative and effective approach used to control the vector population at the larvicidal stage is necessary, because, during this stage the mosquitoes are immobile. This can be done by the application of insecticides to larval habitats (Tennyson et al., Reference Tennyson, Ravindran and Arivoli2012).
The resistance to synthetic pesticides and harmful effects of their accumulation in the environment has created the need for natural and non-persistent insecticides. The essential oils extracted from plants are suitable as they are economically reasonable and have high activity in certain cases (Silva et al., Reference Silva, Doria, Maia, Nunes, Carvalho, Blank, Alves, Marcal and Cavalcanti2008) and are also bio-degradable (Sukumar et al., Reference Sukumar, Perich and Boobar1991).
Eucalyptus is a diverse genus of flowering trees and shrubs in the Myrtle family, Myrtaceae. The oil extracted from eucalyptus leaves possesses allelopathic property and prevents insects from attacking it, thereby, acting as a natural pesticide (Brooker & Kleinig, Reference Brooker and Kleinig1990; Batish et al., Reference Batish, Singh, Kohli and Kaur2008). In assays, such as, fumigation activity (Papachristos & Stamopoulos, Reference Papachristos and Stamopoulos2004) and repellency (Trigg, Reference Trigg1996), the efficiency of eucalyptus oil has been demonstrated (Kumar et al., Reference Kumar, Mishra, Malik and Satya2012).
Downsizing of natural oils to form nanoemulsions could be effective as larvicidal agents (Anjali et al., Reference Anjali, Sharma, Mukherjee and Chandrasekaran2012). Nanoemulsions are emulsions whose droplet size is uniform and extremely small with the size ranging from 20 to 200 nm. Nanoemulsions are metastable and their stability is determined by the method of preparation (Wang et al., Reference Wang, Li, Zhang, Dong and Eastoe2007). Nanoemulsions can be formulated by two kinds of methods such as high-energy and low-energy emulsification methods. High-energy emulsification method comprises high-pressure homogenization and ultrasonication (Yilmaz & Borchert, Reference Yilmaz and Borchert2005; Takegami et al., Reference Takegami, Kitamura, Kawada, Matsumoto, Kitade, Ishida and Nagata2008; Klang et al., Reference Klang, Nadejda, Valenta and Hofer2012). Ultrasonication is most widely used method owing to its ease of use and it is an economical method. The low-energy emulsification technique comprises methods that exploit the chemical properties of a system to convert a microemulsion into a nanoemulsion (Bouchemal et al., Reference Bouchemal, Briancon, Perrier and Fessi2004; Tadros et al., Reference Tadros, Izquierdo, Esquena and Solans2004; Anton et al., Reference Anton, Gayet, Benoit and Saulnier2007; Sole et al., Reference Sole, Pey, Maestro, Gonzalez, Porras, Solans and Gutierrez2010). The use of nanopesticides would be a contemporary measure for the control of pests and reducing the toxic effect of synthetic bulk pesticides on the environment (Suresh Kumar et al., Reference Suresh Kumar, Shiny, Anjali, Jerobin, Goshen, Magdassi, Mukherjee and Chandrasekaran2013). Recently, controlling for vector-borne diseases using neem oil nanoemulsion will be of good alternative against Culex quinquefasciatus compared to the synthetic pesticides (Anjali et al., Reference Anjali, Sharma, Mukherjee and Chandrasekaran2012). The present study was carried out to evaluate the larvicidal activity of eucalyptus oil nanoemulsion against the mosquito larvae. The larvicidal studies were also carried out against Cx. quinquefasciatus mosquito. Early third instars larvae of Cx. quinquefasciatus mosquitoes were exposed for up to 24 h at various concentrations. Target the specific nature of nanoemulsion makes it an ideal larvicide against Cx. quinquefasciatus. Moreover, it has proved to be ineffective against selected test microorganism, Rhizobium leguminosorum.
Materials and methods
Materials
Eucalyptus oil (Eucalyptus globulus) and nutrient agar was purchased from Himedia, India and stored at room temperature under laboratory conditions; Tween 80 (Polyoxyethylene 20 monooleate) was supplied from Sigma, India. Ultrapure water was obtained from Cascada™ Biowater System, Pall Corporation, USA, with a resistivity not less than, 18.2 MΩ cm, was used for the preparation of all solutions. All other chemicals used were of analytical reagent grade.
Microorganism
R. leguminosorum (MTCC 099) was obtained from National Chemical Laboratory (Pune, India). The strain was cultured in a 50 ml nutrient broth at 37 °C for 24 h. Roughly 1 ml of this grown culture was reinoculated again into a 50 ml nutrient broth, and the growth was adjusted to 1×108 colony-forming units (CFU) ml−1 at 600 nm using phosphate-buffered saline (PBS).
Methods
GC–MS analysis
Eucalyptus oil composition was analyzed by GC–MS (GC model: Hp5890, series 2, MS model: 5971 A). Carrier gas used was helium gas at a flow rate of 3.0 ml min−1. The samples were injected with a split ratio of 1:100. Injector temperature was 80 °C and detector temperature was 260 °C. Mass spectra was recorded over 60–260 amu range with 70 eV of ionization energy. The percentage composition was calculated using the peak normalization method assuming equal detector response. Identification of components of oil was done by matching the obtained mass spectra data with NIST library search.
Preparation of nanoemulsion
The oil-in-water nanoemulsion was formulated using eucalyptus oil, non-ionic surfactant (tween 80) and water. The concentration of eucalyptus oil (6%, v/v) was fixed for all the formulations. Initially, coarse emulsion was prepared by adding water to organic phase containing oil and surfactant in different ratios 1:1, 1:2, and 1:3 (v/v) using a magnetic stirrer, which was then subjected to ultrasonic emulsification using a 20 kHz Sonicator (Ultrasonics, USA) with a power output of 750 W. Energy input was given through sonotrode containing a piezoelectric crystal with a probe diameter of 13 mm. Sonicator probe generates disruptive forces that reduce the droplet diameter converting coarse emulsion to nanoemulsion. The characterization of nanoemulsions was carried out and the emulsion stability was investigated.
Characterization of nanoemulsion
Thermodynamic stability study
To prove the stability, formulated emulsion was subjected to different stress tests (Shafiq et al., Reference Shafiq, Shakeel, Talegoankar, Ahmad, Khar and Ali2007).
Centrifugation: The formulated nanoemulsions were centrifuged at 3000 rpm for 30 min and observed for phase separation if any. Stable samples were further analyzed for heating, cooling, and freeze–thaw cycle.
Heating–cooling cycle: This was performed by keeping formulated nanoemulsion at 40 and 4 °C, alternating each temperature for 48 h. The cycle was repeated thrice. This was done to check the stability of nanoemulsion at varying temperature.
Freeze–thaw stress: This was carried out by keeping the nanoemulsion alternatively at −21 and 25 °C for 48 h at each temperature. The cycle was repeated twice. The experiment was performed in triplicates. The formulations that passed the thermodynamic stress tests were taken for further characterization studies.
Droplet size distribution and polydispersity index
The droplet size distribution (analysis by volume) and polydispersity index (PDI) of eucalyptus oil nanoemulsion formulation (1:2) was determined using a 90 plus particle size analyzer (Brookhaven Instruments Corporation, USA). The PDI is a measure of the homogeneity and stability of the droplet size in the nanoemulsion system. PDI values below 0.2 indicate a narrow size distribution and thus provide long-term stability to the formulated nanoemulsion. Prior to experiment, formulated emulsion was diluted with milli-Q (Millipore corporation) double-distilled water to trim down multiple scattering effects.
Morphology of emulsion droplets
To visualize the shape and morphology of the formulated nanoemulsions, transmission electron microscopy (TEM) was carried out. One drop of emulsion was negatively stained with phosphotungstic acid and was positioned on a copper grid. The TEM micrographs were acquired using a transmission electron microscope (Tecnai-10, Phillips) with a tungsten source and operating at 80 kV.
Larvicidal bioassay
Larvae
Mosquito larvae were collected from a water-stagnated area. Species identification was carried out in Zonal Entomological Research Laboratory, Vellore, Tamil Nadu, India. The mosquito larvae were identified as Cx. quinquefasciatus. The collected larvae were acclimatized under proper temperature and humidity (Anjali et al., Reference Anjali, Sudheer Khan, Goshen, Magdassi, Mukherjee and Chandrasekaran2011).
Larvicidal activity
The larvicidal activity of eucalyptus oil nanoemulsion against Culex sp. third instar larvae of Cx. quinquefasciatus mosquitoes were treated with different percent concentrations of nano and bulk eucalyptus oil emulsions, following the standard WHO larval susceptibility test method (WHO, 2005). Twenty larvae of Culex sp. were placed in each of the five 250 ml sterile beaker containing 200 ml of water. After adding larvae to beakers containing 5, 50, 100, 150, and 250 ppm of nanoemulsion (1:2), the bulk emulsion (1:2) was also added in each of the beakers, separately. A control was kept without addition of sample. The larvicidal effect of sample was monitored by recording mortality after 2, 4, 6, 12, and 24 h of the exposure period. The dead larvae were identified when they failed to move after probing with a needle in the siphon or cervical region. Each test was performed in six replicates. The percentages of larval mortality and standard deviation were calculated for each concentration of nanoemulsion and bulk emulsion.
Histological studies
For histological purposes, the treated and untreated third instar larvae of Cx. quinquefasciatus were stored in 10% formalin solution. The material was then embedded and cut with glass knives in a rotary microtome. The sections were then stained with hematoxylin–eosin blue, which is used to study the morphological changes using phase contrast microscope (Carl Zeiss, USA) fitted with a digital camera and compared with the control sample (Al-Mehmadi & Al-Khalaf, Reference Al-Mehmadi and Al-Khalaf2010).
Antibacterial susceptibility: well diffusion method
The susceptibility of bacteria to eucalyptus oil and nanoemulsion was determined by an agar well diffusion method (Lopez et al., Reference Lopez, Sanchez, Batlle and Nerin2005). The bacterial suspension (100 μl of 107–108 CFU ml−1) was applied uniformly on the surface of a nutrient agar plate before making the wells on the plate. The plates were incubated at 37 °C for 24 h, after which, the average diameter of inhibition zone surrounding the well was measured with a ruler with up to 1 mm resolution. The experiments were performed in duplicates.
Preparation of whole body homogenates
The treated and control larvae were taken separately and washed with double-distilled water and then dabbed with a tissue paper to remove the moisture until it was completely dry. About 20 larvae were homogenized along with 2 ml of PBS in an Eppendorf tube. This homogenate was then subjected to centrifugation at 10,000 g for 20 min, after which, the clear supernatant was retrieved and the pellet was discarded. The supernatant was placed on ice until further use for biochemical assays.
Quantitative analysis of biochemical constituents
The protein concentration of the control and treated larval homogenates was analyzed by Bradford method (Reference Bradford1976).
The activity of acetylcholinesterase in the larval homogenate was measured according to a method by Ikezawa & Taguchi, (Reference Ikezawa and Taguchi1981) with slight modification. 50 μl of homogenate was successively mixed with 450 μl of PBS (100 mM, pH 7.4), 50 μl of 10 mM 5-5-dithiobis 2-nitro benzoic acid and 50 μl of 12.5 mM acetylcholine iodide which acts as the substrate. The mixture was then incubated at room temperature for 5 min and at 400 nm the optical density was measured.
The acid and alkaline phosphatase activity of the larval homogenates was measured by a slightly modified version of the method by Asakura (Reference Asakura1978). For the acid phosphatase assay, 100 μl of the larval homogenate was taken with 400 μl of 50 mM sodium acetate buffer (pH 4.0) and 500 μl of 15 mM of p-nitrophenyl phosphate. For the alkaline phosphatise assay, 100 μl of larval homogenate was taken along with 400 μl of 50 mM Tris–HCl buffer (pH 8.0) and 500 μl of 15 mM p-nitrophenyl phosphate. The individual mixtures were incubated for 5 min at 37 °C in water bath. They were then treated with 100 μl of 0.5 N NaOH in order to stop the enzymatic reaction, the mixtures were then centrifuged at 4000 g for 5 min. The optical density of the respective clear supernatants was measured at 440 nm.
Results
GC–MS analysis
The chemical compound present in eucalyptus oil was identified by GC–MS analysis. Eucalyptol was found to be the major component of the oil with 17.73% of total peak area.
Nanoemulsion formulation
A volume of eucalyptus oil (6%), Tween 80 and water was prepared using magnetic stirrer at 250 rpm for 30 min. The average diameter of 1:2 ratio of bulk emulsion was found to be 7224 nm and PDI is 0.890. Also found to be unstable (phase separated) during thermodynamic stability study. This bulk emulsion was further subjected to ultrasonication method and it has shown in fig. 1. It represents the visual appearance of nanoemulsion prepared by sonication with respect to three different ratios (1:3; 1:2; 1:1) as fig. 1a–c. The nanoemulsion of 1:2 ratios was found stable after thermodynamic stabilization cycle. The emulsion showed no phase separation. Even though, the nanoemulsion of the ratio 1:2 is stable; it is preferable to use fresh aliquots of the nanoemulsions.
Fig. 1. Effect of sonication time on the visual appearance of eucalyptus oil nanoemulsions (a) 1:3; (b) 1:2; (c) 1:1.
Characterization of the selected nanoemulsion
Based on the thermodynamic stability study, 1:2 ratio nanoemulsions was selected and average diameter is 9.4 nm and their size distribution is shown in fig. 2. The polydispersity index (PI) of the nanoemulsions is 0.060 which shows that it is uniform. The particles are presented in TEM image. According to TEM evaluation, the particle size ranges from 20 to 40 nm and the droplets are spherical in nature (fig. 3). The nanoemulsion of the ratios 1:1 and 1:3 showed phase separation, thereby, rendering it for particle size analysis.
Fig. 2. A typical particle size distribution measurement by dynamic light scattering.
Fig. 3. TEM image of eucalyptus oil nanoemulsion.
Larvicidal activity of eucalyptus oil nanoemulsion against Cx. quinquefasciatus
The larvicidal activity of eucalyptus oil nanoemulsion (1:2) and bulk emulsion (1:2) was recorded against Cx. quinquefasciatus. The larvicidal activity of the nanoemulsion was compared with the bulk emulsion of the same. It was observed that at a concentration of 250 ppm nanoemulsion, 98% mortality within 4 h of treatment was observed as shown in fig. 4a, whereas, for the bulk emulsion, 100% mortality was observed at 24 h and is shown in fig. 4b. Thus, proves that the nanoemulsion formulation of eucalyptus oil is far more effective than bulk emulsion.
Fig. 4. (a) Mortality rate of eucalyptus oil nanoemulsions against Cx. quinquefasciatus and (b) Mortality rate of eucalyptus oil bulk emulsion against Cx. quinquefasciatus.
Histopathology of larvae of Cx. quinquefasciatus
The histological images (fig. 5) show that the midgut region of the Cx. quinquefasciatus larvae with and without treatment. Fig. 5a showed the control larvae in which the midgut region is intact, whereas, in fig. 5b, the image of the larvae after treatment shows that the peritrophic membrane (PM) and the epithelial cells (EC) of the midgut have been damaged and there is also a leakage of the midgut contents.
Fig. 5. (a) Midgut of Cx. quinquefasciatus larvae; EC – epithelial cells, PM – peritrophic membrane, MC – midgut contents and (b) Midgut of the larvae that have been treated with eucalyptus oil nanoemulsions. The image shows damage to EC and PM and leakage of MC.
Antibacterial activity of nanoemulsion
In order to evaluate the environmental impact of the nanoemulsion, its antimicrobial activity was evaluated for soil bacteria, R. leguminosorum. The tested strain exhibited no zone of inhibition (data not shown). It was observed that the bacterial strain was susceptible to eucalyptus oil and not for nanoemulsion formulations (1:3, 1:2, and 1:1) since; there was no formation of zones in the samples. Therefore, its nano form can be used as an environment friendly larvicidal agent.
Quantification of biochemical constituents
Determination of protein concentration
On exposure of the larvae to eucalyptus oil nanoemulsions, the total protein content has reduced from 0.1223 mg protein ml−1 of homogenate of the controlled larvae to 0.1214 mg protein ml−1 of the treated larval homogenate (fig. 6a). This result demonstrates that there is no significant level found in both control and treated larval homogenate of the total protein content.
Fig. 6. Biochemical assays of control and treated larval homogenates. (a) Protein assay. (b) Acetylcholinesterase assay. (c) Acid assay. (d) Alkaline phosphatase assay.
Acetylcholinesterase assay
As depicted in fig. 6b, eucalyptus oil nanoemulsion affected esterase activities in the larvae of Cx. quinquefasciatus. The level of acetylcholinesterase activity in the control larvae was 0.231 lM acetylcholinesterase released min−1 mg−1 protein, and it gradually decreased to 0.05 lM acetylcholinesterase released min−1 mg−1 protein in the larvae exposed to nanoemulsion for 24 h. The optical density of the larval homogenate of the larvae-treated nanoemulsion showed a reduction of 0.184 when compared with control larvae.
Acid and alkaline phosphatase assay
Exposure to eucalyptus oil nanoemulsion, there was a decrease of 0.13 in the absorbance of the larval homogenate when compared to homogenate of the control larvae, thus, showing a reduction in the acid phosphatase enzyme. In fig. 6c, d, there was a reduction in the levels of alkaline phosphatase enzyme. Also, the difference in absorbance between the control and treated larvae was found to be 0.11. The level of acid phosphatase activity significantly decreased from the control value of 0.45 to 0.322 mM p-nitrophenol released min−1 mg−1 protein upon exposure of the larvae to nanoemulsion for 24 h. Also, the level of alkaline phosphatase decreased from 0.09 to 0.05 mM p-nitrophenol released min−1 mg−1 protein in the third instar larvae of Cx. quinquefasciatus exposed to nanoemulsion.
Discussion
Cx. quinquefasciatus serves as the vector for the nematode Wuchereria bancrofti that causes filariasis. The ideal method of controlling mosquitoes is by targeting them at the stage at which they are vulnerable i.e., the larval stage. Therefore, mosquitoes can be controlled by application of insecticides to their habitat, where, the mobility of the larvae is low (Wiseman & Chapagain, Reference Wiseman and Chapagain2005).
The use of synthetic-based pesticides over the past few years has increased the resistance toward the vectors (Wirth & Georghiou, Reference Wirth and Georghiou1999; Rodriguez et al., Reference Rodriguez, Bisset, Ruiz and Soca2002; Macoris et al., Reference Macoris, Andrighetti, Takaku, Glasser, Garbeloto and Bracco2003), thus, instilling the need for finding alternative ways to control mosquitoes. The plant-based pesticides are in high demand, because, they are sustainable in the environment; they are safe and are also pleasant to use (Maia & Moore, Reference Maia and Moore2011). The plant-based compounds have natural pesticide activity, because, they contain certain bioactive compounds that prevent them from being eaten by herbivores. Although, the main function of these compounds is defense against plant-eating insects, the volatile compounds released as a result of herbivores have mosquito repellent properties (Pichersky & Gershenzon, Reference Pichersky and Gershenzon2002).
Eucalyptus oil is the oil distilled from the leaves of eucalyptus, a genus of the plant family Myrtaceae. In the study carried out by Mandal (Reference Mandal2011), the repellent activity of eucalyptus oil was demonstrated against Cx. quinquefasciatus. Eucalyptus oil is among the essential oils that have been registered as an insect repellent by US Environmental Protection Agency (US EPA) (Katz et al., Reference Katz, Miller and Hebert2008). The seed and leaf extract of eucalyptus oil contain compounds that are toxic to mosquito larvae (Elbanna, Reference Elbanna2006) that are in agreement with the study carried out by Brooker & Kleinig (Reference Brooker and Kleinig1990).
The steam distillation of aromatic plants and their derivatives yield essential oils that are rich in bioactive compounds, which forms non-toxic compounds when they undergo degradation. Since, they do not cause any adverse health effects; they are used as insect repellents and a safe and eco-friendly alternative to synthetic pesticides (Isman, Reference Isman2000). In the past, essential oils have been shown to have good activity against mosquitoes (Gillij et al., Reference Gillij, Gleiser and Zygadlo2008). It was observed that essential oils of many plants exhibited mosquito larvicidal activity (Phasomkusolsil & Soonwera, Reference Phasomkusolsil and Soonwera2010).
A problem that arises with the use of essential oils as pesticides is that their effect is short lasting (Barnard & Xue, Reference Barnard and Xue2004). To overcome this problem, essential oils are formulated into nanoemulsions. It also has technological limitations, such as hydrophobicity, reactivity, and volatility of the bioactive molecules constituting the essential oils (Huang et al., Reference Huang, Yu and Ru2010). This problem may be overcome by formulating Essential oils (EOs) into nanoemulsion which is transparent and can be used in food and beverage products, thereby, decreasing the amount of EOs required (Qian et al., Reference Qian, Decker, Xiao and McClements2012). The emulsions that have droplets which are very small with size ranging from 20 to 200 nm are called nanoemulsions. The method of preparation determines the stability of the formulated nanoemulsion.
The nanoemulsions are formed when the oil phase is dispersed in an aqueous phase and it is stabilized due to the presence of surfactant that forms a layer at the interface of the droplet, thus, separating the oil from aqueous phase owing to the stability of emulsion (Tadros et al., Reference Tadros, Izquierdo, Esquena and Solans2004; McClements et al., Reference McClements, Decker and Weiss2007; Acosta, Reference Acosta2009). The attractive forces between the droplets is proportional to the size of the droplets and so when the size of the droplets are in the nano range, the attractive forces between the droplets are weak, thereby, preventing particle aggregation and helps in making the nanoemulsions more stable (Tadros et al., Reference Tadros, Izquierdo, Esquena and Solans2004; McClements, Reference McClements2005). The stabilization of the nanoemulsions is also dependent on the steric effect of the non-ionic surfactant (Tadros et al., Reference Tadros, Izquierdo, Esquena and Solans2004).
The eucalyptus oil nanoemulsion was prepared in three ratios by varying the volumes of oil, surfactant, and water. The ratios were 1:1, 1:2, and 1:3, respectively. The uniformity and stability of the nanoemulsion can be determined by the PI. The PI is inversely proportional to the uniformity and stability of the nanoemulsion. The lower the PI, more stable the nanoemulsion (Shinoda & Saito, Reference Shinoda and Saito1969).
In this study, eucalyptus oil (6%, v/v) is mixed with a non-ionic surfactant, tween 80 along with water, which is used as an aqueous phase. This emulsion is then subjected to ultrasonication that breaks down the bulk emulsion into an emulsion comprising droplets having the size in nanometer range. This increases the surface area of the droplets, thereby, increasing the reactivity; thus, making nanoemulsions more effective than its bulk counterpart. The larvicidal activity of the nanoemulsion is expected due to the presence of eucalyptol, which is the major component of the eucalyptus oil by GC–MS analysis.
A number of compounds present in relatively high concentrations in the essential oil of E. globulus used in the present study are known to have larvicidal properties. In particular, it is worth to note 1,8-cineole (eucalyptol), which accounted for approximately 17.73% of the total essential oil of E. globulus. Several researchers have reported that the insecticidal and antimicrobial activity of an essential oil is linked to its chemical composition (Inouye et al., Reference Inouye, Yamaguchi and Takizawa2001; Cimanga et al., Reference Cimanga, Kambu, Tona, Apers, De Bruyne, Hermans, Totte, Pieters and Vlietinck2002; Kumar et al., Reference Kumar, Mishra, Malik and Satya2011).
This is the first time that a nanoemulsion formulation using eucalyptus oil with a small droplet size (Z-average diameter) of 9.3 nm has been reported against Cx. quinquefasciatus. The size-based effect was demonstrated in a study conducted by Anjali et al. (Reference Anjali, Sudheer Khan, Goshen, Magdassi, Mukherjee and Chandrasekaran2011), where, nanopermethrin was found to be more effective against larvae of Cx. quinquefasciatus when compared to bulk permethrin. From the same group Anjali et al. (Reference Anjali, Sharma, Mukherjee and Chandrasekaran2012), the neem oil nanoemulsion showed higher mortality rate against Cx. quinquefasciatus larvae which is based on droplet size distribution. Similarly nanoemulsion formulation with use of essential oils was studied for various applications include drug delivery, cosmetics, antimicrobials, and nutraceuticals (Tadros et al., Reference Tadros, Izquierdo, Esquena and Solans2004; Shafiq et al., Reference Shafiq, Shakeel, Talegoankar, Ahmad, Khar and Ali2007; Rao & McClements, Reference Rao and McClements2011; Ziani et al., Reference Ziani, Chang, McLandsborough and McClements2011).
The larvicidal activity of the eucalyptus oil nanoemulsion was compared with the bulk emulsion of the same. The nanoemulsion of the eucalyptus oil was found to be more effective than its bulk counterpart. The observed histopathological effects of the eucalyptus oil nanoemulsion on Cx. quinquefasciatus was in agreement with the results obtained by Suresh Kumar et al. (Reference Suresh Kumar, Shiny, Anjali, Jerobin, Goshen, Magdassi, Mukherjee and Chandrasekaran2013), where, it is stated that the midgut of the Aedes aegypti treated with nanopermethrin showed damage of the PM and EC and leakage of the midgut content.
According to earlier studies, it is shown that the protein concentration of larvae decreases on treatment with biocides that is in agreement with the results of our present study. The larvae and pupae of A. aegypti showed a decrease in the protein concentration levels on exposure to extracts of the kernel of soapnut, Sapindus emarginatus according to a study conducted by Koodalingam et al. (Reference Koodalingam, Mullainadhan and Arumugam2011). The nymphs of the coleopteran, Callosobruchus maculatus, showed a decrease in the levels of protein concentration on exposure to an insecticidal leaf lectin from Bauhinia monandra according to a study conducted by Macedo et al. (Reference Macedo, Freire, Silva and Coelho1997). According to a recent study conducted by Koodalingam et al. (Reference Koodalingam, Mullainadhan, Rajalakshmi, Deepalakshmi and Ammu2012), there was a noticeable decrease in protein concentration levels of the larvae of A. aegypti on exposure to a Bt-based product.
Esterases are the main enzymes that are responsible for resistance mechanism against chemical insecticides in mosquitoes (Brogdan & McAllister, Reference Brogdan and McAllister1998). There was a significant decrease in the levels of acetylcholinesterase in the larvae of A. aegypti that were treated with extract of the kernel of soapnut (Koodalingam et al., Reference Koodalingam, Mullainadhan and Arumugam2011). In a study conducted by Koodalingam et al. (Reference Koodalingam, Mullainadhan, Rajalakshmi, Deepalakshmi and Ammu2012), there was a significant decrease in the levels of acetylcholinesterase in A. aegypti larvae that were treated with the Bt-based product, Vectobar. These findings are in accordance with the results of the present study, where, there is a significant decrease in the levels of acetylcholinesterase activity in the larvae that are treated with the eucalyptus oil nanoemulsion as compared with the untreated larvae.
The hydrolytic cleavage of phosphoric acid esters are catalyzed by acid and alkaline phosphatase enzymes and they play a significant role in metabolism and cell signalling processes (Walter & Schutt, Reference Walter, Schutt and Bergmeyer1974; Torres & Forman, Reference Torres and Forman2006).
The larvae of Cnaphalocrocis medinalis when exposed to plant secondary metabolites showed a decrease in the levels of acid and alkaline phosphatase enzymes (Nathan et al., Reference Nathan, Choi, Paik and Seo2007). These findings are in accordance with the result of the present studies, in which, there is a decrease in the levels of acid and alkaline phosphatase enzymes in the larvae that were treated with eucalyptus oil nanoemulsions.
Conclusion
Eucalyptus oil being a plant-based component is eco-friendly and has natural pesticide activity. The nanoemulsion formulation of eucalyptus oil ensures higher efficacy as a larvicidal agent against Cx. quinquefasciatus when compared to its bulk counterpart. From this study, it can be concluded that eucalyptus oil nanoemulsion can be used as a safe and effective alternative in the control of vector-borne diseases caused by mosquito larvae.
Acknowledgements
The authors acknowledge VIT University for the research fund extended by them for the completion of this work. Authors also thank Mr Gopalrathinam, Senior Entomologist, Zonal Entomological Research Laboratory, Vellore, Tamil Nadu, for identifying Cx. quinquefasciatus mosquito larvae and Mr Suresh Kumar R.S. is greatly acknowledged for histopathology studies of Cx. quinquefasciatus larvae.
