Introduction
Mosquitoes are a significant cause of several vector-borne diseases occurring in India and worldwide, affecting both human and animals (Mehlhorn et al., Reference Mehlhorn, Al-Rasheid, Al-Quraishy and Abdel-Ghaffar2012; Benelli, Reference Benelli2015). Vector-borne diseases such as dengue, filariasis, malaria and Japanese encephalitis lead to 1173 deaths year−1. These diseases cause economic loss and social disturbance (Gopalan & Das Reference Gopalan and Das2009; Becker et al., Reference Becker, Petrić, Zgomba, Boase, Madon, Dahl and Kaiser2010; Dhiman et al., Reference Dhiman, Pahwa, Dhillon and Dash2010). Thus, the control of mosquito-borne diseases is one of the challenging tasks facing the society due to an inadequacy of preventive medicines and vaccines.
Culex tritaeniorhynchus is one of the prime vectors for Japanese encephalitis (JE), which leads to transmission of Japanese encephalitis virus (JEV). JE being one of the major viral encephalitis in Asia is known to cause around 50,000 cases and 10,000 deaths year−1. JEV has shown a tendency to extend to other geographic regions (Tiwari et al., Reference Tiwari, Singh, Tiwari and Dhole2012). The prevalence of the larval form of C. tritaeniorhynchus is mainly seen in paddy fields during the vegetation conditions (Mutheneni et al., Reference Mutheneni, Upadhyayula and Natarajan2014).
The larval source management of mosquito species is the major thrust areas of an integrated pest management system (WHO, 2009). An integrated approach to the larval management relies on an obliteration of the aquatic stages either through biological ways or by chemical control. Larviciding is the commonly used method for mosquito control in different ecological patterns of urban and coastal areas, where breeding sites can be easily traced (Djènontin et al., Reference Djènontin, Pennetier, Zogo, Soukou, Ole-Sangba, Akogbéto, Chandre, Yadav and Corbel2014). Controlling the mosquito larval stage is much easier than managing the adult mosquito population and has the added advantage of the reduced requirement for pesticide application.
However, the use of conventional pesticide formulations for mosquito control has become a major threat to the environment. The overuse of pesticides has contaminated the soil, sediment, and groundwater, besides becoming a threat to non-target species. In the current scenario, the threat of environmental and personal exposure to pesticides combined with the resistance in the insects toward these formulations have resulted in the declined usage of pest and vector control measures (Salahuddin et al., Reference Salahuddin, SitiHajar and Hidayatulfathi2004). Aerosol and emulsion-based pesticide formulations for mosquito control can also cause serious health hazards by entering the human body through respiratory, oral, or dermal route (Kamrin, Reference Kamrin1997).
Recent advancements in nanoformulation technologies enable efficient replacement of conventional pesticide formulations with the nanometric form. The formulated nanopesticides have a greater specificity toward the target species and present less significant environmental hazards (Anjali et al., Reference Anjali, Khan, Margulis-Goshen, Magdassi, Mukherjee and Chandrasekaran2010; Kumar et al., Reference Kumar, Shiny, Anjali, Jerobin, Goshen, Magdassi, Mukherjee and Chandrasekaran2013). Major formulations of nanopesticide involve the encapsulation of pesticidal active ingredient within the polymer matrix (Boehm et al., Reference Boehm, Martinon, Zerrouk, Rump and Fessi2003; Liu et al., Reference Liu, Tong and Prud'homme2008; Margulis-Goshen & Magdassi, Reference Margulis-Goshen and Magdassi2012). In this study, we describe a different process for the preparation of organic nanoparticles by solvent removal from volatile oil-in-water (o/w) microemulsions. The resultant particles contain neither polymeric matrix nor organic solvent and are freely dispersible in water. It is essential for pesticidal formulations to be stable at the natural habitat of the target species such as water-rich environments. The transportability of nanopesticides into natural conditions is strongly influenced by the ability of the nanoparticle to disperse in the aqueous phase. Distinctive subsurface parameters such as pH, temperature, and ionic strength can vary over wide ranges (Metin et al., Reference Metin, Lake, Miranda and Nguyen2011). The soil and water chemistry, as well as the presence of microorganisms in the environment, may also affect the nanopesticide stability and release of active ingredient into the environment. The characterization of nanoparticle dispersions (e.g. particle size, stability, and zeta potential in aqueous conditions) is essential for the bioactivity study (Jiang et al., Reference Jiang, Oberdörster and Biswas2009).
In this study, the effect of physicochemical parameters such as pH and temperature on the nanopermethrin dispersion in paddy field water was evaluated. The lethal implications of the nanopesticide on the physiological and biochemical profile were estimated through histopathological, stress biomarker (enzyme) such as acetylcholinesterase (AChE), glutathione S-transferase (GST), and digestive enzymes such as acid and alkaline phosphatases, respectively.
Materials and methods
Materials
Technical-grade permethrin (96%) was obtained from Tagros Chemicals India Ltd.; ammonium glycyrrhizinate (AG), Sec-butyl alcohol (SecBuOH), and n-butyl acetate, were obtained from Sigma-Aldrich, India. Soybean lecithin with 92% soybean phosphatidylcholine (SbPC) was purchased from Lipoid, Switzerland. Dimethyl sulfoxide (DMSO) was purchased from SRL Chemicals, India. All other chemicals used in the experimentation were of analytical grade.
Formation of nanopermethrin
Formation of nanopermethrin was carried out as reported earlier (Anjali et al., Reference Anjali, Khan, Margulis-Goshen, Magdassi, Mukherjee and Chandrasekaran2010). The preparation of nanopermethrin was commenced using the method of rapid evaporation of solvents from o/w microemulsion. The pesticide-loaded o/w microemulsion was obtained by mixing requisite volume of aqueous and volatile organic phase along with surfactants and co-surfactants. The lyophilization of the o/w microemulsion at 95°C and <1 mbar for 24 h resulted in a water-dispersible powder containing pesticide nanoparticles along with the surfactants. The obtained lyophilized powder consists of 13% permethrin, 29.5% AG, 29.5% SbPC, and 28% sucrose by weight.
Characterization of the nanopermethrin
Particle size determination
The particle size of nanopermethrin was measured using the Nano Particle Analyzer (SZ100, Horiba Scientific, Japan). Before the experimentation, the nanopermethrin powder (10 mg l−1) was prepared in Milli-Q water and analyzed at 25°C. The analysis was performed by placing the samples in vertical cylindrical cuvettes (10 mm diameter), and the intensity of scattered light was estimated at an angle of 90°. The particle size and the corresponding polydispersity index (PDI) value were noted for the nanopermethrin dispersion. Measurement of the particle size was carried out in triplicates.
Zeta potential measurement
The zeta potential of the nanopermethrin particles dispersed in an aqueous medium was measured using Nano Particle Analyzer (SZ100, Horiba, Japan) and determined using the Helmholtz–Smoluchowski equation (Meyer et al., Reference Meyer, Berrut, Goodenough, Rajendram, Pinfield and Povey2006). The zeta potential measurements were also carried out in triplicates.
Collection of paddy field water
The paddy field water was collected from the paddy fields located in Kangeyanallur area Vellore district, Tamil Nadu, India (12.953°N, 79.155°E). The physicochemical parameters (pH, temperature, TDS, DO, salinity, and conductivity) of the collected water samples were determined using Orion™ 5-Star Plus Multiparameter Meter, Thermo Scientific, Singapore.
Stability assessment of nanopermethrin
Preparation of nanopermethrin colloidal dispersion
To examine the behavior nanopermethrin dispersion, the colloidal dispersion of nanopermethrin was prepared using 100 ml of Milli-Q and paddy field water, respectively. The working concentration of 1 mg l−1 nanopermethrin was prepared by diluting the stock nanopermethrin dispersion (10 mg l−1) with the respective test water matrices and used for the further experimentation.
pH and temperature effect on the stability
The stability of nanopermethrin dispersion was analyzed by measuring the time-dependent changes in the particle size at different pH and temperature conditions for 30 days. The size of nanoparticles was determined by dynamic light scattering (DLS) analysis. For determining the effect of pH on stability, 1 mg l−1 concentration of nanopermethrin dispersion was prepared using Milli-Q water (at pH 4, 7, and 10) and their stability was analyzed. The nanopermethrin dispersion (1 mg l−1) stability was also checked at varying temperatures from 5 to 45°C for a 1-month duration.
Zeta potential measurement
The zeta potential of the nanopermethrin dispersion (1 mg l−1) in different water matrices were determined to evaluate the stability of nanopermethrin. The sample was stored at room temperature for the test duration of 30 days, and the zeta potential was measured for every 5 days using Nano Particle Analyzer (SZ100, Horiba Scientific, Japan).
Larvicidal bioassay
Collection and maintenance of target species
Mosquito larvae were collected from the paddy field area located in Kangeyanallur, Vellore, Tamil Nadu (12.953°N, 79.155°E). The taxonomical identification of the larvae was confirmed by the Zonal Entomology Team (ZET), Vellore, Tamil Nadu. Based on the anatomical and other morphological characters of the obtained culicines, they were identified as C. tritaeniorhynchus. The third-instar larvae were screened and acclimatized to a suitable temperature (28 ± 2°C) and humidity (70–80%) conditions with a photoperiod of light: dark:: 14:10 phases (WHO, 2005). The larvae were fed with protein biscuit and yeast mixture in 1:3 ratio.
Larvicidal bioassay of nanopermethrin
The larvicidal bioassay against C. tritaeniorhynchus was carried out according to World Health Organisation (WHO) guidelines (WHO, 2005). The test concentrations ranging from 0.007 to 1 mg l−1 were prepared for nanopermetrhin based on the presence of active ingredient, i.e. permethrin. Similar concentrations were selected for test groups treated with the bulk form of permethrin. Since bulk permethrin is poorly soluble in water, initial dilution was made in DMSO, followed by the subsequent concentration dilutions with water. Twenty-five larvae, each of third instar, were introduced in the respective test concentrations of bulk and nanopermethrin, and the larval mortality was noted down for 24 h. Distilled water without the addition of pesticide and DMSO was kept as the control. The distilled water with the addition of DMSO was used as a vehicle control. The mortality and morbidity of larvae were checked for 24 h by their inability to move after probing the cervical region with a needle. The complete experiments were conducted at room temperature with triplicates for each test concentration. For biochemical assays, larval tissues treated with LC30 and LC50 concentrations of nanopermethrin and bulk permethrin were used.
Histopathological studies
The effect of the nanopermethrin dispersion and its bulk counterpart was assessed by performing histopathological studies on the cross section (CS) and longitudinal section (LS) of the respectively treated larvae. Third-instar larvae of C. tritaeniorhynchus were treated with the LC50 concentrations of nano and bulk permethrin for 24 h. Treated, as well as control larvae were preserved in 10% buffered formalin (pH 7.2) at 4°C. The larval tissues treated with DMSO were used as a vehicle control. The tissues of the larvae were then fixed using Davidson fixative and dehydrated using a graded ethanol serial concentrations. The thin, distinct sections of tissues (5 µm) were cut with the help of a rotary microtome (Leica, RM-2235). The sections were stained using Hemotoxylin & Eosin (H&E) stain (Himedia Chemicals, Mumbai, India) and examined for histopathological alterations using a phase contrast microscope (Leica, DM-2500, Germany). The images were captured at 400× magnification using Leica DFC 295 camera and processed using Leica Application Suite 3.8 software.
Biochemical study of larval tissues
Preparation of larval tissue homogenate
In this series of experiments, some biochemical compounds, including total protein, lipid, carbohydrate, and biomarker enzymes such as AChE, GST, and acid/alkaline phosphatase of C. tritaeniorhynchus larvae that are important regarding physiological responses were evaluated. The LC30 and LC50 concentrations of both bulk and nanopermethrin were used for the treatment. Ten larvae were treated with four replicates for each concentration. After the treatment of larvae for 24 h, the larvae were selected, and the adhered water on the larval surface was removed by blotting with the tissue paper. Later the larval whole body tissue was homogenized using 500 µl of ice-cold phosphate buffer (20 mM, pH 7.0). The prepared homogenate was further centrifuged at 8000 g , 4°C for 20 min and the obtained supernatant was used for further analysis (Revathi et al., Reference Revathi, Chandrasekaran, Thanigaivel, Kirubakaran, Sathish-Narayanan and Senthil-Nathan2013).
Total protein analysis
The total protein content in the larval tissues was estimated using the method described by Bradford (Bradford, Reference Bradford1976). 10 µl of the homogenate samples were mixed with 200 µl of Bradford's reagent and incubated for 5 min. The protein concentration in the larval tissues was spectrophotometrically assayed at 595 nm using BSA (bovine serum albumin) as a standard.
Carbohydrate and lipid determination assay
The total carbohydrate and lipid content present in the larval tissues were estimated using the method described by Yuval et al. (Reference Yuval, Holliday-Hanson and Washing1994). Larval tissue homogenates were mixed with 750 µl of chloroform: methanol (1:2) mixture. The centrifugation of the samples was carried out for 10 min at 8000 rpm. The supernatant of volume 500 µl was collected and dried at 40°C. The dried samples were redissolved in 500 µl of H2SO4 and incubated at 90°C for 10 min in a water bath. Then, 270 µl of vanillin reagent (60 mg vanillin was added to a mixture of 10 ml of water and 40 ml of 85% H3PO4) was added to the samples. The spectrophotometric detection of lipid in the test samples was carried out at 630 nm using ELISA plate reader (Biotek Power Wave, XS2, India).
Carbohydrate extraction from the prepared homogenate was carried out using 750 µl of chloroform: methanol mixture (1:2). 150 µl of samples were taken from the chloroform: methanol extracts and diluted with 100 µl of distilled water along with 500 µl of Anthrone reagent (500 mg of anthrone dissolved in 500 ml of H2SO4). The colored complex was spectrophotometrically assayed at 625 nm using an ELISA plate reader.
GST assay
GST assay was carried out to determine the detoxifying potential of a particular metabolic enzyme present in the larval cells. The 0.1 ml of larval homogenates were mixed with 0.1 ml of phosphate buffer (0.3 M). To these mixtures, 0.1 ml of 0.1 M 1-chloro,2,4, dinitrobenzene was added along with 0.1 ml of 0.2 M reduced glutathione. The absorbance changes were recorded for every 10 min duration with a 30 sec interval. The readings were noted at 340 nm using a UV–vis Spectrophotometer (U-2910, Double beam UV–vis spectrophotometer, Hitachi, Japan).
AChE assay
The neurotoxic potential of nanopesticides was assessed by determining the AChE activity levels in larvae (Ellman et al., Reference Ellman, Courtney, Andres and Featherstone1961). The 100 µl of larval homogenates were diluted using 82.5 µl of distilled water. 12.5 µl of 100 mM phosphate buffer was added to the sample followed by the addition of 5 µl of 0.075 M ATCI (acetyl thiocholine iodide). Sample mixtures were incubated for 10 min at 37°C, and then 25 µl of 0.01 M dithiobis (2-nitrobenzoic acid) (DTNB) reagent was added. The optical density of the yellow-colored product was measured spectrophotometrically at 412 nm.
Acid and alkaline phosphatase tests
The effects of nanopesticide on the gut digestive enzymes were assessed by estimating the alkaline and acid phosphatase activity (Sugumar et al., Reference Sugumar, Clarke, Nirmala, Tyagi, Mukherjee and Chandrasekaran2014). For the assessment of the enzyme alkaline phosphatase, 20 µl of the larval homogenates were added to 80 µl of 50 mM Tris–HCl buffer of pH 9 along with 15 mM of p-nitrophenyl phosphate. The resultant mixtures were incubated at a temperature of 37°C, and the change in the absorbance was assessed spectrophotometrically at 405 nm. For the acid phosphatase activity, 50 mM sodium acetate buffer at pH 4 was used instead of Tris–HCl buffer, and the further steps remained similar as mentioned above.
Statistical analysis
The lethal indices were determined at 95% confidence level (P<0.05) using EPA Probit analysis software. Student's t-test was carried out to check the significant difference obtained between the lethal indices for nanopermethrin and bulk permethrin treatment. The test for the level of significance of the experimental results was carried out using one-way ANOVA (analysis of variance) followed by Tukey's multiple comparison tests. GraphPad Prism 6 was used for the statistical analysis.
Results and discussions
Nanopermethrin formulation
The lyophilization of the microemulsion containing the mixture of an aqueous and a volatile organic phase resulted in the water dispersible powder of nanopermethrin. The nanopermethrin dispersed in Milli-Q water was characterized by its particle size and surface charge using DLS and zeta potential analysis. The mean hydrodynamic size of the nanopermethrin dispersion was found to be 165 ± 0.9 nm with a PDI of 0.341 exhibiting the uniform distribution of the nanopesticide particles. The resultant zeta potential of the nanoparticle dispersion was found to be −66 ± 0.9 mV exhibiting the hydrodynamic stability. From the previous studies conducted on the nanopermethrin (Anjali et al., Reference Anjali, Khan, Margulis-Goshen, Magdassi, Mukherjee and Chandrasekaran2010), it is evident that nanopermethrin powder is amorphous nature. The amorphous nature significantly improves of the nanopowder its solubility in water (Liu et al., Reference Liu, Sun, Hao, Jiang, Zheng and Wang2010), which contributes to its increased dissolution rate and bioavailability. In contrast to the use of synthetic emulsifiers in commercial pesticide formulations, the biosurfactants such as soybean lecithin and ammonium glycyrrhizinate were used for microemulsion formation. It results in the reduction of ecotoxicity and makes it propitious for larvicidal applications (Kumar et al., Reference Kumar, Shiny, Anjali, Jerobin, Goshen, Magdassi, Mukherjee and Chandrasekaran2013).
Stability study of nanopermethrin colloidal dispersion
The stability of nanopermethrin powder (1 mg l−1) dispersed in Milli-Q & paddy field water was studied for the period of 30 days at room temperature. Table 1 describes the physiochemical parameters exhibited by the collected paddy field water. The mean hydrodynamic size of the nanopermethrin particle dispersed in the Milli-Q water was found to be 164.4 ± 0.59 and 172.2 ± 0.75 nm on 1st and 5th day. At longer periods of time, an increase in the particle size was observed gradually reaching the micron level on the day 30. Similarly, the nanopermethrin dispersion in the paddy water exhibited the mean hydrodynamic size of 167.9 ± 0.49 nm on the 1st day and 175.3 ± 1.94 nm on the 5th day. Further, the particle size significantly increased with time reaching micron size at 30th day. Thus, the particle size analysis confirms that the nanopermethrin dispersion in both Milli-Q and paddy field water was experimentally stable for 5 days (fig. 1).
Temporal variation in the particle size of nanopermethrin in Milli-Q and Paddy field water. Corresponding error bar represents standard error of three replicates (SE, n = 3).
Physiochemical parameters of collected Paddy field water.
The stability of nanopermethrin dispersion (1 mg l−1) was further assessed by zeta potential analysis in Milli-Q and paddy field water at room temperature for 30 days. Zeta potential values of nanopermethrin dispersion in Milli-Q water was found to be −65.8 ± 0.4 mV on the first day and increased to −61 ± 0.7 on day 5, reaching to −2.7 ± 0.47 mV on the 30th day. In the same way, the nanopermethrin dispersion in the paddy field water exhibited the zeta potential −35.8 ± 0.4 mV on day 1, later increasing to −30.6 ± 0.28 mV on day 5. Further zeta potential value gradually increased reaching −0.4 mV on day 30 and probably leading to particle aggregation (fig. 2). At this point, nano-sized particles reached micron size, which resulted in the termination of the study. The shift in the particle size is due to the increase in zeta potential absolute values, which lead to reduced electrostatic stabilization of the dispersive system (Freitas & Müller, Reference Freitas and Müller1998). The zeta potential values of greater than −60 mV provide excellent stability to the colloid, while good stability typically necessitates zeta potential values of greater than −30 mV (Riddick, Reference Riddick1968).
Temporal variation in Zeta potential of nanopermethrin particles dispersed in Milli-Q and paddy field water, Corresponding error bars in graphs represents standard error of three replicates (SE , n = 3).
To elucidate the pH influence on the nanopermethrin stability, the particle size analysis was carried out for nanopermethrin dispersion at pH 4, 7, and 10. The nanopermethrin dispersion was more stable in the acidic and neutral conditions compared to the alkaline condition (table 2). As the pH of the dispersion medium increase to high pH (pH 10), the particles gets aggregated and remains settled at the bottom. The instability of nanopermethrin particles in the alkaline medium (pH 10) may arise due to the degradation of the surfactant-like ammonium glycrrhizinate. As ammonium glycrrhizinate being the prime surfactant being used to as a coating agent, the deterioration of this surfactant at the higher pH leads to aggregation of the particles leading to instability. Coiffard et al. (Reference Coiffard, Coiffard, Peigne and de Roeck-Holtzhauer1998) describe the instability of the ammonium glycrrhizinate at the elevated pH of 10. This particular compound tends to exhibit the better stability till pH 9, while the further increase in the alkalinity leads to its degradation. Therefore, the ammonium glycrrhizinate being the essential component in the nanopermethrin dispersion, its degradation results in the instability of the nanopermethrin dispersion.
Effect of pH on the stability of nanopermethrin dispersion
The temperature effect on the stability of nanopermethrin dispersion was also studied at different temperatures of 5, 25, and 45°C, respectively. The particle dispersion of nanopermethrin showed better stability at a lower temperature (5 and 25°C) compared to high temperature (45°C). At 45°C, there was a significant increase in the particle size, resulting in the rapid aggregation of nanoparticles (table 3). As the surfactant-like ammonium glycrrhizinate are present in the nanoformulation of permethrin, its thermodegradation at the high-temperature results in the instability of the nanopermethrin dispersion. The thermodegradation of ammonium glycrrhizinate at the elevated temperature (Coiffard et al., Reference Coiffard, Coiffard, Peigne and de Roeck-Holtzhauer1998), also the active ingredient, i.e. permethrin itself degrades at the higher temperatures (Sharom & Solomon, Reference Sharom and Solomon1981) results in the instability of the nanometric pesticide, i.e. nanopermethrin. The stability and efficacy of nanoparticles are widely affected by different physicochemical properties such as pH and temperature (Metin et al., Reference Metin, Lake, Miranda and Nguyen2011).
Effect of temperature on the stability of nanopermethrin dispersion.
The deterioration of the surfactant coating on the nanopesticide may alter the surface properties of nanopesticide, thereby making it unstable. The stability of nanopesticide might be influenced by the microbial biota present in the soil and water of the agricultural ecosystem (Balson & Felix, Reference Balson, Felix, Karsa and Porter1995). The surfactant gets utilized by microbes as a substrate for their energy and nutrition or may get co-metabolized by microbes through various metabolic reactions (Guang Guo, Reference Guang Guo2004). Bacterial species such as Pseudomonas and Aeromonas sobria tend to have degradation potential against the surfactant and other pesticidal components in the aqueous phase (Doong & Lei, Reference Doong and Lei2003; Lee et al., Reference Lee, Gan, Kim, Kabashima and Crowley2004). These effective parameters resulted in the detritions of the surfactant coat of the nanopesticide making it stable only for 5 days.
Larvicidal activity of nanopermethrin against C. tritaeniorhynchus
The larvicidal activity of nanopermethrin and its bulk counterpart was studied against the JE vector C. tritaeniorhynchus. The 12 and 24 h lethal indices (LC50) for the nanopermethrin was found to be 0.057 and 0.014 mg l−1, while for bulk permethrin was found to be 0.442 and 0.150 mg l−1, respectively (table 4). No mortality was observed in the DMSO treatment and control. In comparison to nanopermethrin exhibiting 90% mortality at a concentration of ≥0.250 mg l−1, bulk permethrin was toxic for the survival of larvae only at concentrations above 0.5 mg l−1 at 24 h.
Larvicidal activity of bulk permethrin and nanopermethrin against the larvae of C. tritaeniorhynchus.
1 Denotes the significant difference (P > 0.05) between the LC50 nanopermethrin (12 h) and bulk permethrin (12 h).
2 Denotes the significant difference (P > 0.05) between the LC50 Nanopermethrin (24 h) and Bulk permethrin (24 h).
The application of nanoparticles as a larvicidal agent is widely seen in the current scenario (Rajakumar & Rahuman, Reference Rajakumar and Rahuman2011). Reduction in the particle size and increase in surface area of the nanopesticides facilitate their easier penetration into the larval body resulting in an enhanced larvicidal potency (Balaji et al., Reference Balaji, Mishra, Kumar, Mukherjee and Chandrasekaran2015a , Reference Balaji, Mishra, Kumar, Ashu, Margulis, Magdassi, Mukherjee and Chandrasekaran b ; Mishra et al., Reference Mishra, Balaji, Swathy, Paari, Kezhiah, Tyagi, Mukherjee and Chandrasekaran2016). Permethrin being a type-I pyrethroid insecticide acts as a neurotoxin resulting in hypersensitivity of the host nervous system. Permethrin blocks the Na+-gated channel of the insect nervous system and induces the continuous firing of impulses in the axons of the insect nervous system (Ray et al., Reference Ray, Ray and Forshaw2000). Pyrethroid pesticide tends to show its effect on both peripheral, as well as the central nervous system of the insects. Anjali et al. (Reference Anjali, Khan, Margulis-Goshen, Magdassi, Mukherjee and Chandrasekaran2010) also reported the similar findings against the Culex quinquifasciatus larvae that propound the increased toxicity permethrin when it is formulated into the nanoform. The active ingredient, i.e. permethrin has a major role in the action exhibited by the nanopesticide, which gets enhanced due to the conversion of the bulk form to the nanoform. The transformation of the bulk pesticide into the nanopesticide leads to change in the morphological characteristics of the compound making it unique as the surface area of the nanoparticle gets increased. The larger surface area tends to provide easy penetration into the cells and has enhanced efficacy as a pesticide. For toxicity studies of any nanomaterial, it is essential to have its accurate characterization at various stages of the formulation and also at pre and post administration to the biological system (Oberdörster et al., Reference Oberdörster, Maynard, Donaldson, Castranova, Fitzpatrick, Ausman, Carter, Karn, Kreyling and Lai2005). The size of the nanoparticles governs their interaction with the biological system, including the ingestion, absorption, distribution, and excretion (Borm et al., Reference Borm, Robbins, Haubold, Kuhlbusch, Fissan, Donaldson, Schins, Stone, Kreyling and Lademann2006; Choi et al., Reference Choi, Liu, Misra, Tanaka, Zimmer, Ipe, Bawendi and Frangioni2007).
Kumar et al. (Reference Kumar, Shiny, Anjali, Jerobin, Goshen, Magdassi, Mukherjee and Chandrasekaran2013) describe the effectiveness of the nanoform of permethrin toward the A. aegypti compared with that of its bulk counterpart. The higher effectiveness of this nanopesticide at the lower concentration defines its potency as a mosquitocidal agent. Similarly Balaji et al. (Reference Balaji, Mishra, Kumar, Ashu, Margulis, Magdassi, Mukherjee and Chandrasekaran2015b ) described the maximum influence of the nanometric form of the permethrin against the dreadful mosquito vectors such as C. tritaeniorhynchus, Aedes aegypti, and Aedes albopictus when compared with its bulk form. The nanoform of pesticides is helpful toward the management of resistance problem occurring worldwide due to overuse of conventional pesticides.
Histopathological studies
Histological studies revealed that the LS and CS sections of control larvae showed a normal morphology of the larval midgut with a layer of epithelial cells resting upon the basement membrane. The integrity of the larval tissues in control, when compared with the pesticide was found unaffected, and the cellular components remain intact. Treatment of the larval tissues with the pesticide resulted in the histological alterations in the epidermis and other cellular components. Farnesi et al. (Reference Farnesi, Brito, Linss, Pelajo-Machado, Valle and Rezende2012) describe the potency of the novaluron pesticide treatment and its effect on the histological disintegration when compared with the control.
The midgut of the nanopermethrin-treated larvae showed damage to the epithelial lining with swollen cells and loss of cellular integrity. The peritrophic membrane was found damaged with leakage in the midgut content in nanopermethrin-treated larvae. The effect of bulk permethrin treatment on larvae was less pronounced with minimal damage to the epithelial cells of the larval midgut region (table 5). There were no such alterations found in the gut region of the larvae exposed to DMSO (figs 3 and 4). The histopathological alterations in the midgut region of the mosquito larvae were more significant in the nanopesticide-treated larvae than its bulk counterpart treatment. The midgut of the insect is an important region where the secretion of the digestive enzymes, as well as the absorption of nutrients, takes place. Also, it is a sensitive indicator of several kinds of toxicants (Sutherland et al., Reference Sutherland, Burgess, Philip, McManus, Watson and Christeller2002). The peritrophic membrane located between the gut lumen and the epithelial layer plays a prominent role in protection from damage and also from several other toxicants (Terra, Reference Terra2001). Vijayakumar et al. (Reference Vijayakumar, Vinoj, Malaikozhundan, Shanthi and Vaseeharan2015) describe the potency of biologically synthesized zinc oxide nanoparticles and efficacy as a larvicidal agent. The treatment of the mosquito larvae (Anopheles stephensi, Culex quinquefasciatus, and C. tritaeniorhynchus) resulted in the disintegration of larval tissues, and its component cells when compared with the control samples. There was the severe devastation of the epithelial cells, peritrophic membrane, and midgut contents were observed in the nanopermethrin-treated larvae due to its nanometric size that helps its easier penetration into the cells leading to histological disintegration.
H&E staining of larval gut region (longitudinal section), objective- 400× magnification. (a) Control midgut with occurrence of normal cellular components such as EC (epithelial cells), PM (peritrophic membrane), MC (midgut content). (b) DMSO-treated midgut (c) bulk permethrin treatment with deformities like damage in EC, PM, and leakage in MC. (d) Nanopermethrin treatment with deformities like damage in EC*, PM*, and leakage in MC*. Note: * mark signifies the severity in deformities.
H&E staining of larval gut region (cross section), objective – 400× magnification. (a) Control midgut with normal cellular components such as EC (epithelial cells), PM (peritrophic membrane), MC (midgut content). (b) DMSO-treated midgut (c) bulk permethrin treatment with deformities like damage in EC, PM, and leakage in MC. (d) Nanopermethrin treatment with deformities like damage in EC*, PM*, and leakage in MC*. Note: * mark signifies the severe deformities.
Semi-quantitative histopathology analysis of control, DMSO, permethrin, and nanopermethrin.
Control and treated larvae with histopathology scores: − (Nil), + (mild effect), ++ (moderate effect), +++ (severe effect).
Biochemical study of larval tissues
The exposure to a xenobiotic can result in the modification in the synthesis of certain metabolites and its associated cellular functionalities. In this perspective, the major functional metabolites such as total protein, carbohydrate, and lipid, present in the whole body of third-instar C. tritaeniorhynchus larvae decreased under the stress conditions due to its exposure to the nanopermethrin.
C. tritaeniorhynchus larvae exposed to LC30 and LC50 concentrations of bulk permethrin, and nanopermethrin led to significant alterations in their total protein, lipid, and carbohydrate content of larvae. Protein, lipid, and carbohydrates are the major proximal cellular biomolecules present in the insect body that have a role in the majority of biochemical reactions. The total protein content of control larvae was found to be 0.317 µg larvae−1. The larvae treated with LC30 and LC50 concentrations of nanopermethrin exhibited significant decrease (P < 0.05) in total protein values as 0.121 and 0.095 µg larvae−1, whereas the total protein in bulk permethrin-treated larvae was found to be 0.191 and 0.175 µg larvae−1, respectively (fig. 5). Similarly, a significant (P < 0.05) difference in the total carbohydrate content (fig. 6) was observed in the nanopermethrin-treated larval tissue (0.207 and 0.19 µg larvae−1) as compared with bulk permethrin (0.264 and 0.243μg larvae−1) and control (0.377 µg larvae−1). The total lipid content for the nanopermethrin-treated larvae was found to be reduced (0.073 and 0.068 µg larvae−1) as compared with the control (0.123 µg larvae−1) at a significance level of P < 0.05. Significant differences (P < 0.05) in the lipid content were observed between bulk permethrin- (0.078, 0.075 µg sample−1) and nanopesticide-treated larvae (0.073 and 0.068 µg sample−1) (fig. 7).
Total protein level in larvae treated with LC30 and LC50 concentrations of bulk permethrin and nanopermethrin. Note: a denotes significant difference (P < 0.05) between nanotreatment and control and bdenotes significance (P < 0.05) between nanotreatment and bulk treatment. Corresponding error bar represents standard error of three replicates (SE, n = 3).
Total carbohydrate level in larvae treated with LC30 and LC50 concentrations of bulk permethrin and nanopermethrin. Note: adenotes significance (P < 0.05) between nanotreatment and control, and bdenotes significance (P < 0.05) between nanotreatment and bulk treatment. Corresponding error bar represents standard error of three replicates (SE, n = 3).
Lipid content in larvae treated with LC30 and LC50 concentrations of bulk permethrin and nanopermethrin. Note: adenotes significance (P < 0.05) between nanotreatment and control, and bdenotes significance (P < 0.05) between nanotreatment and bulk treatment. Corresponding error bar represents standard error of three replicates (SE, n = 3).
Significant reduction in the concentration of the proximal cellular molecules was observed in the nanopermethrin-treated larvae when compared with the control. Proteins play a major role in the transformation and structure of the biomolecules, cuticle scleritization, and synthesis of the diapause and visual pigments (Terra, Reference Terra2001). The depletion of protein content in the nanopermethrin-treated larvae may have occurred due to hindrance in the insect physiology. The loss of protein content in the larvae could be a result of the degradation of the protein into their respective amino acids, which involve the TCA (tricarboxylic acid) cycle to compensate the stress and loss of energy (Schoonhoven, Reference Schoonhoven1982). Ali et al. (Reference Ali, Ali and Shakoori2014) describe the breakdown of the total protein due to the reduction in the hemolymph volume of the insect. The depletion of the total protein level may constitute a physiological mechanism that plays an immense role in the compensatory mechanism occurring in the insect body due to the insecticidal stress (Khosravi & Sendi, Reference Khosravi and Sendi2013). The increase in the uric acid level as a side product of protein catabolism is an example of such compensatory mechanism that occurs due to the depletion in the total protein level.
Similarly, carbohydrates are an important source of insect diet, and the energy is transformed into fats and used for protein production (Khosravi & Sendi, Reference Khosravi and Sendi2013). The reduced carbohydrate level in the insects occurs due to the reduction in the feeding behavior of the insect as a result of stress (Etebari et al., Reference Etebari, Bizhannia, Sorati and Matindoost2007). Carbohydrates as the primary source of energy play a significant role in the physiology of the insects. The rate of the glycogen in the insect tissues is related to the several physiological events occurring inside the body such as reproduction, molt, and flight (Kaufmann & Brown, Reference Kaufmann and Brown2008). Accordingly, the insect body under the stress condition leads to the metabolism of the huge amount of sugar to substantiate the energy need which leads to the depletion of the carbohydrate levels in an organism (Suzuki et al., Reference Suzuki, Sakurai and Iwami2011).
The decrease in the lipid content of the larval body tissues was more significant in the nanopermethrin-treated samples than their corresponding control. The reduction of the lipid content in the larval body may occur due to the dysfunction of the individual hormones responsible for the control of lipid metabolic activities (Locke & Huie, Reference Locke and Huie1981). Canavoso et al. (Reference Canavoso, Jouni, Karnas, Pennington and Wells2001) describe the decrease in the lipogenic activity due to the exposure of insect to insecticide. Shaurub & El-Aziz (Reference Shaurub and El-Aziz2015) reported similar findings of the depletion in the levels of total protein, carbohydrate, and lipid in the third-instar larvae of C. pipens after exposure to lambda-cyhalothrin and lufeneron insecticides.
The effect of nanopesticide on the activities of stress biomarker enzymes of C. tritaeniorhynchus larvae such as AChE, acid/alkaline phosphatase and GST was studied. A significant (P < 0.05) decrease in these biomarker enzyme activities was observed compared with the control and bulk. The toxicity of the pyrethroid pesticide would lead to changes in the activity of AChE enzyme, an essential indicator of neurotoxicity. The AChE activity was significantly reduced to 68.1 and 64.9% in the nanopesticide-treated larvae, whereas to 79.8 and 71.4% in bulk permethrin when compared with the control (fig. 8). Haynes (Reference Haynes1988) describes the neurotoxic behavior of permethrin on the insects leading to depletion in the feeding behavior of the A. aegypti. A similar finding of the neurotoxic behavior of pyrethroid was described by Abou-Donia et al. (Reference Abou-Donia, Goldstein, Dechovskaia, Bullman, Jones, Herrick, Abdel-Rahman and Khan2001), which defines the neurobehavioral deficits due to alterations in the AChE activity in rats.
Activity of AchE in larvae treated with LC30 and LC50 concentrations of bulk permethrin and nanopermethrin. Note: adenotes significance (P < 0.05) between nanotreatment and control, and bdenotes significance (P < 0.05) between nanotreatment and bulk treatment. Corresponding error bar represents standard error of three replicates (SE, n = 3).
Similarly, reduction in the GST activity for nanopermethrin-treated larvae was observed as compared the control and bulk permethrin. The percentage of GST activity for LC30 and LC50 nanopermethrin treated larval tissues were found to be reduced to 64.5 and 20%, whereas the bulk (LC30 and LC50) treated larvae showed 83.6 and 88.7% activity compared with the control (fig. 9). The nanopesticide-treated larvae demonstrated a substantial depletion in the activity of biomarker enzymes than the bulk permethrin-treated samples. The intoxication due to pesticides in the insect body results in the depletion of GST level (Kady et al., Reference Kady, Kamel, Mosleh and Bahght2008).
Activity of GST in larvae treated with LC30 and LC50 concentrations of bulk permethrin and nanopermethrin. Note: adenotes significance (P < 0.05) between nanotreatment & control, and bdenotes significance (P < 0.05) between nanotreatment and bulk treatment. Corresponding error bar represents standard error of three replicates (SE, n = 3).
GST is one of the essential enzymes present in the insect body that acts as a defensive mechanism against several well-known pesticides such as organophosphates and organochlorines (Clark et al., Reference Clark, Shamaan, Sinclair and Dauterman1986). Stimulation of the GST activity is not only seen in insects after the exposure of the organophosphate (Hayaoka & Dauterman, Reference Hayaoka and Dauterman1982; Clark et al., Reference Clark, Shamaan, Sinclair and Dauterman1986) and organochlorines (Lagadic et al., Reference Lagadic, Cuany, Bergé and Echaubard1993), but also after the interaction with pyrethroid molecules (Yu et al., Reference Yu, Robinson and Nation1984; Punzo, Reference Punzo1993). Grant & Matsumura (Reference Grant and Matsumura1989) discuss the alteration in the GST level and its activity due to the interaction with the pyrethroid pesticides. Kostaropoulos et al. (Reference Kostaropoulos, Papadopoulos, Metaxakis, Boukouvala and Papadopoulou-Mourkidou2001) also describe the possible interaction of the GST molecules with the pyrethroid insecticides in a sequestering mechanism that aids to the defense of the organism either in a passive way of detoxification or a facilitating manner. Therefore the determination of the GST level can serve as an important parameter for the toxicity in the insects.
Esterases are enzymes that help in the catalysis of esterase into one acid and alcohol molecule by the addition of one molecule of H2O (Devorshak & Roe, Reference Devorshak and Roe1999). The role of the esterase enzymes in the resistance mechanism of mosquitoes was well documented in the literature (Brogdon & McAllister, Reference Brogdon and McAllister1998). The detoxification process by enzymes consumes much energy resulting in a concomitant reduction or increment in the life duration and reproductive nature of the insects (Devorshak & Roe, Reference Devorshak and Roe1999). The enzyme activities of AChE and GST correlate with their ability to degrade the pesticidal components. Hemingway et al. (Reference Hemingway, Hawkes, McCarroll and Ranson2004) and Selvi et al. (Reference Selvi, Edah, Nazni, Lee and Azahari2007) studied the role of AChE and GST in providing resistance to pyrethroids in the mosquitoes. The increased activity of these biomarker enzymes in the pesticide-treated larvae signifies the development of resistance to pesticides. In this study, significant decrease in the enzyme activity of ACHE and GST was observed in nanopermethrin-treated larvae when compared with the bulk permethrin. The similar findings were obtained by Balaji et al. (Reference Balaji, Mishra, Kumar, Mukherjee and Chandrasekaran2015a ), which describes the effectiveness of the nanometric pesticide nano diethyl phenyl acetamide (DEPA) against the biochemical profile of C. tritaeniorhynchus larvae. The treatment of the mosquito larvae with the nanometric form of DEPA resulted in the significant depletion in the enzymatic activity of AChE and catalase activity. The above findings highlight the importance of nanometric form of permethrin as substitute for the conventional pesticides to overcome the problem of resistance in the mosquitoes.
The levels of digestive enzymes such as acid and alkaline phosphatase were significantly (P < 0.05) reduced in nanopermethrin-treated larvae when compared with the control larvae and bulk treatment. The acid phosphatase activity in the nanopermethrin-treated (LC30 and LC50) larvae decreased to 22.2 and 5.5% when compared with the control, whereas in bulk permethrin treatment, a significant reduction in the enzyme activity was observed in 44.4 and 33.3% at P < 0.05 level of significance. Similarly, there was a decline in the alkaline phosphatase activity to 29 and 12.9% in nanopermethrin-treated samples, whereas in bulk permethrin-treated larvae to 35.4 and 22.5% compared with the control (fig. 10). The substantial reduction in the level of digestive enzymes was observed in the nanopermethrin-treated samples, and these findings were corroborative with the histopathological observations on midgut region.
Digestive enzyme (a) acid phosphatase, (b) alkaline phosphatase activity in larvae treated with LC30 and LC50 concentrations of bulk permethrin and nanopermethrin. Note: a,cdenote significant difference (P < 0.05) between nanotreatment and control, while b,ddenote significant difference (P < 0.05) between nano and bulk treatment. Corresponding error bar represents standard error of three replicates (SE, n = 3).
The toxicity to the midgut tissues resulted in leakage of cellular content and depletion in the levels of the digestive enzyme. The acid phosphatase (E.C 3.1.3.2) and alkaline phosphatase (E.C 3.1.3.1) are digestive enzymes secreted by the gut tissues and the lumen of the insect. The alkaline phosphatase is a primary enzyme of the midgut microvillar membrane, which is found in the dipteran and lepidopteran insects. Their occurrence also reported in the midgut basolateral membranes. The acid phosphatase is present in the cytosol of the insect midgut cells. These digestive enzymes help in the removal of phosphate moieties from the phosphorylated compounds before their absorption (Terra & Ferreira, Reference Terra and Ferreira1994). The hydrolytic cleavage occurring in the phosphoric acid esters is catalyzed by the acid and alkaline phosphatase enzymes, which help in proper metabolism and cell-signaling processes (Broberg & Sahlin, Reference Broberg and Sahlin1989). Acid phosphatase is an example of a lysosomal enzyme; whose activity may get hampered due to the toxicity of the exposure of xenobiotic compounds (Shaurub & El-Aziz, Reference Shaurub and El-Aziz2015). The reduction in the activity of the alkaline phosphatase could be indorsed to the decrease in the enzyme synthesis or the inhibition of the enzyme kinetics due to blockage of the active site due to the insecticidal compound (Shakoori et al., Reference Shakoori, Saleem and Mantle1998). The larvae of C. tritaeniorhynchus exposed to nanopermethrin exhibited a substantial decrease in acid and alkaline phosphatase activities were similar to the findings of Sugumar et al. (Reference Sugumar, Clarke, Nirmala, Tyagi, Mukherjee and Chandrasekaran2014). The report by Sak et al. (Reference Sak, Uckan and Ergin2006) describes the toxicity of cypermethrin to Pimpla turionellaei resulting in the depletion of the cellular biomolecules. The toxicity of the phytoextracts and other pesticides on the biochemical profile of the mosquito larval species occurs due to the lowering down of the feeding and improper digestion (Sharma et al., Reference Sharma, Mohan, Dua and Srivastava2011). Therefore the assessment of the biochemical profile is a helpful strategy, which depicts the severity of the toxicity as a larvicidal agent against the dreadful vectors causing fatal diseases.
Conclusion
A hydrodispersive nanometric powder of permethrin formulated using biological surfactants has the potential to minimize the toxicity load to the environment. The exceptional colloidal stability exhibited by nanopermethrin dispersion under different physicochemical conditions for 5 days favors its application as a larvicidal agent. The larvicidal activity of the pesticide in nanometric form was more efficient than the bulk form. The results of histopathological observation, total composition of cellular biomolecules, and activity of stress biomarker enzymes confirm the effective insecticidal action of nanopermethrin in the larvae. By all these findings it is evident that nanoformulation of the pyrethroid pesticide is an effective tool for controlling the C. tritaeniorhynchus larvae.
Acknowledgements
We acknowledge Vellore Institute of Technology for providing the laboratory space and facilities. We deeply acknowledge ICMR BMS (35/10/2012-BMS) for the financial aid provided by them. We also acknowledge Dr K. Gopalarathinam, Senior Entomologist, ZET, Vellore for his immense help in the identification of the mosquito species.
Conflict of Interest
None.
