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Journal of Asia-Pacific Entomology 19 (2016) 1089–1094

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Larvicidal and pediculicidal activity of synthesized TiO2 nanoparticles using Vitex negundo leaf extract against blood feeding parasites P. Rajiv Gandhi, C. Jayaseelan, E. Vimalkumar, R. Regina Mary ⁎ Division of Nanobiotechnology, Department of Zoology, Auxilium College (Autonomous), Gandhi Nagar ‐ 632 006, Vellore District, Tamil Nadu, India

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Article history: Received 23 August 2016 Revised 26 September 2016 Accepted 1 October 2016 Available online 06 October 2016 Keywords: Vitex negundo Titanium dioxide nanoparticles FTIR XRD Larvicidal Pediculicidal

a b s t r a c t Insecticide resistance and inadequate attention to the application instructions of topical pediculicides are common reasons for treatment failure. Essential oils or plant extracts are good and safe alternatives due to their low toxicity to mammals and easy biodegradability. The present study was carried out to establish the larvicidal and the pediculicidal activity of synthesized titanium dioxide nanoparticles (TiO2 NPs) using the leaf aqueous extract of Vitex negundo (Verbenaceae) against the fourth instar larvae of the malaria vector, Anopheles subpictus Grassi and filariasis vector, Culex quinquefasciatus Say (Diptera: Culicidae) and the head louse, Pediculus humanus capitis De Geer (Phthiraptera: Pediculidae). The synthesized TiO2 NPs were characterized by UV, XRD, FTIR and SEM-EDX. The SEM analyses were clearly indicated that the spherical shape of the synthesized TiO2 NPs. Mosquito larvae and head lice were exposed to varying concentrations of the synthesized TiO2 NPs, V. negundo leaf aqueous extract and titanium tetrachloride (TiCl4) for 24 h. The maximum activity was observed in the synthesized TiO2 NPs against A. subpictus, C. quinquefasciatus and lice, (LC50 = 7.52, 7.23 and 24.32 mg/L; χ2 = 0.161, 2.678 and 4.495; r2 = 0.663, 0.742 and 0.924), respectively. The TiO2 NPs did not exhibit any noticeable toxicity on Poecilia reticulata after 24 h of exposure. The findings revealed that the synthesized TiO2 NPs possess excellent mosquito larvicidal and anti-lice activity. These results suggest that the green synthesis of TiO2 NPs has the potential to be used as an ideal eco-friendly approach for the control of vectors and head lice. © 2016 Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society.

Introduction Malaria is a serious global health challenge, and it has infected millions of people worldwide, with a large number of cases being reported from India. Malaria infects N500 million humans each year, killing approximately 1.2 to 2.7 million per year. About 90% of all malaria cases occur in Africa, as do approximately 90% of the world's malaria-related deaths (Breman et al., 2004; Snow et al., 2005; WHO, 2012). Anopheles subpictus is known to transmit malaria and filariasis. A. subpictus is a complex isomorphic sibling species and is recognized as a vector of malaria, a disease of great socioeconomic importance, and also a vector of some helminth and arboviruses (Chandra et al., 2010). A. subpictus is a potential vector of bancroftian filariasis and fed on microfilaremia carriers harbored Wuchereria bancrofti larvae (Hoedojo et al., 1980; Amerasinghe and Amerasinghe, 1999). Culex quinquefasciatus, a vector of lymphatic filariasis, is widely distributed in tropical zones with around 120 million people infected worldwide and 44 million people having common chronic manifestation (Bernhard et al., 2003). Microfilariae are transmitted to humans by different mosquitoes. Culex species, with a special reference to C. quinquefasciatus, are the most familiar vectors across urban and semi-urban areas of Asia. Globally, 25 ⁎ Corresponding author. E-mail address: [email protected] (R.R. Mary).

million men suffer with genital disease and over 15 million people are afflicted with lymphoedema (WHO, 2014). The harmful effects of chemicals on non-target populations, ever-growing resistance to chemical insecticides along with the recent resurgence of different mosquitoborne diseases have induced scientists to explore alternative, simple, sustainable methods of mosquito control. Head lice (Pediculus humanus capitis) infestation is a major concern in public health. Infestations due to unhygienic conditions are prevalent worldwide and especially common among school children in both developed and developing countries (Gratz, 1997). The P. humanus capitis infections cause skin irritation, pruritus, and sleep loss, as well as occasional secondary bacterial infection from scratching (Rozendaal, 1997). Nanotechnology can be defined as the science and technology involved in the design, synthesis, characterization and application of materials and devices whose smallest functional organization in at least one dimension is on the nanometer scale (Emerich and Thanos, 2003). In the past few years, nanotechnology has grown by leaps and bounds, and this multidisciplinary scientific study is undergoing explosive development (Chan, 2006). Titanium dioxide has a more helpful role in our environmental purification due to its non-toxicity, photocatalytic activity, photo induced super-hydrophilicity and anti-fogging effect. These properties have been applied in removing bacteria and harmful organic materials from water and air, as well as in self-cleaning or self-sterilizing surfaces in medical centers (Ashkarran and Theor,

http://dx.doi.org/10.1016/j.aspen.2016.10.001 1226-8615/© 2016 Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society.

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2011). Green synthesis methods are eco-friendly approach and compatible for pharmaceutical and other biomedical applications, as the toxic chemicals are not used in these methods (Willner and Basnar, 2007). Vitex negundo L. (Verbenaceae) commonly known as nirgundi chiefly occurring throughout India (Watt, 1972; Gupta et al., 2005). Though almost all parts of V. negundo are used, the leaves and the barks are the most important in the field of medicine. The decoction of leaves is considered as tonic, vermifuge and is given with pepper in catarrhal fever (Chandramu et al., 2003). Water extract of mature fresh leaves exhibited anti-inflammatory, analgesic and antihistamine properties (Dharmasiri et al., 2003). The leaves of this plant have been shown mosquito repellent effects (Hebbalkar et al., 1992), as well as antiulcerogenic (Sahni et al., 2001), ant parasitic (Parveen, 1991), antimicrobial (Rusia and Srivastava, 1998) and hepatoprotective potential (De et al., 1993). In the present study, we reported the synthesis of TiO2 NPs using V. negundo leaf extract would be useful in promoting research aiming at the development of new agents for mosquito larvicidal and pediculicidal activity.

the range 500–4000 cm−1 at a resolution of 4 cm−1. These measurements were carried out on a Perkin-Elmer Spectrum-One instrument in the diffuse reflectance mode at a resolution of 4 cm−1 in KBr pellets, and the pellets were mixed with KBr powder and pelletized after drying properly. The pellets were later subjected to FTIR spectroscopy measurement. For the electron microscopic studies, 25 μL of the sample was sputter-coated on copper stub and the images of nanoparticles studied using SEM-EDX (JEOL, model JFC-1600). Parasites rearing and collection A. subpictus and C. quinquefasciatus larvae were collected from the rice field and stagnant water area of Melvisharam (12°56′23″N, 79°14′ 23″E) and identified in Zonal Entomological Research Centre, Vellore (12°55′48″N, 79°7′48″E), Tamil Nadu, to start the colony, and larvae were kept in plastic and enamel trays containing tap water. They were maintained and reared in the laboratory as per the method of Kamaraj et al. (2009).

Materials and methods

Larvicidal bioassay

Materials

The larvicidal activity was assessed by the procedure of WHO (1996). Larvicidal activity of the synthesized TiO2 NPs was performed by placing 20 mosquito larvae into 200 mL of sterilized double distilled water with nanoparticles into a 250-mL beaker (Borosil). The nanoparticle solutions were diluted using double-distilled water according to the desired concentrations (50, 25, 12.5, 6.25 and 3.125 mg/L). Each test included the control group (distilled water) with five replicates for each individual concentration. Mortality was assessed after 24 h to determine the acute toxicities on fourth instar larvae of A. subpictus and C. quinquefasciatus. Larvicidal activity of V. negundo leaf aqueous extract and TiCl4 solution were performed by placing 20 mosquito larvae into 200 mL of sterilized double distilled water with test solution into a 250-mL beaker (Borosil). The test solutions were diluted using double-distilled water according to the desired concentrations (100, 50, 25, 12.5, 6.25 and 3.125 mg/L). The numbers of dead larvae were counted after 24 h of exposure, and the percentage of mortality was reported from the average of five replicates. The experimental media in which 100% mortality of larvae occurs alone were selected for dose-response bioassay. Twenty mosquito larvae were placed into 250-mL glass beakers (Borosil) and set in an environmental chamber at 25 °C with a 16:8-h light/dark cycle. Each beaker containing the mosquito larvae in distilled water was spiked with stock solutions of TiO2 NPs in order to achieve target nominal concentrations of 50, 25, 12.5, 6.25 and 3.125 mg/L with a final volume of 200 mL. In order to compare the mortality of synthesized TiO2 NPs to that of dissolved Ti released, the mosquito larvae were exposed to a range of dissolved Ti concentrations so as to cover the range released from all doses of TiO2 NPs. To avoid settling of particles, especially at higher doses, all treatment solutions were sonicated for an additional 5 min prior to the addition of the mosquito larvae. Since this additional sonication appeared to significantly decrease the settling of particles, the experiment was conducted with synthesized TiO2 NPs without sonication (stirred only).

The healthy leaves of Vitex negundo Linn. (Verbenaceae) were collected from Thandrampet Village, Thiruvannamalai district, Tamil Nadu, (12°9′15″N, 78°56′48″E) India and the taxonomic identification was made by Dr. C. Hema, Department of Botany, Arignar Anna Govt. Arts College for Women, Walajapet, Vellore, India. The voucher specimen was numbered and kept in our research laboratory for further reference. Titanium tetrachloride (TiCl4) (Analytical grade) was purchased from Sigma-Aldrich, India (99.9%) purity. Preparation of V. negundo leaf extract and synthesis of TiO2 NPs The fresh leaves of V. negundo were washed thoroughly in tap water for 10 min in order to remove the dust particles and rinsed briefly in deionized water. The plant leaf broth solution was prepared by taking 10 g of washed and finely cut leaves in a 250-mL Erlenmeyer flask along with 100 mL of deionized water and then boiling the mixture at 60 °C for 5 min. After boiling, the solution was filtered through Whatman no. 1 filter paper. The 20 mL of this broth was added to 80 mL of 5 mM aqueous TiCl4 solution, and the resulting solution became pink in colour. This extract was filtered through nylon mesh (Spectrum) followed by a Millipore hydrophilic filter (0.22 μm) and used for further experiments (Parashar et al., 2009). A control setup was also maintained without the V. negundo extract, and the colour intensity of the extracts was measured at 360 nm for different intervals. Characterization of TiO2 NPs The bioreduction of TiO2 NPs was monitored by sampling the reaction mixture at regular intervals, and the absorption maxima was scanned by UV–vis spectra, at the wavelength of 300–600 nm in Schimadzu 1601 spectrophotometer operated at a resolution of 1 nm. The solution mixture was subjected to centrifugation at 10,000 rpm for 45 min; resulting pellet was dissolved in deionized water and filtered through 0.22-μm Millipore filter. An aliquot of this filtrate containing TiO2 NPs was used for X-ray diffraction (XRD), Fourier transform infrared (FTIR), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). XRD measurements of the V. negundo leaf broth-reduced TiO2 NPs were carried out on films of the respective solutions drop coated onto glass substrates on a Phillips PW 1830 instrument operating at a voltage of 40 kV and a current of 30 mA with CuKα1 radiation. Characterization involved FTIR analysis of the dried powder of TiO2 NPs, by scanning it in

Dose-response bioassay Based on the preliminary screening results, leaf aqueous extract of V. negundo and synthesized TiO2 NPs were subjected to dose-response bioassay for larvicidal activity against the larvae of A. subpictus and C. quinquefasciatus. Different concentrations ranging from 3.125 to 100 mg/L (for aqueous plant extract) and 3.125 to 50 mg/L (for synthesized TiO2 NPs) were prepared for larvicidal activity. The numbers of dead larvae were counted after 24 h of exposure, and the percentage of mortality was reported from the average of five replicates.

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Adults of P. humanus capitis were collected from a population of children between the ages of 3 and 12, with the approval of their guardians, by raking a metal louse comb through sections of the scalp. Adult were obtained and pooled by carefully removing them from the metal teeth of the comb into clean plastic boxes. Once collected, head lice were transported to our laboratory (Picollo et al., 1998, 2000). The children had not been treated with any pediculicide solution for at least the preceding month, using only the louse comb. The head lice were identified by Dr. A. Sangaran, Department of Veterinary Parasitology, Madras Veterinary College, Chennai, Tamil Nadu. Pediculicidal activity The synthesized TiO2 NPs solutions, V. negundo leaf aqueous extract and TiCl4 solution were diluted using double distilled water as a solvent according to the desired concentrations of 100, 50, 25, 12.5, 6.25 and 3.125 mg/L. Each test included a set control group (distilled water) with five replicates for each individual concentration. Each louse was carefully transferred into a glass dish and 0.02 mL of the test solutions were applied directly on the dorsal part of the louse using a 1-mL micropipette. After 15 s of contact with the agent, the louse was transferred into a Petri dish lined with filter paper and observed using a hand lens until dead or otherwise. All the Petri dishes were set aside in a dark chamber at 26 ± 0.5 °C and 70 ± 1% humidity. The elapsed time was recorded for each test agent as the “knockdown” time. The death of the louse was confirmed when there was cessation of motility or waggling of the appendages on touching with a needle. Ten lice were used for each determination (Oladimeji et al., 2000). Data analysis Mean percent larval mortality data were subjected to analysis of variance and compared with Duncan's multiple range tests to determine any differences between plant species and within species and concentration (SPSS, 2007). Prior to analysis, mortality in treatments was corrected for that in controls using the formula of Abbott (1925). LC50 and their associated confidence intervals were estimated from 24-h concentration mortality data using probit analysis (Finney, 1971). Results with p b 0.05 were considered to be statistically significant. Toxicity of TiO2 NPs to non-target fish Poecilia reticulata (Guppy fish) To determine the toxicity of the synthesized TiO2 NPs solutions, a non-target organism P. reticulata was selected. Healthy P. reticulata (Guppy; 2.85 ± 0.82 g) were brought to the aquarium fish farming in Vellore. P. reticulata were acclimatized to the laboratory environment for about 5 days. They were fed with commercial food pellets, and only healthy P. reticulata were used in the experiments. Assessment of toxicity was carried out as per report (Patil et al., 2012). Twenty P. reticulata were placed in a rectangular, glass aquarium containing 1 L water solution in three replicates. Each group of 20 fish was exposed to test solutions at a concentration of 100 mg/L. A control consisting of 20 fish in de-chlorinated tap water, was studied at the same time. The numbers of dead fish were recorded first at 24 h, and the percentage mortalities were recorded. All of these bioassay tests were conducted at a room temperature of approximately 25–28 °C, without aeration or renewal of water.

to dark pink during 25-min incubation period after which there was no significant change occurred. Absorption spectrum of the solution (V. negundo leaf extracts with TiCl4 solution) at different wavelengths ranging from 300 to 600 nm revealed a peak at 360 nm. The formation of TiO2 NPs synthesized using V. negundo leaf extract was analysed by XRD measurements. The Bragg reflections at 2θ values 28.02°, 35.48°, 40.23°, 53.79°, 66.31° and 73.47° can be indexed to the (110), (101), (111), (211), (221), and (311) orientations, respectively, confirming the presence of nano-crystalline nature of TiO2 NPs. There are no peaks in the XRD pattern due to crystallographic impurities which means that the TiO2 NPs obtained had a high purity (Fig. 2). The FTIR spectrum of the synthesized TiO2 NPs shows the band intensities in different regions (3377, 1622, 1412, 1360, 1273, 1041, 774 and 541 cm−1). The spectra showed sharp and strong absorption band at 3377, 1622, 1360 cm−1 and the peaks corresponding to the presence of alcohols, ether, carboxylic acid, esters and may have participated in the process of nanoparticle synthesis (Fig. 3). SEM analyses of the synthesized TiO2 NPs clearly showed spherical shapes, mostly aggregated and having an average size of 93.33 nm with inter-particle distance, which magnified in 10,000 × and 15,000 × (Fig. 4). Energy dispersive X-ray (EDX) spectroscopy confirmed the purity of TiO2 NPs and the percentage of chemical composition of the synthesized TiO2 NPs (Fig. 5). In the present study, the larvicidal activity of the synthesized TiO2 NPs, V. negundo leaf aqueous extract and TiCl4 solution were tested against the fourth instar larvae of A. subpictus and C. quinquefasciatus. After 24 h of exposure, the highest mortality was observed in the synthesized TiO2 NPs against the fourth instar larvae of A. subpictus and C. quinquefasciatus (LC50 = 7.52 and 7.23 mg/L; χ2 = 0.161 and 2.678; r2 = 0.663 and 0.742). After 24 h of exposure, the larvicidal activity of V. negundo leaf aqueous extract showed the good activity against the fourth instar larvae of A. subpictus and C. quinquefasciatus (LC50 = 29.32 and 33.65 mg/L; χ2 = 14.446 and 13.201; r2 = 0.970 and 0.967). The larvicidal activity of the TiCl4 solution was observed against A. subpictus and C. quinquefasciatus (LC50 = 56.59 and 67.89 mg/L; χ2 = 11.472 and 14.588; r2 = 0.996 and 0.998) on 24 h exposure. The chisquare and r2 value was significant at p b 0.05 level. Percent mortality and LC50 were presented in graphical representations (Fig. 6). The pediculicidal activity of the synthesized TiO2 NPs, V. negundo leaf aqueous extract and TiCl4 solution were tested against P. humanus capitis on 24 h exposure. The highest pediculicidal activity was observed in synthesized TiO2 NPs against P. humanus capitis with the LC50 value of 24.32 mg/L (χ2 = 4.495; r2 = 0.924). The V. negundo leaf aqueous extract showed the pediculicidal activity against P. humanus capitis with the LC50 value of 44.20 mg/L (χ2 = 16.172; r2 = 0.983), and the TiCl4 solution showed the pediculicidal activity against P. humanus capitis with the LC50 value of 69.99 mg/L (χ2 = 19.113; r2 = 0.995). All head lice of 1.1 5min 10min 15min 20min 25min

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The colour change was noted by visual observation in the V. negundo leaf extracts when incubated with TiCl4 solution. V. negundo leaf extract without TiCl4 solution did not show any change in colour (Fig. 1). The colour of the extract changed to pink within 5 min, and later, it changed

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Wavelength (nm) Fig. 1. UV–vis absorption spectra of the synthesized TiO2 NPs using V. negundo leaf extract.

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the negative control group survived during the observation period. The chi-square and r2 value was significant at p b 0.05 level. Percent mortality and LC50 were presented in graphical representations (Fig. 6). The toxicity of experimental TiO2 NPs to the larvivorous fish P. reticulata was tested in the laboratory. The TiO2 NPs did not exhibit any noticeable effects on P. reticulata after 24 h of exposure at a concentration of 100 mg/L. Discussion To determine the crystal phase composition of the TiO2 NPs, XRD measurements were carried out over the diffraction angle (2θ) 10– 80°. The main peak of 2θ = 27.42° matches the (110) crystallographic plane of rutile form of TiO2 nanoparticles, indicating that the nanoparticles structure produced is only rutile form and not of anatase or brookite form (Roopan et al., 2012). Similarly, Jayaseelan et al. (2013) reported the XRD pattern of the synthesized TiO2 NPs revealed intense peaks at 27.47°, 31.77°, 36.11°, 41.25°, 54.39°, 56.64° and 69.53° corresponding to 110, 100, 101, 111, 211, 220 and 301 Bragg's reflection, respectively. Which is found to be that of the rutile form as when compared with the JCPDS data (File No. 99-101-0954). When comparing to earlier reports, our results are very similar. The formation of TiO2 NPs synthesized using Nyctanthes arbor-tristis leaves extract was supported by XRD peaks at 25.3°, 37.8°, 47.9, 54.5°, 62.8°, 69.5°, and 75.1° which indexed the planes

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Fig. 4. SEM micrograph of the synthesized TiO2 NPs using V. negundo leaf extract.

101, 004, 200, 105, 204, 116, and 215, respectively, of the cubic face-centered titanium dioxide (Sundrarajan and Gowri, 2011). The FTIR spectrum, firmly suggests the presence of Ti\\O bonds, and the absence of peroxo, and\\OH groups in the final product. A broad intense band at 3430 cm−1 in the spectra was assigned to the N\\H stretching frequency arising from the peptide linkages present in the alkaloids of plant extract using TiO2 (Kasturi et al., 2009; Narayan and Sakthivel, 2008; Shankar et al., 2004). FTIR spectral analysis showed an array of absorbance bands in 400 cm−1–1500 cm−1. Organic functional groups are available in the air dried silver nanoparticles. The spectral bands were prominent for Vitex negundo at 2358 cm−1 (Si\\H silane), 1683 cm−1 (C_O), 1510 cm−1 (nitro compounds), 1458 (C_C, aromatic ring), 1238 cm−1 (C\\O), 1066 cm−1 (alcohols, ether, carboxylic acid, esters), 887 cm−1 (alkenes). This organic group presence is due to silver particle reduction through biological sources (Kathireswari et al., 2014). Therefore, it may be assumed that water-soluble compounds such as flavonoids, terpenoids, and thiamine were the capping ligands of the nanoparticles (Philip, 2010). In the present study, the interaction of nanoparticles with biomolecules of V. negundo showed intense peaks at 3377, 1622, 1412, 1360, 1273, 1041, 774 and 541 cm−1 relative shift in position and intensity distribution were confirmed with FTIR. In the present study, the SEM analysis was carried out to understand the morphology of TiO2 NPs. The SEM images were clearly indicated that the spherical shape of the synthesized TiO2 NPs. SEM micrographs of the synthesized TiO2 NPs from the flower aqueous extract of Calotropis gigantea showed the aggregated, spherical in shape, and with an average of size of 160–220 nm (Marimuthu et al., 2013). In the present study, the larvicidal and pediculicidal activity of the synthesized TiO2 NPs, V. negundo leaf aqueous extract and TiCl4 solution

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Fig. 5. Energy-dispersive X-ray spectroscopy exhibiting the chemical components of the synthesized TiO2 NPs.

were tested against the fourth instar larvae of A. subpictus, C. quinquefasciatus and Pediculus humanus capitis. However, the maximum activity was observed in the synthesized TiO2 NPs using V. negundo leaf aqueous extract compared to the earlier reports. Earlier authors reported that the larvicidal aqueous crude leaf extracts and synthesized silver nanoparticles of Mimosa pudica showed the highest mortality in synthesized Ag NPs against the larvae of A. subpictus and C. quinquefasciatus (LC50 13.90 and 11.73 mg/L; r2 0.411 and 0.286), respectively (Marimuthu et al., 2011). The crude methanol and aqueous leaf of Nelumbo nucifera extracts and synthesized Ag NPs showed the maximum efficacy against the larvae of A. subpictus (LC50 = 8.89, 11.82, and 0.69 ppm; LC90 = 28.65, 36.06, and 2.15 ppm) and against the larvae of C. quinquefasciatus (LC50 9.51, 13.65, and 1.10 ppm; LC90 28.13, 35.83, and 3.59 ppm), respectively (Santhoshkumar et al., 2011). The larvicidal activity of synthesized Ag NPs utilizing an aqueous extract from Eclipta prostrata was observed in crude aqueous and synthesized Ag NPs against C. quinquefasciatus (LC50 27.49 and 4.56 mg/L; LC90 70.38 and 13.14 mg/L) and against A.

subpictus (LC50 27.85 and 5.14 mg/L; LC90 71.45 and 25.68 mg/L), respectively (Rajakumar and Rahuman, 2011). The pediculicidal and the larvicidal activity of the synthesized zinc oxide nanoparticles were observed against Pediculus humanus capitis, and the larvae of A. subpictus and C. quinquefasciatus showed LC50 values of 11.80, 11.14, and 12.39 mg/L, respectively (Kirthi et al., 2011). Similarly, the maximum activity was observed in the aqueous leaf extract of Solanum trilobatum, TiO(OH)2 solutions (bulk) and synthesized TiO2 NPs against P. humanus capitis (LC50 = 35.14, 25.85, and 4.34 mg/L) and against A. subpictus (LC50 = 28.80, 24.01, and 1.94 mg/L) (Rajakumar et al., 2014). In our observation, the synthesized TiO2 NPs using V. negundo leaf aqueous extract showed the promising larvicidal and pediculicidal activity against A. subpictus, C. quinquefasciatus and P. humanus capitis and the TiO2 NPs did not exhibit any toxic effects on P. reticulata after 24 h of exposure. The nontoxicity of synthesized TiO2 NPs towards the non-target fish P. reticulate suggests that the nanoparticles could be used along with this predatory fish in integrated vector control. However, previous studies demonstrated that silver nanoparticles induce embryonic injuries and reduce survival in zebrafish Danio rerio (Griffitt et al., 2008). Conclusion In the present study the larvicidal and pediculocidal activity of the synthesized TiO2 NPs, V. negundo leaf aqueous extract and TiCl4 solution were tested. The V. negundo can be used as an effective, reducing agent for the synthesis of TiO2 NPs. This biological reduction of metal oxide would be a boon for the development of clean, nontoxic, and environmentally acceptable metal oxide nanoparticles. The synthesized TiO2 NPs are hydrophilic in nature, disperse uniformly in water, highly stable, and had significant mosquito larvicidal and pediculocidal activity against A. subpictus, C. quinquefasciatus and P. humanus capitis. Acknowledgement

Fig. 6. Graph showing the LC50 values of the larvicidal and pediculicidal activity of the synthesized TiO2 NPs, V. negundo leaf aqueous extract and TiCl4 solution against the fourth instar larvae of A. subpictus, C. quinquefasciatus and Pediculus humanus capitis.

The authors are grateful to the management of Auxilium College, Principal, and HOD of Zoology for their help and support to carry out this work. We acknowledge the support extended by SAIF (STIC), Cochin for their help towards characterization analysis in my study.

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