One-Pot Fabrication of Silver Nanocrystals Using Nicandra Physalodes:A Novel Route for Mosquito Vector Control with Moderate Toxicity on Non-Target Water Bugs

Research in Veterinary Science 107 (2016) 95–101

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Research in Veterinary Science

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One-pot fabrication of silver nanocrystals using Nicandra physalodes:A novel route for mosquito vector control with moderate toxicity on non-target water bugs

Marimuthu Govindarajan a,⁎, Hanem F. Khater b,ChellasamyPanneerselvamc,GiovanniBenellid,⁎ a Unit of Vector Control, Phytochemistry and Nanotechnology, Department of Zoology, Annamalai University, Annamalainagar, 608 002, Tamil Nadu, India b Department of Parasitology, Faculty of Veterinary Medicine, Benha University, Moshtohor, Toukh, 13736, Egypt c Department of Biology, Faculty of Science, University of Tabuk, Tabuk 71491, Saudi Arabia d Insect Behaviour Group, Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80, 56124 Pisa, Italy article info abstract

Article history: Mosquitoes (Diptera: Culicidae) as vectors for important diseases and parasites causing millions of deaths every Received 12 December 2015 year. The use of synthetic pesticides against Culicidae leads to resistance and environmental concerns. Therefore, Received in revised form 12 May 2016 eco-friendly control tools are a priority. In this research, Nicandra physalodes-mediated synthesis of silver nano- Accepted 30 May 2016 particles (Ag NPs) was conducted, in order to control larval populations of three important mosquito vectors, Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. Biofabricated Ag NPs were characterized using Keywords: UV–vis spectrophotometry, XRD, FTIR spectroscopy, SEM, and TEM analyses. Ag NPs were highly toxic against Biosafety ﬁ μ Dengue the three mosquito vectors. Maximum ef cacy was detected against A. stephensi (LC50 = 12.39 g/mL), followed μ μ Filariasis by Ae. aegypti (LC50 = 13.61 g/mL) and Cx. quinquefasciatus (LC50 = 14.79 g/mL). Interestingly, Ag NPs were Malaria safer for the non-target aquatic organism Diplonychus indicus sharing the same aquatic habitats of mosquito lar-

Green-synthesis vae. LC50 and LC90 values were 1032.81 and 19,076.59 μg/mL, respectively. Overall, our results highlight that N. Zika virus physalodes-fabricated Ag NPs are a promising for development of eco-friendly larvicides against mosquito vectors, with negligible toxicity against non-target aquatic water bugs. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction al., 2016c). In particular, silver nanoparticles can be used for purification of drinking water, degradation of pesticides, to kill human pathogenic Mosquitoes (Diptera: Culicidae) are responsible for transmission of bacteria, as well as novel mosquitocides and antiplasmodial drugs some serious and dreadful pathogens and parasites in tropical and sub- (Kathiresan et al., 2009; Benelli and Mehlhorn, 2016). tropical countries worldwide, including malaria, avian malaria, yellow Recently, a growing number of plants have been successfully used fever, Rift valley fever, dengue, West Nile virus, Zika virus, Japanese en- for efficient and rapid extracellular synthesis of silver, copper and gold cephalitis, Western equine encephalomyelitis, bancroftian and brugian nanoparticles (Govindarajan, 2016). Good examples include cheap ex- filariae, canine heartworm disease (Dirofilaria immitis) and setariosis tracts of neem, Azadirachta indica (Shankar et al., 2004; Murugan et (Setaria spp.) (Benelli and Mehlhorn, 2016; Benelli et al., 2016; al., 2016), Chomelia asiatica (Muthukumaran et al., 2015a), Sida acuta Govindarajan and Benelli, 2016a; Govindarajan et al., 2016a). The use (Veerekumar et al., 2013), Gmelina asiatica (Muthukumaran et al., of synthetic insecticides to control mosquito vectors lead to resistance, 2015b), Barleria cristata (Govindarajan and Benelli, 2016b), Bauhinia adverse environmental effects and high operational costs. Therefore, variegata (Govindarajan et al., 2016d)andClerodendrum chinense eco-friendly control tools are urgently needed (Benelli, 2015a, 2015b; (Govindarajan et al., 2016e). Nanoparticles possess peculiar toxicity Govindarajan et al., 2016b). In recent years, the green processes for mechanisms due to surface modification (Oberdorster et al., 2005), the synthesis of silver nanoparticles evolved into an important branch and this may actively contribute to their excellent mosquitocidal poten- of nanotechnology, due to low cost, simple procedures and absence of tial against Culicidae larvae (Saxena et al., 2010; Benelli, 2016; toxic chemicals or high energy inputs (Benelli, 2016; Govindarajan et Govindarajan et al., 2016f ). However, despite the increasing number of evidences of plant-synthesized mosquitocidal nanoparticles, only moderate efforts have been carried out to shed light on the nanoparticle biotoxicity on non-target organisms sharing the same ecological niche ⁎ Corresponding authors. E-mail addresses: [email protected] (M. Govindarajan), [email protected], of mosquito young instars (see Benelli, 2016; Veerakumar and [email protected] (G. Benelli). Govindarajan, 2014).

Fig. 1. (a) Nicandra physalodes (L.) Gaertn. (Solanaceae). (b) Color intensity of Nicandra physalodes aqueous extract before and after the reduction of silver nitrate (1 mM). The color change indicates Ag+ reduction to elemental nanosilver. (c) UV-visible spectrum of silver nanoparticles after 120 min from the reaction.

Nicandra physalodes (L.) Gaertn. (Solanaceae) is commonly known 2.2. Preparation of plant leaf extract as “shooflyplant”, while the tamil name is “sudakku thakkali”. N. physalodes is an erect herb, with light blue or light purple flowers (Fig. The leaves of N. physalodes were dried in the shade and ground to 1a). In Tibetan medicine, this plant is used for the treatment of diuresis, fine powder in an electric grinder. Aqueous extract was prepared by mydriasis, analgesia, antibacterial and inflammation (Chopra et al., mixing 50 g of dried leaf powder with 500 mL of water (boiled and 1986). Several compounds (i.e. nicaphysalins) have been isolated from cooled distilled water) with constant stirring on a magnetic stirrer. the plant (Kirson et al., 1972), and it has also been confirmed that this The suspension of dried leaf powder in water was left for 3 h and filtered species has insecticidal properties (Olga et al., 1964). However, its through Whatman n. 1 filter paper and the filtrate was stored in an mosquitocidal potential is currently unknown. amber-colored airtight bottle at 10 °C temperature until testing In this research, we reported a method to fabricate silver nanoparti- (Govindarajan et al., 2016b). cles (Ag NPs) using the aqueous leaf extract of the N. physalodes, a cheap and eco-friendly material acting as reducing and stabilizing agent. Ag NPs were characterized by UV–vis spectrophotometry, X-ray diffraction 2.3. Synthesis of silver nanoparticles (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The aqueous extract of fresh leaves was prepared by taking 10 g of The aqueous extract of N. physalodes and the green-synthesized Ag thoroughly washed and finely cut leaves in a 300-mL Erlenmeyer flask NPs were tested for their larvicidal potential against the malaria vector along with 100 mL of sterilized double-distilled water and then boiling Anopheles stephensi, the dengue vector Aedes aegypti and the filariasis the mixture for 5 min before finally decanting it. The extract was filtered vector Culex quinquefasciatus. Furthermore, we evaluated the biotoxicity with Whatman filter paper n. 1, stored at −15 °C and tested within a of N. physalodes aqueous extract and green-synthesized Ag NPs on the week. The filtrate was treated with aqueous 1 mM AgNO3 (21.2 mg of non-target aquatic water bug Diplonychus indicus, which shares the AgNO3 in 125 mL of Milli-Q water) solution in an Erlenmeyer flask same ecological niche of Anopheles, Aedes and Culex mosquito in South and incubated at room temperature. Eighty-eight milliliters of an aque- India. ous solution of 1 mM silver nitrate was reduced using 12 mL of leaf extract at room temperature for 10 min, resulting in a brown–yellow solution indicating the formation of Ag NPs (Govindarajan et al., 2016c). 2. Materials and methods

2.1. Materials 2.4. Characterization of silver nanoparticles

Silver nitrate was purchased from Merck, India. The glassware was The bioreduction of Ag+ ions was monitored using UV–vis spectro- acid-washed thoroughly and then rinsed with Millipore Milli-Q water. photometry (UV-160v, Shimadzu, Japan). Analysis of size, morphology Healthy and fresh leaves of N. physalodes (Fig. 1) were collected from and composition of Ag NPs were performed by scanning electron mi- Nilgiris, Western Ghats (11° 10′ Nto11°45′ N latitude and 76° 14’Eto croscopy (Hitachi S3000 H SEM), and transmission electron microscopy 77° 2′ E longitude), Tamil Nadu State, India. The identity was confirmed (TEM Technite 10 Philips). The purified Ag NPs were examined for the at the Department of Botany, Annamalai University, Annamalai Nagar, presence of biomolecules using FTIR spectrum (Thermo ScientificNico- Tamil Nadu. Voucher specimens were numbered and kept in our labora- let 380 FT-IR Spectrometer) KBr pellets and crystalline Ag NPs were de- tory and are available upon request (ID: AUDZ-400). termined by XRD analysis (Govindarajan et al., 2016c). M. Govindarajan et al. / Research in Veterinary Science 107 (2016) 95–101 97

Fig. 4. Scanning electron microscopy (SEM-60, 000X) of silver nanoparticles synthesized using the Nicandra physalodes leaf extract.

Fig. 2. XRD pattern of silver nanoparticles synthesized using the Nicandra physalodes aqueous extract. aegypti feeding was done from 12 noon to 4.00 p.m. and A. stephensi and C. quinquefasciatus were fed during 6.00 p.m. to 10.00 p.m. A mem- 2.5. Mosquito rearing brane feeder with the bottom end fitted with Parafilm was placed with 2.0 mL of the blood sample (obtained from a slaughter house by Following the method by Govindarajan and Sivakumar (2015),labo- collecting in a heparinized vial and stored at 4 °C) and kept over a netted ratory-bred pathogen-free strains of mosquitoes were reared in the vec- cage of mosquitoes. The blood was stirred continuously using an auto- tor control laboratory, Department of Zoology, Annamalai University. At mated stirring device, and a constant temperature of 37 °C were main- the time of adult feeding, these mosquitoes were 3–4daysoldafter tained using a water jacket circulating system. After feeding, the fully emergences (maintained on raisins and water) and were starved for engorged females were separated and maintained on raisins. Mosqui- 12 h before feeding. Each time, 500 mosquitoes per cage were fed on toes were held at 28 ± 2 °C, 70–85% relative humidity, with a photo pe- blood using a feeding unit fitted with Parafilm as membrane for 4 h. A. riod of 12-h light and 12-h dark.

Fig. 3. FTIR spectrum of silver nanoparticles synthesized using the Nicandra physalodes aqueous leaf extract. 98 M. Govindarajan et al. / Research in Veterinary Science 107 (2016) 95–101

extract and Ag NPs of the potential plant was tested against non-target organism D. indicus. The species were ﬁeld collected and separately maintained in cement tanks (85 cm diameter and 30 cm depth) con- taining water at 27 ± 3 °C and relative humidity 85%. The aqueous extract and Ag NPs of N. physalodes were evaluated at a

concentration of 50 times higher the LC50 dose for mosquito larvae. Ten replicates will be performed for each concentration along with four replicates of untreated controls. The non-target organism was observed for mortality and other abnormalities such as sluggishness and reduced swimming activity after 48 h exposure. The exposed non-target organism was also observed continuously for ten days to understand the post treatment effect of this extract on survival and swimming activity.

2.8. Data analysis

Mortality data were subjected to probit analysis. LC50 and LC90 were calculated using the method by Finney (1971).Inexperimentsevaluat- Fig. 5. Transmission electron microscopy of silver nanoparticles synthesized using the ing biotoxicity on non-target organism, the Suitability Index (SI) was Nicandra physalodes aqueous extract. calculated for each non-target species using the following formula (Deo et al., 1988). 2.6. Acute toxicity against mosquito larvae LC of non‐target organisms SI ¼ 50 : Larvicidal activity of the aqueous extract and Ag NPs from N. LC50of target vector species physalodes was evaluated according to WHO (2005). The aqueous crude extract was tested at 100, 200, 300, 400 and 500 μg/mL while All data were analyzed using the SPSS Statistical Software Package Ag NPs was tested at 6, 12, 18, 24 and 30 μg/mL. Twenty numbers of version 16.0. A probability level of P b 0.05 was used for the significance late III instar larvae were introduced into a 500-mL glass beaker contain- of differences between values. ing 250 mL of dechlorinated water, plus the desired concentration of the leaf extract or Ag NPs. For each concentration, five replicates were per- 3. Results and discussion formed. Larval mortality was recorded at 24 h after exposure, during which no food was given to the larvae. Each test included a set control 3.1. Characterization of silver nanoparticles groups (silver nitrate and distilled water) with five replicates for each individual concentration. The synthesis of Ag NPs was confirmed within 2 h after that the N.

physalodes extract was added to AgNO3 solution. The color of the leaf extract changed from colorless to yellowish brown (Fig. 1b), and this can 2.7. Biotoxicity on non-target organisms be due to excitation of surface plasmon vibrations in green-synthesized Ag NPs (Shankar et al., 2004; Muthukumaran et al., 2015a). The forma- The effect of non-target organism was assessed following the meth- tion of Ag NP was conﬁrmed through the presence of an absorption od by Sivagnaname and Kalyanasundaram (2004). The effect of aqueous peak at 449 nm (Fig. 1c). Our UV–vis results are in agreement with

Table 1 Larvicidal activity of Nicandra physalodes aqueous leaf extract against the mosquito vectors Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.

a 2 Mosquito species Concentration (μg/mL) Mortality (%)±SD LC50 (μg/mL) LC90 (μg/mL) Slope Regression equation χ (d.f.) (LCL-UCL) (LCL-UCL)

A. stephensi 100 29.3 ± 1.2 202.82 404.85 3.47 y = 12.35 + 0.181x 3.411 (4) 200 47.6 ± 0.8 (178.79–223.91) (375.19–443.88) n.s. 300 68.4 ± 1.2 400 89.3 ± 0.4 500 99.1 ± 0.6 A. aegypti 100 25.2 ± 0.8 221.92 429.66 2.93 y = 7.29 + 0.187x 1.737 (4) 200 43.5 ± 0.8 (198.61–242.91) (398.68–470.39) n.s. 300 64.7 ± 0.6 400 86.4 ± 0.4 500 97.3 ± 0.6 C. quinquefasciatus 100 20.6 ± 0.6 242.41 449.33 2.45 y = 1.66 + 0.194x 1.126 (4) 200 39.2 ± 0.8 (220.30–262.91) (417.81–490.68) n.s. 300 61.5 ± 0.8 400 82.4 ± 1.2 500 96.2 ± 0.6

No mortality was observed in the control. SD = standard deviation.

LC50 = lethal concentration that kills 50% of the exposed organisms.

LC90 = lethal concentration that kills 90% of the exposed organisms. UCL = 95% upper confidence limit. LCL = 95% lower confidence limit. χ2 = chi square. d.f. = degrees of freedom. n.s. = not significant (α =0.05). a Values are mean ± SD of five replicates. M. Govindarajan et al. / Research in Veterinary Science 107 (2016) 95–101 99

Table 2 Larvicidal activity of silver nanoparticles synthesized using the Nicandra physalodes leaf extract against the mosquito vectors Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.

a 2 Mosquito species Concentration (μg/mL) Mortality (%)±SD LC50 (μg/mL) LC90 (μg/mL) Slope Regression equation χ (d.f.) (LCL-UCL) (LCL-UCL)

A. stephensi 6 27.5 ± 0.6 12.39 24.24 3.09 y = 10.74 + 3.087x 2.773 (4) 12 46.8 ± 0.4 (10.99–13.62) (22.49–26.52) n.s. 18 69.4 ± 0.6 24 88.6 ± 0.6 30 99.2 ± 0.4 A. aegypti 6 23.4 ± 0.6 13.61 25.68 2.63 y = 5.16 + 3.2x 2.288 (4) 12 42.6 ± 0.8 (12.26 + 14.83) (23.87–28.05) n.s. 18 64.5 ± 0.8 24 85.2 ± 0.4 30 98.1 ± 0.8 C. quinquefasciatus 6 19.8 ± 1.2 14.79 27.26 2.41 y = 0.77 + 3.248x 1.693 (4) 12 38.5 ± 0.8 (13.48–16.03) (25.34–29.77) n.s. 18 61.3 ± 0.6 24 80.2 ± 0.6 30 96.4 ± 0.4

No mortality was observed in the control. SD = standard deviation.

LC50 = lethal concentration that kills 50% of the exposed organisms.

LC90 = lethal concentration that kills 90% of the exposed organisms. UCL = 95% upper confidence limit. LCL = 95% lower confidence limit. χ2 = chi square. d.f. = degrees of freedom. n.s. = not significant (α =0.05). a Values are mean ± SD of five replicates. previous research (Singh et al., 2010; Zargar et al., 2011), where the Ag earlier where Ag NPs biofabricated using Trianthema decandra,showed NPs were observed as stable in solution and also showed little aggrega- interplanar distance corresponds to mixed phase of cubic and hexago- tion. Besides, the Plasmon bands were broadened with an absorption nal structures (Geethalakshmi and Sarada, 2010). tail in longer wavelengths; this could be related to the size distribution The FTIR spectrum of green-synthesized Ag NPs by using N. of Ag NPs (Ahmad et al., 2003). physalodes leaf extract is shown in Fig. 3. In order to identify the biomol- XRD patterns exhibited several size-dependent features leading to ecules responsible for reduction and efficient stabilization of the metal anomalous peak position height and width (Fig. 2). Bragg reflections nanoparticles, it was highlighted that the band at 3216 cm−1 may cor- corresponding to the (111), (200), (220), and (311) sets of lattice planes respond to O–H, as well as to H-bonded alcohols and phenols. were noted. The XRD pattern showed that the Ag NPs formed by the re- Shanmugam et al. (2014) suggested that these bonds could be due to duction of AgNO3 by N. physalodes leaves extract were crystalline in na- the stretching of –OH in proteins, enzymes or polysaccharides present ture. Results showed that the Ag+ of silver nitrate had reduced to Ag0 by in the plant extract. The peak at 2920 cm−1 may be linked with the N. physalodes. Sharp Bragg peaks may be due to the capping agent stabi- presence of carboxylic acids (Li et al., 2007). Shoulder peaks at lizing the nanoparticles. Our findings are in agreement with previous re- 1577 cm−1 probably indicate amide I and amide II, due to carbonyl search conducted on Ag NPs fabricated using the leaf extract of Sida and –NH stretch vibrations in the amide linkages of the proteins. The acuta (Veerekumar et al., 2013). Similar phenomena were reported band at 1380 cm−1 may correspond to C–C stretching of aromatic

Table 3 Biotoxicity of Nicandra physalodes aqueous leaf extract and green-synthesized silver nanoparticles against the non-target water bug Diplonychus indicus, which shares the same ecological niche of Anopheles, Aedes and Culex mosquito vectors.

a 2 Treatment Concentration (μg/mL) Mortality (%)±SD LC50 (μg/mL) LC90 (μg/mL) Slope Regression equation χ (d.f.) (LCL-UCL) (LCL-UCL)

Aqueous leaf extract 9000 26.7 ± 1.2 19,076.59 36,921.45 2.92 y = 21.492 + 0.002 x 5.921 (4) 18,000 47.2 ± 0.8 (17,014.79–20,918.34) (34,271.15–40,399.22) n.s. 27,000 65.4 ± 0.8 36,000 87.2 ± 0.6 45,000 100.0 ± 0.0 Silver nanoparticles 500 28.5 ± 0.8 1032.81 2020.73 3.09 y = 10.84 + 0.037x 5.570 (4) n.s. 1000 46.9 ± 0.8 (916.83–1135.66) (1874.65–2212.49) 1500 67.2 ± 0.6 2000 88.5 ± 0.6 2500 100.0 ± 0.0

No mortality was observed in the control. SD = standard deviation.

LC50 = lethal concentration that kills 50% of the exposed organisms.

LC90 = lethal concentration that kills 90% of the exposed organisms. UCL = 95% upper confidence limit. LCL = 95% lower confidence limit. χ2 = chi square. d.f. = degrees of freedom. n.s. = not significant (α =0.05). a Values are mean ± SD of five replicates. 100 M. Govindarajan et al. / Research in Veterinary Science 107 (2016) 95–101

Table 4 3.3. Biotoxicity on non-target organisms Suitability index/predator safety factor of the non-target organism Diplonychus indicus over young instars of Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus The biotoxicity of N. physalodes aqueous extract and green-synthe- exposed to Nicandra physalodes aqueous leaf extract and green-synthesized silver nanoparticles. sized Ag NPs on non-target organism D. indicus is presented in Table 3. Both toxicity treatments achieved negligible toxicity against D. indicus, Treatment An. stephensi Ae. aegypti C. quinquefasciatus with LC50 values ranged from 1032.81 to 19,076.59 μg/mL (Table 3). Aqueous leaf extract 94.05 85.96 78.69 Focal observations highlighted that longevity and swimming activity Silver nanoparticles 83.35 75.88 69.83 of the study species were not altered for a week after testing. SI indicat- ed that N. physalodes-fabricated Ag NPs were less toxic to the non-target organism tested if compared to the targeted mosquito larval populations (Table 4). Nowadays, moderate knowledge is available about the acute toxicity of mosquitocidal nanoparticles towards non-target aquat- amine. The band at 1114 and 1040 cm−1 probably indicates the pres- ic species (see Benelli, 2016 for a recent review). Notably, Pergularia ence of C–O stretching alcohols, carboxylic acids, esters and ethers. rubra-andPergularia daemia-synthesized Ag NPs did not exhibit any ev- The peak near 663 cm−1 may be assigned to CH out of plane bending vi- ident toxicity effect against Poecilia reticulata fishes, after 48 h of expo- brations of substituted ethylene systems –CH_CH (see also sure to LC50 and LC90 values calculated on IV instar larvae of A. aegypti Veerekumar et al., 2013). The immediate reduction of silver ions in and A. stephensi (Patil et al., 2012). Similarly, Haldar et al. (2013) did the present investigation might be linked with the presence of water- not detected toxicity of Ag NPs produced using dried green fruits of soluble phytochemicals such as flavones, quinones, and organic acids Drypetes. roxburghii against P. reticulata, after 48 h-exposure to LC50 of present in the leaves of N. physalodes. IV instar larvae of A. stephensi and C. quinquefasciatus. Rawani et al. SEM showed that Ag NPs were mostly spherical or with cubic struc- (2013) showed that mosquitocidal Ag NP synthesized using Solanum tures (Fig. 4). We also noted that “capped” Ag NPs were stable in solu- nigrum berry extracts were not toxic against two mosquito predators, tion for at least 8 weeks. As a general trend, the particle shape of Toxorhynchites larvae and Diplonychus annulatum,andChironomus plant-mediated Ag NPs was spherical, with exception of some neem- circumdatus larvae, exposed to lethal concentrations of dry nanoparti- synthesized Ag NPs, which are poly-dispersed, with spherical or flat, cles calculated on A. stephensi and C. quinquefasciatus larvae. Ag NPs fab- plate-like, morphology, and mean size range of 5–35 nm in size ricated using the 2,7.bis [2-[diethylamino]-ethoxy]fluorence isolate (Shankar et al., 2004; Murugan et al., 2016). SEM of Ag NPs fabricated from the Melia azedarach leaves did not show acute toxicity against using Emblica officinalis were also predominantly spherical with an av- Mesocyclops pehpeiensis copepods (Ramanibai and Velayutham, 2015). erage size of 16.8 nm ranging from 7.5 to 25 nm (Ankamwar et al., Interestingly, the exposure to extremely low doses (e.g. 1 ppm) of 2005). Fig. 5 showed the TEM of Ag NPs biosynthesized using N. green-synthesized Ag NPs did not negatively impact the predation effi- physalodes leaf extract. Most of the Ag NPs was roughly circular in ciency of a number of mosquito predators of relevance for mosquito shape with smooth edges. In agreement with our findings, Ag NPs control (Benelli, 2016). from Annona squamosa leaf extract were spherical in shape with an av- erage size ranging from 20 to 100 nm (Vivek et al., 2012) while Thirunavokkarasu et al. (2013) reported spherical nanoparticles with 4. Conclusions size ranging from 8 to 90 nm in Desmodium gangeticum. TEM images showed that the surfaces of the Ag NPs were surrounded by a black Overall, we fabricated silver nanoparticles using a cheap aqueous ex- thin layer, which might be due to the capping organic constituents tract of N. physalodes leaves as reducing and stabilizing agent. The from the plant extract, as also highlighted by Rafiuddin (2013). green-synthesized Ag NPs were mostly spherical in shape, crystalline in nature, with face-centered cubic geometry, and their mean size was 22–44 nm. This research highlighted that N. physalodes-synthesized 3.2. Larvicidal activity against mosquito vectors Ag NPs are easy to produce, stable over time, and can be employed at low dosages to strongly reduce populations of vectors mosquitoes with- In laboratory conditions, the N. physalodes aqueous leaf extract out detrimental effects on predation rates of non-target aquatic organ- showed moderate larvicidal properties against A. stephensi, A. aegypti ism D. indicus. and C. quinquefasciatus;LC50 values were 202.82, 221.92 and 242.41 μg/mL, respectively (Table 1). Recently, a great number of Conflicts of interest plant extracts have been found effective against mosquito larval instars, with a dose-dependent effect (e.g., Veerakumar et al., 2014; Benelli et The authors declare no conflicts of interest. al., 2015a, 2015b, 2015c; Muthukumaran et al., 2015b; Murugan et al., 2016). Furthermore, the N. physalodes-synthesized Ag NPs were highly toxic against A. stephensi, A. aegypti and C. quinquefasciatus larvae; LC50 Compliance with ethical standards values were 12.39, 13.61 and 14.79 μg/mL, respectively (Table 2). In lat- est years, several botanicals and plant-synthesized Ag NPs have been All applicable international and national guidelines for the care and studied for their larvicidal activity against important mosquito vectors use of animals were followed. All procedures performed in studies in- (Benelli, 2015a, 2015b, 2016). For instance, comparable toxicity rates volving animals were in accordance with the ethical standards of the in- have been recently reported for Ag NPs synthesized using Chomelia stitution or practice at which the studies were conducted. asiatica against A. stephensi larvae (LC50 =17.95ppm) (Muthukumaran et al., 2015a). The mortality effect evoked by Ag NPs on mosquito larvae and pupae may be due by the small size of the Ag Acknowledgements NPs, which allows their passage through the insect cuticle and into individual cells, where they interfere with molting and other physiological Two anonymous reviewers improved an earlier version of the man- processes. To our mind, the residual toxicity of silver ions against mos- uscript. The authors would like to thank Professor and Head, Depart- quito larvae covered a little role, since the peak in Fig. 1c was saturated ment of Zoology, Annamalai University for the laboratory facilities after 120 min, indicating complete reduction of silver nitrate provided. We also acknowledge the cooperation of staff members of (Veerakumar et al., 2014; Benelli, 2016). the VCRC (ICMR), Pondicherry. M. Govindarajan et al. / Research in Veterinary Science 107 (2016) 95–101 101

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