Raja Mohamed Sait Thameem Azarudeen 1 Marimuthu Govindarajan 2 Abubucker Amsath. Udaiyan Muthukumaran 2 Giovanni Benelli

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1 J Clust Sci (2017) 28: DOI /s y ORIGINAL PAPER Single-Step Biofabrication of Silver Nanocrystals Using Naregamia alata: A Cost Effective and Eco-Friendly Control Tool in the Fight Against Malaria, Zika Virus and St. Louis Encephalitis Mosquito Vectors Raja Mohamed Sait Thameem Azarudeen 1 Marimuthu Govindarajan 2 Abubucker Amsath 1 Udaiyan Muthukumaran 2 Giovanni Benelli 3 Received: 6 August 2016 / Published online: 25 August 2016 Springer Science+Business Media New York 2016 Abstract Botanical-based nanosynthesis has been recently reported as a cheap alternative for mosquito management. Screening different botanicals as reducing and capping agents led to the production of metal nanoparticles with different biophysical and mosquitocidal features. Here, Naregamia alata-mediated biosynthesis of silver nanoparticles (AgNPs) was conducted. AgNPs were tested on egg, larval and adult populations of three important mosquito vectors, Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. AgNPs were characterized using UV Vis spectrophotometry, FTIR, AFM, SEM, TEM, EDX, and XRD analyses. Maximum larvicidal toxicity was detected against A. stephensi (LC 50 = lg/ml), followed by A. aegypti (LC 50 = lg/ml) and C. quinquefasciatus (LC 50 = lg/ ml). A single treatment with AgNPs tested at 60, 75 and 90 lg/ml led to no egg hatchability. In adulticidal experiments, the maximum efficacy was observed on A. stephensi (LD 50 = lg/ml), followed by A. aegypti (LD 50 = lg/ml) and C. quinquefasciatus (LD 50 = lg/ml), respectively. AgNPs were safer for three non-target mosquito natural enemies, Anisops bouvieri, Diplonychus indicus and Gambusia affinis, with LC 50 ranging from 629 to 2111 lg/ml. Overall, N. alata- Journal of Cluster Science Invited Contribution for the Special Issue Applications of greensynthesized nanoparticles in pharmacology, parasitology and entomology. & Marimuthu Govindarajan drgovind1979@gmail.com & Giovanni Benelli benelli.giovanni@gmail.com Department of Zoology, Khadir Mohideen College, Adirampattinam, Tamil Nadu , India Unit of Vector Control, Phytochemistry and Nanotechnology, Department of Zoology, Annamalai University, Annamalainagar, Tamil Nadu , India Insect Behavior Group, Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80, Pisa, Italy

2 180 R. M. S. T. Azarudeen et al. fabricated AgNPs are a promising and eco-friendly tool against Anopheles, Aedes and Culex mosquito vectors, with negligible toxicity against non-target aquatic organisms. Keywords Green-synthesis AFM SEM TEM Neglected tropical diseases Nanotechnology Introduction Mosquitoes (Diptera: Culicidae) pose an important threat to public health since of their ability to act as vectors of pathogens and parasites causing malaria, lymphatic filariasis, dengue, chikungunya, yellow fever, Zika virus and Japanese encephalitis. These diseases are responsible for mortality, morbidity, economic loss and social disruption [1 3]. Anopheles stephensi is the primary vector of malaria in India and other west Asian countries. Every year, an estimated million new infections and 600,000 cases based on world malaria report 2013 [4] deaths result from malaria worldwide. Malaria is a protozoan infection of erythrocytes caused in human beings by five species of the genus Plasmodium (P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi). In most cases, malaria is transmitted via the bite of an infected female anopheline mosquito, but congenital malaria and acquisition through infected blood transfusion are well described [5]. More than 40 per cent of the world s population-approximately 3 billion people are exposed to malaria in 108 endemic countries [6]. About one million cases of malaria are reported in India every year. Aedes mosquito borne diseases have become one of the major threats to human population. Zika virus infection is a mosquito-borne illness like dengue (DEN) and chikungunya (CHIK) viruses. A lot about the biology of vectors and its role in disease transmission is known but Aedes mosquito-borne infections continue to be the major cause of mortality in many subtropical and tropical countries. The Aedesborne infections like Zika are a potential threat, especially in urban settings where A. aegypti is abundant. The growing population in urban settings also increased the need for potable water, which necessitated storage practices in households, making ideal breeding habitats for A. aegypti mosquitoes that also increase dengue and CHIKV infections. Due to this mosquito-borne viral diseases are going to be a major threat in the 21st century [7]. Culex quinquefasciatus is a vector of many pathogens and parasites of humans, and both domestic and wild animals. Viruses transmitted by this species include West Nile virus, St. Louis encephalitis virus and Western equine encephalitis virus. C. quinquefasciatus is the principal vector of St. Louis encephalitis virus in the southern U.S.A. The virus increases its numbers in birds and then infects mosquitoes feeding on birds during the bird-nesting season in the spring. The mosquito may then transmit the virus to humans. St. Louis encephalitis is agedependent, affecting older humans more than the young. Symptoms of the disease are flu-like and can range from fever and headaches to stiffness and confusion. After a period of several days the brain may begin to swell, accompanied by depression,

3 Single-Step Biofabrication of Silver Nanocrystals Using 181 extreme excitement, sleepiness, or sleeplessness. Humans do not develop high levels of the virus in the blood and therefore are considered dead-end hosts unable to infect mosquitoes [8]. The use of synthetic insecticides to control mosquito vectors lead to resistance, adverse environmental effects and high operational costs [1]. Therefore, ecofriendly control tools are urgently needed [9]. In recent years, the green processes for the synthesis of silver nanoparticles (AgNPs) are evolving into an important branch of nanotechnology, due to low cost, simple procedures and absence of toxic chemicals or energy inputs [9]. In particular, AgNPs can be used for purification of drinking water, degradation of pesticides, to kill human pathogenic bacteria, as well as novel mosquitocides [10]. Recently, a growing number of plants have been successfully used for efficient and rapid extracellular synthesis of silver, copper and gold nanoparticles [11]. Good examples include cheap extracts of neem, Azadirachta indica [12], Pongamia pinnata [13], Feronia elephantum [14], Heliotropium indicum [15], Chomelia asiatica [16], Sida acuta [17], Gmelina asiatica [18], and Barleria cristata [19]. Nanoparticles possess peculiar toxicity mechanisms due to surface modification [20], and this may actively contribute to their excellent mosquitocidal potential against Culicidae larvae [21, 22]. 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 of mosquito young instars [9]. Naregamia alata Weight & Arn. (Meliaceae) an undershurb with pungent, aromatic roots. Distributed throughout South India in all districts up to 900 m. The plant is acid, sweet, cooling, aromatic, alexeteric, vulnerary, emetic, cholagogue, expectorant, depurative and antipyretic. It is useful in the treatments of wounds, ulcers, vitiated conditions of pitta and vata, halitosis, cough, asthma, bronchitis, splenomegaly, scabies, pruritus, dysentery, dyspepsia, catarrh, anaemia and malarial fevers [23]. However, to the best of our knowledge, its mosquitocidal activity is currently unknown. In this research, we reported a route to biosynthesize silver nanoparticles (AgNPs) using the aqueous leaf extract of the N. alata, a cheap and eco-friendly material acting as reducing and stabilizing agent. AgNPs were characterized by UV Vis spectrophotometry, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDX) and transmission electron microscopy (TEM). The aqueous extract of N. alata and the biosynthesized AgNPs were tested for their ovicidal, larvicidal and adulticidal potential against the malaria vector Anopheles stephensi, the dengue and Zika virus vector Aedes aegypti and the filarasis and St. Louis encephalitis vector C. quinquefasciatus. Furthermore, we evaluated the biotoxicity of N. alata aqueous extract and green-synthesized AgNPs on three non-target aquatic natural enemies sharing the same ecological niche of Anopheles and Aedes mosquitoes, Anisops bouvieri, Diplonychus indicus, and Gambusia affinis.

4 182 R. M. S. T. Azarudeen et al. Materials and Methods Materials Silver nitrate was purchased from Merck, India. The glassware was acid-washed thoroughly and then rinsed with Millipore Milli-Q water. Healthy and fresh leaves of N. alata (Fig. 1) were collected from Nilgiris, Western Ghats ( Nto N latitude and Eto E longitude), Tamil Nadu State, India. The identity was confirmed at the Department of Botany, Annamalai University, Annamalai Nagar, Tamil Nadu. Voucher specimens were numbered and kept in our laboratory and are available upon request (ID: AUDZ-815). Preparation of Plant Leaf Extract The leaves of N. alata were dried in the shade and ground to fine powder in an electric grinder. Aqueous extract was prepared by mixing 50 g of dried leaf powder with 500 ml of water (boiled and cooled distilled water) with constant stirring on a magnetic stirrer. The suspension of dried leaf powder in water was left for 3 h and filtered through Whatman no. 1 filter paper and the filtrate was stored in an ambercolored airtight bottle at 10 C temperature until testing. Biosynthesis of Silver Nanoparticles The broth solution of fresh leaves was prepared by taking 10 g of thoroughly washed and finely cut leaves in a 300-ml Erlenmeyer flask along with 100 ml of Fig. 1 Naregamia alata Wight & Arn. (Meliaceae)

5 Single-Step Biofabrication of Silver Nanocrystals Using 183 sterilized double-distilled water and then boiling the mixture for 5 min before finally decanting it. The extract was filtered with Whatman filter paper no. 1, stored at -15 C and tested within a week. The filtrate was treated with aqueous 1 mm AgNO 3 (21.2 mg of AgNO 3 in 125 ml of Milli-Q water) solution in an Erlenmeyer flask and incubated at room temperature. Eighty-eight milliliters of an aqueous 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 AgNPs. Characterization of Silver Nanoparticles The bioreduction of Ag? ions was monitored using a UV Vis spectrophotometer (UV-160v, Shimadzu, Japan). Analysis on size, morphology, agglomeration pattern and dispersed nature of AgNPs were performed by atomic force microscopy (Agilent Technologies AFM- 5500), scanning electron microscopy (Hitachi S3000 H SEM) and transmission electron microscopy (TEM Technite 10 Philips). The purified AgNPs were examined for the presence of biomolecules using FTIR spectroscopy (Thermo Scientific Nicolet 380 FT-IR Spectrometer) KBr pellets and crystalline AgNPs were determined by XRD analysis. Mosquito Rearing Following the method by Govindarajan and Benelli [19, 24, 25], laboratory-bred pathogen-free strains of mosquitoes were reared in the vector control laboratory, Department of Zoology, Annamalai University. At the time of adult feeding, these mosquitoes were 3 4 days old after emergences (maintained on raisins and water) and were starved for 12 h before feeding. Each time, 500 mosquitoes per cage were fed on blood using a feeding unit fitted with Parafilm as membrane for 4 h. A. 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 p.m. A membrane feeder with the bottom end fitted with Parafilm was placed with 2.0 ml of the blood sample (obtained from a slaughter house by collecting in a heparinized vial and stored at 4 C) and kept over a netted cage of mosquitoes. The blood was stirred continuously using an automated stirring device, and a constant temperature of 37 C were maintained using a water jacket circulating system. After feeding, the fully engorged females were separated and maintained on raisins. Mosquitoes were held at 28 ± 2 C, % relative humidity, with a photoperiod of 12-h light and 12-h dark. Acute Toxicity Against Mosquito Larvae Larvicidal activity of the aqueous extract and AgNPs from N. alata was evaluated according to WHO protocol [26]. Based on the wide range and narrow range tests, aqueous crude extract was tested at 80, 160, 240, 320 and 400 lg ml -1

6 184 R. M. S. T. Azarudeen et al. concentrations and AgNPs were tested at 6, 12, 18, 24 and 30 lg ml -1 concentrations. Twenty numbers of late III instar larvae were introduced into a 500-ml glass beaker containing 249 ml of dechlorinated water, and 1 ml of desired concentrations of leaf extract or AgNPs was added. For each concentration, five replicates were performed, for a total of 100 larvae. Larval mortality was recorded at 24 h after exposure, during which no food was given to the larvae. Each test included a set control groups (silver nitrate and distilled water) with five replicates for each individual concentration [24]. Ovicidal Activity To evaluate the ovicidal potential of the leaf extract and AgNPs, the method of Su and Mulla [27] with slight modification by Govindarajan et al. [28] was followed. Eggs were collected from vector control laboratory, Department of Zoology, Annamalai University. The aqueous leaf extracts and silver nanoparticle were to achieve various concentrations ranging from 60 to 360 lg/ml and 15 to 90 lg/ml, respectively. Eggs of these mosquito species (n = 100 for 0 6, 6 12 and h old egg rafts) were exposed to each concentration of leaf aqueous extract and AgNPs. After treatment, the eggs from each concentration were individually transferred to distilled water cups for hatching assessment after counting the eggs under a photomicroscope (Leica, Germany). Each experiment was replicated six times along with appropriate control. The hatch rates were assessed 48 h posttreatment by the following formula. Egg hatchability ð% Þ ¼ Number of hatched larvae 100 Total number of eggs Adulticidal Activity Adulticidal bioassay was performed following the method by WHO [29] with minor modifications by Veerakumar and Govindarajan [30]. Based on the wide range and narrow range tests, aqueous crude extract was tested at 120, 240, 360, 480 and 600 lg ml -1 concentrations, and AgNPs were tested at 15, 30, 45, 60, 75, and 60 lg ml -1 concentrations. Aqueous crude extract and AgNPs were applied on Whatman no. 1 filter papers (size cm). Control papers were treated with silver nitrate and distilled water. Twenty female mosquitoes were collected and gently transferred into a plastic holding tube. The mosquitoes were allowed to acclimatize in the holding tube for 1 h and then exposed to test paper for 1 h. At the end of exposure period, the mosquitoes were transferred back to the holding tube and kept for 24-h recovery period. A pad of cotton soaked with 10 % glucose solution was placed on the mesh screen. Each test included a set control groups (silver nitrate and distilled water) with five replicates for each individual concentration.

7 Single-Step Biofabrication of Silver Nanocrystals Using 185 Biotoxicity on Non-target Mosquito Natural Enemies The effect of non-target organisms was assessed following the method by Sivagnaname and Kalyanasundaram [31]. The effect of aqueous extract and AgNPs of the potential plant was tested against non-target mosquito natural enemies A. bouvieri, D. indicus and G. affinis. The species were field collected and separately maintained in cement tanks (85 cm diameter and 30 cm depth) containing water at 27 ± 3 C and external relative humidity 85 %. The aqueous extract and Ag NP of N. alata were evaluated at concentrations of even 50 times higher the LC 50 dose for mosquito larvae. Ten replicates will be performed for each concentration along with four replicates of untreated controls. The non-target organisms were observed for mortality and other abnormalities such as sluggishness and reduced swimming activity after 48 h exposure. The exposed non-target organisms were also observed continuously for 10 days to understand the post treatment effect of this extract on survival and swimming activity. Data Analysis Larval and adult mortality data were subjected to probit analysis. LC 50 (LD 50 ) and LC 90 (LD 90 ) were calculated using the method by Finney [32]. Ovicidal data were analyzed by two-way ANOVA followed by Tukey s HSD test. In experiments evaluating biotoxicity on non-target organisms, the Suitability Index (SI) was calculated for each non-target species using the following formula [33]. SI ¼ LC 50 of non target organisms LC 50 of target vector species All data were analyzed using the SPSS Statistical Software Package version A probability level of P \ 0.05 was used for the significance of differences between values. Results and Discussion Characterization of Silver Nanoparticles The biosynthesis of AgNPs was confirmed within 2 h after that the N. alata extract was added to AgNO 3 solution. The color of the leaf extract changed from colorless to yellowish brown (Fig. 2a), and this can be due to excitation of surface plasmon vibrations in green-synthesized AgNPs [12]. The formation of AgNPs was confirmed through the presence of an absorption peak at 473 nm (Fig. 2b). Our UV Vis results are in agreement with previous research [34, 35], where the AgNPs were observed as stable in solution and also showed little aggregation. Besides, the plasmon bands were broadened with an absorption tail in longer wavelengths; this could be related to the size distribution of nanoparticles [36]. The crystalline nature of AgNPs was studied by XRD analysis (Fig. 3). The obtained XRD patterns confirmed the crystalline nature of green synthesized

8 186 R. M. S. T. Azarudeen et al. Fig. 2 a Color intensity of Naregamia alata aqueous extract before and after the reduction of silver nitrate (1 mm). The color change indicates Ag? reduction to elemental nanosilver. b UV Vis spectrum of silver nanoparticles after 180 min from the reaction (Color figure online) AgNPs. Four diffraction peaks were observed at 38.6, 45.3, 64.5 and 77.2 represent the (111), (200), (220) and (311), reflections and the face-centered cubic structure of metallic silver, respectively. Our findings are in agreement with previous research conducted on AgNPs green-synthesized using the leaf extract of S. acuta [17]. Similar phenomena were reported earlier where AgNPs biofabricated using Trianthema decandra, showed interplanar distance corresponds to mixed phase of cubic and hexagonal structures [37]. The FTIR spectrum of biosynthesized AgNPs by using N. alata leaf extract is shown in Fig. 4. In order to identify the biomolecules responsible for reduction and efficient stabilization of the metal nanoparticles, it was highlighted that the band at 3148 cm -1 may correspond to O H, as well as to H-bonded alcohols and phenols.

9 Single-Step Biofabrication of Silver Nanocrystals Using 187 Fig. 3 XRD pattern of silver nanoparticles fabricated using the Naregamia alata aqueous extract Shanmugam et al. [38] suggested that these bonds could be due to the stretching of -OH in proteins, enzymes or polysaccharides present in the plant extract. The peak at 2201 cm -1 may be lined with the presence of carboxylic acids [39]. Shoulder peaks at 1554 cm -1 probably indicate that the amide I and amide II arisen, due to carbonyl and NH stretch vibrations in the amide linkages of the proteins, respectively. The band at 1467 and1383 cm -1 may correspond to C C stretching of aromatic amine. The band at 1111 and 1013 cm -1 probably indicate the presence of C O stretching alcohols, carboxylic acids, esters and ethers. The peak near 663 cm -1 may be assigned to CH out of plane bending vibrations of substituted ethylene systems CH=CH. Our findings are in agreement with Ag NP synthesized using the leaf extract of Sida acuta [17]. The immediate reduction of silver ions in the present investigation might be linked with the presence of watersoluble phytochemicals such as flavones, quinones, and organic acids present in the leaves of N. alata. AFM is a primary tool for analyzing size, shape, agglomeration pattern and offers visualizations of three-dimensional views of the nanoparticles unlike the electron microscopes. It has an advantage over combination of high resolution, samples does not have to be conductive and does not require the high-pressure vacuum conditions [40]. 2.5 lm resolution studies of green-synthesized AgNPs with AFM reveal the particles are poly-dispersed, spherical in shape, having the size range from 0 to 5.5 nm and there is no agglomeration observed between the particles (Fig. 5a). Raw data obtained from AFM microscope were treated with a specially designed image processing software (NOVA-TX) to further exploit the 3D image of nanoparticles (Fig. 5b). The average particle size obtained from the corresponding diameter distribution was about 5 nm (Fig. 5c, d).

10 188 R. M. S. T. Azarudeen et al. Fig. 4 FTIR spectrum of silver nanoparticles fabricated using the Naregamia alata aqueous leaf extract

11 Single-Step Biofabrication of Silver Nanocrystals Using 189 SEM of AgNPs showed that AgNPs were mostly spherical or with cubic structures (Fig. 6). We also noted that capped AgNPs were stable in solution for at least 8 weeks, and Fig. 7 showed a representative profile of the spot EDX analysis. As a general trend, the particle shape of plant-mediated AgNPs was spherical, with exception of some neem-synthesized AgNPs. They are polydispersed, with spherical or flat, plate-like, morphology, and mean size range of 5 35 nm in size [12]. SEM images of AgNPs fabricated using Emblica officinalis were also predominantly spherical with an average size of 16.8 nm ranging from 7.5 to 25 nm [41]. Figure 8 showed the TEM of AgNPs biosynthesized using N. alata leaf extract. Among shapes, spheres dominated with an average size of 35 nm, which was slightly higher if compared to AFM observations. Most of the AgNPs was roughly circular in shape with smooth edges. In agreement with these findings, AgNPs from Annona squamosa leaf extract were spherical in shape with an average size ranging from 20 to 100 nm [42] while Thirunavokkarasu et al. [43] reported the fabrication of spherical nanoparticles with size ranging from 8 to 90 nm in Desmodium gangeticum. TEM images showed that the surfaces of the AgNPs were surrounded by a black thin layer of some material, which might be due to the capping organic constituents from the plant extract, as also highlighted by Rafiuddin [44]. Larvicidal Activity Against Mosquito Vectors In laboratory conditions, the N. alata aqueous leaf extract showed moderate larvicidal properties against A. stephensi, A. aegypti and C. quinquefasciatus; LC 50 values were , and lg/ml, respectively (Table 1). Furthermore, the N. alata-synthesized AgNPs were highly toxic against A. stephensi, A. aegypti and C. quinquefasciatus larvae; LC 50 values were 12.40, and lg/ml, respectively (Table 2). N. alata aqueous leaf extract and AgNPs exerted 100 % mortality (zero hatchability) when tested at 240, 300, 360 and 60, 75, 90 lg/ml, respectively. Control eggs showed 100 % egg hatchability (Tables 3, 4). The results of the adulticidal activity of aqueous crude extract and synthesized AgNPs against the adult of A. stephensi, A. aegypti, and C. quinquefasciatus are presented in Tables 5 and 6. Considerable mortality was evident after the treatment of N. alata for all three important vector mosquitoes. At higher concentrations, the adult showed restless movement for some times with abnormal wagging and then died. The rates of mortality were directly proportional to concentration. The maximum efficacy was observed in green synthesized AgNPs against the adult of A. stephensi (LD 50 = lg/ml), followed by A. aegypti (LD 50 = lg/ml), and C. quinquefasciatus (LD 50 = lg/ml) respectively. In agreement with Marimuthu et al. [45] and Govindarajan and Benelli [24], no mortality was observed in the positive and negative controls. Recently, a great number of plant extracts have been found effective against mosquito larval instars, with a dose-dependent effect [17, 18, 22]. In latest years, several plant-synthesized AgNPs have been studied for their larvicidal activity against important mosquito vectors [22]. For instance, comparable toxicity rates have been recently reported for AgNPs synthesized using Chomelia asiatica against

12 190 R. M. S. T. Azarudeen et al. Fig. 5 AFM micrograph of synthesized silver nanoparticles biofabricated using the Naregamia alata extract a 2.5 lm resolution studies nm size, spherical shaped, polydispersed particles, b 3D image of AgNPs analyzed by NOVA-TX software, c histogram showing the particle size distribution, d line graph showing the size distribution of green-synthesized silver nanoparticles

13 Single-Step Biofabrication of Silver Nanocrystals Using 191 Fig. 6 Scanning electron microscopy (SEM) of Naregamia alata-fabricated silver nanoparticles Fig. 7 Energy dispersive X-ray (EDX) spectrum of Naregamia alata-synthesized silver nanoparticles showing presence of different phyto-elements as capping agents A. stephensi larvae (LC 50 = ppm) [16]. The mortality effect evoked by AgNPs on mosquito larvae and pupae may be due by the small size of the AgNPs, which allows their passage through the insect cuticle and into individual cells,

14 192 R. M. S. T. Azarudeen et al. Fig. 8 Trasmission electron microscopy (TEM) of silver nanoparticles fabricated using the Naregamia alata aqueous leaf extract where they interfere with molting and other physiological processes. To our mind, the residual toxicity of silver ions against mosquito larvae covered a little role, since the peak in Fig. 1c was saturated after 120 min, indicating complete reduction of silver nitrate [17]. Biotoxicity on Non-target Organisms The biotoxicity of N. alata aqueous extract and green-synthesized AgNPs on nontarget mosquito predators A. bouvieri, D. indicus and G. affinis is presented in Tables 7 and 8, respectively. Both toxicity treatments achieved negligible toxicity against A. bouvieri, D. indicus and G. affinis, with LC 50 values ranging from 629 to 29,633 lg/ml (Tables 7, 8). Focal observations highlighted that longevity and swimming activity of the study species were not altered for a week after testing. SI indicated that N. alata-fabricated AgNPs were less toxic to the non-target organism tested if compared to the targeted mosquito larval populations (Table 9). Nowadays, moderate knowledge is available about the acute toxicity of mosquitocidal nanoparticles towards non-target aquatic species [22, 46 48]. Notably, Pergularia rubra- and Pergularia daemia-synthesized AgNPs did not exhibit any evident toxicity effect against Poecilia reticulata fishes, after 48 h of exposure to LC 50 and LC 90 values calculated on IV instar larvae of A. aegypti and A. stephensi [49]. Similarly, Haldar et al. [50] did not detected toxicity of AgNPs produced using dried green fruits of D. roxburghii against P. reticulata, after 48 h-

15 Single-Step Biofabrication of Silver Nanocrystals Using 193 Table 1 Larvicidal activity of Naregamia alata aqueous leaf extract against the malaria vector Anopheles stephensi, the Zika virus and dengue vector Aedes aegypti, and the filariasis and St. Louis encephalitis vector Culex quinquefasciatus Mosquito species Concentration (lg/ml) Mortality LC 50 (lg/ml) (LCL UCL) LC 90 (lg/ml) (LCL UCL) Slope Regression equation v 2 (d.f.) (%) ± SD a An. stephensi ± ( ) ( ) 3.01 y = 10.58? 0.233x (4) ± ± ± ± 0.8 Ae. aegypti ± ( ) ( ) 2.75 y = 6.35? 0.237x (4) ± ± ± ± 0.6 Cx. quinquefasciatus ± ( ) ( ) 2.64 y = 2.77? 0.235x (4) ± ± ± ± 0.4 No mortality was observed in the control SD standard deviation, LC 50 lethal concentration that kills 50 % of the exposed organisms, LC 90 lethal concentration that kills 90 % of the exposed organisms, UCL 95 % upper confidence limit, LCL 95 % lower confidence limit, v 2 Chi square, d.f. degrees of freedom, not significant (a = 0.05) Values are mean ± SD of five replicates a

16 194 R. M. S. T. Azarudeen et al. Table 2 Larvicidal activity of biogenic silver nanoparticles fabricated using the Naregamia alata leaf extract against the malaria vector Anopheles stephensi, the Zika virus and dengue vector Aedes aegypti, and the filariasis and St. Louis encephalitis vector Culex quinquefasciatus Mosquito species Concentration (lg/ml) Mortality LC 50 (lg/ml) (LCL UCL) LC 90 (lg/ml) (LCL UCL) Slope Regression equation v 2 (d.f.) (%) ± SD a An. stephensi ± ( ) ( ) 3.04 y = 10.66? 3.087x (4) ± ± ± ± 0.0 Ae. aegypti ± ( ) ( ) 2.67 y = 5.52? 3.183x (4) ± ± ± ± 0.4 Cx. quinquefasciatus ± ( ) ( ) 2.45 y = 1.64? 3.22x (4) ± ± ± ± 0.6 No mortality was observed in the control SD standard deviation, LC 50 lethal concentration that kills 50 % of the exposed organisms, LC 90 lethal concentration that kills 90 % of the exposed organisms, UCL 95 % upper confidence limit, LCL 95 % lower confidence limit, v 2 Chi square, d.f. degrees of freedom, not significant (a = 0.05) Values are mean ± SD of five replicates a

17 Single-Step Biofabrication of Silver Nanocrystals Using 195 Table 3 Ovicidal activity of Naregamia alata aqueous leaf extract against the malaria vector Anopheles stephensi, the Zika virus and dengue vector Aedes aegypti, and the filariasis and St. Louis encephalitis vector Culex quinquefasciatus Mosquito species Age of the egg raft/eggs (h) Egg hatchability (%) F value (d.f.) Control 60 lg/ml 120 lg/ml 180 lg/ml 240 lg/ml 300 lg/ml 360 lg/ml P value An. stephensi ± ± ± 0.7 NH NH NH NH (5) \ ± ± ± 1.2 NH NH NH NH (5) \ ± ± ± ± 0.3 NH NH NH (5) \0.001 Ae. aegypti ± ± ± ± 0.4 NH NH NH (5) \ ± ± ± ± 1.7 NH NH NH (5) \ ± ± ± ± ± 1.9 NH NH (5) \0.001 Cx. quinquefasciatus ± ± ± ± ± 1.3 NH NH (5) \ ± ± ± ± ± 0.6 NH NH (5) \ ± ± ± ± ± ± 0.7 NH (5) \0.001 Within each row, different letters indicate significant differences among values (ANOVA, Tukey s HSD, P \ 0.001) NH no hatchability, d.f. degrees of freedom

18 196 R. M. S. T. Azarudeen et al. Table 4 Ovicidal activity of silver nanoparticles synthesized using the Naregamia alata leaf extract against the malaria vector Anopheles stephensi, the Zika virus and dengue vector Aedes aegypti, and the filariasis and St. Louis encephalitis vector Culex quinquefasciatus Mosquito species Age of the egg raft/eggs (h) Egg hatchability (%) F value (d.f.) Control 15 lg/ml 30 lg/ml 45 lg/ml 60 lg/ml 75 lg/ml 90 lg/ml P value An. stephensi ± ± ± 1.9 NH NH NH NH (5) \ ± ± ± 1.6 NH NH NH NH (5) \ ± ± ± ± 1.5 NH NH NH (5) \0.001 Ae. aegypti ± ± ± ± 1.8 NH NH NH (5) \ ± ± ± ± 1.2 NH NH NH (5) \ ± ± ± ± ± 0.6 NH NH (5) \0.001 Cx. quinquefasciatus ± ± ± ± ± 1.8 NH NH (5) \ ± ± ± ± ± 1.2 NH NH (5) \ ± ± ± ± ± ± 1.4 NH (5) \0.001 Within each row, different letters indicate significant differences among values (ANOVA, Tukey s HSD, P \ 0.001) NH no hatchability, d.f. degrees of freedom

19 Single-Step Biofabrication of Silver Nanocrystals Using 197 Table 5 Adulticidal activity of Naregamia alata aqueous leaf extract against the malaria vector Anopheles stephensi, the Zika virus and dengue vector Aedes aegypti, and the filariasis and St. Louis encephalitis vector Culex quinquefasciatus Mosquito species Concentration (lg/ml) Mortality LD 50 (lg/ml) (LCL UCL) LD 90 (lg/ml) (LCL UCL) Slope Regression equation v 2 (d.f.) (%) ± SD a An. stephensi ± ( ) ( ) 2.88 y = 10.24? 0.156x (4) ± ± ± ± 0.0 Ae. aegypti ± ( ) ( ) 2.56 y = 4.93? 0.161x (4) ± ± ± ± 0.8 Cx. quinquefasciatus ± ( ) ( ) 2.35 y = 0.19? 0.164x (4) ± ± ± ± 1.2 No mortality was observed in the control SD standard deviation, LC 50 lethal concentration that kills 50 % of the exposed organisms, LC 90 lethal concentration that kills 90 % of the exposed organisms, UCL 95 % upper confidence limit, LCL 95 % lower confidence limit, v 2 Chi square, d.f. degrees of freedom, not significant (a = 0.05) Values are mean ± SD of five replicates a

20 198 R. M. S. T. Azarudeen et al. Table 6 Adulticidal activity of silver nanoparticles synthesized using Naregamia alata leaf extract against the malaria vector Anopheles stephensi, the Zika virus and dengue vector Aedes aegypti, and the filariasis and St. Louis encephalitis vector Culex quinquefasciatus Mosquito species Concentration (lg/ml) Mortality LD 50 (lg/ml) (LCL UCL) LD 90 (lg/ml) (LCL UCL) Slope Regression equation v 2 (d.f.) (%) ± SD a An. stephensi ± ( ) ( ) 2.88 y = 9.41? 1.249x (4) ± ± ± ± 0.0 Ae. aegypti ± ( ) ( ) y = 6? 1.248x (4) ± ± ± ± 0.8 Cx. quinquefasciatus ± ( ) ( ) 2.52 y = 1.47? 1.266x (4) ± ± ± ± 0.6 No mortality was observed in the control SD standard deviation, LC 50 lethal concentration that kills 50 % of the exposed organisms, LC 90 lethal concentration that kills 90 % of the exposed organisms, UCL 95 % upper confidence limit, LCL 95 % lower confidence limit, v 2 Chi square, d.f. degrees of freedom, not significant (a = 0.05) Values are mean ± SD of five replicates a

21 Single-Step Biofabrication of Silver Nanocrystals Using 199 Table 7 Acute toxicity of Naregamia alata aqueous leaf extract on three natural enemies of Anopheles, Aedes and Culex mosquito vectors Non-target organism Concentration (lg/ml) Mortality LC50 (lg/ml) (LCL UCL) LC90 (lg/ml) (LCL UCL) Slope Regression equation v 2 (d.f.) (%) ± SD a A. bouvieri ± , ( ,441.93) 20, (18, ,304.61) 3.09 y = 10.52? 0.004x 5.593(4) 10, ± , ± , ± , ± 0.0 D. indicus ± , (13, ,068.81) 28, (26, ,019.99) 2.93 y = 9.89? 0.003x 6.010(4) 14, ± , ± , ± , ± 0.0 G. affinis 14, ± , (26, ,504.24) 57, (53, ,879.81) 2.95 y = 9.43? 0.001x 5.991(4) 28, ± , ± , ± , ± 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, v 2 Chi square, d.f. degrees of freedom, not significant (a = 0.05) Values are mean ± SD of five replicates a

22 200 R. M. S. T. Azarudeen et al. Table 8 Acute toxicity of silver nanoparticles green-synthesized using the Naregamia alata leaf extract on three natural enemies of Anopheles, Aedes and Culex mosquito vectors Non-target organism Concentration (lg/ml) Mortality LC 50 (lg/ml) (LCL UCL) LC 90 (lg/ml) (LCL UCL) Slope Regression equation v 2 (d.f.) (%) ± SD a A.bouvieri ± ( ) ± 1.2 ( ) ± ± ± 0.0 D.indicus ± ( ) ± 0.6 ( ) ± ± ± 0.0 G.affinis ± ± 0.8 ( ) ± ± ± ( ) 2.89 y = 9.6? 0.062x 5.722(4) 2.69 y = 8.36? 0.038x 5.692(4) 2.88 y = 9.35? 0.019x 5.376(4) No mortality was observed in the control SD standard deviation, LC 50 lethal concentration that kills 50 % of the exposed organisms, LC 90 lethal concentration that kills 90 % of the exposed organisms, UCL 95 % upper confidence limit, LCL 95 % lower confidence limit, v 2 Chi square, d.f. degrees of freedom, not significant (a = 0.05) Values are mean ± SD of five replicates a

23 Single-Step Biofabrication of Silver Nanocrystals Using 201 Table 9 Suitability index of different non-target organism over young instars of Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus exposed to Naregamia alata aqueous leaf extract and greensynthesized silver nanoparticles Treatment Non-target organism An. stephensi Ae. aegypti Cx. quinquefasciatus A. bouvieri Naregamia alata extract D. indicus G. affinis A. bouvieri Silver nanoparticles D. indicus G. affinis exposure to LC 50 of IV instar larvae of A. stephensi and C. quinquefasciatus. Rawani et al. [51] showed that mosquitocidal AgNPs synthesized using Solanum nigrum berry extracts were not toxic against two mosquito predators, Toxorhynchites larvae and Diplonychus annulatum, and Chironomus circumdatus larvae, exposed to lethal concentrations of dry nanoparticles calculated on A. stephensi and C. quinquefasciatus larvae. Furthermore, Govindarajan et al. [40] reported that the biotoxicity of M. sylvestris aqueous extract and green-synthesized AgNPs was negligible also on non-target organisms D. indicus and G. affinis, since the toxicity treatments achieved LC 50 values ranging from to 10, lg/ml. Also, the biotoxicity of B. cristata aqueous extract and green-synthesized AgNPs was evaluated on non-target organisms D. indicus, A. bouvieri, and G. affinis with LC 50 values ranging from to lg/ml, respectively [19]. Lastly, Govindarajan et al. [52] investigated the biotoxicity of C. spinarum aqueous extract and green-synthesized AgNPs on non-target organisms D. indicus, A. bouvieri and G. affinis. Toxicity treatments achieved negligible toxicity with LC 50 values ranging from to lg/ml. Concerning sub-lethal effects, G. affinis exposed to nanoparticle-contaminated aquatic environments showed higher predation rates against both An. stephensi and Ae. albopictus larvae. Interestingly, the exposure to extremely low doses (e.g. 1 ppm) of green-synthesized AgNPs did not negatively impact the predation efficiency of a number of mosquito predators of relevance for mosquito control [9]. Conclusions Overall, here we synthesized mosquitocidal AgNPs using a cheap aqueous extract of N. alata leaves as reducing and stabilizing agent. The bio-reduced AgNPs were mostly spherical in shape and crystalline in nature, with their mean size ranging from 5 to 35 nm. This research highlighted that N. alata-synthesized AgNPs are easy to produce, stable over time, and can be employed at low dosages to strongly reduce populations of vectors mosquitoes without detrimental effects on non-target aquatic organisms, such as A. bouvieri, D. indicus, and G. affinis.

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