One-pot green synthesis of silver nanocrystals using Hymenodictyon orixense: a cheap and effective tool against malaria, chikungunya and Japanese encephalitis mosquito vectors?

Abstract

Mosquitoes are important vectors of malaria, dengue, Zika virus and many other parasites and pathogens of public health relevance. Recently, the green nanosynthesis of mosquitocides relying on plant compounds as reducing and stabilizing agents has received growing interest, due to the absence of toxic chemicals and high-energy input. In this research, Hymenodictyon orixense-mediated synthesis of silver nanoparticles (AgNPs) was conducted to control larval populations of the malaria vector Anopheles subpictus, the chikungunya vector Aedes albopictus and the Japanese encephalitis vector Culex tritaeniorhynchus. AgNPs were characterized using UV-visible spectrophotometry, FTIR spectroscopy, EDX and XRD analyses, AFM, SEM and TEM. AgNPs were toxic towards all the mosquito vectors, LC₅₀ values ranged from 17.10 μg ml⁻¹ to 20.08 μg ml⁻¹. Notably, AgNPs were safer to the non-target mosquito predator Diplonychus indicus (LC₅₀ = 833 μg ml⁻¹). Overall, H. orixense-fabricated AgNPs can be considered for the development of novel and safer control tools against mosquito vectors of medical and veterinary importance.

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Introduction

Mosquitoes (Diptera: Culicidae) are responsible for the transmission of important and dreadful pathogens and parasites worldwide, including malaria, dengue, yellow fever, filariasis and Zika virus.¹ Mosquito-borne diseases are endemic in over 100 countries, causing mortality of nearly two million people every year, and at least one million children die of such diseases each year, leaving as many as 2100 million people at risk around the world.^2–4 Chemical insecticides are widely used to control mosquitoes. However, they are often harmful to non-target organisms and human health.^5,6 Therefore, effective and eco-friendly control strategies are urgently needed. The development of plant-borne pesticides with multiple mechanisms of action may be successful for mosquito control.⁷ Current emphasis on bioactivity of plant materials highlighted excellent toxic properties against different developmental instars of a number of Culicidae vectors.⁸

Furthermore, with the progress of nanotechnology, the production of metal nanoparticles for mosquitocidal purposes has been studied.⁹ In recent years, the nanosynthetic methods based on the employment of plant extracts received more attention over chemical, physical and microbial-based ones, due to the absence toxic chemicals and high-energy inputs. Recently, a growing number of plants have been successfully used for efficient and rapid extracellular synthesis of silver, copper, and gold nanoparticles.¹⁰ Good examples include cheap extracts of neem, Azadirachta indica,¹¹ Chomelia asiatica,¹² Sida acuta,¹³ Gmelina asiatica,¹⁴ and Pongamia pinnata.¹⁵ Interestingly, nanoparticles possess peculiar toxicity mechanisms due to surface modification.¹⁶ Silver nanoparticles also have antibacterial, antifungal, antiplasmodial and mosquitocidal properties.¹⁷ 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 toxicity on non-target organisms sharing the same ecological niche of mosquito young instars.⁹

Hymenodictyon orixense (Roxb.) Mabb. (Rubiaceae) is native to tropical Asia and widely distributed in India. It is a tree commonly known as “Vellai kadambu” in Tamil Nadu region. Leaves are decussate, elliptic-ovate, and acute-attenuate at base. Flowers in terminal and axillary paniculate raceme, faintly scented, greenish. Capsules ellipsoid, on recurved pedicels. The stem bark contains tannin, toxic alkaloid, hymenodictine, a bitter substance, aesculin, an apioglucoside of scopoletin, hymexelsin.¹⁸ Anthraquinones, rubiadin and its methyl ether, lucidin, nordamnacanthal, damnacanthal, 2-benzylzanthopurpurin, anthragallol, soranjidol and morindone have been isolated from roots.¹⁹

In this research, we reported a method to synthesize silver nanoparticles (Ag NPs) using the aqueous leaf extract of the H. orixense, a cheap and eco-friendly material acting as reducing and stabilizing agent. Ag NPs were characterized by UV-visible spectrophotometry, Fourier transform infrared spectroscopy (FTIR), energy dispersive X-ray analysis (EDX), X-ray diffraction analysis (XRD), atomic force microscopy (AFM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The aqueous extract of H. orixense and the synthesized Ag NPs were tested for their larvicidal potential against the malaria vector Anopheles subpictus, the chikungunya vector Aedes albopictus and the Japanese encephalitis vector Culex tritaeniorhynchus. Furthermore, we evaluated the biotoxicity of H. orixense aqueous extract and green-synthesized Ag NPs on the non-target aquatic predator Diplonychus indicus, which shares the same ecological niche of Anopheles and Aedes mosquitoes in India.

Experimental

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 H. orixense were collected from Nilgiris, Western Ghats (11° 10′ N to 11° 45′ N latitude and 76° 14′ E to 77° 2′ 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 under request (ID: AUDZ-318).

Preparation of plant leaf extract

The leaves of H. orixense 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 amber-coloured airtight bottle at 10 °C temperature until testing.

Synthesis and bio-physical characterization 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 sterilized double-distilled water and then boiling the mixture at 1000 °C for 5 min before finally decanting it for 30 min. The extract was filtered with Whatman filter paper no. 1, stored at −15 °C, in order to prevent degradation processes, and tested within 7 days. The filtrate was treated with aqueous 1 mM AgNO₃ (21.2 mg of AgNO₃ in 125 ml of Milli-Q water) solution in an Erlenmeyer flask and incubated at room temperature (28 ± 2 °C, R.H. 70–85%). Eighty-eight millilitres 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 Ag NPs.

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 Ag NPs 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 Ag NPs were examined for the presence of biomolecules using FTIR spectroscopy (Thermo Scientific Nicolet 380 FT-IR Spectrometer) KBr pellets and crystalline Ag NPs were determined by XRD analysis.

Mosquito rearing

Following the method by Govindarajan and Benelli²⁰ 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. albopictus feeding was done from 12 noon to 4.00 p.m. and A. subpictus and C. tritaeniorhynchus were fed during 6.00 p.m. to 10.00 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 slaughterhouse 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, 70–85% 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 Ag NPs from H. orixense was evaluated according to WHO protocol.²¹ Based on the wide range and narrow range tests, aqueous crude extract was tested at 50, 100, 150, 200 and 250 μg ml⁻¹ concentrations and Ag NP was tested at 8, 16, 24, 32 and 40 μg 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 Ag NP was added. For each concentration, five replicates were performed. 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 (i.e. silver nitrate tested at the same concentrations of green-fabricated Ag NP, and distilled water) with five replicates for each individual concentration.

Biosafety on non-target mosquito predators

The effect of non-target organisms was assessed following the method by Sivagnaname and Kalyanasundaram.²² The effect of aqueous extract and Ag NPs of the potential plant was tested against non-target organisms D. indicus. The species were field collected and separately maintained in cement tanks (85 cm diameter and 30 cm depth) containing water at 27 ± 3 °C, air relative humidity was 85%.

The aqueous extract and Ag NPs of H. orixense were evaluated at a concentration of even 50 times higher the LC₅₀ dose for mosquito larvae. Ten replicates will be performed for each concentration along with four replicates of untreated controls. Non-target organisms were observed continuously for 10 days to understand the post-treatment effect of the extract and Ag NP on their survival.

Data analysis

Data were analysed using the SPSS Statistical Software Package version 16.0. For all tested species, toxicity data were subjected to probit analysis. LC₅₀ and LC₉₀ were calculated using the method by Finney.²³ For each LC₅₀ and LC₉₀ calculated in acute toxicity assays, the 95% confidence limits, regression equation, slope and chi square values were provided. In experiments evaluating the acute toxicity on non-target organism and the Suitability Index (SI) was calculated for non-target species using the following formula.²⁴

Results and discussion

Synthesis and characterization of silver nanoparticles

The colour of H. orixense aqueous extract changed from yellowish to dark brown after the reduction of silver nitrate (Fig. 1a). The colour change indicates Ag⁺ reduction to elemental nanosilver. Fig. 1b shows the UV-visible spectrum of biosynthesized silver nanoparticles after 180 min from the reaction. An intense broad absorption peak was observed at 449 nm, and it was probably due to surface plasmon resonance (SPR). The SPR peak is very sensitive to the size and shape of the nanoparticles, amount of extract, silver nitrate concentration and the type of biomolecules present in the leaf extract (Fig. 1b). Our UV-Vis results are in agreement with previous research,^10,25,26 where the Ag NPs were observed as stable in solution and also showed little aggregation. Besides, the plasmon bands were broadened with an absorption tail in longer wavelengths, and this may be related to the size distribution of nanoparticles.²⁷

Fig. 1 (a) Color intensity of Hymenodictyon orixense aqueous extract before and after the reduction of silver nitrate (1 mM). The color change indicates Ag⁺ reduction to elemental nanosilver. (b) UV-visible spectrum of silver nanoparticles after 180 min from the reaction.

The crystalline nature of Ag NPs was studied by XRD analysis (Fig. 2). The XRD pattern confirmed the crystalline nature of bio-fabricated Ag NP. Four diffraction peaks were observed at 38.22, 44.37, 64.54 and 77.47 represent the (111), (200), (220) and (311), reflections and the face-centered cubic structure of metallic silver, respectively.⁹ Notably, limited information is available on the phytoconstituents of the extract of H. orixense leaves. Probably, hymenodictine, aesculin, scopoletin and hymexelsin are major compounds, as already reported for the bark of this species.^18,28 The FTIR spectrum of synthesized silver nanoparticles by using H. orixense leaf extract is shown in Fig. 3. It is confirmed the fact that to identify the biomolecules for reduction and efficient stabilization of the metal nanoparticles. The band at 3420.95 cm⁻¹ may correspond to O–H, as also the H-bonded alcohols and phenols. Shanmugam et al.²⁹ suggested that these bonds could be due to the stretching of –OH in proteins, enzymes or polysaccharides present in the extract. The peak at 2922 cm⁻¹ may indicate the presence of carboxylic acids.³⁰ 2851 cm⁻¹ may be linked to the presence of methylene groups (CH₂) close to OH or NH₂ functional groups. Similarly, bands at 1653 cm⁻¹, 1636 cm⁻¹ and 1457 cm⁻¹ could be due to the C‚C stretching of aromatic rings, which may indicate the presence of benzene ring, a band at very low intensity at 1684 cm⁻¹ suggests the ortho-substitution pattern of benzene ring. In addition, shoulder peaks at 1559 cm⁻¹, 1541 cm⁻¹, and 1507 cm⁻¹ probably indicate that the amide I and amide II probably arise due to carbonyl and –NH stretch vibrations in the amide linkages of the proteins, respectively. The band at 1384 cm⁻¹ may correspond to C–C stretching of aromatic amines. The bands at 1111.07 cm⁻¹, and 1019.16 cm⁻¹ might indicate the presence of C–O stretching alcohols, carboxylic acids, esters and ethers, while 693.43 cm⁻¹ probably corresponds to C–H stretching strong vinyl di-substituted alkenes.³¹

Fig. 2 XRD pattern of silver nanoparticles synthesized using the Hymenodictyon orixense aqueous extract.

Fig. 3 FTIR spectrum of silver nanoparticles synthesized using the Hymenodictyon orixense aqueous leaf extract.

AFM is a primary tool for analysing 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. Our 2.5 μm resolution studies of biofabricated synthesized Ag NPs with AFM revealed the particles are poly-dispersed, spherical in shape, with size ranging from 0.3 to 3.5 nm. No agglomeration was observed between the particles (Fig. 4a). Raw data obtained from this AFM microscope were treated with a specially designed image processing software (NOVA-TX) to further exploit the 3D image of nanoparticles (Fig. 4b). The average nanoparticle size obtained from the corresponding diameter distribution was about 2 nm (Fig. 4c and d). SEM of Ag NPs showed that the nanoparticles were mostly spherical (Fig. 5a). Fig. 5b showed the TEM of Ag NP synthesized using H. orixense leaf extract, highlighting that some nanoparticles were of a size slightly bigger (i.e. 25–35 nm) to the mean size reported in AFM size analysis, however, still below 100 nm. We also noted that “capped” Ag NP were stable in solution for at least 8 weeks. In addition, EDX analysis provided information on the chemical analysis at specific locations of the sample (spot EDX). Fig. 6 shows spot EDX analysis, confirming the presence of silver in the sample.

Fig. 4 Atomic force microscopy (AFM) of silver nanoparticles green synthesized from Hymenodictyon orixense, (a) 2.5 μm resolution studies of 0.3 to 3.5 nm size, spherical shaped, poly-dispersed particles, (b) 3D image of silver nanoparticles analyzed by NOVA-TX software, (c) histogram and (d) line graph showing the size distribution of green-synthesized silver nanoparticles.

Fig. 5 (a) Scanning electron micrograph and (b) transmission electron micrograph of the Hymenodictyon orixense-synthesized silver nanoparticles.

Fig. 6 Energy dispersive X-ray (EDX) spectrum of Hymenodictyon orixense-synthesized silver nanoparticles showing the presence of different phyto-elements as capping agents.

As a general trend, the shape of plant-synthesized Ag NPs was spherical, with exception of some neem-synthesized Ag NPs. They are poly-disperse, with spherical or flat, plate-like, morphology, and mean size range of 5–35 nm in size.³² For example, Ag NPs fabricated using Emblica officinalis were also predominantly spherical with an average size of 16.8 nm ranging from 7.5 to 25 nm.³³ Most of the Ag NPs was roughly circular in shape with smooth edges, as in the case of our H. orixense-fabricated Ag NPs. In agreement our these findings, Ag NPs from Annona squamosa leaf extract were spherical in shape with an average size ranging from 20 to 100 nm³⁴ while Thirunavokkarasu et al.³⁵ reported spherical nanoparticles with size ranging from 8 to 90 nm in Desmodium gangeticum. TEM images showed that the surface of the Ag NPs was often surrounded by a grey thin layer of some material, which might be due to the capping organic constituents of the plant broth, as highlighted by Rafiuddin.³⁶

Larvicidal activity against malaria, chikungunya and Japanese encephalitis vectors

In laboratory conditions, the H. orixense aqueous leaf extract showed larvicidal activity against A. subpictus, A. albopictus and C. tritaeniorhynchus; LC₅₀ values were 104.62, 113.88 and 123.55 μg ml⁻¹, for A. subpictus, A. albopictus and C. tritaeniorhynchus, respectively (Table 1). Recently, a growing number of plant extracts have been found effective against C. quinquefasciatus larvae.^37,38 Furthermore, the H. orixense-synthesized Ag NPs were highly toxic against A. subpictus, A. albopictus and C. tritaeniorhynchus larvae; LC₅₀ values were 17.10, 18.74 and 20.08 μg ml⁻¹, A. subpictus, A. albopictus and C. tritaeniorhynchus, respectively (Table 2). Controls treatments, including the bioassays testing Ag⁺ ions at the same concentrations of the tested Ag NP showed no mortality, in agreement with Jayaseelan et al.³⁹ and Marimuthu et al.⁴⁰ In latest years, a growing number of plant-synthesized Ag NP have been studied for their excellent larvicidal activity against important mosquito vectors.^9,10 For instance, comparable toxicity rates have been recently reported for Ag NPs synthesized using Chomelia asiatica against A. stephensi larvae (LC₅₀ = 17.95 ppm).¹² The mortality effect evoked by Ag NPs on mosquito larvae and pupae may be due by the small size of the Ag NPs, which allows their passage through the insect cuticle and into individual cells, where they interfere with moulting and other physiological processes.⁹ The residual toxicity of silver ions against mosquito larvae covered a little role, since the peak in Fig. 1b was saturated after 180 min, indicating complete reduction of silver nitrate.⁴¹

Table 1 Larvicidal activity of the Hymenodictyon orixense aqueous leaf extract against the mosquito vectors Anopheles subpictus, Aedes albopictus and Culex tritaeniorhynchus

Mosquito species	Concentration (μg ml^−1)	Mortality (%) ± SD^a	LC50 (μg ml^−1) (LCL-UCL)	LC90 (μg ml^−1) (LCL-UCL)	Slope	Regression equation	χ^2 (d.f.)
a Values are mean ± SD of five replicates. 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. χ^2 = chi square. d.f. = degrees of freedom. n.s. = not significant (α = 0.05).
A. subpictus	50	27.5 ± 0.4	104.62 (92.82–115.08)	205.84 (190.92–225.46)	3.18	y = 10.45 + 0.368x	2.950 (4) n.s.
	100	46.2 ± 0.8
	150	68.4 ± 1.2
	200	87.3 ± 0.6
	250	99.0 ± 0.8
A. albopictus	50	24.2 ± 0.6	113.88 (102.25–124.42)	219.24 (203.41–240.11)	2.85	y = 6.11 + 0.375x	1.872 (4) n.s.
	100	42.6 ± 1.2
	150	63.7 ± 0.8
	200	84.1 ± 0.4
	250	97.2 ± 0.8
C. tritaeniorhynchus	50	21.4 ± 1.2	123.55 (111.97–134.27)	233.68 (216.64–256.31)	2.65	y = 2.49 + 0.376x	1.349 (4) n.s.
	100	38.6 ± 0.6
	150	60.2 ± 0.8
	200	79.3 ± 0.4
	250	95.1 ± 0.6

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Table 2 Larvicidal activity of silver nanoparticles synthesized using the Hymenodictyon orixense leaf extract against the mosquito vectors Anopheles subpictus, Aedes albopictus and Culex tritaeniorhynchus

Mosquito species	Concentration (μg ml^−1)	Mortality (%) ± SD^a	LC50 (μg ml^−1) (LCL-UCL)	LC90 (μg ml^−1) (LCL-UCL)	Slope	Regression equation	χ^2 (d.f.)
a Values are mean ± SD of five replicates. 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. χ^2 = chi square. d.f. = degrees of freedom. n.s. = not significant (α = 0.05).
A. subpictus	8	25.8 ± 1.2	17.10 (15.26–18.75)	33.16 (30.78–36.27)	2.95	y = 8.94 + 2.335x	3.185 (4) n.s.
	16	46.3 ± 0.8
	24	67.2 ± 0.6
	32	86.5 ± 0.4
	40	99.1 ± 0.8
A. albopictus	8	22.4 ± 0.4	18.74 (16.95–20.39)	35.25 (32.75–38.54)	2.59	y = 3.89 + 2.394x	3.621 (4) n.s.
	16	41.7 ± 1.2
	24	62.8 ± 0.6
	32	81.6 ± 0.8
	40	98.2 ± 0.6
C. tritaeniorhynchus	8	19.3 ± 0.6	20.08 (18.32–21.74)	36.94 (34.33–40.38)	2.39	y = 0.2 + 2.425x	2.708 (4) n.s.
	16	38.6 ± 0.4
	24	59.4 ± 1.2
	32	78.2 ± 0.6
	40	96.5 ± 0.8

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Biosafety on non-target mosquito predators

In our experiments, the toxicity treatments achieved negligible toxicity against D. indicus, with LC₅₀ values ranging from 833.59 to 6233.15 μg ml⁻¹ (Table 3). Focal observations highlighted that longevity the study species was not altered for 10 days after testing. SI indicated that H. orixense-fabricated Ag NP were less toxic to the non-target organism tested if compared to the targeted mosquitoes (Table 4). Currently, moderate knowledge is available about the acute toxicity of mosquitocidal nanoparticles towards non-target aquatic species.⁹ Recently, the genotoxic effect of silver nanoparticles synthesized using neem cake was studied on Carassius auratus using the comet assay and micronucleus frequency test. DNA damage was evaluated on peripheral erythrocytes sampled at different time intervals from the treatment. Interestingly, no significant damages were found at doses below 12 ppm.⁴² Furthermore, Spergularia rubra- and Pergularia daemia-synthesized Ag NP did not exhibit any evident toxicity effect against Poecilia reticulata fishes, after 48 h of exposure to LC₅₀ and LC₉₀ values calculated on IV instar larvae of A. aegypti and A. stephensi.⁴³ Haldar et al.⁴⁴ did not detected toxicity of Ag NP produced using dried green fruits of D. roxburghii against P. reticulata, after 48 h-exposure to LC₅₀ of IV instar larvae of A. stephensi and C. quinquefasciatus. Rawani et al.⁴⁵ showed that mosquitocidal Ag NP 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. Ag NPs biosynthesized using the 2,7-bis[2-[diethylamino]-ethoxy]fluorence isolate from the Melia azedarach leaves did not show acute toxicity against Mesocyclops pehpeiensis copepods.⁴⁶ Interestingly, the exposure to extremely low doses (e.g. 1 ppm) of green-synthesized Ag NP did not negatively influence the predation efficiency of a number of mosquito predators of relevance for mosquito control.^9,41

Table 3 Toxicity of Hymenodictyon orixense aqueous leaf extract and green-synthesized silver nanoparticles against the non-target mosquito predator Diplonychus indicus

Treatment	Concentration (μg ml^−1)	Mortality (%) ± SD^a	LC50 (μg ml^−1) (LCL-UCL)	LC90 (μg ml^−1) (LCL-UCL)	Slope	Regression equation	χ^2 (d.f.)
a Values are mean ± SD of five replicates. 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. χ^2 = chi square. d.f. = degrees of freedom. n.s. = not significant (α = 0.05).
Aqueous leaf extract	3000	26.2 ± 1.2	6233.15 (5563.16–6831.64)	11 942.84 (11 096.82–13 047.17)	2.79	y = 9.67 + 0.006x	4.737 (4) n.s.
	6000	48.3 ± 0.6
	9000	67.5 ± 0.4
	12 000	89.4 ± 0.8
	15 000	100.0 ± 0.0
Silver nanoparticles	400	27.3 ± 0.4	833.59 (741.65–915.37)	1619.86 (1503.63–1772.15)	2.98	y = 10.15 + 0.047x	4.784 (4) n.s.
	800	46.5 ± 0.8
	1200	68.9 ± 1.2
	1600	87.2 ± 0.6
	2000	100.0 ± 0.0

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Table 4 Suitability index of the non-target aquatic organism Diplonychus indicus over young instars of Anopheles subpictus, Aedes albopictus and Culex tritaeniorhynchus exposed to Hymenodictyon orixense aqueous leaf extract and green-synthesized silver nanoparticles

Treatment	A. subpictus	A. albopictus	C. tritaeniorhynchus
Aqueous leaf extract	59.57	54.73	50.45
Silver nanoparticles	48.74	44.48	41.51

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Conclusions

Overall, here we synthesized mosquitocidal silver nanoparticles using a cheap aqueous extract of H. orixense leaves as reducing and stabilizing agent. The bio-reduced Ag NPs were mostly spherical in shape, crystalline in nature, with face-cantered cubic geometry, and their mean size was 25–30 nm. This research highlighted that H. orixense-synthesized Ag NPs are easy to produce, stable over time, and can be employed at low dosages to strongly reduce populations of mosquito vectors, with little detrimental effects on the non-target mosquito predator D. indicus.

Conflicts of interest

The Authors declare no conflicts of interest.

Compliance with ethical standards

All applicable international and national guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Acknowledgements

Dr Igor Medintz and two anonymous reviewers kindly improved an earlier version of the manuscript. The authors would like to thank Professor and Head, Department of Zoology, Annamalai University for the laboratory facilities provided. We also acknowledge the cooperation of staff members of the VCRC (ICMR), Pondicherry.

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