Size-controlled fabrication of silver nanoparticles using the Hedyotis puberula leaf extract: toxicity on mosquito vectors and impact on biological control agents

Abstract

Mosquitoes vector important diseases, including malaria, dengue and Zika virus. The effective control and eradication of the mosquitoes can restrict the spread and severity of these diseases. Here the efficacy of silver nanoparticles (AgNPs) synthesized using the extract of Hedyotis puberula leaves on eggs, larvae and adults of Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. AgNPs were subjected to different biophysical analyses, including UV-Vis spectrophotometry, FTIR, XRD, AFM, SEM, TEM, EDX and DLS analysis. AgNPs were effective against the larvae of A. stephensi (LC₅₀ 16.58 μg ml⁻¹), A. aegypti (LC₅₀ 18.05 μg ml⁻¹) and C. quinquefasciatus (LC₅₀ 19.52 μg ml⁻¹). AgNPs exerted complete egg mortality at 80 μg ml⁻¹ against A. stephensi and at 100 and 120 μg ml⁻¹ against A. aegypti and C. quinquefasciatus, respectively. LC₅₀ of AgNPs on adults of A. stephensi, A. aegypti and C. quinquefasciatus were 33.11, 36.34 and 39.56 μg ml⁻¹, respectively. Both the H. puberula leaf extract and AgNPs were tested against three mosquito biocontrol agents, Anisops bouvieri, Diplonychus indicus and Gambusia affinis. LC₅₀ ranged from 1048 to 33 552 μg ml⁻¹. Overall, the H. puberula aqueous leaf extract can be employed to fabricate eco-friendly AgNPs with mean size of 6–16 nm, highly effective on A. stephensi, A. aegypti and C. quinquefasciatus.

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Introduction

In tropical and subtropical regions worldwide, mosquitoes (Diptera: Culicidae) constitute a major problem for public health.^1,2 They vector severe human and animal diseases such as malaria, dengue and severe dengue, West Nile virus, Japanese encephalitis, yellow fever, chikungunya, filariasis, Zika virus, and St. Louis encephalitis virus.^3,4 All parts of the world population are being affected by vector borne diseases, but in the developing countries control of such vector is becoming an issue difficult to challenge.^5–7 The routine use of synthetic insecticidal products for control of mosquito led to the development of resistant vector strains, as well as to concerns for human health and the environment.^8–10

Nowadays, nanotechnology rapidly evolved due to the use of nanometer size particles. Nanoparticles exhibit novel features, such as extraordinary strength, high chemical reactivity, magnetic properties and electrical conductivity. Current nanotechnology deals with application of such particles in environmental, agricultural and pharmaceutical sciences. Depending to the method of preparation, nanoparticles, nanospheres and/or nanocapsules can be obtained. Although physical and chemical methods are more popular and widely used for the synthesis of nanoparticles, the related environmental toxicity and non-biodegradable nature of the involved products limited their applications. Therefore, the “green” routes for synthesis of nanoparticles from herbal origin are of great interest due to eco-friendliness, economic prospects, feasibility and wide range of applications.^1,11

Green synthesized AgNPs may be a right opt source for mosquito control programs.^1,2,12 Green synthesis of AgNPs has been carried out employing extracts from Sida acuta,¹³ Solanum nigrum,¹⁴ Leucas aspera,¹⁵ Pongamia pinnata,¹⁶ Calotropis gigantean,¹⁷ Delphinium denudatum,¹⁸ Feronia elephantum,¹⁹ Heliotropium indicum,²⁰ Chomelia asiatica,²¹ Gmelina asiatica,²² Barleria cristata,⁵ Naregamia alata,²³ Bougainvillea glabra,²⁴ Carissa carandas,⁷ Hymenodictyon orixense,⁶ Zornia diphylla,²⁵ Nicandra physalodes,²⁶ and Quisqualis indica.²⁷ Particularly, it has been showed that the biophysical features and mosquitocidal activity of the green synthesized nanoparticles varied according to the tested plant extract used as reducing and capping agent.^28–32

Hedyotis puberula, commonly known as “imbural”, is an annual or biennial herb. In India, the plant's extract is used as an antipyretic and expectorant. It is also employed to treat asthma, cold, tuberculosis, and cancer. It is believed that the roots and leaves hold expectorant properties, thus they are used in bronchitis and constipation. Moreover, the leaf extract is used for the treatment of venomous bites.^33–35 Substances contained in the root bark include anthraquinone derivatives such as rubichloric and ruberythric acids, and alizarin.³⁶ Finally, there are reports regarding the antitubercular action of the root of H. puberula,³⁷ as well as of the aerial parts' hepatoprotective properties.³⁸

In the present investigation, we prepared green synthesized AgNPs using the extract of H. puberula leaves. Furthermore, the green fabricated nanoparticles were tested against the eggs, larvae and adults of the Anopheles stephensi, A. aegypti and C. quinquefasciatus. Moreover, the H. puberula aqueous leaf extract and AgNPs were tested against three aquatic biological control agents of mosquito young instars, i.e. Anisops bouvieri, Diplonychus indicus and the mosquito fish Gambusia affinis.

Experimental

Materials

Silver nitrate (AgNO₃) was purchased from Himedia, India. We gathered H. puberula healthy leaves from Nilgiris (11° 10′ N to 11° 45′ N latitude and 76° 14′ E to 77° 2′ E longitude), Southern India.

Preparation of plant extracts

We washed carefully the fresh leaves of H. puberula with tap and distilled water. Then 5 grams of air-dried leaves were finely cut and boiled for 10 minutes in a microwave oven, to get the leaves' extract. Finally, we let the extract cool at room temperature and then we filtered it through Whatman filter paper no. 1.

Green fabrication of Ag nanoparticles

The obtained extract of H. puberula was used for synthesis of AgNPs as a reducing as well as a stabilizing agent. To facilitate the synthesis of AgNPs 10 ml of H. puberula leaf extract was mixed with 90 ml of 1 mM AgNO₃ solution. This reaction mixture was kept at room temperature. Later, the color change was observed to designate the fabrication of colloidal AgNPs.

Characterization of Ag nanoparticles

The green synthesis of AgNPs was confirmed by UV-vis spectroscopy (Hitachi-2001) in a wavelength ranges between 200 and 800 nm at 1 nm resolution. The AgNPs were used for FTIR analysis to identify the functional groups (Perkin-Elmer). The particles size and nature of the green AgNPs were determined by XRD (XPERTPRO). XRD is a rapid analytical method to identify the crystalline structure. Size, surface morphology and composition were studied by AFM (Agilent Technologies AFM-5500), SEM coupled with EDX (Jeol JSM-6490A), and TEM (TEM Technite 10 Philips).

Mosquito rearing

Here we used parasite- and pathogen-free strains of A. stephensi, A. aegypti and C. quinquefasciatus mosquitoes reared in laboratory conditions. We obtained the mosquitoes from a permanent colony kept in Annamalai University's insectary, South India, at 28 ± 2 °C and 85% relative humidity, with a photoperiod of 12 h light and 12 h dark. We fed the larvae with a 3 to 1 ratio of dog biscuits and yeast powder. We fed the adults with a 10% sucrose solution. We periodically fed female mosquitoes with blood, acquired from a slaughter house in a heparinized vial and stored at 4 °C, with the use of a feeding system equipped with Parafilm as a membrane for egg production.^5,6

Larvicidal activity

The larvicidal assays were done following the World Health Organization³⁹ standard protocols with minor modifications.^5–7 The leaf extract of H. puberula and green synthesized AgNPs were prepared at different concentrations, i.e. 90, 180, 270, 360 and 450 μg ml⁻¹ and 8, 16, 24, 32 and 40 μg ml⁻¹, respectively, in double distilled water. Twenty-five reared third instar larvae were transferred to 500 ml beakers and each concentration was tested in five replicates with the control groups (i.e. silver nitrate and distilled water²⁶) under the laboratory conditions. Mortality of the larvae was calculated after 24 h of exposure period. During the exposure period, no food was supplied to the larvae.

Ovicidal activity

Ovicidal activity was studied following the method by Su and Mulla⁴⁰ with slight modifications.⁴¹ The leaf extracts of H. puberula and biosynthesized AgNPs were used to prepare different concentrations, 70, 140, 210, 280, 350, 420 μg ml⁻¹ and 20, 40, 60, 80, 100, 120 μg ml⁻¹, respectively. 0–6, 6–12 and 12–18 h old 100 eggs of A. stephensi, A. aegypti and C. quinquefasciatus were exposed to each dose of leaf extracts of H. puberula and biosynthesized AgNPs. Each concentration was replicated five times. Eggs exposed to AgNO₃ in water served as control. The hatch rate was assessed 48 h post treatment by the following formula.

Adulticidal activity

Followed a slightly modified WHO⁴² standard protocol we assessed the adulticidal activity of H. puberula leaf extract and biosynthesized AgNPs. We prepared different doses of aqueous H. puberula leaf extract and biosynthesized AgNPs, (130–650 μg ml⁻¹ in 130 μg ml⁻¹ increments and 16–80 μg ml⁻¹ in 16 μg ml⁻¹ increments, respectively). We formulated 2.5 ml of the tested concentration on 12 × 15 cm Whatman no. 1 filter paper. We used either water alone or the corresponding aqueous Ag concentration, in the form of AgNO₃, as controls. We dried the impregnated papers for 5 minutes on air, before inserting them into an exposure tube in the WHO testing kit. We introduced 20 blood-starved female mosquitoes into the tube, aged from 2 to 5 days, and kept them for one hour to acclimatize. Then, we transferred the mosquitoes by gentle blowing to the exposure tube, where they were kept for one hour before returning them to the holding tube for recovery. During the recovery phase, the mosquitoes were fed via a cotton pad soaked with a 10% glucose solution. When the 24 hour recovery period ended; we counted the dead mosquitoes and calculated the mortality percentage. We tested each extract two times, with five repetitions for each assay.

Toxicity on mosquito natural enemies

The H. puberula leaf extracts and AgNPs were tested against three predators of mosquito young instars, A. bouvieri, D. indicus, and G. affinis. As per the procedure used by Sivagnaname and Kalyanasundaram⁴³ slightly modified by Govindarajan and Benelli,^5–7 the non-target organisms were exposed to various concentrations of the plant extract and AgNPs. Ten predators were placed in a plastic jar containing 500 ml pond water. The numbers of dead were recorded after 24 h of exposure and percentage mortality was recorded. Each experiment was replicated five times. A set of controls (without the mosquitocidal compounds) for each organism was run at the same time. We also monitored the non-target organisms for mortality and other abnormalities such as reduced swimming activity and sluggishness after exposing them for 48 h. Subsequently, we observed these organisms for a ten-day period to assess the possibility of impacts on swimming activity and survival.

Data analysis

The data were presented in mean ± standard deviation and all the statistical analyses were performed by SPSS version 16.0. The average of the larvae mortality LC₅₀, LC₉₀ and chi-square test was calculated using Finney's method.⁴⁴ In experiments evaluating biotoxicity on mosquito predators, the Suitability Index (SI) was calculated for each non-target species.⁴⁵

Results and discussion

Biosynthesis and characterization of Ag nanoparticles

The formation of AgNPs was done by reduction of Ag⁺ ions into AgNPs with exposure of the leaf extract of H. puberula and the formation was highlighted by the color change of the aqueous suspension. The initial color of the suspension (AgNO₃ and leaf extract) was yellow, after the incubation period, the yellow turned into dark brown in color (Fig. 1a) which indicated surface plasmon resonance (SPR).^46–48 The SPR phenomenon was very sensitive to NP's nature, size and shape, which were formed by their inter particle distance and the surrounding media.^49,50 Fig. 1b represents the UV-vis spectrum of green synthesized AgNPs with higher peak level observed at 445 nm and SPR band also exposed at the same peak without any shifting. Veerakumar et al.¹⁹ observed the same absorption band in Feronia elephantum. Single peak indicated the synthesized particles were uniform in size and shape. So, the formation of AgNPs was attributed to hydrophilic and hydrophobic interaction, which prevents the particles from aggregation by intermolecular forces.⁵¹

Fig. 1 (a) Color intensity of the Hedyotis puberula 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.

XRD analysis was employed to study the crystalline nature of the AgNPs. Fig. 2 shows the XRD pattern of AgNPs and peak values at 2 h degrees of 37.80°, 43.25°, 64.85° and 76.15° corresponding to (111), (200), (220), and (311) planes of AgNPs. All the degrees of the peaks corresponded to a face centered cubic (FCC) crystalline structure. The intense peak 37.20° represented a high degree of crystallinity.^52,53

Fig. 2 XRD pattern of silver nanoparticles biofabricated using the Hedyotis puberula aqueous leaf extract.

FTIR spectroscopy identified the functional groups of the synthesized AgNPs. FTIR spectrum indicated the clear peaks with (3417.98, 2922.58, 2853.13, 2358.43, 1746.75, 1730.54, 1574.44, 1538.98, 1470.68, 1384.41, 1114.35, 869.29, 791.31, 661.15 and 617.59 cm⁻¹) different values (Fig. 3). These peak valuescould indicate the presence of functional groups like amides (N–H stretching 3417.98 cm⁻¹), aliphatic groups (cyclic CH₂ – 2922.58 cm⁻¹), methyl groups (bend CH₂–CH₃ stretching 1384.41 cm⁻¹), aliphatic amine groups (C–N stretching 1114.35 cm⁻¹), alkyl halides groups (C–Cl stretching 869.29 and 717.07 cm⁻¹) and alkyl halides (C–Br stretching 661.15 cm⁻¹ and 617.59 cm⁻¹).^54,55 The terpenoid groups have a high potential to convert the aldehyde groups into carboxylic acids in the Ag⁺ solution. Additionally, amide groups could also indicate the presence of some enzymes, which may be responsible for the synthesis of metal particles. Further, polyphenols have been also reported for their potential to reduce silver ions.⁵⁶

Fig. 3 FTIR spectrum of silver nanoparticles biofabricated using the Hedyotis puberula aqueous leaf extract.

As outlined in Fig. 4a, the biosynthesized AgNPs are spherical, poly-dispersed, and sized between 3.59 and 7.18 nm, as shown via 2.5 μm resolution studies with AFM. We treated the raw data produced by the AFM microscope with NOVA-TX to shed light on the AgNPs 3D structure (Fig. 4b). The corresponding diameter distribution revealed a mean particle size of approximately 6.46 nm (Fig. 4c and d). SEM assays showed slightly bigger AgNPs, which sometimes reached 50–60 nm (Fig. 5). AgNPs were mostly spherical with different sizes but a small number of anisotropic nanostructures such as nanotriangles, a few nanorods, hexagonal and polygonal nanoprisms were also observed. The uniform size distribution of nanoparticles in this study indicated the efficient stabilization of nanoparticles while the large size and/or anisotropic shapes of some nanoparticles might be due to the aggregation of smaller nanoparticles.⁵⁷

Fig. 4 AFM micrograph of silver nanoparticles biofabricated using the Hedyotis puberula extract (a) 2.5 μm resolution studies 0 to 7 nm size, spherical shaped, poly-dispersed particles, (b) 3D image of Ag nanoparticles analyzed by NOVA-TX software, (c) histogram showing the particle size distribution, (d) line graph showing the size distribution of green-synthesized Ag nanoparticles.

Fig. 5 Scanning electron microscopy (SEM) of Hedyotis puberula-fabricated silver nanoparticles.

The EDX analysis confirmed the presence of metallic Ag in the samples (Fig. 6). Strong signals were observed from AgNPs at approximately 2.7 keV. The appearance of strong signals for C and O were also due to the presence of bioorganic molecules that were involved in capping the Ag-NPs. The signals for CL and K were due to the X-ray emission from different bio-molecules of the H. puberula. The appearance of elemental Ag in the EDX analysis supported the XRD results, which indicated the reduction of metal cations to elemental form.⁵⁸

Fig. 6 Energy dispersive X-ray (EDX) spectrum of Hedyotis puberula-synthesized silver nanoparticles.

Moreover, we examined the size of the synthesized AgNPs using TEM. In support of our AFM data, TEM micrographs showed that well-dispersed particles were significantly spherical in shape (Fig. 7) and the particle size ranged from 5 nm to 21 nm, with an average size of 12 nm.^59,60 Dynamic light scattering (DLS) measurements were done to determine the size of the AgNPs formed. The particle size distribution curve from DLS analysis is shown in Fig. 8. Still in agreement with AFM and TEM data, the size of the particles ranged from 3 nm to 8 nm, with average particle size of 5 nm.⁶¹

Fig. 7 Transmission electron microscopy (TEM) of silver nanoparticles biofabricated using the Hedyotis puberula aqueous leaf extract.

Fig. 8 Dynamic light scattering (DLS) analysis showing the size distribution of silver nanoparticles biosynthesized using the aqueous leaf extract of Hedyotis puberula.

Acute toxicity against mosquito eggs, larvae and adults

We tested the H. puberula leaf extracts and AgNPs against the vectors A. stephensi, A. aegypti and C. quinquefasciatus (Tables 1 and 2). AgNPs of H. puberula showed higher larvicidal activity if compared to the plant extract alone (Table 2). The larvicidal activity after the 24 h was LC₅₀ = 16.58; LC₉₀ = 32.11 μg ml⁻¹ on A. stephensi, LC₅₀ = 18.05; LC₉₀ = 34.04 μg ml⁻¹ on A. aegypti and LC₅₀ = 19.52; LC₉₀ = 36.07 μg ml⁻¹ on C. quinquefasciatus. No mortality was observed in controls, as recently pointed out by Govindarajan and Benelli.^6,7 Tables 3 and 4 report the mean egg hatchability of A. stephensi, A. aegypti and C. quinquefasciatus. The AgNPs exerted zero hatchability (100% mortality) when tested at 80, 100 and 120 μg ml⁻¹, respectively. No mortality was observed in controls. The adulticidal activity of H. puberula leaf extracts and AgNPs on female A. stephensi, A. aegypti and C. quinquefasciatus at is presented in Tables 5 and 6. The AgNPs was found to be the most effective adulticidal agent, with the LD₅₀ values of 33.11, 36.34 and 39.56 μg ml⁻¹, respectively. No mortality was observed in controls.

Table 1 Larvicidal activity of the Hedyotis puberula aqueous leaf extract against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus

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, SD = standard deviation, χ^2 = chi square, d.f. = degrees of freedom, n.s. = not significant (α = 0.05).
A. stephensi	90	29.6 ± 0.4	182.67 (160.41–202.13)	369.97 (342.55–406.15)	3.83	y = 12.81 + 0.199x	2.225 (4)
	180	47.2 ± 1.2					n.s.
	270	68.5 ± 0.6
	360	89.3 ± 0.8
	450	98.1 ± 1.2
A. aegypti	90	24.8 ± 1.2	199.14 (178.18–217.99)	385.34 (357.65–421.73)	2.92	y = 7.36 + 0.208x	1.190 (4)
	180	43.5 ± 0.6					n.s.
	270	66.4 ± 0.8
	360	85.9 ± 0.4
	450	97.3 ± 1.2
C. quinquefasciatus	90	20.6 ± 0.6	217.67 (197.73–236.15)	404.10 (375.73–441.33)	2.47	y = 1.85 + 0.216x	1.140 (4)
	180	39.2 ± 0.8					n.s.
	270	62.4 ± 1.2
	360	81.9 ± 0.4
	450	96.3 ± 0.8

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Table 2 Larvicidal activity of silver nanoparticles biosynthesized using the Hedyotis puberula leaf extract against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus

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, SD = standard deviation, χ^2 = chi square, d.f. = degrees of freedom, n.s. = not significant (α = 0.05).
A. stephensi	8	26.2 ± 0.8	16.58 (14.75–18.19)	32.11 (29.81–35.11)	2.94	y = 10.33 + 2.331x	4.962 (4)
	16	49.5 ± 1.2					n.s.
	24	67.3 ± 0.6
	32	88.4 ± 0.4
	40	100.0 ± 0.0
A. aegypti	8	22.8 ± 0.8	18.05 (16.27–19.67)	34.04 (31.64–37.18)	2.62	y = 5.47 + 2.396x	4.372 (4)
	16	45.2 ± 0.6					n.s.
	24	63.5 ± 1.2
	32	84.3 ± 0.4
	40	99.1 ± 0.6
C. quinquefasciatus	8	19.5 ± 0.6	19.52 (17.75–21.15)	36.07 (33.54–39.40)	2.43	y = 1.47 + 2.426x	3.170 (4)
	16	41.9 ± 0.4					n.s.
	24	59.3 ± 1.2
	32	80.6 ± 0.8
	40	97.2 ± 0.4

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Table 3 Ovicidal activity of the Hedyotis puberula aqueous leaf extract against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus^a

Mosquito species	Age of the egg raft/eggs (h)	Egg hatchability (%)							F value (d.f.)	P value
		Control	70 μg ml^−1	140 μg ml^−1	210 μg ml^−1	280 μg ml^−1	350 μg ml^−1	420 μg ml^−1
a Values are mean ± SD of five replicates, NH = no hatchability, d.f. = degrees of freedom. Within each row, different letters indicate significant differences among values (ANOVA, Tukey's HSD, P < 0.001).
A. stephensi	0–6	100 ± 0.0	29.2 ± 1.9	18.4 ± 1.5	NH	NH	NH	NH	121.52 (5)	<0.001
	6–12	100 ± 0.0	36.7 ± 1.3	20.6 ± 1.7	NH	NH	NH	NH	138.63 (5)	<0.001
	12–18	100 ± 0.0	48.9 ± 1.8	37.5 ± 1.4	21.7 ± 0.8	NH	NH	NH	145.87 (5)	<0.001
A. aegypti	0–6	100 ± 0.0	47.5 ± 1.4	36.3 ± 1.2	18.4 ± 1.7	NH	NH	NH	112.14 (5)	<0.001
	6–12	100 ± 0.0	58.3 ± 1.6	45.8 ± 1.3	21.7 ± 1.9	NH	NH	NH	129.34 (5)	<0.001
	12–18	100 ± 0.0	66.2 ± 1.9	53.6 ± 1.6	36.3 ± 1.5	23.4 ± 1.2	NH	NH	152.82 (5)	<0.001
C. quinquefasciatus	0–6	100 ± 0.0	68.7 ± 0.4	55.9 ± 1.5	38.2 ± 1.3	19.7 ± 1.6	NH	NH	127.91 (5)	<0.001
	6–12	100 ± 0.0	76.9 ± 1.2	67.8 ± 1.4	43.5 ± 0.6	23.8 ± 1.3	NH	NH	141.38 (5)	<0.001
	12–18	100 ± 0.0	86.1 ± 0.3	76.2 ± 1.3	57.4 ± 1.9	37.5 ± 1.4	22.9 ± 1.6	NH	158.39 (5)	<0.001

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Table 4 Ovicidal activity of silver nanoparticles biosynthesized using the Hedyotis puberula leaf extract against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus^a

Mosquito species	Age of the egg raft/eggs (h)	Egg hatchability (%)							F value (d.f.)	P value
		Control	20 (μg ml^−1)	40 (μg ml^−1)	60 (μg ml^−1)	80 (μg ml^−1)	100 (μg ml^−1)	120 (μg ml^−1)
a Values are mean ± SD of five replicates, NH = no hatchability, d.f. = degrees of freedom. Within each row, different letters indicate significant differences among values (ANOVA, Tukey's HSD, P < 0.001).
A. stephensi	0–6	100 ± 0.0	28.4 ± 1.6	16.3 ± 1.7	NH	NH	NH	NH	158.96 (5)	<0.001
	6–12	100 ± 0.0	35.7 ± 1.2	20.4 ± 1.6	NH	NH	NH	NH	142.97 (5)	<0.001
	12–18	100 ± 0.0	44.8 ± 0.5	29.3 ± 1.9	18.4 ± 1.8	NH	NH	NH	128.76 (5)	<0.001
A. aegypti	0–6	100 ± 0.0	49.2 ± 1.3	28.7 ± 1.5	19.5 ± 1.7	NH	NH	NH	174.29 (5)	<0.001
	6–12	100 ± 0.0	54.8 ± 1.7	36.9 ± 0.3	22.8 ± 1.6	NH	NH	NH	158.75 (5)	<0.001
	12–18	100 ± 0.0	59.4 ± 1.8	45.6 ± 1.5	32.6 ± 1.9	21.4 ± 0.8	NH	NH	136.74 (5)	<0.001
C. quinquefasciatus	0–6	100 ± 0.0	56.7 ± 1.3	48.9 ± 1.8	27.9 ± 1.3	19.6 ± 1.7	NH	NH	142.91 (5)	<0.001
	6–12	100 ± 0.0	74.8 ± 1.5	59.3 ± 1.4	38.5 ± 1.4	22.4 ± 1.2	NH	NH	137.37 (5)	<0.001
	12–18	100 ± 0.0	86.6 ± 1.2	69.4 ± 1.2	56.3 ± 1.5	34.8 ± 0.6	19.5 ± 1.3	NH	122.92 (5)	<0.001

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Table 5 Adulticidal activity of the Hedyotis puberula aqueous leaf extract against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus

Mosquito species	Concentration (μg ml^−1)	Mortality (%) ± SD^a	LD50 (μg ml^−1) (LCL–UCL)	LD90 (μg ml^−1) (LCL–UCL)	Slope	Regression equation	χ^2 (d.f.)
a Values are mean ± SD of five replicates, SD = standard deviation, χ^2 = chi square, d.f. = degrees of freedom, n.s. = not significant (α = 0.05).
A. stephensi	130	28.4 ± 1.2	269.40 (239.27–296.15)	526.61 (488.57–576.56)	3.07	y = 10.66 + 0.142x	5.528 (4)
	260	46.6 ± 0.8					n.s.
	390	67.3 ± 0.6
	520	88.2 ± 0.4
	650	100.0 ± 0.0
A. aegypti	130	23.8 ± 0.8	295.65 (266.99–321.82)	554.42 (515.48–605.37)	2.56	y = 4.77 + 0.148x	4.734 (4)
	260	42.5 ± 0.6					n.s.
	390	63.1 ± 1.2
	520	84.6 ± 0.4
	650	99.2 ± 0.6
C. quinquefasciatus	130	20.5 ± 0.4	321.29 (293.34–347.47)	585.42 (544.88–638.46)	2.32	y = −0.12 + 0.152x	2.703 (4)
	260	37.2 ± 0.6					n.s.
	390	59.4 ± 1.2
	520	81.6 ± 0.8
	650	97.1 ± 0.6

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Table 6 Adulticidal activity of silver nanoparticles biosynthesized using the Hedyotis puberula leaf extract against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus

Mosquito species	Concentration (μg ml^−1)	Mortality (%) ± SD^a	LD50 (μg ml^−1) (LCL–UCL)	LD90 (μg ml^−1) (LCL–UCL)	Slope	Regression equation	χ^2 (d.f.)
a Values are mean ± SD of five replicates, SD = standard deviation, χ^2 = chi square, d.f. = degrees of freedom, n.s. = not significant (α = 0.05).
A. stephensi	16	27.3 ± 0.8	33.11 (29.46–36.35)	64.17 (59.58–70.19)	2.94	y = 10.39 + 1.164x	5.728 (4)
	32	48.5 ± 0.6					n.s.
	48	66.2 ± 1.2
	64	89.4 ± 0.4
	80	100.0 ± 0.0
A. aegypti	16	23.4 ± 0.6	36.34 (32.69–39.66)	69.35 (64.39–75.87)	2.74	y = 5.83 + 1.182x	3.517 (4)
	32	44.8 ± 1.2					n.s.
	48	62.1 ± 0.8
	64	84.3 ± 0.4
	80	98.2 ± 1.2
C. quinquefasciatus	16	20.1 ± 0.4	39.56 (35.93–42.93)	74.08 (68.74–81.14)	2.54	y = 1.93 + 1.188x	3.054 (4)
	32	41.6 ± 0.6					n.s.
	48	57.4 ± 0.8
	64	79.5 ± 1.2
	80	96.2 ± 0.6

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Recently, a number of green-synthesized nanoparticles have been tested against the three mosquito vectors selected in the research.^1,2,62–64 For example, Srinivasan et al.⁴⁸ reported the larvicidal activity of biosynthesized AgNPs using Avicennia marina leaf extract against A. aegypti (LC₅₀ = 4.374 and LC₉₀ = 4.928 ppm) and A. stephensi (LC₅₀ = 7.40 and LC₉₀ = 9.865 ppm). The larvicidal activity of Ficus racemosa-synthesized AgNPs was noted against C. quinquefasciatus, with LC₅₀ = 67.72 and LC₉₀ = 63.70 ppm.⁶⁵ The larvicidal activity Tinospora cordifolia-fabricated AgNPs was studied against fourth instar larvae of C. quinquefasciatus, with LC₅₀ = 6.96 ppm.⁶⁶ The AgNPs fabricated using Eclipta prostrata showed maximum larvicidal activity against the fourth instar larvae of C. quinquefasciatus (LC₅₀ = 27.49 and LC₉₀ = 70.38) and A. subpictus (LC₅₀ = 27.85 and LC₉₀ = 71.45 ppm).⁶⁷ In the present investigation, the larvicidal activity of AgNPs was higher activity on A. stephensi if compared to A. aegypti and C. quinquefasciatus. Vector management is one of the major issues due to the capacity of resistance against the usual insecticides. Therefore, the development of newer and effective insecticides is urgently needed.^1,2,68 In this framework, green synthesized nanoparticles are easy to produce, stable for a long time and highly effective against a number of important mosquito species. However, more information about their mechanisms of action against insects are urgently needed.³⁰

Biotoxicity on mosquito natural enemies

The acute toxicity of H. puberula aqueous leaf extract and synthesized-AgNPs towards A. bouvieri, D. indicus, and G. affinis is presented in Tables 7 and 8. The AgNPs had little toxicity on G. affinis, D. indicus, and A. bouvieri, with LC₅₀ of 2704, 1658 and 1048 μg ml⁻¹. G. affinis was less susceptible to the AgNPs if compared to D. indicus, and A. bouvieri. SI/PSF indicated that the H. puberula leaf extracts and biosynthesized-AgNPs are less harmful to predatory fishes than other mosquito predators tested (Table 9). No changes in the survival rate and swimming activity of the non-target organisms were observed within 48 h post exposure. The AgNPs is non-toxic up to a concentration of 250 μg ml⁻¹ to the non-target predatory fish G. affinis and the water bugs D. indicus, and A. bouvieri.

Table 7 Biotoxicity of the Hedyotis puberula aqueous leaf extract against several biocontrol agents of Anopheles, Aedes and Culex mosquito vectors

Non-target organism	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, SD = standard deviation, χ^2 = chi square, d.f. = degrees of freedom, n.s. = not significant (α = 0.05).
A. bouvieri	6000	29.6 ± 1.2	12 364.98 (10 955.09–13 612.47)	24 372.65 (22 597.61–26 707.44)	3.21	y = 11.24 + 0.003x	6.068 (4)
	12 000	45.8 ± 0.8					n.s.
	18 000	67.4 ± 0.6
	24 000	88.2 ± 0.4
	30 000	100.0 ± 0.0
D. indicus	9000	27.2 ± 0.8	18 747.76 (16 647.76–20 610.21)	36 708.68 (34 053.35–40 197.65)	3.10	y = 10.58 + 0.002x	5.406 (4)
	18 000	48.3 ± 1.2					n.s.
	27 000	66.9 ± 0.6
	36 000	87.1 ± 0.4
	45 000	100.0 ± 0.0
G. affinis	16 000	26.4 ± 0.4	33 552.24 (29 871.31–36 828.76)	65 127.06 (60 459.27–71 243.83)	2.97	y = 9.87 + 0.001x	4.805 (4)
	32 000	47.2 ± 0.8					n.s.
	48 000	68.9 ± 1.2
	64 000	86.3 ± 0.6
	80 000	100.0 ± 0.0

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Table 8 Biotoxicity of green-synthesized silver nanoparticles using the Hedyotis puberula leaf extract against several biocontrol agents of Anopheles, Aedes and Culex mosquito vectors

Non-target organism	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, SD = standard deviation, χ^2 = chi square, d.f. = degrees of freedom, n.s. = not significant (α = 0.05).
A. bouvieri	500	26.4 ± 1.2	1048.08 (934.09–1149.72)	2026.20 (1881.29–2216.01)	2.91	y = 9.73 + 0.037x	5.303 (4)
	1000	48.2 ± 0.8					n.s.
	1500	66.5 ± 0.6
	2000	87.9 ± 0.4
	2500	100.0 ± 0.0
D. indicus	800	28.5 ± 0.6	1658.82 (1476.45–1821.24)	3216.53 (2985.72–3518.85)	2.95	y = 10.24 + 0.023x	5.722 (4)
	1600	45.8 ± 0.8					n.s.
	2400	67.3 ± 1.2
	3200	89.2 ± 0.6
	4000	100.0 ± 0.0
G. affinis	1300	29.3 ± 0.8	2704.29 (2394.52–2978.18)	5360.04 (4966.61–5879.30)	3.32	y = 11.17 + 0.014x	6.906 (4)
	2600	46.9 ± 1.2					n.s.
	3900	65.1 ± 0.6
	5200	87.4 ± 0.4
	6500	100.0 ± 0.0

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Table 9 Suitability index of different mosquito natural enemies over young instars of Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus exposed to Hedyotis puberula aqueous leaf extract and green-synthesized silver nanoparticles

Treatment	Non-target organism	A. stephensi	A. aegypti	C. quinquefasciatus
Aqueous leaf extract	A. bouvieri	67.69	62.09	56.80
	D. indicus	102.63	94.14	86.12
	G. affinis	183.67	168.48	154.14
Silver nanoparticles	A. bouvieri	63.21	58.06	53.69
	D. indicus	100.04	91.90	84.98
	G. affinis	163.10	149.82	138.53

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As regards to other biopesticides recently studied, it has been reported that the Heracleum sprengelianum essential oil and its main chemical compounds are eco-friendly for the mosquito predators A. bouvieri, D. indicus and G. affinis, with the LC₅₀ ranging from 206 to 4219 μg ml⁻¹.⁶⁹ Moreover, the aqueous extract and biosynthesized AgNPs of Quisqualis indica had a moderate biotoxic effect on two mosquito predators A. bouvieri (LC₅₀ 653 μg ml⁻¹) and G. affinis (LC₅₀ 2183 μg ml⁻¹).²⁷ Additionally, the B. cristata aqueous extract and the biosynthesized AgNPs were evaluated on three mosquito predators A. bouvieri, D. indicus, and G. affinis, yielding LC₅₀ values from 633.26 to 8595.89 μg ml⁻¹, respectively.⁵ Finally, the aqueous extract and biosynthesized silver nanoparticles of Carissa spinarum was assessed on the non-target organisms D. indicus, A. bouvieri and G. affinis, yielding minimal toxicity, as shown by the obtained LC₅₀ values that ranged from 424.09 to 6402.68 μg ml⁻¹.⁶⁴

Conclusions

There is a worldwide abundance of phytochemicals in plant species, which may be investigated to replace chemical pesticides. Green synthesis of biopesticides may become a viable alternative to replace synthetic insecticides, as these agents are shown to be safe, low-cost, and have high availability worldwide, with special reference to poor and marginalized Asian and African countries. In this study, we easily biosynthesized silver nanoparticles at room temperature from an inexpensive leaf extract of H. puberula. The AgNPs we obtained were spherically-shaped, sized from 10 to 16 nm, with crystalline nature. Our results show that the aqueous leaf extract of H. puberula can be safely employed to biosynthesize AgNPs with high efficacy against A. stephensi, A. aegypti and C. quinquefasciatus mosquito vectors.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

The authors are thankful to the Head of the Department of Zoology, Khadir Mohideen College and Annamalai University for the facilities provided to carry out this work. The authors extend their sincere appreciations to the Deanship of Scientific Research at King Saud University for its funding this Prolific Research Group (PRG-1437-36).

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