Introduction

Arthropods are important vectors of pathogens and parasites of public health relevance (Benelli et al. 2016a, b). In particular, recent outbreaks of mosquito-borne diseases such as dengue, chikungunya, West Nile, Japanese encephalitis, and Zika virus underlined the key role of mosquito control strategies (Benelli and Mehlhorn 2016; Ward and Benelli 2017). However, the application of chemical insecticides leads to pesticide resistance, toxicity to non-target organisms, as well as environmental and human health concerns (Mehlhorn et al. 2012; Naqqash et al. 2016; Govindarajan et al. 2016a, b, c, d, e). Therefore, current research focuses on biopesticides and repellents formulated using plant extracts and essential oils as novel and safer alternatives for mosquito control (Maguranyi et al. 2009; Mathew et al. 2009; Semmler et al. 2009; Zhu and Tian 2011, 2013; Benelli 2015a, b; Benelli et al. 2014, 2015a, b, c; Govindarajan 2016; Pavela and Benelli 2016a, b; Govindarajan and Benelli 2016a, b, c).

Recently, green synthesis of metal nanoparticles using cheap and abundant plant extracts and related metabolites showed a number of important advantages, ranging from reduced energy inputs to avoidance of highly toxic chemical for nanosynthesis (Bar et al. 2009; Parashar et al. 2009; Zhang et al. 2011; Vivek et al. 2012; Benelli 2016a, b, c, d; Yugandhar and Savithramma 2016). In particular, silver is well known for its antimicrobial activity, widely exploited in medicine and industrial processes (Reda et al. 2011; Zargar et al. 2011; Vijayakumar et al. 2013). Among biological methods, the use of plants for synthesis of metal nanoparticles attracts wide research attention since it is a quick, cheap, and one-step method (Kowshik et al. 2003; Madhiyazhagan et al. 2015; Murugan et al. 2015a, b, c; Roni et al. 2015; Subramaniam et al. 2015; Sujitha et al. 2015; Benelli 2016a, b, c, d; Chandramohan et al. 2016).

Green-synthesized Ag NPs have been produced using extracts from a wide number of plant species as reducing and capping agents. Good examples are Emblica officinalis (Ankamwar et al. 2005), Aloe vera (Chandran et al. 2006), Cinnamomum camphora (Huang et al. 2007), Cinnamon zeylanicum (Sathishkumar et al. 2009a), Azadirachta indica (Tripathi et al. 2009), Glycine max (Vivekanandhan et al. 2009), Camellia sinensis (Begum et al. 2009), Ocimum sanctum (Ahmad et al. 2010), Pongamia pinnata (Raut et al. 2010), Allium sativum (Rastogi and Arunachalam 2011), Tagetes erecta (Krishnamurthy et al. 2012), Cocos nucifera (Roopan et al. 2013), Desmodium gangeticum (Thirunavokkarasu et al. 2013), Andrographis paniculata (Kotakadi et al. 2014), and Hibiscus sabdariffa (Thovhogi et al. 2015). Effective mosquitocidal activity on Anopheles stephensi, Anopheles subpictus, Aedes aegypti, Aedes albopictus, Culex tritaeniorhynchus, and Culex quinquefasciatus has been reported for Ag NPs fabricated using extracts from Eclipta prostrata (Rajakumar and Abdul Rahuman 2011), Plumeria rubra (Patil et al. 2012a), Pergularia daemia (Patil et al. 2012b), Drypetes roxburghii (Haldar et al. 2013), Sida acuta (Veerakumar et al. 2013), Pongamia pinnata (Naik et al. 2014), Feronia elephantum (Veerakumar et al. 2014a), Leucas aspera (Suganya et al. 2014), Heliotropium indicum (Veerakumar et al. 2014b), Bauhinia variegata (Govindarajan et al. 2016f), Clerodendrum chinense (Govindarajan et al. 2016g), Malva sylvestris (Govindarajan et al. 2016h), Mussaenda glabra (Govindarajan et al. 2016i), and Zornia diphylla (Govindarajan et al. 2016c). In addition, the efficacy of green-synthesized Ag NPs has been reported as effective in reducing young instars populations of mosquito vectors even in the field (Suresh et al. 2015).

Interestingly, the biophysical features and bioactivity of green-fabricated NPs strongly differ when different botanicals are employed for nanosynthesis, highlighting the importance of screening local botanical resources for the biofabrication of nanomosquitocides (Benelli 2016a, b). Acacia caesia (L.) Willd. (Mimosaceae) is widely distributed in hills of the Western Ghats, around the altitude of 500 m a.s.l. Medicinal value of this plant lies in bioactive phytochemical constituents (Pullaiah 2006) including alkaloids, flavonoids, glycosides, and saponins. A. caesia extracts exhibited antimicrobial and antioxidant activity (Sathishkumar et al. 2009b). Leaves are used in the treatment of asthma, skin diseases (Paulsamy et al. 2010), menstrual disorder (Pullaiah 2006), and scabies (Thambiraj and Paulsamy 2010). To our best of knowledge, the mosquitocidal properties of A. caesia have not been investigated.

In this research, A. caesia leaf extract was used to synthesize Ag NPs with ovicidal, larvicidal, and adulticidal toxicities against three mosquito vectors, malaria vector An. subpictus, chikungunya and Zika virus vector Ae. albopictus, and Japanese encephalitis vector Cx. tritaeniorhynchus. Ag NPs were analyzed by various biophysical methods, including spectroscopy (UV-visible spectrophotometry, XRD, FTIR, EDX) and microscopy (SEM, TEM, atomic force microscopy (AFM)) analyses. Furthermore, toxicity of A. caesia aqueous leaf extract and A. caesia-fabricated Ag NPs was evaluated on three non-target biological control agents, i.e., Anisops bouvieri, Diplonychus indicus, and Gambusia affinis, which predate on Culicidae young instars.

Materials and methods

Collection of materials

Analytical grade AgNO3 was from Merck (India), glassware was washed with acid and rinsed afterwards with Millipore Milli-Q water. A. caesia leaves were collected from Western Ghats, India (10° 10′ 23.1″ N 77° 03′ 41.5″ E) in June 2016. Plant taxonomic identification was conducted by Prof. V. Venkatesalu, Annamalai University, India.

Preparation of A. caesia leaf extracts

Leaves were thoroughly washed with tap water, rinsed with Millipore-Milli-Q water, and left to dry out in the shade, before grinding them to fine powder. Aqueous extract was prepared by adding 50 g of the aforementioned leaf powder to 0.5 l of distilled water, under continuous stirring. After 3 h, the suspension was left for 3 h and then filtered using a Whatman no. 1 filter paper. The filtrate was stored at 10 °C until the assays.

Biogenic synthesis and characterization of silver nanoparticles

Ten milliliters of the A. caesia leaf extract were mixed with 90 ml of 1 mM AgNO3 solution and heated in a water bath, set at 80 °C for 10 min. A color change from yellow to brown designates the formation of colloidal Ag NPs. To track formation of Ag NPs, a UV-Vis spectrophotometry (UV-160v) was used, with 200–800 nm wavelength and a 1-nm resolution (Shimadzu UV 1700, Japan). To examine size, morphology, and composition of the resulting Ag NPs, EDX, TEM (Technite 10 Philips), AFM (Agilent Technologies AFM-5500), and SEM (Hitachi S3000 H) were used. To determine whether the purified Ag NPs were capped by Acacia extract-borne metabolites, FTIR spectroscopy (Thermo Scientific Nicolet 380) was employed, while XRD shed light on the possible presence of Ag nanocrystals.

Mosquito rearing

Pathogen- and parasite-free species of the three mosquito species were reared continuously for several generations at Annamalai University (Govindarajan and Benelli 2016a). They were maintained at 28 ± 2 °C, 70–85% R.H., with a photoperiod of 12-h light and 12-h dark. To feed the larvae, a mixture of yeast powder and dog biscuits at a ratio of 1 to 3 was used. When adult feeding was about to take place, 3 to 4 days had passed after emergence, and the subjects were fed only on raisins and water during that time, whereas a 12-h starvation period preceded feeding. Adult feeding involved 500 mosquitoes per cage being fed on blood for 4 h, via a Parafilm-fitted feeding unit. Ae. albopictus were fed during the 12.00 to 16.00 h period, whereas An. subpictus and Cx. tritaeniorhynchus were from 18.00 to 22.00 h (see Govindarajan and Benelli 2016a, b).

Larvicidal activity

The protocol by World Health Organization (2005) was employed to assess whether A. caesia aqueous extract and biosynthesized Ag NPs exerted acute larvicidal activity on the three mosquito vectors. During the experiment, 20 late III instar larvae were added in 250 ml of dechlorinated water plus the leaf extract (five doses, ranging from 60 to 300 μg ml−1, in 60 μg ml−1 increments) or Ag NPs (five doses, from 5 to 25 μg ml−1, in 5 μg ml−1 increments); for each tested dose, 5 replicates were conducted. Mortality was assessed after 24 h, during which the larvae were not fed. For every test, a corresponding test of control groups was conducted, containing AgNO3 and distilled water, with five repetitions (Govindarajan and Benelli 2016b).

Ovicidal activity

In agreement with Su and Mulla (1998), for each mosquito vector, 100 eggs were exposed to six concentrations of the leaf extract, ranging from 150 to 900 μg ml−1 in 150 μg ml−1 increments, and an equal number of silver nitrate concentrations, ranging from 15 to 90 μg ml−1 in 15 μg ml−1 increments. After exposure for 24 h, the eggs were counted under the microscope and then assessed for hatching after being transferred to cups containing distilled water. Six repetitions per each concentration were carried out, accompanied by untreated controls, and estimated the hatch rates 48 h after treatment by using the following equation:

$$ \mathrm{Hatchability}\ \left(\%\right)=\frac{\mathrm{Number}\ \mathrm{of}\ \mathrm{hatched}\ \mathrm{larvae}}{\mathrm{Total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{eggs}}\times 100 $$

Adulticidal activity

To assess the adulticidal activity of extract and Ag NPs, the World Health Organization (1981) standard method was followed. Several concentrations of both the leaf extract and Ag NPs, in the range of 9–500 μg ml−1, were tested, following the same process that was described previously, using 12 × 15 cm-sized Whatman no. 1 filter papers treated with 5 ml of aqueous solution (Govindarajan and Sivakumar 2011). Control papers were treated with distilled water or aqueous AgNO3. Twenty female mosquitoes were selected and gently inserted them into a plastic container. Mosquitoes were let acclimatize in the container for 1 h, followed by exposure to the test paper for another hour. When the exposure period ended, we returned the test subjects to the plastic container and left them for 24 h to recover. After the recovery period, a piece of cotton soaked in a 10% glucose solution was inserted in the mesh screen, then we assessed the mosquito mortality and repeated each experiment five times.

Toxicity on non-target aquatic insects and fishes

Following Sivagnaname and Kalyanasundaram (2004) with minor changes by Govindarajan and Benelli (2016b), we evaluated the acute toxicity of aqueous leaf extract and Ag NPs on A. bouvieri, D. indicus, and G. affinis. We tested concentrations of leaf extract and Ag NPs up to 50 times higher than the calculated LC50 for the mosquito larvae, and the experiment for every test concentration was repeated 10 times, accompanied by four repetitions for the corresponding untreated controls. Then, mortality was noted and any other abnormal behavior (e.g., sluggishness or decreased swimming activity) was also observed after 48 h of exposure. After exposure, survival rates and swimming activity were monitored for ten additional days in order to investigate the incidence of any residual post-treatment effect of extract and Ag NPs.

Data analysis

Probit analysis was used to investigate the mortality data (Benelli 2017). The method by Finney (1971) was used to determine LC50 (LD50) and LC90 (LD90). To assess the toxicity of the extract and silver nanoparticles on non-target organisms, suitability index (SI) was estimated for every individual non-target species via the formula below (Deo et al. 1988).

$$ \mathrm{SI}=\frac{{\mathrm{LC}}_{50}\ \mathrm{of}\ \mathrm{non}\hbox{-} \mathrm{target}\ \mathrm{organisms}}{{\mathrm{LC}}_{50}\ \mathrm{of}\ \mathrm{target}\ \mathrm{vector}\ \mathrm{species}} $$

The SPSS Statistical Software Package version 16.0 was used for data analysis.

Results and discussion

Biophysical characterization of silver nanoparticles

When A. caesia leaf extract was mixed with aqueous solution of Ag+ ions, the color of plant extract changed from yellow to dark brown within 180 min (Fig. 1a) (see also Sathishkumar et al. 2009a, b). Figure 1b shows the UV-Vis spectrum of Ag NP suspension, showing a main absorption peak at 473 nm. The presence of broad resonance indicated an aggregated structure of Ag NPs in the suspension (Rashmi and Preeti 2009; Jayaseelan et al. 2011). Furthermore, Ag NPs fabricated in the present investigation showed a crystalline structure, as evidenced by peaks at 2θ values of 38.52°, 43.64°, 65.37°, and 78.62° corresponding to 111, 200, 220, and 311 facets of face-centered cubic crystalline structure (Fig. 2a) (Kalishwaralal et al. 2008; Marimuthu et al. 2011; Kumar et al. 2012). The presence of elemental nanosilver in the material was confirmed by EDX analysis (Fig. 2b). Indeed, Ag NPs exhibited typical optical absorption peak at 3 keV, due to surface plasmon resonance (SPR) (Magudapatty et al. 2001; Dinesh et al. 2015).

Fig. 1
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a Color intensity of Acacia caesia aqueous extract before and after the reduction of silver nitrate (1 mM). b UV-visible spectrum of Ag nanoparticles after 180 min

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Fig. 2
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a XRD pattern of Ag nanoparticles rapidly synthesized employing the Acacia caesia aqueous extract. b EDX spectrum of green-fabricated Ag nanoparticles

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FTIR analysis was carried out to identify the possible bioreducing molecules in the leaf extract of A. caesia. Figure 3 shows the FTIR spectrum of Ag NPs synthesized from the aqueous leaf extract of A. caesia. The intense peak at 3380.22 cm−1 may be ascribed to the O-H stretch of alcohols or phenols (Gopinath et al. 2013; Velayutham et al. 2013). The peak at 2920.33 and 2847.40 cm−1 probably indicate to CH stretching (Sanghi and Verma 2009; Ramanibai and Velayutham 2015). The band at 1578.70 cm−1 may correspond to carboxylic group N-H bending (Shivshankar et al. 2003). The peak at 1383.90 cm−1 may be assigned to C-O stretching while 824.93 cm−1 could indicate to C-Cl stretching alkyl halides. The peak at 546.54 cm−1 was probably linked to C-H stretching strong vinyl di-substituted alkenes (Angajala et al. 2014). Overall, the carbonyl and hydroxyl groups present in tannins, flavonoids, alkaloids, steroids, and glycosides are the main groups responsible for reducing Ag+ to Ag0 as well as capping and stabilizing the Ag NPs.

Fig. 3
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FTIR spectrum of biogenic Ag nanoparticles synthesized using the Acacia caesia aqueous leaf extract

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A. caesia-synthesized Ag NPs were characterized by SEM and TEM analyses. The nanoparticles were predominantly spherical in shape (Fig. 4a). Our results on spherical shapes of green-synthesized Ag NPs are in agreement with Elavazhagan and Arunachalam (2011) as well as to Suresh et al. (2015), while a limited number of reported showed the synthesis of cubic or flat Ag NPs (recently reviewed by Benelli 2016a). In addition, TEM (Fig. 4b) confirmed that nanoparticles are spherical in shape and polydispersed without significant agglomeration; mean sizes ranged from 20 to 46 nm (Krishnamurthy et al. 2012; Kotakadi et al. 2014). AFM data analyzed by NOVA-TX software highlighted that A. caesia-synthesized Ag NPs showed a size ranging from 10.1 to 90.5 nm, with most of them falling within the range 40.2 to 70.4 nm (Fig. 5). At variance with our results, AFM studies on Hymenodictyon oxirense, Carissa carandas, and M. sylvestris-fabricated Ag NPs showed lower size ranges (i.e., mean size of 0.7–2.93, 1.65–7.44, and 3.5–13.9 nm, respectively) (Govindarjan and Benelli 2016b, c; Govindarajan et al. 2016h). This highlights the key role of screening different plant products as sources of reducing agents for nanosynthesis of mosquitocidal products.

Fig. 4
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SEM and TEM micrographs of Acacia caesia-synthesized Ag nanoparticles

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Fig. 5
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AFM micrograph of Ag nanoparticles fabricated using the aqueous extract of Acacia caesia leaves. a Studies of spherical poly-dispersed particles (2.5-μm resolution). 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 nano-Ag

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Toxicity against mosquito vectors

The employ of natural products as reducing agents to fabricate insecticidal nanocomposites is gaining research attention worldwide, due to low costs and high effectiveness (Benelli 2016a, b). In our study, the A. caesia leaf extract and synthesized Ag NPs exhibited dose-dependent acute larvicidal efficacy against all tested mosquito species (Tables 1 and 2, respectively). Compared to the aqueous extract, Ag NPs exhibited high effectiveness against An. subpictus (LC50 = 10.33 μg/ml) followed by Ae. albopictus (LC50 = 11.32 μg/ml), and Cx. tritaeniorhynchus (LC50 = 12.35 μg/ml) (Table 2). In ovicidal assays, egg hatchability was inversely proportional to the concentration of extract and Ag NPs and directly proportional to the eggs (Tables 3 and 4). Ag NPs completely inhibited egg hatchability on the three vectors at 60, 75, and 90 μg/ml, respectively. Control eggs showed 100% hatchability. Results of the adulticidal activity of A. caesia leaf extract and synthesized Ag NPs are presented in Tables 5 and 6. Highest adulticidal activity was observed for green-synthesized Ag NPs against An. subpictus (LD50 = 18.66 μg/ml), Ae. albopictus (LD50 = 20.94 μg/ml), and Cx. tritaeniorhynchus (LD50 = 22.63 μg/ml) (Tables 5 and 6).

Table 1 Acute toxicity of the Acacia caesia aqueous leaf extract on third instar larvae of Anopheles subpictus, Aedes albopictus, and Culex tritaeniorhynchus
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Table 2 Acute toxicity of silver nanoparticles fabricated using the Acacia caesia leaf extract on third instar larvae of Anopheles subpictus, Aedes albopictus, and tCulex tritaeniorhynchus
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Table 3 Ovicidal effectiveness of the Acacia caesia aqueous leaf extract on Anopheles subpictus, tAedes albopictus, and tCulex tritaeniorhynchus
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Table 4 Ovicidal effectiveness of silver nanoparticles fabricated using the Acacia caesia leaf extract on Anopheles subpictus, Aedes albopictus, and Culex tritaeniorhynchus
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Table 5 Adulticidal effectiveness of Acacia caesia aqueous leaf extract on Anopheles subpictus, Aedes albopictus, and Culex tritaeniorhynchus
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Table 6 Adulticidal effectiveness of silver nanoparticles fabricated using the Acacia caesia leaf extract on Anopheles subpictus, Aedes albopictus, and Culex tritaeniorhynchus
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In latest years, a growing number of green-fabricated metal nanoparticles have been produced for mosquitocidal purposes, showing that the biophysical properties of the produced nanoparticles are strongly affected by the botanicals used as reducing agents (Murugan et al. 2016a, b; Suresh et al. 2015; Panneerselvam et al. 2016; Subramaniam et al. 2016; see Benelli 2016a, b for reviews). Unfortunately, knowledge on their mechanism(s) of action is patchy and still requires further research (Benelli 2016b, d). In addition, scarce information is available on the ovicidal potential of metal nanoparticles fabricated using plant-borne products. On the other hand, more knowledge has been recently reported concerning the larvicidal and adulticidal potential of nanoformulated mosquitocidals (Veerakumar et al. 2014a, b; see reviews by Benelli 2016a, d). For instance, Raman et al. (2012) reported on the larvicidal activity of Ag NPs biosynthesized using Pithecellobium dulce. These nanoparticles showed effective larvicidal activity against the filariasis vector C. quinquefasciatus (LC50 = 21.56 mg l−1) due to high surface-to-volume ratio. Ag NPs biosynthesized using the aqueous extract of Solanum nigrum showed LC50 values of 1.33, 1.59, and 1.56 ppm for dry leaves, fresh leaves, and berries on the malaria vector An. stephensi (Rawani et al. 2013). Veerekumar et al. (2013) investigated the toxicity of Ag NPs synthesized from S. acuta leaves and their larvicidal activity on An. stephensi (LC50 = 21.92 μg ml−1), Ae. aegypti (LC50 = 23.96 μg ml−1), and Cx. quinquefasciatus (LC50 = 26.13 μg ml−1). Later on, Kumar et al. (2014) investigated the larvicidal potential of the Morinda tinctoria leaf aqueous extract and biosynthesized Ag NPs against the larvae of Cx. quinquefasciatus. Ag NP LC50 was 1.442 ppm against Cx. quinquefasciatus. Soni and Prakash (2014) reported the synthesis of effective Ag NPs using the leaf and bark extract of neem, A. indica. The biosynthesized Ag NPs were tested as larvicides, pupicides, and adulticides against An. stephensi and Cx. quinquefasciatus. The larvae of Cx. quinquefasciatus showed a 100% mortality rate after 30 min of exposure. In pupicidal assays against Cx. quinquefasciatus, the LC50 value was 4 μg ml−1. In the case of adult mosquitoes, LC50 of 1.06 μL cm−2 was obtained after 4 h of exposure. Notably, it has been argued that, after Ag NPs reached the larvae midgut epithelial membrane, enzymes were inactivated and generated peroxides lead to cell death (see also Benelli 2016a).

Toxicity on non-target aquatic insects and fishes

Here, the acute biotoxicity of A. caesia aqueous extract and green-synthesized Ag NPs was investigated testing them on three aquatic predators of mosquito young instars, including the important biocontrol agent G. affinis. Nanoparticle-based treatment achieved moderate toxicity against A. bouvieri, D. indicus, and G. affinis, with LC50 ranging from 684 to 2245 μg/ml (Tables 7 and 8). In addition, our continuous observations (lasting 10-day post-treatment) evidenced that longevity and swimming activity of the three biocontrol agents were not influenced for at least 10 days after testing. SI indicated that A. caesia-fabricated Ag NPs were less toxic to the non-target organism tested if compared to the targeted larval populations of the three mosquito vectors (Table 9).

Table 7 Acute toxicity of the Acacia caesia aqueous leaf extract on three non-target aquatic biocontrol organisms predating mosquito young instars
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Table 8 Acute toxicity of biogenic silver nanoparticles fabricated using the Acacia caesia leaf extract on three non-target aquatic biocontrol organisms predating mosquito young instars
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Table 9 Suitability index of three non-target aquatic biocontrol agents over young instars of Anopheles subpictus, tAedes albopictus, and Culex tritaeniorhynchus exposed to the Acacia caesia aqueous leaf extract and green silver nanoparticles
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While extensive research has focused on the mosquitocidal properties of plant mediated-synthesized Ag NPs, their impact against non-target mosquito predators has been evaluated only in a limited number of studies (Muthukumaran et al. 2015; Benelli 2016a; Govindarajan and Benelli 2016a; Pavela and Govindarajan 2016; Mahesh Kumar et al. 2016). For instance, Govindarajan et al. (2016h) recently showed little biotoxicity of M. sylvestris-synthesized AgNPs on non-target aquatic organisms D. indicus (LC50 = 813.16 μg/ml) and G. affinis (LC50 = 1044.52 μg/ml). In addition, an effective option for effective mosquito control may be the employ of biological control agents of mosquito young instars in the presence of ultra-low quantities of nanoformulated botanicals, which boost their predation rates (Murugan et al. 2015b; Benelli and Mehlhorn 2016).

Conclusions

Overall, the employ of natural products as reducing agents to fabricate insecticidal nanocomposites is gaining research attention worldwide, due to low costs, quick synthesis routes, and high effectiveness. Interestingly, biophysical features of green-synthesized nanoparticles strongly differ when different botanicals are employed for nanosynthesis, pointing out the value of screening local botanical resources as reducing and capping agents for nanomosquitocide production. In this study, we highlighted that a cheap A. caesia leaf extract can be used for effective and eco-friendly fabrication of Ag NPs with larvicidal, ovicidal, and adulticidal toxicity against three important mosquito vectors of medical and veterinary relevance, including the invasive Zika virus vector Ae. albopictus. XRD, SEM, TEM, and AFM showed that the obtained Ag NPs were crystalline, mostly spherical, and with a mean size of 40.2–70.4 nm. Notably, if compared to mosquito larvae, A. caesia-fabricated Ag NPs were risk-free to three mosquito predators. Overall, our study pointed out the promising utility of A. caesia as an abundant and cheap bioresource to synthesize Ag NPs effective against mosquito young instars and adults, with moderate impact on non-target aquatic biocontrol agents controlling young instar populations of Culicidae vectors.