Green-synthesised nanoparticles from Melia azedarach seeds and the cyclopoid crustacean Cyclops vernalis: an eco-friendly route to control the malaria vector Anopheles stephensi?

Abstract The impact of green-synthesised mosquitocidal nanoparticles on non-target aquatic predators is poorly studied. In this research, we proposed a single-step method to synthesise silver nanoparticles (Ag NP) using the seed extract of Melia azedarach. Ag NP were characterised using a variety of biophysical methods, including UV–vis spectrophotometry, scanning electron microscopy, energy-dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy. In laboratory assays on Anopheles stephensi, Ag NP showed LC50 ranging from 2.897 (I instar larvae) to 14.548 ppm (pupae). In the field, the application of Ag NP (10 × LC50) lead to complete elimination of larval populations after 72 h. The application of Ag NP in the aquatic environment did not show negative adverse effects on predatory efficiency of the mosquito natural enemy Cyclops vernalis. Overall, this study highlights the concrete possibility to employ M. azedarach-synthesised Ag NP on young instars of malaria vectors.


Introduction
Mosquitoes (Diptera: Culicidae) represent a key threat for millions of humans worldwide, since they act as vectors for malaria, dengue, yellow fever, West Nile virus, Japanese encephalitis and filariasis (Mehlhorn et al. 2012;Benelli 2015a). According to the latest estimates, there were about 198 million cases of malaria in 2013 and an estimated 584,000 deaths. Malaria mortality rates have fallen by 47% globally since 2000, and by 54% in the African region. Most deaths occur among children living in Africa, where a child dies every minute from malaria. Malaria is caused by Plasmodium parasites; they are vectored to people and animals through the bites of infected Anopheles mosquitoes, which bite mainly between dusk and dawn (WHO 2014).
Culicidae larvae and pupae are usually targeted using organophosphorus insecticides, insect growth regulators and microbial control agents. Indoors residual spraying and insecticide-treated bed nets are also employed to reduce transmission of malaria in tropical countries. However, synthetic chemicals have strong negative effects on human health and the environment, and induce resistance in a number of mosquito species (Benelli 2015a). In this scenario, eco-friendly control tools are urgently needed. In latest years, huge efforts have been carried out to investigate the efficacy of botanical products on mosquito vectors (Benelli 2015b); many plant-borne compounds have been reported as effective on Culicidae, acting as adulticidal, larvicidal, ovicidal, oviposition deterrent, growth and/or reproduction inhibitors and/or adult repellents (e.g. Nicoletti et al. 2012;Benelli et al. 2013;Benelli, Bedini, et al. 2015;. Melia azedarach, commonly known as white cedar, is a deciduous tree native to north-western India. In traditional medicine this plant is recognised for its helmintic, antilithiatic, diuretic and antiseptic properties. Furthermore, its seed oil showed good insecticidal activity on a number of insect pests, and this seems to be mainly due to a number of limonoids with ovicidal, larvicidal, growth-regulating and ovideterrent properties (D'Ambrosio & Guerriero 2002;Banchio et al. 2003;Wandscheer et al. 2004;Coria et al. 2008).
Nanobiotechnologies have the potential to revolutionise a wide array of applications, including drug delivery, diagnostics, imaging, sensing, gene delivery, artificial implants, tissue engineering and pest management (elechiguerra et al. 2005). The plant-mediated biosynthesis (i.e. "green synthesis") of nanoparticles is advantageous over chemical and physical methods, since it is cheap, single-step, does not require high pressure, energy, temperature and the use of highly toxic chemicals (Huang et al. 2007;Song and Kim 2009;Rahimi-Nasrabadi et al. 2014;Piryaei et al. 2015). In particular, a growing number of plants and fungi have proposed for efficient and rapid extracellular synthesis of metal nanoparticles (Rajan et al. 2015), which showed excellent mosquitocidal properties, also under field conditions (e.g. Amerasan et al. 2015;Dinesh et al. 2015;Suresh et al. 2015; Benelli 2016 for a review).
In this study, we reported a novel method to synthesise silver nanoparticles (Ag NP) using the seed extract of M. azedarach, a cheap, non-toxic and eco-friendly material, that worked as reducing and stabilising agent during the biosynthesis. Ag NP were characterised by UV-vis spectrophotometry, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SeM) and energy dispersive X-ray analysis (eDX). We investigated the larvicidal and pupicidal properties of M. azedarach seed extract and green-synthesised Ag NP on the malaria vector Anopheles stephensi. Furthermore, we evaluated the predation efficiency of the cyclopoid copepod Cyclops vernalis on second and third instar larvae of A. stephensi, both under normal laboratory conditions and in Ag NP-contaminated aquatic environment.

Green-synthesis and characterisation of silver nanoparticles
When the AgNO 3 aqueous solution was added to the M. azedarach seed extract, the colour changed from pale yellow to dark brown, indicating the reduction from Ag + to Ag 0 , and the formation of Ag NP (Supplementary Material Figure S1 a and b). The absorption spectra of Ag NP at different time intervals showed highly symmetric single band absorption peaks. A maximum absorption peak was observed at 420 nm (Supplementary Material Figure S1c); it steadily increased with reaction time, and saturated after 150 min indicating complete reduction of the silver nitrate. Ag NP have free electrons generating a surface plasmon resonance absorption band, due to the combined vibration of electrons of metal nanoparticles in resonance with the light wave (Noginov et al. 2007; see also Ramanibai & Velayutham 2015).
SeM (Supplementary Material Figure S2) showed that Ag NP were highly aggregated, with a mean size of 30-60 nm. For SeM analysis, the reaction mixture was air-dried on silicon wafers. As a result, a coffee-ring phenomenon was observed. Indeed, it is well known that when liquids containing fine particles evaporate on a flat surface, solid particles accumulate along the outer edge (Chen & evans 2009). eDX pattern (Supplementary Material Figure S3) showed the chemical composition of M. azedarach-synthesised Ag NP. It showed a strong silver signal, confirming the presence of metallic silver, in agreement with UV-vis results. Furthermore, the weak signals linked to oxygen are due to the occurrence of secondary metabolites present in the aqueous seed extract of M. azedarach. These metabolites are extremely important for nanobiosynthesis. Indeed, they often surround metallic nanoparticles with a thin layer of "capping" organic material from the plant leaf broth, and this enhances the Ag NP stability in the solution for several weeks after synthesis Suresh et al. 2015).
The FTIR spectrum of M. azedarach-synthesised Ag NP (Supplementary Material Figure S4) exhibited prominent peaks at 463 cm −1 (C-H bending alkenes), 596.97 cm −1 (C-O stretching alcohols), 657.36 cm −1 (N-H bending amines), 1638.68 and 2120.98 cm −1 (O-H stretching carboxylic acids) and 3347.53 cm −1 (N-H stretching due to amines group). In agreement with our results, the FTIR spectrum of aqueous Ag NP prepared from the Nerium oleander leaf extract shows transmittance peaks at 509.12, 1077.05, 1638.68, 2736.49, 2120.92 and 3347.53 cm −1 (Sathyavathi et al. 2010). Notably, M. azederach seeds contain a considerable amount of water-soluble furfural and furfural derivatives (Ntalli et al. 2010); these compounds may contribute to the reduction of Ag + to Ag 0 while carbonyl groups from amino acid residues could act as capping agents, preventing agglomeration of Ag NP, and thereby contribute to stabilise the medium (Amerasan et al. 2015;Murugan, Priyanka, et al. 2015;Suresh et al. 2015). The tested Ag NP were extremely stable in the water for more than 4 weeks.

Mosquitocidal assays on A. stephensi
Under laboratory conditions, the M. azedarach seed extract was toxic on A. stephensi young instars (Supplementary Material Table S1). LC 50 values were 46.073 (I instar larvae), 70.686 (II), 118.006 (III), 201.265 (IV) and 253.871 ppm (pupae). A dose-dependent effect was found, as previously described for other botanical-based mosquitocidals (Panneerselvam et al. 2013;Benelli et al. 2013;Benelli, Bedini, et al. 2015;. This result is in agreement with previous research, showing that the toxicity of M. azedarach seed extract on different mosquito vectors is mainly due to the presence of several limonoids (Senthil Nathan et al. 2006;Coria et al. 2008).
Furthermore, M. azedarach-synthesised Ag NP were highly toxic on A. stephensi. LC 50 were 2.897(I), 4.800(II), 6.554(III), 9.732(IV) and 14.548 ppm (pupae) (Supplementary Material Table  S2). Recently, a growing number of green-synthesised Ag NP showed comparable larvicidal and pupicidal toxicity on different mosquito vectors. For example, Suresh et al. (2015) highlighted that Ag NP synthesised using the aqueous extract of Phyllanthus niruri are highly effective on larvae and pupae of the dengue vector A. aegypti, with LC 50 values ranging from 3.90 ppm (I) to 13.04 ppm (pupae). Similarly, Ag NP produced using the filamentous fungus Cochliobolus lunatus were toxic on A. aegypti and A. stephensi larvae with LC 50 values ranging from 1.29(II) to 1.58 ppm (IV) and from 1.17 (II) to1.41 ppm (IV), respectively (Salunkhe et al. 2011). Moreover, low doses of Caulerpa scalpelliformis-synthesised Ag NP were toxic on the filariasis vector Culex quinquefasciatus, with LC 50 values ranging from 3.08 (I) to 7.33 ppm (pupae) .
In the field, the application of M. azedarach seed extract or Ag NP (10 × LC 50 ) leads to the complete elimination of A. stephensi larval populations after 72 h (Supplementary Material  Table S3). These results evidenced that plant extracts may be useful candidates to reduce larval populations of mosquito vectors in rural areas of the world. Indeed, plant extracts and essential oils are often cheap, easy to prepare and really effective on Culicidae (Benelli et al. 2013;Dinesh et al. 2015). For instance, Panneerselvam et al. (2013) reported that the leaf extract of Euphorbia hirta is highly effective in field trials on A. stephensi, since it leads to larval density reduction of 13.17, 37.64 and 84.00% after 24, 48 and 72 h, respectively. Different mechanisms of action have been proposed to explain the efficacy of plant-borne molecules on mosquito larvae. The thin film of oily substances from plant extracts on the water surface cuts of oxygen supply to mosquito larvae. In addition, a number of polar compounds may dissolve into the water and penetrate the larvae through the respiratory tube, killing them by suffocation and/or by poisoning. As regards to the field effectiveness of M. azedarach-synthesised Ag NP, we hypothesise that the good mortality rates exerted by green-synthesised Ag NP on larval populations of malaria mosquitoes may be due to the small size of Ag NP, which allows the passage through the insect's cuticle and even into individual cells, where they interfere with moulting and other physiological processes .

Impact of sub-lethal doses of silver nanoparticles on C. vernalis predation
Interestingly, the predatory efficiency of copepod C. vernalis was not reduced after a mosquitocidal treatment with sub-lethal doses of M. azedarach-synthesised Ag NP. Under standard laboratory conditions, the predation of C. vernalis after 24 h was 92.5 and 71.0%, on II and III instar A. stephensi larvae, respectively. In Ag NP-contaminated environment, the predation of C. vernalis was 96.5 (II) and 75.5% (III) (  (Ramanibai & Velayutham 2015). Furthermore, little knowledge is available about how sub-lethal low dosages of green-synthesised nanoparticles may impact al traits of aquatic organisms sharing the same ecological niche of mosquitoes, such as their predators (Murugan, Benelli, Ayyapan, et al. 2015;Murugan, Priyanka, et al. 2015;Murugan, Sanoopa, et al. 2015;Murugan, Venus, et al. 2015). Notably, these investigations recently unveiled fascinating scenarios. For instance,  showed that very low doses (i.e. 1 ppm) of lemongrass-synthesised gold nanoparticles may control malaria and dengue mosquitoes boosting copepod predation on early instar mosquito larvae in a gold nanoparticle 'contaminated' environment.

Conclusions
Overall, our study highlights the effectiveness of M. azedarach seed extract as a reducing and stabilising agent for the production of mosquitocidal Ag NP. Furthermore, the application of sub-lethal doses of Ag NP in the aquatic environment did not show negative adverse effects on predatory efficiency of the mosquito natural enemy C. vernalis. At variance with the recent work of Ramanibai and Velayutham (2015), where Ag NP have been synthesised using a 2,7.bis[2-[diethylamino]-ethoxy]fluorence isolate from the M. azedarach leaves, we believe that our green route is a simpler biosynthetical approach for Ag NP production, since the M. azedarach seeds are cheap and easy to find in many developing countries. This highlights the concrete possibility to employ M. azedarach-synthesised Ag NP to control young instar populations of the malaria vector A. stephensi. Table 1. Predation efficiency of the cyclopoid copepod C. vernalis against larvae of the malaria vector A. stephensi. experiments were conducted under standard laboratory conditions and post-treatment with an ultra-low dosage of M. azedarach-synthesised silver nanoparticles (i.e. 1 ppm).
Notes: Predation rates are means ± sd of five replicates (20 C. vernalis vs. 200 mosquito per replication). No predation in control (i.e. clean water without C. vernalis). Within each column, means followed by the same letter are not significantly different (p < 0.05).

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.

Informed consent
Informed consent was obtained from all individual participants included in the study.