Single-Step Biofabrication of Silver Nanocrystals Using Naregamia … · 2017. 8. 26. · Annamalai University, Annamalainagar, Tamil Nadu 608 002, India 3 Insect Behavior Group,

ORI GIN AL PA PER

Single-Step Biofabrication of Silver Nanocrystals UsingNaregamia alata: A Cost Effective and Eco-FriendlyControl Tool in the Fight Against Malaria, Zika Virusand St. Louis Encephalitis Mosquito Vectors

Raja Mohamed Sait Thameem Azarudeen1 •

Marimuthu Govindarajan2 • Abubucker Amsath1 •

Udaiyan Muthukumaran2 • Giovanni Benelli3

Received: 6 August 2016 / Published online: 25 August 2016

� Springer Science+Business Media New York 2016

Abstract Botanical-based nanosynthesis has been recently reported as a cheap

alternative for mosquito management. Screening different botanicals as reducing and

capping agents led to the production of metal nanoparticles with different biophys-

ical and mosquitocidal features. Here, Naregamia alata-mediated biosynthesis of

silver nanoparticles (AgNPs) was conducted. AgNPs were tested on egg, larval and

adult populations of three important mosquito vectors, Anopheles stephensi, Aedes

aegypti and Culex quinquefasciatus. AgNPs were characterized using UV–Vis

spectrophotometry, FTIR, AFM, SEM, TEM, EDX, and XRD analyses. Maximum

larvicidal toxicity was detected against A. stephensi (LC50 = 12.40 lg/ml), followed

by A. aegypti (LC50 = 13.57 lg/ml) and C. quinquefasciatus (LC50 = 14.84 lg/

ml). A single treatment with AgNPs tested at 60, 75 and 90 lg/ml led to no egg

hatchability. In adulticidal experiments, the maximum efficacy was observed on

A. stephensi (LD50 = 31.60 lg/ml), followed by A. aegypti (LD50 = 34.31 lg/ml)

and C. quinquefasciatus (LD50 = 37.52 lg/ml), respectively. AgNPs were safer for

three non-target mosquito natural enemies, Anisops bouvieri, Diplonychus indicus

and Gambusia affinis, with LC50 ranging from 629 to 2111 lg/ml. Overall, N. alata-

Journal of Cluster Science—Invited Contribution for the Special Issue ‘‘Applications of green-

synthesized nanoparticles in pharmacology, parasitology and entomology’’.

& Marimuthu Govindarajan

[email protected]

& Giovanni Benelli

[email protected]

1 Department of Zoology, Khadir Mohideen College, Adirampattinam, Tamil Nadu 614701, India

2 Unit of Vector Control, Phytochemistry and Nanotechnology, Department of Zoology,

Annamalai University, Annamalainagar, Tamil Nadu 608 002, India

3 Insect Behavior Group, Department of Agriculture, Food and Environment, University of Pisa,

via del Borghetto 80, 56124 Pisa, Italy

123

J Clust Sci (2017) 28:179–203

DOI 10.1007/s10876-016-1067-y

http://crossmark.crossref.org/dialog/?doi=10.1007/s10876-016-1067-y&domain=pdf

http://crossmark.crossref.org/dialog/?doi=10.1007/s10876-016-1067-y&domain=pdf

fabricated AgNPs are a promising and eco-friendly tool against Anopheles, Aedes

and Culex mosquito vectors, with negligible toxicity against non-target aquatic

organisms.

Keywords Green-synthesis � AFM � SEM � TEM � Neglected tropical diseases �Nanotechnology

Introduction

Mosquitoes (Diptera: Culicidae) pose an important threat to public health since of

their ability to act as vectors of pathogens and parasites causing malaria, lymphatic

filariasis, dengue, chikungunya, yellow fever, Zika virus and Japanese encephalitis.

These diseases are responsible for mortality, morbidity, economic loss and social

disruption [1–3]. Anopheles stephensi is the primary vector of malaria in India and

other west Asian countries. Every year, an estimated 300–500 million new

infections and 600,000 cases based on world malaria report 2013 [4] deaths result

from malaria worldwide. Malaria is a protozoan infection of erythrocytes caused in

human beings by five species of the genus Plasmodium (P. falciparum, P. vivax, P.

ovale, P. malariae, and P. knowlesi). In most cases, malaria is transmitted via the

bite of an infected female anopheline mosquito, but congenital malaria and

acquisition through infected blood transfusion are well described [5]. More than 40

per cent of the world’s population-approximately 3 billion people are exposed to

malaria in 108 endemic countries [6]. About one million cases of malaria are

reported in India every year.

Aedes mosquito borne diseases have become one of the major threats to human

population. Zika virus infection is a mosquito-borne illness like dengue (DEN) and

chikungunya (CHIK) viruses. A lot about the biology of vectors and its role in

disease transmission is known but Aedes mosquito-borne infections continue to be

the major cause of mortality in many subtropical and tropical countries. The Aedes-

borne infections like Zika are a potential threat, especially in urban settings where

A. aegypti is abundant. The growing population in urban settings also increased the

need for potable water, which necessitated storage practices in households, making

ideal breeding habitats for A. aegypti mosquitoes that also increase dengue and

CHIKV infections. Due to this mosquito-borne viral diseases are going to be a major

threat in the 21st century [7].

Culex quinquefasciatus is a vector of many pathogens and parasites of humans,

and both domestic and wild animals. Viruses transmitted by this species include

West Nile virus, St. Louis encephalitis virus and Western equine encephalitis virus.

C. quinquefasciatus is the principal vector of St. Louis encephalitis virus in the

southern U.S.A. The virus increases its numbers in birds and then infects

mosquitoes feeding on birds during the bird-nesting season in the spring. The

mosquito may then transmit the virus to humans. St. Louis encephalitis is age-

dependent, affecting older humans more than the young. Symptoms of the disease

are flu-like and can range from fever and headaches to stiffness and confusion. After

a period of several days the brain may begin to swell, accompanied by depression,

180 R. M. S. T. Azarudeen et al.

123

extreme excitement, sleepiness, or sleeplessness. Humans do not develop high

levels of the virus in the blood and therefore are considered dead-end hosts unable to

infect mosquitoes [8].

The use of synthetic insecticides to control mosquito vectors lead to resistance,

adverse environmental effects and high operational costs [1]. Therefore, eco-

friendly control tools are urgently needed [9]. In recent years, the green processes

for the synthesis of silver nanoparticles (AgNPs) are evolving into an important

branch of nanotechnology, due to low cost, simple procedures and absence of toxic

chemicals or energy inputs [9]. In particular, AgNPs can be used for purification of

drinking water, degradation of pesticides, to kill human pathogenic bacteria, as well

as novel mosquitocides [10].

Recently, a growing number of plants have been successfully used for efficient

and rapid extracellular synthesis of silver, copper and gold nanoparticles [11]. Good

examples include cheap extracts of neem, Azadirachta indica [12], Pongamia

pinnata [13], Feronia elephantum [14], Heliotropium indicum [15], Chomelia

asiatica [16], Sida acuta [17], Gmelina asiatica [18], and Barleria cristata [19].

Nanoparticles possess peculiar toxicity mechanisms due to surface modification

[20], and this may actively contribute to their excellent mosquitocidal potential

against Culicidae larvae [21, 22]. However, despite the increasing number of

evidences of plant-synthesized mosquitocidal nanoparticles, only moderate efforts

have been carried out to shed light on the nanoparticle biotoxicity on non-target

organisms sharing the same ecological niche of mosquito young instars [9].

Naregamia alata Weight & Arn. (Meliaceae) an undershurb with pungent,

aromatic roots. Distributed throughout South India in all districts up to 900 m. The

plant is acid, sweet, cooling, aromatic, alexeteric, vulnerary, emetic, cholagogue,

expectorant, depurative and antipyretic. It is useful in the treatments of wounds,

ulcers, vitiated conditions of pitta and vata, halitosis, cough, asthma, bronchitis,

splenomegaly, scabies, pruritus, dysentery, dyspepsia, catarrh, anaemia and malarial

fevers [23]. However, to the best of our knowledge, its mosquitocidal activity is

currently unknown.

In this research, we reported a route to biosynthesize silver nanoparticles

(AgNPs) using the aqueous leaf extract of the N. alata, a cheap and eco-friendly

material acting as reducing and stabilizing agent. AgNPs were characterized by

UV–Vis spectrophotometry, X-ray diffraction (XRD), Fourier transform infrared

spectroscopy (FTIR), atomic force microscopy (AFM), scanning electron micro-

scopy (SEM), energy-dispersive X-ray analysis (EDX) and transmission electron

microscopy (TEM). The aqueous extract of N. alata and the biosynthesized AgNPs

were tested for their ovicidal, larvicidal and adulticidal potential against the malaria

vector Anopheles stephensi, the dengue and Zika virus vector Aedes aegypti and the

filarasis and St. Louis encephalitis vector C. quinquefasciatus. Furthermore, we

evaluated the biotoxicity of N. alata aqueous extract and green-synthesized AgNPs

on three non-target aquatic natural enemies sharing the same ecological niche of

Anopheles and Aedes mosquitoes, Anisops bouvieri, Diplonychus indicus, and

Gambusia affinis.

Single-Step Biofabrication of Silver Nanocrystals Using… 181

123

Materials and Methods

Materials

Silver nitrate was purchased from Merck, India. The glassware was acid-washed

thoroughly and then rinsed with Millipore Milli-Q water. Healthy and fresh leaves

of N. alata (Fig. 1) were collected from Nilgiris, Western Ghats (11� 100N to 11�450N latitude and 76� 140E to 77� 20E longitude), Tamil Nadu State, India. The

identity was confirmed at the Department of Botany, Annamalai University,

Annamalai Nagar, Tamil Nadu. Voucher specimens were numbered and kept in our

laboratory and are available upon request (ID: AUDZ-815).

Preparation of Plant Leaf Extract

The leaves of N. alata were dried in the shade and ground to fine powder in an

electric grinder. Aqueous extract was prepared by mixing 50 g of dried leaf powder

with 500 ml of water (boiled and cooled distilled water) with constant stirring on a

magnetic stirrer. The suspension of dried leaf powder in water was left for 3 h and

filtered through Whatman no. 1 filter paper and the filtrate was stored in an amber-

colored airtight bottle at 10 �C temperature until testing.

Biosynthesis of Silver Nanoparticles

The broth solution of fresh leaves was prepared by taking 10 g of thoroughly

washed and finely cut leaves in a 300-ml Erlenmeyer flask along with 100 ml of

Fig. 1 Naregamia alata Wight& Arn. (Meliaceae)


123

sterilized double-distilled water and then boiling the mixture for 5 min before

finally decanting it. The extract was filtered with Whatman filter paper no. 1, stored

at -15 �C and tested within a week. The filtrate was treated with aqueous 1 mM

AgNO3 (21.2 mg of AgNO3 in 125 ml of Milli-Q water) solution in an Erlenmeyer

flask and incubated at room temperature. Eighty-eight milliliters of an aqueous

solution of 1 mM silver nitrate was reduced using 12 ml of leaf extract at room

temperature for 10 min, resulting in a brown–yellow solution indicating the

formation of AgNPs.

Characterization of Silver Nanoparticles

The bioreduction of Ag? ions was monitored using a UV–Vis spectrophotometer

(UV-160v, Shimadzu, Japan). Analysis on size, morphology, agglomeration pattern

and dispersed nature of AgNPs were performed by atomic force microscopy

(Agilent Technologies AFM- 5500), scanning electron microscopy (Hitachi S3000

H SEM) and transmission electron microscopy (TEM Technite 10 Philips). The

purified AgNPs were examined for the presence of biomolecules using FTIR

spectroscopy (Thermo Scientific Nicolet 380 FT-IR Spectrometer) KBr pellets and

crystalline AgNPs were determined by XRD analysis.

Mosquito Rearing

Following the method by Govindarajan and Benelli [19, 24, 25], laboratory-bred

pathogen-free strains of mosquitoes were reared in the vector control laboratory,

Department of Zoology, Annamalai University. At the time of adult feeding, these

mosquitoes were 3–4 days old after emergences (maintained on raisins and water)

and were starved for 12 h before feeding. Each time, 500 mosquitoes per cage were

fed on blood using a feeding unit fitted with Parafilm as membrane for 4 h. A.

aegypti feeding was done from 12 noon to 4.00 p.m. and A. stephensi and C.

quinquefasciatus were fed during 6.00 p.m. to 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 slaughter house by collecting in a heparinized vial and stored at

4 �C) and kept over a netted cage of mosquitoes. The blood was stirred continuously

using an automated stirring device, and a constant temperature of 37 �C were

maintained using a water jacket circulating system. After feeding, the fully

engorged females were separated and maintained on raisins. Mosquitoes were held

at 28 ± 2 �C, 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 AgNPs from N. alata was evaluated

according to WHO protocol [26]. Based on the wide range and narrow range tests,

aqueous crude extract was tested at 80, 160, 240, 320 and 400 lg ml-1


123

concentrations and AgNPs were tested at 6, 12, 18, 24 and 30 lg ml-1

concentrations. Twenty numbers of late III instar larvae were introduced into a

500-ml glass beaker containing 249 ml of dechlorinated water, and 1 ml of desired

concentrations of leaf extract or AgNPs was added. For each concentration, five

replicates were performed, for a total of 100 larvae. Larval mortality was recorded

at 24 h after exposure, during which no food was given to the larvae. Each test

included a set control groups (silver nitrate and distilled water) with five replicates

for each individual concentration [24].

Ovicidal Activity

To evaluate the ovicidal potential of the leaf extract and AgNPs, the method of Su

and Mulla [27] with slight modification by Govindarajan et al. [28] was followed.

Eggs were collected from vector control laboratory, Department of Zoology,

Annamalai University. The aqueous leaf extracts and silver nanoparticle were to

achieve various concentrations ranging from 60 to 360 lg/ml and 15 to 90 lg/ml,

respectively. Eggs of these mosquito species (n = 100 for 0–6, 6–12 and 12–18 h

old egg rafts) were exposed to each concentration of leaf aqueous extract and

AgNPs. After treatment, the eggs from each concentration were individually

transferred to distilled water cups for hatching assessment after counting the eggs

under a photomicroscope (Leica, Germany). Each experiment was replicated six

times along with appropriate control. The hatch rates were assessed 48 h post-

treatment by the following formula.

Egg hatchability %ð Þ ¼ Number of hatched larvae

Total number of eggs� 100

Adulticidal Activity

Adulticidal bioassay was performed following the method by WHO [29] with minor

modifications by Veerakumar and Govindarajan [30]. Based on the wide range and

narrow range tests, aqueous crude extract was tested at 120, 240, 360, 480 and

600 lg ml-1 concentrations, and AgNPs were tested at 15, 30, 45, 60, 75, and

60 lg ml-1 concentrations. Aqueous crude extract and AgNPs were applied on

Whatman no. 1 filter papers (size 12 9 15 cm). Control papers were treated with

silver nitrate and distilled water. Twenty female mosquitoes were collected and

gently transferred into a plastic holding tube. The mosquitoes were allowed to

acclimatize in the holding tube for 1 h and then exposed to test paper for 1 h. At the

end of exposure period, the mosquitoes were transferred back to the holding tube

and kept for 24-h recovery period. A pad of cotton soaked with 10 % glucose

solution was placed on the mesh screen. Each test included a set control groups

(silver nitrate and distilled water) with five replicates for each individual

concentration.


123

Biotoxicity on Non-target Mosquito Natural Enemies

The effect of non-target organisms was assessed following the method by

Sivagnaname and Kalyanasundaram [31]. The effect of aqueous extract and AgNPs

of the potential plant was tested against non-target mosquito natural enemies A.

bouvieri, D. indicus and G. affinis. The species were field collected and separately

maintained in cement tanks (85 cm diameter and 30 cm depth) containing water at

27 ± 3�C and external relative humidity 85 %.

The aqueous extract and Ag NP of N. alata were evaluated at concentrations of

even 50 times higher the LC50 dose for mosquito larvae. Ten replicates will be

performed for each concentration along with four replicates of untreated controls.

The non-target organisms were observed for mortality and other abnormalities such

as sluggishness and reduced swimming activity after 48 h exposure. The exposed

non-target organisms were also observed continuously for 10 days to understand the

post treatment effect of this extract on survival and swimming activity.

Data Analysis

Larval and adult mortality data were subjected to probit analysis. LC50 (LD50) and

LC90 (LD90) were calculated using the method by Finney [32]. Ovicidal data were

analyzed by two-way ANOVA followed by Tukey’s HSD test. In experiments

evaluating biotoxicity on non-target organisms, the Suitability Index (SI) was

calculated for each non-target species using the following formula [33].

SI ¼ LC50 of non � target organisms

LC50 of target vector species

All data were analyzed using the SPSS Statistical Software Package version 16.0.

A probability level of P\ 0.05 was used for the significance of differences between

values.

Results and Discussion

Characterization of Silver Nanoparticles

The biosynthesis of AgNPs was confirmed within 2 h after that the N. alata extract

was added to AgNO3 solution. The color of the leaf extract changed from colorless

to yellowish brown (Fig. 2a), and this can be due to excitation of surface plasmon

vibrations in green-synthesized AgNPs [12]. The formation of AgNPs was

confirmed through the presence of an absorption peak at 473 nm (Fig. 2b). Our

UV–Vis results are in agreement with previous research [34, 35], where the AgNPs

were observed as stable in solution and also showed little aggregation. Besides, the

plasmon bands were broadened with an absorption tail in longer wavelengths; this

could be related to the size distribution of nanoparticles [36].

The crystalline nature of AgNPs was studied by XRD analysis (Fig. 3). The

obtained XRD patterns confirmed the crystalline nature of green synthesized


123

AgNPs. Four diffraction peaks were observed at 38.6, 45.3, 64.5 and 77.2 represent

the (111), (200), (220) and (311), reflections and the face-centered cubic structure of

metallic silver, respectively. Our findings are in agreement with previous research

conducted on AgNPs green-synthesized using the leaf extract of S. acuta [17].

Similar phenomena were reported earlier where AgNPs biofabricated using

Trianthema decandra, showed interplanar distance corresponds to mixed phase of

cubic and hexagonal structures [37].

The FTIR spectrum of biosynthesized AgNPs by using N. alata leaf extract is

shown in Fig. 4. In order to identify the biomolecules responsible for reduction and

efficient stabilization of the metal nanoparticles, it was highlighted that the band at

3148 cm-1 may correspond to O–H, as well as to H-bonded alcohols and phenols.

Fig. 2 a Color intensity ofNaregamia alata aqueousextract before and after thereduction of silver nitrate(1 mM). The color changeindicates Ag? reduction toelemental nanosilver. b UV–Visspectrum of silver nanoparticlesafter 180 min from the reaction(Color figure online)


123

Shanmugam et al. [38] suggested that these bonds could be due to the stretching of

-OH in proteins, enzymes or polysaccharides present in the plant extract. The peak

at 2201 cm-1 may be lined with the presence of carboxylic acids [39]. Shoulder

peaks at 1554 cm-1 probably indicate that the amide I and amide II arisen, due to

carbonyl and –NH stretch vibrations in the amide linkages of the proteins,

respectively. The band at 1467 and1383 cm-1 may correspond to C–C stretching

of aromatic amine. The band at 1111 and 1013 cm-1 probably indicate the

presence of C–O stretching alcohols, carboxylic acids, esters and ethers. The peak

near 663 cm-1 may be assigned to CH out of plane bending vibrations of

substituted ethylene systems –CH=CH. Our findings are in agreement with Ag NP

synthesized using the leaf extract of Sida acuta [17]. The immediate reduction of

silver ions in the present investigation might be linked with the presence of water-

soluble phytochemicals such as flavones, quinones, and organic acids present in

the leaves of N. alata.

AFM is a primary tool for analyzing size, shape, agglomeration pattern and offers

visualizations of three-dimensional views of the nanoparticles unlike the electron

microscopes. It has an advantage over combination of high resolution, samples does

not have to be conductive and does not require the high-pressure vacuum conditions

[40]. 2.5 lm resolution studies of green-synthesized AgNPs with AFM reveal the

particles are poly-dispersed, spherical in shape, having the size range from 0 to

5.5 nm and there is no agglomeration observed between the particles (Fig. 5a). Raw

data obtained from AFM microscope were treated with a specially designed image

processing software (NOVA-TX) to further exploit the 3D image of nanoparticles

(Fig. 5b). The average particle size obtained from the corresponding diameter

distribution was about 5 nm (Fig. 5c, d).

Fig. 3 XRD pattern of silver nanoparticles fabricated using the Naregamia alata aqueous extract


123

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123

SEM of AgNPs showed that AgNPs were mostly spherical or with cubic

structures (Fig. 6). We also noted that ‘‘capped’’ AgNPs were stable in solution for

at least 8 weeks, and Fig. 7 showed a representative profile of the spot EDX

analysis. As a general trend, the particle shape of plant-mediated AgNPs was

spherical, with exception of some neem-synthesized AgNPs. They are poly-

dispersed, with spherical or flat, plate-like, morphology, and mean size range of

5–35 nm in size [12]. SEM images of AgNPs fabricated using Emblica officinalis

were also predominantly spherical with an average size of 16.8 nm ranging from 7.5

to 25 nm [41]. Figure 8 showed the TEM of AgNPs biosynthesized using N. alata

leaf extract. Among shapes, spheres dominated with an average size of 35 nm,

which was slightly higher if compared to AFM observations. Most of the AgNPs

was roughly circular in shape with smooth edges. In agreement with these findings,

AgNPs from Annona squamosa leaf extract were spherical in shape with an average

size ranging from 20 to 100 nm [42] while Thirunavokkarasu et al. [43] reported the

fabrication of spherical nanoparticles with size ranging from 8 to 90 nm in

Desmodium gangeticum. TEM images showed that the surfaces of the AgNPs were

surrounded by a black thin layer of some material, which might be due to the

capping organic constituents from the plant extract, as also highlighted by Rafiuddin

[44].

Larvicidal Activity Against Mosquito Vectors

In laboratory conditions, the N. alata aqueous leaf extract showed moderate

larvicidal properties against A. stephensi, A. aegypti and C. quinquefasciatus; LC50

values were 165.15, 179.17 and 196.48 lg/ml, respectively (Table 1). Furthermore,

the N. alata-synthesized AgNPs were highly toxic against A. stephensi, A. aegypti

and C. quinquefasciatus larvae; LC50 values were 12.40, 13.57 and 14.84 lg/ml,

respectively (Table 2). N. alata aqueous leaf extract and AgNPs exerted 100 %

mortality (zero hatchability) when tested at 240, 300, 360 and 60, 75, 90 lg/ml,

respectively. Control eggs showed 100 % egg hatchability (Tables 3, 4). The results

of the adulticidal activity of aqueous crude extract and synthesized AgNPs against

the adult of A. stephensi, A. aegypti, and C. quinquefasciatus are presented in

Tables 5 and 6. Considerable mortality was evident after the treatment of N. alata

for all three important vector mosquitoes. At higher concentrations, the adult

showed restless movement for some times with abnormal wagging and then died.

The rates of mortality were directly proportional to concentration. The maximum

efficacy was observed in green synthesized AgNPs against the adult of A. stephensi

(LD50 = 31.60 lg/ml), followed by A. aegypti (LD50 = 34.31 lg/ml), and C.

quinquefasciatus (LD50 = 37.52 lg/ml) respectively. In agreement with Marimuthu

et al. [45] and Govindarajan and Benelli [24], no mortality was observed in the

positive and negative controls.

Recently, a great number of plant extracts have been found effective against

mosquito larval instars, with a dose-dependent effect [17, 18, 22]. In latest years,

several plant-synthesized AgNPs have been studied for their larvicidal activity

against important mosquito vectors [22]. For instance, comparable toxicity rates

have been recently reported for AgNPs synthesized using Chomelia asiatica against


123

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A. stephensi larvae (LC50 = 17.95 ppm) [16]. The mortality effect evoked by

AgNPs on mosquito larvae and pupae may be due by the small size of the AgNPs,

which allows their passage through the insect cuticle and into individual cells,

Fig. 6 Scanning electron microscopy (SEM) of Naregamia alata-fabricated silver nanoparticles

Fig. 7 Energy dispersive X-ray (EDX) spectrum of Naregamia alata-synthesized silver nanoparticlesshowing presence of different phyto-elements as capping agents


123

where they interfere with molting and other physiological processes. To our mind,

the residual toxicity of silver ions against mosquito larvae covered a little role, since

the peak in Fig. 1c was saturated after 120 min, indicating complete reduction of

silver nitrate [17].

Biotoxicity on Non-target Organisms

The biotoxicity of N. alata aqueous extract and green-synthesized AgNPs on non-

target mosquito predators A. bouvieri, D. indicus and G. affinis is presented in

Tables 7 and 8, respectively. Both toxicity treatments achieved negligible toxicity

against A. bouvieri, D. indicus and G. affinis, with LC50 values ranging from 629 to

29,633 lg/ml (Tables 7, 8). Focal observations highlighted that longevity and

swimming activity of the study species were not altered for a week after testing. SI

indicated that N. alata-fabricated AgNPs were less toxic to the non-target organism

tested if compared to the targeted mosquito larval populations (Table 9).

Nowadays, moderate knowledge is available about the acute toxicity of

mosquitocidal nanoparticles towards non-target aquatic species [22, 46–48].

Notably, Pergularia rubra- and Pergularia daemia-synthesized AgNPs did not

exhibit any evident toxicity effect against Poecilia reticulata fishes, after 48 h of

exposure to LC50 and LC90 values calculated on IV instar larvae of A. aegypti and A.

stephensi [49]. Similarly, Haldar et al. [50] did not detected toxicity of AgNPs

produced using dried green fruits of D. roxburghii against P. reticulata, after 48 h-

Fig. 8 Trasmission electron microscopy (TEM) of silver nanoparticles fabricated using the Naregamiaalata aqueous leaf extract


123

Table

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(lg

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40

09

9.5

±0

.8

Ae.

aegypti

80

23

.4±

1.2

17

9.1

7(1

60

.93

–1

95

.66)

34

2.2

1(3

17

.86

–3

74

.16

)2

.75

y=

6.3

5?

0.2

37

x2

.349

(4)

n.s

.1

60

44

.6±

0.8

24

06

5.3

±0

.6

32

08

4.5

±0

.4

40

09

8.2

±0

.6

Cx.quinquefasciatus

80

20

.6±

0.4

19

6.4

8(1

78

.01

–2

13

.56)

37

1.5

0(3

44

.48

–4

07

.39

)2

.64

y=

2.7

7?

0.2

35

x2

.602

(4)

n.s

.1

60

41

.2±

1.2

24

05

9.8

±0

.8

32

07

8.3

±0

.6

40

09

6.1

0±

0.4

No

mort

alit

yw

aso

bse

rved

inth

eco

ntr

ol

SD

stan

dar

dd

evia

tio

n,LC50

leth

alco

nce

ntr

atio

nth

atkil

ls50

%o

fth

eex

pose

dorg

anis

ms,LC90

leth

alco

nce

ntr

atio

nth

atk

ills

90

%o

fth

eex

po

sed

org

anis

ms,UCL

95

%

up

per

con

fid

ence

lim

it,LCL

95

%lo

wer

con

fid

ence

lim

it,v2

Chi

squ

are,

d.f.

deg

rees

of

free

dom

,n.s.

no

tsi

gn

ifica

nt

(a=

0.0

5)

aV

alu

esar

em

ean±

SD

of

five

repli

cate

s


123

Table

2L

arvic

idal

acti

vit

yof

bio

gen

icsi

lver

nan

opar

ticl

esfa

bri

cate

dusi

ng

theNaregamia

alata

leaf

extr

act

agai

nst

the

mal

aria

vec

torAnopheles

stephensi,

the

Zik

a

vir

us

and

den

gue

vec

torAedes

aegypti,

and

the

fila

rias

isan

dS

t.L

ou

isen

cep

hal

itis

vec


Mosq

uit

osp

ecie

sC

once

ntr

atio

n

(lg

/ml)

Mo

rtal

ity

(%)±

SD

aL

C50

(lg

/ml)

(LC

L–

UC

L)

LC

90

(lg/m

l)(L

CL

–U

CL

)S

lope

Reg

ress

ion

equat

ion

v2(d.f.)

An.stephensi

62

8.5

±1

.21

2.4

0(1

1.0

2–

13

.63

)2

4.2

0(2

2.4

6–

26

.49

)3

.04

y=

10

.66?

3.0

87

x5

.38

7(4

)

n.s

.1

24

6.3

±0

.6

18

67

.8±

0.8

24

88

.5±

0.4

30

10

0.0

±0

.0

Ae.

aegypti

62

4.1

±0

.41

3.5

7(1

2.2

2–

14

.79

)2

5.7

1(2

3.8

9–

28

.09

)2

.67

y=

5.5

2?

3.1

83

x2

.92

9(4

)

n.s

.1

24

2.8

±0

.8

18

63

.4±

0.6

24

85

.6±

1.2

30

98

.2±

0.4

Cx.quinquefasciatus

62

1.3

±0

.61

4.8

4(1

3.5

1–

16

.08

)2

7.4

9(2

5.5

4–

30

.06

)2

.45

y=

1.6

4?

3.2

2x

2.5

71

(4)

n.s

.1

23

8.6

±0

.4

18

60

.2±

1.2

24

81

.4±

0.8

30

96

.5±

0.6

No

mort

alit

yw

aso

bse

rved

inth

eco

ntr

ol

SD

stan

dar

dd

evia

tio

n,LC50

leth

alco

nce

ntr

atio

nth

atkil

ls50

%o

fth

eex

pose

dorg

anis

ms,LC90

leth

alco

nce

ntr

atio

nth

atk

ills

90

%o

fth

eex

po

sed

org

anis

ms,UCL

95

%

up

per

con

fid

ence

lim

it,LCL

95

%lo

wer

con

fid

ence

lim

it,v2

Chi

squ

are,

d.f.

deg

rees

of

free

dom

,n.s.

no

tsi

gn

ifica

nt

(a=

0.0

5)

aV

alu

esar

em

ean±

SD

of

five

repli

cate

s


123

Table

3O

vic

idal

acti

vit

yofNaregamia

alata

aqu

eous

leaf

extr

act

agai

nst

the

mal

aria

vec

torAnopheles

stephensi,

the

Zik

av

iru

san

dd

eng

ue

vec

torAedes

aegypti,

and

the

fila

rias

isan

dS

t.L

ou

isen

cep

hal

itis

vec


Mo

squ

ito

spec

ies

Ag

eo

fth

eeg

g

raft

/eggs

(h)

Egg

hat

chab

ilit

y(%

)F

val

ue

(d.f.)

Pv

alue

Co

ntr

ol

60lg

/ml

12

0lg

/ml

18

0lg

/ml

24

0lg

/ml

30

0l

g/m

l3

60l

g/m

l

An.stephensi

0–

61

00±

0.0

27

.4±

1.5

18

.6±

0.7

NH

NH

NH

NH

11

8.2

4(5

)\

0.0

01

6–

12

10

0±

0.0

37

.4±

1.9

22

.8±

1.2

NH

NH

NH

NH

12

5.5

4(5

)\

0.0

01

12

–18

10

0±

0.0

49

.6±

1.7

38

.3±

1.5

20

.6±

0.3

NH

NH

NH

13

2.3

6(5

)\

0.0

01

Ae.

aegypti

0–

61

00±

0.0

42

.7±

1.4

28

.5±

1.9

19

.5±

0.4

NH

NH

NH

16

1.4

7(5

)\

0.0

01

6–

12

10

0±

0.0

56

.3±

1.8

39

.4±

1.5

22

.5±

1.7

NH

NH

NH

12

6.1

5(5

)\

0.0

01

12

–18

10

0±

0.0

68

.4±

0.2

56

.8±

1.6

39

.7±

0.4

20

.6±

1.9

NH

NH

16

0.9

5(5

)\

0.0

01

Cx.

quinquefasciatus

0–

61

00±

0.0

69

.8±

1.9

47

.4±

0.7

32

.2±

1.5

18

.5±

1.3

NH

NH

11

4.2

6(5

)\

0.0

01

6–

12

10

0±

0.0

75

.3±

1.5

62

.8±

0.3

45

.6±

1.8

27

.2±

0.6

NH

NH

13

3.5

6(5

)\

0.0

01

12

–18

10

0±

0.0

87

.6±

0.2

75

.2±

1.6

57

.6±

1.9

42

.8±

1.4

23

.5±

0.7

NH

12

5.7

5(5

)\

0.0

01

Wit

hin

each

row

,d

iffe

ren

tle

tter

sin

dic

ate

sign

ifica

nt

dif

fere

nce

sam

on

gv

alu

es(A

NO

VA

,T

uk

ey’s

HS

D,

P\

0.0

01

)

NH

no

hat

chab

ilit

y,d.f.

deg

rees

of

free

dom


123

Table

4O

vic

idal

acti

vit

yof

silv

ernan

opar

ticl

essy

nth

esiz

edusi

ng

theNaregamia

alata

leaf

extr

act

agai

nst

the

mal

aria

vec

torAnopheles

stephensi,

the

Zik

av

iru

san

d

den

gue

vec

torAedes

aegypti,

and

the

fila

rias

isan

dS

t.L

ou

isen

cep

hal

itis

vec


Mo

squ

ito

spec

ies

Ag

eo

fth

eeg

g

raft

/eggs

(h)

Egg

hat

chab

ilit

y(%

)F

val

ue

(d.f.)

Pv

alue

Co

ntr

ol

15lg

/ml

30lg

/ml

45lg

/ml

60l

g/m

l7

5l

g/m

l9

0l

g/m

l

An.stephensi

0–

61

00±

0.0

28

.5±

1.6

16

.7±

1.9

NH

NH

NH

NH

16

2.5

8(5

)\

0.0

01

6–

12

10

0±

0.0

34

.7±

1.7

22

.4±

1.6

NH

NH

NH

NH

15

5.2

6(5

)\

0.0

01

12

–18

10

0±

0.0

46

.1±

0.2

34

.8±

0.3

20

.6±

1.5

NH

NH

NH

13

6.2

5(5

)\

0.0

01

Ae.

aegypti

0–

61

00±

0.0

41

.3±

1.7

27

.4±

1.6

18

.7±

1.8

NH

NH

NH

18

5.3

7(5

)\

0.0

01

6–

12

10

0±

0.0

49

.7±

1.3

39

.6±

0.8

23

.5±

1.2

NH

NH

NH

16

1.3

9(5

)\

0.0

01

12

–18

10

0±

0.0

64

.6±

1.8

53

.9±

1.4

38

.4±

1.9

21

.4±

0.6

NH

NH

13

7.5

6(5

)\

0.0

01

Cx.

quinquefasciatus

0–

61

00±

0.0

65

.8±

1.9

48

.3±

1.6

26

.9±

1.4

19

.2±

1.8

NH

NH

15

7.7

3(5

)\

0.0

01

6–

12

10

0±

0.0

75

.5±

1.4

56

.4±

0.5

39

.6±

1.7

24

.3±

1.2

NH

NH

14

6.1

4(5

)\

0.0

01

12

–18

10

0±

0.0

87

.9±

1.2

69

.6±

1.3

58

.2±

0.5

37

.5±

1.6

23

.7±

1.4

NH

16

3.9

1(5

)\

0.0

01

Wit

hin

each

row

,d

iffe

ren

tle

tter

sin

dic

ate

sign

ifica

nt

dif

fere

nce

sam

on

gv

alu

es(A

NO

VA

,T

uk

ey’s

HS

D,

P\

0.0

01

)

NH

no

hat

chab

ilit

y,d.f.

deg

rees

of

free

dom


123

Table

5A

dult

icid

alac

tivit

yofNaregamia

alata

aqu

eous

leaf

extr

act

agai

nst

the

mal

aria

vec

torAnopheles

stephensi,th

eZ

ika

vir

us

and

den

gue

vec

torAedes

aegypti,an

d

the

fila

rias

isan

dS

t.L

ou

isen

cep

hal

itis

vec


Mo

squ

ito

spec

ies

Con

cen

trat

ion

(lg

/ml)

Mo

rtal

ity

(%)±

SD

aL

D50

(lg

/ml)

(LC

L–

UC

L)

LD

90

(lg/m

l)(L

CL

–U

CL

)S

lope

Reg

ress

ion

equat

ion

v2(d

.f.)

An.stephensi

12

02

7.6

±1

.22

47

.59

(22

0.5

1–

271

.71

)4

77

.69

(44

3.6

4–

522

.21

)2

.88

y=

10

.24?

0.1

56

x4

.732

(4)

n.s

.2

40

46

.8±

0.8

36

06

8.5

±0

.4

48

08

9.4

±0

.6

60

01

00

.0±

0.0

Ae.

aegypti

12

02

3.9

±0

.62

71

.25

(24

4.8

3–

295

.35

)5

08

.78

(47

3.1

4–

555

.34

)2

.56

y=

4.9

3?

0.1

61

x3

.502

(4)

n.s

.2

40

42

.5±

0.8

36

06

3.2

±1

.2

48

08

6.4

±0

.4

60

09

8.6

±0

.8

Cx.

quinquefasciatus

12

01

9.8

±0

.42

96

.43

(27

0.3

7–

320

.78

)5

42

.55

(50

4.8

5–

591

.92

)2

.35

y=

0.1

9?

0.1

64

x1

.372

(4)

n.s

.2

40

38

.2±

1.2

36

05

9.6

±0

.8

48

08

2.3

±0

.6

60

09

6.1

±1

.2

No

mort

alit

yw

aso

bse

rved

inth

eco

ntr

ol

SD

stan

dar

dd

evia

tio

n,LC50

leth

alco

nce

ntr

atio

nth

atkil

ls50

%o

fth

eex

pose

dorg

anis

ms,LC90

leth

alco

nce

ntr

atio

nth

atk

ills

90

%o

fth

eex

po

sed

org

anis

ms,UCL

95

%

up

per

con

fid

ence

lim

it,LCL

95

%lo

wer

con

fid

ence

lim

it,v2

Chi

squ

are,

d.f.

deg

rees

of

free

dom

,n.s.

no

tsi

gn

ifica

nt

(a=

0.0

5)

aV

alu

esar

em

ean±

SD

of

five

repli

cate

s


123

Table

6A

du

ltic

idal

acti

vit

yo

fsi

lver

nan

opar

ticl

essy

nth

esiz

edu

sin

gNaregamia

alata

leaf

extr

act

agai

nst

the

mal

aria

vec

torAnopheles

stephensi,

the

Zik

av

iru

san

d

den

gue

vec

torAedes

aegypti,

and

the

fila

rias

isan

dS

t.L

ou

isen

cep

hal

itis

vec


Mo

squ

ito

spec

ies

Con

cen

trat

ion

(lg

/ml)

Mo

rtal

ity

(%)±

SD

aL

D50

(lg

/ml)

(LC

L–

UC

L)

LD

90

(lg/m

l)(L

CL

–U

CL

)S

lope

Reg

ress

ion

equat

ion

v2(d

.f.)

An.stephensi

15

26

.4±

1.2

31

.60

(28

.19–

34

.65

)6

0.9

4(5

6.5

9–

66

.65

)2

.88

y=

9.4

1?

1.2

49

x6

.03

8(4

)

n.s

.3

04

8.2

±0

.8

45

65

.1±

0.6

60

88

.3±

0.4

75

10

0.0

±0

.0

Ae.

aegypti

15

23

.7±

0.4

34

.31

(30

.84–

37

.46

)6

5.8

9(6

1.1

2–

72

.19

)2

.82

2y=

6?

1.2

48

x4

.76

7(4

)

n.s

.3

04

5.3

±0

.8

45

60

.1±

1.2

60

83

.5±

0.6

75

98

.2±

0.8

Cx.

quinquefasciatus

15

19

.6±

0.6

37

.52

(34

.11–

40

.69

)7

0.0

6(6

4.9

9–

76

.77

)2

.52

y=

1.4

7?

1.2

66

x4

.13

2(4

)

n.s

.3

04

1.8

±0

.4

45

56

.3±

1.2

60

78

.1±

0.8

75

96

.4±

0.6

No

mort

alit

yw

aso

bse

rved

inth

eco

ntr

ol

SD

stan

dar

dd

evia

tio

n,LC50

leth

alco

nce

ntr

atio

nth

atkil

ls50

%o

fth

eex

pose

dorg

anis

ms,LC90

leth

alco

nce

ntr

atio

nth

atkil

ls90

%o

fth

eex

pose

dorg

anis

ms,UCL

95

%

up

per

con

fid

ence

lim

it,LCL

95

%lo

wer

con

fid

ence

lim

it,v2

Chi

squ

are,

d.f.

deg

rees

of

free

dom

,n.s.

no

tsi

gn

ifica

nt

(a=

0.0

5)

aV

alu

esar

em

ean±

SD

of

five

repli

cate

s


123

Table

7A

cute

tox

icit

yo

fNaregamia

alata

aqu

eous

leaf

extr

act

on

thre

en

atu

ral

enem

ies

ofAnopheles,Aedes

andCulex

mosq

uit

ov

ecto

rs

No

n-t

arg

et

org

anis

m

Conce

ntr

atio

n

(lg

/ml)

Mo

rtal

ity

(%)±

SD

aL

C50

(lg

/ml)

(LC

L–

UC

L)

LC

90

(lg/m

l)(L

CL

–U

CL

)S

lope

Reg

ress

ion

equat

ion

v2(d.f.)

A.bouvieri

50

00

28

.3±

1.2

10

,40

9.2

6(9

24

6.1

7–

11

,441

.93

)2

0,3

68

.43

(18

,894

.54

–2

2,3

04

.61

)3

.09

y=

10

.52?

0.0

04x

5.5

93

(4)

n.s

.1

0,0

00

46

.4±

0.8

15

,00

06

7.2

±0

.6

20

,00

08

7.6

±0

.4

25

,00

01

00

.0±

0.0

D.indicus

70

00

26

.9±

0.6

14

,64

4.2

9(1

3,0

46

.01–

16

,06

8.8

1)

28

,35

5.3

1(2

6,3

22

.43

–3

1,0

19

.99

)2

.93

y=

9.8

9?

0.0

03

x6

.010

(4)

n.s

.1

4,0

00

48

.3±

1.2

21

,00

06

5.4

±0

.8

28

,00

08

8.6

±0

.6

35

,00

01

00

.0±

0.0

G.affinis

14

,00

02

7.4

±0

.42

9,6

33

.74

(26

,41

9.2

6–

32

,50

4.2

4)

57

,45

4.9

5(5

3,3

22

.36

–6

2,8

79

.81

)2

.95

y=

9.4

3?

0.0

01

x5

.991

(4)

n.s

.2

8,0

00

46

.2±

0.6

42

,00

06

5.7

±1

.2

56

,00

08

7.3

±0

.8

70

,00

01

00

.0±

0.0

No

mort

alit

yw

aso

bse

rved

inth

eco

ntr

ol

SD

stan

dar

dd

evia

tio

n,LC50

leth

alco

nce

ntr

atio

nth

atkil

ls50

%o

fth

eex

pose

dorg

anis

ms,LC90

leth

alco

nce

ntr

atio

nth

atk

ills

90

%o

fth

eex

po

sed

org

anis

ms,UCL

95

%

up

per

con

fid

ence

lim

it,LCL

95

%lo

wer

con

fid

ence

lim

it,v2

Chi

squ

are,

d.f.

deg

rees

of

free

dom

,n.s.

no

tsi

gn

ifica

nt

(a=

0.0

5)

aV

alu

esar

em

ean±

SD

of

five

repli

cate

s


123

Table

8A

cute

toxic

ity

of

silv

ern

ano

par

ticl

esg

reen

-sy

nth

esiz

edu

sin

gth

eNaregamia

alata

leaf

extr

act

on

thre

en

atu

ral

enem

ies

ofAnopheles,Aedes

andCulex

mo

squ

ito

vec

tors

No

n-t

arg

eto

rgan

ism

Con

cen

trat

ion

(lg

/ml)

Mo

rtal

ity

(%)±

SD

aL

C50

(lg

/ml)

(LC

L–

UC

L)

LC

90

(lg/m

l)(L

CL

–U

CL

)S

lope

Reg

ress

ion

equat

ion

v2(d.f.)

A.bouvieri

30

02

6.5

±0

.46

29

.58

(56

1.4

5–

69

0.3

9)

12

14.9

5

(11

28.1

4–

13

28.6

6)

2.8

9y=

9.6

?0

.062x

5.7

22

(4)

n.s

.6

00

48

.2±

1.2

90

06

5.7

±0

.6

12

00

88

.4±

0.8

15

00

10

0.0

±0

.0

D.indicus

50

02

7.4

±0

.41

05

8.3

1(9

48

.12

–11

57

.48

)2

01

1.1

4

(18

69.2

5–

21

96.2

4)

2.6

9y=

8.3

6?

0.0

38

x5

.692

(4)

n.s

.1

00

04

3.8

±0

.6

15

00

67

.3±

1.2

20

00

89

.2±

0.8

25

00

10

0.0

±0

.0

G.affinis

10

00

25

.7±

0.6

21

11.3

4

(18

83.6

5–

23

14.6

4)

40

73.0

7

(37

82.2

6–

44

54.0

1)

2.8

8y=

9.3

5?

0.0

19

x5

.376

(4)

n.s

.2

00

04

8.5

±0

.8

30

00

66

.3±

1.2

40

00

87

.2±

0.4

50

00

10

0.0

±0

.0

No

mort

alit

yw

aso

bse

rved

inth

eco

ntr

ol

SD

stan

dar

dd

evia

tio

n,LC50

leth

alco

nce

ntr

atio

nth

atkil

ls50

%o

fth

eex

pose

dorg

anis

ms,LC90

leth

alco

nce

ntr

atio

nth

atk

ills

90

%o

fth

eex

po

sed

org

anis

ms,UCL

95

%

up

per

con

fid

ence

lim

it,LCL

95

%lo

wer

con

fid

ence

lim

it,v2

Chi

squ

are,

d.f.

deg

rees

of

free

dom

,n.s.

no

tsi

gn

ifica

nt

(a=

0.0

5)

aV

alu

esar

em

ean±

SD

of

five

repli

cate

s


123

exposure to LC50 of IV instar larvae of A. stephensi and C. quinquefasciatus.

Rawani et al. [51] showed that mosquitocidal AgNPs synthesized using Solanum

nigrum berry extracts were not toxic against two mosquito predators, Toxorhyn-

chites larvae and Diplonychus annulatum, and Chironomus circumdatus larvae,

exposed to lethal concentrations of dry nanoparticles calculated on A. stephensi and

C. quinquefasciatus larvae. Furthermore, Govindarajan et al. [40] reported that the

biotoxicity of M. sylvestris aqueous extract and green-synthesized AgNPs was

negligible also on non-target organisms D. indicus and G. affinis, since the toxicity

treatments achieved LC50 values ranging from 813.16 to 10,459.13 lg/ml. Also, the

biotoxicity of B. cristata aqueous extract and green-synthesized AgNPs was

evaluated on non-target organisms D. indicus, A. bouvieri, and G. affinis with LC50

values ranging from 633.26 to 8595.89 lg/ml, respectively [19]. Lastly, Govin-

darajan et al. [52] investigated the biotoxicity of C. spinarum aqueous extract and

green-synthesized AgNPs on non-target organisms D. indicus, A. bouvieri and G.

affinis. Toxicity treatments achieved negligible toxicity with LC50 values ranging

from 424.09 to 6402.68 lg/ml. Concerning sub-lethal effects, G. affinis exposed to

nanoparticle-contaminated aquatic environments showed higher predation rates

against both An. stephensi and Ae. albopictus larvae. Interestingly, the exposure to

extremely low doses (e.g. 1 ppm) of green-synthesized AgNPs did not negatively

impact the predation efficiency of a number of mosquito predators of relevance for

mosquito control [9].

Conclusions

Overall, here we synthesized mosquitocidal AgNPs using a cheap aqueous extract of

N. alata leaves as reducing and stabilizing agent. The bio-reduced AgNPs were

mostly spherical in shape and crystalline in nature, with their mean size ranging

from 5 to 35 nm. This research highlighted that N. alata-synthesized AgNPs are

easy to produce, stable over time, and can be employed at low dosages to strongly

reduce populations of vectors mosquitoes without detrimental effects on non-target

aquatic organisms, such as A. bouvieri, D. indicus, and G. affinis.

Table 9 Suitability index of different non-target organism over young instars of Anopheles stephensi,

Aedes aegypti and Culex quinquefasciatus exposed to Naregamia alata aqueous leaf extract and green-

synthesized silver nanoparticles

Treatment Non-target organism An. stephensi Ae. aegypti Cx. quinquefasciatus

A. bouvieri 63.02 58.09 52.97

Naregamia alata extract D. indicus 88.67 81.73 74.53

G. affinis 179.43 165.39 150.82

A. bouvieri 50.77 46.39 42.42

Silver nanoparticles D. indicus 85.34 77.98 71.31

G. affinis 170.26 155.58 142.27


123

Acknowledgments The authors are thankful to the Head of the Department of Zoology, Khadir

Mohideen College for the facilities provided to carry out this work. The authors would like to thank

Professor and Head, Department of Zoology, Annamalai University for the laboratory facilities provided.

The authors would also like to acknowledge the cooperation of staff members of the VCRC (ICMR),

Pondicherry.

Conflicts of interest The Authors declare no conflicts of interest.

Research Involving Animal Participants 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.

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Single-Step Biofabrication of Silver Nanocrystals Using Naregamia … · 2017. 8. 26. · Annamalai University, Annamalainagar, Tamil Nadu 608 002, India 3 Insect Behavior Group,