Parasitol Res DOI 10.

1007/s00436-011-2689-5

ORIGINAL PAPER

Feeding deterrent activity of synthesized silver nanoparticles using Manilkara zapota leaf extract against the house fly, Musca domestica (Diptera: Muscidae)
Chinnaperumal Kamaraj & Govindasamy Rajakumar & Abdul Abdul Rahuman & Kanayairam Velayutham & Asokan Bagavan & Abdul Abduz Zahir & Gandhi Elango

Received: 19 September 2011 / Accepted: 5 October 2011 # Springer-Verlag 2011

Abstract With a greater awareness of the hazards associated with the use of synthetic organic insecticides, there has been an urgent need to explore suitable alternative products for pest control. Musca domestica is ubiquitous insect that has the potential to spread a variety of pathogens to humans and livestock. They are mechanical carriers of more than hundred human and animal intestinal diseases and are responsible for protozoan, bacterial, helminthic, and viral infections. The present work aimed to investigate the feeding deterrent activity of synthesized silver nanoparticles (Ag NPs) using leaf aqueous extract of Manilkara zapota against M. domestica. The synthesized Ag NPs were recorded from UVvis spectrum at 421 nm and scanning electron microscopy confirm the biosynthesis and characterization of Ag NPs with spherical and oval in shape and size of 70140 nm. The FTIR analysis of the purified nanoparticles showed the presence of bands 1,079, 1,383, 1,627, 2,353, and 2,648 cm1, which were complete synthesis of AgNPs; the XRD pattern of AgNPs showed diffraction peaks at 2 values of 38.06, 44.37, 64.51, and 77.31 sets of lattice planes were observed (111), (200), (220), and (311) facts of silver, respectively. Adult flies were exposed to different concentrations of the aqueous extract of synthesized Ag NPs, 1 mM silver nitrate (AgNO3) solution and aqueous extract of

M. zapota for 1, 2, and 3 h; however, AgNPs showed 72% mortality in 1 h, 89% mortality was found in 2 h, and 100% mortality was found in 3 h exposure at the concentration of 10 mg/mL and the leaf aqueous extract showed 32% mortality in 1 h, 48% mortality was found in 2 h, and 83% mortality was found in 3 h exposure at concentration of 50 mg/mL. The most efficient activity was observed in synthesized Ag NPs against M. domestica (LD50 = 3.64 mg/mL; LD90 =7.74 mg/mL), the moderate activity reported in the aqueous extract of M. zapota (LD50 = 28.35 mg/mL; LD90 =89.19 mg/mL) and nil activity were observed in AgNO3 solution at 3 h exposure time at 10 mg/mL. Dimethyl 2, 2-dichlorovinyl phosphate (DDVP) was used as a positive control and showed the LD50 value of 3.38 mL/L. These results suggest that the synthesized Ag NPs have the potential to be used as an ideal eco-friendly approach for the control of the adult of M. domestica. This method is considered as a new approach to control sanitary pest. Therefore, this study provides first report on the feeding deterrent activity of synthesized Ag NPs against housefly.

Introduction The housefly, Musca domestica, is one of the most common insects intimately associated with human settlements. In rural areas, houseflies cause irritation to livestock, leading to decreased efficiency in various ways. They also invade urban areas and are a major nuisance in residential areas and business complexes (Axtell and Arend 1990). Flies feed and breed on decaying matter, human waste, and foods, and are vectors of pathogens such as bacteria, protozoa, viruses and metazoan parasites (Barin et al. 2010).The

Chinnaperumal Kamaraj and Govindasamy Rajakumar contributed equally to this work. C. Kamaraj : G. Rajakumar : A. A. Rahuman (*) : K. Velayutham : A. Bagavan : A. A. Zahir : G. Elango Unit of Nanotechnology and Bioactive Natural Products, Post Graduate and Research Department of Zoology, C. Abdul Hakeem College, Melvisharam, 632 509, Vellore District, Tamil Nadu, India e-mail: abdulrahuman6@hotmail.com

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housefly is categorized as an important contributing factor in the dissemination of various infectious food-borne diseases, disease in humans and animals, such as cholera, typhoid, shigellosis, bacillary dysentery, tuberculosis, anthrax ophthalmia, and infantile diarrhea, as well as the presence of parasitic worms (Iwasa et al. 1999; Olsen et al. 2001). Pathogenic organisms are picked up by flies from garbage, sewage, and from other sources of filth, and carried on their mouthparts, through their vomitus, feces, and contaminated external body parts to human and animal food (Fotedar 2001; Zurek et al. 2001). Housefly management relies heavily on sanitation, screening measures, and pesticide application. However, these are often difficult to implement because of high labor costs and the impracticability of screening methods. Among pesticides, organochlorines, organophosphates, and more recently, pyrethroids have been used for housefly control. However, houseflies can develop resistance to these pesticides (Shono and Scott 2003; Srinivasan et al. 2008) and health and environmental risks are associated with these compounds. Due to the disadvantages associated with such synthetic pesticides, including development of pesticide resistant strains, ecological imbalances, and harm to non-target organisms, there is a renewed effort to develop substances of plant origin which are considered to be more environments friendly due to their innate biodegradability and lower toxicity to most organisms (Frederich et al. 2002). Many pesticides are poorly soluble in water for which large amounts of organic solvents are required to solubilize them. Most of the organic solvents are hostile to the environment. The resistivities of insect pests to chemical insecticides increase in costs of materials, and problems of environmental/personal exposure have hampered the control of vectors and other insects (Salahuddin et al. 2004). A major challenge for manufacturers of natural products is to maintain a commercial product of consistent composition related to the expected variation in chemical composition of the essential oils with location, cultivar, season, and over time (Heukelbach et al. 2006). A conventional method for fly control in the short term is the use of insecticides. Nevertheless, the widespread and massive applications of chemical insecticides frequently produce risks in the development of insect resistance and residues to humans and to the environment (Farnham 1973; Scott et al. 2000). Accordingly, botanical insecticides, a botanical type based on natural plant compounds, are expected to become the possible application, representing selective, efficacious, and toxicologically safe insecticides. Several reports have shown the efficacy of natural compounds on insects. With regard to flies, the majority of studies have been found to assess the crude extracts from many botanical sources (Pavela 2004; Pavela and Teixeira da Silva 2006).

In fact, many researchers have reported on the effectiveness of plant extracts or essential oils against insects (Amer and Mehlhorn 2006a, b, c; Rahuman et al. 2008a, b, c). These limitations therefore necessitate the search for new control method which may replace these synthetic insecticides. The synthesized nanoparticles, which are less likely to cause ecological damage, have been identified as potential replacement of synthetic chemical insecticides; hence the need to use green synthesized silver nanoparticles (Ag NPs) for the control of disease houseflies. Targeting the adult stage control is a critical point in eradicating houseflies, because of their inability to move from the habitats. The current study aimed at evaluating the feeding deterrent efficacy of synthesized Ag NPs against M. domestica in the laboratory. One approach to arrest their aggregation is by capping the nanoparticles with organic molecules which can be facilitated through extracellular phytosynthesis, plantmediated biosynthesis of metallic nanoparticles that has drawn a great deal of attention in recent years due to its cost-effectiveness, simplicity, and eco-friendliness rendering this new branch of nanotechnology a competitive edge over other techniques (Narayanan and Sakthivel 2008; Huang et al. 2007). Ag NPs may be released into the environment from discharges at the point of production, from erosion of engineered materials in household products (anti-bacterial coatings and silver-impregnated water filters), and from washing or disposal of silver-containing products (Benn and Westerhoff 2008). Using plants for nanoparticle synthesis can be advantageous over other biological processes because it eliminates the elaborate process of maintaining cell cultures and can also be suitably scaled up for large-scale nanoparticle synthesis (Shankar et al. 2004). Synthesis of nanoparticles using microorganisms or plants can potentially eliminate this problem by making the nanoparticles more biocompatible. Nanoparticles, generally considered as particles with a size of up to 100 nm, exhibit completely new or improved properties as compared to the larger particles of the bulk material that they are composed of based on specific characteristics such as size, distribution, and morphology (Willems and van den Wildenberg 2005). The silver nanoparticles are reported to possess antibacterial (Sathishkumar et al. 2009), anti-viral (Rogers et al. 2008), and anti-fungal activity (Panacek et al. 2009). In recent years, the biosynthetic method using plant extracts has received more attention than chemical and physical methods, and even than the use of microbes, for the nanoscale metal synthesis due to the absence of any requirement to maintain an aseptic environment. Nanoparticles have attracted considerable attention owing to their various applications. Synthesis of nanoparticles using plants or microorganisms can potentially eliminate this problem by making the nanoparticles more bio-compatible. Indeed, over the past several

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years, plants, algae, fungi, bacteria, and viruses have been used for low-cost, energy-efficient, and nontoxic production of metallic nanoparticles (Thakkar et al. 2009). Use of plant extract for the synthesis of nanoparticles could be advantageous over other environmentally benign biological processes by eliminating the elaborate process of maintaining cell cultures. Recently green silver nanoparticles have been synthesized using various natural products like Nelumbo nucifera (Santhoshkumar et al. 2011), Ocimum sanctum (Ahmad et al. 2010), Eclipta prostrata (Rajakumar and Rahuman 2011), Pongamia pinnata (Raut et al. 2010), and Cinnamon zeylanicum (Sathishkumar et al. 2009). Manilkara zapota L. (Sapotaceae) is evergreen, glabrous tree with a milky juice. It is cultivated throughout India. The bark is antibiotic, astringent, and febrifuge. It is used as tonic and the decoction is given in diarrhea and peludism (Anjaria et al. 2002). The ethyl acetate extract of stem bark of M. zapota was applied on larvae and adult of red flour beetle, Tribolium castaneum effectively control after 72 h of exposure (Osman et al. 2011), the aqueous extract inhibited 50% activity against Aspergillus ochraceus and Aspergillus tamarii (Satish et al. 2007), and the seed acetone extract was found to be active against both gram-positive and gram-negative organisms (Kothari and Seshadri 2010). Polyphenols were isolated from the fruit methanol extract of M. zapota (Ma et al. 2003) and these biomolecules acted as a probable stabilizer for the synthesized silver nanoparticles and chitosan-coated tea polyphenols nanoparticles (Moulton et al. 2010; Liang et al. 2011). The compounds attached with the silver nanoparticles could be polyphenols with aromatic ring and bound amide region (Krishnan and Maru 2006; OCoinceanainn et al. 2009; Susanto et al. 2009). The objective of this study, slow reduction of the aqueous silver ions along with the shape directing effects of the constituents of the M. zapota extract play a key role in the formation of the silver nanoparticles with spherical and oval in shape tested against the feeding deterrent of adult of M. domestica.

Synthesis of silver nanoparticles by M. zapota leaf extract M. zapota leaves (Fig. 1) were washed thoroughly in tap water for 10 min in order to remove the dust particles and rinsed briefly in deionized water. The plant leaf broth solution was prepared by taking 10 g of washed and finely cut leaves in a 250-mL Erlenmeyer flask along with 100 mL of deionized water and then boiling the mixture at 60C for 5 min. After boiling, the solution was decanted, and 12 mL of this broth was added to 88 mL of 1 mM aqueous AgNO3 solution and the resulting solution became brown in color. This extract was filtered through nylon mesh (spectrum), followed by Millipore hydrophilic filter (0.22 m), and used for further experiments (Santhoshkumar et al. 2011). When the aqueous leaf M. zapota extract with AgNO3 the color was changed in to light brown and the color intensity was measured at 421 nm for different intervals (1, 2, 3, 4 and 5 hr respectively). Housefly collection and maintenance Colonies of M. domestica adults were collected from sugarcane juice shop, Melvisharam, using a sweep net. The flies were transferred to a small cage and then reared in entomological cages (303030 cm) at 26 (1)C under a 12:12 light/dark cycle and 70% humidity. Adult flies were provided with water and fed with 1:1 (v/v, approximately) mixture of granulated sugar and powdered milk. Bran and milk were prepared at a weight ratio of 1:3 and 100 g of this mixture was placed on a plastic plate as an oviposition site (Palacios et al. 2009). Bioassay of housefly The bioassay test against M. domestica was performed as previously reported (Palacios et al. 2009). Twenty adult houseflies (male and female, 45 days old) were placed in a glass jar. One hundred millilitres of sterilized double

Materials and methods Preparation of M. zapota leaf aqueous extract 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 (Minjas and Sarda 1986). The suspension of dried leaf powder in water was left for 3 h, filtered through Whatman no. 1 filter paper, and the filtrate was stored in amber colored air tight bottle at 10C and used within a week.

Fig. 1 Manilkara zapota L. (Sapotaceae) and its leaves

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distilled water with different concentrations (2, 4, 6, 8, and 10 mg/mL) of synthesized Ag NPs was mixed with 10 g of feeding mixture and stirred few minutes in 100 mL beaker (Borosil). The nanoparticle suspension was dipped in cotton yarn (1.2 dm3) fitted with a screw cap and 7 cm length of cotton yarn suspended from the centre of the internal face of the cap. Different dosages of synthesized Ag NPs were used to the yarn and similarly aqueous plant extract was used in different concentrations at 10 to 50.0 mg/mL to produce a range of mortality from 10% to 100%. The control vessel had feeding mixture and distilled water on the cotton yarn. The jars were then sealed tightly and kept in a room at 261C for 24 h. Each test was replicated three times. Mortality in each group was assessed after 1, 2, and 3 h of exposure by softly stimulating each fly with the tip of a needle. Flies that did not respond were considered dead. Silver nitrate (AgNO3) solution (1 mM) was used in all experiments in the same conditions were tested in three replicates. To avoid settling of particles especially at higher doses, all treatment solutions were stirred for an additional 5 min prior to the experiment. DDVP was used as a positive control (10 mL of DDVP was diluted in 1 L of distilled water). Characterization of silver nanoparticles The bioreduction of Ag NPs was monitored by sampling the reaction mixture at regular intervals and the absorption maxima was scanned by UVvis spectra, at the wavelength of 200700 nm in Schimadzu 1601 spectrophotometer operated at a resolution of 1 nm. Silver (Ag) nanoparticles exhibit unique and tunable optical properties on account of their surface plasmon resonance (SPR), dependent on shape, size, and size distribution of the nanoparticles. The reduction of Ag + ions was monitored by measuring the UVvisible spectra of the solutions after diluting a small aliquot (0.2 mL) of the sample 20 times. The solution mixture was subjected to centrifugation at 10,000 rpm for 45 min; resulting pellet was dissolved in deionized water and filtered through 0.22 m Millipore filter. An aliquot of this filtrate containing Ag NPs was used for XRD, FTIR and scanning electron microscopy (SEM), and EDX analysis. XRD measurements of the M. zapota leaf broth reduced Ag nanoparticles were carried out on films of the respective solutions drop-coated onto glass substrates on a Phillips PW 1830 instrument operating at a voltage of 40 kV and a current of 30 mA with CuK1 radiation. Characterization involved FTIR analysis of the dried powder of Ag NPs by scanning it in the range of 3504,000 cm1 at a resolution of 4 cm1. These measurements were carried out on a PerkinElmer Spectrum One instrument in the diffuse

reflectance mode at a resolution of 4 cm1 in KBr pellets and the pellets were mixed with KBr powder and pelletized after drying properly. The pellets were later subjected to FTIR spectroscopy measurement. For electron microscopic studies, 25 L of sample was sputter coated on copper stub and the images of nanoparticles were studied using SEMEDX (JEOL, Model JFC-1600). Data analysis Adult mortality counts were made after 1, 2, and 3 h exposure. Mean percent adult mortality data were subjected to analysis of variance and compared with Duncans multiple range tests to determine any differences between plant species and within species and concentration (SPSS 2007).The average adult mortality data were subjected to probit analysis for calculating LD50 and LD90 and other statistics at 95% fiducial limits of upper confidence limit and lower confidence limit and chi-square values were calculated using the software developed by Reddy et al. (1992).

Results In the present study, the toxic effect was evaluated on adult houseflies after 1, 2, and 3 h of exposure; the results of the mortality produced by aqueous leaf extract of M. zapota, 1 mM silver nitrate solution and synthesized Ag NPs against adult fly of M. domestica are provided in Table 1. AgNPs showed 72% mortality in 1 h, 89% mortality was found in 2 h, and 100% mortality was found in 3 h exposure at concentration of 10 mg/mL and the leaf aqueous extract showed 32% mortality in 1 h, 48% mortality was found in 2 h, and 83% mortality was found in 3 h exposure at concentration of 50 mg/mL. The efficient mortality was observed in aqueous crude leaf extracts of M. zapota and synthesized AgNPs against the adult of M. domestica (LD50 =28.35 and 3.64 mg/mL; LD90 =89.19 and 7.74 mg/mL) at a 3-h exposure. Silver nitrate solution did not show any mortality at the concentration of 10 mg/mL for 3 h of exposure. DDVP was selected as a positive control and showed with 100% mortality at10 mL/L in 1, 2, and 3 h; LD50 value of 3.38 1 h, 4.27 in 2 h, and 6.29 mL/L in 3 h (Table 1). In the present study, the maximum activity was observed in Ag NPs and the moderate activity was observed in aqueous leaf extract of M. zapota and the activity was 7.8 times more in the synthesized Ag NPs than the leaf aqueous extract of M. zapota. The color intensity of the leaf extracts of M. zapota incubated with silver ions at the beginning of reaction and after 5 h of reaction. The color change was noted by visual observation in the leaf extracts when incubated with

Parasitol Res Table 1 LD50 and LD90 of aqueous and synthesized silver nanoparticles using leaf extract of Manilkara zapota against Musca domestica Test materials Concentration (mg/mL) 1 h% Mortalitya SD 2 h% Mortalitya SD 3 h% Mortalitya SD LD50 SE (UCLLCL) (mg/mL) 28.351.50 (31.3225.39) LD90 SE (UCLLCL) (mg/mL) 89.1910.37 (109.5268.87) 2 (df=4)

Plant aqueous extract

Ag NPs

DDVP (mL/L)

50 40 30 20 10 10 8 6 4 2 10 8 6 4 2 10 8 6 4 2 10 8 6 4 2 10

321.34 281.62 161.04 100.26 40.82 721.87 562.16 341.04 201.26 120.14 1000.00 821.48 681.24 521.86 341.08

481.42 361.86 281.28 141.32 61.08 890.18 681.42 421.60 281.08 160.26

831.68 581.20 401.62 261.42 181.82 1000.00 861.68 681.24 421.62 200.64

15.32

3.640.16 (3.963.32)

7.740.47 (8.696.39)

17.00

3.380.17 (3.703.05)

7.540.45 (8.426.65)

17.19

1000.00 941.84 761.20 602.06 421.64

4.270.87 (18.299.76)

12.281.56 (28.1512.48)

16.81

1000.00 1000.00 861.04 741.86 561.42

6.290.86 (24.2813.41)

20.682.89 (32.2018.87)

14.27

Silver nitrate Significant at P<0.05 level

0.00

LD50 lethal dose that kills 50% of the exposed adult, LD90 lethal dose that kills 90% of the exposed adult, UCL upper confidence limit, LCL lower confidence limit, 2 chi-square, df degree of freedom, DDVP dimethyl 2,2-dichlorovinyl phosphate
a

Mean value of three replicates

AgNO3 solution. Figure 2 displays the UVvis spectra of stem solution as a function of reaction time. The strong

Fig. 2 UVvis spectra of aqueous silver nitrate with M. zapota leaf extract at different time intervals

resonance centered at 421 nm was clearly observed and increased in intensity with time. It might arise from the excitation of longitudinal plasmon vibrations in Ag NPs in the solution. Reduction of silver ions present in the aqueous solution of silver complex during the reaction with the ingredients present in the leaves of M. zapota extract observed by the UVvis spectroscopy revealed the presence of Ag NPs may be correlated with the UVvis spectra. FTIR analysis confirmed that the bioreduction of Ag+ ions to Ag NPs is due to the reduction by capping material of plant extract. The FTIR spectrum showed peaks at 1,079, 1,383, 1,627, 2,353, and 2,648 cm1, respectively, which shows the complete synthesis of Ag NPs (Fig. 3). The XRD (Fig. 4) shows the number of Bragg reflections with 2 values of 38.06, 44.37, 64.51, and 77.31 sets of lattice planes that were observed which may be indexed to the (111), (200), (220), and (311) facts of silver, respectively. XRD pattern thus clearly illustrates that

Parasitol Res Fig. 3 FTIR spectrum of silver nanoparticles synthesized by M. zapota leaf extract

the Ag NPs formed in the present synthesis were crystalline in nature. This indicates that the nanoparticles are abundant in (111) plane. Thus, diffraction intensity of (111) plane should be greatly enhanced in comparison to that of other planes. The value of the pure silver lattice constant have been estimated to be =4.081, a value that is consistent with =4.0862 A reported by the JCPDS file no4-0787. The SEM micrograph (Fig. 5a, b) of synthesized nanoparticles was granular, spherical structures with size range of 70140 nm. The rough morphology of the NPs provides excellent larvicidal activity for the synthesized nanoparticles. The EDX attachment present with the SEM was known to provide information on the chemical analysis of the fields being investigated or the composition at specific locations (spot EDX). Figure 6 shows a representative profile of the spot EDX analysis and obtained by focusing on AgNPs.

Discussion Many plant extracts have shown potential insecticidal activity against houseflies. In the present study, the feeding
Fig. 4 XRD pattern of silver nanoparticles synthesized using leaves of M. zapota leaf extract

deterrent activity of aqueous leaf extracts and synthesized Ag NPs of M. zapota were noted. Earlier authors reported that the larvicidal effect of aqueous crude leaf extracts, silver nitrate, and synthesized silver nanoparticles of Mimosa pudica showed that the highest mortality was found in synthesized AgNPs against the larvae of Anopheles subpictus (LC50 =8.89, 11.82, and 0.69 ppm) and against the larvae of Culex quinquefasciatus (LC50 =9.51, 13.65, and 1.10 ppm; Marimuthu et al. 2011). The highest mortality was found in methanol, aqueous extracts of Nelumbo nucifera, and synthesized AgNPs tested against the larvae of A. subpictus (LC50 =8.89, 11.82, and 0.69 ppm; LC90 =28.65, 36.06, and 2.15 ppm) and against the larvae of C. quinquefasciatus (LC50 =9.51, 13.65, and 1.10 ppm; LC90 = 28.13, 35.83, and 3.59 ppm), respectively (Santhoshkumar et al. 2010). The maximum activity was observed in the synthesized Ag NPs using Tinospora cordifolia extract against lice, Pediculus humanus capitis, fourth instar larvae of A. subpictus and C. quinquefasciatus (LC50 =12.46, 6.43 and 6.96 mg/L), respectively (Jayaseelan et al. 2011). The aqueous leaf extract of Campsis grandiflora that was evaluated against M. domestica adults showed 80% mortality

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Fig. 5 SEM micrograph showing the morphological characteristics of silver nanoparticles synthesized using leaves of M. zapota leaf extract. a Lower magnification. b Higher magnification

with 5.75 g/fly topical application (Pandey 2006). Mohottalage et al. (2007) have reported that the insecticidal activity of Piper betle leaf oil tested against the adult M. domestica indicated LC50 values of 10.3 and 8.7 mg/dm after 24 and 48 h exposure and the oil extracted from Cymbopogon nardus showed LC50 values of 26.5 and 24.2 mg/dm after 24 and 48 h exposure. The essential oils of six plant species, Mentha piperita, Mentha citrata, Eucalyptus globulus, Cymbopogon citratus, Vetiver zizanoides, and Curcuma longa were screened for repellent, larvicidal, and pupicidal activities against the housefly, M. domestica. In repellency bioassays, M. piperita (repellency concentration (RC) 84, 61.0 g/cm2) was found to be most effective, followed by E. globulus (RC84, 214.5 g/cm2) and C. citratus (RC84, 289.2 g/cm2), formulated M. piperita and E. globulus showed RC84 values of 1.6 and 4.1 g/cm2. The M. piperita and E. globulus achieved larval mortality with LC50 values in 72 h at 5.12 and 6.09 g/cm2, and the pupicidal bioassays, crude oils of M. piperita and E. globulus suppressed the emergence of adult flies by 100%, respectively (Kumar et al. 2011). The toxicity of Griffonia simplicifolia seed extract and Zanthoxylum xanthoxyloides root extract was effective against M. domestica adults with LD50 values of 0.28 and 0.35 g/fly for a 24-h topical application (Bisseleua et al. 2008). The coumarin compounds were isolated from Ageratum conyzoides showed effective insecticidal activity against M. domestica after 12 and 24 h topical application (LD50 =2.28 and 1.18 mg g1and LD90 =14.07 and 3.67 mg g1; Moreira et al. 2007), respectively. In the present observation, the synthesized silver nanoparticles showed effective active compound compared with the previous authors report. Formation of Ag NPs from 1 mM solution of silver nitrate was confirmed by using UVvis spectral analysis.

Fig. 6 EDX showing the chemical constituents of the synthesized nanoparticles

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Metal nanoparticles silver have free electrons, which give rise to a SPR absorption band (Noginov et al. 2003) and due to the combined vibration of electrons of metal nanoparticles in resonance with the light wave. Ag NPs exhibit yellowish brown color in aqueous solution due to excitation of surface plasmon vibrations in Ag NPs (Krishnaraj et al. 2010). Ag nanoparticles were observed to be stable in solution and show very little aggregation. Besides, the plasmon bands were broadened with an absorption tail in the longer wavelengths, which may be due to the size distribution of the particles (Ahmad et al. 2003). The FTIR spectrum was observed in the frequency of 1,079 (CO), 1,383 (CH2 and CH3 deformation), 1,627 (C=C), 2,014 (aromatic combination bonds), 2,353 (aliphatic cyanide/nitrile), and 2,648 cm1 (Thiols (SH stretch); John 2000). These compounds may be responsible for production of Ag NPs from leaves of M. zapota. The sharpening of the peaks clearly indicates that the particles were in the nanoregime. Dubey et al. (2009) reported the size of silver nanocrystallites as estimated from the full width at half maximum of the (111) peak of silver using the Scherrer formula was 2060 nm. The XRD pattern of pure silver ions was known to display peaks at 2=7.9, 11.4, 17.8, 30.38, and 44 (Gong et al. 2007). When the metal nanoparticles form in solution, they must be stabilized against the van der Waals forces of attraction which may otherwise cause coagulation. The particle shape of plant-mediated Ag NPs were mostly spherical with exception of neem (Azaddirachita indica) which yielded polydisperse particles both with spherical and flat plate-like morphology 535 nm in size (Shankar et al. 2004). SEM images of Ag NPs from Emblica officinalis were also predominantly spherical with an average size of 16.8 nm ranging from 7.5 to 25 nm (Ankamwar et al. 2005). The stability of the Ag NPs can be attributed to the formation of silver electride that may form a thin layer on the aqueous surface of the reaction mixture. The protein present further is believed to cap the Ag NPs formed, restricting the agglomeration of the particles and thus checking the size and shape. Presumably biosynthetic products or reduced cofactors play an important role in the reduction of respective salts to nanoparticles. It seems quite probable that the phenols play an important part in the reduction of ions to Ag NPs as the concept of antioxidant action of phenol compounds is not new. It is becoming clear that there are some important differences between testing the effects of soluble chemicals and the toxic potential of nanoparticles. The present green synthesis shows that the environmentally benign and renewable source of M. zapota used as an effective reducing agent for the synthesis of Ag NPs. This biological reduction of metal would be boon for the development of clean, nontoxic and environmentally acceptable green

approach to produce metal nanoparticles, involving organisms even ranging higher plants. This is the first report on the feeding deterrent activity of synthesized silver nanoparticles against M. domestica. In conclusion, the present study clearly demonstrated the feeding deterrent potential of synthesized AgNPs from M. zapota as a medicinal plant. It has been shown that the synthesized AgNPs may produce a great range of biological effects on M. domestica and can be a potent candidate in integrated pest management programs for controlling such pests. However, isolation of the active compounds from M. zapota plant and further trial assay of synthesized AgNPs in the field conditions is underway to explore its suitability for such application.
Acknowledgments The authors are grateful to C. Abdul Hakeem College Management, Dr. W. Abdul Hameed, Principal, and Dr. Hameed Abdul Razack, HOD of Zoology Department, for providing the facilities to carry out this work.

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