Parasitol Res (2014) 113:3843–3851 DOI 10.1007/s00436-014-4052-0
ORIGINAL PAPER
Optimization and synthesis of silver nanoparticles using Isaria fumosorosea against human vector mosquitoes A. Najitha Banu & C. Balasubramanian
Received: 20 June 2014 / Accepted: 23 July 2014 / Published online: 3 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The efficacy of silver generated larvicide with the help of entomopathogenic fungi, Isaria fumosorosea (Ifr) against major vector mosquitoes Culex quinquefasciatus and Aedes aegypti. The Ifr-silver nanoparticles (AgNPs) were characterized structurally and functionally using UV-visible spectrophotometer followed by scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy and Fourier transform infrared (FTIR) spectra. The optimum pH (alkaline), temperature (30 °C) and agitation (150 rpm) for AgNP synthesis and its stability were confirmed through colour change. Ae. aegypti larvae (I–IV instars) were found highly susceptible to synthesized AgNPs than the larvae of Cx. quinquefasciatus. However, the mortality rate was indirectly proportional to the larval instar and the concentration. The lethal concentration that kills 50 % of the exposed larvae (LC50) and lethal concentration that kills 90 % of the exposed larvae (LC90) values of the tested concentration are 0.240, 0 0.075.337, 0.430, 0.652 and 1.219, 2.210, 2.453, 2.916; 0.065, 0.075, 0.098, 0.137 and 0.558, 0.709, 0.949, 1.278 ppm with respect to 0.03 to 1.00 ppm of Ifr-AgNPs against first, second, third and fourth instars of Cx. quinquefasciatus and Ae. aegypti, respectively. This is the first report for synthesis of AgNPs using Ifr against human vector mosquitoes. Hence, Ifr-AgNPs would be significantly used as a potent mosquito larvicide.
Keywords Isaria fumosorosea . Silver nanoparticles . Toxicity . LC50 and LC90
A. N. Banu (*) : C. Balasubramanian Department of Zoology and Microbiology, Thiagarajar College (Autonomous), Madurai, Tamil Nadu 625009, India e-mail: [email protected]
Introduction One of the major public health concerns in developing countries is vector-borne diseases. The genera Aedes, Anopheles and Culex adults transmit serious human diseases; viz, malaria, filariasis, encephalitis, dengue and recently chikungunya are the major mosquito-borne diseases in India. In the current situation of global warming and contaminated fresh water bodies, a number of mosquitoes are markedly increasing in concurrence with a high incidence of dengue fever (Halstead 2007). Mosquitoes constitute the most important single family of insects from the stand point of human health. In this regard, Aedes aegypti (Diptera: Culicidae) is the main vector of chikungunya and dengue fever (Sourisseau et al. 2007). Till 10th October 2012, 151 districts of eight states/provinces of India have been affected by chikungunya fever (Pialoux et al. 2007; Yang et al. 2009). According to WHO report of year 2009, two fifth of world population is under risk of dengue infection (WHO index) and in year 2010, 28,292 cases of infection and 108 deaths were reported in India (NVBDCP 2011). The incidence of dengue has grown dramatically around the world in recent decades over 2.5 billion people (40 % of the world’s population) at risk from dengue. WHO currently estimates that there may be 50– 100 million dengue infections worldwide every year (WHO 2012). Dengue cases were reported in the year 2012, November 27, the total number of dengue cases in India around 35,066 and 216 deaths. The highest numbers of dengue cases were recorded in the country for the year 2012 to 2013 till November 15, around 9,249 cases in Tamil Nadu and West Bengal 6,067 cases. Lymphatic filariasis is a serious public health problem in India, constituting one third of the infected population in the world (WHO 1997). Culex quinquefasciatus is a vector of lymphatic filariasis, which affects 120 million people worldwide, and approximately 400 million people are at risk of
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contracting filariasis, resulting in an annual economic loss of US$1.5 billion (WHO 2002). Malaria now is responsible for illness in more than an estimated 300 million people, resulting in 1 million deaths per year (WHO 2007). Lymphatic filariasis, commonly known as elephantiasis, is a painful and profoundly disfiguring disease. An estimated 120 million people in tropical and subtropical areas of the world are infected with lymphatic filariasis; of these, almost 25 million men have genital disease (most commonly hydrocele) and almost 15 million, mostly women, have elephantiasis of the leg. Approximately 66 % of those at risk of infection live in the WHO South-East Asia Region and 33 % in the African Region (WHO 2010). More importantly, the incidences of resistance to larvicides of mosquito larvae have been reported by Braga et al. (2004) and Melo-Santos et al. (2010).Thus, attempts to develop novel materials as mosquito larvicides are still necessary. Among the biological organism so far used for the mosquito control programmes, bacteria such as Bacillus species are known for its mosquito larvicidal effect. Of which, Bacillus thuringiensis var. israelensis and Bacillus sphaericus are effective, but serious resistance as high as 50,000-fold has evolved where B. sphaericus is used against Culex mosquitoes (Soni and Prakash 2011). Recently, the laboratory resistance in the mosquitoes has been demonstrated to some isolates of B. thuringiensis (Surendran and Vennison 2011; CadavidRestrepo et al. 2012; Chenniappan and Ayyadurai 2012). Many organisms synthesize silver nanoparticles (AgNps) extracellularly and intracellularly, among which Verticillium spp. (Mukherjee et al. 2001), Aspergillus fumigatus (Bhainsa and D’Souza 2006), Fusarium oxysporum (Anil Kumar et al. 2007; Sonal et al. 2013), Klebsiella pneumoniae, (Shahverdi et al. 2007), Bacillus licheniformis (Kalimuthu et al. 2008), Escherichia coli, Enterobacter cloacae (Minaein et al. 2008), Bacillus megaterium (Saravanan et al. 2011), Pseudomonas proteolytica, Pseudomonas meridiana, Arthrobacter kerguelensis, Bacillus indicus (Shivaji et al. 2011), Aspergillus niger (Soni and Prakash 2013), B. thuringiensis (Jain et al. 2010; Najitha Banu et al. 2014), Bacillus strain CS 11 (Das et al. 2014) and Beauveria bassiana (Najitha Banu and Balasubramanian 2014) have been reported extensively. The nanoparticles encoded by secondary metabolites of bacteria and entomopathogenic fungi are a novel tool to havoc the larval population from the nuisance where vector species cause endemic diseases. This approach has been successfully adopted in vector control programme in future and also thus the reduction of xenobiotic chemicals loads in the environment directly (Najitha Banu and Balasubramanian 2014). Recently, the new era was developed for controlling mosquito population by using biologically synthesized AgNps treated against the mosquito larvae. Hence, the present study was aimed to evaluate the fungi-synthesized AgNps against the human vector mosquitoes.
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Fig. 1 Isaria fumosorosea (Ifr)
Materials and methods Isolation and identification of entomopathogenic fungi The entomopathogenic fungi Isaria fumosorosea (Ifr) was isolated from rhizosphere soil, around Madurai, TN, India according to the method of Haraprasad et al. 2001. After 7 days of incubation, pure culture of Ifr was subcultured into PDA by streak plate method. The isolated culture was identified by slide culture method subjected to lactophenol cotton blue staining observed under the light microscope (Labomed). Extracellular synthesis of AgNPs The pure culture of Ifr was freshly inoculated on a liquid media containing (g/L) KH 2 PO 4 7.0, K 2 HPO 4 2.0, MgSO4 ·H2O 0.1, (NH4)2SO4 1.0, yeast extract 0.6 and glucose 10.0 in an Erlenmeyer flask. The flask containing medium was incubated in orbitary shaker at 150 rpm in 25±2 °C (Neolab Instruments, Mumbai, India) for 72 h. The biomass was harvested after 72 h of growth by sieving through a Whatman No. 1 filter paper (Whatman International Ltd, England), followed by extensive wash
Fig. 2 High-density mycelium in specific media
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a
b
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c
Fig. 3 Photographs of a Ifr-AgNPs, b mycelial extract and c 1 mM AgNO3
with distilled water to remove any medium components from the biomass. Twenty gram of fresh and clean biomass was taken into Erlenmeyer flask containing 200 mL Milli-Q water, and the flask was incubated at 25 °C for 72 h and agitated in the same condition as described earlier. After incubation, the cell filtrates were obtained by passing it through the Whatman No. 1 filter paper. Cell filtrate (50 mL) was taken into 250-mL Erlenmeyer flask and mixed with 1 mM AgNO 3 (Laboratory Reagent, Reachem Laboratory Chemicals Private Ltd, Madras, Tamil Nadu, India) (0.017 g/100 mL) as final concentration. The flasks were incubated at 25 °C in a dark condition for 120 h and agitated in the same condition as described earlier. The control was maintained separately (without the addition of AgNO3), only cell filtrate with the experimental flask. The AgNPs turned into brownish yellow colour solution, and it was stored in screw-capped vials under ambient condition for further experiment.
Fig. 5 SEM image of Ifr-AgNPs
PerkinElmer (Lambda 35) spectrophotometer. The spectra were recorded in the region 200–800 nm at a scan rate of 240 nm s−1. The atomic composition of nanoparticles was confirmed the energy-dispersive X-ray spectroscopy (EDX; Oxford instrument, INCA PentaFET x3, Karunya University, Coimbatore, Tamil Nadu, India) coupled with scanning electron microscope (SEM) (Model-JEOL, JSM-6390). Further characterization of Ag bionanoparticles implicated with Fourier transform infrared (FTIR) spectra was recorded using Bruker Tensor-27 FT-IR spectrometer with OPUS software in the range 4,000–400 cm−1, at a resolution of 4 cm−1. The pellet for analysis was made by taking equal amounts of IfrAgNPs and KBr (1:1 ratio); the background calibrations have been carried out using pure KBr pellet.
Characterization of AgNPs
XRD analysis
The UV-Vis spectra analysis of the freshly prepared nanoparticles was recorded with water/methanol as reference using the
AgNps were checked by X-ray diffractometer (XRD). The pre-prepared dry power was collected for the determination of the formation of Ag nanoparticles by an
Fig. 4 UV-Visible spectra of silver nanoparticles
Fig. 6 EDX spectrum of Ifr-synthesized silver nanoparticles
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Fig. 7 XRD spectrum of Ifr-synthesized silver nanoparticles
Counts 64
NA-IRF
36
16
4
0 20
30
40
50
60
70
Position [°2Theta] (Copper (Cu))
X’Pert PRO X-ray diffractometer operated at a voltage of 40 kV and current of 30 mM with Cu Kα radiation in a θ–2θ configuration. The crystallite domain sizes were calculated from the width of the XRD peaks, assuming that they are free from non-uniform strains, using the Debye-Scherrer formula (Cullity 1978). D ¼ 0:94λ=βcosθ where D is the average crystallite domain size perpendicular to the reflecting planes, λ is the X-ray wavelength, β is the full width at half maximum (FWHM), and β is the diffraction angle. To eliminate additional instrumental broadening the FWHM from a large grained sample, βCorrected ¼ FWHM2 sample−FWHM2 Si ½
Optimization and stabilization of Ifr-AgNPs Mycelial extracts were prepared as for the same procedure mentioned above. Different hydrogen ion concentrations, Table 1 Characteristics of silver nanoparticles synthesized by Ifr-AgNPs
temperature and agitation (like acidic (pH 4.5), neutral (pH 6.5) and alkali (8.5); below room temperature (20± 2 °C), room temperature (30±2 °C) and above room temperature (40±2 °C); and 90, 120, 150 rpm) were prepared in 250-mL Erlenmeyer flask containing 50 mL of mycelial extract, and 1 mM AgNO3 was added into the flask. The change of colour was noted and initial characterization was done through UV-Vis spectra. After colour change, Ifr-AgNPs were stored in screw-capped vials at room temperature, and stability was checked through UV-Vis spectra at different month intervals. Laboratory evaluation of Ifr-synthesized AgNPs against Cx. quinquefasciatus and Ae. aegypti Cx. quinquefasciatus and Ae. aegypti egg rafts were collected from Thiagarajar College campus and reared in the laboratory condition supplemented with dog biscuits and yeast extract in the ratio of 3:1. Bioassay was conducted with Ifr-synthesized AgNps against first, second, third and fourth instar larvae of Cx. quinquefasciatus and Ae. aegypti based on a method of
Characteristics Structural
Functional
UV-Vis spectra (nm)
SEM (Particles Size (nm)
EDX (peak range, KeV)
XRD angle
410
51.31–111.02
3–4
2θ of the intense peak (°) 28.16 32.56 46.48 64.71
Lattice plane 111 122 200 220
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85 80
3500
3000
2500 2000 Wavenumber cm-1
C:\Documents and Settings\PHY CHEM LAB\Desktop\meas\Sample description.16
1500
Sample description
1000
629.08 580.78 548.23 472.35 435.43
896.97 836.09
1111.46 1056.00
1248.12
1411.49 1383.63
1735.82 1671.25 1595.78
2426.91 2355.55 2326.31
2891.10
3166.88
3287.97
3919.39 3890.07 3871.46 3840.84 3790.98 3768.85 3724.79 3693.51 3666.02
65
70
75
Transmittance [%]
90
95
Fig. 8 FTIR spectrum of Ifr-AgNPs
500
Instrument type and / or access 27/01/2014
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World Health Organization (WHO 2005) with minor modifications. For bioassay, 25 larvae/concentration/replication were transferred into 250-mL glass beaker (Borosil®) containing 0.03 to 1.0-ppm concentration. Five replications of IfrAgNPs were maintained separately; each was covered with a mosquito net. The set-up was maintained at 27±2 °C and 77± 4 % RH. The mortality of mosquito larvae was noted at 24-hr intervals with and without Ifr-AgNPs. Statistical analysis Mortality data was subjected to probit analysis to predict the lethal concentration that kills 50 % of the exposed larvae (LC50), lethal concentration that kills 90 % of the exposed larvae (LC90), chi-square, slope and intercept values by using EPA 1.5. Percentage mortality was also calculated for the mortality data using Excel 2007.
Characterization and optimization Ifr-AgNPs The high-density mycelial balls were formed in a specific medium (Fig. 2). Filtrate along with silver nitrate (AgNO3) was subjected to reduction reaction. After 72 h of incubation, the colour changed from pale yellow to dark brown indicating the formation of AgNPs (Fig. 3). UV-Vis spectroscopy is an initial characterization step for analyzing the formation of AgNPs in aqueous solution. The characteristic surface plasmon absorption band was observed at 410 nm after 72 h. Excitation spectra for AgNPs synthesized from AgNO3 were also observed and are presented in Fig. 4. The AgNPs has also been well defined in SEM imaging (Fig. 5), and different sizes and spherical-shaped particles ranging from 51.31 to 111.02 nm and characteristic peak at 3—4 keV in EDX were obtained which indicates the reduction of Ag+ to Ag0 (Fig. 6). The dry powder of the AgNps was used for XRD analyses.
Table 2 FTIR functional groups analysis
Results
Vibration assignment/ functional groups
Observed wave number (cm−1)
Visible intensity
The present investigation, I. fumosorosea (Ifr), was isolated from soil (Fig. 1). Mycelia extracts were used as a starting material for the synthesis of silver nanoparticles (Ifr-AgNPs). The larvicidal potentials were tested against the different instars of Cx. quinquefasciatus and Ae. Aegypti; details of their efficacy are presented vide infra.
O–H stretch ═C–H stretch C–H –C–H bending C═O
3,287.97 3,166.88 2,891.10 1,671.25 1,383.63
Strong broad beak Medium beak Strong broad beak Variable Very strong beak
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The control thin films of the mycelial extract as well as the AgNO3 did not show the characteristic peaks. The diffracted intensities were recorded from 10° to 90° at 2θ angles. XRD analysis (Fig. 7 and Table 1) showed four strong intense peaks at 2θ values of 28.16°, 32.56°, 46.48° and 64.71° assigned to the 111, 122, 200 and 220 planes of a faced centre cubic structure of AgNps. The target was Cu Kα with a wavelength of 1.54056 Å. The XRD pattern indicated that the nanoparticles had a cubic structure. No peaks of the XRD pattern of Ag and other substance appeared, indicating that the AgNps had a high purity. The observed peak broadening and noise were probably related to the effect of nanosized particles and the presence of various crystalline biological macromolecules in the mycelial extract. The obtained results showed that Ag+ had definitely been reduced by Ifr-mycelial extract under reaction conditions. FTIR spectroscopy analysis was carried out to identify the biomolecules responsible for the reduction of Ag+ ions and capping of the bioreduced AgNps synthesized by fungal cell filtrate where located at about 3,287.97; 3,166.88; 2,891.10; 1,671.25 and 1,383.63 in the region 4,000–400 cm−1 (Fig. 8 and Table 2). The FTIR spectral analysis revealed the presence of O–H stretch strong broad beak, ═C–H stretch medium beak, C–H strong broad beak, –C–H bending variable and C ═O very strong beak, which may be present between the amino acid residues and protein synthesized during Ifr-AgNPs. Further, the culture filtrate was optimized, and results are shown in Table 3. The colour was immediately changed at the pH adjusted at alkaline condition. At the same time, agitation in 150 rpm and the temperature at 30 °C were optimum condition for AgNPs confirmed through colour change. Stability was confirmed through characterization at monthly intervals, but there were no changes in the surface plasmon absorption bands as well as there was no deposition in aqueous solution after 1 year in room temperature.
Toxicity of Ifr-AgNPs against Cx. quinquefasciatus and Ae. aegypti larval instars The Ifr-AgNPs were found more susceptible against all larval instars of Cx. quinquefasciatus and Ae. aegypti. Ae. aegypti was found highly susceptible to the synthesized
Table 3 Optimization of Ifr-AgNps at 24 h
Parameters
Optimum condition
pH Temperature (°C) Agitation (rpm)
8.5 30±2 150
Table 4 Larvicidal activity (in ppm) of I. fumosorosea (Ifr)-synthesized silver nanoparticles (Ifr-AgNPs) against Cx. quinquefasciatus Concentration (ppm)
% Mortality of larval instars First instar
Second instar
Third instar
Fourth instar
0.03 0.06 0.09 0.12 0.15 0.25
6.00±0.70 10.0±1.14 18.4±1.14 28.0±0.83 40.4±1.67 56.4±0.70
4.8±0.83 7.2±0.83 15.2±1.30 22.8±2.28 38.0±1.87 50.4±1.14
2.0±0.44 6.0±0.70 10.6±1.14 16.0±1.58 22.0±2.07 44.0±1.22
0.00±0.00 1.6±0.54 3.2±0.83 6.4±0.89 12.8±1.30 24.0±1.58
0.50 1.00 Control
76.0±2.16 81.6±1.94 0.00±0.00
59.2±1.64 72±1.22 0.00±0.00
56.8±1.92 67.2±1.94 0.00±0.00
42.4±1.14 60.8±1.30 0.00±0.00
% Percent mortality values are means±SD
AgNPs than the larvae of Cx. quinquefasciatus. The first instar of Ae. aegypti has shown 100 % mortality after 24 h. While, the fourth instar larvae were less susceptible to the synthesized AgNPs. However, the mortality rate was indirectly proportional to the larval instar, and the concentration was directly proportional to the mortality (Tables 4 and 5). The LC50 and LC90 values of the tested concentration are 0.240, 0 0.075.337, 0.430, 0.652 and 1.219, 2.210, 2.453, 2.916; 0.065, 0.075, 0.098, 0.137 and 0.558, 0.709, 0.949, 1.278 ppm with respect to 0.03 to 1.00 ppm of Ifr-AgNPs against first, second, third and fourth instars of Cx. quinquefasciatus and Ae. aegypti, respectively. The chi-square values at 0.05 significant level, confidential limit and intercept slope are shown in Tables 6 and 7.
Table 5 Larvicidal activity (in ppm) of I. fumosorosea (Ifr)-synthesized silver nanoparticles (Ifr-AgNPs) against Ae. aegypti Concentration (ppm)
0.03 0.06 0.09 0.12 0.15 0.25 0.50 1.00 Control
% Mortality of larval instars First instar
Second instar
Third instar
Fourth instar
39.2±1.64 47.2±0.83 52.0±0.70 60.0±1.58 72.0±1.22 77.6±0.89 84.8±1.30 100±0.00 0.00±0.00
35.2±1.48 43.2±1.09 51.2±1.14 57.6±1.14 68.4±1.51 74.4±1.14 79.2±0.83 99.2±0.44 0.00±0.00
30.4±1.14 40.0±1.00 46.4±2.07 48.8±1.48 58.4±1.14 70.4±1.14 79.2±0.83 95.2±0.83 0.00±0.00
25.6±2.80 31.2±2.46 37.6±1.14 43.2±0.83 49.6±1.14 60.8±0.83 76.8±0.83 92±0.70 0.00±0.00
% Percent mortality values are means±SD
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Table 6 LC50, LC90, intercept, chi-Square and slope values of I. fumosorosea (Ifr)-synthesized silver nanoparticles (Ifr-AgNPs) against Cx. quinquefasciatus Larval instar
LC50 (lower and upper confidence limit)
LC90 (lower and upper confidence limit)
Chi-square
Intercept
Slope
First instar Second instar Third instar Fourth instar
0.240 (0.211–0.276) 0.337 (0.288–0.404) 0.430 (0.366–0.520) 0.652 (0.551–0.802)
1.219 (0.941–1.697) 2.210 (1.566–3.482) 2.453 (1.752–3.822) 2.916 (2.096–4.520)
7.645* 11.299* 6.962* 2.982*
6.125 5.741 5.621 5.365
1.815 1.568 1.694 1.970
LC50 lethal concentration that kills 50 % of the exposed larvae, LC90 lethal concentration that kills 90 % of the exposed larvae *Significant at 0.05 % level
Discussion The various microscopic filamentous fungi (ascomycetes, fungi imperfecti, etc.) and other fungi produced about 6,400 bioactive compounds (Berdy 2005; Siddhardha et al. 2012). S e v e n s p e c i e s o f f u n g i , n a m e l y, C l a d o s p o r i u m spherospermum, Cladosporium oxysporum, Chaetomium indicum, Gilmaniella subornata and Penicillium purpurogenum were screened for their ability to produce secondary metabolites. The crude extracts of fungi were evaluated for antimicrobial and larvicidal activity. Extracellularly produced nanoparticles were stabilized by the proteins and reducing agents secreted by the fungus. There were reports that some high-molecular-weight proteins including the NADH-dependent reductase are released by fungal biomass in nanoparticle synthesis and stabilization. However, emission band produced by fluorescence spectra indicate the native form of these proteins present in the solution as well as bound to the surface of nanoparticles (Macdonald and Smith 1996). In the present investigation, AgNps were produced extracellularly using entomopathogenic fungi Ifr against major vector mosquitoes. The filtrated was treated with AgNO3, the reaction started after 24 h of incubation in dark condition, with change in colour of filtrate from pale yellow to brownish yellow, indicating the formation of silver bionanoparticles which correlated with the results obtained by Mukherjee et al. 2001; Soni and Prakash 2012; Najitha Banu et al. 2014 and Najitha Banu and Balasubramanian 2014. In addition, they have given a
characteristic band at 410 nm, while no absorption band was observed in both controls (positive and negative). Thus, it indicates the complete reduction of silver ions to turn from pale yellow to dark brown colour in aqueous solution due to excitation of surface plasmon vibration in AgNps (Vigneshwaran et al. 2007). However, the bioreduction of the Ag+ could be associated with metabolic processes utilizing nitrate by reducing nitrate to nitrite and ammonium (Lengke et al. 2007). The extracellular biosynthesis of AgNps using the filamentous fungus A. fumigates and entomopathogenic fungi B. bassiana has been investigated by Bhainsa and D’Souza (2006), Ingle et al. (2008) and Najitha Banu and Balasubramanian (2014). The present study reveals that the presence of nanoparticles was confirmed by scanning electron microscope and the particles size ranges between 51.31 and 111.02 nm. The EDX has confirmed the presence of elemental silver by the sharp peaks at a range of 3–4 keV which is typical for the absorption of metallic AgNps. The X-ray diffraction pattern of pure silver ions is known to display peaks at 2θ values of 07.9°, 11.4°, 17.8°, 30.38°, 44° and 23.2250, 27.8800, 32.1833 (Gong et al. 2007; Najitha Banu and Balasubramanian 2014). Similarly, the study reveals the characteristic peaks at 28.16°, 32.56°, 46.48° and 64.71°, which correspond to the (111), (122), (200), (220) (Bragg reflection) and reflections of silver. Therefore, X-ray diffraction results also suggest that crystallization of bioorganic phase occurs on the surface of AgNps. FTIR spectra clearly indicate that the biomolecules especially proteins present in filtrate are responsible for synthesis and stabilization of AgNps (Dhanasekaran and Thangaraj 2013).
Table 7 LC50, LC90, intercept, chi-square and slope value of I. fumosorosea (Ifr)-synthesized silver nanoparticles (Ifr-AgNPs) against Ae. aegypti Larval instar
LC50 (lower and upper confidence limit)
LC90 (lower and upper confidence limit)
Chi-square
Intercept
Slope
First instar Second instar
0.065 (0.052–0.078) 0.075 (0.061–0.090)
0.558 (0.426–0.806) 0.709 (0.526–1.063)
11.907* 12.341*
6.628 6.478
1.371 1.315
Third instar Fourth instar
0.098 (0.081–0.115) 0.137 (0.116–0.161)
0.949 (0.687–1.476) 1.278 (0.908–2.032)
6.102* 6.157*
6.311 6.140
1.296 1.322
LC50 lethal concentration that kills 50 % of the exposed larvae, LC90 lethal concentration that kills 90 % of the exposed larvae *Significant at 0.05 % level
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Sanghi and Verma (2009) have investigated the ability of Coriolus versicolor in formation of monodisperse spherical AgNps; the time taken for production of AgNps was reduced from 72 to 1 h under alkaline conditions (pH 10). It was indicated that alkaline conditions might be involved in bioreduction of silver ions (Iravani et al. 2014). Similar results were obtained in the present investigation that when the culture filtrate is mixed with AgNO3 solution, immediately, the colour will change, and it was confirmed by the bioreduction that has occurred in an aqueous solution. Extracellular synthesis of AgNps was reported using fungus like A. niger, Chrysosporium tropicum, Penicillium sp., and A. niger 2587, B. bassiana, (Soni and Prakash 2012, 2013; Dhanasekaran and Thangaraj 2013; Najitha Banu and Balasubramanian 2014). The use of nanoparticles in insects and their potential in insect pest management have been focused by Bhattacharyya et al. (2010). The larvicidal potential of AgNps synthesized using fungus Cochliobolus lunatus against Ae. aegypti and Anopheles stephensi has been observed (Salunkhe et al. 2011). Soni and Prakash (2013) reported on the potentiality of AgNPs synthesized by a fungus F. oxysporum and found LC50 and LC90 values of 8, 6, 4; 12.30, 12.58, 11.48 against first, second and fourth instar larvae of Cx. quinquefasciatus, An. stephensi and Ae. aegypti, respectively, which are much higher to the concentration in the present investigation. The LC50 and LC90 values are 0.240 to 0.652 and 1.219 to 2.916, respectively, in all larval instars (I–IV) of Cx. quinquefasciatus and for Ae. aegypti 0.065 to 0.137 and 0.558 to 1.278 ppm. Similarly, Santhoshkumar et al. (2011) also obtained LC50 and LC90 values of 0.69 and 1.10 ppm as well as 2.15 and 3.59 ppm of AgNPs synthesized by leaf extract of Nelumbo nucifera against Cx. quinquefasciatus and Anopheles subpictus which were analogous to the results obtained in the present study. Subarani et al. (2013) reported that the Vinca rosea-synthesized AgNps did not exhibit any noticeable toxicity on Poecilia reticulata after 24, 48, and 72 h of exposure. From the above results, we suggest that myco-synthesized AgNps are potential larvicidal and eco-friendly agent for controlling mosquito population. Acknowledgment The authors gratefully acknowledge the Management, Thiagarajar College (Autonomous), Madurai, for providing the facilities to perform the research works in the PG and Research Department of Zoology and Microbiology. Author ANB thank UGC-MANF, India, for the financial support and CRME (ICMR), Madurai, who kindly supplied eggs and larvae required during our work. We thank the Department of Chemistry, Madras University and Karunya University, Coimbatore, for the instrumental analysis.
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ORIGINAL PAPER
Optimization and synthesis of silver nanoparticles using Isaria fumosorosea against human vector mosquitoes A. Najitha Banu & C. Balasubramanian
Received: 20 June 2014 / Accepted: 23 July 2014 / Published online: 3 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The efficacy of silver generated larvicide with the help of entomopathogenic fungi, Isaria fumosorosea (Ifr) against major vector mosquitoes Culex quinquefasciatus and Aedes aegypti. The Ifr-silver nanoparticles (AgNPs) were characterized structurally and functionally using UV-visible spectrophotometer followed by scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy and Fourier transform infrared (FTIR) spectra. The optimum pH (alkaline), temperature (30 °C) and agitation (150 rpm) for AgNP synthesis and its stability were confirmed through colour change. Ae. aegypti larvae (I–IV instars) were found highly susceptible to synthesized AgNPs than the larvae of Cx. quinquefasciatus. However, the mortality rate was indirectly proportional to the larval instar and the concentration. The lethal concentration that kills 50 % of the exposed larvae (LC50) and lethal concentration that kills 90 % of the exposed larvae (LC90) values of the tested concentration are 0.240, 0 0.075.337, 0.430, 0.652 and 1.219, 2.210, 2.453, 2.916; 0.065, 0.075, 0.098, 0.137 and 0.558, 0.709, 0.949, 1.278 ppm with respect to 0.03 to 1.00 ppm of Ifr-AgNPs against first, second, third and fourth instars of Cx. quinquefasciatus and Ae. aegypti, respectively. This is the first report for synthesis of AgNPs using Ifr against human vector mosquitoes. Hence, Ifr-AgNPs would be significantly used as a potent mosquito larvicide.
Keywords Isaria fumosorosea . Silver nanoparticles . Toxicity . LC50 and LC90
A. N. Banu (*) : C. Balasubramanian Department of Zoology and Microbiology, Thiagarajar College (Autonomous), Madurai, Tamil Nadu 625009, India e-mail: [email protected]
Introduction One of the major public health concerns in developing countries is vector-borne diseases. The genera Aedes, Anopheles and Culex adults transmit serious human diseases; viz, malaria, filariasis, encephalitis, dengue and recently chikungunya are the major mosquito-borne diseases in India. In the current situation of global warming and contaminated fresh water bodies, a number of mosquitoes are markedly increasing in concurrence with a high incidence of dengue fever (Halstead 2007). Mosquitoes constitute the most important single family of insects from the stand point of human health. In this regard, Aedes aegypti (Diptera: Culicidae) is the main vector of chikungunya and dengue fever (Sourisseau et al. 2007). Till 10th October 2012, 151 districts of eight states/provinces of India have been affected by chikungunya fever (Pialoux et al. 2007; Yang et al. 2009). According to WHO report of year 2009, two fifth of world population is under risk of dengue infection (WHO index) and in year 2010, 28,292 cases of infection and 108 deaths were reported in India (NVBDCP 2011). The incidence of dengue has grown dramatically around the world in recent decades over 2.5 billion people (40 % of the world’s population) at risk from dengue. WHO currently estimates that there may be 50– 100 million dengue infections worldwide every year (WHO 2012). Dengue cases were reported in the year 2012, November 27, the total number of dengue cases in India around 35,066 and 216 deaths. The highest numbers of dengue cases were recorded in the country for the year 2012 to 2013 till November 15, around 9,249 cases in Tamil Nadu and West Bengal 6,067 cases. Lymphatic filariasis is a serious public health problem in India, constituting one third of the infected population in the world (WHO 1997). Culex quinquefasciatus is a vector of lymphatic filariasis, which affects 120 million people worldwide, and approximately 400 million people are at risk of
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contracting filariasis, resulting in an annual economic loss of US$1.5 billion (WHO 2002). Malaria now is responsible for illness in more than an estimated 300 million people, resulting in 1 million deaths per year (WHO 2007). Lymphatic filariasis, commonly known as elephantiasis, is a painful and profoundly disfiguring disease. An estimated 120 million people in tropical and subtropical areas of the world are infected with lymphatic filariasis; of these, almost 25 million men have genital disease (most commonly hydrocele) and almost 15 million, mostly women, have elephantiasis of the leg. Approximately 66 % of those at risk of infection live in the WHO South-East Asia Region and 33 % in the African Region (WHO 2010). More importantly, the incidences of resistance to larvicides of mosquito larvae have been reported by Braga et al. (2004) and Melo-Santos et al. (2010).Thus, attempts to develop novel materials as mosquito larvicides are still necessary. Among the biological organism so far used for the mosquito control programmes, bacteria such as Bacillus species are known for its mosquito larvicidal effect. Of which, Bacillus thuringiensis var. israelensis and Bacillus sphaericus are effective, but serious resistance as high as 50,000-fold has evolved where B. sphaericus is used against Culex mosquitoes (Soni and Prakash 2011). Recently, the laboratory resistance in the mosquitoes has been demonstrated to some isolates of B. thuringiensis (Surendran and Vennison 2011; CadavidRestrepo et al. 2012; Chenniappan and Ayyadurai 2012). Many organisms synthesize silver nanoparticles (AgNps) extracellularly and intracellularly, among which Verticillium spp. (Mukherjee et al. 2001), Aspergillus fumigatus (Bhainsa and D’Souza 2006), Fusarium oxysporum (Anil Kumar et al. 2007; Sonal et al. 2013), Klebsiella pneumoniae, (Shahverdi et al. 2007), Bacillus licheniformis (Kalimuthu et al. 2008), Escherichia coli, Enterobacter cloacae (Minaein et al. 2008), Bacillus megaterium (Saravanan et al. 2011), Pseudomonas proteolytica, Pseudomonas meridiana, Arthrobacter kerguelensis, Bacillus indicus (Shivaji et al. 2011), Aspergillus niger (Soni and Prakash 2013), B. thuringiensis (Jain et al. 2010; Najitha Banu et al. 2014), Bacillus strain CS 11 (Das et al. 2014) and Beauveria bassiana (Najitha Banu and Balasubramanian 2014) have been reported extensively. The nanoparticles encoded by secondary metabolites of bacteria and entomopathogenic fungi are a novel tool to havoc the larval population from the nuisance where vector species cause endemic diseases. This approach has been successfully adopted in vector control programme in future and also thus the reduction of xenobiotic chemicals loads in the environment directly (Najitha Banu and Balasubramanian 2014). Recently, the new era was developed for controlling mosquito population by using biologically synthesized AgNps treated against the mosquito larvae. Hence, the present study was aimed to evaluate the fungi-synthesized AgNps against the human vector mosquitoes.
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Fig. 1 Isaria fumosorosea (Ifr)
Materials and methods Isolation and identification of entomopathogenic fungi The entomopathogenic fungi Isaria fumosorosea (Ifr) was isolated from rhizosphere soil, around Madurai, TN, India according to the method of Haraprasad et al. 2001. After 7 days of incubation, pure culture of Ifr was subcultured into PDA by streak plate method. The isolated culture was identified by slide culture method subjected to lactophenol cotton blue staining observed under the light microscope (Labomed). Extracellular synthesis of AgNPs The pure culture of Ifr was freshly inoculated on a liquid media containing (g/L) KH 2 PO 4 7.0, K 2 HPO 4 2.0, MgSO4 ·H2O 0.1, (NH4)2SO4 1.0, yeast extract 0.6 and glucose 10.0 in an Erlenmeyer flask. The flask containing medium was incubated in orbitary shaker at 150 rpm in 25±2 °C (Neolab Instruments, Mumbai, India) for 72 h. The biomass was harvested after 72 h of growth by sieving through a Whatman No. 1 filter paper (Whatman International Ltd, England), followed by extensive wash
Fig. 2 High-density mycelium in specific media
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a
b
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c
Fig. 3 Photographs of a Ifr-AgNPs, b mycelial extract and c 1 mM AgNO3
with distilled water to remove any medium components from the biomass. Twenty gram of fresh and clean biomass was taken into Erlenmeyer flask containing 200 mL Milli-Q water, and the flask was incubated at 25 °C for 72 h and agitated in the same condition as described earlier. After incubation, the cell filtrates were obtained by passing it through the Whatman No. 1 filter paper. Cell filtrate (50 mL) was taken into 250-mL Erlenmeyer flask and mixed with 1 mM AgNO 3 (Laboratory Reagent, Reachem Laboratory Chemicals Private Ltd, Madras, Tamil Nadu, India) (0.017 g/100 mL) as final concentration. The flasks were incubated at 25 °C in a dark condition for 120 h and agitated in the same condition as described earlier. The control was maintained separately (without the addition of AgNO3), only cell filtrate with the experimental flask. The AgNPs turned into brownish yellow colour solution, and it was stored in screw-capped vials under ambient condition for further experiment.
Fig. 5 SEM image of Ifr-AgNPs
PerkinElmer (Lambda 35) spectrophotometer. The spectra were recorded in the region 200–800 nm at a scan rate of 240 nm s−1. The atomic composition of nanoparticles was confirmed the energy-dispersive X-ray spectroscopy (EDX; Oxford instrument, INCA PentaFET x3, Karunya University, Coimbatore, Tamil Nadu, India) coupled with scanning electron microscope (SEM) (Model-JEOL, JSM-6390). Further characterization of Ag bionanoparticles implicated with Fourier transform infrared (FTIR) spectra was recorded using Bruker Tensor-27 FT-IR spectrometer with OPUS software in the range 4,000–400 cm−1, at a resolution of 4 cm−1. The pellet for analysis was made by taking equal amounts of IfrAgNPs and KBr (1:1 ratio); the background calibrations have been carried out using pure KBr pellet.
Characterization of AgNPs
XRD analysis
The UV-Vis spectra analysis of the freshly prepared nanoparticles was recorded with water/methanol as reference using the
AgNps were checked by X-ray diffractometer (XRD). The pre-prepared dry power was collected for the determination of the formation of Ag nanoparticles by an
Fig. 4 UV-Visible spectra of silver nanoparticles
Fig. 6 EDX spectrum of Ifr-synthesized silver nanoparticles
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Fig. 7 XRD spectrum of Ifr-synthesized silver nanoparticles
Counts 64
NA-IRF
36
16
4
0 20
30
40
50
60
70
Position [°2Theta] (Copper (Cu))
X’Pert PRO X-ray diffractometer operated at a voltage of 40 kV and current of 30 mM with Cu Kα radiation in a θ–2θ configuration. The crystallite domain sizes were calculated from the width of the XRD peaks, assuming that they are free from non-uniform strains, using the Debye-Scherrer formula (Cullity 1978). D ¼ 0:94λ=βcosθ where D is the average crystallite domain size perpendicular to the reflecting planes, λ is the X-ray wavelength, β is the full width at half maximum (FWHM), and β is the diffraction angle. To eliminate additional instrumental broadening the FWHM from a large grained sample, βCorrected ¼ FWHM2 sample−FWHM2 Si ½
Optimization and stabilization of Ifr-AgNPs Mycelial extracts were prepared as for the same procedure mentioned above. Different hydrogen ion concentrations, Table 1 Characteristics of silver nanoparticles synthesized by Ifr-AgNPs
temperature and agitation (like acidic (pH 4.5), neutral (pH 6.5) and alkali (8.5); below room temperature (20± 2 °C), room temperature (30±2 °C) and above room temperature (40±2 °C); and 90, 120, 150 rpm) were prepared in 250-mL Erlenmeyer flask containing 50 mL of mycelial extract, and 1 mM AgNO3 was added into the flask. The change of colour was noted and initial characterization was done through UV-Vis spectra. After colour change, Ifr-AgNPs were stored in screw-capped vials at room temperature, and stability was checked through UV-Vis spectra at different month intervals. Laboratory evaluation of Ifr-synthesized AgNPs against Cx. quinquefasciatus and Ae. aegypti Cx. quinquefasciatus and Ae. aegypti egg rafts were collected from Thiagarajar College campus and reared in the laboratory condition supplemented with dog biscuits and yeast extract in the ratio of 3:1. Bioassay was conducted with Ifr-synthesized AgNps against first, second, third and fourth instar larvae of Cx. quinquefasciatus and Ae. aegypti based on a method of
Characteristics Structural
Functional
UV-Vis spectra (nm)
SEM (Particles Size (nm)
EDX (peak range, KeV)
XRD angle
410
51.31–111.02
3–4
2θ of the intense peak (°) 28.16 32.56 46.48 64.71
Lattice plane 111 122 200 220
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85 80
3500
3000
2500 2000 Wavenumber cm-1
C:\Documents and Settings\PHY CHEM LAB\Desktop\meas\Sample description.16
1500
Sample description
1000
629.08 580.78 548.23 472.35 435.43
896.97 836.09
1111.46 1056.00
1248.12
1411.49 1383.63
1735.82 1671.25 1595.78
2426.91 2355.55 2326.31
2891.10
3166.88
3287.97
3919.39 3890.07 3871.46 3840.84 3790.98 3768.85 3724.79 3693.51 3666.02
65
70
75
Transmittance [%]
90
95
Fig. 8 FTIR spectrum of Ifr-AgNPs
500
Instrument type and / or access 27/01/2014
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World Health Organization (WHO 2005) with minor modifications. For bioassay, 25 larvae/concentration/replication were transferred into 250-mL glass beaker (Borosil®) containing 0.03 to 1.0-ppm concentration. Five replications of IfrAgNPs were maintained separately; each was covered with a mosquito net. The set-up was maintained at 27±2 °C and 77± 4 % RH. The mortality of mosquito larvae was noted at 24-hr intervals with and without Ifr-AgNPs. Statistical analysis Mortality data was subjected to probit analysis to predict the lethal concentration that kills 50 % of the exposed larvae (LC50), lethal concentration that kills 90 % of the exposed larvae (LC90), chi-square, slope and intercept values by using EPA 1.5. Percentage mortality was also calculated for the mortality data using Excel 2007.
Characterization and optimization Ifr-AgNPs The high-density mycelial balls were formed in a specific medium (Fig. 2). Filtrate along with silver nitrate (AgNO3) was subjected to reduction reaction. After 72 h of incubation, the colour changed from pale yellow to dark brown indicating the formation of AgNPs (Fig. 3). UV-Vis spectroscopy is an initial characterization step for analyzing the formation of AgNPs in aqueous solution. The characteristic surface plasmon absorption band was observed at 410 nm after 72 h. Excitation spectra for AgNPs synthesized from AgNO3 were also observed and are presented in Fig. 4. The AgNPs has also been well defined in SEM imaging (Fig. 5), and different sizes and spherical-shaped particles ranging from 51.31 to 111.02 nm and characteristic peak at 3—4 keV in EDX were obtained which indicates the reduction of Ag+ to Ag0 (Fig. 6). The dry powder of the AgNps was used for XRD analyses.
Table 2 FTIR functional groups analysis
Results
Vibration assignment/ functional groups
Observed wave number (cm−1)
Visible intensity
The present investigation, I. fumosorosea (Ifr), was isolated from soil (Fig. 1). Mycelia extracts were used as a starting material for the synthesis of silver nanoparticles (Ifr-AgNPs). The larvicidal potentials were tested against the different instars of Cx. quinquefasciatus and Ae. Aegypti; details of their efficacy are presented vide infra.
O–H stretch ═C–H stretch C–H –C–H bending C═O
3,287.97 3,166.88 2,891.10 1,671.25 1,383.63
Strong broad beak Medium beak Strong broad beak Variable Very strong beak
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The control thin films of the mycelial extract as well as the AgNO3 did not show the characteristic peaks. The diffracted intensities were recorded from 10° to 90° at 2θ angles. XRD analysis (Fig. 7 and Table 1) showed four strong intense peaks at 2θ values of 28.16°, 32.56°, 46.48° and 64.71° assigned to the 111, 122, 200 and 220 planes of a faced centre cubic structure of AgNps. The target was Cu Kα with a wavelength of 1.54056 Å. The XRD pattern indicated that the nanoparticles had a cubic structure. No peaks of the XRD pattern of Ag and other substance appeared, indicating that the AgNps had a high purity. The observed peak broadening and noise were probably related to the effect of nanosized particles and the presence of various crystalline biological macromolecules in the mycelial extract. The obtained results showed that Ag+ had definitely been reduced by Ifr-mycelial extract under reaction conditions. FTIR spectroscopy analysis was carried out to identify the biomolecules responsible for the reduction of Ag+ ions and capping of the bioreduced AgNps synthesized by fungal cell filtrate where located at about 3,287.97; 3,166.88; 2,891.10; 1,671.25 and 1,383.63 in the region 4,000–400 cm−1 (Fig. 8 and Table 2). The FTIR spectral analysis revealed the presence of O–H stretch strong broad beak, ═C–H stretch medium beak, C–H strong broad beak, –C–H bending variable and C ═O very strong beak, which may be present between the amino acid residues and protein synthesized during Ifr-AgNPs. Further, the culture filtrate was optimized, and results are shown in Table 3. The colour was immediately changed at the pH adjusted at alkaline condition. At the same time, agitation in 150 rpm and the temperature at 30 °C were optimum condition for AgNPs confirmed through colour change. Stability was confirmed through characterization at monthly intervals, but there were no changes in the surface plasmon absorption bands as well as there was no deposition in aqueous solution after 1 year in room temperature.
Toxicity of Ifr-AgNPs against Cx. quinquefasciatus and Ae. aegypti larval instars The Ifr-AgNPs were found more susceptible against all larval instars of Cx. quinquefasciatus and Ae. aegypti. Ae. aegypti was found highly susceptible to the synthesized
Table 3 Optimization of Ifr-AgNps at 24 h
Parameters
Optimum condition
pH Temperature (°C) Agitation (rpm)
8.5 30±2 150
Table 4 Larvicidal activity (in ppm) of I. fumosorosea (Ifr)-synthesized silver nanoparticles (Ifr-AgNPs) against Cx. quinquefasciatus Concentration (ppm)
% Mortality of larval instars First instar
Second instar
Third instar
Fourth instar
0.03 0.06 0.09 0.12 0.15 0.25
6.00±0.70 10.0±1.14 18.4±1.14 28.0±0.83 40.4±1.67 56.4±0.70
4.8±0.83 7.2±0.83 15.2±1.30 22.8±2.28 38.0±1.87 50.4±1.14
2.0±0.44 6.0±0.70 10.6±1.14 16.0±1.58 22.0±2.07 44.0±1.22
0.00±0.00 1.6±0.54 3.2±0.83 6.4±0.89 12.8±1.30 24.0±1.58
0.50 1.00 Control
76.0±2.16 81.6±1.94 0.00±0.00
59.2±1.64 72±1.22 0.00±0.00
56.8±1.92 67.2±1.94 0.00±0.00
42.4±1.14 60.8±1.30 0.00±0.00
% Percent mortality values are means±SD
AgNPs than the larvae of Cx. quinquefasciatus. The first instar of Ae. aegypti has shown 100 % mortality after 24 h. While, the fourth instar larvae were less susceptible to the synthesized AgNPs. However, the mortality rate was indirectly proportional to the larval instar, and the concentration was directly proportional to the mortality (Tables 4 and 5). The LC50 and LC90 values of the tested concentration are 0.240, 0 0.075.337, 0.430, 0.652 and 1.219, 2.210, 2.453, 2.916; 0.065, 0.075, 0.098, 0.137 and 0.558, 0.709, 0.949, 1.278 ppm with respect to 0.03 to 1.00 ppm of Ifr-AgNPs against first, second, third and fourth instars of Cx. quinquefasciatus and Ae. aegypti, respectively. The chi-square values at 0.05 significant level, confidential limit and intercept slope are shown in Tables 6 and 7.
Table 5 Larvicidal activity (in ppm) of I. fumosorosea (Ifr)-synthesized silver nanoparticles (Ifr-AgNPs) against Ae. aegypti Concentration (ppm)
0.03 0.06 0.09 0.12 0.15 0.25 0.50 1.00 Control
% Mortality of larval instars First instar
Second instar
Third instar
Fourth instar
39.2±1.64 47.2±0.83 52.0±0.70 60.0±1.58 72.0±1.22 77.6±0.89 84.8±1.30 100±0.00 0.00±0.00
35.2±1.48 43.2±1.09 51.2±1.14 57.6±1.14 68.4±1.51 74.4±1.14 79.2±0.83 99.2±0.44 0.00±0.00
30.4±1.14 40.0±1.00 46.4±2.07 48.8±1.48 58.4±1.14 70.4±1.14 79.2±0.83 95.2±0.83 0.00±0.00
25.6±2.80 31.2±2.46 37.6±1.14 43.2±0.83 49.6±1.14 60.8±0.83 76.8±0.83 92±0.70 0.00±0.00
% Percent mortality values are means±SD
Parasitol Res (2014) 113:3843–3851
3849
Table 6 LC50, LC90, intercept, chi-Square and slope values of I. fumosorosea (Ifr)-synthesized silver nanoparticles (Ifr-AgNPs) against Cx. quinquefasciatus Larval instar
LC50 (lower and upper confidence limit)
LC90 (lower and upper confidence limit)
Chi-square
Intercept
Slope
First instar Second instar Third instar Fourth instar
0.240 (0.211–0.276) 0.337 (0.288–0.404) 0.430 (0.366–0.520) 0.652 (0.551–0.802)
1.219 (0.941–1.697) 2.210 (1.566–3.482) 2.453 (1.752–3.822) 2.916 (2.096–4.520)
7.645* 11.299* 6.962* 2.982*
6.125 5.741 5.621 5.365
1.815 1.568 1.694 1.970
LC50 lethal concentration that kills 50 % of the exposed larvae, LC90 lethal concentration that kills 90 % of the exposed larvae *Significant at 0.05 % level
Discussion The various microscopic filamentous fungi (ascomycetes, fungi imperfecti, etc.) and other fungi produced about 6,400 bioactive compounds (Berdy 2005; Siddhardha et al. 2012). S e v e n s p e c i e s o f f u n g i , n a m e l y, C l a d o s p o r i u m spherospermum, Cladosporium oxysporum, Chaetomium indicum, Gilmaniella subornata and Penicillium purpurogenum were screened for their ability to produce secondary metabolites. The crude extracts of fungi were evaluated for antimicrobial and larvicidal activity. Extracellularly produced nanoparticles were stabilized by the proteins and reducing agents secreted by the fungus. There were reports that some high-molecular-weight proteins including the NADH-dependent reductase are released by fungal biomass in nanoparticle synthesis and stabilization. However, emission band produced by fluorescence spectra indicate the native form of these proteins present in the solution as well as bound to the surface of nanoparticles (Macdonald and Smith 1996). In the present investigation, AgNps were produced extracellularly using entomopathogenic fungi Ifr against major vector mosquitoes. The filtrated was treated with AgNO3, the reaction started after 24 h of incubation in dark condition, with change in colour of filtrate from pale yellow to brownish yellow, indicating the formation of silver bionanoparticles which correlated with the results obtained by Mukherjee et al. 2001; Soni and Prakash 2012; Najitha Banu et al. 2014 and Najitha Banu and Balasubramanian 2014. In addition, they have given a
characteristic band at 410 nm, while no absorption band was observed in both controls (positive and negative). Thus, it indicates the complete reduction of silver ions to turn from pale yellow to dark brown colour in aqueous solution due to excitation of surface plasmon vibration in AgNps (Vigneshwaran et al. 2007). However, the bioreduction of the Ag+ could be associated with metabolic processes utilizing nitrate by reducing nitrate to nitrite and ammonium (Lengke et al. 2007). The extracellular biosynthesis of AgNps using the filamentous fungus A. fumigates and entomopathogenic fungi B. bassiana has been investigated by Bhainsa and D’Souza (2006), Ingle et al. (2008) and Najitha Banu and Balasubramanian (2014). The present study reveals that the presence of nanoparticles was confirmed by scanning electron microscope and the particles size ranges between 51.31 and 111.02 nm. The EDX has confirmed the presence of elemental silver by the sharp peaks at a range of 3–4 keV which is typical for the absorption of metallic AgNps. The X-ray diffraction pattern of pure silver ions is known to display peaks at 2θ values of 07.9°, 11.4°, 17.8°, 30.38°, 44° and 23.2250, 27.8800, 32.1833 (Gong et al. 2007; Najitha Banu and Balasubramanian 2014). Similarly, the study reveals the characteristic peaks at 28.16°, 32.56°, 46.48° and 64.71°, which correspond to the (111), (122), (200), (220) (Bragg reflection) and reflections of silver. Therefore, X-ray diffraction results also suggest that crystallization of bioorganic phase occurs on the surface of AgNps. FTIR spectra clearly indicate that the biomolecules especially proteins present in filtrate are responsible for synthesis and stabilization of AgNps (Dhanasekaran and Thangaraj 2013).
Table 7 LC50, LC90, intercept, chi-square and slope value of I. fumosorosea (Ifr)-synthesized silver nanoparticles (Ifr-AgNPs) against Ae. aegypti Larval instar
LC50 (lower and upper confidence limit)
LC90 (lower and upper confidence limit)
Chi-square
Intercept
Slope
First instar Second instar
0.065 (0.052–0.078) 0.075 (0.061–0.090)
0.558 (0.426–0.806) 0.709 (0.526–1.063)
11.907* 12.341*
6.628 6.478
1.371 1.315
Third instar Fourth instar
0.098 (0.081–0.115) 0.137 (0.116–0.161)
0.949 (0.687–1.476) 1.278 (0.908–2.032)
6.102* 6.157*
6.311 6.140
1.296 1.322
LC50 lethal concentration that kills 50 % of the exposed larvae, LC90 lethal concentration that kills 90 % of the exposed larvae *Significant at 0.05 % level
3850
Sanghi and Verma (2009) have investigated the ability of Coriolus versicolor in formation of monodisperse spherical AgNps; the time taken for production of AgNps was reduced from 72 to 1 h under alkaline conditions (pH 10). It was indicated that alkaline conditions might be involved in bioreduction of silver ions (Iravani et al. 2014). Similar results were obtained in the present investigation that when the culture filtrate is mixed with AgNO3 solution, immediately, the colour will change, and it was confirmed by the bioreduction that has occurred in an aqueous solution. Extracellular synthesis of AgNps was reported using fungus like A. niger, Chrysosporium tropicum, Penicillium sp., and A. niger 2587, B. bassiana, (Soni and Prakash 2012, 2013; Dhanasekaran and Thangaraj 2013; Najitha Banu and Balasubramanian 2014). The use of nanoparticles in insects and their potential in insect pest management have been focused by Bhattacharyya et al. (2010). The larvicidal potential of AgNps synthesized using fungus Cochliobolus lunatus against Ae. aegypti and Anopheles stephensi has been observed (Salunkhe et al. 2011). Soni and Prakash (2013) reported on the potentiality of AgNPs synthesized by a fungus F. oxysporum and found LC50 and LC90 values of 8, 6, 4; 12.30, 12.58, 11.48 against first, second and fourth instar larvae of Cx. quinquefasciatus, An. stephensi and Ae. aegypti, respectively, which are much higher to the concentration in the present investigation. The LC50 and LC90 values are 0.240 to 0.652 and 1.219 to 2.916, respectively, in all larval instars (I–IV) of Cx. quinquefasciatus and for Ae. aegypti 0.065 to 0.137 and 0.558 to 1.278 ppm. Similarly, Santhoshkumar et al. (2011) also obtained LC50 and LC90 values of 0.69 and 1.10 ppm as well as 2.15 and 3.59 ppm of AgNPs synthesized by leaf extract of Nelumbo nucifera against Cx. quinquefasciatus and Anopheles subpictus which were analogous to the results obtained in the present study. Subarani et al. (2013) reported that the Vinca rosea-synthesized AgNps did not exhibit any noticeable toxicity on Poecilia reticulata after 24, 48, and 72 h of exposure. From the above results, we suggest that myco-synthesized AgNps are potential larvicidal and eco-friendly agent for controlling mosquito population. Acknowledgment The authors gratefully acknowledge the Management, Thiagarajar College (Autonomous), Madurai, for providing the facilities to perform the research works in the PG and Research Department of Zoology and Microbiology. Author ANB thank UGC-MANF, India, for the financial support and CRME (ICMR), Madurai, who kindly supplied eggs and larvae required during our work. We thank the Department of Chemistry, Madras University and Karunya University, Coimbatore, for the instrumental analysis.
Parasitol Res (2014) 113:3843–3851
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