Parasitol Res DOI 10.1007/s00436-014-3948-z
ORIGINAL PAPER
Myco-synthesis of silver nanoparticles using Beauveria bassiana against dengue vector, Aedes aegypti (Diptera: Culicidae) A. Najitha Banu & C. Balasubramanian
Received: 7 April 2014 / Accepted: 5 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The efficacy of silver synthesized biolarvicide with the help of entomopathogenic fungus, Beauveria bassiana, was assessed against the different larval instars of dengue vector, Aedes aegypti. The silver nanoparticles were observed and characterized by a scanning electron microscope (SEM) and energy-dispersive X-ray (EDX). A surface plasmon resonance band was observed at 420 nm in UV-vis spectrophotometer. The characterization was confirmed by shape (spherical), size 36.88–60.93 nm, and EDX spectral peak at 3 keV of silver nanoparticles. The synthesized silver nanoparticles have been tested against the different larval instars of Ae. aegypti at different concentrations for a period of 24 h. Ae. aegypti larvae were found more susceptible to the synthesized silver nanoparticles. The LC50 and LC90 values are 0.79 and 1.09 ppm with respect to the Ae. aegypti treated with B. bassiana (Bb) silver nanoparticles (AgNPs). First and second instar larvae of Ae. aegypti have shown cent percent mortality while third and fourth instars found 50.0, 56.6, 70.0, 80.0, and 86.6 and 52.4, 60.0, 68.5, 76.0, and 83.3 % mortality at 24 h of exposure in 0.06 and 1.00 ppm, respectively. It is suggested that the entomopathogenic fungus synthesized silver nanoparticles would be appropriate for environmentally safer and greener approach for new leeway in vector control strategy through a biological process.
Keywords Silver nanoparticle . Beauveria bassiana . Pathogenicity . Percent cumulative mortality . Aedes aegypti
A. N. Banu (*) : C. Balasubramanian Postgraduate and Research Department of Zoology and Microbiology, Thiagarajar College (Autonomous), Madurai 625009, Tamil Nadu, India e-mail: [email protected]
Introduction Aedes aegypti is a carrier of dengue fever virus causing dengue, chikungunya, and dengue hemorrhagic fever (Yang et al. 2009). According to the WHO report of the year 2009, two fifth of the world population is under risk of dengue infection (WHO index) and in the year 2010, 28,292 cases of infection and 108 deaths were reported tin 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 there may be 50–100 million dengue infections worldwide every year (WHO 2012). Dengue cases reported 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. Biological control is used as an alternative to currently employed larvicides for minimizing the mosquito population which provides an effective and environmentally friendly approach to bring down mosquitoes’ population under bottom level. Unfortunately, the mosquitoes were developed resistance against the chemical larvicides (Cadavid-Restrepo et al. 2012; Chenniappan and Ayyadurai 2012). Currently, fungi are being exploited for the production of nanoparticle synthesis. Fungi have shown that environmentally benign and renewable source can be used as an effective reducing agent for synthesis of silver nanoparticles (AgNPs). This biological reduction of metal could be utilized for a clean, nontoxic, and environmentally acceptable “green” approach to produce metal nanoparticles. It is well known that some microbes such as yeast (Mourato et al. 2011), fungi (Soni and Prakash 2013), and bacteria (Najitha Banu et al. 2014) are potentially useful in the preparation of metal nanoparticles under normal temperature and pressure. Many species of fungi have been used for
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nanoparticle production, including Verticillium spp. (Mukherjee et al. 2001), Aspergillus fumigates (Bhainsa and D’Souza 2006) Aspergillus niger (Soni and Prakash 2013), and Fusarium oxysporum (Sonal et al. 2013). In the recent decade, mosquitoes were defended to the chemical pesticides; some bacterial toxins (Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis) (Tetreau 2013) are novel environmentally safer to havoc vectors at bottom level. The study aims to evaluate the entomopathogenic fungi, Beauveria bassiana-synthesized silver nanoparticles against the dengue vector.
Materials and methods Isolation and identification of entomopathogenic fungi The entomopathogenic fungi B. bassiana was isolated from infected coffee berry borer, Hypothenemus hampei, Thandikudi, Dindigul district, TN, India. The cadaver was placed on potato dextrose agar (PDA (Hi-Media)) supplemented with streptomycin (1 mg/100 mL) and incubated for 7 days at 27± 2 °C (Haraprasad et al. 2001). After 7 days of incubation, pure culture of B. bassiana was subcultured into PDA by streak plate method. The isolated culture was identified by slide culture method with subjected to lactophenol cotton blue staining observed under the light microscope (Labomed).
and agitated in the same condition as described earlier. The control was maintained (without the addition of AgNO3, only cell filtrate) with the experimental flask. The AgNPs turned brownish yellow color solution and it was stored in screw capped vials under ambient condition for further experiment.
Characterization of AgNPs The UV-vis analysis of the freshly prepared nanoparticles was recorded with water/methanol as reference using the Perkin Elmer (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 (EDX-Oxford instrument, INCA PENTAFET X3, Karunya University, Coimbatore, Tamilnadu, India) spectroscopy coupled with scanning electron microscope (SEM) (Model-JEOL.JSM-6390). Further characterization of Ag bionanoparticles implicated with Fourier Transform Infrared (FTIR) spectra were recorded using Bruker Tensor-27 FTIR 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 B. bassiana (Bb)-AgNPs and KBr (1:1 ratio); the background calibrations have been carried out using pure KBr pellet.
Extracellular synthesis of AgNPs
X-Ray diffraction analysis
The pure culture of B. bassiana was freshly inoculated on a liquid media containing (g L−1) KH2PO4 7.0, K2HPO4 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 an 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 Whatman No. 1 filter paper (Whatman International Ltd, England), followed by an extensive wash with distilled water to remove any medium components from the biomass. Twenty grams of fresh and clean biomass was taken into an Erlenmeyer flask containing 200 mL milli-Q water, and the flask was incubated at 25ºC for 72 h and agitated in the same condition as described earlier. After incubation, the cell filtrates were obtained by passing it through Whatman No. 1 filter paper. Cell filtrate (50 mL) was taken into a 250-mL Erlenmeyer flask and mixed with 1 mM AgNO3 (Laboratory Reagent, Reachem laboratory chemicals Private Ltd, Madras, Tamil Nadu, India) (0.017 g/100 mL) as the final concentration. The flasks were incubated at 25 °C in a dark condition for 120 h
Silver nanoparticles 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 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 size was calculated from the width of the XRD peaks, assuming that they are free from nonuniform strains, using the Debye-Scherer 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
1 2
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Fig. 1 Beauveria bassiana (Bb)
Laboratory evaluation of Bb synthesized AgNPs against Ae. aegypti Ae. aegypti eggs 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 Bb-synthesized silver nanoparticles against first, second, third, and fourth instar larvae of Ae. aegypti based on a method of the World Health Organization (WHO 2005) with minor modifications. For bioassay, 25 larvae/concentrations/replications were transferred into a 250-mL glass beaker (Borosil ®) containing 0.06 to 1.0 ppm concentration. Five replications of Bb-AgNPs were maintained separately, each was covered with a mosquito net. The setup was maintained at 27±2 °C and 77±4 % RH. The mortality of mosquito larvae was noted at 24-h intervals with and without Bb-AgNPs.
Fig. 2 Microscopic view of Bb
Fig. 3 Photographs of (a) Bb-AgNPs, (b) 1 mM AgNO3
Statistical analysis Morality data was subjected to probit analysis to predict the LC50 and LC90 values by using SPSS 16.0. Percentage mortality was also calculated for the mortality data using Excel 2007.
Results In the present investigation, B. bassiana (Bb) was isolated from H. hampei (Fig. 1) and it confirmed through
Fig. 4 UV-Visible spectra of silver nanoparticles
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Fig. 5 SEM image of Bb-AgNPs
microscopically (Fig. 2). Mycelia extracts were used as a starting material for the synthesis of silver nanoparticles (Bb-AgNPs). The larvicidal potentials were tested against the different instars of Ae. Aegypti and details of their efficacy are presented vide infra. Characterization of Bb-AgNPs The culture filtrate along with silver nitrate (AgNO3) was subjected to the reduction, and its color was turned into yellowish brown to dark brown solution without any deposition indicating the formation of AgNPs (Fig. 3). UV-vis spectroscopy is an initial characterization step for analyzing
Fig. 6 EDX spectrum of Bbsynthesized silver nanoparticles
the formation of AgNPs in aqueous solution. The characteristics surface plasmon absorption band was observed at 420 nm after 24 h. Excitation spectra for AgNPs synthesized from AgNO3 were also observed and is presented in Fig. 4. The AgNPs has also been well defined in SEM imaging (Fig. 5); different sizes and spherical-shaped particles ranging from 36.88 to 60.93 nm and characteristic peak at 3 keV in EDX were obtained which indicates the reduction of Ag+ to Ag0 (Fig. 6). The dry powder of the silver nanoparticles was used for XRD analyses. The control thin films of the leaf 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 three strong intense peaks at 2θ values of 31.79°, 45.55°, and 56.58° assigned to the 111, 200, and 220 planes of a faced center cubic structure of silver nanoparticles. The target was Cu Kα with a wavelength of 1.54056 A°. The XRD pattern indicated that the nanoparticles had a cubic structure. No peak of the XRD pattern of Ag and other substance appears, indicating that the silver nanoparticles 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 mycellial extract. The obtained results showed that Ag+ had definitely been reduced by Bb-mycellilal extract under reaction conditions. FTIR spectroscopy analysis were carried out to identify the biomolecules responsible for the reduction of Ag+ ions and capping of the bioreduced silver nanoparticles synthesized by fungal cell filtrate where located at about 580.32, 624.18, 835.33, 1,057.92, 1,383.57,1,650.66, 2,940.63, and 3,434.70 in the
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I (Counts)
XRD XRD- 856
Group: Out-
Data:
Bb> Profile
4000 3000 2000 1000
(Counts) I
Smoothing Profile 4000 3000 2000
I (Counts)
1000
B.G. Subtract Profile 4000 3000 2000
I (Counts)
1000
Ka1 Profile 4000 3000 2000 1000
(Counts) I
Peak 4000 3000 2000 1000 0 10
20
30
Fig. 7 XRD spectrum of Bb-synthesized silver nanoparticles
40
50
60
70
80 90 Theta - 2Theta (deg)
Parasitol Res Table 1 Characteristics of silver nanoparticles synthesized by BbAgNPs Structural
concentration at 24 h intervals. The mortality rate is direct proportional to the concentrations (Table 3).
Functional
UV-vis spectra SEM (Particles EDX (Peak XRD Angle (nm) size, nm) range) 2θ of the intense Lacttice peak (deg) Plane 3 KeV
31.7912 45.5542
111 200
56.5825
220
region 4,000–400 cm−1 (Fig. 8 and Table 2). The FTIR spectral analysis revealed the presence of C=C, =C-H, O-H, C=O, N-H functional groups, which may be present between the amino acid residues and protein synthesized during Bb-AgNPs.
Toxicity of Bb-AgNPs against Ae. aegypti larval instars
90 Transmittance [%] 60 70 80 50
3500
3000
2500 2000 Wavenumber cm-1
1500
1000
624.18 580.32
835.33
1052.92
1383.57
2940.63
3434.70
40
Fig. 8 FTIR spectrum of BbAgNPs
100
The efficacy of the Bb-AgNPs as assessed against Ae. aegypti indicates the higher susceptibility towards Bb-AgNPs. The LC50 and LC90 values of the tested concentration are 0.79 and 1.09 ppm with respect to 0.06, 0.12, 0.25, 0.50, and 1.00 ppm of Bb-AgNPs. The 100 % mortality was observed at 21-h intervals in first and second instar larvae of Ae. aegypti for the tested concentrations. The percent mortality of the third and fourth instars was 50.0, 56.6, 70.0, and 80.0 and 52.4, 60.0, 68.5, 76.0, and 83.3 % with respect to the above ppm
1650.66
36.88–60.93
In recent years, silver nanoparticles play a major role in antibacterial, antifungal, and insect control programs. The development of novel technology was in the field of insect control particularly mosquito control due to the resistance behavior of the mosquitoes. Plant materials which synthesized silver nanoparticles also used for the mosquito control (Borase et al. 2013) are more popular. Even though the microorganisms synthesized silver nanoparticles both extracellularly and intracellularly by Pseudomonas stulzeri AG259 (Tanja et al. 1999), Klepsiella pneumoniae (Ahmad et al. 2007), Bacillus licheniformis (Kalimuthu et al. 2008), Escherichia coli (Gurunathan et al. 2009a, b), Staphylococcus aureus (Nanda an d Sa rav ana n 20 09) a nd Brevib acte rium ca sei (Kalishwaralal et al. 2010), Phychrophilic bacteria (Shivaji et al. 2011), Pseudomonas aeruginosa (Jeevan et al. 2012), and B. thuringiensis (Najitha Banu et al. 2014), entomopathogenic fungus Chrysosporium tropicum (Soni and Prakash 2012), A. niger 2587 (Soni and Prakash 2013), A. niger fungal strain (MTCC 10180) (Singh et al. 2013), and F. oxysporum (Sonal et al. 2013) has been investigated. In the present investigations, B. bassaina was used for synthesis of silver nanoparticles extracellularly for the control dengue vector Ae. aegypti. The filtrated was treated with AgNO3; the reaction started after 24 h of incubation in dark condition, with a change in color of filtrate from pale yellow to brownish yellow, indicating the formation of silver
2426.22
420
Discussion
500
Parasitol Res Table 2 FTIR functional groups analysis
α in-plane bending, β out-of plane bending, ω wagging, τ twisting, γ stretching, δ bending, γs symmetric stretching, γas asymmetric stretching
Vibration assignment/functional groups
Observed wave number (cm−1)
Visible intensity
O-H (stretch, H-bonded) CH3 γas+CH 2 γas+CH γ+CH (Ketones) γ+OH γ OH γ+CH 3 γ+CH 2 γ NH α+NO 2 γas+NO γ CH 3 δ+OH α+C-O γ+COO - γs+C-N γ+NO 2 γs+ SO 2 γas+C=S γ+C-F γ Ring γ+CH α+C-CHO (skeletal)+C-N γ+N-N γ+ SO 3 γs+S=O γ+C-F γ CH out of plane bending CH β+C-O-C γ+OCN (deformation)+N-H ω+N-H τ+ O-N γ+C-S γ+C-Cl γ C-I stretch
3434.70 2940.63 2426.22 1650.66 1383.57
Strong broad Weak Weak Sharp Sharp
1057.92
Broad
835.33 624.18
Weak Weak
580.32
Weak
bionanoparticles which correlated with the results obtained by Mukherjee et al. (2001), Soni and Prakash (2012), and Najitha Banu et al. (2014). In addition, they have given a characteristic band at 420 nm. While no absorption band was observed in both controls (positive and negative). Thus, it indicates the complete reduction of silver ions to turn yellowish brown color in aqueous solution due to excitation of surface plasmon vibration in silver nanoparticles (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 reduction of silver ions by F. oxysporum strains has been attributed to a nitrate-dependent reductase and a shuttle quinine extracellular process. The extracellular biosynthesis of silver nanoparticles using the filamentous fungus Aspergillus fumigatus has been investigated by Bhainsa and D’Souza (2006) and Ingle et al. (2008). The present study reveals the presence of nanoparticles was confirmed by scanning electron microscope and the particles size ranges between 36.88–60.93 nm. The EDX has Table 3 Larvicidal activity (in parts per million) of Beauveria bassiana synthesized silver nanoparticles (Bb-AgNPs) against different instar of Aedes aegypti Concentration (ppm)
Larval instars % mortality I
Control 0.06 0.12 0.25 0.50 1.00
II
III
IV
**
0.00±0.00 50.0±2.5 56.6±0.9 70.0±0.9 80.0±0.5
00.00 52.4±0.5 60.0±0.6 68.5±0.5 76.0±0.9
86.6±0.9
83.3±1
**
LC50
LC90
0.79
1.09
**100 % mortality LC50 lethal concentration that kills 50 % of the exposed larvae, LC90 lethal concentration that kills 90 % of the exposed larvae, percent cumulative mortality values are means±SD
confirmed the presence of elemental silver by the sharp peaks at a range of 3 keV which is typical for the absorption of metallic silver nanoparticles. 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°, and 44° (Gong et al. 2007). Similarly, in the present study, the characteristic peaks at 23.2250, 27.8800, and 32.1833 which correspond to the 111, 200, and 220 (Bragg reflection) and reflections of silver were obtained. Therefore, X-ray diffraction results also suggest that crystallization of bioorganic phase occurs on the surface of silver nanoparticles. FTIR spectra clearly indicate that the biomolecules especially proteins present in filtrate are responsible for synthesis and stabilization of AgNPs (Dhanasekaran and Thangaraj 2013). The use of nanoparticles in insects and their potential in insect pest management have been focused by Bhattacharyya et al. (2010). The larvicidal potentials of silver nanoparticles synthesized using fungus Cochliobolus lunatus against Ae. aegypti and Anopheles stephensi have been observed (Salunkhe et al. 2011). Recently, the silver and gold nanoparticles synthesized with C. tropicum have been tested as a larvicide against the Ae. aegypti larvae (Soni and Prakash 2012). 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, and 11.48 against first, second, and fourth instar larvae of Culex quinquefasciatus, An. stephensi, and Ae. aegypti, which are much higher to the concentration in the present investigation. The LC50 and LC90 values are 0.79 and 1.09 ppm synthesized by Bb-AgNPs against first, second, third, and fourth instar larvae of Ae. aegypti, respectively. 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
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Cx. quinquefasciatus and Anopheles subpictus which was analogous to the results obtained in the present study. Subarani et al. (2013) reported that the Vinca rosea-synthesized silver nanoparticles did not exhibit any noticeable toxicity on Poecilia reticulata after 24, 48, and 72 h of exposure. These results suggest that the synthesized AgNPs have the potential to be used as an ideal eco-friendly approach for the control of the Ae. aegypti larvae. Thus, the present investigation highlighted the potential of the B. bassiana mycellial extract that has keyed up the synthesized silver nanoparticles as a potential biolarvicidal agent for the dengue vector Ae. aegypti. This is probably the first report with synthesized silver nanoparticles using entomopathogenic fungus B. bassiana for control of Ae. aegypti. The fungus-mediated silver nanoparticles have rapid impact on vector mosquito population and thus conclude that the fungus-synthesized silver nanoparticles may perhaps be a better, environmentally safer, and greener approach for vector control strategy, so that the further investigation was required to find out the mass production, formulation, field application, patent, and commercialization of the Bb-AgNPs as biolarvicides. Acknowledgments 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. The author (ANB) thank UGCMANF, India, for the financial support and CRME (ICMR), Madurai, 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
Myco-synthesis of silver nanoparticles using Beauveria bassiana against dengue vector, Aedes aegypti (Diptera: Culicidae) A. Najitha Banu & C. Balasubramanian
Received: 7 April 2014 / Accepted: 5 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The efficacy of silver synthesized biolarvicide with the help of entomopathogenic fungus, Beauveria bassiana, was assessed against the different larval instars of dengue vector, Aedes aegypti. The silver nanoparticles were observed and characterized by a scanning electron microscope (SEM) and energy-dispersive X-ray (EDX). A surface plasmon resonance band was observed at 420 nm in UV-vis spectrophotometer. The characterization was confirmed by shape (spherical), size 36.88–60.93 nm, and EDX spectral peak at 3 keV of silver nanoparticles. The synthesized silver nanoparticles have been tested against the different larval instars of Ae. aegypti at different concentrations for a period of 24 h. Ae. aegypti larvae were found more susceptible to the synthesized silver nanoparticles. The LC50 and LC90 values are 0.79 and 1.09 ppm with respect to the Ae. aegypti treated with B. bassiana (Bb) silver nanoparticles (AgNPs). First and second instar larvae of Ae. aegypti have shown cent percent mortality while third and fourth instars found 50.0, 56.6, 70.0, 80.0, and 86.6 and 52.4, 60.0, 68.5, 76.0, and 83.3 % mortality at 24 h of exposure in 0.06 and 1.00 ppm, respectively. It is suggested that the entomopathogenic fungus synthesized silver nanoparticles would be appropriate for environmentally safer and greener approach for new leeway in vector control strategy through a biological process.
Keywords Silver nanoparticle . Beauveria bassiana . Pathogenicity . Percent cumulative mortality . Aedes aegypti
A. N. Banu (*) : C. Balasubramanian Postgraduate and Research Department of Zoology and Microbiology, Thiagarajar College (Autonomous), Madurai 625009, Tamil Nadu, India e-mail: [email protected]
Introduction Aedes aegypti is a carrier of dengue fever virus causing dengue, chikungunya, and dengue hemorrhagic fever (Yang et al. 2009). According to the WHO report of the year 2009, two fifth of the world population is under risk of dengue infection (WHO index) and in the year 2010, 28,292 cases of infection and 108 deaths were reported tin 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 there may be 50–100 million dengue infections worldwide every year (WHO 2012). Dengue cases reported 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. Biological control is used as an alternative to currently employed larvicides for minimizing the mosquito population which provides an effective and environmentally friendly approach to bring down mosquitoes’ population under bottom level. Unfortunately, the mosquitoes were developed resistance against the chemical larvicides (Cadavid-Restrepo et al. 2012; Chenniappan and Ayyadurai 2012). Currently, fungi are being exploited for the production of nanoparticle synthesis. Fungi have shown that environmentally benign and renewable source can be used as an effective reducing agent for synthesis of silver nanoparticles (AgNPs). This biological reduction of metal could be utilized for a clean, nontoxic, and environmentally acceptable “green” approach to produce metal nanoparticles. It is well known that some microbes such as yeast (Mourato et al. 2011), fungi (Soni and Prakash 2013), and bacteria (Najitha Banu et al. 2014) are potentially useful in the preparation of metal nanoparticles under normal temperature and pressure. Many species of fungi have been used for
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nanoparticle production, including Verticillium spp. (Mukherjee et al. 2001), Aspergillus fumigates (Bhainsa and D’Souza 2006) Aspergillus niger (Soni and Prakash 2013), and Fusarium oxysporum (Sonal et al. 2013). In the recent decade, mosquitoes were defended to the chemical pesticides; some bacterial toxins (Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis) (Tetreau 2013) are novel environmentally safer to havoc vectors at bottom level. The study aims to evaluate the entomopathogenic fungi, Beauveria bassiana-synthesized silver nanoparticles against the dengue vector.
Materials and methods Isolation and identification of entomopathogenic fungi The entomopathogenic fungi B. bassiana was isolated from infected coffee berry borer, Hypothenemus hampei, Thandikudi, Dindigul district, TN, India. The cadaver was placed on potato dextrose agar (PDA (Hi-Media)) supplemented with streptomycin (1 mg/100 mL) and incubated for 7 days at 27± 2 °C (Haraprasad et al. 2001). After 7 days of incubation, pure culture of B. bassiana was subcultured into PDA by streak plate method. The isolated culture was identified by slide culture method with subjected to lactophenol cotton blue staining observed under the light microscope (Labomed).
and agitated in the same condition as described earlier. The control was maintained (without the addition of AgNO3, only cell filtrate) with the experimental flask. The AgNPs turned brownish yellow color solution and it was stored in screw capped vials under ambient condition for further experiment.
Characterization of AgNPs The UV-vis analysis of the freshly prepared nanoparticles was recorded with water/methanol as reference using the Perkin Elmer (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 (EDX-Oxford instrument, INCA PENTAFET X3, Karunya University, Coimbatore, Tamilnadu, India) spectroscopy coupled with scanning electron microscope (SEM) (Model-JEOL.JSM-6390). Further characterization of Ag bionanoparticles implicated with Fourier Transform Infrared (FTIR) spectra were recorded using Bruker Tensor-27 FTIR 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 B. bassiana (Bb)-AgNPs and KBr (1:1 ratio); the background calibrations have been carried out using pure KBr pellet.
Extracellular synthesis of AgNPs
X-Ray diffraction analysis
The pure culture of B. bassiana was freshly inoculated on a liquid media containing (g L−1) KH2PO4 7.0, K2HPO4 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 an 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 Whatman No. 1 filter paper (Whatman International Ltd, England), followed by an extensive wash with distilled water to remove any medium components from the biomass. Twenty grams of fresh and clean biomass was taken into an Erlenmeyer flask containing 200 mL milli-Q water, and the flask was incubated at 25ºC for 72 h and agitated in the same condition as described earlier. After incubation, the cell filtrates were obtained by passing it through Whatman No. 1 filter paper. Cell filtrate (50 mL) was taken into a 250-mL Erlenmeyer flask and mixed with 1 mM AgNO3 (Laboratory Reagent, Reachem laboratory chemicals Private Ltd, Madras, Tamil Nadu, India) (0.017 g/100 mL) as the final concentration. The flasks were incubated at 25 °C in a dark condition for 120 h
Silver nanoparticles 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 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 size was calculated from the width of the XRD peaks, assuming that they are free from nonuniform strains, using the Debye-Scherer 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
1 2
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Fig. 1 Beauveria bassiana (Bb)
Laboratory evaluation of Bb synthesized AgNPs against Ae. aegypti Ae. aegypti eggs 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 Bb-synthesized silver nanoparticles against first, second, third, and fourth instar larvae of Ae. aegypti based on a method of the World Health Organization (WHO 2005) with minor modifications. For bioassay, 25 larvae/concentrations/replications were transferred into a 250-mL glass beaker (Borosil ®) containing 0.06 to 1.0 ppm concentration. Five replications of Bb-AgNPs were maintained separately, each was covered with a mosquito net. The setup was maintained at 27±2 °C and 77±4 % RH. The mortality of mosquito larvae was noted at 24-h intervals with and without Bb-AgNPs.
Fig. 2 Microscopic view of Bb
Fig. 3 Photographs of (a) Bb-AgNPs, (b) 1 mM AgNO3
Statistical analysis Morality data was subjected to probit analysis to predict the LC50 and LC90 values by using SPSS 16.0. Percentage mortality was also calculated for the mortality data using Excel 2007.
Results In the present investigation, B. bassiana (Bb) was isolated from H. hampei (Fig. 1) and it confirmed through
Fig. 4 UV-Visible spectra of silver nanoparticles
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Fig. 5 SEM image of Bb-AgNPs
microscopically (Fig. 2). Mycelia extracts were used as a starting material for the synthesis of silver nanoparticles (Bb-AgNPs). The larvicidal potentials were tested against the different instars of Ae. Aegypti and details of their efficacy are presented vide infra. Characterization of Bb-AgNPs The culture filtrate along with silver nitrate (AgNO3) was subjected to the reduction, and its color was turned into yellowish brown to dark brown solution without any deposition indicating the formation of AgNPs (Fig. 3). UV-vis spectroscopy is an initial characterization step for analyzing
Fig. 6 EDX spectrum of Bbsynthesized silver nanoparticles
the formation of AgNPs in aqueous solution. The characteristics surface plasmon absorption band was observed at 420 nm after 24 h. Excitation spectra for AgNPs synthesized from AgNO3 were also observed and is presented in Fig. 4. The AgNPs has also been well defined in SEM imaging (Fig. 5); different sizes and spherical-shaped particles ranging from 36.88 to 60.93 nm and characteristic peak at 3 keV in EDX were obtained which indicates the reduction of Ag+ to Ag0 (Fig. 6). The dry powder of the silver nanoparticles was used for XRD analyses. The control thin films of the leaf 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 three strong intense peaks at 2θ values of 31.79°, 45.55°, and 56.58° assigned to the 111, 200, and 220 planes of a faced center cubic structure of silver nanoparticles. The target was Cu Kα with a wavelength of 1.54056 A°. The XRD pattern indicated that the nanoparticles had a cubic structure. No peak of the XRD pattern of Ag and other substance appears, indicating that the silver nanoparticles 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 mycellial extract. The obtained results showed that Ag+ had definitely been reduced by Bb-mycellilal extract under reaction conditions. FTIR spectroscopy analysis were carried out to identify the biomolecules responsible for the reduction of Ag+ ions and capping of the bioreduced silver nanoparticles synthesized by fungal cell filtrate where located at about 580.32, 624.18, 835.33, 1,057.92, 1,383.57,1,650.66, 2,940.63, and 3,434.70 in the
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I (Counts)
XRD XRD- 856
Group: Out-
Data:
Bb> Profile
4000 3000 2000 1000
(Counts) I
Smoothing Profile 4000 3000 2000
I (Counts)
1000
B.G. Subtract Profile 4000 3000 2000
I (Counts)
1000
Ka1 Profile 4000 3000 2000 1000
(Counts) I
Peak 4000 3000 2000 1000 0 10
20
30
Fig. 7 XRD spectrum of Bb-synthesized silver nanoparticles
40
50
60
70
80 90 Theta - 2Theta (deg)
Parasitol Res Table 1 Characteristics of silver nanoparticles synthesized by BbAgNPs Structural
concentration at 24 h intervals. The mortality rate is direct proportional to the concentrations (Table 3).
Functional
UV-vis spectra SEM (Particles EDX (Peak XRD Angle (nm) size, nm) range) 2θ of the intense Lacttice peak (deg) Plane 3 KeV
31.7912 45.5542
111 200
56.5825
220
region 4,000–400 cm−1 (Fig. 8 and Table 2). The FTIR spectral analysis revealed the presence of C=C, =C-H, O-H, C=O, N-H functional groups, which may be present between the amino acid residues and protein synthesized during Bb-AgNPs.
Toxicity of Bb-AgNPs against Ae. aegypti larval instars
90 Transmittance [%] 60 70 80 50
3500
3000
2500 2000 Wavenumber cm-1
1500
1000
624.18 580.32
835.33
1052.92
1383.57
2940.63
3434.70
40
Fig. 8 FTIR spectrum of BbAgNPs
100
The efficacy of the Bb-AgNPs as assessed against Ae. aegypti indicates the higher susceptibility towards Bb-AgNPs. The LC50 and LC90 values of the tested concentration are 0.79 and 1.09 ppm with respect to 0.06, 0.12, 0.25, 0.50, and 1.00 ppm of Bb-AgNPs. The 100 % mortality was observed at 21-h intervals in first and second instar larvae of Ae. aegypti for the tested concentrations. The percent mortality of the third and fourth instars was 50.0, 56.6, 70.0, and 80.0 and 52.4, 60.0, 68.5, 76.0, and 83.3 % with respect to the above ppm
1650.66
36.88–60.93
In recent years, silver nanoparticles play a major role in antibacterial, antifungal, and insect control programs. The development of novel technology was in the field of insect control particularly mosquito control due to the resistance behavior of the mosquitoes. Plant materials which synthesized silver nanoparticles also used for the mosquito control (Borase et al. 2013) are more popular. Even though the microorganisms synthesized silver nanoparticles both extracellularly and intracellularly by Pseudomonas stulzeri AG259 (Tanja et al. 1999), Klepsiella pneumoniae (Ahmad et al. 2007), Bacillus licheniformis (Kalimuthu et al. 2008), Escherichia coli (Gurunathan et al. 2009a, b), Staphylococcus aureus (Nanda an d Sa rav ana n 20 09) a nd Brevib acte rium ca sei (Kalishwaralal et al. 2010), Phychrophilic bacteria (Shivaji et al. 2011), Pseudomonas aeruginosa (Jeevan et al. 2012), and B. thuringiensis (Najitha Banu et al. 2014), entomopathogenic fungus Chrysosporium tropicum (Soni and Prakash 2012), A. niger 2587 (Soni and Prakash 2013), A. niger fungal strain (MTCC 10180) (Singh et al. 2013), and F. oxysporum (Sonal et al. 2013) has been investigated. In the present investigations, B. bassaina was used for synthesis of silver nanoparticles extracellularly for the control dengue vector Ae. aegypti. The filtrated was treated with AgNO3; the reaction started after 24 h of incubation in dark condition, with a change in color of filtrate from pale yellow to brownish yellow, indicating the formation of silver
2426.22
420
Discussion
500
Parasitol Res Table 2 FTIR functional groups analysis
α in-plane bending, β out-of plane bending, ω wagging, τ twisting, γ stretching, δ bending, γs symmetric stretching, γas asymmetric stretching
Vibration assignment/functional groups
Observed wave number (cm−1)
Visible intensity
O-H (stretch, H-bonded) CH3 γas+CH 2 γas+CH γ+CH (Ketones) γ+OH γ OH γ+CH 3 γ+CH 2 γ NH α+NO 2 γas+NO γ CH 3 δ+OH α+C-O γ+COO - γs+C-N γ+NO 2 γs+ SO 2 γas+C=S γ+C-F γ Ring γ+CH α+C-CHO (skeletal)+C-N γ+N-N γ+ SO 3 γs+S=O γ+C-F γ CH out of plane bending CH β+C-O-C γ+OCN (deformation)+N-H ω+N-H τ+ O-N γ+C-S γ+C-Cl γ C-I stretch
3434.70 2940.63 2426.22 1650.66 1383.57
Strong broad Weak Weak Sharp Sharp
1057.92
Broad
835.33 624.18
Weak Weak
580.32
Weak
bionanoparticles which correlated with the results obtained by Mukherjee et al. (2001), Soni and Prakash (2012), and Najitha Banu et al. (2014). In addition, they have given a characteristic band at 420 nm. While no absorption band was observed in both controls (positive and negative). Thus, it indicates the complete reduction of silver ions to turn yellowish brown color in aqueous solution due to excitation of surface plasmon vibration in silver nanoparticles (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 reduction of silver ions by F. oxysporum strains has been attributed to a nitrate-dependent reductase and a shuttle quinine extracellular process. The extracellular biosynthesis of silver nanoparticles using the filamentous fungus Aspergillus fumigatus has been investigated by Bhainsa and D’Souza (2006) and Ingle et al. (2008). The present study reveals the presence of nanoparticles was confirmed by scanning electron microscope and the particles size ranges between 36.88–60.93 nm. The EDX has Table 3 Larvicidal activity (in parts per million) of Beauveria bassiana synthesized silver nanoparticles (Bb-AgNPs) against different instar of Aedes aegypti Concentration (ppm)
Larval instars % mortality I
Control 0.06 0.12 0.25 0.50 1.00
II
III
IV
**
0.00±0.00 50.0±2.5 56.6±0.9 70.0±0.9 80.0±0.5
00.00 52.4±0.5 60.0±0.6 68.5±0.5 76.0±0.9
86.6±0.9
83.3±1
**
LC50
LC90
0.79
1.09
**100 % mortality LC50 lethal concentration that kills 50 % of the exposed larvae, LC90 lethal concentration that kills 90 % of the exposed larvae, percent cumulative mortality values are means±SD
confirmed the presence of elemental silver by the sharp peaks at a range of 3 keV which is typical for the absorption of metallic silver nanoparticles. 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°, and 44° (Gong et al. 2007). Similarly, in the present study, the characteristic peaks at 23.2250, 27.8800, and 32.1833 which correspond to the 111, 200, and 220 (Bragg reflection) and reflections of silver were obtained. Therefore, X-ray diffraction results also suggest that crystallization of bioorganic phase occurs on the surface of silver nanoparticles. FTIR spectra clearly indicate that the biomolecules especially proteins present in filtrate are responsible for synthesis and stabilization of AgNPs (Dhanasekaran and Thangaraj 2013). The use of nanoparticles in insects and their potential in insect pest management have been focused by Bhattacharyya et al. (2010). The larvicidal potentials of silver nanoparticles synthesized using fungus Cochliobolus lunatus against Ae. aegypti and Anopheles stephensi have been observed (Salunkhe et al. 2011). Recently, the silver and gold nanoparticles synthesized with C. tropicum have been tested as a larvicide against the Ae. aegypti larvae (Soni and Prakash 2012). 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, and 11.48 against first, second, and fourth instar larvae of Culex quinquefasciatus, An. stephensi, and Ae. aegypti, which are much higher to the concentration in the present investigation. The LC50 and LC90 values are 0.79 and 1.09 ppm synthesized by Bb-AgNPs against first, second, third, and fourth instar larvae of Ae. aegypti, respectively. 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
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Cx. quinquefasciatus and Anopheles subpictus which was analogous to the results obtained in the present study. Subarani et al. (2013) reported that the Vinca rosea-synthesized silver nanoparticles did not exhibit any noticeable toxicity on Poecilia reticulata after 24, 48, and 72 h of exposure. These results suggest that the synthesized AgNPs have the potential to be used as an ideal eco-friendly approach for the control of the Ae. aegypti larvae. Thus, the present investigation highlighted the potential of the B. bassiana mycellial extract that has keyed up the synthesized silver nanoparticles as a potential biolarvicidal agent for the dengue vector Ae. aegypti. This is probably the first report with synthesized silver nanoparticles using entomopathogenic fungus B. bassiana for control of Ae. aegypti. The fungus-mediated silver nanoparticles have rapid impact on vector mosquito population and thus conclude that the fungus-synthesized silver nanoparticles may perhaps be a better, environmentally safer, and greener approach for vector control strategy, so that the further investigation was required to find out the mass production, formulation, field application, patent, and commercialization of the Bb-AgNPs as biolarvicides. Acknowledgments 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. The author (ANB) thank UGCMANF, India, for the financial support and CRME (ICMR), Madurai, 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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