Possible Mosquito Control by Silver Nanoparticles Synthesized by Entomopathogenic Fungus Beauveria bassiana

 

Marwah Amer Qamandar, Maan Abdul Azeez Shafeeq

Department of Biology, College of Science, University of Al-Mustansiriyah, Iraq

*Corresponding Author E-mail: maanalsalihi@uomustansirivah.edu.iq

 

ABSTRACT:

Objective: we have synthesized the silver nanoparticles (AgNPs) by using the soil fungus Beauveria bassiana, the results recorded from UV-vis spectrophotometer and scanning electron microscopy (SEM) support the biosynthesis and characterization of AgNPs. The synthesized silver nanoparticles have also been tested against the larvae and pupae of Culex pipiens.

Methods: The synthesized silver nanoparticles tested against larvae and pupae of Culex pipiens, the efficacy test was achieved at different concentrations (150, 200 and 250 ppm)

The results: second larval instar of Cx. pipiens were high susceptible to synthesized AgNPs while, pupae were found less susceptible to synthesized AgNPs as the highest mortality rate were 98.88%, 95.55%, and 94.44% for second, third and fourth larval in star respectively at concentration 250ppm after 24 h, and 50.00% for pupa at the same concentration after 48 h.

Conclusion: The results described in this study open the possibility for further investigations of the efficacy of larvicidal and properties of natural products, the beneficial point of silver nanoparticles as larvicides is that the insecticides resistance, environmental pollution, toxic hazards to humans and other non-target organism due to the overuse of pesticides can be overcome

 

KEYWORDS: culex pipiens, Beauveria bassiana, Bio control, Silver Nanoparticles, morphological abnormalities.

 

 


1. INTRODUCTION:

Culex mosquitoes are painful and persistent biters transmit filariasis, the lymphatic filariasis is a neglected tropical disease, more than 1.3 billion people in 72 countries worldwide are threaten by this disease, usually know as elephantiasis, and over 120 million people currently infect, with about 40 million disfigure by this disease [1]. Genus Beauveria belongs to phylum Ascomycota, more precisely to the order Hypocreales and family Cordycipitaceae. Beauveria is a worldwide anamorphic genus of soil-borne entomopathogenic fungi, usually facultative necrotroph [2], but that can also exists as saprotroph or as a plant endophyte [3].

B. bassiana is one of the most widespread entomopathogenic fungi but it is important feature that B. bassiana is sensitive to the disturbance effects of cultivation [4]. Fungi of genus Beauveria are also recognize for their production of biologically active secondary metabolites as polyketides (e.g., oosporein, bassianin and tennelin), non-peptide pigments and other metabolites (e.g., oxalic acid). Non-ribosomal synthesized peptide antibiotics (e.g., beauvericin, bassianolides and beauverilides). These secondary metabolites are essentially pathogen to insect and can be applying in agriculture [5] [6] [7].In present times, the advanced approach of nanotechnology and microemulsion are the effective way for mosquito’s control, very fine droplets of size 1-100 nm in diameter apply for insect control [8] found  that the nano-particles could be useful for development of new insecticide, silver nanoparticles exhibit high larvicidal activity as compared to gold nanoparticles, the biological control uses as an alternative method employed for larvicides to minimize the mosquito population which offers an effective and environmentally friendly approach to bring down mosquitoes’ population under bottom level, unluckily the mosquitoes develop resistance against the chemical larvicides [9] [10].

 

MATERIALS AND METHODS:

Mosquitoes rearing:

Larvae of Cx. pipiens are collected froma water pools in College of Education for pure science / Ibn Al-Haitham as the insect is identified in Natural Historic Museum and Research Center /Baghdad University, larvae are reared in animal house /Biology department /College of science /University of Al-Mustansiriyah began on February until April 2017, larvae are fed with fish diet which contain  protein (18-50%), lipid (10-25%), carbohydrate (15-20%), ash ( 8.5%), phosphorus (1.5%), water ( 10%), and trace amounts of vitamins, and minerals in the plastic containers with 50 ml capacity filled with water exposed to sun light for 72 h to remove chlorine, when larvae became pupae, they are transferred to (30cm × 30cm × 30cm) organdy cloth cages tied onto iron frame, water is refreshed every three days for prevent scum formation on the water surface [11].When pupae became adult plastic container is replaced by another one for egg collection, male mosquitoes are fed by cotton layers are soaked in 10% sugar solution, then placed in petri dishes, cotton layers must change daily, females are fed by blood meals of pigeon by shaving animal's chest, then linked its parts to prevent movement and put above the cage and hanged overnight tightly as a source of blood[12]. After 2-3 days the gravid females are oviposited their first batch of eggs, egg rafts are collected in clean container containing dechlorinated water and food, and will hatch 1–2 days after deposition, larvae are passed through four instars, the experiment is performed at mean room temperature 27±2°C measured by using thermometer, with 12:12 (L: D) photoperiod and repeated for three generations to eliminate from chemical pesticides [13]. The 2nd, 3rd, 4th instar larvae and pupae are collected and transferred to experimental cups.

 

Fungal Strain, Preparation of Broth and Culture of B. bassiana:

The fungal strain of B. bassiana was obtained from the directorate of agriculture and research / Ministry of Higher Education and Scientific Research, and was routinely maintained in the laboratory on Potato Dextrose Agar (PDA) at 25°C.The broth of B. bassiana was prepared for culture of following the method [14]. 1000ml Erlenmeyer flask each containing 500 ml of Potato Dextrose Broth was autoclaved at 121ºC and 15 psi for 15 min. The broth was supplemented with chloramphenicol (250mg/L) as a bacteriostatic agent. B. bassiana colonies grown on PDA plates were transferred to each flask by inoculation needle. The conical flask inoculated with B.bassiana was incubated at 25°Cfor 14 days [15].

 

 

Fig (1): Beauveria bassiana on PDAFig (2): Beauveria bassiana on PDB

 

Synthesis and Characterization of AgNPs:

After 14 days incubation, the fungal biomass is isolated from the medium by filtration through Whatman filter paper NO1, biomass is washed 3 times in distilled water to remove any nutrient media that may interact with silver ions, 20g of wet biomass is took into conical flask containing 500ml of deionized water and incubated for 120 h at 25±2°C and 80±5% relative humidity, the components of aqueous solution are separated by passing it through Whatman filter paper NO1 and Millipore filter 0.2μm [16].

 

Fig (3): Biomass of Beauveria bassiana

 

Molecular weight of AgNo3:Ag=107.87, N=14, O=16 = 107.87 + 14 + 16 × 3 = 169.87, so the molar mass of AgNo3 is 169.87, therefore 1000ml of  50Mm contains 169.87 × 50 /1000= 8.4935g of AgNo3 is added to 1000ml of deionized water, 800ml of this solution is added to 200ml of cell filters and heated at 60°C for 10 min, shacked with magnetic hot plate stirrer, pH is adjusted to 7 by adding drops of  NaOH solution with continuous measurement of pH until it became neutral, the flask is incubated at 25°C and 80±5% relative humidity in a dark condition for 120h, the control is kept under same conditions, separately (without addition of AgNO3, only cell filtrate), AgNPs are turned brownish yellow color solution as shows in fig (6) [17]. The proteins, enzymes and other compounds exist in the fungal liquid acted as reducing agents and are responsible for conversion of silver nitrate to silver nanoparticles [18].

 

Efficacy Study of Synthesized AgNPs by using the B. bassiana against the Larvae and Pupae of Culex pipiens:

Larvicidal and pupicidal activity of synthesized AgNPs against the larvae and pupae Cx. pipiens is assessed by using the standard method [19]. To test the larvicidal and the pupicidal activity of synthesized AgNPs, 10 larvae of each in star 2nd, 3rd, 4th and 10 pupae of Cx. pipiens were separately placed in a 60ml plastic cup containing 50ml of dechlorinated water and a different concentrations of AgNPs (150, 200 and 250 ppm) which transferred by pipette, in the similar way. The control (without AgNPs) is used to test natural mortality. Nine replicates are used for each tested concentration on each in star larva and pupa. All the plastic cups are incubated at a temperature of 27±2˚C and 12:12 (L: D) photoperiod, the mortality which is determined after 24 and 48 h of treatment.

 

Statistical Analysis:

Statistical analysis of data is performed by using SAS (Statistical Analysis System-version 9.1). The mortality rates are compared by using the Chi-square test. P < 0.05 is considered statistically significant.

 

RESULTS:

UV-Vis Spectrophotometer and SEM Analysis of Synthesized AgNPs:

UV-Vis spectra (Figure3) reveals that Beauveria bassiana plays an important role in the formation of AgNPs, the AgNPs are subjected to ultra-sonication at room temperature and their surface plasmon resonance is recorded at 234nm.

 

 

Fig (4): Transmittance Spectrum of Beauveria bassiana Silver Nanoparticles

Scanning electron micrograph is enabled visualization and shape of the silver nanoparticles (Figure 5) clearly revealed clustered and irregular, the rough morphology of AgNPs are provided excellent larvicidal activity for the synthesized nanoparticles.

 

 

Fig(5): SEM Image of Beauveria bassiana Silver Nanoparticles (320x)

 

X-Ray diffraction analysis:

Table (1) shows a summary of the X-ray characterization of B. bassiana silver nanoparticles, Scherer's equation is used to calculate the average crystallite size of AgNPs nanoparticles is around 49nm. Nanoparticles are particles had one or more dimensions of the order of 100nm or less [20]. The XRD patterns of pure industrial synthetic nanoparticles show in figure (6). The crystal shape of them was pure and there was no trace of another material also, a strong diffraction peaks were 14.03, 28.05 and 32.67 attributed to crystallographic planes.

 

Table (1): Powder X-ray Diffraction Data of Beauveria bassiana Silver Nanoparticles

2Theta (deg)

β (deg)

D (nm)

σ x10-4

lines-2.m-4

σ x 1014 lines/m2

28.05

0.35

23.27295

14.88853

18.46279

14.03

0.33

24.19577

14.32068

17.0813

32.67

0.10

82.36097

4.20709

1.474202

 

 

Fig (6): XRD Pattern of Beauveria bassiana Silver Nanoparticles

 

FTIR Analysis of Synthesized Ag NPs:

FTIR analysis is carried out to recognize the possible interactions between silver and bioactive molecules, which may be responsible for the synthesis and steadiness (capping) of silver nanoparticles, FTIR spectroscopy is used to identify the functional groups of the active compounds based on the peak value in the infrared region [21] FTIR spectra clearly showed that the biomolecules especially proteins existing in the filtrate are responsible for the synthesis and stabilization of silver nanoparticles [22].

 

FTIR analysis of the AgNPs showed the presence of prominent bands due to C-C (1600),the O-H group (2300), C-H SP2 (3000) and C-H SP3 alkanes (3300)        cm-1. Figure (7) which depend on the oxidation degree of (AgNPs).

 

 

Fig (7): FTIR Spectra of Beauveria bassiana Silver Nanoparticles

 

Table (3) shows the highest larval mortality 98.88% is found in a concentration 250ppm and the lowest larval mortality 95.55% is found in a concentration 150ppm after 24 h of exposure, the chi-square value was 1.80 and non-significant at p > 0.05 level.

 

Table (3): Effect of Different Concentrations of Beauveria bassiana Silver Nanoparticleson Second Larval instar of Culex pipiens at        24 h

Mortality rate %

Dead No.

Total No.

Concentration(ppm)

95.55

86

90

150

96.66

87

90

200

98.88

89

90

250

1.80

 

 

Chi square

0.40 NS

 

 

P-value

NS: Non-significant

 

Table (4) shows the highest larval mortality 95.55% is found in concentration 250 ppm and the lowest larval mortality 91.11% is found in concentration 150 ppm after 24 h of exposure; the chi-square value was 1.47 and non-significant at p > 0.05 level.

Table (4): Effect of Different Concentrations of Beauveria bassiana Silver Nanoparticles on Third Larval Instar of Culex pipiens at         24 h

Mortality rate %

Dead No.

Total No.

Concentration(ppm)

91.11

82

90

150

92.22

83

90

200

95.55

86

90

250

1.47

 

 

Chi square

0.47NS

 

 

P-value

NS: Non-significant

 

Table (5) shows the highest larval mortality 94.44% is found in a concentration 250 ppm and the lowest larval mortality 91.11% is found in a concentration 150 ppm after 24 h of exposure; the chi-square value was 0.79 and non-significant at p > 0.05 level.

 

Table (5): Effect of Different Concentrations of Beauveria bassiana Silver Nanoparticles on Fourth Larval Instar of Culex pipiens at  24 h

Mortality rate %

Dead No.

Total No.

Concentration(ppm)

91.11

82

90

150

93.33

84

90

200

94.44

85

90

250

0.79

 

 

Chi square

0.67 NS

 

 

P-value

NS: Non-significant

 

Table (6) shows the highest pupa mortality 38.88% is found in concentration 250ppm and the lowest larval mortality 33.33% is found in concentration 150ppm after 24h of exposure; the chi-square value was 0.61 and non-significant at p > 0.05 level.

 

Table (6): Effect of Different Concentrations of Beauveria bassiana Silver Nanoparticleson Pupa of Culex pipiens at 24 h

Mortality rate %

Dead No.

Total No.

Concentration(ppm)

33.33

30

90

150

35.55

32

90

200

38.88

35

90

250

0.61

 

 

Chi square

0.73 NS

 

 

P-value

NS: Non-significant

 

Table (7) shows the highest pupa mortality 50.00% is found in concentration 250ppm and the lowest larval mortality 47.77% is found in concentration 150ppm after 48 h of exposure; the chi-square value was 0.08 and non-significant at p > 0.05 level.

 

Table (7): Effect of Different Concentrations of Beauveria bassiana Silver Nanoparticleson Pupa of Culex pipiens at 48 h

Mortality rate %

Dead No.

Total No.

Concentration (ppm)

47.77

43

90

150

48.88

44

90

200

50.00

45

90

250

0.08

 

 

Chi square

0.95 NS

 

 

P-value

NS: Non-significant

Mortality rates (98.88%, 95.55%, 94.44% and 38.88%) are observed at 250ppm concentration against second, third, fourth larval in star and pupa respectively, after 24 h of exposure while at 48h mortality rate is 50.00% is observed in the same concentration against pupa, no mortality observed in control, the results don’t agree with the result obtained by [23] Who mentioned the 100% mortality is observed at 21 h intervals in second in star larvae of Ae. aegypti at 0.50ppm concentration. The percent mortality of the third and fourth instars was 50.0%, 56.6%, 70.0%, 80.0% and 86.6%, and 52.4%, 60.0%, 68.5%, 76.0%, and 83.3% at a concentration 0.06, 0.12, 0.25, 0.50, and 1.00ppm of B. bassiana silver nanoparticles at 24 h intervals, also results don’t agree with[24] who observed mortality rates 10.00%, 50.00%, 65.00%, 90.00% and 95.00% in second in star larvae of Cx. quinquefasciatus, and 60.00%, 70.00%, 80.00%, 90.00% and 100% in third in star larvae, 0.00% mortality rate in fourth in star larvae, after 1 h of exposure to 2, 4, 6, 8, 10 and 12ppm of Aspergillus niger silver nanoparticles while the mortality rates of pupa were 40.00%, 45.00%, 50.00%, 65.00%, 70.00% and 80.00% after 20 h of exposure to the same concentration of A. niger silver nanoparticles. In addition the results don’t agree with [25]who reported the percentage mortality of second, third, fourth larval instar and pupa of Cx. quinquefasciatus, respectively, were 100%, 96%, 92.00% and 86.00% at 25ppm concentration of Penecillium verucosum silver nanoparticles while at a concentration 50ppm the mortality rates were 99.00%, 94.00%, 94.00% and 91.00% of second, third, fourth larval in star and pupa of Cx.  quinquefasciatus. The silver nanoparticles in the intracellular space can bind to sulphur containing proteins or to phosphorus containing compounds like DNA, leading to the denaturation of some organelles and enzymes. Subsequently, the decrease in a membrane permeability and disturbance in proton motive force causes the loss of cellular function and finally cell death [26]. The stability of the silver nanoparticles can be attributed to the formation of a silver electrode that can create a thin layer on the aqueous surface of the reaction mixture. This silver electrode possibly can change the silver to Nano silver, the protein present further is believed to cap the silver nanoparticles formed, restricting the agglomeration of the particles and as a result checking the size and shape. The precise mechanism of the formation of these nanoparticles in these biological media is unknown. Presumably biosynthetic products or reduced cofactors play an essential role in the reduction of respective salts to nanoparticles. It seems quite probable that the phenols play an essential part in the reduction of ions to AgNPs, the structure changes were observed in the thorax and abdomen,  cause important damage in the anal papillae region as well as cuticle layer, these structural distortions in the anal papillae and cuticle because of close relation with the death of exposure larval by silver nanoparticles, In addition to larvae which were to have the ability to transformation to the pupal stage have severe distortions in the head, thorax, and abdomen as shown in figures (9,11 and13), as well as the pupae lost the ability to breathe because of the lack of a trumpet as shown in fig (15), structural deformations of anal papillae were regarded one of the most essential reasons of the treated mosquito larvae's death [27].

 

 

Fig (8): Normal Second Instar Larva of Culex pipiens (10x)

 

 

Fig (9): Infected Second Instar Larva Primary Infection Sites in the Larval Bodies are the Head and Anal Region, and that the Main Route of Infection is Through the Cuticle (10x).

 

 

Fig (10):Normal third instar larva of Culex pipiens (10x)

 

 

(11): Infected Third Instar Larva Abnormal Black Fragile Body (10x)

 

Fig (12): Normal Fourth Instar Larva of Culex pipiens (10x)

 

 

Fig (13): Infected Fourth Instar Larva Abnormal Black Fragile Body (10x)

 

 

Fig (14): Normal Pupa of Culex pipiens (10x)

 

 

Fig (15): Infected Fourth Instar the Treated Pupae Showed Considerable Morphological Changes in the Whole Body in the Cuticle Layer and Abnormal Black Body (10x)

 

CONCLUSION:

The current study of green synthesis shows that the environmentally benign and renewable source of B. bassiana is used as an effective reducing agent for the synthesis of AgNPs. This biological reduction of silver nanoparticles could be a boon for the development of a clean, nontoxic, and environmentally acceptable green method to production of AgNPs. The biological applications of the nanoparticles showed that they have high larvicidal efficacy against second, third, fourth instar larvae and pupae of Cx. pipiens. The few concentrations of silver nanoparticles have no effect on larvae and pupae mosquitoes because of high insect resistance to toxic chemical insecticides, while high concentrations have an effect on larvae and pupae.

 

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Received on 12.12.2017        Modified on 03.01.2018

Accepted on 20.02.2018        © RJPT All right reserved

Research J. Pharm. and Tech. 2018; 11(3): 1058-1064.

DOI: 10.5958/0974-360X.2018.00198.1