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Larvicidal Potential of Fungi Based Silver Nanoparticles Against Culex Quinquefasciatus Larvae (II and III Instar)

Brindha Durairaj*, Santhoshkumar Muthu and Priya Shanthi

Department of Biochemistry, PSG College ofrtsnd Science, Coimbatore- 641014, Tamilnadu. India

*Corresponding Author:
Brindha Durairaj
Department of Biochemistry, PSG College ofrtsnd Science, Coimbatore- 641014, Tamilnadu. India

Received date: 05/08/2014; Revised date: 21/08/2014; Accepted date: 26/08/2014

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Silver nanoparticles (AgNPs) were synthesized using Penicillium notatum and further characterized by UV-visible spectrophotometer, Scanning Electron Microscope (SEM), Energy Dispersive X-Ray Spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy to support the nanoparticles biosynthesis by fungi. The synthesized AgNPs were further investigated for its antibacterial and larvicidal activity in mosquitoes. AgNPs treatment caused considerable mortality rate against 2nd and 3rd instar larvae of Culix quinquefasciatus after 24 hours exposure. However, higher concentration of AgNPs was required to induce mortality against 3rd instar than 2nd instar. In general, lipid and protein contents were found to be reduced in larval tissues after AgNPs treatment; whereas level of carbohydrate was found to be increased. AgNPs exhibited antibacterial activity against Staphylococcus aureus, Escherichia coli, Salmonella typhimurium and Salmonella shigella. Characterization studies reveal that Penicillium notatum biologically synthesized silver nanoparticles by reducing silver nitrate into nanosized silver ions. It can be concluded that rapid synthesis of fungi based silver nanoparticles will be helpful in developing a biological process for mosquito control using nanotechnology.


Silver nitrate, Penicillium notatum, Culix quinquefasciatus, vector control, filariasis


Mosquitoes continue to be world's number one vectors to transmit humanndnimal diseases;ndre prominent nuisance insect evenfter massive efforts of eradication or control [1]. Culex mosquitoes persistently bitend cause pain to both humanndnimals. Culex quinquefasciatusre especially responsible for the spread of filariasis. Lymphatic filariasis knowns elephantiasis has largelyffected nearly 1.4 billion people living in 73 countries Worldwide [2]. World health organization (WHO) has proclaimed that mosquitoesre human enemy number 1nd presently estimated that 50- 100 millions individualsreffected by the mosquito borne diseases [3]. Several methods including the use of chemical pesticidesredapted to controlnd eradicate mosquito population in theffected regions. However, synthetic chemical pesticides cause more harm to the environment, human lifend other non target organismss theyre not easily degradable [4]. Inddition, Culex quinquefasciatus mosquitoes unfortunately tend to develop more resistancegainst larvicides thatre currentlyvailable. Fungi based biosynthesis of nanoparticle is one of the best biological methods to prepare silver nanoparticles [5]. Hence, silver nanoparticles, the benign materials exhibit numerous benefits in terms of eco-friendlinessnd better compatibility for the eradication of mosquitoest their larval stage [6]. The mechanism of larvicidalctivity of both naturalnd synthetic pesticides is still not clear to support the scientific evidences in vector control strategy. Therefore, investigation of basic biochemical parameters (protein, carbohydratend lipid contents) in the pesticide treated insects is the need of the hour [7].

Pathogenic bacteria influence general quality of lifend oral health leading to chronic conditionsnd systemic diseases [8]. Therefore, the present work was designated to investigate thentimicrobial effect of silver nanoparticlesgainst microbes suchs Staphylococcusureus, Escherichia coli, Salmonella typhimuriumnd Salmonella shigella.

Material and Methods

Synthesisnd characterization of silver nanoparticles

Synthesis of silver nanoparticles: Penicillium notatum culture was grown inppropriate medium until it reached stationary phase.fter 7 days, fungi culture was separatednd grown in new medium for 3 days to prepare pure culture.fter incubation, filtrate of pure fungi culture (4.5 ml) wasdded with 0.5ml of 1mMgNO3nd incubated in dark condition in shaker for biosynthesis of silver nanoparticles.

Characterization of nanoparticles

UV-Vis spectralnalysis was performed by using UV-Vis spectrophotometer, UV159 (Elico), equipped with matched quartz cuvettes. The reduction of pure silver ions was monitored by measuring the UV-Vis spectrum of the reaction mediumt 24 hours of incubation using smallliquot of the samplet 200-800nm. The synthesized nanoparticles were characterized by Scanning Electron Microscope (SEM), Energy Dispersive X-Ray Spectroscopynd Fourier Transform Infrared (FTIR) spectroscopy.

Screening of larvicidalctivity of silver nanoparticle

2ndnd 3rd in star larvae of Culex quinquefasciatus were procured from National Centre for Disease Control, Mettupalam, Coimbatore, Tamilnadu. The larvae were maintained in trays containing distilled waternd supplied with yeast. The larvicidalctivity of fungi synthesizedgNPs was evaluateds described by World Health Organization method with slight modifications [9]. Different test concentrations ofgNP for 2nd instar larvae (0.5ppm 0.7ppm, 0.9ppm, 1.1ppmnd 1.3ppm)nd 3rd instar larvae (1ppm, 2ppm, 3ppm, 4ppmnd 5ppm) in 200ml of distilled water were prepared. Five replicates each containing 20 larvae was subjected to larvicidal bioassay forll the test concentrationnd control group (distilled water). Mortality rate was recordedfter 24 hour exposure period.

Estimation of biochemical parameters

After the treatment of 24 hours, larvae were removed from the test solutionnd washed with chilled normal saline. Larval tissue homogenate (10%) was prepared in 0.25M chilled sucrose solution by homogenizer. The homogenate was centrifugedt 700x g for 10 minutes to removell the cell debris. Supernatant wasdopted for estimation of total carbohydrates, lipids, proteins,lkaline phosphatasendcid phosphatase.ll the parameters were carried out in triplicate.

Estimation of total proteins

Lowry’s method wasdopted to estimate protein content in the larvae. Reaction of protein with Folin-Coicalteu become purple blue proportional to themount of proteinsnd readt 620 nm. Further protein concentration was calculated with optical density [10].

Estimation of total carbohydrates

Carbohydrate was estimateds described in the method of Nelson [11]. Proteins were removed from the tissue homogenatend the filtrate containing glucose onlys reducing substrate was heated withlkaline copper reagentnd subsequently treated withrsenomolybdate reagent. The blue color thus developed was readt 540 nmnd protein content was calculated.

Estimation of lipids

Total lipids present in the larval tissue were estimated following the method of Bragdon [12]. Lipid content was separated from the non-lipid components by chloroform- methanol solutionnd lipid in thequeous phase was reduced by sulphuriccid- dichromate mixture. The resultant green colour was measuredt 600 nmnd the concentration of lipid was calculated.

Antimicrobialctivity ofgNP

In-vitrontimicrobial screening of silver nanoparticles was performed by disc diffusion method using Staphylococcusureus, Escherichia coli, Salmonella typhimuriumnd Salmonella shigella [13]. Muller Hintongar (MHA) obtained from Hi-media (Mumbai) was used for the preparation of medium. Kanamycin (30μg per disc) was useds positive control.

Results and Discusion

UV- Visible Spectroscopy

Reduction of Silver nitrate into Silver nanoparticles during exposure of fungi was indicated by gradual increase in color development from colorless tolmost reddish brown (Figure 1). Silver nanoparticles (AgNPs) synthesized by P.notatum were primarily characterized by UV- Visible Spectroscopy. gNPs present typical spectrum having maximumbsorption in the range of 250 to 400 nm. The spectra ttribute to the surface Plasmon response (SPR) properties of metal nanoparticles when electronsre conducted ongNPs surface. These uniquend tunable optical properties due to the SPR depend on size, shapend distribution of nano sized particles of silver ions [14]. Reduction of silver ions is particularly measurednd monitored using UV-Visible spectrumnalysis with diluted silver nanoparticle sample (Figure 2).


Figure 1: Reduction of Silver Nitrate to Silver Nanoparticles by P.notatum [A- fungal filtrate B- AgNO nanoparticles, C- 2.0 mM AgNO3].


Figure 2: UV-Visible Spectrum indicating the presence of nanosized silver ions.


Figure 3: Fourier Transform Infrared Spectroscopy spectra

The spectrum exhibits the bandt 1635.64 cm-1 corresponding to primarymide groups (strong peak); similarly the presence of bandst 1381.64 cm-1 represents the nitro compounds including primary (CN)nd secondarymines (NH) stretch vibration of proteins. Strong bands of phenyl ring compounds indicate the occurrence of proteins with silver nanoparticles. The role of proteins is necessary for the reductionnd cappinground the nanoparticles synthesized by P.notatum. FTIRnalysis clearly suggests that proteinnd other bioorganic compound from P.notatum might be involved in the formationnd stabilization ofgNPs. P.notatum releases the extra cellular proteinsnd enzyme molecule to stabilize nano silver ions inqueous medium [15].

Scanning Electron Microscopynalysis

Characterization of silver nanoparticles using SEMnalysis revealed that theverage size of Silver ions ranged from 70 nm to 90 nm. SEM resultslso demonstrate the existence of rodsnd hexagonal shapes ofgNPs synthesized by P.notatum.


Figure 4: Scanning Electron Microscope picture of silver nanoparticles

Energy Dispersive X-Ray Spectroscopy

The EDAX spectrum exhibits the peaks indicating the silver, chloride, calcium, sodiumnd oxygen species. This proves the generation of silver nanoparticleslong with other ions by thection of fungi in the medium.


Figure 5: EDAX spectra of silver nanoparticles synthesized by P.notatum

Antibacterialctivity of Silver Nanoparticles

Thentibacterialctivity of silver nanoparticles was determinedt 5 different concentrationsgainst four bacterial Strains (Salmonella thyphinurium, Staphylococcusuries, Escherichia colind Salmonella shigella) by disk diffusion methodnd results were summarized in Table-1. Thentibacterialctivity was found to be increased with increase in concentration ofgNPs. The maximum zone of inhibition of 18mm was exhibitedt 0.05μg ofgNPsgainst E.coli followed by S.auries, Salmonella, Shigella with zone of inhibition of 14 mm, 17.4 mm 17mm respectively. Salmonella, Shigelland E.coli were more susceptible than S.auries. The effectiventibacterialctivity is due to the silver nanoparticles which overcome the barriers of bacterial cell wallnd therefore inhibits the bacterial growth [16].


Table 1: Antibacterial activity of silver nanoparticles

Larvicidalctivity ofgNPs

2ndnd 3rd instar stage of Culex quinquefasciatus larvae were treated with increasing concentration (0.5, 0.7, 0.9, 1.1nd 1.3 ppm) of silver nanoparticles to screen the larval mortality. 76.4% mortality was noted when treated with 0.5 ppmgNP against 2nd instar larvae; whereas the percentage mortality was found to increase to 89.7%t 1.3ppm ofgNPs treatment (Table 2). The LC50nd LC90 values for 2nd instar larvae were found to be 0.44ppmnd 1.13ppm respectively.


Table 2: Percentage mortality and biochemical changes in treated II instar larvae

Similarly the mortality rate of 3rd instar larvae was evaluated by treating with higher concentration range (1, 2, 3, 4,nd 5 ppm). The range for the 3rd instar larvae was fixed based on the observation in pilot study.gNP exhibited only 40% mortalitygainst 3rd instar larvaet 1ppm concentration; however the mortality percentage was found to increase in concentration dependant manner (Table 3). The maximum larvicidalctivity (92%) was found to be noticed when treated with 5ppmgNP. The LC50nd LC90 values of Silver nanoparticlesgainst 3rd instar larvae were noteds 2.3ppmnd 4.4 ppm respectively.


Table 3: Percentage mortality and biochemical changes in treated III instar larvae

From the result obtained, it is very clear that highest concentration is required to induce maximum mortalitygainst 3rd instar larvae when compared to 2nd instar larvae. This could be due to the structuralnd functional development 2nd instar grows tottain 3rd instar stage. It is concluded that increased silver nanoparticle concentration is suggested to exert consistent toxicitygainst developed larval stage.

Kovendan etl demonstrated that 20% mortality rate was exerted upon treatment of 20ppm plant extractgainst 1st instar larvae. The same extract was found to exhibit 89% mortalityt 100ppm concentration [17]. Many studies have proven that percentage mortality in mosquito larvae is directly proportional to the concentration of insecticides that induce toxicity [18]. Potential of silver nanoparticles synthesized by wide range of fungi suchs Diatum capillum, Chryosposium tropicumnd Penicillium notatum have been published by Christina 2013nd Thangaraj ramasamy 2013. Earlier researchers have clearly demonstrated that 0.9 ppmnd 4 ppmgNPs exhibited 85% mortalitygainst 2ndnd 3rd instar larvae respectively. This clearly indicates the dose dependent toxicity of the nanoparticlesgainst two different stages of mosquito larvae [19].

AgNP induced changes in Biochemical parameters

Carbohydrate content in the 3rd instar larval tissue was found to increase from 13.3 mg/g (control) to 19.33 mg/gfter treating with 5ppmgNPs. Similarly, the level of carbohydrate in 2nd instar larval tissues was observed to be 14.67 mg/g when treated with 1.3ppmgNP (Table 2 and 3). It was noticed that carbohydrate content was relatively high in the treated larvae than in the untreated larvae. These observations signify that level of carbohydrate in thegNP treated larval tissues increased in both 2ndnd 3rd instar larvae of mosquitoes when treated with increasing concentration of silver nanoparticles. The larvae might be unable tossimilate the food thereby increasing the level of carbohydrate in their tissues [20]. The larvicidal stress induced bygNP might have enhanced glycogenolysis leading to the hyperglycemia [21].

After treatment with silver nanoparticles, lipid level was found to be 90%nd 73% in 3rdnd 2nd instar larval tissues when compared with control larvae. The significant reduction in the lipid content indicates the negative effect of thegNP on the lipid metabolismnd lipid peroxidation. Stress induced in the mosquito larvae might be responsible for theltered energy metabolism leading to increased lipid catabolismnd declined lipid level [22, 23]. The similar negative effect was observed in malathion treated insects in which lipid depletion of oocytes, fat bodiesnd haemolymph was majorly noticed [24].

Total protein content was found to be decreased in both 2ndnd 3rd instar mosquito larvae when treated with different doses ofgNP. However, the reduction in the level of protein was comparatively lesser in 3rd stage larvae of Culex quinquefasciatus. Body wall of larvaenddult mosquitoes is made up of chitinnd other proteins whichre the structuralnd functional features. In our study, we observed that treatment withgNP caused damagend rupture in the dead larvae. The structural deformities observed might be due to the diminished protein profile in thegNPs treated larvae when compared with untreated control mosquito larvae [25-26]. Insecticidal interference in the hormones which regulate the protein synthesis might lead to the disturbance in the normal protein metabolism, rupturend destruction in the larval body [23]. Nanoparticles induced intoxicationnd growth retardation in larval stage of mosquitoes were found to be correlated with biochemical changes particularly in decrease or increase of the total protein, lipidnd carbohydrates toscertain functionalnd physiological interactions [27-31].


P. notatum based silver nanoparticles induced consistent mortalitygainst 2ndnd 3rd stage of Culix quinquefasciatus larvae. Biochemical parameters suchs lipid, proteinnd carbohydrate level werelso foundto beltered in the larval tissues upon intoxication with silver nanoparticles. The synthesized silver nanoparticles werelso found to possess considerable inhibitory effectgainst bacterial growth. In conclusion, the study indicates that P.notatum synthesized silver nanoparticles can be used to control the vectornd vector borne diseases.


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