Received 21 May 2015; revised 07 July 2017

Transcription

1 Indian Journal of Experimental Biology Vol. 56, January 2018, pp Larvicidal efficacy of silver nanoparticles synthesized biologically using Swietenia mahagoni (L.) Jacq. leaf extract against Anopheles stephensi, Culex quinquefasciatus and Cx. vishnui group Utpal Adhikari 1, Kuntal Bhattacharya 1,3, Partha Mitra 2 & Goutam Chandra 1 * 1 Mosquito, Microbiology and Nanotechnology Research Units, Parasitology Laboratory, Department of Zoology; 2 Department of Physics, The University of Burdwan, Burdwan , West Bengal, India 3 Department of Zoology, Durgapur Government College, Paschim Barddhaman , West Bengal, India Received 21 May 2015; revised 07 July 2017 Mosquito borne diseases are a global crisis, particularly in developing countries. Non-availability of apposite vaccines against these diseases has lead to sole dependence on the vector managerial steps for dropping the incidences. In the present study, we tried to evaluate the larvicidal potential of biologically synthesized silver nanoparticles (Ag NP) using aqueous leaf extracts of Swietenia mahagoni (L.) Jacq. against third instar larvae of Anopheles stephensi, Culex quinquefasciatus and Culex vishnui group. Aqueous extract of leaves reduced the aqueous silver ions to produce stable Ag NP. The characterization of synthesized nanoparticles was done through UV-Vis spectrum, Transmission electron microscope (TEM), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscope. Third instar larvae of three mosquito species namely An. stephensi, Cx. quinquefasciatus and Cx. vishnui group were exposed to different concentrations of synthesized nanoparticles for 24, 48and 72 h. TEM measured the range of nanoparticle size as 8-9 nm whereas XRD measured as 6 nm. Cent percent mortality of larvae of An. stephensi was recorded at 80 ppm at 48 h. About 96 and 80% mortality of Cx. vishnui group and Cx. quinquefasciatus larvae respectively were noted at 80 ppm after 72 h of exposure. The result of regression analysis proved that the mortality rate (Y) was positively correlated with the period of exposure (X) and regression coefficients were close to one. Toxicity study on non-target species showed no injurious activity during experimental period. Results indicate, possibly a first report on mosquito larvicidal effect of Ag NP synthesized using S. mahagoni leaf extract which may be used to effectively control the larval forms of three important vector mosquitoes. Keywords: Encephalitis, Elephantiasis, Lymphatic filariasis, Malaria, Mosquitoes, West Indian mahogany, Vector control Mosquitoes are potential vectors of several severe human diseases like malaria, lymphatic filariasis, dengue, DHF, chikungunya, yellow fever, encephalitis, etc. 1. Worldwide, about 860 million people are reported to be suffering from lymphatic filariasis 2. In malaria, about 216 million people are reported suffering from 91 countries, and from India, it is estimated to be 13 million cases 3,4. The malaria incidence in India according to WHO 2016 report is 18.6 per 1000 population 5. Japanese encephalitis is also one of the most common diseases in India 6. While chemical insecticides are effective in minimizing mosquito population, they lead to development of resistance and resurgence of the vector insects 7. They are non-biodegradable and causes harm to non-target organisms 8. *Correspondence: Phone: (Mob.) goutamchandra63@yahoo.co.in In recent years, scientists have focused on new herbal insecticides which are biodegradable and nontoxic to non-targets In current context, green nanotechnology opens a new horizon in the field of biocontrol of mosquitoes 15,16. Nanoparticles have been applied in biological, chemical, physical, industrial, pharmaceutical and environmental sciences. Applications of nanotechnology have been extended in the field of mosquito control by the synthesis of silver nanoparticles (Ag NP) from plant extracts 17. In the present study, we evaluated the larvicidal role of Ag NP, synthesized using Swietenia mahagoni leaf extract and AgNO 3, against three species of vector mosquitoes, Anopheles stephensi, Culex vishnui group and Culex quinquefasciatus including determination of possible structure of this biologically synthesized nanoparticle. Materials and Methods Fresh, mature leaves of Swietenia mahagoni (L.) Jacq. were collected from the campus of the

2 ADHIKARI et al.: MOSQUITO CONTROL BY SILVER NANO PARTICLES 15 University of Burdwan. After proper identification of the plant, the voucher specimen was deposited in the Department of Zoology (BUZGU-125), The University of Burdwan, West Bengal, India. The silver nitrate (AgNO 3, AR 99.9%) of Merck, India was used without further purification. De-ionized and double distilled water was utilized for preparation of extracts and solutions. The eggs of An. stephensi, Cx. quinquefasciatus and Cx. vishnui group were collected from fresh water of underground and overhead tanks of Kolkata metropolis, cemented drains, and rice fields surrounding the University campus, respectively. They were reared in the laboratory. The larvae were fed with artificial food (dried yeast powder and powder of dog biscuits in the ratio of 1:3). Preparation of leaf extract Collected leaves of S. mahagoni were thoroughly rinsed with distilled water and dried in paper towel. Then the leaves were cut into small pieces by sharp knife. The aqueous leaf extract was prepared by mixing 10 g of the leaves and 200 ml double distilled water taken in a glass conical flask, followed by vigorous shaking with a vortex stirrer (Spinix, India) for an hour. Synthesis of silver nanoparticles A stock solution of AgNO 3 was prepared. Twenty milliliters of the silver nitrate stock solution was mixed with 25 ml of the leaf extracts of S. mahagoni, and the volume was adjusted with deionized water to 250 ml so that final concentration of Ag + ion become M and leaf extract concentration remained 10% (v/v) of the stock 15. The mixture was stirred with a vortex stirrer for 5 min and then warmed at 50 C in a thermo stated water bath for 8 h. The resultant dark brown coloured solution was allowed to cool for overnight in air tight container. The final nano colloidal solution was subjected to repeated centrifugation (twice) to get rid of any un-interacted biological molecules at rpm for 15 min in Remi Research centrifuge instrument. The final pellet was collected, dried in vacuum desiccators and stored for future use. Characterization of nanoparticles The characterizations of the nano particles, synthesized from S. mahagoni, were done through the application of modern scientific instruments such as UV-vis spectroscope, TEM, XRD and FTIR spectroscope. Ag NP shows a characteristic surface plasmon resonance (SPR) absorption in the UV-visible region 18. The nanoparticle formation kinetics and the SPR absorption spectra of the water dispersed ultracentrifuged pellet of Ag NP were studied on a UV-Vis spectrophotometer (UV 1800 series, Japan) functioned at a resolution of 1 nm within 190 to 1100 nm wavelength range. Transmission electron microscope (TEM) studies of the particles were carried out at an accelerated voltage of 200 kv using a Philips CM200 TEM equipped with a LaB6 source. The pellet of purified ultracentrifuged nanoparticles was dusted and subjected to X-ray diffraction analysis (Bruker D8 advance) with a parallel beam optics attachment. The X-ray diffraction profiles of the samples were recorded using Ni-filtered CuK -radiation ( = Å) from a highly stabilized and automated Philips X-ray generator (PW 1830) operated at 30 ma current and 35 kv voltage. The X-ray diffraction pattern of the sample should reveal the formation of phase pure silver. From the X-ray diffraction pattern, the particle size ( D) can be found using the Scherrer s formula 19,20 given below: k D cos where k is a constant which is approximately 0.9 and β in radians represents the broadening in X-ray diffraction. The full angular width at a point where the intensity has fallen to half its maximum value i.e. full width at half maximum intensity (FWHM) is a measure of broadening of X -ray peaks 19. The Fourier transform infrared (FTIR) analysis was done with water dispersed pellet of the ultracentrifuged Ag NP sample produced from S. mahagoni leaf extract to get an idea about the chemical framework of nano particles by a FTIR spectrometer (Perkin Elmer Lx ). The scanning range was cm -1 at a resolution of 4 cm -1. Larvicidal bioassay The bioassay experiments were carried out according to standard WHO procedure 21 with moderate modifications. Synthesized nanoparticles were treated on 3 rd instars larvae of An. stephensi, Cx. quinquefasciatus and Cx. Vishnui group separately for bioassay experiment. Twenty five larvae of each species were kept separately in glass Petridishes (150 ml capacity/9 cm diameter)

3 16 INDIAN J EXP BIOL, JANUARY 2018 containing 100 ml of disinfected tap water. Each of four concentrations of nanoparticles (20, 40, 60 and 80 ppm) was applied separately into Petridishes. The experiments were repeated thrice for accuracy against each species of larva. They were maintained in 29±2 C and 82±1% RH in a photoperiod of 14:10 h light and dark cycle. Tap water was used in the control experiment without any nano particle against each concentration. Larval mortalities were noted after 24, 48 and 72 h of exposure. Effect on non-target organisms Non-target organisms, such as fish (Gambusia affinis), dragonfly nymph (Diplonychus annulatum) and tad pole of Bufo sp. were collected from field and acclimatized for 3 days in the laboratory. Batches of 25 fishes, dragonfly nymphs and tadpoles were placed in three separate pots, respectively, each containing 500 ml of dechlorinated tap water. In each pot, Ag NP at LC 50 concentration of third instars An. stephensi larvae was dissolved. At the same time, the controls with similar set without Ag NP were studied. Experiments on non-targets were done thrice on three separate days. The mortalities or any abnormal activities were studied up to 72 h. Statistical analysis The computer software STAT PLUS 2007 (Trial version) and MS EXCEL 2003 were used to calculate the LC 50, LC 90, regression equations (Y=mortality, X=concentration), regression coefficient values, mean mortality and standard error, etc. The percentage of corrected mortality was analyzed by Abbott s formula 22. Result and Discussion UV-Vis absorption spectra of the Ag NP showed absorption peak at 450 nm in Fig. 1. TEM analysis proved the presence of nanoparticles in the analyzed sample (Fig. 2). The size of the synthesized nanoparticles was within 8-9 nm range through TEM measurement. XRD is a non-destructive analytical method to uniquely identify the crystalline phases present and to study the structural properties. It is shown in Fig. 3. The major peaks at and are in good agreement with the joint committee on powder diffraction standard (JCPDS) data respective to silver structure 23. The other relatively low intensity peaks at 54.4, 57.1 and also agrees well with JCPDS data. Using the observed value of o and utilizing the known value of Å and measuring the values of β, Fig. 1 UV-Vis spectrum graph. Fig. 2 Transmission electron microscopic view of synthesized nanoparticle. Fig. 3 XRD analysis of synthesized nanoparticle. the particle size value comes out to be ~58 Å. Similarly, for o, the particle size value comes out to be ~62 Å. Thus, the average value of particle size in the sample is ~ 60 Å or 6 nm. The X-ray value is on the lower side comparable to TEM value since instrumental and strain broadening was not taken into account. Scherrer equation only considers peak broadening. If the instrumental and strain broadening was taken into consideration, the FWHM value would have been lowered and the particle size would have been higher. FTIR analysis of synthesized nano particles and its respective functional group is shown in Fig. 4 and Table 1, respectively that proves that specific functional group is related to the active principle.

4 ADHIKARI et al.: MOSQUITO CONTROL BY SILVER NANO PARTICLES 17 The larvicidal activity of silver nanoparticles against larvae of An. stephensi, Cx. vishnui group and Cx. quinquefasciatus are presented in Table 2. Log probit analysis and regression analysis of larvicidal activity of Ag NP of S. mahagoni leaf extract were done. LC 50 and LC 90 values, regression equations and R 2 values for the 3 rd instars larvae of these three species are presented in Table 3. The results of the present study indicate that the mortality rate of 3 rd instars of An. stephensi, Cx. vishnui group and Cx. quinquefasciatus at 80 ppm concentration were significantly higher than the mortality rates at 20 ppm, 40 ppm and 60 ppm concentrations of Ag NP of S. mahagoni leaf extract at 24, 48 and 72 h of exposure. Higher mortality rate was also recorded in all cases at 72 h bioassay than those at 24 and 48 h. The results of regression analyses revealed that the mortality rate (Y) was positively correlated with the period of exposure (X) having a regression coefficient close to one in each case. The results of log probit analyses (95% confidence level) revealed that LC 50 values gradually decreased with the exposure period. Cent percent mortality was observed in Ag NP at 80 ppm at 72 h of exposure on 3 rd instar larvae of An. stephensi. In control experiments, no larval mortality was observed. The results of toxicity test on non target organisms such as fish (Gambusia affinis), dragonfly nymph (Diplonychus annulatum), and tad pole of Bufo indicated no mortality after 24 h. The viewpoint of using herbal products for synthesizing silver nanoparticles (Ag NP) was to Table 1 Probable groups present in Swietenia mahagoni leaf extract (FTIR Fig. 4) Absorption Spectra (cm -1 ) Probable Groups S Phenolic Group (Ar-0H) Alkanes/Carboxylic acid Alkanes Aldehyde (C=O Stretch) Amide (NH/NH 2 Out of plane) Alkanes/ Aromatic Stretch Alkanes/Nitro compounds (-CH 3 /-NO) Amines Amines Aromatic Amines Table 2 Efficacy of synthesized Ag NPs on 3 rd instars larvae of Anopheles stephensi, Culex vishnui group and Cx. quinquefasciatus mosquitoes (Mean ± Standard errors) Third instar larvae Conc. of different species Mortality ± SE (ppm) of mosquitoes 24 h 48 h 72 h Anopheles stephensi Culex vishnui group Culex quinquefasciatus ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.00 Fig. 4 FTIR analysis of synthesized nanoparticle.

5 18 INDIAN J EXP BIOL, JANUARY 2018 Table 3 Log probit and regression analyses of larvicidal efficacy of synthesized Ag NPs on 3 rd instars larvae of An. stephensi, Cx. vishnui and Cx. quinquefasciatus Third instar larvae of different Hours species of mosquitoes Anopheles stephensi Culex vishnui group Culex quinquefasciatus LC 50 (ppm) LC 90 (ppm) Regression equation R 2 value 24 h Y=0.51X h Y=0.61X h Y=0.53X h Y=0.68X h Y=0.65X h Y=0.66X h Y=0.60X h Y=0.45X h Y=0.45X control three mosquito species. Different phytochemical constituents have been fractionated from diverse plant 24 sources and used against mosquitoes as repellent, oviposition deterrent 25, larvicidal 13, pupicidal 26 and adulticidal agents 27. However, green synthesized nanoparticles are modern tool facilitating the development of a most effective and environmentally safe mosquito control programmes 28. Nanotechnology has an ample relevance in vector and pest management in the form of nanocapsules. The application boundary of nanotechnology has been extended in the field of mosquito control by the synthesis of silver nanoparticles from plant extracts. Rajakumar et al. 29 first used silver nanoparticles synthesized from leaf extract of Eclipta prostrata against 4 th instar larvae of Cx. quinquefasciatus and An. subpictus. Silver nanoparticles were synthesized by Nelumbo nucifera leaf extract and its larvicidal activity was reported against Cx. quinquefasciatus and An. subpictus 1. The larvicidal activity of Ag NP synthesized from aqueous fruit extract of Drypetes roxburghii against the larvae of An. stephensi and Cx. quinquefasciatus was also reported 15. Elemike et al. 30 studied the larvicidal efficacy of synthesized Ag NP from nanoparticles using aqueous leaf extract of Eupatorium odoratum leaf against Cx. quinquefasciatus and estimated the LC50 value for third instars was 95.9 ppm after 24 h of exposure. The nanoparticles generally bind with the phosphate of DNA or RNA and sulphur bearing protein 31. The larvicidal efficacy of Ag NP had been reported 1,31 Adhikari et al. 14, reported the larvicidal potentiality of S. mahagoni leaves crude extract against 3 rd instar larvae of Cx. vishnui group where cent percent larval death was noticed at 0.4% concentration after 72 h of exposure period with LC 50 and LC 90 values of 0.05 and 0.28%, respectively. The present study is important as the synthesized nanoparticles prepared by the treatment of AgNO 3 with aqueous extract of leaves of S. mahagoni have larvicidal potentiality against An. stephensi, Cx. vishnui group and Cx. quinquefasciatus. Cent percent mortality of An. stephensi larvae was achieved using aqueous Ag NP after 48 h of exposure. This finding is important in vector control. The toxicity test of the synthesized nanoparticles on nontargets (Gambusia affinis, Diplonychus annulatum and tad pole of Bufo) did not show any abnormal activity or sluggishness and no mortality was observed in control experiments. The use of green nanoparticles to control mosquito larvae is rapid and ecofriendly approach. It is the first attempt to synthesize Ag NP with the help of S. mahagoni and to study their effect on An. stephensi, Cx. vishnui group and Cx. quinquefasciatus larvae. Acknowledgement The authors are grateful to the University Grants Commission (UGC), New Delhi for providing instruments through DRS programmes. References 1 Santhoshkumar T, Rahuman AA, Rajakumar G, Marimuthu S, Bagavan A, Jayaseelan C, Zahir AA, Elango G & Kamaraj C, Synthesis of silver nanoparticles using Nelumbo nucifera leaf extract and its larvicidal activity against malaria and filariasis vectors. Parasitol Res, 108 (2011) WHO Lymphatic filariasis Fact sheet As accessed 25 December The World Malaria Report bitstream/10665/259492/1/ eng.pdf As accessed 25 December Malaria. Global Health Observatory (GHO) data, India: country profiles, (World Health Organization), profile_ind_en.pdf. As accessed 25 December World Health Statistics 2017: monitoring health for the SDGs, Sustainable Development Goals, (World Health Organization), /255336/1/ eng.pdf. As accessed 25 December Hati A K, Vector of Japanese Encephalites in India and their bionomics and control. Bull Cal Sch Trop Med, 29 (1981) Liu N, Xu Q, Zhu F & Zhang L, Pyrethroid resistance in mosquitoes. Insect Sci, 13 (2006) Yang YC, Lee SG, Lee HK, Kim MK, Lee SH & Lee HS, A piperidine amide extracted from Piper longum L. fruit shows activity against Aedes aegypti mosquito larvae. J Agric Food Chem, 50 (2002) Bhattacharya K & Chandra G, Bioactivity of Acyranthes aspera (Amaranthaceae) Foliage against the Japanese

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