J Arthropod-Borne Dis, June 2020, 14(2): 162–172 M Waris et al.: Evaluation of … 162 http://jad.tums.ac.ir Published Online: June 30, 2020 Original Article Evaluation of Larvicidal Efficacy of Ricinus communis (Castor) Plant Extract and Synthesized Green Silver Nanoparticles against Aedes albopictus Muhammad Waris, *Shabab Nasir, Azhar Rasule, Iqra Yousaf Department of Zoology, Government College University, Faisalabad, Pakistan (Received 14 Nov 2018; accepted 19 May 2020) Abstract Background: Aedes mosquitoes are the most important group of vectors having ability of transmitting pathogens in- cluding arboviruses that can cause serious diseases like Chikungunya fever, Dengue fever and Zika virus in human. Biosynthesis and the use of green silver nanoparticles (AgNPs) is an important step in the search of reliable and eco- friendly control of these vectors. Methods: In this study an aqueous leaves extract of Ricinus communis (castor) and silver nanoparticles (AgNPs) syn- thesized from this extract were evaluated as larvicidal agent for 2 nd and 3 rd instar larvae of the Aedes albopictus. Differ- ent concentrations (50, 100, 150, 200 and 250ppm) of plant extract and synthesized nanoparticles were prepared and applied on second and third instar larvae. The percent mortality was noted after 6, 12, 18, 24, 30, 36, 42 and 48H of exposure and subjected to probit analysis to calculate LC50 and LC90. Results: Synthesized Ag + nanoparticles were characterized by UV-Vis spectroscopy, Fourier transform infrared spec- troscopy (FT-IR), and energy-dispersive X-ray spectroscopy (XRD). The nanoparticles were more toxic against larvae of Ae. albopictus with LC50 value (49.43ppm) and LC90 value (93.65ppm) for 2 nd instar larvae and LC50 (84.98ppm) and LC90 (163.89ppm) for 3 rd instar larvae as compared to the plant extract (149.58ppm, 268.93ppm) and (155.58ppm, 279.93ppm) for 2 nd and 3 rd instar larvae of Ae. albopictus respectively after 48H. Conclusion: Our results suggest the extract of R. communis and synthesized nanoparticles as excellent replacement of chemical pesticides to control the vector mosquitoes. Keywords: Dengue mosquito; Larvicidal; Ricinus communis; Mosquito larvae; AgNPs Introduction Mosquitoes cause a serious threat to public health (1–2). Vector-borne diseases such as ma- laria, dengue, chikungunya, Zika virus, Japanese encephalitis, filariasis, are being spread by mos- quitoes (3). These diseases are found all over the world and cause millions of deaths annu- ally (4). Pakistan is at the great risk of vector- borne diseases especially dengue due to it's over crowded cities, insanitation and poor vac- cination. In Pakistan, dengue cases are report- ed throughout the year but situation, usually, become worst in the post monsoon period (5). Pakistan had the worst dengue epidemic in 2011, during which more than 20,000 cases and 300 deaths were reported officially. In Khy- ber Pakhtunkhwa of Pakistan, July to end of September 2017 a total of, 52 926 cases of den- gue fever including 38 deaths were reported (6) Chikungunya virus was detected in 1983 (7) and more than 4000 cases have confirmed through qualitative RT-PCR. Zika virus has reached near border areas in neighboring countries like China and India, so outbreak of the disease may oc- cur in Pakistan (8). The easy solution to avoid mosquito-borne diseases is the management of mosquito pop- ulation. This management through chemicals causes health risks to human beings, environ- mental pollution and insecticidal resistance in mosquitoes (9). This prompted the need of searching for new chemical compounds and alternative strategies, as novel biological tools. So, medicinal plants can be used as an alter- nate for this purpose because these plants have *Corresponding author: Dr Shabab Nasir, E-mail: flourenceshabab@yahoo.com mailto:flourenceshabab@yahoo.com J Arthropod-Borne Dis, June 2020, 14(2): 162–172 M Waris et al.: Evaluation of … 163 http://jad.tums.ac.ir Published Online: June 30, 2020 many types of target specific, rapidly biodegrada- ble, ecofriendly, and less toxic to human health larvicidal phytochemicals such as saponins, iso- flavonoids, tannins, terpenes, steroids, etc. (10- 11). Plants are good source of bioactive insec- ticidal phytochemicals that can kill mosquito larvae with high mortality rate (12-14) by bring- ing changes in development, midgut epitheli- um (15), mutation in DNA and production of reactive oxygen species (16-17). They have different mechanisms of action that reduce the chance of resistance development in mosqui- toes (18). Biologists have begun the use these phyto- chemicals as larvicides to control the mosqui- toes (19). One step ahead, green silver nano- particles (AgNPs) synthesized from plant ex- tracts are proved more toxic than phytochemi- cal as larvicides (20). Green silver nanoparti- cles (AgNPs) have high larvicidal effect because of small size ranging 1–100nm (21) and large surface area. These characteristics of AgNPs made them a unique larvicide at very low con- centrations. These have been tested in a varie- ty of entomological research because these are safe, low cost, easily available and have a sim- ple easy biosynthesis process (21-22). Ricinus communis (castor) plant belongs to a big fam- ily Euphorbiaceae contains nearly about 300 genera and 7500 species. Ricinus communis (castor) is a flowering plant, has high medici- nal value for healthy human life. This plant is used as laxative, fungicide, anti-oxidant, anti- asthmatic, antiulcer, wound healing, insecticidal and larvicidal agent. It has important phyto- chemicals like glycosides, alkaloids, flavonoids, steroids and saponins. that are helpful in con- trolling mosquitoes (23-24) (Fig. 1). Due to these reasons, the present study was designed to evaluate the larvicidal potential of the plant extract and AgNPs synthesized from this ex- tract of R. communis (castor) against 2 nd and 3 rd larvae of Ae. albopictus under laboratory conditions. UV-Vis spectroscopy analysis, Pow- dered X-ray diffraction (PXRD) and Fourier Transform Infrared Radiation (FTIR) spec- troscopy were used to confirm the biosynthe- sis of AgNPs. Materials and Methods Preparation of leaf extract Healthy and fresh leaves of the R. com- munis (castor) plants were collected (hand plucked) from the old campus of University of Agriculture Faisalabad, Pakistan during the month of May, 2017. Leaves were cleaned with cloth and washed with tap water to re- move dust. Then the leaves were dried in shady place at room temperature and grinded in an electric grinder (Anex Germany) (25). Fifty grams powder of leaves was mixed with 250ml acetone as solvent in the Soxhlet appa- ratus and boiled gently at boiling point range 55.5–56.5 ºC for complete extraction (8h) and stored at 4 ºC (26). Preparation of Green AgNPs Silver nitrate (AgNO3) of Sigma was pur- chased and 1mM solution of silver nitrate (AgNO3) was prepared in 250mL Erlenmeyer flask in the darkness to avoid action of light. 10ml acetone plant extracts of R. communis (castor) was put in 250ml conical flask having 90ml of 1mM silver nitrate solution. Two to three drops of 1% NaOH were added for the adjustment of pH at 8 and mixed continuously by magnetic stirrer. This mixture was kept at 40 °C for one hour under clear sky condition for irradiation. Colour change of the solution indicated the formation of AgNPs. Reaction completed on attaining reddish brown colour. Solution was cooled and stored in amber bot- tle at 4 °C, then centrifuged for three times at 5000rpm for 20 minutes to obtain pellets. Pu- rified suspension was made by dissolving pel- lets in double distilled water and was frozen for further use (27). Characterization of AgNPs. The biosynthesized silver nanoparticles were characterized by UV-Vis spectroscopy ana- J Arthropod-Borne Dis, June 2020, 14(2): 162–172 M Waris et al.: Evaluation of … 164 http://jad.tums.ac.ir Published Online: June 30, 2020 lysis, Powdered X-ray diffraction (PXRD) and Fourier Transform Infrared Radiation (FTIR) spectroscopy assistance through High Tech central laboratory of Government College Uni- versity Faisalabad. UV-Vis absorbance spectroscopy. To monitor formation of the green silver nanoparticles, absorption spectra were taken at a scanning speed of 200 to 800nm using a Cary 60 double beam UV-Vis spectropho- tometer (Spectramax M3 molecular devices) operating at the resolution of 1nm. UV-Vis spectra were recorded after 15 and 30 minutes (28). Powdered X-ray Diffraction (PXRD) The shape of structure and size of the sil- ver nanoparticles was calculated through dif- fracted intensities at 40kV voltage and 30mA current with the range of 0°–80° 2θ in CuKα radiation (Rigaku, Ultima IV, and X-ray dif- fractometer system). Fourier Transform Infrared (FTIR) Spec- troscopy The residue solution of 100ml was centri- fuged at 5,000rpm for 10 minutes after reac- tion to remove impurities. To obtain pure pel- let of AgNPs the supernatant was again cen- trifuged at 10000rpm for 60 minutes. All measurements were carried out in the range of 400–4000cm -1 at a resolution of 4cm -1 (29). Fresh samples having volume of 1–2ml in aqueous form were sent for FTIR Analysis to Hi-Tech Lab, Government College University Faisalabad. Collection and rearing of mosquitoes Larvae and pupae were collected with dip- pers from a forest near Faisalabad, Punjab, Pakistan (31° 25' 7.3740'' N and 73° 4' 44.7924'' E, 192 meters above the sea level). Collected immature stages of mosquito were brought back to the Zoology Lab, department of Zoology, Government College University, Faisalabad, inside beakers closed with muslin cloth. Larvae and pupae of Ae. albopictus were identified with the help of identification keys (30–31), reared to adults in 1000ml beakers containing water under lab conditions at 25±5 °C and 80±5% RH (32). Adults fur- ther reared in separate glass cages. Male adults were fed with 10% sugar solution and females with blood on live white rats in sepa- rate glass cages for egg laying (33). Larvae emerged from the eggs were reared in batches of 300 each, in 1200ml deionized water in stainless steel trays (35x 30x 5cm) for the bi- oassays (34). Fifth generation larvae were used for the bioassay. Bioassay Group of 20 actively swimming 2 nd and 3 rd instar larvae (identified from the shed cuticle and from the size and colour of the larvae) of Ae. albopictus were released in 250ml beaker containing 200ml distilled water separately. Five concentrations including 50, 100, 150, 200 and 250ppm of larvicidal solution of R. communis extract and green AgNPs synthe- sized from the extract were prepared using distilled water and subjected for mortality as- says separately. Bioassay was conducted at 27±3 °C, 80±3% relative humidity (RH) and a photoperiod of 16: 8 (L: D) hours (35). The control was set up with dechlorinated tap wa- ter and five replications were done for each treatment. Mortality rates were calculated us- ing the WHO (3) bioassay protocol with slight modifications. The percentage mortalities were corrected by using Abbott’s formula (36). The average larval mortality data was sub- jected to probit analysis using Minitab -17 statistical software (2017) for calculating le- thal concentration 50% (LC50) and 90% (LC90) of larvae and for getting dose and time mortality regression lines. J Arthropod-Borne Dis, June 2020, 14(2): 162–172 M Waris et al.: Evaluation of … 165 http://jad.tums.ac.ir Published Online: June 30, 2020 Results Synthesis of Silver Nanoparticles Formation of green AgNPs through the re- duction of the silver metal ions by the extract of R. communis that turned the colour of mix- ture (plant extract +AgNO3 Solution) into reddish brown in 1H at 40 ºC (Fig. 2). UV-Vis spectrum of silver nanoparticles The progress of the reaction between Ag + and the leaf extract was monitored by UV– visible spectra of silver nanoparticles in aque- ous solution with different reaction times that are shown (Fig. 3). It was observed from the figure that localized surface plasmon reso- nance band showed maximum absorbance at 430nm after 30 minutes of reaction time. Powdered X-ray Diffraction (PXRD) Studies Result of PXRD (Fig. 4) showed intense silver nanoparticle (AgNPs) diffraction peaks at 38.10, 44.46, 64.45, 77.51, and 81.60, cor- responding to facets 113, 202, 221, 310, and 223 of the face-centered cubic crystal structure. Fourier Transform Infrared Radiation Spec- troscopy (FTIR) Analysis The FTIR spectra of silver nanoparticles prepared from the R. communis leaf extract (Fig. 5) showed transmittance peaks at 1263.2, 978.6, 849.1, 710.5, 662.8, 502.7, and 435.6/cm. The carbonyl group formed amino acid residues which capped the silver nano- particles indicated by these peaks. These resi- dues prevent from agglomeration of AgNPs, and made the medium stable. FTIR clearly indicate role of proteins and other compounds of leaf extract in the formation and stabiliza- tion of AgNPs. Larvicidal activity of leaf extracts and syn- thesized silver nanoparticles The results of the larvicidal activity of leaf extract of R. communis (Castor) and synthe- sized AgNPs with different concentrations (50–250ppm) after different exposure times (6, 12, 18, 24, 30, 36, 42 and 48H) showed a dose and time-dependent toxic effects against 2 nd and 3 rd instar larvae of Ae. albopictus. No mortality was observed in the control group. AgNPs synthesized from R. communis showed 100% mortality for all the exposed larvae after 36H at the concentration of 250ppm (Table 1). The synthesized AgNPs showed least values of LC50 (49.43ppm) and LC90 (93.65ppm) after 48H with regression equation Y= -1.208+0.1521x against 2 nd in- star larvae of Ae. albopictus. Similarly for the 3 rd instar larvae, the least values of LC50 and LC90 were 84.98 and 163.89ppm respectively after 48H with regression equation Y= - 1.072+0.1461x as shown in Table 1. The mortality rate of 2 nd and 3 rd instar lar- vae of Ae. albopictus was noted as 98 and 96% respectively after 48H at 250ppm con- centration of R. communis leaves extract (Ta- ble 2). The least values of LC50 and LC90 were 149.57 and 268.92ppm for 2 nd instar lar- vae and 155.57 and 279.92ppm for 3 rd instar respectively with regression equations Y= - 1.16+0.129x and Y= -1.210+0.113x after 48H. The extract of R. communis exhibited prominent larvicidal activity against the 2 nd instar larvae of Ae. albopictus. All the con- centrations of plant extracts used in the pre- sent study exhibited repellency activity. Fig. 1. Ricinus communis (castor) plant (original pho- to) J Arthropod-Borne Dis, June 2020, 14(2): 162–172 M Waris et al.: Evaluation of … 166 http://jad.tums.ac.ir Published Online: June 30, 2020 Table 1. Larvicidal activity of AgNPs synthesized from R.communis against Ae. albopictus larvae Time Larval instars %Mor- tal- ity±SD Lethal con- centration FL at 95% C.I Chi- square P value Regression equation 6H 2 nd 10±0.27 LC50 523.02 384.98-1129.65 3.52 0.319 Y= -2.30+0.0044x LC90 813.34 564.21-1925.07 3.52 0.319 Y= -2.30+0.0044x 3 rd 9±0.27 LC50 565.14 400.52-1494.50 3.72 0.292 Y= -2.27+0.0040x LC90 883.53 589.09-2568.35 3.72 0.292 Y= -2.27+0.0040x 12H 2 nd 15±0.24 LC50 471.43 363.43-827.90 2.16 0.539 Y= -2.19+0.0046x LC90 747.16 543.88-1430.95 2.16 0.539 Y= -2.19+0.0046x 3 rd 10±0.21 LC50 513.08 380.19-1034.82 1.34 0.712 Y= -.96+0.0038x LC90 847.98 591.32-1873.27 1.34 0.712 Y= -1.96+0.0038x 18H 2 nd 20±0.20 LC50 437.37 344.45-706.71 0.54 0.909 Y= -1.96+0.0038x LC90 728.90 541.17-1285.97 0.54 0.909 Y= -1.96+0.0038x 3 rd 16±0.21 LC50 513.08 380.19-1034.82 0.13 0.718 Y= -.96+0.0038x LC90 847.98 591.32-1873.27 0.13 0.718 Y= -1.96+0.0038x 24H 2 nd 25±0.19 LC50 384.70 316.42-547.57 0.84 0.809 Y= -1.89+0.0049x LC90 645.16 501.53-998.63 0.84 0.809 Y= -1.96+0.0038x 3 rd 20±0.19 LC50 455.96 351.97-786.41 0.17 0.918 Y= -1.83+0.0040x LC90 774.01 561.84-1463.36 0.17 0.918 Y= -1.83+0.0040x 30H 2 nd 50±0.16 LC50 245.24 222.17-280.11 0.34 0.809 Y= -1.70+0.0059x LC90 430.12 526.76-372.84 0.34 0.809 Y= -1.70+0.0059x 3 rd 35±0.19 LC50 339.95 291.79-435.60 0.17 0.244 Y= -1.99+0.0058x LC90 558.36 455.79-770.54 0.17 0.244 Y= -1.99+0.0058x 36H 2 nd 70±0.15 LC50 188.19 172.39-207.37 0.77 0.809 Y= -1.42+0.0075x LC90 357.11 317.50-418.58 0.77 0.809 Y= -1.42+0.0075x 3 rd 50±0.16 LC50 242.61 220.36-275.70 4.17 0.253 Y= -1.719+0.0070x LC90 423.45 368.57-514.79 4.17 0.253 Y= -1.719+0.0070x 42H 2 nd 100±0.15 LC50 104.99 90.40-117.45 5.77 0.033 Y= -1.43+0.1545x LC90 217.85 202.21-238.34 5.77 0.033 Y= -1.43+0.1545x 3 rd 95±0.14 LC50 115.02 103.75-125.31 1.67 0.000 Y= -1.077+0.0108x LC90 229.82 211.09-225.49 1.67 0.00 Y= -1.077+0.0108x 48H 2 nd 100±0.12 LC50 49.43 37.51-59.44 6.77 0.032 Y= -1.208+0.1521x LC90 93.65 81.06-110.33 6.77 0.032 Y= -1.208+0.1521x 3 rd 100±0.12 LC50 84.98 70.40-101.45 1.27 0.303 Y= -1.077+0.1461x LC90 163.89 151.09-175.49 1.27 0.303 Y= -1.077+0.1461x LC50: lethal concentration that kills 50% of the exposed larvae; LC90: lethal concentration that kills 90% of the exposed larvae. J Arthropod-Borne Dis, June 2020, 14(2): 162–172 M Waris et al.: Evaluation of … 167 http://jad.tums.ac.ir Published Online: June 30, 2020 Table 2. Larvicidal activity of leaf extracts of R. communis against Ae.s albopictus larvae Time Larval instars %Mor- tality±SD Lethal con- centration FL at 95% C.I Chi- square P value Regression equation 6H 2 nd 9±0.28 LC50 572.94 403.52-1584.71 5.12 0.163 Y= -2.31+0.004x LC90 889.79 589.68-2705.85 5.12 0.163 Y= -2.31+0.004x 3 rd 6±0.27 LC50 725.56 448.49-1927.47 4.22 0.238 Y= -2.19+0.003x LC90 1148.36 664.68-2035.2 4.22 0.238 Y= -2.19+0.003x 12H 2 nd 15±0.22 LC50 537.82 403.69-1002.15 1.35 0.717 Y= -2.06+0.041x LC90 778.85 559.72-1546.13 1.35 0.717 Y= -2.06+0.041x 3 rd 11±0.27 LC50 725.56 448.49-1927.47 4.22 0.238 Y= -2.19+0.003x LC90 1148.31 664.68-2035.20 4.22 0.238 Y= -2.19+0.003x 18H 2 nd 25±0.20 LC50 407.93 329.78-608.36 0.92 0.81 Y= -2.06+0.041x LC90 679.10 518.69-1101.86 0.92 0.81 Y= -2.06+0.041x 3 rd 18±0.19 LC50 507.42 374.13-1045.96 0.06 0.995 Y= -1.79+0.003x LC90 869.61 601.13-1974.48 0.06 0.995 Y= -1.79+0.003x 24H 2 nd 30±0.16 LC50 313.16 265.99-411.52 2.08 0.554 Y= -1.48+0.005x LC90 583.50 465.24-845.33 2.08 0.554 Y= -2.06+0.041x 3 rd 22±0.19 LC50 470.34 356.20-869.28 0.49 0.921 Y= -1.18+0.004x LC90 820.08 582.04-1670.4 0.49 0.921 Y= -1.18+0.004x 30H 2 nd 40±0.16 LC50 313.16 265.99-411.52 2.08 0.554 Y= -1.48+0.005x LC90 583.50 465.24-845.33 2.08 0.554 Y= -1.48+0.041x 3 rd 30±0.18 LC50 370.21 303.81-530.08 0.054 0.997 Y= -1.65+0.004x LC90 656.65 506.69-1031.3 0.054 0.997 Y= -1.65+0.004x 36H 2 nd 50±0.16 LC50 258.97 229.21-309.36 0.67 0.880 Y= -1.46+0.006x LC90 485.52 406.70-633.31 0.67 0.880 Y= -1.46+0.046x 3 rd 40±0.17 LC50 317.15 272.97-403.39 2.05 0.532 Y= -1.71+0.005x LC90 554.63 451.92-766.05 2.05 0.532 Y= -1.71+0.005x 42H 2 nd 88±0.15 LC50 197.93 176.04-209.75 3.26 0.114 Y= -1.51+0.153x LC90 323.98 301.79-336.20 3.26 0.114 Y= -1.51+0.153x 3 rd 87±0.15 LC50 276.36 244.35-288.73 0.55 0.832 Y= -1.517+0.010x LC90 372.02 350.53-401.52 0.55 0.832 Y= -1.517+0.010x 48H 2 nd 98±0.14 LC50 149.57 138.29-159.74 2.06 0.518 Y= -1.16+0.129x LC90 268.92 254.26-280.00 2.06 0.518 Y= -1.16+0.129x 3 rd 96±0.15 LC50 155.57 139.29-170.74 1.65 0.102 Y= -1.210+0.113x LC90 279.92 252.26-295.00 1.65 0.102 Y= -1.210+0.113x LC50: lethal concentration that kills 50% of the exposed larvae; LC90: lethal concentration that kills 90% of the exposed larvae J Arthropod-Borne Dis, June 2020, 14(2): 162–172 M Waris et al.: Evaluation of … 168 http://jad.tums.ac.ir Published Online: June 30, 2020 Fig. 2. Bioreduction of AgNO3 into AgNPs using plant extract (colour change) Fig. 3. Ultraviolet-Visible spectra of silver nanoparticles synthesized by treating R. communis leaf extract with 1 mM AgNO3 solution Fig. 4. Powdered X-ray diffraction J Arthropod-Borne Dis, June 2020, 14(2): 162–172 M Waris et al.: Evaluation of … 169 http://jad.tums.ac.ir Published Online: June 30, 2020 Fig. 5. Fourier Transform Infrared Radiation spectra of AgNPs synthesized from leaf extract of R. communis (Castor) Discussion Nanotechnology is an emerging technol- ogy in modern era that enables scientists to synthesize particles of different sizes, forms and components. Hence synthesized nanopar- ticles of gold, silver and platinum are being used for insect vectors control and in pharma- ceutical industries (17). During current study, change in colour (reddish brown) of the solu- tion indicated the formation of AgNPs due to the reduction of silver metal ions by the ex- tract of R. communis and was confirmed by the localized surface plasmon resonance band absorbance at 430nm after 30 minutes of re- action time. Previous studies also indicated the similar colour changes and maximum ab- sorbance at 430nm (37). Our result of PXRD indicated intense silver nanoparticle (AgNPs) diffraction peaks at 38.10, 44.46, 64.45, 77.51 and 81.60 corresponding to facets 113, 202, 221, 310 and 223 of the face-centered cubic crystal structure. Sathyavathi et al. (25) also reported diffraction peaks at 44.50, 52.20, and 76.7, which correspond to the 111, 200, and 222 facets of the face-centered cubic crystal structure. XRD result of silver nanoparticles reported by Nirmala et al. (38) is also close to the cited results. The FTIR spectra of current study showed transmittance peaks at 1263.2, 978.6, 849.1, 710.5, 662.8, 502.7, and 435.6/cm. The car- bonyl group formed amino acid residues which capped the silver nanoparticles indicated by these peaks. These residues prevent from ag- glomeration of AgNPs, and made the medium stable. FTIR clearly indicate role of proteins and other compounds of leaf extract in the formation and stabilization of AgNPs (37–40). In our study, the R. communis AgNPs showed 100% mortality at 250ppm for Ae. albopictus after 48H, with LC50 and LC90 values 49.43, 93.65ppm and 84.98, 163.89ppm for 2 nd and 3 rd instar larvae respectively, while LC50 and LC90 values of the leaf extract of R. communis after 48h exposure were 149.57, 268.92ppm and 155.57, 279.92ppm for 2 nd and 3 rd instar larvae respectively. These results clearly indi- cated that R. communis AgNPs were more potent than leaf extract of R. communis due to less LC50 and LC90 values. These results are in line with the results of other scientists who also reported more potency of AgNPs than simple plant extracts (38–39). Karthikeyan et al. (39) also reported the toxicity of AgNPs synthesized from Euphor- bia hirta leaf extract against An.stephensi 1 st to 4 th instar larvae with LC50 (10.14, 16.82, 21.51, and 27.89ppm, respectively) after 24H. This high potency of AgNPs was due to high surface area-to-volume ratio, that imparts dif- ferent biological and catalytic activities in them (30–40). Hemant et al. (40) also re- ported the potency of AgNPs of Euphorbia J Arthropod-Borne Dis, June 2020, 14(2): 162–172 M Waris et al.: Evaluation of … 170 http://jad.tums.ac.ir Published Online: June 30, 2020 tirucalli against 2 nd and 4 th instar larvae with least LC50 values (3.50 to 7.01ppm) and (4.44 to 8.74ppm) respectively after 24 hrs. Our findings are at par with the previous findings that simple leaf extracts are less po- tent than AgNPs. The larvicidal effects of leaves extracts of R. communis showed the LC50 val- ues of 1091.44, 1364.58 and 1445.44ppm against 2 nd , 3 rd and 4 th larval instars of Cx. quinquefasciatus (41). Basheer (42) also used R. communis extracts through different sol- vents and found ethyl acetate extract more potent than others against 3 rd instar larvae of Ae. albopictus. He also noted that LC50 values decreased with time. These results are similar with our findings. Mandal (43) also noted that R. communis seed extract exhibited larvicidal effects with 100% mortality at concentrations 32–64μg/mL, with LC50 value 16.84μg/mL for Ae. albopictus larvae. All previous scientists studied either plant extracts or their AgNPs separately but the present study compared the plant extract with its AgNPs. Cited results are close to our findings but not exactly same due to using different plant, mosquito species, lar- val stage and solvent for plant extraction. Results of our study suggested that the leaf extract of R. communis is toxic to Ae. al- bopictus larvae and toxicity increased when extract combined with AgNPs. Our results clearly proved the excellent larvicidal efficacy of R. communis against Ae. albopictus. Conclusion It is concluded from our findings that, the leaf extract and synthesized silver nanoparti- cles of R. communis had excellent potential for killing the of mosquito larvae. The appli- cation of this plant extract along with silver nanoparticle on mosquito breeding places surely decrease the population of vector mos- quitoes, control many dreadful diseases and prevent environmental pollution. References 1. Rozendaal JA (1997) Mosquitos and other biting Diptera vectors of malaria leish- maniasis filariasis onchocerciasis den- gue yellow fever and other diseases. In: Rozendaal JA (Eds): Vector Control Methods for Use by Individuals and Communities, World Health Organiza- tion, Geneva, Switzerland, pp. 7–177. 2. Service M (2004) Medical Entomology for Students. 3 rd edition, Cambridge Uni- versity Press, UK. 3. WHO (2006) Pesticides and their applica- tion for the control of vectors and pests of public health importance. World Health Organization, Geneva, Switzer- land, p.114. 4. Rajkumar G, Rahuman A (2011) Larvicidal activity of synthesized silver nanoparti- cles using Eclipta prostrata leaf extract against filariasis and malaria vectors. Acta Trop. 118: 196–203. 5. Jahan F (2017) Dengue Fever (DF) in Paki- stan. Asia Pacific Fam Med. 10(1): 1–4. 6. WHO (2017) EMRO Weekly Epidemio- logical Monitor. 10: 40. 7. Darwish MA, Hoogstraal H, Roerts TJ, Ahmed IP, Omar F (1983) A sero-epi- demiological survey for certain arbo- viruses (Togaviridae) in Pakistan. Trans R Soc Trop Med Hyg. 77: 442–445. 8. Rauf M, Fatima-tuz-Zahra, Sobia M, Azra M, Shameem B (2017) Outbreak of chikungunya in Pakistan. Lancet Infect Dis. 17: 258. 9. Isman MB (2006) Botanical insecticides deterrents and repellents in modern ag- riculture and an increasingly regulated world. Ann Rev Entomol. 51: 45–66. 10. Zhu J, Zeng X, Neal OM, Schultz G (2008) Mosquito larvicidal activity of botanical based mosquito repellents. J Amer Mosq Cont Assoc. 2: 161–168. 11. Ghosh A, Chowdhury N, Chandra G (2012) Plant extracts as potential mosquito lar- J Arthropod-Borne Dis, June 2020, 14(2): 162–172 M Waris et al.: Evaluation of … 171 http://jad.tums.ac.ir Published Online: June 30, 2020 vicides. Ind J Med Plants Res. 13: 581– 598. 12. Khair-ul-Bariyah S, Ahmed D, Ikram M (2015) Ocimum basilicum: a review on phytochemical and pharmacological stud- ies. Pak J Chem. 2: 78–85. 13. Mdoe FP, Cheng SS, Msangi S, Nkwen- gulila G, Chang ST, Kweka EJ (2014) Activity of Cinnamomum osmophloeum leaf essential oil against Anopheles gam- biae. Parasite Vect. 7: 209. 1–6. 14. Pavela R (2016) History presence and perspective of using plant extracts as commercial botanical insecticides and farm products for protection against in- sects - A review. Plant Protect Sci. 52: 229–241. 15. Al-Mekhlafi FA (2018) Larvicidal ovi- cidal activities and histopathological al- terations induced by Carum copticum (Apiaceae) extract against Culex pipiens (Diptera: Culicidae). Saudi J Biol Sci. 25: 52–56. 16. Arjunan N, Murugan K, Rejeeth C, Mad- hiyazhagan, Barnard D (2012) Green synthesis of silver nanoparticles for the control of mosquito vectors of malaria, filariasis, and dengue. Vector Borne Zo- onot Dis. 12: 262–268. 17. Subramaniam J, Murugan K, Panneerselvam C (2015) Ecofriendly control of malaria and arbovirus vectors using the mosqui- tofish Gambusia affinis and ultra-low dosages of Mimusops elengi-synthesized silver nanoparticles: towards an integra- tive approach. Env Sci Poll Res. 22: 20067–20083. 18. Okumu FO, Knols BG, Fillinger U (2007) Larvicidal effects of a neem (Azadirachta indica) oil formulation on the malaria vector Anopheles gambiae. Malar J. 6: 63. 1–8. 19. Eliman MA, Elmalik KH, Ali FS (2009) Efficacy of leaves extract of Calotropis procera Ait. (Asclepiadaceae) in con- trolling Anopheles arabiensis and Culex quinoquefasciatus mosquito. Saudi J Biol Sci. 16: 95–100. 20. Bilal H, Hassan SA (2012) Plants second- ary metabolites for mosquito control. Asian Pac J Trop Dis. 2: 168–168. 21. Borase H, Patil C, Patil R (2013) Phyto- synthesized silver nanoparticles: a po- tent mosquito biolarvicidal agent. J Na- nomed Biotech Dis. 3(1): 1–7. 22. Muthukumaran U, Govindarajan U, Ra- jeswary M, Hoti S (2015) Synthesis and characterization of silver nanoparticles using Gmelina asiatica leaf extract against filariasis, dengue, and malaria vector mos- quitoes. Parasitol Res. 114: 1817–1827. 23. Jitendra J, Ashish KG (2012) Ricinus com- munis Linn: A phytopharmacological Re- view. Int J Pharm Pharmaceutical Sci. 4: 0975–1491. 24. Palanivelu J, Kunjumon MM, Suresh A, Nair A, Ramalingam C (2015) Green synthesis of silver nanoparticles from Dracaena mahatma leaf extract and its antimicrobial activity. J Pharmaceutical Sci Res. 7: 690–695. 25. Satyavani K, Ramanathan T, Gurudeeban S (2011) Plant mediated synthesis of bi- omedical silver nanoparticles by leaf ex- tract of Citrullus colosynthis. Res J Nano Tech. 1: 95–101. 26. Vogel AI (1978) Text Book of Practical Organic Chemistry. The English Lan- guage Book Society and Longman, Lon- don. pp. 1540. 27. Sathyavathi R, Krishna KB, Venugopal Rao M (2010) Biosynthesis of silver na- noparticles using Coriandrum sativum leaf extract and their application in non- linear optics. Adv Sci Letters. 3: 1–6. 28. Rajesh WR, Jaya RL, Niranjan SK, Vijay DM, Sahelebrao BK (2009) Phyto syn- thesis of silver nanoparticles using Glir- icidia sepium (Jaeq). Current Nanosci. 5: 117–122. 29. Vivek M, Kumar PS, Steffi S, Sudha S (2011) Biogenic silver nanoparticles by J Arthropod-Borne Dis, June 2020, 14(2): 162–172 M Waris et al.: Evaluation of … 172 http://jad.tums.ac.ir Published Online: June 30, 2020 Gelidiella acerosa extract and their an- tifungal effects. Avicenna J Medical Bi- otech. 3: 143–148. 30. Barraud PJ (1934) The Fauna of British India, including Ceylon and Burma. Dip- tera family Culicidae.Tribe Megarhinini and Culicini. Taylor Francis, London, pp. 463. 31. Qasim M, Naeem M, Bodlah I (2014) Mos- quito (Diptera: Culicidae) of Murree Hills, Punjab, Pakistan. Pak J Zool. 46: 523– 529. 32. Akram W, Khan HAA, Hafeez F, Bilal H, Kim YK, Lee JJ (2010) Potential of cit- rus seed extracts against dengue fever mosquito, Aedes albopictus (Skuse) (Cu- licdae: Dipetra). Pak J Bot. 42: 3343– 3348. 33. Arivoli S, Ravindran KJ, Raveen R, Ten- nyson S (2012) Larvicidal activity of botanicals against the filarial vector Cu- lex quinquefasciatus Say (Diptera: Cu- licidae). Int J Zool Res. 2: 13–17. 34. Murthy JM, Rani PU (2009) Biological activity of certain botanical extracts as larvicides against the yellow fever mos- quito, Aedes aegypti. J Biopest. 2: 72–76. 35. Soni S, Prakash S (2013) Possible mos- quito control by silver nanoparticles syn- thesized by soil fungus (Aspergillus niger). Adv Nanoparticles. 2: 125–132. 36. Abbott WS (1925) A method of compu- ting the effectiveness of insecticide. J Econ Entomol.18: 265–267. 37. Ahmed S, Ahmad M, Swami BL, Ikram S (2016) A review on plants extract medi- ated synthesis of silver nanoparticles for antimicrobial applications: a green ex- pertise. J Adv Res. 7: 17–28. 38. Jinu U, Rajakumaran S, Senthil-Nathan S, Geetha N, Venkatachalam P (2018) Po- tential larvicidal activity of silver nano- hybrids synthesized using leaf extracts of Cleistanthus collinus (Roxb.) Benth. ex Hook.f. and Strychnos nuxvomica L. nuxvomica against dengue, Chikungu- nya and Zika vectors. Physiol Mol Plant Pathol. 101: 163–171. 39. Karthikeyan AP, Kadarkarai M, Chel- lasamy P (2012) Biolarvicidal and pupi- cidal potential of silver nanoparticles synthesized using Euphorbia hirta against Anopheles stephensi Liston (Diptera: Cu- licidae) Parasitol Res. 11: 997–1006. 40. Hemant PB, Chandrashekhar DP, Rahul BS (2013) Phyto-synthesized silver na- noparticles: a potent mosquito biolarvi- cidal agent. J Nanomed Biotherapeutic Dis. 3: 1. 41. Elimam AM, Elmalik KH, Ali FS (2009) Larvicidal, adult emergence inhibition and oviposition deterrent effects of foli- age extract from Ricinus communis L. against Anopheles arabiensis and Culex quinquefasciatus in Sudan. Trop Bio- med. 26(2): 130–139. 42. Basheer AGM (2014) Ricinus communis (CASTOR) as larvicide on Anopheles arabiensis Patton. Int J Adv Pha Biol Chem. 3: 2277–4688. 43. Mandal S (2010) Exploration of larvicidal and adult emergence inhibition activi- ties of Ricinus communis seed extract against three potential mosquito vectors in Kolkata, India. Asian Pac J Trop Med. 3(8): 605–609.