Evaluation of Larvicidal Efficacy of Ricinus communis (Castor) Plant Extract and Synthesized Green Silver Nanoparticles against Aedes albopictus

Background: Aedes mosquitoes are the most important group of vectors having ability of transmitting pathogens including 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 ecofriendly control of these vectors. Methods: In this study an aqueous leaves extract of Ricinus communis (castor) and silver nanoparticles (AgNPs) synthesized from this extract were evaluated as larvicidal agent for 2nd and 3rd instar larvae of the Aedes albopictus. Different 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 spectroscopy (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 2nd instar larvae and LC50 (84.98ppm) and LC90 (163.89ppm) for 3rd instar larvae as compared to the plant extract (149.58ppm, 268.93ppm) and (155.58ppm, 279.93ppm) for 2nd and 3rd 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.


Introduction
Mosquitoes cause a serious threat to public health (1)(2). Vector-borne diseases such as malaria, dengue, chikungunya, Zika virus, Japanese encephalitis, filariasis, are being spread by mosquitoes (3). These diseases are found all over the world and cause millions of deaths annually (4). Pakistan is at the great risk of vectorborne diseases especially dengue due to it's over crowded cities, insanitation and poor vaccination. In Pakistan, dengue cases are reported 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 Khyber 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 occur in Pakistan (8).
The easy solution to avoid mosquito-borne diseases is the management of mosquito population. This management through chemicals causes health risks to human beings, environmental 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 alternate for this purpose because these plants have many types of target specific, rapidly biodegradable, ecofriendly, and less toxic to human health larvicidal phytochemicals such as saponins, isoflavonoids, tannins, terpenes, steroids, etc. (10)(11). Plants are good source of bioactive insecticidal phytochemicals that can kill mosquito larvae with high mortality rate (12)(13)(14) by bringing changes in development, midgut epithelium (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 mosquitoes (18).
Biologists have begun the use these phytochemicals as larvicides to control the mosquitoes (19). One step ahead, green silver nanoparticles (AgNPs) synthesized from plant extracts are proved more toxic than phytochemical as larvicides (20). Green silver nanoparticles (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 concentrations. These have been tested in a variety of entomological research because these are safe, low cost, easily available and have a simple easy biosynthesis process (21)(22). Ricinus communis (castor) plant belongs to a big family Euphorbiaceae contains nearly about 300 genera and 7500 species. Ricinus communis (castor) is a flowering plant, has high medicinal value for healthy human life. This plant is used as laxative, fungicide, anti-oxidant, antiasthmatic, antiulcer, wound healing, insecticidal and larvicidal agent. It has important phytochemicals like glycosides, alkaloids, flavonoids, steroids and saponins. that are helpful in controlling 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 extract of R. communis (castor) against 2 nd and 3 rd larvae of Ae. albopictus under laboratory conditions. UV-Vis spectroscopy analysis, Powdered X-ray diffraction (PXRD) and Fourier Transform Infrared Radiation (FTIR) spec-troscopy were used to confirm the biosynthesis of AgNPs.

Preparation of leaf extract
Healthy and fresh leaves of the R. communis (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 remove 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 apparatus 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 purchased 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 bottle at 4 °C, then centrifuged for three times at 5000rpm for 20 minutes to obtain pellets. Purified suspension was made by dissolving pellets 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-lysis, Powdered X-ray diffraction (PXRD) and Fourier Transform Infrared Radiation (FTIR) spectroscopy assistance through High Tech central laboratory of Government College University 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 spectrophotometer (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 silver nanoparticles was calculated through diffracted intensities at 40kV voltage and 30mA current with the range of 0°-80° 2θ in CuKα radiation (Rigaku, Ultima IV, and X-ray diffractometer system).

Fourier Transform Infrared (FTIR) Spectroscopy
The residue solution of 100ml was centrifuged at 5,000rpm for 10 minutes after reaction to remove impurities. To obtain pure pellet of AgNPs the supernatant was again centrifuged 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 dippers 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 further reared in separate glass cages. Male adults were fed with 10% sugar solution and females with blood on live white rats in separate 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 bioassays (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 synthesized from the extract were prepared using distilled water and subjected for mortality assays 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 water and five replications were done for each treatment. Mortality rates were calculated using 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 subjected to probit analysis using Minitab -17 statistical software (2017) for calculating lethal concentration 50% (LC50) and 90% (LC90) of larvae and for getting dose and time mortality regression lines.

Synthesis of Silver Nanoparticles
Formation of green AgNPs through the reduction of the silver metal ions by the extract of R. communis that turned the colour of mixture (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 UVvisible spectra of silver nanoparticles in aqueous solution with different reaction times that are shown (Fig. 3). It was observed from the figure that localized surface plasmon resonance band showed maximum absorbance at 430nm after 30 minutes of reaction time.

Fourier Transform Infrared Radiation Spectroscopy (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 nanoparticles indicated by these peaks. These residues 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 stabilization of AgNPs.

Larvicidal activity of leaf extracts and synthesized silver nanoparticles
The results of the larvicidal activity of leaf extract of R. communis (Castor) and synthesized 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 ( The mortality rate of 2 nd and 3 rd instar larvae of Ae. albopictus was noted as 98 and 96% respectively after 48H at 250ppm concentration of R. communis leaves extract (Table 2). The least values of LC50 and LC90 were 149.57 and 268.92ppm for 2 nd instar larvae 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 concentrations of plant extracts used in the present study exhibited repellency activity.  LC50: lethal concentration that kills 50% of the exposed larvae; LC90: lethal concentration that kills 90% of the exposed larvae.  LC50: lethal concentration that kills 50% of the exposed larvae; LC90: lethal concentration that kills 90% of the exposed larvae http://jad.tums.ac.ir Published Online: June 30, 2020

Discussion
Nanotechnology is an emerging technology in modern era that enables scientists to synthesize particles of different sizes, forms and components. Hence synthesized nanoparticles of gold, silver and platinum are being used for insect vectors control and in pharmaceutical industries (17). During current study, change in colour (reddish brown) of the solution indicated the formation of AgNPs due to the reduction of silver metal ions by the extract of R. communis and was confirmed by the localized surface plasmon resonance band absorbance at 430nm after 30 minutes of reaction time. Previous studies also indicated the similar colour changes and maximum absorbance at 430nm (37) (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 carbonyl group formed amino acid residues which capped the silver nanoparticles indicated by these peaks. These residues 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 stabilization of AgNPs (37)(38)(39)(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 indicated 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 Euphorbia 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 different biological and catalytic activities in them (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) 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 potent than AgNPs. The larvicidal effects of leaves extracts of R. communis showed the LC50 values 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 solvents 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, larval stage and solvent for plant extraction.
Results of our study suggested that the leaf extract of R. communis is toxic to Ae. albopictus 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 nanoparticles of R. communis had excellent potential for killing the of mosquito larvae. The application of this plant extract along with silver nanoparticle on mosquito breeding places surely decrease the population of vector mosquitoes, control many dreadful diseases and prevent environmental pollution.