Ipomoea batatas (Convolvulaceae)-mediated synthesis of silver nanoparticles for controlling mosquito vectors of Aedes albopictus, Anopheles stephensi, and Culex quinquefasciatus (Diptera:Culicidae)

Abstract

We proposed an effective and eco-friendly control of dengue, malaria, and filariasis-causing vectors. We tested Ipomoea batatas leaves-mediated silver nanoparticles (AgNPs) against first to fourth instar larvae and adults of Aedes albopictus, Anopheles stephensi, and Culex quinquefasciatus at different concentrations. The synthesized AgNPs showed broad spectrum of larvicidal and adulticidal effects after 48 h of exposure. The characterization of synthesized AgNPs was done using various spectral and microscopy analyses. The maximum efficacy was observed in synthesized AgNPs against the adult of Ae. albopictus with the LC₅₀ and LC₉₀ values were 10.069 and 15.657 μg/mL, respectively, followed by others.

Keywords:

• Ipomoea batatas
• larvicide
• adulticidal
• spectral analysis

Introduction

Mosquitoes act as a vector for most of the life-threatening diseases like malaria, dengue, chikungunya, filariasis, encephalitis, West Nile virus infection, etc., in almost all tropical and subtropical countries and many parts of the world (Kager 2002). Mosquitoes are cause allergic responses in human that include local skin and systemic reactions (Peng et al. 1999). Mosquitoes transmit several human diseases, causing millions of deaths every year. India reports 1.4 million malarial cases and about 173 deaths; 1.4 million suspected and 11,985 confirmed chikungunya cases; 5000 Japanese encephalitis cases and approximately 1000 deaths; and 383 dengue cases and 6 deaths during 2006 and 2007 (Kovendan et al. 2012; Pavela and Benelli 2016). Mosquito-borne diseases are prevalent in more than 100 countries across the world and infecting over 70 crore peoples every year globally and 4 crore of the Indian populations.

Aedes albopictus is a small, dark mosquito with a white dorsal stripe and banded legs. They are strongly attracted to bite humans, cats, dogs, squirrels, deer, and other mammals, as well as birds. They will bite any exposed skin surface. Ae. albopictus is a very aggressive daytime biter (Benelli et al. 2015a; Marcondes and Ximenes 2015). Its peak feeding times are during the early morning and late afternoon, and its considered to be a vector of dengue fever, a disease endemic to Southeast Asia, Africa, and America (Benelli et al. 2015c; Maillard et al. 1993; Vasilakis et al. 2007). More than 50 million peoples are at risk of dengue virus exposure worldwide. Annually, there are 5 million cases of dengue, 2 million infections, and 12,000 deaths (Kovendan et al. 2012). Dengue is the most significant mosquito-spread viral disease and a major international public health concern. It is a self-limiting disease found in tropical and subtropical regions around the world, predominantly in urban and semi-urban areas. DF or DHF is caused by dengue virus which belongs to genus Flavivirus, family Flaviviridae, and includes serotypes 1, 2, 3, and 4 (Den-1, Den-2, Den-3, and Den-4) (Benelli 2016b; Benelli and Mehlhorn 2016c; WHO 2010). The WHO estimates that around 2.5 billion people are at risk of dengue. Infections have dramatically increased in recent decades due to increased urbanization, trade, and travel. No effective drug or vaccine is available so far. The only solution is to prevent the disease carrying mosquito from breeding and biting to humans.

Anopheles species are the most important species as they are a capable vector for malaria parasites. Anopheles stephensi is responsible for the transmission of malaria in urban regions of India (Benelli and Mehlhorn 2016c; Rahman et al. 1989). In India, malaria is still the most important cause of morbidity and mortality with approximately 2–3 million new cases rising every year (Sharma et al. 2009). It begins with a bite from an infected female Anopheles mosquito, which introduces the protists through saliva into the circulatory system. Malaria causes symptoms that typically include fever and headache, which in severe cases can progress to coma or death. About 3.3 billion people – half of the world’s population – are at risk of malaria. In 2010, about 216 million malaria cases (with an uncertainty range of 149–274 million) and estimated 655,000 malaria deaths (with an uncertainty range of 537,000–907,000 occurred in World Wide). Increased prevention and control measures have led to a reduction in malaria mortality rates by more than 25% globally since 2000 and by 33% in the WHO African Region (WHO 2012b). Culex quinquefasciatus, a vector of lymphatic filariasis, is widely distributed in tropical Zones with around 120 million people infected worldwide and 44 million people having common chronic manifestation (Benelli et al. 2015a; Bernhard et al. 2003). Culex mosquitoes are painful and persistent biters and are responsible for filariasis (Conti et al. 2010). Lymphatic filariasis is a neglected tropical disease. More than 1.3 billion people in 72 countries worldwide are threatened by lymphatic filariasis, commonly known as elephantiasis. Over 120 million people are currently infected, with about 40 million disfigured and incapacitated by the disease (WHO 2012a, 2012b). It is a potential vector of bancroftian filariasis and fed on microfilaremia carriers harboured Wuchereria bancrofti larvae, found in waste water disposal systems, and irrigated sites (Mukhtar et al. 2003).

Mosquito larvicide, nowadays, includes an organophosphate temephos, methoprene, phytochemicals, soil bacterium, and fungal species. However, the high amount of synthetic chemical larvicides could lead to long-term residual effect to the environment and chronic effects on non-target organisms. Mosquito control is being strengthened in many areas, yet there are significant challenges, including increased mosquito resistance to insecticides and a lack of alternate, cost-effective, and safe insecticides. Nanoparticles play an important role in pharmaceutical, industrial, and biotechnological applications like a drug delivery, diagnostics, image sensing, artificial implants, and tissue engineering (Mishra et al. 2013). Nowadays metallic nanoparticles are mostly prepared from noble metals viz., platinum (Pt), gold (Au), silver (Ag), Zinc (Zn), Copper (Cu), and lead (Pb); among those, Ag is the metal of choice in the field of biological system (Kunjiappan et al. 2014). Plant constituents such as phenols, flavonoids, terpenoids, tannins, glycosides, alkaloids, proteins, and steroids are capable of reducing metal ions into nanoparticles in a single-step green synthesis process and ensures excellent control over size distribution and crystallinity of the nanoparticles (Velu et al. 2015).

Green-synthesized silver nanoparticles (AgNPs) have been recently proposed as newer and safer control tools against mosquito vector of medical and veterinary importance (Benelli 2016a; Murugan et al. 2015a). The synthesized AgNPs using aerial extract of Ammannia baccifera (Suman et al. 2013) showed significant toxic effects against the larvae of An. subpictus and Ae .aegypti. Many plant species have been used in nanotechnology for AgNPs production, which were stable and have significant mosquito larvicidal activity against Cx. quinquefasciatus viz: Mimosa pudica (Marimuthu et al. 2010), Tinospora cordifolia (Jayaseelan et al. 2011), Eclipta prostrata (Rajakumar and Rahuman 2011), Nelumbo nucifera (Santhoshkumar et al. 2011), Sida acuta (Veerakumar et al. 2013), Heliotropium indicum (Veerakumar et al. 2014b), and Cinnamomum zeylanicum (Soni and Prakash 2014), respectively.

Ipomoea batatas commonly known as “Sweet potato” is a perennial dicotyledonous plant belonging to the family Convolvulaceae. It is large, starchy sweet-tasting tuberous root as vegetable (FAO 1992). Sweet potato is an important food crop in tropical and sub-tropical countries (Tewe et al. 2003). I. batatas is an herbaceous perennial vine bearing, simple, entire, and alternate heart-shaped or palmately lobed leaves (Gad and George 2009), and it contains protein (0.46% to 2.93%), dietary fiber (0.49% to 4.71%), lipid (0.06% to 0.48%), and ash (0.31% to 1.06%) and essential minerals nutrients such as Ca, P, Mg, Na, K, S, Fe, Cu, Zn, Mn, Al and vitamin A, thiamine, riboflavin, niacin, ascorbic acid, and many other functional compounds (Woolfe 1992). The plant tops used in the manufacture of alcohol, vinegar, lactic acid, yeast, acetone, and young leaves used as baby food particularly in Southeast Asia and East Asia (Ma et al. 2003). The leaves contain cyanide, tannins, oxalate, and phytic acid as antinutrients and a couple of minerals (Igwenyi et al. 2011). Biological activity of I. batatas reported as anti-mutagenic, anti-diabetic (Ijaola et al. 2014), anti-bacterial, anti-fungal (Kumar et al. 2014), anti-inflammatory, anti-oxidant (Hamsa and Kuttan, 2011), cardiovascular (Yamakawa et al. 2007), hypertension (Nagamine et al. 2014), hyperglycemia and diabetes (Yoshimoto et al. 2002), wound healing (Panda and Sonkamble 2012), cytotoxic (Lin et al. 2008; Yu et al. 2013), anti-tumor, Ca2 + antagonist (Pàska et al. 1999), antiulcer (Hermes et al. 2013), cardiovascular (Jabeen and Aslam 2013), hepatoprotective, immunomodulatory, and anticancer activities (Amin et al. 2012), respectively. Considering the above information in mind, the present study AgNPs were synthesized using the I. batatas (Convolvulaceae) leaf extract as reducing and stabilizing agent. The AgNPs were characterized by UV–visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), Energy dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD). The I. batatas leaf aqueous-mediated AgNPs were tested for its larvicidal and adulticidal potential against the An. stephensi (malaria vector), Ae. albopictus (dengue vector), and Cx. quinquefasciatus (Japanese encephalitis vector).

Materials and methods

Collection of plant material

Fresh and healthy leaves of Ipomoea batatas (Convolvulaceae) (Figure 1) were collected from Vettrilaikaranur kattuvalavu, (Latitude 11°00′N, Longitude 78°00′E) Salem, and the taxonomic identification was done by Dr. D. Natarajan, Assistant Professor, Department of Biotechnology, School of Biosciences, Periyar University, Salem. The voucher specimen was numbered and kept in NDRL research laboratory for further reference. Silver nitrate was purchased from Hi-Media chemicals Pvt. Ltd, Mumbai, India.

Figure 1. Ipomoea batatas – a close view.

Preparation of aqueous plant extract

The fresh and healthy plant leaves were washed thoroughly in tap water rinsed in distilled water, dried with paper towel, and shade-dried at room temperature (35 ± 1 °C). These dried leaves were powdered mechanically using electrical mixer. Aqueous extract was prepared by mixing 10 g of dried powder in 100 mL of double-distilled water. This suspension was mixed well and left for 5 h without disturbance, then filtered through Whattman filter paper (No. 1), and the filtrate was used to check the larvicidal and adulticidal activity against the target mosquito vectors (Minjas and Sarda 1986).

Collection of mosquitoes

Different larval instars and pupa of Ae. albopictus, An. stephensi, and Cx. quiquefasciatus were collected from the Centre for Research in Medical Entomology (CRME), Madurai, and were brought to the laboratory without any disturbances. These larvae and pupae were maintained in enamel trays containing deionized water and allowed to feed on brewer’s yeast, and dog biscuits (3:1 ratio) in the laboratory at room temperature for 24 h; before starting of the experiment, the laboratory mosquitoes were maintained according to the modified method of Kamaraj et al. (2009). All the experiments were carried out at 27 ± 2 °C and 75–85% relative humidity under 14:10 light and dark cycles. Pupae were transferred from the trays to a plastic cup (10 × 5 × 3 cm) containing tap water and were maintained in an insectary tray (45 × 45 × 40 cm) where adults emerged. Adults were maintained in plastic cages and were provided with 10% sucrose solution in a jar with a cotton wick. Glass petridish with 50 mL of tap water lined with filter paper was kept inside the cage for oviposition (Lalrotluanga et al. 2012; Rahuman et al. 2007).

Synthesis of silver nanoparticles from leaves extract

Aqueous leaves extract of I. batatas was prepared by placing 10 g of chopped fresh leaves in a 250-mL Erlenmeyer flask and boiled with 100 mL of sterile double-distilled water upto 20 min at 60 °C in a water bath. The aqueous extract was passed through Whatman filter paper (No.1), and the filtrate (aqueous leaf extract) was stored at 4 °C and used within 3 days. About 10 mL of aqueous leaf extract was treated with 90 mL of prepared 1 mM of Ag NO₃ solution incubated in dark at room temperature. The aqueous solution of 1 mM of Ag NO₃ was greatly reduced from Ag⁺to Ag° by aqueous leaf extract leading to change of pale yellow to dark brown indicate the synthesis (Parashar et al. 2009).

Characterizations of green synthesized silver nanoparticles

UV-visible spectroscopy analysis

The bio-reduction of silver ions to silver was monitored by measuring the UV-Visible spectrum of the reaction mixture (silver nitrate + aqueous leaf extract). The samples were diluted with 2 mL of double-distilled water and subsequently analyzed by UV– vis spectroscopy at regular time intervals using a quartz cuvette with water as a reference (Rajesh et al. 2009), and surface plasmon resonance (SPR) of AgNPs was characterized using UV–vis spectrophotometer (Shimadzu 1601 model, Japan) at resolution of 1 nm between 200 and 800 nm (Dash et al. 2014).

X-ray diffraction analysis

To determine the nature and size of the synthesized AgNPs, X-ray diffraction (XRD) was performed. For this, the reaction mixture was centrifuged (20 000 × g, 15 min, 4 °C), and the pellet was dissolved in deionized sterile water and washed thrice in the same by centrifugation. The powder form of the sample was recovered and coated on the XRD grid; the spectra were recorded at a voltage of 40 kV and a current of 30 mA with Cu Kα1 radiation using Philips PW 1050/37 model. The diffracted intensities were recorded from 10° to 90° at 2θ angles. The full-width at half-maximum (FWHM) from three different peaks were used in Scherrer’s equation to determine the average crystallite size of the nanoparticles (Rao and Biswas 2009).

Fourier transform infrared spectroscopy

Dried metallic powder of the AgNPs was subjected to analyze the presence of possible biofunctional groups for the reduction of Ag + ions resulting in formation of AgNPs. The solid residue containing AgNP solution was dispersed in sterile deionized water for three times to remove the unattached biological impurities. The pure residue was oven dried in overnight at 60 °C. The obtained powder was subjected to FTIR measurement using a PerkinElmer Spectrum one instrumental resolution of 4 cm⁻¹ in KBr pellets (Vivek et al. 2011).

Scanning electron microscope

The particle size and microstructure were deliberate by high resolution SEM (instrument from Nikon, Japan) (Chattopadhyay et al. 2013a). In brief, AgNPs were suspended in deionized water at a concentration of 1 mg/mL and then sonicated using a sonicator bath until the sample form a homogenous suspension. For size measurement, the sonicated stock solution of AgNPs (1 mg/mL) was diluted 20 times. Then, one drop of sonicated aqueous solution was taken on a glass plate and allowed to dry overnight at room temperature under a sterilized condition. Then, the sample was gold coated and images were taken. SEM was used to characterize the size and shape of AgNPs from samples.

Transmission electron microscope

The size and structure of synthesized AgNPs were characterized by TEM in a JEOL 3010, Japan, operating at 200 kV according to the modified method of Chattopadhyay et al. (2012). In brief, AgNPs were suspended in deionized water at a concentration of 1 mg/mL. Then, the sample was sonicated using a sonicator bath until sample form a homogenous suspension. For size measurement, sonicated stock solution of all AgNPs (0.5 mg/mL) was diluted for 10 times. TEM was used to characterize the size and shape of the AgNPs. A drop of the aqueous AgNPs suspension to make a thin layer on to carbon-coated copper grid (300 mesh size) by slow evaporation and then allowed to dry in vacuum at 25 °C overnight to obtain TEM images.

Energy dispersive X-ray spectroscopy

This technique determines the elemental composition of a sample. In this study, EDX used to confirm the presence of silver in the particles as well as to detect the other elemental compositions of the synthesized silver particles. The particle solution was diluted 100-fold in water, and a drop of 10-μL diluted solution was placed on a carbon stub and air-dried. The EDX spectrum was obtained at an acceleration voltage of 20 kV and collected for 19 s. Mapping was completed using pseudo-colors to represent the two-dimensional spatial distribution of energy emissions of the chemical elements present in the sample. This analysis was done using JEOL JSM 6360 equipped with an EDX (energy dispersive X-ray) analyzer (Majumdar et al. 2013).

Larvicidal activity

The larvicidal activity of green synthesized AgNPs was evaluated using WHO protocol (2005) with slight modifications. To test different concentrations of synthesized AgNPs in 200 mL capacity autoclaved Borosil glass beakers, bioefficacy test was conducted against the larvae of target vector at five different concentrations of synthesized AgNPs (25, 50, 75, 100, and 125 μg/mL). To find out the larvicidal activity of synthesized AgNPs, 20 larvae were exposed to each test at different concentrations of extracts. Similarly, each test included a set of control group (Distilled water) with five replicates for each individual concentration of extract. Mortality rate was recorded after 48 h of exposure period. The dead larvae in five replicates were combined and expressed as a percentage of larval mortality for each concentration. The corrected mortality was calculated by Abbott’s formula (Abbott 1925): $\begin{array}{c}\multicolumn{1}{c}{\text{Corrected mortality}\hspace{1em}}\\ \multicolumn{1}{c}{\mathrm{\hspace{1em}\hspace{1em}\hspace{1em}}=\hspace{1em}\frac{\text{Observed mortality in treatment}-\text{Observed mortality in control}}{100-\mathrm{Control}\mathrm{mortality}}\hspace{1em}\times 100}\end{array}$ $\text{Percentage mortality}\hspace{1em}=\hspace{1em}\frac{\text{Number of dead larvae}}{\text{Number of larvae introduced}}$

Dose-response bioassay

Synthesized AgNPs were subjected to a dose-response bioassay for larvicidal activity against Ae. albopictus, An. stephensi, and Cx. quinquefaciatus. Different concentrations (25, 50, 75, 100, and 125 μg/mL) of synthesized AgNPs were prepared for larvicidal activity. The numbers of dead larvae were counted after 48 h of exposure, and the percent mortality was calculated from the average of five replicates. However, at the end of 48 h, the selected test sample turned out to be equal in their toxic potential; this assay was performed as per the modified method of Selvaraj Mohana Roopan et al. (2013).

Adulticidal activity

Adulticidal bioassay of samples was performed by the modified WHO protocol (1981). Based on the wide range and narrow range tests, and AgNPs were tested at 100, 200, 300, 400, 500, and 600 μg/mL concentrations (Veerakumar and Govindarajan 2014a). AgNPs were applied on Whatmann no. 1 filter paper (size 12 × 15 cm). Control papers were treated with silver nitrate and distilled water. Twenty mosquitoes were collected and gently transferred into a glass holding tube. The mosquitoes were allowed to acclimatize in the holding tube for1 h, and then exposed to test paper for 1 h. At the end of exposure period, the mosquitoes were transferred back to the holding tube and kept for 24-h recovery period. A pad of cotton soaked with 10% glucose solution was placed on the mesh screen. Each test includes a set control groups (distilled water) with five replicates for each individual concentration. The lethal concentrations [lethal dose (LD₅₀ and LD₉₀)] were calculated by probit analysis (Finney 1971).

Statistical analysis

The average larvae and adult mortality data were subjected to probit analysis for calculating LD₅₀, LD₉₀, and other statistics at 95% confidence limits of upper and lower confidence limits, and chi-square values and data were calculated using the Statistical Package of Social Sciences IBM 20.0 software (SPSS Inc., Chicago, IL). Results with P < .05 were considered to be statistically significant.

Results

Synthesis and characterizations of I. Batatas-mediated AgNPs

The color change was noted by visual observation in the I. batatas leaf aqueous extract when incubated with AgNO₃ solution. I. batatas leaf extract without added AgNO₃ did not show any change in color. The aqueous extract mixed with silver nitrate changed to light brown within an hour, and later, it was changed into dark brown during 24-h incubation period, after which no significant changes occurred (Figure 2(A,B)). The absorption spectrum of I. batatas leaf extract at different wave lengths ranging from 200 to 800 nm revealed a peak at 410 nm (Figure 2(C)). XRD spectrum of I. batatas leaf extract reduced AgNPs and revealed the five distinct diffraction peaks observed in the spectra at 2θ values of 27.8°, 32.22°, 46.22°, 54.8°, and 76.6° attributed to (111), (200), (202), (211), (220), and (311) of the cubic silver (Figure 3). The lattice constant calculated from the diffraction spectrum was a = 4100 Å, and the resultant data were coordinated with the database Joint Committee on Powder Diffraction Standards (JCPDS) file no. 01-087-0717. The resultant XRD spectrum clearly suggests that the AgNPs synthesized from the leaf aqueous extract of I. batatas was crystalline in nature. The average crystal size of AgNPs was calculated by applying Scherrer’s equation and found to be the size of 29 nm.

Figure 2. Formation of AgNPs from I. batatas A: AgNO₃ solution, B: AgNO₃+ I. batatas leaf extract at 0 h, C: AgNPs from I. batatas at 24 h. (Dark purple coloration indicates the formation of AgNPs).

Figure 3. XRD spectrum of AgNPs synthesized from leaf extract of I. batatas.

FTIR has become a significant tool in understanding the involvement of functional groups in relation linkage of metal particles and biomolecules. In the current study, FTIR measurements were performed to identify the promising functional group of biomolecules responsible for capping and stabilizing the AgNPs. FTIR spectral peaks suggest the occurrence of bands relevant to alcohols OH stretching (3966.48 cm⁻¹), A weak band appears around 3771.09 cm⁻¹ may be due to the stretching frequency of the O-H bond possibly arising from the protein present in the sample. The weak bands at 3319.28 cm⁻¹ may be assigned due to N-H stretching of the primary amines which possibly arise from the sugars and protein present in the extract. A peak around 2923.14 cm⁻¹ can be assigned to the alkanes C-H stretching. The prominent peak around 2048.36 cm⁻¹ alkynes could be attributed to a peak responsible for reduction of silver nitrate into silver metal. The peaks at 1639.90 cm⁻¹ may correspond to carbonyls correlating to proteins. The peaks at 1243.69 cm⁻¹ can be attributed to the C-N stretching frequency arising from the aliphatic amines. The absorption peak at 1079.00 cm⁻¹ may be due to carboxylic acids strong bonds. The bands at 764.31 cm⁻¹ indicate the presence of N-H stretching primary amine. A peak was observed around 705.34 cm⁻¹ that could be assigned to C-H stretching vibration of alkane (Table 1; Figure 4). After reduction, the AgNPs were precipitated at the bottom of conical flask. This precipitate was washed out twice with sterile double-distilled water and then analyzed by employing SEM. According to the SEM analysis, the morphology of the AgNPs was observed in spherical shape and aggregate form (Figure 5). The samples of AgNPs synthesized using I. batatas liquids were prepared by placing a drop of reaction mixture over copper grid and allowing water to evaporate in sterile condition. TEM highlighted the presence of spherical and oval shapes of AgNPs, with 20–50 nm-sized AgNPs were observed (Figure 6). Energy dispersive x-ray spectroscopy (EDX) results show silver (Ag) signal arise from the Ag nanoparticles, and the atomic percentage of silver is 58.44%. Except for Ag, there are some other peaks observed in EDX. The atomic percentage of Carbon (C), Chlorine (Cl), and Copper (Cu) are 6.79%, 1.63%, and 34.76%, respectively. The carbon (C) signal obtained from the adsorbed components of the leaf extract as well as coating material of the instrument. Except copper, other elements have a very low atomic percentages compared to silver, and suggests the formation of pure AgNPs (Figure 7).

Figure 4. FT-IR spectrum of synthesized AgNPs from aqueous leaf extract of I. batatas.

Figure 5. Scanning electron microscopic observation of synthesized AgNPs from I. batatas.

Figure 6. TEM image of AgNPs synthesized by I. batatas leaf extract. (Scale bars, 20 and 50 nm).

Figure 7. Energy dispersive X-ray (EDX) spectrum of AgNPs from I. batatas.

Table 1. FTIR spectrum of I. batatas leaf extract.

S. No	Observed wave’s number cm^−1	Functional groups	Bonding pattern
1.	3966.48	Alcohol (strong, sharp)	O–H stretch free hydroxyl
2.	3771.09	Phenols (strong, sharp)	O–H stretch free hydroxyl
3.	3319.28	Primary amines (medium)	N–H stretch
4.	2923.14	Alkanes (medium)	C–H stretch
5.	2048.36	Alkynes (weak)	–C = C– stretch
6.	1639.90	Carbonyls (general, strong)	C = O stretch
7.	1243.69	Aliphatic amines (medium)	C–N stretch
8.	1152.69	Alkyl halides	C–H Wagging
9.	1079.00	Carboxylic acids (strong)	O–H
10.	764.31	Primary amines (medium)	N–H stretch
11.	705.34	Alkanes (broad, medium)	C–H stretch
12.	615.53	Alkyl halides (medium)	C–Br stretch

The larvicidal bioassay of I. batatas synthesized AgNPs was observed at various concentrations against the first to fourth-instar larvae of the mosquito vectors Ae. albopictus, An. stephensi, and Cx. quinquefasciatus. Considerable mortality was evident after the treatment of I. batatas synthesized AgNPs for all larval instars. It was observed that Cx. quinquefasciatus is the most susceptible strain followed by An. stephensi and Ae. albopictus, respectively. The mortality rate was increased based on the concentration of extract; for example, in the An. stephensi first-instar stage at 25 μg/mL concentration, the larval mortality was 34.3% whereas at 125 μg/mL concentration, it was increased to 81.0%. The LC₅₀ and LC₉₀ values were represented as follows: LC₅₀ 4.03, 4.305, 3.490, 3.16; LC₉₀ 6.42, 6.794, 5.84, and 5.192 μg/mL, respectively (Table 2). A similar observation has been noted in Ae. albopictus; the mortality rate was totally concentration dependent. In first-instar stage at 25 μg/mL concentration, the larval mortality was 29.0%, whereas at 125 μg/mL concentration, it was increased to 74.0%. Similarly, fourth-instar larvae stage at 25 μg/mL concentration, the larval mortality was 34.6%, while at 125 μg/mL concentration, it was improved to 82.3%. The LC₅₀ and LC₉₀ values were presented as follows: LC₅₀ value of the first instar of Ae. albopictus, i.e. LC₅₀ 8.62, 5.95, 6.40, 4.74; LC₉₀ 13.52, 9.27, 9.80, 7.46 μg/mL (Table 3). In the control group, no mortality could be recorded. The larvae of Cx. quinquefasciatus were found highly susceptible to the synthesized AgNPs. The time-dependant effect shows the maximum mortality (>85%) at 125 μg/mL against fourth-instar larvae of Cx. quinquefasciatus. At higher concentration, mortality starts within the first 18 h of exposure. More than 50% mortality occurs within the first 24 h. The LC₅₀ and LC₉₀ values were represented as follows: Cx. quinquefasciatus LC₅₀ 7.55, 8.02, 5.63, 6.31; LC₉₀ 11.77, 12.03, 8.60, and 9.04 μg/mL, respectively (Table 4). The decreasing concentrations of AgNPs (after 48 h) of the lived larvae of mosquitoes were pupated or emerged as adults before they died. The results of larvicidal activity clearly show that the percentage of mortality being directly proportional to the concentration of the AgNPs. The outcome of study proves that concentration of extract plays a significant role in larvicidal activity.

Table 2. I. batatas AgNPs against different larvae stages of An. stephensi.

Mosquito name	Larva stages	Concentrationsof AgNPs (μg/mL)	48-h mortality(%), ±SD^a	LC50 (μg/mL)(LCL–UCL)	LC90 (μg/mL)(LCL–UCL)	χ^2 (df = 5)
An. stephensi	First instar	Control	0.0 ± 0.0
		25	34.3 ± 2.0
		50	48.0 ± 1.0	4.030 (0.853–8.510)	6.420 (1.751–12.044)	3.179
		75	56.3 ± 1.5			n.s
		100	67.6 ± 1.5
		125	81.0 ± 1.0
	Second instar	Control	0.0 ± 0.0
		25	33.0 ± 1.0
		50	53.0 ± 1.0	4.305 (0.981–8.860)	6.794 (1.972–12.472)	5.800
		75	61.0 ± 1.0			n.s
		100	76.0 ± 1.0
		125	85.6 ± 1.5
	Third instar	Control	0.0 ± 0.0
		25	34.3 ± 2.0
		50	41.0 ± 1.0	3.490 (0.517–8.123)	5.848 (1.205–11.820)	5.266
		75	54.3 ± 1.5			n.s
		100	65.3 ± 1.5
		125	86.3 ± 1.5
	Fourth instar	Control	0.0 ± 0.0
		25	35.0 ± 1.0
		50	47.6 ± 1.5		5.192 (1.100–10.587)	7.429
		75	61.6 ± 1.5	3.161(0.497–7.357)		n.s
		100	68.3 ± 1.5
		125	89.0 ± 1.0

Control (deionized water) – nil mortality. ^aValues are mean ± SD of five replicates.

LCL: lower confidence limits; UCL: upper confidence limits; χ²: chi-square test; df = degrees of freedom; n.s.: not significant at α = 0.05 level.

Table 3. Larvicidal activity of green synthesized I. batatas AgNPs against different instar larvae of Ae. albopictus.

Mosquito name	Larva stages	Concentrations ofAgNPs (μg/mL)	48-h mortality(%), ±SD^a	LC50 (μg/mL)(LCL–UCL)	LC90 (μg/mL)(LCL–UCL)	χ^2 (df = 5)
Ae. albopictus	First instar	Control	0.0 ± 0.0
		25	29.0 ± 1.0
		50	36.3 ± 1.0	8.629 (2.616–15.331)	13.520 (5.242–21.469)	3.416
		75	44.0 ± 1.0			n.s
		100	67.6 ± 2.5
		125	74.0 ± 1.0
	Second instar	Control	0.0 ± 0.0
		25	33.0 ± 1.0
		50	41.3 ± 1.5
		75	52.0 ± 2.0	5.959 (1.662–11.253)	9.274 (3.244–15.718)	3.103
		100	61.6 ± 1.5			n.s
		125	71.6 ± 1.5
	Third instar	Control	0.0 ± 0.0
		25	32.3 ± 1.5
		50	43.0 ± 1.0
		75	52.3 ± 1.2	6.409 (2.006–11.646)	9.801 (3.760–16.108)	8.683
		100	66.0 ± 2.6			n.s
		125	77.3 ± 1.5
	Fourth instar	Control	0.0 ± 0.0
		25	34.6 ± 1.5
		50	46.6 ± 1.5	4.749 (1.158–9.506)	7.465 (2.306–13.353)	7.005
		75	61.0 ± 1.7			n.s
		100	76.0 ± 1.0
		125	82.3 ± 2.5

Control (deionized water) – nil mortality. ^aValues are mean ± SD of five replicates.

LCL: lower confidence limits; UCL: upper confidence limits; χ²: chi-square test; df = degrees of freedom; n.s.: not significant at α = 0.05 level.

Table 4. I. batatas AgNPs against different larvae stages of Cx. quinquefasciatus.

Mosquito name	Larva stages	Concentrations ofAgNPs (μg/mL)	48-h mortality(%), ±SD^a	LC50 (μg/mL)(LCL–UCL)	LC90 (μg/mL)(LCL–UCL)	χ^2 (df = 5)
Cx. quinquefasciatus	First instar	Control	0.0 ± 0.0
		25	27.6 ± 0.5
		50	35.3 ± 1.1	7.559 (2.281–(13.643)	11.772 (4.478–19.054)	4.944
		75	47.0 ± 1.0			n.s
		100	60.0 ± 1.0
		125	73.6 ± 2.0
	Second instar	Control	0.0 ± 0.0
		25	32.3 ± 0.5
		50	35.0 ± 1.0	8.027 (2.915–13.694)	12.037 (5.256–18.715)	8.198
		75	47.6 ± 0.5			n.s
		100	64.0 ± 3.6
		125	77.6 ± 0.5
	Third instar	Control	0.0 ± 0.0
		25	35.3 ± 1.1
		50	47.3 ± 0.5
		75	52.6 ± 0.5	5.637 (1.681–10.514)	8.605 (3.140–14.520)	2.731
		100	75.0 ± 2.6			n.s
		125	83 ± 4.3
	Fourth instar	Control	0.0 ± 0.0
		25	38.0 ± 1.0
		50	54.0 ± 1.0
		75	64 ± 1.0	6.317 (2.505–10.676)	9.047 (4.122–14.173)	4.790
		100	85.0 ± 4.5			n.s
		125	90.0 ± 2.6

Control (deionized water) – nil mortality. ^aValues are mean ± SD of five replicates.

LCL: lower confidence limits; UCL: upper confidence limits; χ²: chi-square test; df = degrees of freedom; n.s.: not significant at α = 0.05 level.

The results of adulticidal activity of synthesized AgNPs of I. batatas against the Ae. albopictus, An. stephensi, and Cx. quinquefasciatus adults (shown in Table 5). Among three vectors tested, the highest mortality was observed in followed by Ae. albopictus, An. stephensi, and Cx. quinquefasciatus. At higher concentrations, the adult showed restless movement for some times with irregular wagging and died. The rates of mortality were directly relative to the concentrations. The maximum efficacy was observed in synthesized AgNPs against the adult LC₅₀ and LC₉₀ values were 10.069 and 15.657 μg/mL, Ae. albopictus; 17.578 and 26.040 μg/mL, An. stephensi; 12.568 and 19.510 μg/mL, respectively. No mortality was observed in the control (χ² value was significant at the P < .05 level).

Table 5. Adulticidal activity of I. batatas aqueous leaf extract synthesized AgNPs against Ae. albopictus, An. stephensi, and Cx. quinquefasciatus.

Mosquitoes	Concentrations ofAgNPs (μg/mL)	48-h mortality(%), ±SD^a	LC50 (μg/mL) (LCL–UCL)	LC90 (μg/mL) (LCL–UCL)	χ^2 (df = 5)
Ae. albopictus	Control	0.0 ± 0.0
	100	43.0 ± 2.6
	200	64.6 ± 1.5
	300	81.0 ± 1.5	17.578 (5.227–33.953)	26.040 (9.155–46.056)	19.253
	400	92.0 ± 2.0			n.s
	500	94.0 ± 2.6
	600	98.3 ± 1.5
An. stephensi	Control	0.0 ± 0.0
	100	43.6 ± 1.5
	200	60.3 ± 1.5
	300	73.0 ± 2.0	12.568 (2.624–27.776)	19.510 (5.060–38.752)	5.425
	400	79.0 ± 1.0			n.s
	500	84.0 ± 1.0
	600	94.6 ± 0.5
Cx. quinquefasciatus	Control	0.0 ± 0.0
	100	44.0 ± 1.0
	200	66.0 ± 1.0	10.069 (1.776–23.755)	15.657 (3.468–33.096)	11.108
	300	76.0 ± 1.0			n.s
	400	84.0 ± 1.0
	500	95.0 ± 1.0
	600	99.0 ± 1.0

Control (deionized water) – nil mortality. ^aValues are mean ± SD of five replicates.

LCL: lower confidence limits; UCL: upper confidence limits; χ²: chi-square test; df = degrees of freedom; n.s.: not significant at α = 0.05 level.

Discussion

The nanoparticle synthesis results of AgNPs solution with leaf aqueous extract may be easily observed after bioreduction of Ag⁺was taken into further AgNPs confirmation using UV–Visible absorption spectrum of I. batatas highlighted changes in color intensity. After 24 h, the UV-spectrum had showed a maximum absorption peak at 410 nm. This result is agreement with earlier work using different plant species, such as Aloe vera (Dinesh et al. 2015), Phyllanthus niruri (Suresh et al. 2015), Moringa oleifera (Sujitha et al. 2015), and Caulerpa scalpelliformis (Murugan et al. 2015b). FTIR spectra clearly indicate that the biomolecules particularly proteins present in aqueous leaf extract are responsible for synthesis and stabilization of AgNPs (Dhanasekaran and Thangaraj 2013). The functional groups such as alcohol, amines, amides, alkanes, aliphatic, and halides confirmed their presence in AgNPs, and these are the main classes in most of the functional groups. The similar kind of results were obtained from the present study; very strong broad absorption peaks at 3966.48 cm⁻¹ (O–H stretch and sharp); very strong and sharp peaks at 3771.09 cm⁻¹ (phenols) were observed. The FTIR results indicate that the secondary structure of the proteins is not affect as a consequence of reaction with the Ag + ions or binding with the AgNPs. This result suggests that the biological molecules could probably perform a function for the formation and stabilization of AgNPs in an aqueous solution. It is well known that proteins can bind to AgNPs through free amine groups in the proteins (Gole et al. 2001) and therefore stabilization of the silver by surface-bound protein (Logeswari et al. 2013).

In XRD analysis of plant-mediated synthesized AgNPs showed intense peaks at values of 27.8°, 32.22°, 46.22°, 54.8°, and 76.6°. They indexed the full width half maximum (111), (200), (202), (211), (220), and (311) regarded as an attributive indicator of biosynthesized silver nanocrystallites. The XRD spectrum results of present study agreed with the Bragg’s reflection of silver nanocrystals (Ouda 2014), as well as with other XRD report of AgNPs were done by Suresh et al. (2015). Vivek et al. (2011) observed that the SEM analysis of AgNPs synthesized by the help of Gelidiella acerosa extract having an average mean size of the AgNPs and seems to be spherical in morphology. Similarly, the present results show the scanning electron microscopic observation has confirmed the occurrence of spherical shape nanoparticle in the sample. EDX spectroscopy revealed a strong signal in the Ag region, which confirm the presence of elemental silver. Similarly, Amkamwar et al. (2005) reported the strong signal in AgNPs synthesized by Embilica officinalis.

The present results express that the I. batatas AgNPs were effective against the all larvae stages of selected mosquitoes. Highest mortality rate was observed in Cx. quinquefasciatus, followed by An. stephensi and Ae. albopictus. A dose-dependent effect was observed in the entire investigation and this statement was supported by previous reports that were performed by Amer and Mehlhorn (2006), Bagavan et al. (2009), Subramaniam et al. (2015); Murugan et al. (2016) stated that an increasing number of plant-mediated synthesized AgNPs showed larvicidal toxicity against different mosquito vectors; Marimuthu et al. (2010) studied the larvicidal effect of M. pudica aqueous leaf synthesized AgNPs, showing highest mortality for synthesized AgNPs against the larvae of An. subpictus (LC₅₀ 8.89, 11.82, and 0.69 ppm) and Cx. quinquefasciatus (LC₅₀ 9.51, 13.65, and 1.10 ppm). Srinivasan et al. (2014) reported the larvicidal activity of phyto-synthesized AgNPs of Avicennia marina leaf aqueous extract against Ae. aegypti (LC₅₀ 4.374 and LC₉₀ 4.928 ppm) and An. stephensi (LC₅₀ 7.40 and LC₉₀ 9.865 ppm). The larvicidal activity of synthesized AgNPs of Ficus racemosa was evidence against Cx. quinquefasciatus and Cx. gelidus (LC₅₀ 67.72 and LC₉₀ 63.70 ppm). The better larvicidal activity was observed in AgNPs of Tinospora cordifolia against fourth-instar larvae of An. subpictus and Cx. quinquefasciatus LC₅₀ 6.43 ppm. The AgNPs showed (using Eclipta prostrata) highest efficacy of larvicidal activity against the fourth-instar larvae of Cx. quinquefasciatus (LC₅₀ 27.49 and LC₉₀ 70.38) and An. subpictus (LC₅₀ 27.85 and LC₉₀ 71.45 ppm) (Velayutham et al. 2013). A dose-dependent effect was found as earlier described for other plant-borne compounds (Benelli 2015b). The findings of present study was comparable with the report of Muthukumaran et al. (2015) proved the mosquito larvicidal action of AgNPs fabricated using Gmelina asiatica against Cx. quinquefasciatus (LC₅₀ 27.83 μg/mL), Ae. aegypti (LC₅₀ 25.77 μg/mL), and An. stephensi (LC₅₀ 22.44 μg/mL). Similarly, Govindarajan et al. (2016) who find out the toxicity of Hugonia mystax-synthesized AgNPs exhibited larval toxicity against Cx. quinquefasciatus (LC₅₀ 17.46 μg/mL), Ae. aegypti (LC₅₀ 15.86 μg/mL), and An. stephensi (LC₅₀ 14.45 μg/mL).

The mosquitocidal properties of AgNPs may be due to structural deformation of genetic material, digestive tract enzymes, and generation of reactive oxygen species (Patil et al. 2012) and non-target aquatic species (Benelli 2016a). The extensive larvicidal toxicity against mosquito vectors could be due to the synergistic insecticidal action of AgNPs and capping biomolecules (Rajan et al. 2015). It has been hypothesized that the toxicity of AgNPs against targeted vectors possibly will be attributed to the small size of these nanoparticles, which allows passageway through the insect cuticle and into individual cells where they interfere with molting and disturb other biochemical processes (Arjunan et al. 2012).

The adults emerged from all the treatments were deformed due to inhibition and disturbance of regular physiological and metabolic processes (Murugan et al. 1996). Our results have shown that synthesized AgNPs of I. batatas have significant adulticidal activity against the Cx. quinquefasciatus, Ae. Albopictus, and An. stephensi. The results are comparable with an earlier report by Veerakumar et al. (2014c) who reported that the maximum adultcidal activity observed in Feronia elephantum leaf extract synthesized AgNPs against filariasis, malaria, and dengue vectors with the following lethal dose values of An. stephensi had LD₅₀ and LD₉₀ values of 18.041 and 32.575 μg/mL; Ae. aegypti had LD₅₀ and LD₉₀ values of 20.399 and 37.534 μg/mL; and Cx. quinquefasciatus had LD₅₀ and LD₉₀ values of 21.798 and 39.596 μg/mL, respectively. Similarly, Veerakumar et al. (2014b) reported Heliotropium indicum synthesized AgNPs against the adult of An. stephensi (lethal dose LD₅₀ 26.712 μg/mL; LD₉₀ 49.061 μg/mL), Ae. aegypti (LD₅₀ 29.626 μg/mL; LD₉₀ 54.269 μg/mL), and Cx. quinquefasciatus (LD₅₀ 32.077 μg/mL; LD₉₀ 58.426 μg/mL), respectively. On the other hand, Suresh et al. (2015) investigated that adultcidal activity of AgNPs synthesized using an aqueous extract from Phyllanthus niruri, against Ae. aegypti resulted better LC₅₀ and LC₉₀ values (LC₅₀ 422.29 and LC₉₀ 23.58 ppm/mL).

Conclusions

In conclusion, the synthesized AgNPs were confirmed by analyzing the excitation of SPR using UV–Vis spectrophotometer at 410 nm. The results of the FTIR were used to identify the possible biomolecules like an aliphatic amine and primary amine groups which are responsible for the presence of the enzymes are responsible for the reduction synthesis and stabilization of the silver metal ions. Bio-fabricated AgNPs were mostly revealed in spherical shape; crystalline in nature, with face-centered cubic geometry, and their mean size was 20–50 nm. AgNPs have admirable larvicidal activity against third-instar larvae of three tested mosquito vectors, with LC₅₀ values ranging from 3.49 to 6.40 μg/mL. The maximum efficacy was observed in synthesized AgNPs against the adult of Ae. albopictus LC₅₀ and LC₉₀ values were 10.069 and 15.657 μg/mL; An. stephensi 17.578 and 26.040 μg/mL; Cx. quinquefasciatus 12.568 and 19.510 μg/mL, respectively. Green synthesized AgNPs as promising representative for vector control and we oberved the morphological changes in the cuticular membrane of the fourth-instar larvae due to the toxicity of the AgNPs.

Acknowledgements

The authors would like to thanks Periyar University, Department of Biotechnology, Natural Drug Research Laboratory, Salem, Tamil Nadu, India, for providing the excellent facilities to carry out the work. We acknowledge the support extended by SAIF, IIT Madras for TEM and EDX studies. We wish to acknowledge St Joseph’s College, Trichy for helping FTIR analysis. The XRD analysis of the AgNPs sample was carried out in the Department of Physics, Periyar University, Salem, Tamil Nadu is highly acknowledged.

Disclosure statement

The authors declare that they have no conflict of interests.