Larvicidal Activity of Silver Nanoparticles Synthesized Using Extracts of Ambrosia arborescens ( Asteraceae ) to Control Aedes aegypti L . ( Diptera : Culicidae )

Exact and Natural Sciences Department, Center for Research on Health in Latin America, Pontificia Universidad Católica del Ecuador, Quito, Ecuador Exact and Natural Sciences Department, Laboratory of Nanotechnology, Pontificia Universidad Católica del Ecuador, Quito, Ecuador Department of Chemistry, University of the Balearic Islands, Palma de Mallorca, Balearic Islands, Spain Universidad de las Fuerzas Armadas ESPE, Sangolquı́, Ecuador


Introduction
Over the last decades, climate change, population growth, deforestation, habitat invasion, and insecticide resistance have contributed to the emergence, reemergence, and dispersion of several vector-borne diseases.Among these, those transmitted by the mosquito Aedes aegypti (Diptera: Culicidae), which include dengue fever, yellow fever, chikungunya, and Zika, represent some of the major challenges of public health in a vast region of our planet, affecting the lives of hundreds of millions of people every year [1,2].
Originally native to Africa, Ae. aegypti is a strongly anthropophilic and sinanthropic species which, due to its capacity to thrive in human settlements, has been able to expand its distribution all along the tropical and subtropical regions of the globe [3].Female Ae. aegypti become arbovirus vectors after ingesting an infectious blood meal.Once the pathogen starts being secreted with the insect's saliva, the mosquito becomes infective and will transmit the pathogen every time it blood feeds on a susceptible host, for the rest of its life [4,5].
With the exception of yellow fever, for which an efficient vaccine has been available since the 1940's [6], no vaccine is currently commercially available against the viral diseases transmitted by Ae. aegypti.erefore, prevention of these diseases is mainly achieved through mosquito population control.Traditionally, most control programs have relied heavily on the use of chemical pesticides, targeting both adults and larvae [7,8].Unfortunately, the persistent and in some cases indiscriminate use of these chemicals has resulted in a reduction of their efficacy due to the dramatic emergence of resistant insect populations during the last decades [9].
Larvicides are among the main tools in our arsenal to control mosquito populations.e most widely used larvicides are organophosphates such as temephos, growth inhibitors, and bacterial insecticides such as Bacillus thuringiensis israelensis (Bti) and Bacillus sphaericus [8,10,11].Because larvicides must be applied to either natural or artificial bodies of water, they must be harmless to fish and other nontarget organisms, including humans, potentially associated with these water reservoirs [12].erefore, the development of new biodegradable, eco-friendly, and specific larvicides is of paramount importance for future control strategies.
Larvicides derived from botanical extracts constitute a new and promising category of pesticide, due to their reduced toxicity for nontarget species and low environmental pollution [13].Unlike conventional insecticides, which normally contain one specific active agent, plantderived larvicides usually contain a combination of several chemical compounds that work synergistically, targeting different biological processes and therefore reducing the likelihood of resistance development in their targets [14].An interesting alternative to strictly botanic larvicides is the production of silver nanoparticles (AgNPs) synthesized using plant extracts as reducing, stabilizing, and capping agents [15].
is technology combines the microbicide properties of silver, the insecticidal activity of the selected plant, and a high efficiency due to the favorable surface area to volume ratio due to the small size of the particles (1-100 nm) [16,17].In combination, these characteristics allow AgNPs to achieve their insecticide effect at very low concentrations (often ≥30 mg/L) [14].
Due to its location, geography, and climate, Ecuador is widely regarded as a hotspot for biodiversity [18].Among the large variety of plant species present in this country, at least 34 have been reported to be used as insecticides by local populations [19].Among these, Ambrosia arborescens (Mill.)stands out as a species used by various populations for insecticidal purposes, including the elimination of fleas, lice, flies, and other insects [19]. is plant, commonly known as "marco," "marku," "altamisa," or "artemisia," is a species native to Ecuador that belongs to the family Asteraceae and is found in the Andean region of the country, between 2000 and 3500 m.a.s.l [20].e plant can range in size from a subshrub to a small tree and presents a pungent odor, and its stems contain an aqueous bitter latex [21].
In this context, we attempted to evaluate the potential of A. arborescens as a larvicide against Ae.aegypti, both as an aqueous extract and in combination with AgNPs.

Materials and Methods
2.1.Plant Specimens.Fresh leaves of A. arborescens were collected in a small, family-owned farm in the Calacalí parish (geographic coordinates: 78 °30′55.7″S,00 °00′32.7″O),which is located approximately 17 km north of Ecuador's capital city, Quito, at an altitude of 2980 m.a.s.l. e plot where the plant was growing had not received any kind of pesticide or fertilizer for at least one year previous to our collection date (R. Zambrano, personal communication).
A botanical voucher specimen (Figure 1) was prepared and deposited at the Center for Research on Health in Latin America (CISeAL) as a reference.

Preparation of A. arborescens Extract.
e leaves of A. arborescens were cleaned of any visible debris and dried in shade for seven days at room temperature.Subsequently, they were dried in an oven at 40 °C for 48 hours; dried sample was ground in a mortar.To prepare the extract, 1 g of vegetable material and 20 mL of distilled water were added.
e mixture was placed under constant stirring for one hour at 60 °C ± 1 °C; after this time, the extract was filtered and stored at 4 °C until use.

Synthesis of Silver Nanoparticles.
e best conditions were previously optimized using 1 mM silver nitrate Sigma-Aldrich ® solution.Twenty mL of this solution was taken, and 2 mL of the extract of A. arborescens were added dropwise.Two to three drops of 1% NaOH were added to adjust the pH to 8. is mixture was stirred for 50 minutes at a temperature of 50 °C ± 1 °C.e colloidal solution was cooled and stored in amber containers at 4 °C for further analysis.e nanoparticles were analyzed on a Cary 60 double beam spectrophotometer from Agilent Technologies.e resolution of the equipment was one nm, and the measurement range was from 350 to 800 nm.Dilutions 1 to 10 were made with distilled water from colloidal solutions obtained from the synthesis process.

FTIR Spectroscopy.
Infrared spectroscopy analyses were performed on a Spectrum BX PerkinElmer with ATR Miracle Pike coupling under 4 cm −1 resolution conditions, ranging from 4000 cm −1 to 500 cm −1 , and ten scans per sample.Both the extract and the synthesized nanoparticles were dried in the oven at 60 °C for 24 hours.

STEM Analysis.
e STEM analysis was carried out on a FEG-SEM Tescan Mira 3 device, using a voltage of 30 kV.Nanoparticles were observed in colloidal solution.

SEM-EDX Analysis. SEM analysis was performed in a Phenom
ProX scanning electron microscope equipped with an EDX-detector operating at 10 kV and Prosuite-EDS software.Nanoparticles were read in solid state over a pin of carbon.

Flame Atomic Absorption Spectroscopy for Silver
Quantification.
e silver content was analyzed on a Perki-nElmer AAnalyst 400 atomic absorption spectrophotometer.A flux of 10.0 L/min of air and 2.5 L/min of acetylene were used for the flame.For the reading of the samples, once the performance of the method was verified, and a 1 : 100 dilution with distilled water of the obtained colloidal solutions was made.

Mosquito Rearing.
e strain of Ae. aegypti used for this work was collected in 2015 in the Ecuadorian city of Puerto Francisco de Orellana, Orellana Province, and has since been continuously maintained under standard insectary conditions (28 ± 1 °C temperature, 80 ± 10% relative humidity, and 12 h light/12 h darkness photoperiod) at CISeAL [22].
Eggs were hatched in water that had been previously boiled and allowed to cool down to room temperature.Larvae were fed on finely ground fish food flakes, following the feeding regime developed by Carvalho et al. [23].e larvae were maintained at the aforementioned standard insectary conditions until reaching the 3rd instar, when they were used for experimentation [24].

Larvicidal Assays.
In order to test the larvicidal activity of the aqueous extract and the plant-synthesized AgNPs of A. arborescens, we followed the protocol proposed by the WHO [24].Briefly, each bioassay consisted of a set of four experimental groups (one for each concentration tested) and a control group.Within each one of these groups, the basic testing unit (i.e., technical replicate) was a plastic well containing 25 third instar larvae in either 200 mL of the test solution at the desired concentration (for experimental groups) or distilled water (for control groups).Each group contained four technical replicates, for a total of 500 larvae per bioassay ((100 larvae per concentration ×4 concentrations) + (100 larvae per control group)).e entire bioassay was repeated five times.
During bioassays, larvae were maintained at standard insectary conditions (28 ± 1 °C temperature, 80 ± 10% relative humidity, and 12 h light/12 h darkness photoperiod) [22].No food was provided during this period.Mortality was recorded 24 hours after the beginning of each bioassay.Specimens were considered as dead when, following stimulation by touch, they either did not move at all or moved sluggishly and were unable to rise towards the surface of the rearing medium [24].
To establish the concentration that results lethal to 50% of individuals (LC 50 ), concentration gradients were established for both the aqueous plant extract and the plantsynthesized AgNPs.
e upper and lower limits of these gradients were established by preliminary experiments (data not shown).Concentrations tested for the aqueous plant extract were 1500, 3000, 4500, and 6000 ppm.Concentrations tested for the plant-synthesized AgNPs tests were 0.2, 0.3, 0.4, and 0.5 ppm.

Data Analysis.
Using the mortality results obtained from the five bioassay replicates, we calculated the mean mortality per dose.With these values, we performed dosemortality regressions using a log-probit model [25] in order to calculate the LC 50 and LC 90 values.Calculations were done using the R-software for statistical computing [26], and codes within the package MASS containing the material are discussed in [27].

UV-Vis Spectroscopy.
Parameters for this assay were previously optimized using 1.0 mM silver nitrate heated at 50 °C for 50 minutes, at pH 8, with 2 mL of aqueous A. arborescens extract [28].e UV spectrum of the synthesized nanoparticles is shown in Figure 2.
e wavelength of maximum absorbance was 414 nm, and this value, according to Pradeep's description [29], corresponds to an average nanoparticle-core shell size of 10 to 14 nm.It can be evidenced by the shape of the spectrum that there is a high dispersion of particle size, since the peak is wide but symmetrical.Peak spreading may also indicate that there are wide spaces between the nanoparticles [30].

FTIR Spectroscopy.
Fourier transform infrared spectroscopy (FTIR) was used to verify that the silver nanoparticles were indeed coated with the A. arborescens extract used for their synthesis.is is important because the extract does not only act as a reducing agent, but its organic fraction overlays or functionalize nanoparticles.us, the compounds responsible for the reduction of silver nitrate can be Journal of Nanotechnology identi ed.In Figure 3, we the spectra of silver nitrate, A. arborescens extract, and nanoparticles synthesized with A. arborescens extract.
e similarity between the spectra of the synthesized nanoparticles and the extract indicates that the organic residue of the extract is kept intact.Similar bands are present in both spectra around 1570 cm −1 corresponding to the stretching of the C C group of the aromatic rings, in addition to a band at approximately 1050 cm −1 of CO bonds within the alcohols.ese data con rm the adequate coating of nanoparticles with the extract.Once again, the extract not only ful lls the function of reducing agent but also acts as a stabilizing medium.
is saves additional use of other reagents.
In the spectra of the A. arborescens extract, there is a band at 1337.47 cm −1 that corresponds to the O-H bond characteristic of the phenolic compounds [31].In the nanoparticle spectra, this band is not present; however, the high intensity band around 3280 cm −1 is maintained.is di erence occurs because the OH groups of the phenolic compounds do not react in their entirety.Rais [32] indicates that only one out of four hydroxyl groups react, which is why the band of the OH groups at 3200 cm −1 is not a ected by the reaction.e remaining hydroxyl groups are attached to the nanoparticles by electrostatic attraction, coating them.A colorimetric test using FeCl 3 showed the color di erence between the extract and the silver nanoparticles-A.arborescens extract.

STEM Analysis.
As shown in Figure 4, the silver nanoparticles obtained are spherical and dispersed with an average size of 14 ± 6 nm.STEM image shows the action of aqueous extract on the nanoparticles as a stabilization agent.Anandalakshmi et al. [33] reported similar results in terms of particle size with diameters of 13 to 61 nm; this study was carried out with natural extract whose predominant components were polyphenols, which is why it resembles the synthesis performed with A. arborescens extract.Ndikau et al. [34] not only got similar size particles but also they found out that silver nanoparticles synthesized with plant extracts have smaller sizes than those synthesized with sodium citrate.
In STEM, a direct projection of the nanoparticles was observed and due to the low di raction of the conjugated molecule these do not appear on the electron microscope images and so you only observe the core shell of the nanoparticles. is is quite useful in green chemistry as normally the UV-Vis methods give an overestimation of the nanoparticle size and then the di erence in size between UV-Vis and STEM gives the average size value of the capping agent.A complete description of this e ect and an extensive list of relevant references can be found in [35].

SEM-EDX.
Figure 5 shows the micrograph of silver nanoparticles-A.arborescens extract powder measured by SEM. e image shows the dispersion of the nanoparticles onto the carbon pin.EDX spectrum shows three peaks of silver located below 4 kV.e rest of elements are present in the organic A. arborescens extracts, con rmed by FAAS.

Flame Atomic Absorption Spectroscopy for Silver Quanti cation.
e silver content of silver nanoparticles synthesis with A. arborescens extract was determined in order to verify that the silver content did not change to determine the yield of the reaction.e amount of theoretical silver that should be obtained was calculated as a function of the amount of silver nitrate in the initial solution and the volume of extract added.An average concentration of 90.73 ppm silver in the synthesized nanoparticles was obtained compared with initial 98.01 ppm present in the silver nitrate. is amount of silver equals a reaction yield of 92.52%, which is acceptable for synthesis on a smaller scale.In his doctoral thesis, Li [36] tested di erent phenols and mixtures of phenols as reducing agents and obtained yields in the range of 75 to 90%.

Larvicidal Activity of Aqueous Extract and Synthesized
AgNPs.Mortality values observed following all bioassays are shown in Table 1.Both the aqueous extract and the plantsynthesized AgNPs showed a dose-dependent toxic e ect against Ae.aegypti larvae.No mortality was observed in the control groups.2).However, the combination of this extract with silver nanoparticles greatly potentiated its toxicity to Ae. aegypti larvae: LC 50 and LC 90 values for plant-synthesized AgNPs were 0.28 ppm and 0.43 ppm (Table 2), respectively.χ 2 value was not signi cant at p ≤ 0.05 level.
Furthermore, exposure to aqueous extract at a concentration of 6000 ppm resulted in an average mortality of 97% of individuals, while exposure to 0.5 ppm plant-synthesized AgNPs resulted in an average mortality of 93.4% of individuals (Table 1).

Discussion
Our study suggests that the leaves of A. arborescens contain water-soluble chemicals which are toxic to Ae. aegypti larvae.Furthermore, our results show that the toxicity of such chemicals is greatly enhanced when they are combined with AgNPs.
It has been reported that plants belonging to the Asteraceae family can present an elevated toxicity due to the  presence of a wide range of chemical agents against herbivores, such as sesquiterpenic lactones, monotherpenes, terpenoids, and polyacetylenic resins [37].Within this family, the genus Ambrosia presents a remarkable physiological plasticity that allows it to adapt to a wide range of environmental conditions [38], a characteristic that might be explained, at least partially, by the secondary metabolites produced by the species of this taxon [39].All members of the Ambrosia genus present a high content of sesquiterpenic lactones which possesses antibacterial, cytotoxic, and antifungal properties [40,41].Regarding A. arborescens, former studies have reported damsines, coronophilines, and psilostachynes as the main sesquiterpenic lactones in this species [39,42,43].It seems plausible that the lethal effect observed is, at least in part, due to these kinds of compounds.
Several studies have reported on the larvicidal activity of plants of the Asteraceae family against Ae.aegypti.For example, Govindarajan and Karuppannan [44] used the methanolic extract of Eclipta alba against third instar larvae, obtaining a LC 50 of 127.64 ppm.Álvarez et al. [45] reported that the LC 50 of the ethereal extracts of Heli oppositifolia and Jaegeria hirta are 41 ppm and 24 ppm, respectively.Sukhthankar et al. [46] used a methanolic extract of the leaves of Chromolaena odorata, obtaining a LC 50 of 138 ppm, and Tennyson et al. [47] tested hexane and ethyl acetate extracts of Ageratum houstonianum, obtaining LC 50 values of 8889.13 ppm and 1952.12ppm, respectively.Furthermore, and as far as we are aware, only two previous reports exist regarding the toxicity of plants within the Ambrosia genus against mosquito larvae: (a) Eisa [48] tested the aqueous extract of A. marítima leaves against field-collected Culex larvae, obtaining a LC 50 of 1349 ppm, and (b) De La Torre et al. [49] tested the ethereal extract made with the aerial parts of A. confertifolia against Ae.aegypti, obtaining a LC 50 of 185.6 ppm.
In this context, the LC 50 value of 1844.61 ppm observed in our study for A. arborescens aqueous extract is within the range observed with similar plant extracts against mosquito larvae.However, one of the most interesting and striking aspects of our study was the exponential potentiation of the lethal effect we observed when the aqueous extract was used to synthetize AgNPs, reaching a LC 50 of 0.28 ppm, which represents a >6 000-fold increase in toxicity.Although this is consistent with other reports which have reported an enhanced efficiency of plant-synthesized AgNPs in comparison with the plant extracts by themselves [50], it is worth mentioning that the LC 50 reported in our work is, as far as we are aware, the lowest reported for AgNPs synthesized from any member of the Asteraceae family [51,52].
e physiological basis for the high toxicity of plantsynthesized AgNPs remains an open question.It has been suggested that a key factor is their ability to permeate through the invertebrate exoskeleton and penetrate into the insect's cells, where they bind macromolecules such as proteins and DNA, altering their structure and therefore their functionality [53,54].Interestingly, it has also been reported that doses of plant-synthesized AgNPs which result lethal to several species of mosquito larvae have little or no effect on other nontarget species, including other aquatic arthropod species and fish [55,56], suggesting that at least some mosquito species are particularly susceptible to the lethal effect of AgNPs.At the moment, the reasons behind this phenomenon remain unknown.
It is, therefore, important to stress that more research is needed to identify the mechanisms by which AgNPs exert their toxic effect on their intended target species.is information will be crucial to establish whether the use of nanoparticles for mosquito control could have any unintended negative impact on either the environment or the health of the human population.Furthermore, any field application of this technology must be preceded by extensive laboratory and semifield research, exploring aspects such as residuality, bioaccumulation, and long-term effects of exposure to AgNPs on relevant biological systems.

Conclusions
In summary, our study shows that the aqueous extract obtained from the leaves of A. arborescens presents a clear insecticidal effect against Ae.aegypti larvae.Furthermore, this effect is greatly potentiated when this extract is used for the synthesis of AgNPs.erefore, our results suggest that A. arborescens has the potential to be used for the development of novel pesticides.However, the said development must be complemented by studies aimed at defining key aspects, such as the nature of the specific molecules responsible for the insecticidal effect observed, the potential effects of these chemicals in both the environment and in human health, and the field logistics required for the use of such a novel insecticide.
Neira supervised the experimental design and performance of bioassays and contributed to the analysis of experimental results.Fernanda Pilaquinga, Flavia Domenech, and Danny Ganchala performed the synthesis and characterization of silver nanoparticles.Alexis Debut performed STEM analysis.Bianca Morejón, Marco Neira, Fernanda Pilaquinga, and Flavia Domenech wrote the manuscript.All authors reviewed and approved this manuscript.Bianca Morejón and Fernanda Pilaquinga contributed equally to this work.

Figure 4 :Figure 5 :
Figure 4: STEM image (a) and frequency histogram (b) of average shape of silver nanoparticles-A.arborescens extract.

Table 1 :
Larvicidal activity of aqueous extracts and silver nanoparticles synthesized using Ambrosia arborescens against Aedes aegypti third instar larvae.
SD: standard deviation; a values are mean ± SD of ve trials.