Efficacy and side effects of bio-fabricated sardine fish scale silver nanoparticles against malarial vector Anopheles stephensi

Mosquitoes are a great menace for humankind since they transmit pathogenic organisms causing Malaria, Dengue, Chikungunya, Elephantiasis and Japanese encephalitis. There is an urgent need to discover new and novel biological tools to mitigate mosquito-borne diseases. To develop bioinsecticides through newly developed nanotechnology is another option in the present research scenario. In this study we synthesize and characterize sardine fish scales with silver nitrate by adopting various instrumental techniques such as UV- and FTIR-spectroscopy, energy-dispersive X-ray (EDAX), X-ray diffraction analyses (XRD) and scanning electron microscopy (SEM). Toxicity bioassays were conducted with young developmental stages of mosquito vectors. Significant mortality appeared after different life stages of mosquito vectors (young larval and pupal instars were exposed to the nanomaterials). LC50 values were 13.261 ppm for young first instar larvae and 32.182 ppm for pupae. Feeding and predatory potential of G. affinis, before and after exposure to nanoparticles against mosquito larval (I & II) instars of the mosquitoes showed promising results in laboratory experiments. Feeding potential of mosquito fish without nanoparticle treatment was 79.7% and 70.55% for the first and second instar larval populations respectively. At the nanoparticle-exposed situation the predatory efficiency of mosquitofish was 94.15% and 84.3%, respectively. Antioxidant enzymes like (SOD), (CAT), and (LPO) were estimated in the gill region of sardine fish in control and experimental waters. A significant reduction of egg hatchability was evident after nanoparticle application. It became evident from this study that the nano-fabricated materials provide suitable tools to control the malaria vector Anopheles stephensi in the aquatic phase of its life cycle. This finding suggests an effective novel approach to mosquito control.


Materials and methods
We confirm that all experimental protocols were approved by Institutional Animal Ethical Committee, Bharathiar University, Coimbatore, India.
Fish scale extraction procedure. Fish scale materials (Sardinella longiceps) were procured from the fish market, Ukkadam, Coimbatore, India. Fish scales were transferred to the Department of Zoology, Bharathiar University, were washed thoroughly for discarded abdicable wreckages, air-dried, and stored at − 60 ℃ while waiting for the next process for no more than 30 days; AgNO 3 (silver nitrate ≥ 99.0% pure-≤ 0.01% not HCl), SOD, CAT, and LPO enzyme analytical kits and other chemicals were purchased from Sigma Chemicals in the USA.

Bio-synthesis and characterization of fish scale bio-fabricated silver nanoparticles (SFS-AgNPs).
For the synthesis of bio-fabricated fish scale silver nanoparticles, we adopted the procedure of Murugan et al. 19 which was previously described and slightly modified by Muthumari et al. 32 and Dinesh et al. 33 . Initially, the Sardine Fish Scale extract was made by adding 10 g of SFS-grains to 100 mL double distilled water which was then stewed at 60 ℃ for 20 min in the filtered extract used to prepare the SFS-AgNPs. The filtered SFS aqueous extract of 10 mL with different concentrations (10%, 20%, 30% and 40%) was added separately to 10 mL of 1 mM AgNO 3 aqueous solution and boiled at 70 ℃ for 10 min followed by incubation at room temperature for cooling the solution 19 . After 24 h the solution of dark brown sediments indicated the formation and presence of the AgNPs. Then physiochemical characterizations were done viz., UV-vis, SEM, EDX, FTIR and XRD analyses 34 . SFS-AgNPs were authenticated by UV-vis, followed by SEM, EDX, FT-IR and XRD. UV-vis spectrophotometry enables the user to illustrate the shape and size of nanofabricated materials in water solution 35 .
Mosquitocidal properties of SFS-AgNPs on the malarial vector A. stephensi L.. The eggs and young larval and pupal mosquitoes were maintained and cultured and pathogen-free colonies were maintained at laboratory conditions (27 ± 2 °C, 75-85% R.H. and 14 h:10 h (L:D) photoperiod) following Subramaniam and Murugan 36 and Subramaniam et al. 37,38 . We have followed the procedure by Murugan et al. 10,39,40 where five replications were made. In each replicate, there were twenty-five larvae/pupae in the two hundred fifty millilitre of double distilled water including the required concentration of sardine fish scale extract (ppm) and SFS-AgNPs (ppm), respectively. Ovicidal efficiency was elaborated using methods by Mullai  Predatory potential of G. affinis. G. affinis was maintained in the laboratory in a definite size of the fish tank and cultured fishes were used to test the predatory potential of mosquitofish. Different larval stages (I-IV) of A. stephensi were used for a predatory bioassay by Murugan et al. 11,44 and Subramaniam et al. 45 . The feeding potential of the fishes was recorded with the decrease in doses for I-IV larvae of A. stephensi i.e. 1/3 of the LC 50 values of SFS extract (Table 1) and SFS-AgNPs (Table 2). Experiments were replicated five times and larvae were replaced daily and the experiments were monitored periodically (predatory/prey potential) the predatory efficiency was calculated by using formula originally defined by Murugan et al. 19,34 and Subramaniam et al. 46,47 . Collection and preparation of samples for enzyme assays. At the completion of each experimental period (1,2,4,8,16 and 28 days) fishes from control, Sardine fish scale extract and sardine fish scale silver nanoparticles were used for treatments (treatment I, II and III) at desired concentrations (2,4,6,8 and 10 ppm) respectively. Blood samples of organs (gills) were collected and stored at cold conditions. The tissues were used to evaluate enzymological parameters (LPO, SOD and CAT). Lipid peroxidation (LPO) was investigated following the methods described and modified by Gupta and Verma 48 . Chemical concentrations were estimated by the procedure of Chen et al. 49 . CAT activities were studied after Bao et al. 50  www.nature.com/scientificreports/ The following enzymes, SOD, CAT and LPO were estimated in the gill region after the exposure of nanoparticles at different dose levels.
Data analysis. Lethal concentrations (LC 50 and LC 90 ) were determined by probit analysis using mortality data 51 . The SPSS statistical package 16.0 version was used for all sample analysis calculations. All data were subjected to Analysis of variance. The means were separated using Duncan's multiple range test modified by Alder and Rossler 52 . Antioxidant enzyme studies SOD and calculated data were shown as means ± SE, percentage changes were compared by the means of treated data against their controls. Data were considered significant at (P < 0.01) and (P < 0.05) levels.
We wish to confirm that all methods were carried out in accordance with relevant guidelines and regulations of Bharathiar University, India and for the manuscript entitled "Efficacy of bio-fabricated Sardine fish scale silver nanoparticles against malarial vector and their effect on antioxidant enzymes of mosquito fish, Gambusia affinis" which was submitted to Scientific Reports.
We are confirming that the study was carried out in compliance with the ARRIVE guidelines for the experimentations and further experimental protocol framed by the of Bharathiar University, India and for the manuscript entitled "Efficacy of bio-fabricated Sardine fish scales silver nanoparticles against malarial vector and their effect on antioxidant enzymes of mosquito fish, Gambusia affinis", which was submitted to Scientific Reports.

Results and discussion
Physicochemical characterization of nanoparticles by instrumentation techniques. Nanoparticles mediated with the aid of nanobiotechnology have provided a series of purposes viz., electronics, physics, drug delivery, stem cell therapy 16 . Nanostructured materials and its possessions must be examined in advanced practice. Regularly to designate the physicochemical representation of the unknown NPs, several corresponding techniques should promptly be employed (i.e. SEM, EDAX, FT-IR spectroscopy and X-ray diffraction studies) 6,53 as we did in the present study.
The reaction time of fish scale extract was visualized in the UV-visualization spectrum and provided in Fig. 2. The color intensity of the SFS extract incubated with AgNO 3 solution at the start of the treatment and after 120 min of reaction is providing a peak at 460 nm (Fig. 2). Similarly, biofabrication of AgNPs was determined via a chromatic indicator represented by color change of the reaction substrate, from light green to brown which showed the biotransformation of Ag + ion to Ag 054, 55 . The UV-vis spectra of fish scale extracts as a function of reaction time provided a strong resonance at about 410 nm which increased in intensity with time. The physically powerful Plasmon resonance centering at 400 nm increased in strength with time. This is explained with the excitation of longitudinal Plasmon vibrations of AgNPs in solution 56 .
Scanning electron microscope (SEM) was used to characterize the morphology of the AgNPs and to observe the dissimilarities in size, capability for accumulation, and stability of experimental conditions 57 . The physical structure of AgNps was visualized by adopting the SEM analysis which procured hexagonal nanostructures as presented in Fig. 3. Santhosh Kumar et al. 58 demonstrated by SEM that the morphologies of Ag-Nps were unique and uniform in size from 25 to 80 nm. Our results agreed with Murugan et al. 14 that the visual part of morphology of chitosan-fabricated AgNP was studied by FE-SEM and showed spherically-shaped particles ranging from 30 to 50 nm.
EDX spectra provided quantitative evidence and the occurrence of elements involved in the materialization of SFS-AgNPs synthesized at 25 °C and 80 °C (Fig. 4). The silver atoms in the SFS-AgNPs and those from oxygen, silver and chloride provided strong signals. Our results agree with Murugan et al. 19 who showed that metallic silver could be confirmed by strong silver signals. The results of Energy-dispersive X-ray (EDX) spectra provided evidence from Centroceras calvulatum extract and Sonneratia alba synthesized AgNPs, respectively 10,21 .
The structure and crystal lattice of the SFS-AgNPs (Sardine fish scale synthesized silver nanoparticles-see  Fourier transformed infrared spectroscopy (FTIR) is another technique to determine physiochemical parameters and elucidate the structure of nanomaterials, based on the electromagnetic wavelength within the midinfrared region (4000-400 cm −1 ) 62 . The FTIR analysis represents a non-invasive, value added, economical and simple method to classify the function of biological elements in the contraction of silver nitrate to silver 63 . FTIR spectrum analysis could be applied to confirm the interaction among the presence of amine groups in the combined nanostructures of sardine fish scale extract and silver nitrate. Figure 6 shows spectral maxima that reveal carbonyl groups such as from polyphenols such as N-O stretching (nitroxide compound), S=O stretching (sulfoxide), C=C stretching (alkene), C=O stretching (carboxylic), C-H stretching (alkene), N-H stretching (secondary amine), respectively. Altogether the data of the present study indicate that particles secured by Ag Nanomaterials had free and bound alkane and amine groups 64 .  www.nature.com/scientificreports/ Similarly, many researchers have studied that biofabricated nanoparticles are possessing different functional groups, including alkene and alkane 65,66 , methylene 67 , aromatic amine 68 , and carboxylic acid 33 , carboxyl groups or/in amide groups 67 were revealed in earlier studies as reducing agents in the biofabrication of AgNPs.
Nanoparticles as mosquito larvicides. Sardine fish scale extract and its biosynthesized silver nanoparticles with various concentrations expressed their toxicity against young instars and pupal populations of the malarial vector. However, the toxic effect was significantly higher in biosynthesized silver nanoparticles and these results are shown in Tables 1 and 2. For example, in the case of fish scale extract treatment of the fourth instar larvae, its LC 50 was 170.38 ppm and for fish scale biosynthesized silver nanoparticles the LC 50 was 22.85 ppm, respectively. Moreover, most of the biogenic nanostructures are spherical in shape 69 with only some exceptionally being of oval shape 70 . Similar to earlier studies, the fish scale nanoparticles in the present study are also spherical and provided greater larval and pupal toxicity to mosquitoes 40 . Nanoparticles as mosquito ovicides. Sardine fish scale extract and sardine fish scale biosynthesized nanoparticles greatly affected the hatchability of eggs of mosquito vectors (Table 3). There was no hatching (NH) occurring at 150 ppm sardine fish scale extract whereas the sardine fish scale biosynthesized nanoparticles pro-  www.nature.com/scientificreports/ vided no hatchability (NH), even at the lowest dose group of 40 ppm. Madhiyazhagan et al. 71 conducted experiments against malarial, filarial, and dengue vector and Sargassum muticum-fabricated silver nanostructures with a single dose (30 μg/mL) expressing significant ovicidal activities. Furthermore, Rajaganesh et al. 72 found that 25 μg/mL administration led to 100% inhibition of hatchability by the exposure to fern (Dicranopteris linearis)fabricated nanostructures.

Predatory efficiency of G. affinis combined with SFS extract and SFS-AgNPs against A. stephensi. Predatory fish showed a high predation activity against malarial vectors and the first instar larvae
were the most preferred food for the mosquito fish, G. affinis. In our predation experiments, predatory efficiency was 79.70% (first instar), 70.55% (second instar), 62.75% (third instar) and 46.30% (fourth instar), respectively (Table 4). Notably, predatory efficacy of G. affinis after application of silver nanoparticles at their elevated dose level reached 94.15%, 84.30%, 71.05% and 56.50%, from first to fourth instar larvae, respectively ( Table 4).
Effect of nanoparticles against non-target aquatic organisms. Silver nanoparticles were tested before for their acute toxicity 55 . Most of the biosynthesized silver nanoparticles did not show any toxicity against any aquatic organism 73 . For instance, plant synthesized silver nanoparticles did not show any toxic impact compared to fabricated nanomaterials to the predatory fish Poecilia reticulata after an exposure for 48 h. Earlier, Murugan et al. 21 reported that a treatment with S. alba synthesized AgNP increased neither the consumption of predatory fish nor of guppy fish (Poecilia reticulata) for mosquito larvae.
Antioxidant enzyme response to nanoparticle contaminated environments for non-target mosquito fish. After exposure of fish to different doses of fish scale biofabricated silver nanoparticles there was a changing profile of enzyme activities in the gill region of the fish. Sodium dismutase (SOD) is a oxyradical scavenger and is mainly involved in the catalytic activity and liable for dismutation of exceedingly abundant superoxide radicals. A significant increase in the activity of SOD was noted (Fig. 7). This may be due to the  www.nature.com/scientificreports/ synthesis of newer enzymes in the body system or enrichment of already existing enzymes loaded in the tissues at different concentrations. Interestingly, the level initially increased during 1, 2, 4 and 8 days and its level has been resumed very near to the uncontaminated biofabricated nanoparticles of fish scales. A similar study was conducted after the treatment of Cu nanoparticles at 25.6 μg L −1 after a 24 h treatment of gills of the mytilid clams B. azoricus 74 and M. galloprovincialis after exposure to 5-25 μg L −1 Cu after 7 day treatment 75 . Catalase (CAT) activation provides another stress/antioxidant system of animals. This enzyme converts the free radicals H 2 O 2 to H 2 O and O 2 in the animal defense system. In our experiment after exposure to fish scale fabricated nanoparticles at 2, 4, 6, 8 and 10 ppm, the antioxidant enzyme (CAT) had slightly increased its activities up to day 1, 2 and 4 where it showed considerably elevated activities (Fig. 8). Yilmaz et al. 76 reported peroxisome distributions in the gill region and its functional role on molecular H 2 O 2 which is metabolized to molecular O 2 and H 2 O.
LPO is yet another very important indicator of oxidative enzymes in the cellular metabolism and it is the end product or outcome of oxidative deterioration of membrane lipids. LPO serves as a marker or oxidative enzyme and it has been extensively used as a biomarker for cellular activities 77 . Normally this enzyme (LPO) is evaluated/ estimated by measuring the concentration of malondialdehyde (MDA) (Fig. 9). The higher activities and overaccumulation of the enzyme malondialdehyde not only damages cells but also triggers the process of apoptosis 78 . In the current study there was no change in the gills after nanoparticle exposure. However, a significant elevation of this enzyme was noted after 1, 2, 4 and 8 days.

Conclusion
In conclusion, Sardine fish scale fabricated nanomaterials are hydrophilic, can uniformly disperse in aqueous media, have moderate toxic effects and provide mosquitocidal properties against the malarial vector Anopheles stephensi. The biosynthesized silver nanoparticles of this study are easy to prepare with high stability and provide pronounced toxic effects on target mosquito vectors. At the same time the nanoparticles used here enhance Table 4. Predatory efficiency on mosquito A. stephensi by the teleost fish Gambusia affinis. Predation rates are represented by means ± SD of four replicates (1 fish vs 200 mosquitoes per replicate). Control was clean water without predators within each column, means followed by the same letter are not. Significantly different (P < 0.05).  www.nature.com/scientificreports/ the feeding activities of mosquito fish in an environment that is nanoparticle contaminated to feed on target mosquito larvae. The present paper also revealed that physiological stress extended to changed enzymological activity levels of the mosquito fish G. affinis in a nanoparticle treated environment. www.nature.com/scientificreports/