Datura metel-synthesized silver nanoparticles magnify ... - Springer Link

2 downloads 0 Views 875KB Size Report
Sep 4, 2015 - ORIGINAL PAPER. Datura metel-synthesized silver nanoparticles magnify predation of dragonfly nymphs against the malaria vector Anopheles.
Parasitol Res (2015) 114:4645–4654 DOI 10.1007/s00436-015-4710-x

ORIGINAL PAPER

Datura metel-synthesized silver nanoparticles magnify predation of dragonfly nymphs against the malaria vector Anopheles stephensi Kadarkarai Murugan 1 & Devakumar Dinesh 1 & Prabhu Jenil Kumar 1 & Chellasamy Panneerselvam 1 & Jayapal Subramaniam 1 & Pari Madhiyazhagan 1 & Udaiyan Suresh 1 & Marcello Nicoletti 2 & Abdullah A. Alarfaj 3 & Murugan A. Munusamy 3 & Akon Higuchi 4 & Heinz Mehlhorn 5 & Giovanni Benelli 6

Received: 21 August 2015 / Accepted: 26 August 2015 / Published online: 4 September 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Malaria is a life-threatening disease caused by parasites transmitted to people and animals through the bites of infected mosquitoes. The employ of synthetic insecticides to control Anopheles populations leads to high operational costs, non-target effects, and induced resistance. Recently, plantborne compounds have been proposed for efficient and rapid extracellular synthesis of mosquitocidal nanoparticles. However, their impact against predators of mosquito larvae has been poorly studied. In this study, we synthesized silver nanoparticles (AgNPs) using the Datura metel leaf extract as reducing and stabilizing agent. The biosynthesis of AgNPs was confirmed analyzing the excitation of surface plasmon resonance using ultraviolet–visible (UV–vis) spectroscopy. Scanning electron microscopy (SEM) showed the clustered and irregular shapes of AgNPs, with a mean size of 40–

* Giovanni Benelli [email protected]; [email protected] 1

Division of Entomology, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore 641046, Tamil Nadu, India

2

Department of Environmental Biology, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy

3

Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia

4

Department of Reproduction, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan

5

Department of Parasitology, Heinrich Heine University, Düsseldorf, Germany

6

Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80, 56124 Pisa, Italy

60 nm. The presence of silver was determined by energydispersive X-ray (EDX) spectroscopy. Fourier transform infrared (FTIR) spectroscopy analysis investigated the identity of secondary metabolites, which may be acting as AgNP capping agents. In laboratory, LC50 of D. metel extract against Anopheles stephensi ranged from 34.693 ppm (I instar larvae) to 81.500 ppm (pupae). LC 50 of AgNP ranged from 2.969 ppm (I instar larvae) to 6.755 ppm (pupae). Under standard laboratory conditions, the predation efficiency of Anax immaculifrons nymphs after 24 h was 75.5 % (II instar larvae) and 53.5 % (III instar larvae). In AgNP-contaminated environment, predation rates were boosted to 95.5 and 78 %, respectively. Our results documented that D. metel-synthesized AgNP might be employed at rather low doses to reduce larval populations of malaria vectors, without detrimental effects on behavioral traits of young instars of the dragonfly Anax immaculifrons. Keywords Anax immaculifrons . Anopheles stephensi . Biological control . Green synthesis . Mosquito-borne diseases . Nanobiotechnologies . Non-target effects . Biosafety

Introduction Arthropods are highly effective vectors of important pathogens and parasites, which may hit as epidemics or pandemics in the increasing world populations of humans and animals. Mosquitoes (Diptera: Culicidae) represent a key threat for millions of organisms worldwide, since they act as vectors of the agents of malaria, dengue, yellow fever, West Nile virus fever, Japanese encephalitis, and filariasis (Mehlhorn et al.

4646

2012; Benelli 2015a). According to the latest estimates, there were at least 198 million cases of malaria in 2013 and an estimated 584,000 deaths. Malaria mortality rates have fallen by 47 % globally since 2000, and by 54 % in the African region, but are still high. Most deaths occur among children living in Africa, where a child dies every minute from malaria (WHO 2014). Human malaria is caused by Plasmodium parasites, vectored to people through the bites of infected female Anopheles mosquitoes, which bite mainly between dusk and dawn (Jensen and Mehlhorn 2009; WHO 2014). People entering into regions where malaria risk exists may protect themselves by use of chemical- or plant-derived repellents (Mehlhorn et al. 2005; Mehlhorn 2011; 2012; Amer and Mehlhorn 2006a, b), while people living in endemic regions have to protect themselves by several strategies at the same time, since the daily infection rates of mosquitoes may be extremely high (Amer and Mehlhorn 2006c, d; Semmler et al. 2009). Anopheles populations are usually targeted using organophosphates, insect growth regulators, and microbial control agents. However, such synthetic pesticides have negative effects on human health, struggle severely the environment, and in addition induce resistance already existing in a number of mosquito species (e.g., Robert and Olson 1989; Wattanachai and Tintanon 1999; Liu et al. 2005). In an eco-friendly scenario, renewed interest has been devoted to the potential of sterile insect technique (SIT) for suppression of mosquito vectors (Oliva et al. 2014). SIT has been recently combined with auto-dissemination (i.e., adult females contaminated with juvenile hormones to treat breeding habitats), a technique proved efficient to control Aedes species but that cannot be used at large scales. This has led to the development of a new control concept, named Bboosted SIT,^ which might enable the area-wide eradication of mosquitoes and other vectors of medical and veterinary importance (Bouyer and Lefrancois 2014; Benelli 2015a). Furthermore, huge efforts have been carried out to investigate the efficacy of botanical products against mosquito vectors. Many plantborne compounds have been reported as effective against different species of Culicidae, acting as adulticidal, larvicidal, ovicidal, oviposition deterrents, growth and/or reproduction inhibitors, and/or adult repellents (see Benelli 2015b, Benelli et al. 2015, and Pavela 2015 for dedicated reviews). Biological control of mosquito larval populations using aquatic predators, such as insects, fishes, copepods, and tadpoles, also received attention (Murugan et al. 2011; Bowatte et al. 2013; Murugan et al. 2015a, b, c, d). Odonate young instars are voracious predators of mosquito larvae both in controlled settings and natural habitats (Fincke et al. 1997; Lacey and Orr 1994; Stav et al. 2000; Yanoviak 2004; see Kumar and Hwang 2006 for a review). Immature dragonflies (Odonata) occupy a great diversity of aquatic habitats but are generally most abundant in lowland streams and ponds.

Parasitol Res (2015) 114:4645–4654

Overall, odonate young instars are an important part of aquatic food webs, and the aquatic instars of mosquitoes comprise significant parts of the diet of different Odonata species (Ward 1992; Westfall and May 1996). Nanoparticles have the potential to revolutionize a wide array of applications, including drug delivery, diagnostics, imaging, sensing, gene delivery, artificial implants, tissue engineering, and pest management (Morones et al. 2005; Dinesh et al. 2015). The plant-mediated biosynthesis (i.e., green synthesis) of nanoparticles offers significant advantages over chemical and physical methods, since it is cheap, needs only single steps, and does not require high grades of pressure, energy, and temperature or the use of highly toxic chemicals (Goodsell 2004). A growing number of plants and fungi have been proposed for efficient and rapid extracellular synthesis of silver and gold nanoparticles (e.g., Shankar et al. 2004; Song and Kim 2009; Priyadarshini et al. 2012; Ponarulselvam et al. 2012; Rajan et al. 2015), which showed excellent mosquitocidal properties, also in field conditions (Soni and Prakash 2012; Dinesh et al.2015; Suresh et al. 2015; see Benelli 2015c for review). However, while extensive efforts have been conducted to investigate non-target effects of nanoparticles against aquatic organisms (Oberdorster et al. 2006; Baun et al. 2008; Fabrega et al. 2011; Park et al. 2014), little has been done to shed light on the impact of green-synthesized mosquitocidal nanoparticles on behavioral traits of aquatic arthropods on toxicity against arthropod predators sharing the same ecological niche as mosquitoes (Murugan et al. 2015a, b, c, d; Benelli 2015c). In detail, little information is available on how synthesized green nanoparticles affect the predatory efficiency of odonates catching mosquitoes (Murugan et al. 2015e). Datura metel L. (Solanaceae) is a plant widely employed in Indian and Chinese traditional medicine (Rajesh and Sharma 2002). It contains the alkaloids hyoscyamine, hyoscine, atropine anisodamine, and anisodine, which are mainly used as sedative, antispasmodic, antihelminthic, and mydriatic agents (Priya et al. 2002). Rajasekharreddy et al. (2010) reported the extracellular production of silver and gold nanoparticles using D. metel by the sunlight exposure method. Similarly, good nanoparticles, in terms of size and morphology features, have been obtained from the leaf extracts of D. metel by Kesharwani et al. (2009). To the best of our knowledge, no information is available about the mosquitocidal potential of D. metel-synthesized nanoparticles. In this study, we biosynthesized silver nanoparticles (AgNPs) using the D. metel leaf extract as reducing and stabilizing agent. AgNPs were characterized by ultraviolet–visible (UV–vis spectroscopy), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and energy-dispersive X-ray (EDX) spectroscopy. We investigated the larvicidal and pupicidal properties of the D. metel leaf extract and green-synthesized AgNPs against the malaria

Parasitol Res (2015) 114:4645–4654

vector Anopheles stephensi. Furthermore, we evaluated the predation efficiency of the dragonfly nymphs, Anax immaculifrons, against second and third instar larvae of Anopheles stephensi, both in normal laboratory conditions and in AgNP-contaminated aquatic environment.

Materials and methods Plant material Plants of D. metel were collected from campus of the Bharathiar University (Coimbatore, India). Voucher specimens were stored in our laboratories (Bharathiar University, Coimbatore) and are available under request (ID: DATMET1-4). Mosquito rearing Experiments were conducted using a laboratory-reared strain of Anopheles stephensi. The strain was originally established as described by Dinesh et al. (2015). Batches of 100–110 eggs were transferred to 18 cmL×13 cmW× 4 cm D enamel trays containing 500 mL of water where they were allowed to hatch at laboratory conditions [27± 2 °C and 75–85 % relative humidity (R.H.); 14:10 (light/ dark, L/D)] photoperiod. Larvae were fed daily with 5 g of ground dog biscuits (Pedigree, USA) and brewers yeast (Sigma-Aldrich, Germany) in a 3:1 ratio. Larvae and pupae were used for acute toxicity experiments. Furthermore, each container was placed inside a cubic chiffon cage (90×90×90 cm) to wait for adult emergence. Adults were fed ad libitum on 10 % (w/v) sucrose solution. Five days after emergence, the adults were deprived of sugar feeding for 12 h and then supplied with artificial blood feeding. The blood meal was furnished, by means of a professional heating blood (lamb blood), at a fixed temperature of 38 °C and provided with a membrane of cow gut. After 30 min, the blood meal was removed, due to blood drying phenomena, and gut membrane was substituted by a fresh one for the following utilization (Nicoletti et al. 2012). D. metel-mediated synthesis of silver nanoparticles D. metel leaves were washed with distilled water and dried in shade for 2 days at 25 °C. Leaf extract was prepared by placing 10 g of finely cut leaves in a 300-mL Erlenmeyer flask filled with 100 mL of sterile distilled

Percentage mortality ¼

4647

water. The mixture was boiled for 5 min, decanted, and stored at −4 °C. Within 5 days, the D. metel extract was treated with aqueous 1 mM AgNO3 solution (0.16987 mg/ mL) in an Erlenmeyer flask and incubated for 72 h at 25 °C. AgNO 3 was purchased from the Precision Scientific Co. (Coimbatore, India). Color change indicated the formation of AgNP, since aqueous silver ions were reduced by the D. metel extract generating stable AgNP in water. The effect of reaction time on synthesis rate and particle size of AgNP was studied carrying out the reaction in water bath at 95 °C from 10 min to 4 h with reflux. Characterization of D. metel-synthesized silver nanoparticles The biosynthesis of AgNP was confirmed by sampling the reaction mixture at regular intervals; absorption maxima was scanned by ultraviolet–visible (UV–vis) spectroscopy at the wavelength of 350–600 nm in a UV-3600 Shimadzu spectrophotometer operating at a resolution of 1 nm. The reaction mixture was subjected to centrifugation at 15,000 rpm for 20 min. The resulting pellet was dissolved in de-ionized water and filtered through a Millipore filter (0.45 μm). After freeze-drying of the purified AgNP, their structure, and composition were analyzed by a 10-kV Ultra High Resolution SEM (FEI QUANTA 200) and EDX. The surface groups of AgNP were qualitatively confirmed by FTIR spectroscopy, using a PerkinElmer spectrum 2000 FTIR spectrophotometer. Mosquitocidal assays against Anopheles stephensi Following the methods reported by Dinesh et al. (2015), 25 Anopheles stephensi larvae (I, II, III, or IV instar) or pupae were placed for 24 h in a glass beaker filled with 250 mL of de-chlorinated water plus D. metel aqueous leaf extract (20, 40, 60, 80, and 100 ppm) or D. metelsynthesized AgNP (2, 4, 6, 8, and 10 ppm). Larval food (0.5 mg) was provided for each tested concentration. Each concentration was replicated five times against all instars. Control mosquitoes were stored for 24 h in glass beakers filled with 250 mL of dechlorinated water. No mortality was observed in the controls. During the treatments, percentage mortality was calculated as follows:

 .  number of dead individuals number of treated individuals *100

4648

Parasitol Res (2015) 114:4645–4654

Predation efficiency of dragonfly nymphs Anax immaculifrons nymphs were collected from small water ponds in Coimbatore (Tamil Nadu, India) and transferred to laboratory conditions [27±2 °C and 75–85 % R.H.;14:10 (L/D)] photoperiod. Here the predation efficiency of Anax immaculifrons nymphs was assessed against II and III instar larvae of Anopheles stephensi. For each replicate, 200 mosquito larvae were introduced, with Anax immaculifrons

Predation efficiency ¼

nymph, in plastic cups (5 L) containing de-chlorinated water. For each instar, five replicates were conducted. Control was done with 5 L of de-chlorinated water without dragonfly nymphs. All experimental arenas were checked after 12 and 24 h, and the number of preys consumed by dragonfly nymphs was recorded. After each checking over time, the predated mosquito larvae were replaced by new ones. Predatory efficiency was calculated using the following formula:

h . . i number of consumed mosquitoes number of predators total number of mosquitoes *100

Predation efficiency of dragonfly nymphs in an aquatic environment contaminated with silver nanoparticles

Results and discussion Synthesis and characterization of silver nanoparticles

Here the predation efficiency of Anax immaculifrons nymphs was assessed against Anopheles stephensi larvae, after a mosquitocidal treatment with AgNP. Both for II and III instar larvae, 200 mosquitoes were introduced with 1 dragonfly nymph in a 5-L plastic cups filled with dechlorinated water plus 1 mL of the desired concentration of nanoparticles (i.e., 1 ppm, one third of the LC50 calculated against I instar larvae of Anopheles stephensi). For II and III instar larvae, five replicates were conducted. Control was dechlorinated water without dragonfly nymphs. All experimental arenas were checked after 12 and 24 h, and the number of preys consumed by odonate nymphs was recorded. Predation efficiency was calculated using the above mentioned formula.

When the AgNO3 solution was added to the D. metel leaf extract, the color changed from yellowish to dark brown color, indicating the reduction from Ag+ to Ag0, and the formation of AgNP (Fig. 1a, b). The absorption spectra of AgNP at different time intervals showed highly symmetric single band absorption. A maximum absorption peak was observed at 432 nm (Fig. 1c), which is characteristic of Ag and probably arises from the excitation of longitudinal plasmon vibrations of AgNP in the solution (Ukiya et al. 2001; Shankar et al. 2004; Dhas et al. 2014). The broadness of the peak is a good indicator of the size of the nanoparticles. As the particle size increases, the peak becomes narrower with a decreased bandwidth (Petit et al. 1993; Kong and Jang 2006). In agreement with our results, Savithramma et al. (2011) observed the that

Data analysis 2.2

30 min 60 min 120 min

c

2.0 1.8

a

1.6

Transmitance (%)

Mosquito toxicity data were subjected to ANOVA with two factors (i.e., the targeted mosquito instar and the tested dosage). Means were separated using Tukey’s HSD test. The average mosquito mortality data were subjected to probit analysis. LC50 and LC90 were calculated using the method by Finney (1971). Data were analyzed using the SPSS 22.0 software (SPSS Inc., Chicago, IL, USA). A probability level of P