1. Introduction
Amongst all arthropods, mosquitoes are the most abundant blemished insect for spreading many noxious tropical and subtropical diseases like filariasis, yellow fever, malaria, dengue, different types of encephalitis like Japanese encephalitis and many more diseases resulting millions of deaths yearly. Apart from mortality, mosquito bites result into angioedema, intense itching, redness of skin and swellings. Disease transmission path includes broad regions around the equator especially much of sub Saharan Africa, Asia and the Americas. Anopheles stephensi spread the noxious disease malaria in urban areas mainly. WHO has reported 219 million malarial cases with 1.2 million deaths in 2010 (Nayyar et al., 2012). There are about 10,000 malaria cases per year in Western Europe and 1300–1500 in the United States (Taylor et al., 2012). Rainfall, warm temperatures and stagnant water bodies provide ideal habitats for mosquito larvae, so, malaria is prevalent in tropical and sub tropical regions. In urban area An. stephensi chooses different water-bodies for breeding, primarily in overhead tanks, artificial containers, walls and ground level water tanks (Jeyabalan et al., 2003). Till now many malarial cases are unreported and precise data is unavailable in many rural areas.

To prevent such types of malevolent diseases and to reduce fatality thereof, it is indispensable to find a way to control the mosquito population. For the purpose of vector control plentiful simulated insecticides have been developed and used against the vector and achieved a significant success, but it contains some alarming drawbacks like non-selectiveness and non-biodegradability which leads to toxic hazards (Wattal et al., 1981). Nevertheless indiscriminate use of commercial insecticides results genetic resistance (Devine, 2007) to pesticides among vector species, arbitrary use of those results some adverse effect on ecosystem, environment and to human health as well (Lee et al., 2001). For long term applications it is more difficult to rely upon the obtainable mosquitocidal agents after the outcome of bio pesticide resistance (Tabashnik, 1994), so, finding of eco-friendly and target specific insecticides specially from plant origin (Rawani et al., 2009; Chowdhury et al., 2009; Chakraborty et al., 2013) are apparent in a standard manner.

Nelumbo nucifera, the holy lotus found throughout India is a large aquatic medicinal plant with stout, creeping rhizome. Anti-diarrheal and antimicrobial properties (Mukherjee, 2002) of N. nucifera have been reported. Anti-inflammatory and anti-tumour effects; mediated by the presence of betulinic acid, a steroidal pentacyclic triterpenoid of this plant are also well documented (Mukherjee et al. 1997; Chou et al., 2000). This plant is well recognized for antioxidant (Ling et al., 2005) anti-arrhythmic (Yu and Hu, 1997) properties.

Though diverse parts of N. nucifera have been stepped to discover several medicinal inimitabilities, seed coat has not yet been reported to contain mosquitocidal agents.

2. Material and Methods
2.1 Assemblage of plant materials
Fresh seedpods of Nelumbo nucifera (Nymphaeaceae) were collected randomly from the local ponds of Burdwan (23°16´N, 87°54´E), West Bengal, India during September to November 2012. The voucher specimen was numbered (voucher no. GCASR-03) and kept as a herbarium in the Department of Zoology, The University of Burdwan.

2.2 Nurturing the larvae and colony set up
Larvae of An. stephensi were collected from Kolkata and for further bioassay experiments larval colony is maintained under laboratory condition. The larvae were periodically fed with a diet containing powder of Brewer yeast, dog biscuits and algae mixture in 3:1:1 ratio (Kamaraj et al., 2011a) and kept in plastic trays filled with tap water in a germ free condition. Pupae were collected from the trays and transferred to insectary (45×45×40 cm) for adult immergence. During the rearing of adults, they continuously provided a 10% sucrose solution with multivitamin syrup in a container with a cotton wick. The cotton was changed every day and kept moist with the solution. The mosquitoes were identified by the identification keys of Christophers (1933), Barraud (1934) and Chandra G (2000). In a different glass cage, the adults of An. stephensi were reared, on the 5^th day; adults were given a blood meal from a non-motile shaved pigeon resting on the cages overnight for blood-feeding by females. Glass Petri dishes filled with 100 mL of tap water were kept inside the cage for oviposition. The eggs were unperturbed and allowed to hatch under laboratory conditions. In laboratory the colony of An. stephensi were maintained by repeating the process again and again. Mosquito colony was kept at 27±2°C, 75–85% relative humidity (RH), with a photoperiod of 14:10 h light/dark cycle.

2.3 Crude extracts procurements
Fresh and young seed coats of N. nucifera were rinsed in tap water followed by distilled water and soaked on a paper towel. Then the unspotted seed coats were minced by electric grinder and the liquid was filtered by Whatman’s no-1 filter paper. The filtrate solution was considered as the stock crude solution (100% concentration) and stored in refrigerator for future use.

2.4 Differential solvent extraction
For solvent extraction, fresh and clean seed coats of N. nucifera were dried for few days in shed. 120 g dried seed coats of N. nucifera were put into the ‘thimble’ of the Soxhlet apparatus while 1200 ml solvent was loaded into the ‘still pot’ following 1:10 ratio. Three different solvents in a non-polar to polar fashion viz. petroleum ether, ethyl acetate and water were passed through the column one after another. The extraction phase was set for 8 hours a day with a maximum extraction period of 72 hours for a particular solvent. The extracts were collected from solvent chamber and concentrated through evaporation in a rotary evaporator and the residue obtained was stored at 4°C in a refrigerator.

2.5 Dose dependent larvicidal bioassay
Trusting the WHO standard protocol (WHO/ VBC, 1981) the larvicidal bioassay was executed at the Mosquito, Microbiology and Nano Technology Research Units, Parasitology Laboratory, The University of Burdwan. Instars wise 25 larvae of A. stephensi were affianced in Petri-dishes of 9 cm diameter (150 mL capacity) filled with 100 mL of tap water. From 0.1% to 0.5% crude extractives were added in different Petri dishes while 60 ppm to 180 ppm solvent extracts was given in different Petri dishes for performing larvicidal bioassay. Each experiment was performed in triplicate (n=75) with a set of controls. Petri dishes were retained at room temperature (29 ± 2°C), within 88±2% relative humidity range for a total observation period of 72 hours. The larvae were considered dead when they didn’t exhibit any movement by the pricking with a sharp needle in the siphon or cervical region or they were unable to achieve the water surface (Macedo et al., 1997). The no. of dead wrigglers was calculated every 24 hours period up to 72 hours and percentage mortality was recorded from the average value of three replicates. The mortality of 48 h and 72 h were expressed by additions of the mortalities of 24 h and 48 h, respectively. Initially all the solvent extractives were screened against 3rd instars larvae and then the experiment was elaborated with the best active fraction against all the instars.

2.6 Effects on non-target populations
The little creatures sharing the same environment with mosquitoes are considered as the most fatal risk group. Vulnerability of these organisms to seed coat extractives was figured out on Chironomus circumdatus larvae (insect). They were exposed to concentration level of LC₅₀ value (72 hours of post-exposure) of 3^rd instars larvae to examine the mortality and other irregularities such as tardiness of swimming activity up to 72 h of exposure.

2.7 Statistical analyses
The percentage mortalities (%M) were precised by Abbott’s formula (Abott WS, 1925) during the observation of larvicidal potentiality of the seed coat extracts. Judicious determination of LC₅₀ and LC₉₀ values of crude and solvent extracts were carried out through Log-probit and regression analyses. Further statistical calculations were done through ANOVA analyses using instars, hours of exposure and concentrations as three random variables to validate the significance between the above parameters and larval mortality.

3. Results
Different corporal characteristics and percentage yields for each solvent were significantly different from the other solvents as shown in Table 1. Larval mortalities at different concentrations of crude extractives of N. nucifera seed coats against An. stephensi wrigglers were presented instars wise in Table 2. The mortality percentages using all the three different extractives against 3^rd instars larvae were presented in Figure 1. Ethyl acetate extract was found to be the choicest larvicide, through several trials, amongst all the extracts of N. nucifera seed coats. The larval mortality of An. stephensi using ethyl acetate extractives was depicted in Table 3. Log probit analyses clearly indicated augmentations in LC₅₀ and LC₉₀ values (at 95% confidence level) vis-à-vis late stages larvae which were subsequently subordinated with increase in post-exposure period (from 24 hours to 72 hours) (Table 4). All through the experiment the concentration of ethyl acetate extractives were found to be positively correlated with rate of mortality having a regression coefficient nearly 1. Further analyses using completely randomized three way ANOVA regarding concentration (C), hour (H) and instars (I) as three parameters revealed the statistical significance of the larvicidal effect of ethyl acetate extractive against An. stephensi (Table 5). The non-target populations were found in normal condition after 72 h of post-exposure.

Table 1 Differential yields and some physical characters of N. nucifera seed coat extracts

Table 2 Percent mortality of An. stephensi larvae using crude extracts of N. nucifera seed coats

Figure 1 Graphical presentation of the larval mortality using all the three different extracts against 3rd instars larvae of An. stephensi

Table 3 Percent mortality of An. stephensi larvae using ethyl acetate extracts of N. nucifera seed coats

Table 4 Assessment of LC50 and LC90 values of ethyl acetate extract of N. nucifera through log-probit and regression analyses

Table 5 Completely randomized three way ANOVA analysis using concentration (C), hour (H) and instars (I) as three parameters

 
4. Discussion
Incapability to control of mosquito borne diseases remains a big dispute even today and indeed most challenging. Though personal protection from mosquito bites through several means are always advised, it is very little to control the menace. The best approach to shrink mosquito population is wrigglers’ control. Employment of cost-effective, biodegradable natural insecticides from botanicals has given importance, recently, for this purpose. Mosquito larvicidal potentiality of several plants is verified by a number of workers (Singha et. al., 2012; Bhattacharya and Chandra, 2013, 2014; Kundu et al., 2013). Plant constituents are well established as pupicidal (Rawani et al., 2012), repellent, adulticidal and smoke toxic (Singha et al., 2011, Chowdhury et al., 2007) against different mosquito species. The present study well conferred the target specific larvicidal activity of N. nucifera seed coats against An. stephensi. Govindarajan reported that methanol extract of Andrographis paniculata, Eclipta alba, and Cardiospermum halicacabum are very much effective against the larvae of An. stephensi with the median lethal concentration values of 79.68, 112.56, and 133.01 mg/L respectively. However, we found that the LC₅₀ value of 3^rd instars larvae was 50.27 mg/L (ppm) using ethyl acetate extracts of N. nucifera seed coat. Adhikari et al. (2012) reported the larvicidal activity of glacial acetic acid, an organic acid, against An. stephensi. They recorded 100% mortality of 3rd instars in 0.5% solution. Ethyl acetate extracts of Annona squamosa bark was reported to be larvicidal against the 4^th instars larvae of An. stephensi (Kamaraj et. al., 2010) where LC₉₀ values varied in between 70.38-210.68 ppm. N. nucifera leaf extract using methanol as solvent was found to be mosquitocidal against early 4^th instars An. stephensi larvae (Kamaraj et. al., 2011b) with the median lethal concentration 37.49 ppm following a period of 48 hours. We noticed the LC₅₀ value of 94.98 ppm against mature 4^th instars of An. stephensi larvae when the observation period was set at 48 hours. Ethyl acetate extracts of N. nucifera seed coats showed very promising responses against the 1^st and 2^nd instars of An. stephensi with the median lethal concentration values of 34.09 ppm and 43.74 ppm respectively. Entire larval populations of 1^st and 2^nd instars were diminished following a post-exposure period of 72 hours with no significant detrimental effect on non-target population. This ascertained the selective toxicity of the extract under study with meticulousness. High mortality percentage against 3^rd instars and a moderate mortality value against 4^th instars (Table 3) pertained to the suitability of this active fraction as mosquitocidal bio-pesticide against An. stephensi.

In concise, the findings of the present study reveal that the seed coat extracts of N. nucifera posses larvicidal activity against An. stephensi. Other laboratory investigations are mandatory for enlightening the actual chemical amalgam responsible for larvicidal activity. This veteran activity may again be evaluated against An. stephensi larvae in field conditions. For successfully utilization of this novel mosquitocidal source through commercial formulation detail research is necessary.

Conflict of interest statement
We pronounce that we don’t have any conflict of interest.

Acknowledgements
The authors are indebted to Professor Dr. A. Mukhopadhyay, Botany Department, The University of Burdwan, for his kind help in plant species identifications. We are grateful to UGC DRS for providing financial assistance.

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