Introduction
Pathogens of many dreadfull diseases like filariasis, Japanese encephalities, malaria, dengue, dengu haemorrhagic fever, yellow fever, etc are transmitted by several mosquito species belonging to genus Culex, Anopheles, and Aedes and million of death occur every year (Rahuman et al., 2008; ICMR Bulletin, 2003). Culex quinquefasciatus Say, 1823 is the principal vector of lymphatic filariasis in many tropical countries. 120 million people are infected worldwide with lymphatic filariasis and common chronic menifestations were observed among 44 million people (Bernhard et al., 2003). To control mosquito borne diseases, one of the best approach within many is to kill mosquito larvae at its larval habitats (Kumar and Maneemegalai, 2008). Application of synthetic insecticides to control pests and vectors causing diseases of human beings have environmental impact and toxicity (Maragathavalli et al., 2012). To avoid these problems, use of insecticides of plant origin are needed to control pests and vectors as plant based insecticides are easily degradable in the environment and their sources are renewable (Ilahi et al., 2012). Many researchers worked with extracts of different solvents of several plants to identify their larvicidal activity against different species of mosquitoes (Mallick et al., 2014; Singha et al., 2012; Hossain et al., 2011; Chakraborty et al., 2013; Chowdhury et al., 2008) . Methanol and butanol leaf extracts of Annona reticulata Linn. showed antibacterial activity against both gram negative and gram positive bacterial strains (Jansi Rani et al., 2013). Larvicidal activity of methanol leaf extract of A. reticulata against Cx. quinquefasciatus has been reported (Nayak, 2014). Larvicidal efficacy of acetone leaf extracts of A. reticulata against Aedes aegypti, Anopheles stephensi and Cx. quinquefasciatus has been reported (Mallick et al., 2015). Larvicidal activities of stem bark extracts of A. reticulata against Cx. quinquefasciatus has also been reported recently (Mallick and Chandra, 2015). Root extracts of A. reticulata has been exploited for the first time to kill Cx. quinquefasciatus larvae during the present piece of work.

A. reticulata is commonly named as Bullock’s Heart, Ramphal, Sitaphala, Sarifa, and Custard Apple and the plant is found all over India. Leaves are used as antihelminthic, styptic, insecticides etc. Bark of the plant is used as antidysentric and vermifuge (Ranjini and Jothi Nisha, 2013; Zaman and Pathak, 2013). Ethanol root extract of A. reticulata showed distinct inhibitory effect against human cancer cell lines at the dose range of 10-40 μg/ml but aqueous extract exhibited lower activity at same doses (Suresh et al., 2011).

1 Results
Mortality percent increased with increase in concentrations and time of exposure of the crude root extract of the plant. No mortality were noticed on negative control treatments (Table 1). Table 2 depicts the mortality percent of 3rd instar larvae of Cx. quinquefasciatus at different concentrations of petroleum ether, hexane, and ethyl acetate solvent root extracts of the plant. Among petroleum ether, hexane, and ethyl acetate root extracts of the plant, ethyl acetate root extract showed best larvicidal activity. No mortality were observed on ethanol treated control experiments. LC₅₀ and LC₉₀ values were lowest in case of ethyl acetate root extract of the plant than petroleum ether, and hexane root extracts after 24, 48, and 72 h of exposures (Table 3). Mortality percent for 1^st- 4^th instars larvae of Cx. quinquefasciatus at different concentrations of ethyl acetate root extract of the plant was presented in Table 4. 1^st instar larvae were most susceptible to ethyl acetate root extract. Only at 1 ppm concentration 100% mortality was observed after 48 h of exposure against 1^st instar larvae . 2^nd and 3^rd instars larvae showed 100% mortality at 1 and 2 ppm concentrations after 72 and 48 h of post exposure respectively. But no  mortality were noticed on ethanol treated control experiments. Table 5 and table 6 represented the LC₅₀ and LC₉₀ values, regression equations, R² (co efficient of determination) values of crude and ethyl acetate root extracts respectively. LC₅₀ and LC₉₀ values gradually decreased with time of exposure. values of R² were close to one in all most all cases indicating a positive corelation between concentration of the extracts and mortality percent. No mortality and abnormal behaviour were observed on non target organisms after application of ethyl acetate root extract at LC₅₀ values of 3^rd instar larvae after 24 h of exposure. Preliminary phytochemical analyses showed the presence of alkaloids, steroids, terpenoids, flavanoids, anthraquinones, tannins and phenols but absence of saponins. The larvicidal activity was found statistacally significance (p < 0.05) through completely randomised ANOVA analysis (Table 7).

Table 1 Mortality percent of different instars of Culex quinquefasciatus exposed to different concentrations of crude root extract of Annona reticulata (Mean mortality percent ± Standard error)

Table 2 Mortality percent of 3rd instar larvae of Culex quinquefasciatus exposed to different concentrations of different solvent root extracts of Annona reticulata (Mean mortality percent ± Standard error)

Table 3 LC50 and LC90 values (95% confidence level) of Petroleum ether, Hexane, and Ethyl acetate solvent root extracts of Annona reticulata through Log Probit analyses against 3rd larval instar of Culex quinquefasciatus

Table 4 Mortality percent of different instars of Culex quinquefasciatus exposed to different concentrations of ethyl acetate root extract of Annona reticulata (Mean mortality percent ± Standard error)

Table 5 Log probit and regression analyses of larvicidal activity of crude root extract of Annona reticulata against different larval instars of Culex quinquefasciatus LC = Lethal Concentration, R2 = Co efficient of determination

Table 6 Log probit and regression analyses of larvicidal activity of ethyl acetate root extract of Annona reticulata against different larval instars of Culex quinquefasciatus LC = Lethal Concentration,      R2 = Co efficient of determination

Table 7 Completely randomized three ways ANOVA analyses using instars (I) of Cx. quinquefasciatus, hours (H), and Concentrations of ethyl acetate root extract of Annona reticulata (C) as three independent parameter

2 Discussion
Many researchers worked with different plant extracts to unfold their larvicidal activities on different mosquitoes species and to find out the active chemical compound (s) involved in larvicidal activity present in different extracts of different plants. Present study is unique one as 100% mortality were observed with root extracts of A. reticulata at very low concentrations. Many authors reported the larvicidal activity of different solvent root extracts of many plant parts. Some authors also reported the larvicidal efficacy of ethyl acetate extracts of many plant parts. Ilahi and Ullah, 2013 reported the larvicidal activity of different parts of Artemisia vulgaris Linn. against 3^rd and 4^th instar larvae of  Cx. quinquefasciatus upto 24 h of exposure period and LC₅₀ values of methanol extracts of root, stem, and leaf were 9141.0, 2224.2, and 803.2 ppm respectively. Jayaraman et al., 2015 reported the larvicidal activity of hexane, chloroform, ethyl acetate, acetone, and methanol extracts of seven aromatic plants against Cx. quinquefasciatus, Aedes aegypti, and Anophelis stephensi. The ethyl acetate extract of Chloroxylon swetenia showed remarkable larvicidal activity against three mosquito species, of which LC₅₀ and LC₉₀ values of Cx. quinquefasciatus were 194.22 and 458.83 ppm after 12 h of exposure period. Nganjiwa et al., 2015 worked with ethanolic leaf and root extracts of Balanites aegyptica, Calotropis procera, and Eucalyptus globulus against 4^th instar larvae of mosquito species upto 24 h of exposure period. At 10 ppm concentration ethanolic root extracts of Balanites aegyptica, Calotropis procera, and Eucalyptus globulus showed 64.67, 73.67, and 65.00% mortality having LC₅₀ values 7.24, 6.61, and 6.92 ppm respectively. Rawani et al., 2010 reported the larvicidal activity of leaf ethyl acetate extract of Solanum nigram and showed 100% mortality at 50 ppm concentration with LC₅₀ value17.04 ppm after 24 h of exposure. So comparing the result of above mentioned works with the result of present study, our work is better than their works. So it may be concluded that crude and different solvent root extracts of Annona reticulata can be used effectively to control Cx. quinquefasciatus mosquito larvae in their breeding places because different extracts work at very low concentrations. Further research is needed to know the name (s) and chemical structure (s) of actual compound (s) which is responsible for larvicidal activity.

3 Materials and Methods
3.1 Collection of plant parts
After proper identification of the A. reticulata plant, roots of near about one to two years aged plants were collected during September and October, 2014 from Burdwan town, West Bengal, India (23◦16ꞌ N, 87◦54ꞌ E) and the voucher specimen (No.GCZSM-4) of the plant was kept as herbarium to Mosquito, Microbiology and Nanotechnology Research Units, Parasitology Laboratory, Department of Zoology, The University of Burdwan, West Bengal, India. After collection, roots were washed with tap water and then rinsed with distilled water and dried on paper towel.

3.2 Preparation of crude extract
Washed roots were cut into small pieces and crushed with mechanical grinder and juice was filtered by Whatman No. 1 filter paper and the filtrate served as stock crude test solution. From stock crude test solution required graded concentrations (0.01- 0.05%) of crude extract was prepared through dilution with tap water.

3.3 Preparation of solvent extracts
Washed roots were cut into small pieces and dried for 15-18 days in shade with gentle blowing of air. Dried small pieces of roots (150 g) were packed into the thimble of Soxhlet apparatus and passed petroleum ether, hexane, and ethyl acetate solvents (Volume of each solvent 1500 ml). For each solvent extraction, fresh root samples were used and the extraction period of different solvents were 72 h. Pooled extracts were concentrated by rotary evaporator and semisolid extracts of different solvents were obtained and stored at refrigerator at 4℃ for further use. 0.05 g semi solid extract of different solvents were dissolved initially on 5 ml of ethanol and then added 95 ml of distilled water to obtain 100 ml of stock test solution of different extracts. So stock solution of different solvent extracts were prepared on 5% ethanol. From stock test solutions, 1, 2, 4, and 8 ppm concentrations of petroleum ether, hexane, and ethyl acetate solvent root extracts of the plant were prepared through dilution with tap water which were used for larvicidal bioassay experiments.

3.4 Test mosquito
Cx. quinquefasciatus were collected from cemented drains of Burdwan town and identified properly and the larvae were kept in plastic tray with tap water. Larvae were well maintained in the laboratory at 27± 2℃ temparature and 85% relative humidity under 14:10 light and dark cycles in a day. The larvae were fed with powdered mixtutre of dog biscuits and dried Brewer’s yeast powdered (Ratio 3:1). Pupae were transferred from plastic tray to a plastic bowl containing tap water and bowl was kept in mosquito cage (30×30×30 cm³) where adults were emerged. Adults were provided with glucose solution (10%) in a plastic bowl with a cotton wick. Adults were provided blood meal from restrained pigeon on day five. Plastic bowl with 100 ml of tap water were kept in the cage for oviposition. Next generation larvae were used for bioassay.

3.5 Larvicidal bioassay
Larvicidal bioassay were done according to the protocal of WHO with slight modification (WHO, 2005). 30 larvae were put in plastic bowls containing 100 ml of test solutions of different concentrations of crude extract (0.01- 0.05%) and different concentrations of solvent extracts (1, 2, 4, and 8 ppm). Negative control experiments were set on 100 ml of tap water only and ethanol treated control experiments were set on 100 ml of tap water with 0.5 ml of ethanol (positive control). Each set of experiment was replicated three fold with three replicates of controls  at laboratory condition on separate three days. The percent mortality was recorded after 24, 48 and 72 h of post exposure cumulatively. Larvae were identified dead when they could not move after touching the siphon or cervical area with a fine brush.

3.6 Effect on Non Target Organisms
Non target organisms are animals which live in the same place where larvae of mosquito grow. The effect of ethyl acetate solvent extracts at LC₅₀ value of 3^rd instar larvae of Cx. quinquefasciatus after 24 h of exposure were observed upto 72 h on Diplonychus annulatum, Chironomus circumdatus larvae (insect) and tadpole larvae of toad.

3.7 Qualitative Phytochemical analyses of Root Extract of A. reticulata
Aqueous and ethanolic root extracts (charcoal filtered) of A. reticulata were tested for qualitative phytochemical analyses following the standard protocal (Trease and Evans, 1989)  with slight modification.

3.7.1 Detection of Tannin and Phenolic Compounds
2 ml of aqueous extract were taken in a clean test tube and was added 0.5 ml of ferric chloride solution. The colour of solution change into blue green  indicate the presence of tannin and phenolic compounds.

3.7.2 Detection of Flavanoids
2 ml of aqueous extract was taken in a test tube and was added few drops of NaOH solution. Intence colour formation occur which become colourless on addition of dilute HCL and indication of the presence of flavanoids.

3.7.3 Detection of Alkaloids
2 ml of ethanolic extract were taken in a clean test tube and added few drops of 2N HCL. Then mixed 1 or 2 drops of Mayer’s reagent [1.36 g of mercuric chloride dissolve in 60 ml of H₂O and then this solution pour in potasium iodide solution (5 g potasium iodide dissolve in 100 ml of water)]. Appearence cream  or pale yellow colour precipitation indicate the presence of alkaloids.

3.7 4 Detection of Terpenoids (Salkowski test)
2 ml of ethanolic extract was taken in a test tube and then added 2 ml of chloroform and 3 ml of concentrated H₂SO₄ carefully by the interior wall of the test tube. A reddish brown colouration of the interface was formed which indicate the presence of terpenoids.

3.7.5 Detection of Steroids
2 ml of ethanolic extract was taken in a test tube and then 5 ml of chloroform and 5 ml  concentrated H₂SO₄ was added carefully by the interior wall of the test tube. The upper layer turns red and H2SO4 layer showed yellow with green fluorescence. This indicate the presence of steroids.

3.7.6 Detection of Anthraquinones
2 ml of aqueous extract was added to 2 ml of 2N HCL and NH3 in a clean test tube. The appearence of pink red was turned blue violet indicating the presence of anthrocyanines.

3.7.7 Test of Saponins (Frothing test)
10 ml of aqueous extract were taken in a test tube and was shaking vigorously. Persistence of frothing that is the indication of the presence of saponines in the sample.

3.8 Statistical analyses
MS EXCELL 2007 and the computer software STAT PLUS 2009 (trial version) were used to calculate the mean mortality percent, standard error, LC₅₀, LC₉₀, regression equation (Y= mortality, X= concentration), coefficient of determination (R²) and ANOVA.

Acknowledgement
Authors are thankfull to Eminent Professor, Dr. A Mukhopadhyay, Department of Botany, The University of Burdwan, Burdwan, West Bengal, India for identification of the plant species. We are also grateful to UGC DRS for providing financial supports.

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