1 Introduction
Different mosquito borne diseases like malaria, filariasis, Japanese encephalitis, dengue, dengue hemorrhagic fever, yellow fever, chikungunya etc. are transmitted by different mosquito species and these diseases are the world's most hazardous ailments causing millions of death annually (ICMR Bulletin, 2003). About two-fifth of the world’s population are at the risk of exposure to dengue (Rigau, 1998). The vector which is responsible for the transmission of this dengue, dengue hemorrhagic fever and dengue shock syndrome is Ae. aegypti (Hendarto and Hadinegoro, 1992; Pancharoen, 2002). Another dreadful disease is malaria, caused by Plasmodium species transmitted by urban vector, An. stephensi. According to WHO, 207 million malarial cases have been reported and around 6.3 million deaths occurred in the year 2012 (Caraballo, 2012: WHO, 2014). Pathogens of human lymphatic filariasis Wuchereria bancrofti are transmitted by Cx. quinquefasciatus and about 44 million people out of 120 million persons worldwide show chronic manifestation in the tropical region (Bernhard, 2003). Thereof to avert such types of spiteful diseases and to reduce fatal accident, it is a requisite to find a way to control the mosquito population. Assassinating the mosquito larvae in their breeding places before emerging into adults is the suitable technique to control mosquito. Synthetic insecticides have been used as mosquito larvicidal agents in different regions for the last 30 years (Chavasse and Yap, 1997) but unremitting treatment resulted in lower efficacy of the chemical insecticides and development of resistance in the mosquito populations. Moreover the harmful effects on human health and hazardous impact on environment (De Omena et al., 2007) led to the search of innovative mosquitocidal agent from novel sources especially from botanical origin. Plant based insecticides became the alternative sources to avoid these problems because they are easily biodegradable causing little or no effect on non target populations including human beings (Bhattacharya et al., 2014a, 2014b; Mallick et al., 2014; Singh et al., 2015), cost effective and easy to handle (Chowdhury et al., 2007; Bhattacharya and Chandra, 2013, 2014; Singha et al., 2011, Singha Ray et al., 2014).

Annona reticulata Linn. commonly known as bullock’s heart or ramphal plant is found all over India. In folk medicinal system of Bangladesh, plant parts of A. reticulata are used for the treatment of tumor, fever, epilepsy, dysentery etc (Suresh et al., 2011). Effect of methanol extract of leaves of this plant has been worked out only on 4^th instar larvae of Cx. quinquefasciatus (Nayak, 2014). But no work on the effect of other solvent extracts (besides methanol) of leaves of A. reticulata on Cx. quinquefasciatus and other species of mosquitoes has been examined so far. Present study has been designed to evaluate the effect of A. reticulata as a potent larvicidal agent against three test mosquito species.

2. Material and Methods
2.1 Collection of Plant Materials
Fresh mature green leaves of A. reticulata were collected during September and October, 2013 from Burdwan town, West Bengal, India and the voucher specimen (GCZSM-4) was conserved in the Department of Zoology, The University of Burdwan, West Bengal, India. The leaves were rinsed with distilled water, soaked on paper towel and used for the experiment.

2.2 Preparation of Solvent Extract
Observing excellent results after preliminary experiments with crude extract against larvae of 3 species of mosquitoes mentioned, fresh mature leaves of A. reticulata were air dried in shade for 9-10 days and chopped finely. To prepare the solvent extract of leaves of the plant, 200 g finely chopped leaves were put in the thimble of the soxhlet apparatus and acetone (2000 ml) on the still pot in 1:10 ratio. The period of extraction was 72 h with 8 h maximum a day. The elute was extracted and concentrated by rotary evaporator. Graded concentration of the extract (ranging from 0.5, 1, 2, 4, 6, 9, and 12 ppm) were prepared for larvicidal bioassay experiments. 0.045 g acetone extract was first dissolved in 5 ml of ethanol and 85 ml of distilled water was added afterwards to get the stock solution (i.e. 0.045 g acetone extract were dissolved in 90 ml of 5.55 % ethanol) . From stock test solution, required graded concentration (0.5, 1, 2, 4, 6, 9, and 12 ppm) were made freshly by adding required volume of tap water.

2.3 Test Mosquito
The present study was done at Mosquito, Microbiology and Nanotechnology Research Units, Parasitology Laboratory, Department of  Zoology, The University of Burdwan, Burdwan (23◦16ꞌ N, 87◦54ꞌ E) West Bengal, India. Larvae of three mosquito species were taken from colonies maintained in the laboratory for bioassay experiments. Mosquito colonies were kept free from insecticides, repellents and exposure to pathogens. The mosquito larvae were fed with artificial food (mixture of dog biscuits and dried yeast powder in 3:1 ratio).

2.4 Larvicidal Bioassay:
The larvicidal bioassay was done following the standard protocol of WHO (2005) with slight modification. Twenty five larvae of all instars of test mosquitoes were put in different plastic bowls of 225 ml capacity containing each with 100 ml of test solution of different concentration of acetone extract (0.5, 1, 2, 4, 6, 9 and 12 ppm) to investigate the mortality percent at different time period of exposures. Concentration of acetone leaf extract ranging 0.5, 1, 2, 4 and 6 ppm were used against Cx. quinquefasciatus, 0.5, 1, 2, 4, 6 and 9 ppm for An. stephensi and 0.5, 1, 2, 4, 6, 9 and 12 ppm for Ae. aegypti respectively for the experiment. Ethanol treated controls were set up for control assay. Larval mortalities were recorded after 24, 48 and 72 h of exposure cumulatively. Dead larvae were identified when they failed to move after checking with fine brush in the siphon or cervical region. The experiments were replicated three times on separate three days under laboratory conditions at 25-30◦c and 80-90% relative humidity.

2.5 Effect on Non Target Organisms
The consequence of acetone extract of A. reticulata leaves were also investigated on non target organisms like Diplonychus annulatum, Chironomus circumdatus larvae and tadpole larvae of toad with LC₅₀ at 24 h of 3^rd instar larvae of Cx. quinquefasciatus to observe the mortality and other abnormalities such as sluggishness and reduced swimming activity up to 72 h of post exposure.

2.6 Statistical Analysis
The computer software STAT PLUS 2009 - trial version and MS EXCEL 2007 were used to calculate the LC₅₀, LC₉₀, regression equation (Y= mortality, X = concentration), coefficient of determination (R²), mean mortality percent, Standard error. Further statistical justifications were established through ANOVA analyses using instars, hours and concentrations as three random variables to authenticate the implication between the above parameters and larval mortality.

3 Results
Percent mortalities of different instars of three mosquitoes are presented in tabulated form (Table 1-3). After 72 h of exposure at 12 ppm concentration 100% mortality was observed for 1^st, 2^nd and 3^rd larval instars respectively where as 4th instar larvae showed 46.66% mortality against Ae. aegypti (Table 1). At 9 ppm concentration after 72 h of exposure highest mortality was recorded for 2^nd, 3^rd and 4^th larval instars of which 100% mortality was noticed for 2^nd and 3^rd instars larvae and 1^st instar larvae showed 100% mortality after 48 h of post exposure against An. stephensi (Table 2). After 48 h of exposure 100% mortality was recorded at 6 ppm concentration for 1^st, 2^nd and 3^rd instars larvae respectively and after 72 h of exposure 83.33% mortality was recorded for 4^th instar larvae against Cx. quinquefasciatus (Table 3). Control experiments (ethanol treated) were set up for all the said mosquito species but no larval death was recognized. LC₅₀, and LC₉₀ values, regression equations and R² values for all larval instars of Ae.aegypti, An. stephensi and Cx. quinquefasciatus after 24, 48 and 72 h of exposure were tabulated in Table 4-6 respectively. It was observed that LC₅₀ and LC₉₀ values (at 95% confidence level) gradually decreased with increase in period of exposure and the mortality percentages (Y) were positively correlated with the concentration of extract (X) as coefficient of determination values (R²) were close to 1 in all cases. It was also noted that LC₅₀ and LC₉₀ values for a period of 24, 48 and 72 h were lowest in case of 1^st instar larvae of the afore said mosquitoes. Further, among all the tested species, LC₅₀ and LC₉₀ values for all instars larvae of Cx. quinquefasciatus were lowest. Non target organisms did not exhibit any abnormal behavior after exposure to LC₅₀ at 24 h of 3^rd instar larvae of Cx. quinquefasciatus. ANOVA analysis established statistical significance of larval mortality (p<0.05) (Table 7-9) in terms of concentrations, instars and time of exposure collectively against all the mosquitoes.

Table 1 Mortality percent of different larval instars of Aedes aegypti exposed to different concentrations of acetone leaf extract of Annona reticulata (Mean mortality percent ± Standard error) Note: Control: no mortality (for all instars)

Table 2 Mortality percent of different larval instars of Anopheles stephensi exposed to different concentrations of acetone leaf extract of Annona reticulata (Mean mortality percent ± Standard error) Note: Control: No mortality (for all instars)

Table 3 Mortality percent of different larval instars of Culex quinquefasciatus exposed to different concentrations of acetone leaf extract of Annona reticulata (Mean mortality percent ± Standard error) Note: Control: no mortality (for all instars)

Table 4 Log probit and regression analyses of larvicidal activity of acetone leaf extract of Annona reticulata against different larval instars of Aedes aegypti Note: LC: Lethal Concentration, R2= Coefficient of determination, X= Concentration Y= Mortality

Table 5 Log probit and regression analyses of larvicidal activity of acetone leaf extract of Annona reticulata against different larval instars forms of Anopheles stephensi Note: LC: Lethal Concentration, R2= Coefficient of determination, X= Concentration Y= Mortality

Table 6 Log probit and regression analyses of larvicidal activity of acetone leaf extract of Annona reticulata against different larval instars forms of Culex quinquefasciatus Note: LC: Lethal Concentration, R2= Coefficient of determination, X= Concentration Y= Mortality

Table 7 Completely randomized three way ANOVA analyses of larvicidal activity against Aedes aegypti using instars (I), hour (H), and concentration (C) as three independent parameters

Table 8 Completely randomized three way ANOVA analyses of larvicidal activity against Anopheles stephensi using instars (I), hour (H), and concentration (C) as three independent parameters

Table 9 Completely randomized three way ANOVA analyses of larvicidal activity against Culex quinquefasciatus using instars (I), hour (H), and concentration (C) as three independent parameters

4 Discussions
The practice of synthetic insecticides to control mosquitoes proved hazardous due to its adverse impact on ecospheres, non biodegradability nature, toxicity, and resistancy among different species of mosquitoes. So it is necessary to resolve another way to overcome these problems. Insecticides of plant origin are biodegradable, ecofriendly, target specific and moreover have no toxic effects on environment (Chakraborty et al., 2013; Singha and Chandra, 2011; Hossain et al., 2011). The present study revealed that acetone leaf extract of A. reticulata has a great potency as larvicidal agent to control different species of mosquitoes. All the species under the study showed 100% mortality at remarkably low doses. Several authors reported the mosquitoes larvicidal activity with different solvent extracts derived from several plants parts. (Rawani et al., 2012) reported that application of petroleum ether extract of Carica papaya showed 100% mortality at 50 ppm dose and 96.67% mortality at 100 ppm dose against 3^rd instar larvae of Cx. quinquefasciatus and An. stephensi after 72 h of exposure respectively and LC₅₀ and LC₉₀ values of 24 h of exposure were 31.16, 341.86 ppm and 18.39, 250.76 ppm against 3^rd instar larvae of Cx. quinquefasciatus and An. Stephensi respectively. (Manzoor et al., 2013) investigated the larvicidal activity of essential oils of five plant species against late 3^rd instar larva of Ae. Aegypti and Cx. quinquefasciatus upto 24 h of exposure and they observed the highest larvicidal activity of Ocimum basilicum against larvae of Ae. aegypti and Cx. quinquefasciatus with LC₅₀ values 75.35 and 92.30 ppm against larvae of Ae. aegypti and Cx. Quinquefasciatus respectively. Singha et al., 2012 reported 100% mortality at 400 ppm concentration of acetone leaf extract of Holoptelea integrifolia after 72 h of exposure with LC₅₀ value of 24 h of exposure i.e. 283.7960 ppm against 3^rd instar larvae of Cx. vishnui. Singh et al., 2006 reported that hexane extract of fruits of Momordica charantia has larvicidal activity against 4^th instar larvae of An. stephensi Ae. aegypti and Cx. quinquefasciatus with LC₅₀ values of 24 h of exposure were 66.05, 96.11, and 122.45 ppm respectively. But, acetone leaf extract of A. reticulata showed lower mortality percent and lower LC50 and LC90 values against Ae. aegypti, An. stephensi and Cx. quinquefasciatus than their work. Cx. quinquefasciatus was found to be more susceptible to acetone extract than An. stephensi and Ae. aegypti. On the other hand An. stephensi is more susceptible to acetone extract than Ae. aegypti (Table 4, 5 and 6). Further investigations are needed to know the actual active ingredient of acetone extract of leaves of A. reticulata responsible for their activities as mosquito larvicidal agents. In brief, the result of the current research brings out that acetone extract of leaves of A. reticulata demonstrate notable larvicidal activity against different mosquito species.

Conflict of Interest Statement
We declare that we don’t have any conflict of interest.

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

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