Introduction

Many dreadful diseases are transmitted by different mosquito species. Culex quinquefasciatus Say, 1823 (Cx. quinquefasciatus) is the main vector of filarial diseases, and more than 100 million people per year become infected in more than 110 countries in the tropics by filariasis (WHO, 2006). Even now no vaccine is available to prevent this disease. To reduce the incidence of filariasis, eradication and controlling of mosquito vector is needed, mainly by applying insecticides to mosquito larval habitats (Arivoli and Tennyson, 2012). The wide spread use of synthetic insecticides for controlling pests and vectors, which transmit human diseases, have their toxicity on environments (Bonius, 2003). Plant products, which are easily degradable, are considered as one of the safest sources for controlling insect vectors and pests (Arivoli et al., 2012). Varieties of plants with insecticidal activities have been reported by many researchers to control mosquito larvae, pupae, and adult mosquitoes (Halder and Chandra, 2011; Hossain et al., 2011; Singha et al., 2011; Singha and Chandra, 2011; Halder et al., 2013; Mallick et al., 2014). The Genus, Annona consists of 119 species and are widely distributed in the tropical and sub-tropical countries. They are mainly trees and shrubs. The bark extract of A. reticulata is used as tonic (Thang et al., 2013). A. reticulata is also known as Ramphala. Plant becomes leaf less after maturity of fruits (Jani et al., 2012). Its native land is South America and West Indies and is cultivated in Pakistan and Bangladesh. It is used for the treatment (in Ayurvedic system) of various diseases like cardiac problems, dysentery, constipation, haemorrhage, worm infestation etc. (Kaleem et al., 2006) It is reported that leaves of A. reticulata plant have mosquito larvicidal activity (Nayek, 2014; Mallick et al., 2015; Mallick et al., 2015; Mallick and Chandra, 2015; Bavani et. al., 2015). Aqueous and ethanol root extracts of A. reticulata exhibited inhibitory effect against human cancer cell lines (range of concentrations between 10 and 40 µg/ml) (Suresh et  al., 2011). Cytotoxic gamma-lactone acetogenin, cis-/trans-isomurisolenin, along with six known cytotoxic acetogenins, annoreticuin, annoreticuin-9-one, bullatacin, squamocin, cis-/trans-bullatacinone and cis-/trans-murisolinone had been isolated from ethyl acetate seed extract of A. reticulata. Some of the compounds showed potent cytotoxic activity against cancer cell lines (Cheng et al., 1998). The present study was an attempt to examine the larvicidal efficacy of root and stem bark extracts of the plant, Annona reticulata against filarial vector, Culex quinquefasciatus.

 

1 Results

Table 1 depicts the mortality percent of chloroform: methanol (v/v 1:1), acetone, and ethanol solvent root extracts of A. reticulata against early 3^rd instar larvae of Cx. quinquefasciatus for a period of 24, 48, and 72 h of exposure. Percent mortality increased with increase in concentration of extract and time of exposure. LC₅₀ values of chloroform: methanol (v/v 1:1), acetone, and ethanol solvent root extracts were 0.4854, 0.9795 and 1.1368 ppm respectively against 3^rd instar larvae of Cx. quinquefasciatus after 24 h of exposure and corresponding LC₉₀ values were 1.7485, 2.5758 and 1.8354 ppm respectively. So, chloroform: methanol (v/v 1:1) solvent root extract was the most potent larvicidal agent among tested three solvent root extracts. Table 2 presents the percent mortality of chloroform: methanol (v/v 1:1), acetone, and ethanol solvent stem bark extracts of A. reticulata against early 3^rd instar larvae of Cx. quinquefasciatus and LC₅₀ and LC₉₀ values were 0.5397, 0.8199, and 1.3371 ppm, and 1.3747, 1.9983 and 3.1998 ppm respectively after 24 h of exposure. Table 3 and Table 4 show the percent mortality of chloroform: methanol (v/v 1:1) solvent root and stem bark extract with very low concentrations against 1^st – 4^th instars larvae of Cx. quinquefasciatus. Percent mortality increased with increase in concentrations of extracts and time of exposure. 1^st instar larvae were most susceptible to chloroform: methanol (v/v 1:1) solvent root and stem bark extracts and showed 100% mortality with very low concentrations. Table 5 and Table 6 present the LC₅₀, LC₉₀, regression equations and R² values of larvicidal activity of chloroform: methanol (v/v 1:1) solvent root and stem bark extract respectively. LC₅₀ and LC₉₀ values gradually decreased with time of exposure and there was strong correlation between percent mortality and concentration of the extract of root and stem bark as R² values were close to 1 in most cases. Three way factorial ANOVA established statistical significance of larval mortality of Cx. quinquefasciatus (p <0.05) in terms of instars, times and concentrations of root and stem bark extract of A. reticulata (Table 7 and Table 8). Tested non- target organisms did not respond to the chloroform: methanol (v/v 1:1) solvent root and stem bark extracts.

 

Table 1 Percent mortality of early 3rd instar larvae of Culex quinquefasciatus exposed to different concentrations of different solvent root extracts of Annona reticulata (Mean percent mortality ± Standard error)

 

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

 

Table 3 Percent mortality of different instars of Culex quinquefasciatus exposed to different concentrations of chloroform: methanol (v/v 1:1) root extract of Annona reticulata (Mean percent mortality ± Standard error)

 

Table 4 Percent mortality of different instars of Culex quinquefasciatus exposed to different concentrations of chloroform: methanol (v: v 1:1) stem bark extract of Annona reticulata (Mean mortality percent ± Standard error)

 

Table 5 Log probit and regression analyses of larvicidal activity of chloroform: methanol (v/v 1:1) root extract of Annona reticulata against different larval instars of Culex quinquefasciatus

 

Table 6 Log probit and regression analyses of larvicidal activity of chloroform: methanol (v/ v 1:1) stem bark extract of Annona reticulata against different larval instars of Culex quinquefasciatus

 

Table 7 Completely randomized three way ANOVA analyses using instars (I) of Culex quinquefasciatus, hours (H), and concentrations of chloroform: methanol (v: v 1:1) root extract of Annona reticulata (C) as three independent parameters

 

Table 8 Completely randomized three way ANOVA analyses using instars (I) of Culex quinquefasciatus, hours (H), and concentration of chloroform: methanol (v/v 1:1) stem bark extract of Annona reticulata (C) as three independent parameter

 

2 Discussions

Chloroform: methanol (v/v 1:1), acetone, and ethanol solvent root and stem bark extracts of the plant (A. reticulata) have excellent larvicidal efficacy with very low dosages against Cx. quinquefasciatus mosquito. Among the three solvent extracts of root and stem bark of the plant, chloroform: methanol (v/v 1:1) solvent extract possess best larvicidal activity against Cx. quinquefasciatus. Several researchers worked with root and stem bark extract of different plants and showed larvicidal activity against different mosquito species. Mallick and Chandra (2015) worked with stem bark of A. reticulata with petroleum ether, hexane and ethyl acetate extracts and showed larvicidal activity against 3^rd instar larvae of Cx. quinquefasciatus with LC₅₀ values of 15.2967, 14.0390, and 1.0902 ppm respectively after 48 h of exposure. Present study revealed that chloroform: methanol (v/v 1:1), acetone, and ethanol solvent stem bark extracts of A. reticulata showed LC₅₀ values of 0.5397, 0.8199 and 1.3371 ppm after 24 h of exposure against early 3^rd instar larvae of Cx. quinquefasciatus indicating again chloroform: methanol (v/v 1:1) as the best extractive. Nikkon et al. (2009) reported the larvicidal efficacy of stem and fruits of Duranta repens against the Cx. quinquefasciatus mosquito and showed LC₅₀ values of chloroform extract of stem of D. repens as 10.75 14.06, 26.63, 36.53 ppm against 1^st - 4^th instars larvae respectively. Kholhring (2011) reported the larvicidal activity of ethanolic root bark extract of Millettia pachycarpa against late 3^rd or early 4^th instars larvae of Aedes aegypti and showed 100% mortality at a dose 200 mg/L  having LC₅₀ value of 98.47 mg/L after 24 h of post exposure. Mallick and Chandra (2015)  reported the larvicidal activity of petroleum ether, hexane and ethyl acetate root extract of A. reticulata against 3^rd instar larvae of Cx. quinquefasciatus mosquito species having LC₅₀ values 10.4990, 9.7022, and 0.9191 ppm after 24 h of exposure respectively. However, chloroform: methanol (v/v 1:1), acetone, and ethanol solvent root extracts of A. reticulata showed LC₅₀ values of 0.4854, 0.9795 and 1.1368 ppm respectively after 24 h of exposure against early 3^rd instar larvae of Cx. quinquefasciatus mosquito species. So, among all the solvent root extracts of A. reticulata, chloroform: methanol (v/v 1:1) solvent root extract was the best extract as larvicidal agent.

 

3 Conclusion

Excellent results of the present study clearly indicate that the chloroform: methanol (v/v 1:1) root and stem bark extracts of A. reticulata can effectively be used as larvicidal agents against Cx. quinquefasciatus mosquito. Further study is needed to find out the active chemical compound (S) involved in larvicidal activity of chloroform: methanol (v/v 1:1) root and stem bark extracts of A. reticulata and also their effects on other mosquito species.

 

4 Materials and Methods

4.1 Collection of plant materials

Root and stem bark of A. reticulata plant (aged about one to four years) were collected during the month of September and October, 2014 from Burdwan town, West Bengal, India (23°16' N, 87°54' E). The plant was identified properly and the voucher specimen was deposited to the herbarium (voucher no. GCZSM-4) of the Mosquito, Microbiology and Nanotechnology Research Units, Parasitology Laboratory, Department of Zoology, The University of Burdwan, West Bengal, India. Root and stem bark of the plant were washed clearly with running tap water and then rinsed with distilled water. 

 

4.2 Preparation of solvent extracts

Cleaned root and stem bark of the plant were cut into small bits separately and dried in shade for 15-18 days. 150 g shade dried small bits of root as well as stem bark was packed into the column of Soxhlet apparatus separately and each solvent (1500 ml) of chloroform: methanol (1:1 v/v), acetone, and ethanol was passed through the sample packed column of Soxhlet apparatus. For each solvent extraction, fresh dried root as well as stem bark of the plant was used. Extraction period for dried root and stem bark of the plant with each solvent was 72 h. Then each solvent extractive of root and stem bark were kept on beakers after filtering with Whatman No. 1 filter paper separately. After evaporation of each solvent, semi solid extractives of root and stem bark of each of chloroform: methanol (v/v 1:1), acetone, and ethanol solvents were obtained. Semi solid extractives of each solvent of root and stem bark were stored in a refrigerator at 4º C for further larvicidal bioassay experiments.

 

4.3 Preparation of different concentrations for larvicidal bio-assay experiments

After preliminary trialing, 1, 2, 4, and 8 ppm concentrations of each of chloroform: methanol (1:1 v/v), acetone, and ethanol solvent root and stem bark extracts were used against early 3^rd instar larvae of Cx. quinquefasciatus for larvicidal bioassay experiments. 0.4, 0.6, 0.8, and 1.0 ppm concentrations of chloroform: methanol (1:1 v/v) root and stem bark extracts were used against early 1^st - 4^th instars larvae of Cx. quinquefasciatus for larvicidal bioassay experiments. 0.05 gm each of different semisolid chloroform: methanol (1:1 v/v), acetone and ethanol solvent root and stem bark extracts were dissolved initially in 5 ml of ethanol and then added 95 ml of distilled water to obtain 100 ml of stock test solution of different tested solvent extracts. So stock solution of different solvent extracts of root and stem bark were prepared on 5% ethanol. From stock test solution, tested different concentrations of root as well as stem bark extracts were prepared through dilution with tap water.

 

4.4 Test mosquito species

Larvae of Cx quinquefasciatus were collected from drains of Burdwan town and identified properly. Larvae were kept in a plastic tray with tap water. Powdered mixtutre of dog biscuits and dried Brewer’s yeast powdered in the ratio 3:1 were supplied for their feeding. 27±2°C temperature, 85% relative humidity, and 14:10 light and dark cycles in a day were well maintained in the laboratory. Larvae transformed into pupae and were transferred from plastic tray to 500 ml beaker containing tap water and the beaker 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.

 

4.5 Larvicidal bioassays

Larvicidal bioassays were done according to the standard protocol of WHO with suitable modification (WHO, 2005). Thirty larvae were put in plastic bowls containing 100 ml of test solutions of different concentrations ( mentioned earlier) of each of different solvent (mentioned earlier) extracts of root and stem bark. Ethanol treated control experiments were set on 100 ml of tap water with 0.5 ml of ethanol. Each set of experiment was replicated three times with three replicates of control 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 unabled to move after touching the siphon or cervical region with a fine brush.

 

4.6 Effect on non target organisms

The effect of chloroform: methanol (1:1 v/v) root and stem bark extracts of A. reticulata were investigated on non target organisms like Chironomus circumdatus larvae and tadpoles of toad with LC₅₀ value of chloroform: methanol (1:1 v/v) root and stem bark extracts of A. reticulata against 3^rd instar larvae of Cx. quinquefasciatus after 24 h post exposure period to note the mortality and other abnormalities up to 72 h of post exposure.

 

4.7 Statistical analyses

STAT PLUS 2009 - Trial version (computer software), and MS EXCEL - 2007 were used to calculate the LC₅₀, LC₉₀ (95% confidence level) through Log probit analyses, regression equations, coefficient of determinations (R²), ANOVA, mean mortality percent, and standard error.

 

Acknowledgement

We are thankful to Professor Dr. A Mukhopadhyay, Department of Botany, The University of Burdwan, for his kind help for identification of the plant species. We are also grateful to UGC-DRS for providing financial support.

 

Conflict of interest

We have no conflict of interest.

 

References

Arivoli S., John R.K., Raveen R., and Tennyson S., 2012, Larvicidal activity of botanicals against the filarial vector Culex quinquefasciatus Say (Diptera: Culicidae), Int. J. Res. Zoo., 2(1): 13-17

 

Arivoli S., and Tennyson S., 2012, Larvicidal efficacy of Strychnos nuxvomica Linn. (Loganiaceae) leaf extracts against the filarial vector Culex quinquefasciatus Say (Diptera: Culicidae), World J. Zoo., 7 (1): 06-11

 

Bavani G., Srimathi A., Bhuvana R., and Karthikeyan J., 2015, Mosquito larvicidal efficacy of the leaf extracts of Annona reticulata against Aedes aegypti, Int. J. Curr. Microbiol. App. Sci., 4(8): 132-140 

 

Bounias M., 2003, Etiological factors and mechanism involved in relationships between pesticide exposure and cancer, J. Env. Biol., 24(1): 1-8

 

Cheng F.R., Chen J.L., Chiu H.F., Wu M.J., and Wu Y.C., 1998, Acetogenins from seeds of A. reticulata, 47(6): 1057-61

 

Halder K.M., Ghosh P., and Chandra G., 2011, Evaluation of target specific larvicidal activity of the leaf extract of Typhonium trilobatum against Culex quinquefasciatus Say, Asian Pac. J. Trop. Biomed., 1(2): S199-S203 

http://dx.doi.org/10.1016/S2221-1691(11)60156-1

 

Halder K.M, Halder B., and Chandra G., 2013, Fabrication, characterization and mosquito larvicidal bioassay of silver nanoparticles synthesized from aqueous fruit extract of putranjiva, Drypetes roxburghii (Wall.), Parasitol. Res., 112(4): 1451-9 

http://dx.doi.org/10.1007/s00436-013-3288-4

 

Hossain E., Rawani A., Chandra G., Mandal S.C., and Gupta J.K., 2011, Larvicidal activity of Dregea volubilis and Bombax malabaricum leaf extracts against the filarial vector Culex quinquefasciatus, Asian Pac. J. Trop. Med., 4: 436-441

http://dx.doi.org/10.1016/S1995-7645(11)60121-1

 

Jani S., Harisha C.R., and Mohaddesi B., 2012, Detailed comparative pharmacognostical study of Annona squamosa Linn. and Annona reticulata Linn. Leaves, JPSI, 1(5): 34-38 

 

Kaleem M., Asif M., Ahmad Q.U., and Bano B., 2006, Antidiabetic and antioxidant activity of Annona squamosa extract in streptozotocin induced diabetic rats, Singapore Med. J., 47:670-675

 

Kholhring L., 2011, Mosquitocidal activity of Millettia pachycarpa on the larvae and eggs of Aedes aegypti, Ann. Biol. Res., 2(3): 217-222

 

Mallick S., and Chandra G., 2015, Larvicidal activities of extracts of stem bark of Annona reticulata against filarial vector Culex quinquefasciatus, Int. J. Pharm. Bio. Sci., 6(3): (B): 1347-1356

 

Mallick S., and Chandra G., 2015, larvicidal, pupicidal, oviposition deterrent activity and smoke toxicity of mature leaf extracts of Annona reticulata Linn. against filarial vector Culex quinquefasciatus Say, Int. J. Pharm. Bio. Sci., 6(4): (B): 244-253

 

Mallick S., and Chandra G., 2015, Larvicidal potentiality of root extracts of Annona reticulata Linn. against the filarial vector Culex quinquefasciatus Say (Diptera: Culicidae). J. Mosq. Res., 5(10): 1-7

 

Mallick S., Banerjee R., and Chandra G., 2015, Mosquito larvicidal potential of ethanol leaf extract of the plant Annona reticulata. against Aedes aegypti L. and Culex quinquefasciatus Say (Diptera: Culicidae), J. Mosq. Res., 5(19): 1-7

 

Mallick S., Bhattacharya K., and Chandra G., 2014, Mosquito larvicidal potentiality of wild turmeric, Curcuma aromatica rhizome extracts against Japanese Encephalitis vector Culex vishnui group, J. Mosq. Res., 4(19): 1-6

 

Mallick S., Mukherjee D., and Chandra G., 2015, Evaluation of larvicidal efficacy of acetone leaf extracts of Annona reticulata Linn. against Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus (Diptera: Culicidae), J. Mosq. Res., 5(9): 1-7 

 

Nayak J.B., 2014, Efficacy of crude extract of Annona reticulata and Pongomya piñata as larvicidal for the management of filarial vector Culex quinquefasciatus Say Diptera: Culicidae.  Int. J. Res. Bot., 4(1): 1-5

 

Nikkon F., Saud Z.A., Hossain K., Parvin M.S., and Haque M.E., 2009, Larvicidal effects of stem and fruits of Duranta repens against the mosquito Culex quinquefasciatus, Int. J. Pharm. Tech. Res., 1(4): 1709-1713 

 

Singha S., and Chandra G., 2011, Mosquito larvicidal activity of some common spices and vegetable waste on Culex quinquefasciatus and Anopheles stephensi, Asian Pac. J. Trop.Med., 4(4): 288-293

http://dx.doi.org/10.1016/S1995-7645(11)60088-6 

 

Suresh H.M., Shivakumar B., Hemlatha K., Heroor S.S., Hugar D.S., and Rao K.R., 2011, In vitro antiproliferative activity of Annona reticulate roots on human cancer cell lines, Pharmacognosy Res., 3(1): 9-12

 

Thang T.D., Kuo P.C., Huang G.J., Hung N.H., Huang BS, Yang M.L., Luong N.X., and Wu T.S., 2013, Chemical constituent from leaves of Annona reticulata and their inhibitory effects on NO production, Molecules, 18: 4477-4486 

http://dx.doi.org/10.3390/molecules18044477

 

World Health Organization, 2005, Guidelines for laboratory and field testing of mosquito larvicides. Geneva: WHO (WHO/CDS/WHOPES/GCDPP/2005.13) 

 

World Health Organization, 2006, Global program to eliminate lymphatic filariasis, Wkly. Epidemiol. Rec., 81: 221-32