1 Introduction                                                                  

Mosquito is one of the most notorious creatures of this world, causes nuisance by its bites and transmits deadly diseases like malaria, filariasis, yellow fever, dengue, Japanese encephalitis etc. and contribute significantly to poverty and social hindrance in tropical countries (Jang et al., 2002). Japanese encephalitis is a vector borne viral disease caused by JE virus (JEV). In a zoonotic cycle, JEV is maintained both in enzootic and epizootic cycles. In JEV life cycle, pigs are involved as the major amplifying reservoir host, water birds as carriers and mosquitoes as vectors. JEV is a major cause of viral encephalitis in South- East Asia, affecting more than 50,000 patients annually (Mackenzie et al, 2007). The Cx. vishnui groups of mosquitoes consisting of Cx. vishnui Theobald, Cx. pseudovishnui Colless and Cx. tritaeniorhynchus Giles have been implicated as major vectors of JE virus.  However from 16 species of mosquitoes JEV has been isolated in India (Solomon, 2007).

To combat the devastating scenario of fatal diseases, the control of mosquito population is essential. To the common people, killing of adult mosquitoes is the most common and easy way to control mosquito population. In ancient time, a wide range of insecticides were in a practice to control the mosquito population in adult stage. But now-a-days this attempt has been shifted towards larval control before they become able to transmit the serious diseases. So controlling mosquitoes in their immature stages is more rational than destroying adults and also easy since their breeding habitat is water. In the cosmo tropical areas public health authorities have been using hazardous synthetic insecticides as the principal means to control the high density of mosquito population. Biological control methods include the exploitation of natural enemies of targeted mosquitoes and also biological toxins to achieve effective vector management (Ansari et al., 2000). Among them medicinal plant products are the best alternative to synthetic chemical insecticides (Shaalan et al., 2005; Bhattacharya and Chandra, 2013; Singha and Chandra, 2011; Hossain et al., 2011). The plant products have several advantages as they showed their efficacy at very low test concentrations towards the target species, there is very less to no ill-effect on non-target organisms, field application is very easy, and there is no environmental pollution due to persistence as they are easily degradable (Rawani et al., 2012; Haldar et al., 2011; Bhattacharya and Chandra, 2014; Chowdhury et al., 2008a; Chowdhury et al., 2008b). Synergistic effect of two or more plant (Singha et al., 2011) or synthesized nano particles from plants (Haldar et al., 2013; Rawani et al., 2013a) also gives an alternating way in mosquito control programme. There is no or little evidence of resistance development in target mosquitoes.

A. auriculiformis, belongs to fabaceae family is raised as an ornamental plant, as a shade tree and it is also raised on plantations for fuel wood throughout southeast Asia, Oceania and in Sudan. Its wood is good for making paper, furniture and tools. It contains tannin useful in animal hide tanning. In India, its wood and charcoal are widely used for fuel. Gum from the tree is sold commercially, but it is said not to be as useful as gum arabic. The tree is used to make an analgesic by indigenous Australians. Extracts of A. auriculiformis heartwood inhibit fungi that attack wood. The fruit is irregular in shape, 1 to 3 inches in length, dry and hard covering, brown in colour. Seeds are rich source of triterpenoid saponin especially Acaciaside A (Ac-A) and Acaciacide-B (Ac-B). Ac-B isolated from seed extract shows spermicidal activity (anti fertility) in lower concentration (Pakrashi et al., 1991; Pal et al., 2009) and most importantly inhibits transmission of HIV (Kabir et al., 2008) without any mutagenic effect. Bark extract of   A. auriculiformis also shows some pesticidal activity.

The purpose of the present study was to evaluate the mosquitocidal activity of five solvent extracts of fruits of A. auriculiformis against Cx. vishnui group.

2 Material and Method          

2.1 Collection of Plant material

Fresh and matured fruits of A. auriculiformis were harvested randomly during April – June, 2011, at outskirts of Burdwan (23°16'N, 87°54'E). The plant was identified and submitted as herbarium (Voucher No: GCMB-06) in the Department of Zoology, The University of Burdwan. Fruits were cleaned with tap water, washed with distilled water and soaked on a paper towel.

2.2 Collection and rearing of larvae

The eggs of Cx. vishnui group were collected from rice field near the University campus. They were reared and maintained at 28 ± 2°C temperature and 85% RH in Mosquito Microbiology and Nanotechnology Research units, Parasitology laboratory, Department of Zoology, The University of Burdwan. The eggs were immersed in dechlorinated tap water in enamel tray of 30 cm diameter. The hatched larvae were fed with artificial food (dried yeast powder and powder of dog biscuits in the ratio of 1:3). They were maintained at 28 ± 2ºC. The transformed pupae were separated manually with a glass dropper into a 500 ml beaker with water and introduced into adult cages of 12.6” X 10” X 6” for adult emergence. A cotton ball soaked in 10% glucose solution was used for glucose meal of adult mosquitoes and was periodically blood fed on immobilized pigeon.

The adults were identified by Barraud (1934), Christophers (1933) and Chandra (2000).  Eggs laid were similarly reared and 1st generation laboratory breed larvae were used for bioassay and control experiments.

2.3 Preparation of different solvent extracts

Air dried 250 g mature fruits of A. auriculiformis were crushed and put in the thimble of Soxhlet apparatus and five different solvents, namely petroleum ether, n-hexane, chloroform: methanol (1:1, v/v), acetone and absolute alcohol were applied one after another (extraction period 72 h for each solvent with 8 h maximum a day). The extracts were collected separately, and the column of the Soxhlet apparatus was washed with 200 ml of water and 100 ml of a similar solvent as an eluent after each type of solvent extraction procedure. The eluted material of each type of extract was concentrated by evaporation in a rotary evaporator.

2.4 Dose response larvicidal bioassay

The larvicidal bioassay followed the World Health Organization (1981) standard protocols. Twenty five 3^rd instars larvae of Cx. vishnui group were put in separate glass Petri-dishes (9 cm diameter/150 mL capacity) containing 100 mL of tap water. Three concentrations of each solvent extracts (100 ppm, 200 ppm and 300 ppm) were applied into separate Petri-dishes to investigate the mortality. Dried yeast powder (920 mg) was added in each Petri dish as larval food. A similar type of bioassay was conducted with only distilled water without any of the solvent extracts of the matured fruits against 3^rd larval instars of Cx. vishnui group separately as control. Larval mortalities were recorded after 24 h, 48 h and 72 h of exposure. The experiments were repeated thrice and maintained at 25-30°C and 80-90% relative humidity.

2.5 Effect on non-target organism

The effect of ethyl acetate solvent extract was also studied on non-target organisms such as Daphnia sp, Diplonychus annulatum and Chironomus circumdatus larvae. Non targets were exposed to lethal concentration (LC₅₀ value of chloroform: methanol (1:1v/v) solvent extract at 24h against 3^rd instar larvae) to observe mortality and other abnormalities such as sluggishness and reduced swimming activity up to 72 h of post exposure.

2.6 Statistical analyses

The percentage mortality observed (% M) was corrected using Abbott’s formula (Abbott, 1925). Statistical analysis include the LC₅₀, LC₉₀ regression equation (Y=mortality; X=concentration) and regression coefficient value. ANOVA was carried out using different concentration, different instars and hours as variable to justify the significance between the above parameter and mortality rate.

3 Result

Among the five solvent extracts, the highest mortality (76% mortality) was observed in chloroform: methanol (1:1v/v) solvent extract at 300 ppm concentration against 3^rd larval instars and was significantly (p<0.05) higher than 100 ppm, 200 ppm concentrations at 24 h, 48 h and 72 h of exposure (Table 1).

The order of efficacies of different solvent extracts on 3^rd instars larvae of Cx. vishnui group mosquito were chloroform: methanol (1:1 v/v) > absolute alcohol > n-Hexane > acetone > petroleum ether. The result of log probit analysis and regression analysis showed that LC₅₀ values gradually decreased with exposure periods and mortality rate (Y) was positively correlated with concentration of exposure (X) having  regression coefficient (R) value close to one (Table 2).

Result of three way ANOVA analyses using different solvent extract, concentration and hours of exposures as variable are presented in Table 3.

The Tukey’s test revealed significant difference in mortality rates in different solvent extracts (Table 4) indicating significant difference in mortality rates between all three days of exposures in all solvent extracts (Table 5). No mortality and changes in swimming activity of the non-target organisms, e.g. Daphnia sp., D. annulatum and C. circumdatus were observed within 72 h of post-exposure to a certain concentration of chloroform: methanol (1:1v/v) solvent extract.

4 Discussion

Use of synthetic chemical insecticides brought many problems like persistence in the environment, development of resistance in the insect, bioaccumulations etc. More a function of frequency of use and persistence there is more development of resistance. So, there is an urge to find an alternative way that can substitute the use of chemical insecticides, which are effective, biodegradable, reducing the likelihood of resistance development. To combat with this problem, medicinal plants now a day are in more demands. They are extensively used in traditional medicine practices worldwide because of availability, low cost, proven efficiency and negligible side effects (Martin-Bettolo, 1980; Singh et al., 2015). Phytochemical based research works have been effectively published in many well established journals (Bhattacharya et al., 2014; Singh Ray et al., 2014; Mukherjee et al., 2015). These plants are not only used as a biocontrol agent but known to contain physiologically active principles that have been explored, incorporated and exploited in traditional medicine for the treatment of various diseases including ulcers in human and animals also (Sokmen et al., 1999; Kelmanson et al., 2000; Srinivasan et al., 2001; Chen et al., 1989). The beneficial medicinal effects of plant materials typically result from the combination of secondary products present in the plant. The present study evaluates the larvicidal activity of five solvent extracts against Cx. vishnui group. Highest mortality was observed at 300 ppm concentration of chloroform: methanol (1:1 v/v) solvent extract with a LC₅₀ value of 117.27 ppm. The activity of chloroform: methanol (1:1 v/v) extract of many other plants has been reported by several authors. Chowdhury et al., (2009) reported activity of Chloroform: methanol extract (1:1) of leaf of Solanum villosum against Anopheles subpictus. All the graded concentrations (30, 50, 100 and 200 ppm) of chloroform: methanol extracts (1:1) showed significant (P < 0.05) larval mortality. LC₅₀ values for all instars were between 24.20 and 33.73 ppm after 24 h and between 23.47 and 30.63 ppm after 48 h of exposure period. Similarly the activity of chloroform: methanol extracts (1:1) of leaf of Cestrum diurnum was tested against An. stephensi by Ghosh and Chandra (2006). The LC₅₀ value of the active ingredient was determined as 0.70, 0.89, 0.90 and 1.03 mg/100mL for 1st, 2nd, 3rd and 4^th instars larvae respectively in 24 h study period against An. stephensi. Ghosh et al., (2008) reported the efficacy of chloroform: methanol extract (1:1) of leaf of C. diurnum against Cx. quinquefasciatus. The LC₅₀ value calculated was 0.29, 0.35, 0.57 and 0.65 % for 1st, 2nd, 3rd and 4th instar larvae at 24 h exposure period. Chowdhury et al., (2008b) also studied the activity of chloroform: methanol extract (1:1) of berry of Solanum villosum against Ae. aegypti. The lowest LC₅₀ value calculated was 5.97 ppm at 72 h of bioassay experiment. Rawani et al., (2013b) studied the crude and chloroform: methanol (1:1 v/v) extracts of fresh, mature, green berries of S. nigrum against Cx. quinquefasciatus. In the chloroform: methanol (1:1, v/v) solvent extract, the maximum mortality was recorded at a concentration of 120 μg/mL. The log probit analysis (95% confidence level) recorded lowest LC₅₀ value at 72 h of exposure. Singha and Chandra, (2011) evaluated efficacy of chloroform: methanol (1:1 v/v) extract of four vegetable waste viz. Allium sativum, Cuminum cyminum, Zingiber offinale, Curcuma longa and Solanum tuberosum germinated tuber against the 3^rd  instar larvae of Cx. quinquefasciatus and An. stephensi larvae. At 75 ppm concentration of chloroform: methanol (1:1 v/v) extract of Curcuma longa showed highest mortality to 3^rd instars larvae of both Cx. quinquefasciatus and An. stephensi. The practice of only mosquito control would  not bring out the desired results against vector control, importance should be given to the broad aspect of mosquito control approaches comprises of the use of insecticides, biocontrol agents and environmental managements.

In conclusion, fruit of A. auriculiformis offers a potential larvicidal agent against Cx. vishnui group larvae. However, further studies are required to know the final active ingredient and also evaluate their mode of action. Field trials are also needed to recommend A. auriculiformis as a potent mosquitocidal agent.

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 identification.

References

Abbott W.S., 1925, A method of computing the effectiveness of an insecticide, Journal of Economic Entomology, 18(2): 265-267

http://dx.doi.org/10.1093/jee/18.2.265a

Ansari M.A, Vasudevan P., Tandon M., Razdan R.K., 2000, Larvicidal and mosquito repellent action of peppermint (Mentha piperita) oil, Bioresourse Technology, 71(3): 267-71

http://dx.doi.org/10.1016/S0960-8524(99)00079-6

Barraud P.J., 1934, The fauna of British India, including Ceylon and Burma. Diptera vol- IV. Family Culicidae. Tribes Megarhinini and Culicini. London: Taylor and Francis: p. 463

Bhattacharya K., Chandra G., 2013, Bioactivity of Acyranthes aspera (Amaranthaceae) Foliage against the Japanese Encephalitis Vector Culex vishnui Group, Journal of Mosquito Research, 3(13): 89-96

Bhattacharya K., Chandra G., 2014, Bioactivity of Acyranthes aspera (Amaranthaceae) Foliage against the Japanese Encephalitis Vector Culex vishnui Group, Journal of Mosquito Research, 3(13): 89-96

Bhattacharya K., Burman S., Nandi S., Roy P., Chatterjee D., Chandra G., 2014, Phytochemical extractions from the leaves of Ravenala madagascariensis from Sundarban area and its effect on southern house mosquito Culex quinquefasciatus Say (1823) larvae, Journal of Mosquito Research, 4(12): 1-6  doi: 10.5376/jmr.2014.04.0012

http://dx.doi.org/10.5376/jmr.2014.04.0012

Chandra G., 2000, Mosquito, Calcutta : Sribhumi Publication Co., p 1-102.

Chen C.P, Lin C.C, Namba T., 1989, Screening of Taiwanese crude drugs for the antibacterial activity against Streptococcus mutans, Journal of  Ethnopharmacology, 27: 285- 295

http://dx.doi.org/10.1016/0378-8741(89)90003-2

Chowdhury N., Laskar S., Chandra G., 2008a, Mosquito larvicidal and antimicrobial activity of protein of Solanum villosum leaves, BMC Complementary and Alternative Medicine, 8: 62

http://dx.doi.org/10.1186/1472-6882-8-62

Chowdhury N., Ghosh P., Chandra G., 2008b, Mosquito larvicidal activities of Solanum villosum berry extract against the dengue vector Stegomyia aegypti, BMC Complementary and Alternative Medicine, 8: 10

http://dx.doi.org/10.1186/1472-6882-8-10

Chowdhury N., Chatterjee S.K., Laskar S., Chandra G., 2009, Larvicidal activity of Solanum villosum Mill (Solanaceae: Solanales) leaves to Anopheles subpictus Grassi (Diptera: Culicidae) with effect on non-target Chironomus circumdatus Kieffer (Diptera: Chironomidae), Journal of Pest Science, 82: 13-8

http://dx.doi.org/10.1007/s10340-008-0213-1

Christophers S.R., 1933, The fauna of British India, including Ceylon and Burma. Diptera vol. IV. Family Culicidae. Tribes Anophelini. London: Taylor and Francis. p. 371

Ghosh A., Chandra G., 2006, Biocontrol efficacy of Cestrum diurnum (L.) (Solanales: Solanaceae) against the larval forms of stephensi, Natural Product Research, 20 : 371-9

http://dx.doi.org/10.1080/14786410600661575

Ghosh A., Chowdhury N., Chandra G., 2008, Laboratory evaluation of a phytosteroid compound of mature leaves of Day Jasmine (Solanaceae: Solanales) against larvae of Culex quinquefasciatus (Diptera: Culicidae) and nontarget organisms, Parasitology Research, 103: 271-7

http://dx.doi.org/10.1007/s00436-008-0963-y

Haldar K.M, Ghosh P., Chandra G., 2011, Evaluation of target specific larvicidal activity of the leaf extract of Typhonium trilobatum against Culex quinquefasciatus Say, Asian Pacific Journal of Tropical Biomedicine,  S199-S203

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

Haldar K.M, Haldar B., Chandra G., 2013, Fabrication, characterization and mosquito larvicidal bioassay of silver nanoparticles synthesized from aqueous fruit extract of putranjiva, Drypetes roxburghii (Wall.), Parasitology Research, DOI 10.1007/s00436-013-3288-4

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

Hossain E., Rawani A., Chandra G., Mandal S.C., Gupta J.K., 2011, Larvicidal activity of Dregea volvubilis and Bombax malabaricum leaf extracts against the filarial vector Culex quinquefasciatus, Asian Pacific Journal of Tropical Medicine, 436- 441

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

Jang Y.S., Baek B.R., Yang Y.C., Kim M.K., Lee H.S., 2002, Larvicidal  activity of leguminous seeds and grains against Aedes  aegypti and Culex pipiens pallens, Journal of American Mosquito Control Association, 18: 210-213

Kabir S.N., Ray H.N., Pal B.C., Mitra D., 2008, Pharmaceutical composition having virucidal and spermicidal activity. United State Patent and Trademark Office (USPTO).  Application #: 20080300197

Kelmanson J.E., Jager A.K., Van Staden J., 2000, Zulu medicinal plants with antibacterial activity, Journal of Ethnopharmacology, 69: 241- 246

http://dx.doi.org/10.1016/S0378-8741(99)00147-6

Mackenzie J.S., Williams D.T., Smith D.W., 2007, Japanese encephalitis virus: the geographic distribution, incidence, and spread of a virus with a propensity to emerge in new areas. In: Tabor E, editor. Emerging Viruses in Human Populations. Elsevier, Amsterdam, pp. 201–268

Martin-Bettolo G.B., 1980, Present aspect of the use of medicinal plants in traditional medicine, Journal of Ethnopharmacology, 2: 5-7

 http://dx.doi.org/10.1016/0378-8741(80)90021-5

Mukherjee D., Bhattacharya K., Chandra G., 2015, Extracts of edible pods of Moringa oleifera lam. (Moringaceae) as novel antibacterial agent against some pathogenic bacteria, International Journal of Pharma and Bio Science, 6(3):  513-520

Pakrashi A., Ray H., Pal B.C., Mahato S.B., 1991, Sperm immobilizing effect of triterpene saponins from Acacia auriculiformis, Contraception, 43(5):475–483

http://dx.doi.org/10.1016/0010-7824(91)90137-5

Pal D.P., Chakraborty H.N., Ray B.C., Pal D., Mitra A., Kabir S.N., 2009, Acaciaside-B-enriched fraction of Acacia auriculiformis is a prospective spermicide with no mutagenic property, Reproduction, 138:453–462

http://dx.doi.org/10.1530/REP-09-0034

Rawani A., Ghosh A., Laskar S., Chandra G., 2012, Aliphatic Amide from Seeds of Carica papaya as Mosquito Larvicide, Pupicide, Adulticide, Repellent and Smoke Toxicant, Journal of Mosquito Research, 2(2): 8-18. doi: 10.5376/jmr. 2012.02.0002

Rawani A., Ghosh A., Chandra G., 2013a, Mosquito larvicidal and antimicrobial activity of synthesized nano-crystalline silver particles using leaves and green berry extract of Solanum nigrum L, Acta Tropica, 128: 613-622

http://dx.doi.org/10.1016/j.actatropica.2013.09.007

Rawani A., Chowdhury N., Ghosh  A., Laskar S., Chandra G., 2013b, Mosquito larvicidal activity of Solanum nigrum berry extracts, Indian Journal of Medical Research, 137: 972-976

Shaalan E.A.S., Canyonb D., Younesc M.W.F., Abdel-Wahaba H., Mansoura A.H., 2005, A review of botanical phytochemicals with mosquitocidal potential, Environment International, 31: 1149–66

http://dx.doi.org/10.1016/j.envint.2005.03.003

Singha S., Chandra G., 2011, Mosquito larvicidal activity of some common spices and vegetable waste on Culex quinquefasciatus and Anopheles stephens, Asian Pacific J.ournal of Tropical Biomedicine, 288-293

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

Singha S., Banerjee S., Chandra G., 2011, Synergistic effect of Corton caudatus (fruits) and Tiliacora acuminate (flowers) extracts against filarial vector Culex quinquefasciatus, Asian Pacific Journal of Tropical  Biomedicine, s159-164

http://dx.doi.org/10.1016/S2221-1691(11)60147-0

Singh Ray A., Bhattacharya K., Singh A., Chandra G., 2014, Larvicidal Activity of Nelumbo nucifera Gaertn. (Nymphaeaceae) against Anopheles stephensi (Liston 1901) and its Effect on Non-target Organisms, Journal of Mosquito Research,  4(10): 1-7

Singh A., Bhattacharya K., Chandra G., 2015, Efficacy Of Nicotiana Plumbaginifolia (Solanaceae) Leaf Extracts As Larvicide Against Malarial Vector Anopheles Stephensi Liston 1901, International Journal of Pharma and Bio Science, 6(1): (B) 860- 868

Sokmen A., Jones B.M., Erturk M., 1999, The in vitro antimicrobial activity of Turkish medicinal plants, Journal of Ethnopharmacology, 67: 79-86

http://dx.doi.org/10.1016/S0378-8741(98)00189-5

Solomon T., 2007, Flavivirus encephalitis. The New England Journal of Medicine, 351: 370-8

http://dx.doi.org/10.1056/NEJMra030476

Srinivasan D., Nathan S., Suresh T., Perumalsamy O., 2001, Antimicrobial activity of certain Indian medicinal plants used in folkloric medicine, Journal of Ethnopharmacology, 74: 217-220

http://dx.doi.org/10.1016/S0378-8741(00)00345-7

World Health Organization., 1981, Instructions for determining the susceptibility or resistance of mosquito larvae to insecticides. WHO/VBC., 81: 807