Introduction

The vector mosquitoes and their infamous contribution in transmitting life threatening diseases like malaria, Japanese encephalitis, dengue, filariasis, yellow fever, etc are well-recognized. Culex vishnui group mosquitoes are considered as primary vectors for transmitting Japanese encephalitis. The natural reservoir for maintenance of Japanese encephalitis virus is birds of the family Ardeidae (herons and egrets) and pigs are amplifying hosts, from them mosquitoes get infected. Humans are vulnerable to Japanese encephalitis which is a primary public health concern in Asia. Literature survey on mosquitocidal studies on different mosquitoes reveals that studies on this specific culicines are limited which encouraged us to focus on the control strategy of Culex vishnui group mosquitoes.

Apart from food and clothes, thousands of life-saving drugs and medicines have been produced from plant sources which are increasing our average life expectancy. Plants have also been proved to be useful solution to get relief from the detrimental insects. Changsang (2005) reported about 344 different plant species along with their positive impact in this respective issue. Ghosh et al. 2012 reviewed almost 133 plant species having a potentiality to control different species of mosquitoes. Botanically derived chemicals have been projected as efficient armaments in mosquito control program as they have shown to function as general toxicant, repellents, growth and reproductive inhibitors, and oviposition deterrent to them. Other methods like synergistic effect of two or more plant (Singha et al., 2011) or the plant derived nano particles (Haldar et al., 2013) also gives a surprising result in mosquito control programme. Moreover, these plant derived solutions have advantages over synthetic poisonous chemicals as they are readily biodegradable thus eco-friendly, less harmful to non-target organisms and comparatively cheap.

Alangium salvifolium is a monogeneric plant belonging to the family Alangiiaceae (Figure 1). The shrub as well as the tree having a wide range of size from 3 feet to 12 feet and found mainly in Western Africa, Madagascar, Southern and Eastern Asia (China, Malaysia, Indonesia, India, and Philippines), tropical Australia, the western Pacific Ocean islands and New Caledonia (Balakrishnan et al., 2010) leaves are alternate, oblong, unequal, acute or rounded at base, lanceolate or oval, glabrous above and pubescent on veins beneath acuminate and obtuse at apex with 3-6 pairs of oblique veins. Leaves of the plant contain the alkaloids like ankorine, marckidine, marckine, tubulosine, alangicine, cephaeline, psychotrine, deoxytubulosine, alangimarckine, dehydroprotoemetine etc; steroids and triterpenoids. Leaves also yield monoterpenoid lactam, alangiside, loganic acid, venoterpine, dl-salsoline and isocephaeline; alkaloids. Seeds and bark contain alkaloids alangin A and B, alanginine. The plant is a very familiar folk medicine in Asia (Jain et al., 2005) reputed in curing several harmful illnesses like antifertility (Murugan et al., 2000), anti-inflammatory (Porchezhian et al., 2001), antimicrobial (Pandian et al., 2006), antioxidant (Jain et al., 2010), antitumor (Nahar et al., 2012) and anti-ulceric (Subudhi et al., 2012). Present study revels potentiality of this discussed plant Alangium salvifolium as controlling agent and smoke toxicant against the mosquitoes of Cx. vishnui group.

Figure 1 Leaves of A. salvifolium

1 Results

Both the crude and Chloroform: Methanol extracts of the plant leaves showed positive result in killing of the mosquito larvae. Findings showed that the mortality was highest at 0.5% crude concentration of leaf at 24 h in case of first larval instar followed by 2^nd and 3^rd, while 4^th instars having almost 50% mortality in same concentration (Figure 2-5). The LC₅₀ and LC₉₀ values determined by Log-Probit analysis (at 95% confidence level) also showed the positive result against Cx. vishnui group by gradual decrease in value with the exposer period of bioassay. From regression equation it was evident that for all four larval instars Y (mortality rate, dependent variable) was positively related to its corresponding X (dose, independent variable) and the values of  R² in all cases were nearer to 1 which indicates that the rate of mortality linearly increased with the increasing dose (Table 1). Figure 6 presents pupicidal activity of the crude leaf extract. The result reflects that 20% mortality in pupae was caused by 0.5% concentration of leaf extract. The Chloroform-Methanol extract showed considerable mortality of 3^rd instar larvae. The LC₅₀ value of the chloroform: methanol solvent extract was 48.89 ppm, while LC₉₀ value was 71.779 ppm at 24 h bioassay period (Table 2). Smoke toxicity effect of A. salvifolium leaf against blood fed adult Cx. vishnui group mosquitoes showed an impressive result. In three hours bioassay experiment, out of 500 adult mosquitoes 160 were died. This is attractive when compared to the commercial mosquito coil which shows 100% mortality. The effect is listed in Table 3. The crude leaf extract had no negative influence on survival rate and swimming activity of the two non-target organisms that share the same habitat of Cx. vishnui group mosquito larvae. In qualitative phytochemical analysis for secondary phytochemicals terpenoids, amino acid, free glycoside bound anthroquinones, tannins, flavonoids, and steroids were found to be present.

Figure 2 Effect of crude extract on first instar larvae

Figure 3 Effect of crude extract on second instar larvae

Figure 4 Effect of crude extract on third instar larvae

Figure 5 Effect of crude extract on fourth instar larvae

Figure 6 Effect of crude extract on pupae

Table 1 Log probit analysis and regression analysis of larvicidal activity of leaf crude extract against different larval instars of Culex vishnui group

Table 2 Efficacy of Alangium salvifolium leaf in Chloroform:Methanol solvent extract at different concentration on third instar of Culex vishnui group

Table 3 Smoke toxicity effect of Alangium salvifolium leaf powder, commercial mosquito coil and control mosquito coil without leaf extract on Culex vishnui group

2 Discussion

Organophosphate, organochlorides etc are common insecticides having swift effect in eliminating the vectors from the nature; at the same time they contribute prominent harmful effects on environment. To remove the setback of the synthetic chemical insecticides plant-based insecticides have renewed their importance in pest management including mosquito control. Several plant species have been established to have mosquitocidal potentiality it also includes some of common spices (Cuminum cyminum, Allium sativum, Zingiber offinale, Curcuma longa) and vegetables waste (Solanum tuberosum germinated tuber) reported by Singha et.al. (2011). Different plants contain complex chemicals with unique biological activities (Chowdhury et al., 2009; Haldar et al., 2011; Haldar et al., 2015). Jain et al. (2010) reported that aqueous and alcoholic extracts of the plant A. salvifolium showed a great effect against the Gram-positive bacteria (Staphylococcus aureus, Bacilus subtilis, Staphylococcus epidermis and Micrococcus luteus) and Gram-negative bacteria (Enterobacter aerogens, Escherichia coli, Salmonella typhi and Shigella dysenteriae). According to Porchezhian et al. (2001) this plant is well known for its analgesic and anti-inflammatory effect. Here in this experiment we have found that this plant is effective against the mosquitoes of Cx. vishnui group. We examined the effect of the crude extract on 1^st, 2^nd, 3^rd, and 4^th instar larvae of Cx. vishnui group and found that it works very efficiently on those larvae in respect to percent mortality. We found that the crude extract of the leaf showed 100% mortality in 0.5% concentration in case of third instars and first instars larvae while in second and fourth instar larvae it was 86.67 and 56.67 % respectively (24 hrs.). This study showed that the mortality of mosquito larvae of Cx. vishnui group exposed to the plant extracts increased with concentration of extracts as well as time of exposure as also supported by Obomanu et al. (2006).

Chowdhury et al. (2009) reported that in case of chloroform: methanol (v/v 1:1) extract of leaves of Solanum villosum against Anopheles subpictus 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. In case of Cestrum diurnum against Anopheles stephensi the LC₅₀ value of the active ingredient was determined as 0.70, 0.89, 0.90 and 1.03 mg/100 mL, for 1st, 2nd, 3rd and 4th instar larva respectively in 24 h study period (Ghosh and Chandra, 2006). On another study, Hossain et al. 2011 reported mosquito larvicidal potentiality of two plant Dregea volubilis and Bombax malabaricum against Culex quinquefasciatus. They reported that the Metahnolic extract of these plant having LC50 and LC90 value 56.97 ppm and 48.85 ppm respectively. In our study for the chloroform: methanol (v/v 1:1)  extract the LC₅₀ value is 48.89 ppm and the LC₉₀ is 71.799 ppm which are though slightly higher concentrations  than those reported by Ghosh and Chandra (2006) but better result is expected if it is purified.

Subramonia Thangam and Kathiresan (1992) smoke from burning various dry plant materials have been used since early times to deter insects, especially mosquitoes. In recent time also we use various types of commercial mosquito coils. Liu et al. (1987) reported the adverse effect of commonly used mosquito coils in Asia and South America. Emitted smoke from the burning mosquito coil contains submicron particles (i.e. diameter less than 1 μm) coated with considerable amount of heavy metals, allethrin and a wide range of organic vapors, such as phenol and o-cresol. Focusing on the adverse effects of smoke from commercial mosquito coils scientists attempted to invent plant based mosquito coils. Aarthi et al. (2010) characterized Spathodea campanulata as a potential smoke toxicant against Anopheles stephensi adults. It also has a goodanti-ovipositional response. Murugan et al. (2007) worked on the same field and selected two plants namely Albizzia amara and Ocimum basilicum to report that A. amara was more effective against Ades aegypti than the O. basilicum as a smoke toxicant. Present study we recorded almost 32% death of adult mosquito population during toxicity test of smoke originated from A. salvifolium. The data revealed the utility of this coil as an eco-friendly measure to control the adult mosquito to some extent. As during the present experiment on non-target organisms negative effect was not established it also could be incorporated in integrated pest management. Further research is required in this field for isolation and characterization active ingredients and exploration of the mechanism of action and field application.

3 Materials and Methods

3.1 Culture of test mosquito

Eggs of Cx. vishnui group mosquitoes were collected from the rice fields adjacent to university campus by random sweeping of net. They were hatched in laboratory. 1^st instar larvae were collected, reared and cultured with sufficient amount of food (crushed dog biscuit mixed with dried yeast in 3:1 ratio) upto pupal stage. Pupae were kept in a separate net chamber (for adult emergence). Sterilized wet cotton dipped into sucrose solution was put into the cage as food source of the adults. Adult females were periodically blood-fed on restrained rats.

3.2 Preparation of Crude phyto extracts

Experiment was conducted following the methodology of Haldar et al. (2010). Fresh, mature and green leaves of A. salvifolium were randomly harvested from the campus of The University of Burdwan, West Bengal, India. Collected leaves were rinsed well with distilled water and dried on paper towel. Crude extract were prepared by crushing the fresh leaves in an electric blender followed by the filtration of the extract with Whatman No.1 filter paper. The clean filtrate was used as stock solution (100% conc.) for this bioassay experiment. Required concentrations (0.1, 0.2, 0.3, 0.4, and 0.5%) were prepared through mixing of stock solution with sterile distilled water in suitable amount.

3.3 Preparation of solvent extract

Fresh leaves were harvested in large amount, cleaned and kept in a cool dark place for complete air drying. 150 gm of dried leaf were crushed and then placed in a Soxhlet apparatus for solvent extraction (72 h) in Chloroform: Methanol (1:1, v/v). After extraction the sample was collected and evaporated to accumulate the semi-solid remnant which was graded in concentrations of 40, 50, 60 and 70 ppm.

3.4 Larvicidal bioassay

All four instars (1^st, 2^nd, 3^rd, and 4^th) of Cx. vishnui group were experimented with crude and chloroform-methanol extracts for 3^rd instar larvae to evaluate the mortality rate at different concentrations. Extracts were transferred to separate sterile Petri dishes (9 cm diameter/150 ml capacity) containing five concentrations of crude extract (0.1, 0.2, 0.3, 0.4 and 0.5 %) and four concentrations of chloroform-methanol extract (40, 50, 60 70 ppm) with 100 ml of distilled water to study the rate of larval mortalities with a control treatment filled only with distilled water. Exact numbers of 25 mosquito larvae of each instar were given separately to the dishes.  Mortalities were recorded after 24 h, 48 h, and 72 h of exposures to plant extract respectively. The whole experiments were conducted at the temperature range of 25-30⁰C and relative humidity of 80-90% with three successive replicates.

3.5 Pupicidal bioassay

One day old pupae were selected for this experiment. Following the same procedure as larvicidal bioassay this experiment was set up. Crude extract was transferred to separate sterile glass dishes (9 cm diameter/150 ml capacity) containing five concentrations of crude extract (0.1, 0.2, 0.3, 0.4, and 0.5 %) with 100 ml of distilled water to study the percentage of pupal mortalities with a control treatment filled only by distilled water.

3.6 Preparation of mosquito coil

According to Singha et al. 2011 this experiment was carried out. Dried leaf of A. salvifolium was finely crushed. 2 gm of crushed leaf was mixed well with equal amount of sawdust and charcoal powder. A thick paste was made by pouring required amount of distilled water. Paste was shaped as a 4 mm thick mosquito coil for experimental use. A control coil was made by equal amount of sawdust and charcoal powder in exact size and shape without the plant material.

3.7 Smoke toxicity test

A glass chamber (140 cm×120 cm×60 cm) was used for this experiment having a single little door (6cm×6cm). Five hundred well-fed adult male and female mosquitoes were exposed to the smoke of burning coils for 3.30 h and activities of the mosquitoes were recorded after every 30 minutes interval. This same experiment was done for three times.

The survived blood fed mosquitoes were reared in a mosquito cage. To authenticate the data, two separate sets of control experiments were carried out, one with the control coil and another with the commercialized mosquito coil.

3.8 Effect on non target organisms

Non target organisms such as Diplonychus annulatum (adult) and Chironomus circumdatus (3^rd instar larvae) were collected from the same rice field and maintained at Mosquito, Microbiology and Nanotechnology Research Units, Parasitology laboratory, The University of Burdwan. Two co-existers of the mosquito larvae who used to share the same habitat were exposed to appropriate lethal concentration of Chloroform: Methanol solvent (as this solvent shows the highest mortality) extract for 24 h to observe the mortality and other abnormalities including sluggishness and reduced swimming activity up to 72 h of post exposure.

3.9 Phytochemical analysis of the plant extract

Phytochemical analysis of the leaf extract was carried out according to the methodology of Ghosh et al. (2011). One or more phytochemicals usually play an active role in killing mosquito larvae. Phytochemicals for whom search was made include saponins, terpenoids, alkaloids, steroids, flavonoids, tannin, cardiac glycosides and free glycoside bound anthroquinones.

3.10 Statistical Analysis

To evaluate the data in statistical way, we used mainly MS Excel 2007 for calculating mean mortality and standard error and Stat Plus 2007 Professional software for Log-Probit analysis and Regression Equation and Regression Coefficient.

4 Conclusion

A. salvifolium offers a potential botanical agent for control of Cx. vishnui group larvae, pupae and adult mosquitoes as well. There is no side effect of this plant extract against non target organism. So, this product can be used as a harmless mosquito larvicide. Further research is also needed to identify the active ingredient of the plant products.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgements

The authors are grateful to Dr. Ambarish Mukherjee, Professor of Botany, The University of Burdwan, for identification of the plant. University Grants Commission is acknowledged for providing financial and instrumental support through Grant no F.17-88 (SA-I) UGC and UGC- SAP. We also want to acknowledge the financial support received from the Eastern Regional Office (ERO) of University Grants Commission (UGC), New Delhi through a Minor Research Project sanctioned to Mr. Rajendra Prasad Mondal to do this work [Grant No. F. PSW-004/13-14 (ERO) ID No. WB1-009; S. No. 219563].

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