Larvicidal, smoke toxicity, repellency and adult emergence inhibition effects of leaf extracts of Swietenia mahagoni Linnaeus against Anopheles stephensi Liston (Diptera: Culicidae)

Medically important human blood sucking mosquitoes are the most prevalent vectors of various communicable diseases such as malaria, lymphatic filariasis, dengue hemorrhagic fever, Japanese encephalitis, yellow fever etc. Malaria causes human mortality[1] and it is the major health problems in tropical regions. Only malaria kills nearly 3 million people yearly[2]. In India, till now malaria is the most dangerous disease, as approximately 2-3 million new cases are prescribed every year[3]. Synthetic insecticides used to check mosquito population are toxic, non biodegradable and harmful to environmental health. Recently, researches on mosquito control are focused to promote plant products which are cost effective, easy to handle and safe for PEER REVIEW ABSTRACT


Introduction
Medically important human blood sucking mosquitoes are the most prevalent vectors of various communicable diseases such as malaria, lymphatic filariasis, dengue hemorrhagic fever, Japanese encephalitis, yellow fever etc. Malaria causes human mortality [1] and it is the major health problems in tropical regions. Only malaria kills nearly 3 million people yearly [2] . In India, till now malaria is the most dangerous disease, as approximately 2-3 million new cases are prescribed every year [3] .
Synthetic insecticides used to check mosquito population are toxic, non biodegradable and harmful to environmental health. Recently, researches on mosquito control are focused to promote plant products which are cost effective, easy to handle and safe for S280 environment. Studies of literatures have focused on larvicidal, oviposition deterrent and adult emergence inhibition effects of botanicals on Anopheles arabiensis, Anopheles stephensi (A. stephensi), Aedes aegypti, Culex quinquefasciatus [4,5] . Mahagoni, the common name of Swietenia mahagoni (S. mahagoni), is a large, branched and very hard tree. This medicinal plant has antioxidant and hepatoprotective properties [6,7] . The objective of the present study was to observe the larvicidal, smoke toxicity, repellency and adult emergence inhibition effects of crude and different solvent extracts of leaves of S. mahagoni against the malaria vector An. stephensi.

Test mosquitoes
The study was performed at Burdwan (23°16' N, 87°54' E), West Bengal, India. An. stephensi larvae were collected from fresh water of underground and overhead tanks of Kolkata metropolis and reared in large plastic tray (15 L) with artificial foods in laboratory. The study period was from March 2011 to February 2012.

Preparation of crude extract
Mature, green leaves of S. mahagoni were randomly collected from plants growing in the University of Burdwan campus. After proper identification of the plant, the voucher specimen was deposited in the Department of Zoology (BUZGU-125), the University of Burdwan, WB, India. Fresh leaves were washed in distilled water and soaked in paper towel. The leaves were chopped into pieces and crushed with a mortar and pestle and filtered by Whatman No. 1 filter paper. The fresh filtrate was used as stock solution (100% concentration) for further bioassay experiments.

Preparation of solvent extracts
Shade dried leaves (200 g) were kept in a Soxhlet apparatus and extracts were prepared using petroleum ether, benzene, ethyl acetate, chloroform: methanol (1:1 v/ v), acetone and absolute alcohol applying one after another on same sample. The extraction period for each solvent was 72 h. The solid residue of each extract was used for preparation of graded concentration.

Dose response larvicidal bioassay
During larvicidal bioassay, World Health Organization s t a n d a r d p r o t o c o l s [8] w e r e f o l l o w e d w i t h s l i g h t modifications. Five concentrations of crude extract (0.1%, 0.2%, 0.3%, 0.4% and 0.5%) and five concentrations (40, 50, 60, 70 and 80 mg/L) of each of the six solvent extracts were applied separately in 100 mL water in Petri dishes (9 cm diameter/150 mL capacity). Twenty five larvae were placed in each Petri dish. Larval mortalities were noted after 24 h, 48 h and 72 h of exposures. During screening test with all the six solvent extracts, good result was obtained with ethyl acetate extract. Hence further experiment was conducted with ethyl acetate extract with 3 replicates. Only tap water was used in the control Petri dishes.

Preparation of mosquito coils and smoke toxicity test
Mosquito coils were prepared following the method of Vineetha et al. with some modifications [9] . Each coil was prepared with 4 g shade dried leaf powder, 2 g saw dust and 2 g charcoal. All the materials were thoroughly mixed and distilled water was added to form a paste. Coils were prepared with the paste and dried in shade for smoke toxicity test. Three equally sized one-door glass chambers (140 cm伊120 cm伊60 cm) were used for smoke toxicity test. Smoke toxicity was tested with commercial mosquito coils, mosquito coils composed of powder of plant leaves and mosquito coils prepared without any plant parts (control). A total of 275 well blood fed mosquitoes were released into the chamber and they were exposed to the smoke released from burning coil for 2 h. The effect of smoke toxicity was observed upon adult mosquitoes from 15 min to 2 h with 15 min intervals (i.e. 15 min, 30 min, 45 min, 1 h, 1h and 15 min, 1h and 30 min, 1h and 45 min and 2h).

Mosquito repellency assay
Repellency of adult An. stephensi was tested with crude and all the solvent extracts. About 250 unfed adult mosquitoes were introduced into a wooden cage (30 cm伊30 cm伊30 cm) which was covered with white, clean cloth. The control hand (i.e. naked hand or treated with six solvents only) and the treated hand (i.e. hand treated with crude or solvent extracts of S. mahagoni) were inserted into the cage at a time and were kept for 10 min at an interval of 30 min. The experiment was continued up to 2 h and 30 min. During screening test with crude and all the six solvent extracts good result was obtained with crude extract only. Hence further experiment was conducted with crude extract with 3 replicates.

Adult emergence inhibition
W orld H ealth O rganization standard protocol was followed to assay the rate of inhibition in adult emergence [10] . Late third instar larvae were exposed to all the solvent extracts. During screening test with all the six solvent extracts good result was obtained with ethyl acetate. Hence further experiment was conducted with ethyl acetate extract with 3 replicates. Briefly, 10 mg/ L, 20 mg/L, 30 mg/L, 40 mg/L and 50 mg/L extracts were dissolved in 100 mL distilled water in each case. The adult emergence inhibition can be expressed (%) based on the number of larvae that did not metamorphose successfully into adults. The experiment was terminated when all the larvae or pupae in controls had died or hatched into adults.

Results
Results of larvicidal bioassay with crude and ethyl acetate extracts of mature leaves are presented in Table 1 to 4.    About 97% mortalities of third instar larvae were found both in crude (0.5% concentration) and ethyl acetate (80 mg/L concentration) extracts of mature leaves after 72 h of exposure. Log probit analysis and regression analysis of larvicidal effects of crude and ethyl acetate extracts of mature leaves against third instar larvae revealed that the mortality rate (Y) was positively correlated with the concentration of exposure (X). The results of log probit analysis (at 95% confidence level) expressed that LC 50 and LC 90 values gradually decreased with exposure times and had the lowest value at 72 h of exposure. The sequences of smoke toxicity were: commercial mosquito coils>mosquito coils composed of powder of plant leaves>mosquito coils without any plant parts (Table 5).  Table 6 expressed adult emergence inhibition. Highest inhibition was found at 50 mg/L concentration of ethyl acetate extract. Table 7 revealed that adult emergence inhibition ( Y) was positively correlated with the concentration of extract (X) and regression coefficient (R 2 ) is close to one. Crude extract of plant leaves showed 100% repellency on treated hand up to 2 h but continuous mosquito biting occurred on control hand (Table 8).

Discussions
From ecological point of view, synthetic pesticides are non biodegradable, having residual hazards and so harmful [11] . Those promote insect resistant to pesticides and are not cost effective. Larval control is the best strategy to reduce mosquito population due to their low mobility. Several plant species revealed their mosquito larvicidal potentiality [12][13][14][15] . Plant derived pesticides are easily bio degradable, cost effective, not harmful to environment and reduce environmental pollution. Depending upon polarity of solvent, the polar solvent will extract polar molecules and non polar solvent will extract non polar molecules [16] . Non polar solvents such as petroleum ether and hexane (polarity index of 0.1) mainly extract out essential oils [17] , moderately polar solvent like chloroform (polarity index of 4.1) generally extracts out steroids [18] and alkaloid [19] etc. The present study revealed the successful use of ethyl acetate extract of leaves of S. mahagoni to kill An. stephensi larvae to a great extent. Several workers used ethyl acetate extract of different plants for larvicidal activity [20,21] . Burning of mosquito coils is a traditional but significant method to repel or kill adult mosquitoes. Different synthetic, chemicals such as octa chlorodipropyl ether and bischloromethyl ether etc., which are used in mosquito coils are toxic and harmful. Octa chlorodipropyl ether is a significant genotoxic agent [22] . On the contrary, plant derived coils are mostly safe and non toxic [23] . Mosquito coils prepared by S. mahagoni leaf dust showed satisfactory result of mosquito repellency and mosquito death up to 2 h. The botanical, bioproducts are safe for personal use [24] . Crude extract of S. mahagoni leaves may be used as mosquito repellent as it provided effective repellency up to two and half hours. An interesting method of mosquito control is to keep the mosquito in their larval or pupal stage i.e. inhibition in adult emergence. Ethyl acetate extract of S. mahagoni leaves inhibited adult emergence successfully up to certain time.
Leaf extracts of S. mahagoni with different solvents offer a treasury of excellent quality in having larvicidal, smoke toxicity, repellency and adult emergence inhibition at a time against An. stephensi. The result of the present study may open a new horizon of research in mosquito control effect of plants against malaria vectors.

Background
Mosquito control through botanicals is a very important avenue of research. Mosquito control in larval stage is most essential as adults are very difficult to control due to their wide distribution. The study have immense importance because here a single plant have been reported with various activities, such as larval control, repellency, adult inhibition etc. So the application of the plant in mosquito control programme must be beneficial in future.

Research frontiers
The study reported the application of crude and solvent extract in the larval forms of malaria vector. The present study also reported the smoke toxicity, repellency and adult emergence inhibition effects of leaf extracts of S. mahagoni against An. stephensi.

Related reports
The results presented in the text are in agreement with the other studies where several plant extracts have been reported for their mosquito larvicidal activity. The choices of solvents are also appropriate.

Innovations & breakthroughs
The study reported the multiple effects of a plant extract against larval form of malaria vector for the first time.

Applications
In further study, if the author can isolate and identify the bioactive principle responsible for the larval mortality, it will have immense importance in mosquito control programme through biological agent.

Peer review
The present study reported the application of crude and solvent extract of S. mahagoni against larval forms of malaria vector. This study also reported the smoke toxicity, repellency and adult emergence inhibition effects of leaf extracts of the same plant. The results are very promising due to low lethal toxicity value and good regression coefficient. It has a high inhibition and repellency effect as well.