Test mosquitoes
The present study was conducted at Burdwan (23°16′ N, 87°54′ E), West Bengal, India. Larvae of Cx. vishnui group and An. subpictus were collected from nearer rice fields. They were kept separate in different plastic trays and fed with a mixture of dog biscuits and yeast powder at the ratio of 3:1. The larvae were kept free from exposure to pathogens, insecticides, or repellants and maintained at 25–30 °C. The transformed pupae were separated manually with a glass dropper into a glass beaker (500 mL) filled with normal tap water. The beaker was kept in glass cages for emergence of adult mosquitoes. A cotton ball soaked in 10% glucose solution was used for glucose meal of adult mosquitoes and was periodically blood fed on immobilized pigeon. Eggs laid were similarly reared and 1st generation laboratory bred larvae were used for bioassay and control experiments.
Preparation of solvent extract
We harvested fresh mature leaves of S. nigrum during study period (June–July 2012) from outskirts of Burdwan (23°16′ N, 87°54′ E) having voucher specimen No. GCZAR-1 (the herbarium was deposited in the Department of Zoology, The University of Burdwan, West Bengal, India) and authenticated by Dr. Ambarish Mukherjee, Professor of Botany, The University of Burdwan, West Bengal, India. The leaves were initially rinsed with distilled water, dried on paper towel and then shade dried for 7–8 days. The dried leaves were cut into small pieces and 200 gm of them were put in the thimble of Soxhlet apparatus. 2000 ml of chloroform: methanol (1:1 v/v) was then loaded into the still pot. The extraction period was 72 h and the temperature was <40 °C. The extract was collected in a beaker and evaporated in a rotary evaporator (RV8. IKA). Then the sample was lyophilized to get powdered samples for the further bioassay experiment.
Column chromatography analysis
The column was made grease free using 5% potassium hydroxide (KOH) in ethanol followed by washing with absolute alcohol and then dried with a dryer. Eighty grams of silica gel for column (Merck, India) was taken on a dry filter paper. The column was packed with the silica gel. Then the dried 10 gm sample obtained from chloroform: methanol (1:1 v/v) solvent extract was added in the column along with 5 ml of chloroform: methanol (1:1 v/v). Then the column was eluted with single and mixtures of organic solvents with increased polarity like petroleum ether, petroleum ether: benzene (1:1 v/v), benzene, benzene: chloroform (1:1 v/v), chloroform, chloroform: methanol (1:1 v/v), methanol, methanol: acetone (1:1 v/v), acetone, acetone: absolute alcohol and absolute alcohol. The flow rate was 2 ml/min and each of the fractions was collected in separate test tubes. After preparative Thin Layer Chromatography, fractions with similar Rf values were combined in a single test tube.
Thin-layer chromatography (TLC) analysis
The bioactive fractions were monitored by thin-layer chromatography on silica gel “G” (Merck, India) coated (0.5 mm thickness) plates using chloroform: methanol (1:1 v/v) as a mobile phase. After progress, plates were dried in the air and then placed in an iodine chamber (21 × 21 × 9 cm) for 1 min. The plate was removed and the main band appeared on the fractions of similar Rf values were mixed together and used as apparently purified compound. The compounds having mosquitocidal effect were further subjected to one-dimensional preparative TLC using solvent system chloroform: methanol (1:1 v/v) as mobile phase. The band containing bioactive toxic principle was detected by keeping the plate in the iodine vapor chamber.
The distance of the run of the developing solvent from the bottom of the plate was measured and the run of the sample spot was also measured. The Rf value was then calculated using formula:
$${\text{Rf}} = {\text{distance}}\,{\text{of}}\,{\text{the}}\,{\text{spot}}\,{\text{centre}}\,{\text{from}}\,{\text{the}}\,{\text{start}}\,{\text{point}}/\,{\text{distance}}\,{\text{of}}\,{\text{the}}\,{\text{solvent}}\,{\text{run}}\,{\text{from}}\,{\text{the}}\,{\text{start}}\,{\text{point}}.$$
Bioassay with active ingredients
The said band was scrapped from TLC plates and dissolved in 20 ml of absolute alcohol and heated in a water bath (60–65 °C) for 5 min. Clear solutions were taken in separate conical flasks discarding the precipitate including silica gel. After evaporation of alcohol, the solid mass present at the bottom of the conical flask was scrapped and weighed. The fraction was dissolved in distilled water to make different concentrations with the help of micropipette. During the experiment, stock solutions were prepared having concentrations of 25, 45, 60 mg/L. From those stock solutions, 100 ml were taken in each experiment. For the bioassay experiment, 25 third instar larvae of Cx. vishnui group and An. subpictus were introduced separately into different Petri dishes containing graded nominal concentrations of active ingredient (25, 45, 60 mg/L). All the larval instar were taken in respective Petri-dishes and 20 mg of larval food (powdered mixture of dog biscuits and dried yeast powder in the ratio of 3:1.) was added per Petri-dish. The experiment was kept in a 12 h light/dark setting. The mortality rate was recorded after 24, 48 and 72 h of post-exposure [25]. The data on mortality in 48 and 72 h were expressed by the addition of the mortality at 24 and 48 h, respectively. Dead larvae were identified when they failed to move after probing with a needle in the siphon or cervical region. The experiments were conducted on three different days with 3 replicates on each day for each instar larvae (n = 9) at 25–30 °C and 80–90% relative humidity. A set of control experiment (without having the test solution) using tap water for each instar larvae was also run parallel on each day of the experiment (n = 3).
IR and GC–MS analyses of bio active principle
A portion of dried sample containing active ingredient was subjected to infrared (IR) spectroscopy. For FTIR analysis, the sample was kept in vacuum desiccators over potassium hydroxide (KOH) pellets for 48 h, and then IR spectral analyses were carried out on a Perkin-Elmer FT-IR Spectrometer (Model: Spectrecee RX1) using potassium bromide (KBr) plates. GC–MS analysis was carried out an SHIMADZU QP 2010T which comprised of an auto sampler and gas chromatography interfaced to a mass spectrometer (GC–MS) instrument employing the following condition: capillary column—624 ms (30 m × 0.32 mm × 1.8 m) operating in an electronic mode at 70 eV; helium (99.999%) was used as the carrier gas at a constant flow of 1.491 ml/min and injection volume of 1.0 ml, injector temperature was 140 °C; ion source temperature of 200 °C. The oven temperature was programmed from 45 °C. Mass spectra were taken at 70 eV.
Effect on non target organisms
Effect of bioactive compound isolated from mature leaves of S. nigrum was tested against non-target organisms (NTO) including Daphnia sp. and Diplonychus annulatum (predatory water-bug). For acclimation to the laboratory, each of them was kept in an environment similar to their natural habitat. As per the procedure used by Suwannee et al. [26], the non targets were exposed to LC50 (at 24 h for early 3rd instar larvae) of the bioactive principle. Twenty five Daphnia sp. were placed in 200 ml pond water in a 500 ml beaker. Twenty five early 3rd instar nymph of Diplonychus annulatum were kept in pond water in a 12.6 × 10 × 6 inch plastic tray. Numbers of dead NTO were recorded after 24, 48 and 72 h of exposures and percentage mortality was recorded. The experiments were conducted on three different days with 3 replicates on each day for each organism (n = 9) was run parallel. A set of control (without having the test solution) was also run parallel on each day of the experiment (n = 3) and average mortality rates were tabulated.
Statistical analysis
The percentage mortality observed (M%) was corrected using Abbott’s formula [27] during the observation of the larvicidal potentiality of plant extract. Statistical analysis of the experimental data was performed using the computer software Statplus 2007 and MS EXCEL 2003 to find the regression equations (Y = mortality; X = concentrations) and regression coefficient values. Probit analysis was done by Statplus 2007 software to find out LC50 and LC90 values. Completely randomized three-way factorial ANOVA using different concentrations, different mosquito species and hours as variables was performed by SPSS 11.0. In bioassay experiment, nominal values are used for statistical analysis as the actual exposed concentrations are about 10% of the nominal values.