MATERIALS AND METHODS. 4.1 Materials: Mosquito: Anopheles stephensi Moringa oelifera leaves. This mosquito is classified as follows:

Transcription

1 MATERIALS AND METHODS 4.1 Materials: Mosquito: Anopheles stephensi This mosquito is classified as follows: Phylum- Arthropoda Class- Insecta Order- Diptera Family- Culicidae Fig.3- Anopheles stephensi Genus- Anopheles Species- stephensi Habit and Habitat In rural areas, the larvae of Anopheles stephensi may exist in many aquatic habitats, such as ponds, streams, swamps, marshes and other sources of standing water. They may also occupy smaller environments, such as: tree holes, leaf axils and man-made containers. Anopheles stephensi breeds in a number of different water bodies in urban areas, but predominantly in artificial containers, walls, overhead tanks and ground level water tank Moringa oelifera leaves Moringa oelifera is the most widely cultivated species of a monogeneric family, the Morangaceae that is native to the sub Himalayan tracts of India, Pakistan, Bangladesh and Afghanistan. It is considered one of the world s most useful trees, as almost every part of the tree can be use for food or some other beneficial properties. Fig.4- Moringa oelifera leaves 8

2 CLASSIFICATION Kingdom: Plantae Division: Class: Order: Family: Genus: Species: Magnoliophyta Magnoliopsida Capparidales Moringaceae Moringa oleifera Pure Saponin Pure saponin (RM 405) CAS was purchased from HIMEDIA (Mumbai). The larvicidal efficacy of pure saponin against Anopheles stephensi was compared with the saponin containing extract from leaves of Moringa oelifera. 4.2 Methods Collection of eggs, larvae and pupae Larvae of mosquito were collected from Dayalbagh campus (Botanical garden). Then larvae were separated in different instars. Fig.5- Sample collection site in Dayalbagh campus 9

3 4.2.2 Collection of Plant sample Leaves of Moringa oelifera was collected from the Dayalbagh campus. After complete cleaning and ringing with water, leaves were air dried at room temperature and kept away sun and high temperature to avoid destroying active compounds. The dry leaves grinded in coarse powder using high capacity grinding machine after this stored in air tight container with necessary markings for identification and kept in cool, dark and dry place for the investigation Preparation of extract The powder of Moringa oelifera leaves was successively extracted in a Soxhlet extractor for 72 hrs at elevated temperature using polar solvent (Methanol) because saponins are more polar solvent. Fig.6- Soxhlet assembly Qualitative test of saponin in crude leaves extract of Moringa oelifera This test help to confirm the presence of saponin in the extract of Moringa oelifera leaves. 10

4 The extract was diluted with distil water and made up to 20 ml. The suspension was shaken in a graduated cylinder for 15 min. 2cm layer of foam indicated the presence of saponin Test of larvicidal efficacy of extract and pure saponin against mosquito larvae The testing of crude saponin extract of Moringa oelifera leaves against mosquito larvae was done as per the WHO protocol in the laboratory conditions Preparation Methodology and dose application of extract and pure saponin Fresh extract was prepared at the time of efficacy test against larvae. First, set the dose for mosquito larvae through the screening test. Then doses producing and found 50, 90, 99% mortalities by the probit analysis (Finney 1971). The experiments were replicated three times to validated results. 4.3 Statistical and computational methods: Statistical technique regarding estimation of median effective dose have been employed, Finney (1971) Data Analysis Experimental tests that demonstrated more than 20% control mortality were discarded and repeated. When control mortality reached between 5 and 20%, the mortality observed was corrected by Abbott s formula (Abbott 1925). %M = Test mortality - %control of Mortality % control of mortality Data were subjected to probit analysis. Procedure to calculate the probit of kill and LC 50, LC 90 and LC The tested dose was entered in the first column of table A in suitable units. 2. In the column headed x enter for each dose its log value. 11

5 Dose(ppm) Log dose x No. of Mosquitoes N No. effected r Corrected mortality (%) p Empirical probit Expected probit Y 3. In the column headed n and r were entered for each dose, the number of subjects tested and the number affected. 4. Percentage response, p = 100 r / n calculated to the nearest whole number after deducting the control mortality (%). 5. Probit of each p, from table 1 (Finney, D.J., 1981 Probit Analysis) entered as EPERICAL PROBIT. 6. Empirical probit were plotted against x. A provisional straight line was drawn to fill the points judging its position on the line. 7. Y the expected probit, entered in the column read as the coordinated to the provisional line at each x used in the experiment till one decimal place. Log Dose x Weighting coefficient w Nw Working probit Y X XX y yy Xy 8. From each column of table 2 (Finney, D.J., 1981 Probit Analysis), weighting coefficient for each y, read multiple by the corresponding n and entered in the column. 9. The working probit, y from table 4 (Finney, D.J., 1981 Probit Analysis), entered in table corresponding to Y and p. 10. The values of nw, x and y, xx, yy and xy was calculated. Each column was summed in the end. 11. nwx is divided by nw to give X. 12

6 12. nwy is divided by nw to give Y. 13. Values of b obtained by b = xy/ xx 14. Hence the relation between probit and doe was estimated as Y =- Y + b (X- X ) Other formulas are: LC 50 = X Y / b LC 90 = X Y / b LC 99 = X Y / b Total or % Mortality = T C/T 100 C = Control T = Test The control mortality will adjust by using Abbott s formula which as follows- Mortality (%) = X Y / 100 Y 100 Where, X = The percentage mortality in the treated sample Y = The percentage mortality in the control. 13