Zhao Xing Liang (4S2) Shi Guan Ming (4S1) Project ID: 1-094

1. Investigating the larvicidal activity of phytoecdysteroids , found in Asian subspecies of Spinaciaoleraca, on Tenebriomolitor Zhao Xing Liang (4S2) Shi Guan Ming (4S1) Project ID: 1-094

2. Background

3. Background

4. Background

5. Objectives

6. Rationale So Some larvae have acquired resistance against conventional methods (Rangel et al, 2009). Therefore could be used to exterminate mealworm larvae in food items

7. Hypothesis • Phytoecdysteroids, extracted from Spinaciaoleracea, cause a high rate of mortality and deformities in Tenebriomolitor larvae .

8. Variables

9. Materials and apparatus for extraction of Phytoecdysteroids

10. Materials and apparatus for larvicidalassay on Tenebriomolitor

11. Overview of Method

12. Set up for Larvicidal Assay

13. Timeline

14. References • Adler, J. H., Grebenok, R. J. (1999). Occurrence, biosynthesis, and putative role of ecdysteroids in plants. Critical Reviews in Biochemistry and Molecular Biology, 34(4), 253-264. • Bakrim, A., Maria, A., Sayah, F., Lafont, R., Takvorian, N. (2008). Ecdysteroids in spinach (Spinaciaoleracea L.): Biosynthesis, transport and regulation of levels. Plant Physiology and Biochemistry, 844-854. • Grebenok, R.J., Galbraith, D.W., Benveniste, I., & Feyereisen, R. (1996). Ecdysone 20-monooxygenase, a cytochrome p450 enzyme from spinach, Spinaciaoleracea. Phytochemistry, 42(4), 927-933.The Insect Process of Molting. (2010). Retrieved from http://www.insectidentification.org • Malausa, T., Salles M., Marquet V., Guillemaud T., Alla, S., Marion-Poll, F., Lapchin L. (2006). Within-species variability of the response to 20-hydroxyecdysone in peach-potato aphid (Myzuspersicaesulzer), Phytochemistry, 52, 480-486. • Savolainen, V., Wuest, J., Lafont, R., Connat, J. L. (1995). Effects of ingested phytoecdysteroids in the female soft tick Ornithodorosmoubata. Phytochemistry. 51, 596-600. • Schmelz, E. A., Grebenok, R. J., Ohnmeiss, T. E., Bowers, W. S. (2002). Interactions between Spinaciaoleracea and Bradysia impatiens: a role for phytoecdysteroids. Archives of Insect Biochemistry and Physiology, 51, (204- 221). • University of Arizona. (1997). Darkling Beetle/Mealworm Information. Retrieved from September 26, 2010 http://insected.arizona.edu/mealinfo.htm • University of California (2009). Mealworms and Darkling Beetles (Tenebrio beetle). Retrieved September 26, 2010 from http://lhsfoss.org/fossweb/teachers/materials/plantanimal/tenebriobeetles.html

15. Thank You Questions?

16. Detailed Methods

17. Methodology (Extraction) • Fractionated precipitation • Dried plant of 6g is extracted with Methanol at a mass-volume ratio of 1:10 ( 60ml methanol needed) • After extraction, the methanolic solution is split into 3 parts. (20 – 21 ml each) • The first part of the solution (20ml) is mixed with half the volume of acetone (11ml) while the second part (20ml) is mixed with same volume of acetone (22ml) and the last part is mixed with twice the volume of acetone (40ml). • The resulting solution is then filtered and the residue is removed. • The residue is washed with the same ratio of methanol and acetone as step 1 and 3 respectively • The washing solution is then added to the filtrate. • The solutions are then evaporated. • The crude extracts are redissolved in methanol at the same mass-volume ratio of 1:10. • Step 2-8 is repeated 2 more times.

18. Methodology (Extraction) • Solvent-solvent distribution • After precipitation, the crude extracts are dissolved in 50% aqueous methanol (specific numbers needed – add until everything dissolves) • Hexane (how much?) is added to the solution to extract the non-polar compounds in the precipitate. • The aqueous methanol phase (bottom) is separated (using separating funnel) and then evaporated to dryness. • The resulting residue is dissolved in pure methanol. • The methanolic solution is mixed with aluminium oxide and the suspension was evaporated to dryness with a rotary evaporator.

19. Methodology (Extraction) • Chromatography • The alumina is eluted with a hybrid of Dichloromethane- 96% Ethanol solution of ratio 9:1 and 8:2. (need to do 2 times) • A cotton wool of mass of 0.2 g was placed at the bottom of the column to prevent alumina from flowing out. • 70-90g (subject to experimental changes) of Alumina is mixed with the eluent. • The mixture of alumina and eluent was stirred and poured into the column until is 75% full. • The bands in the mobile phases are collected in different beakers for further tests.

20. Methodology (Extraction)) • Purification of Ecdysteroids • For further purification, the ecdysteriods are separated by reversed-phase chromatography • In reversed-phase chromatography, octyl silane is used as the stationary phase (being non-polar) instead of silica/alumina. • A cotton wool of mass of 0.2 g was placed at the bottom of the column to prevent alumina from flowing out. • 70- 90g (subject to experimental changes) Octyl Silane is mixed with the eluent. • To control the flow of the mobile phase, a vacuum will be used at the outlet. • Different concentrations of methanol are used as eluents in this chromatography. • Stepwise gradient elution is used with an increase of 5% of methanol content in each step. • Different bands of ecdysteroids will be formed on the stationary phase. • The specific hormone, 20-hydroxyecdysone is isolated with methanol of 35-40% purity.

21. Methodology (Extraction) • Preparation of phytoecdysteroids for bioassay • Evaporate the solution to dryness • Dissolve the phytoecdysteroids in water to the desired concentration

22. Methodology (Bio-Assay) • 10 last-instar mealworm larvae are placed in a box per setup. • A determined amount of extracts are sprayed onto the mealworms. • The mealworm larvae are left to develop for 30 days. • The deformities and mortality rate of the mealworm larvae is recorded after a day, 15 days and 30 days. • Step 1 to 4 is repeated with the extracts being injected or fed to the mealworm larvae.