Investigating the larvacidal activity of phytoecdysteroids found in Asian subspecies of Spinacia oleraca on

Tenebrio molitor

Zhao Xing Liang (4S2)Shi Guan Ming (4S1)

BackgroundMolting

• Process that arthropods undergo • The shedding and re-growth of a new

exoskeleton (The Insect Process of Molting, 2010)

Ecdysteroids

• Insect molting hormones• regulate growth, reproduction, and

development (Grebenok, Galbraith, Benveniste, Feyereisen, 1996)

Phyto-ecdysteroids •Exact replica of ecdystroids produced by plants•Protective mechanism•Disrupt development of insects

Background

- Spinacia oleracea (Spinach)

- Chenopodiaceae family - Produces ecdysteroids

structurally similar to those produced by

arthropods

- Tenebrio molitor (yellow mealworm)- Order Coleoptera- Viewed as pests in

countries such as Mexico (University of California,

2009).

Objectives

Find the most

effective way in

extracting phytoecdy

steroids from

Spinacia oleracea

Investigate the

larvicidal activity of

phytoecdysteroids by

using Tenebrio

molitor as an

indicator

Develop a novel

insecticide that is both

effective and

environmentally

friendly

Rationale

Larvicide

Effective

Biodegradable, environmentally friendly

Phytoecdysteroids are beneficial to human

health (Mária Báthori et al, 2005).

Some larvae have acquired resistance against conventional

methods (Rangel et al, 2009).

So Therefore

could be used to exterminate

mealworm larvae in food items

Hypothesis

• The larvae of Tenebrio molitor, exposed to phytoecdysteroids extracted from Spinacia oleracea, will have a higher rate of mortality and deformities.

Variables

Controlled Independent Dependent

• Species of mealworm larvae

• Culture conditions

• Extraction Method

•Concentration of extracts

• Mortality Rate of T. molitor

• Observable deformities of T.

molitor

Materials and apparatus for extraction of Phytoecdysteroids

Materials• Methanol• Hexane• Dichloromethane• Acetone• Ethanol of 96% purity• Alumina• Octyl silane• Cotton wool

Apparatus• Vacuum pump• Column

Materials and apparatus for Bioassay on Tenebrio Molitor

Materials• Boxes• Mealworm feed• Cotton wool• Petri dishes

Apparatus• Spraying apparatus• Micro syringe

Overview of MethodologyPurchase,

cutting and drying of Spinacia oleracea

Fractionated precipitation

Solvent-solvent distribution

Column chromatographyHPLC

Reverse phase chromatography

Bioassay

Set up for BioassayConcentration

of phyto-ecdysteroids

Method foradministration Number of Meal worms

Solvent Control Petri Dish 1 Petri Dish 2 Petri Dish 3

1

Injection 10 10 10 10

Spraying 10 10 10 10

Ingestion 10 10 10 10

2

Injection 10 10 10 10

Spraying 10 10 10 10

Ingestion 10 10 10 10

3

Injection 10 10 10 10

Spraying 10 10 10 10

Ingestion 10 10 10 10Negative

Control N/A 10

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 (Spinacia oleracea 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, Spinacia oleracea. 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 (Myzus persicae sulzer), Phytochemistry, 52, 480-486.

• Savolainen, V., Wuest, J., Lafont, R., Connat, J. L. (1995). Effects of ingested phytoecdysteroids in the female soft tick Ornithodoros moubata. Phytochemistry. 51, 596-600.

• Schmelz, E. A., Grebenok, R. J., Ohnmeiss, T. E., Bowers, W. S. (2002). Interactions between Spinacia oleracea 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

Thank YouQuestions?

Detailed Methodology

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.

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.

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.

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.

Methodology (Extraction)

• Preparation of phytoecdysteroids for bioassay

• Evaporate the solution to dryness• Dissolve the phytoecdysteroids in water to the

desired concentration

Methodology (Bio-Assay)

1. 10 last-instar mealworm larvae are placed in a box per setup.

2. A determined amount of extracts are sprayed onto the mealworms.

3. The mealworm larvae are left to develop for 30 days.4. The deformities and mortality rate of the mealworm

larvae is recorded after a day, 15 days and 30 days.5. Step 1 to 4 is repeated with the extracts being injected

or fed to the mealworm larvae.