1. Investigating the Anti- fungal Properties of Subterranean Termites Yasamin Sharifzadeh, Katie Gorick, Wong Hong Jie, Samuel Lau

2. Background Variety of termites have been tested to have anti-fungal properties. Anti-fungal compounds origins are unknown Possible sources of the anti-fungal compounds: Hemolymph Termite symbionts (protozoa & bacteria used for cellulose digestion)

3. Our project Objectives Hypothesis Overview of project

4. Objectives To investigate the possible sources of anti-fungal compounds found in two termite species Reticulitermes flavipes (native to North America) Coptetermes gestroi (native to Asia) Possible analysis of extent of anti-fungal properties and compare the differences between American and Asian termites Parts of termite to be investigated: Total termite system (ground up) Hemolymph with protozoa and bacteria Hemolymph without protozoa and bacteria Hemolymph without protozoa but with bacteria Protozoa system Bacteria (surface, inner and surface)

5. Overview TermitesHemolymph Hemolymph w/o protozoa and bacteria Hemolymph w/ bacteria and no protozoa Protozoa system Bacteria Total, ground- up system

6. Hypothesis The hemolymph contains anti-fungal compounds for both American and Asian termites The protozoa in the hemolymph is the main source of anti- fungal compounds for both American and Asian termites

7. Methodology Body fluid extraction (Total system) Hemolymph extraction Hemolymph w/o protozoa and bacteria Hemolymph w/ bacteria but no protozoa Anti-fungal assay

8. Body fluid extraction (Total system) Materials Scalpel Forceps Phosphate buffer solution Petri dishes Homogenizer Dropper Distilled water Micro filter tubes Termites

9. Body fluid extraction (Total system) 1. Wash the termites in a beaker 2. Filter termites with a sieve and repeat washing for 2 more times 3. Crush the termites with a homogenizer 4. Homogenize using the phosphate buffer solution 5. Centrifuge to pellet tissue debris 6. Collect the supernatant 7. Place the supernatant in micro centrifuge tubes 8. Centrifuge at 1300rpm for 10 minutes 9. The body fluids are now separated 10. Extract the supernatant and filter it with a micro- filter

10. Hemolymph extraction Materials Water 70% aqueous solution of ethanol Freezer Scalpel Forceps Buffer solution containing a melanization- inhibitory agent Beaker

11. Hemolymph extraction Anesthetize Immerse the insect bodies in water maintained at 0 ˚C to 25˚C to anesthetize them Freezing Immerse the insect bodies in a cooling medium, one in which the insect bodies can be frozen without the cooling medium freezing, for example ethanol aqueous solutions such as 70% aqueous solution of ethanol or methanol Freeze at -30 ˚C to freeze the hemolymph Cutting Remove the frozen insect bodies from the cooling medium and cut off the abdominal legs of the insect bodies

12. Hemolymph extraction (cont’d) Thawing Thaw the frozen insect bodies in a buffer solution containing a melanization-inhibitory agent (such as phenyl thiourea, sodium thiosulfate, ascorbic acid, cysteine, penicillamine, thiopronine, captopryl or other reducing agents and oxidase inhibitors.)The thawing step is carried out at a temperature of not more than the ordinary temperature. Discharge Allow the hemolymph to be discharged into the buffer solution

13. Hemolymph (cont’d) By filtering – 0.25 Hemolymph (without protozoa and bacteria) By filtering - 0.45 Hemolymph (with bacteria, but no protozoa)

14. Anti-fungal assay Materials Scalpel Forceps Petri dishes Aspergillus niger strain Sterile filter papers Potato dextrose agar (PDA) Potato dextrose broth (PDB) Membrane hyphae filter

15. Anti-fungal assay Sterilize working area with UV light and ethanol Mix Xml of the body fluids of termites with Yml of sterile molten PDA Pour the molten PDA from step 2 onto a petri dish and let it harden Using sterile forceps, cut out cubes of the cultures of A.niger and place them face down Conduct triplicates For the control, we will be using PDA mixed with PDB, Incubate the dish and observe for results Method ONE

16. Anti-fungal assay Culture A. niger in PDB Pass the cultured A. niger through a membrane-hyphae filter to remove the hyphae from the spores. Use a hemocytometer to calculate the number of spores per unit area in the PBS. Add termite extract into the A. Niger (without the hyphae) suspended in PDB. For the control, we will be adding in PBS Leave the mixture (of A. Niger and termite extracts) for 2 days Overlay the mixture onto PDA in petri dish. Spread the mixture over the dish evenly with a spreader. Incubate the dish and observe for inhibition. Method TWO

17. Anti-fungal assay PERCENTAGE Calculate and obtain the percentage of mould growth on the plates COUNTING Counted the number of squares of mould growth GRID Lay a grid transparancy over agar plates

18. Timeline AOSHCI Nov Finalize minor details, ie. which chemicals to use, what concentrations to use etc Dec- Jan Experiments Break/ Research Feb- Mar Experiments Mar- Apr Apr- May May- JunExperiments/ Exams Jun-JulySummer BreakExperiments/ Break July- August Conclude project 21 Aug: Projects Competition finals