Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
33rd SHELL-SINGAPORE YOUTH SCIENCE FESTIVAL
SCIENCE FAIR 2010
Level:
Upper Secondary
Category:
Life Sciences
Project Title:
Investigating the anti-fungal properties of subterranean termites
Team Members:
Samuel Lau Yu Yi (Leader)
Wong Hong Jie
Tan Wei Jie
Sponsor Teacher(s):
Mrs Koh-Oon Hsiu Leng
School:
Hwa Chong Institution (High School Section)
1
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
Investigating the
anti-fungal properties of
subterranean termites
Research Paper
Samuel Lau Yu Yi
Wong Hong Jie
Tan Wei Jie
Hwa Chong Institution (High School)
Mentor: Mrs Koh-Oon Hsiu Leng
2
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
1. Contents Page
Item No.
Item
Page No.
-
Cover page
1, 2
1
Contents page
3
2
Abstract
4
-
4
3
Declaration of Degree of Guidance from External
Mentors
Introduction (Literature review)
4&5
Purpose/ Hypothesis of project
6
6
Methods
7 - 10
7
Results
11 – 18
8
Analysis
19
9
Relevance to Practical Application
20
10
Conclusion
20
11
Reference
21
12
Acknowledgements
21
5, 6
3
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
2. Abstract
With the overreliance of air-conditioners in affluent countries, improper
maintenance could lead to the formation of dust and mould, and our health can be
compromised. Termites feed on decaying wood that inevitably has mould growing on
them, but termites seemed unaffected by the mould, thus suggesting that they most
likely have anti-fungal properties that could render the mould harmless to them.
Hence, this project aims to 1) investigate whether termites can be harvested,
2) investigate whether the body fluids of termites possess anti-fungal properties and
3) investigate the main source of anti-fungal properties. If successful, body fluids of
termites can be used as a potential natural inhibitor of mould in air-conditioners.
To investigate whether termites can be harvested, we set up traps around our
school compound.
To investigate whether body fluids of termites have anti-fungal properties,
termites are washed, homogenized, and centrifuged using phosphate buffer solution.
The supernatant was used to test for anti-fungal properties. We used an anti-fungal
assay method whereby body fluids are mixed with molten PDA, poured onto petri-dish
and allowed to harden. Next, cubes of mould (A. niger and T. kanei) were cut out from
cultures and placed face down on the PDA mixed with body fluids. If anti-fungal
properties were present, there would be decreased mould growth.
To investigate the main source of anti-fungal properties in termites, we
immersed termites in PBS of different pH and pass them through different micro-filter
pore sizes.
Results have shown that 1) termites can be harvested, 2) body fluids of termites
have anti-fungal properties against Aspergillus niger and Trichophyton kanei
4
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
summerbell, and 3) the main source of anti-fungal properties in termites is symbiotic
bacteria in the hemolymph of termites.
Declaration of Degree of Guidance from External Mentors
ORIGIN Exterminators Pte Ltd. gave us the inspiration to start off our project, supplied
us with termites and shared with us methods of harvesting termites. Aardvark Pestkare
Pte Ltd supplied us with termites as well.
3. Introduction
Background
According to a Straits Times article titled “Air-conditioning can spread germs”
(February 26, 2009) many of us in tropical Singapore rely heavily on air-conditioners for
comfort. However, our health can be compromised when an air-conditioning system is
not properly maintained, resulting in the formation of dust and mould, causing
symptoms of allergic rhinitis and also worsen asthma. Mould spores, if disturbed, may
be dispersed through the air and then inhaled by occupants (Lankarge, 2003).
Accumulation of pollutants such as mould might also cause sick building syndrome,
leading to symptoms like headaches, irritation in the eyes, nose or throat, dry or itchy
skin, dizziness and even death. (Hwang S.W., 2009)
In addition, mould is usually hard to remove. Most people would resort to using
chlorine bleach; however, it is not an effective or lasting killer of mould and mould
spores. Bleach is good only for changing the color of the mould.
Termites
The subterranean termite Coptotermes gestroi (C.gestroi) is a native species of
Southeast Asia (Araújo, 1958). Subterranean termites build shelter tubes and nest in
5
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
the soil or on the sides of trees or building constructions and rely principally on soil for
moisture. (Yupaporn, 1996)
The blood, or hemolymph, circulates round the body, bathing tissues directly. It
consists of fluid plasma in which blood cells, hemocytes, are suspended. The plasma,
because of its function of maintaining the tissues throughout the body, contains many
chemicals. (Chapman, 1998).
The protozoa in termite gut help digest cellulose eaten, and they themselves are
aided by a variety of symbiotic bacteria both inside them and embedded in their
surface. (Radek, 1999)
There are more than 100 different bacteria species present in the guts of
termites, and most are not found anywhere else on earth. These microbes play many
roles in the survival of termites. They can remove H2 and CO2 that is released by
protozoa from the gut of the termite. They also fight off other bacteria, acting as an
immune system aside from the termite’s hemolymph (Radek, 1999).
Anti-fungal properties of termites
Termites feed on decaying wood that inevitably has mould growing on them.
However, termites seemed unaffected by the mould, thus suggesting that they most
likely have anti-fungal properties that could render the mould harmless.
Possible candidates of the anti-fungal properties include the protozoa or bacteria
in hemolymph of the termite, or the protozoa or bacteria in the termite gut.
In our study, strains of mould included Aspergillus niger (A. niger) and
Trichophyton kanei summerbell (T. kanei). As for the species of termites, we used
Coptotermes gestroi (C. gestroi), Rhinotermes spp., one of the genus Macrotermes,
and another of genus Nasutitermes.
6
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
4. Purpose
The purpose of our project includes:
1. To investigate whether termites can be harvested.
2. To investigate whether the body fluids of termites possess anti-fungal properties.
3. To investigate the main source of anti-fungal properties in termites.
5. Hypothesis
1. Termites can be harvested.
2. The body fluids of termites possess anti-fungal properties.
3. The main source of anti-fungal properties of termites is the protozoa present in
the hemolymph.
6. Methods
6. 1 Harvesting termites
Origins Exterminators Pte Ltd supplied us with a batch of termites to work on,
and shared with us a method to harvest termites from trees within Hwa Chong
Institution campus. A hole at the bottom of a plastic box was cut, and thick stacks of
toilet paper was folded and placed inside the box. Next, water was poured in to wet the
toilet paper (Fig 1).
Trees that have signs of termite infestation, implied by the formation of mud trails
on trees were located. Mud trails (Fig 2) were broken and the box was placed such that
the broken part of the mud trail comes into contact with the toilet paper inside the box
(Fig 3). Finally, the box was taped around the tree trunk firmly with cloth tape, ensuring
that it would not fall off even in the event of rain (Fig 4).
7
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
(Fig 1, top left): Water was poured in to wet the toilet paper in a plastic box with a hole at the
bottom.
(Fig 2, top right): Mud trails formed on trees were broken.
(Fig 3, bottom left): The box was placed such that the broken part of the mud trail comes into
contact with the toilet paper inside the box.
(Fig 4, bottom right): The box was taped around the tree trunk firmly with cloth tape to hold it
firmly.
6.2 Extraction of body fluids of termites
First of all, termites were removed from wood debris (Fig 5). To obtain the body
fluids of termites, the termites were crushed using a mortar and pester with PBS as a
solvent (Fig 6). 10g of termites were mixed with 30g of sterile PBS for each set of
experiment. They were then homogenized and centrifuged to pellet tissue debris. The
supernatant was then collected and placed in eppendorf tubes and centrifuged once
more at 1300 rpm for approximately 10 minutes (Fig 7). The supernatant was collected
and passed through micro-filter tubes to obtain the sterile-filtered body fluids of termites
(Fig 8).
8
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
(Fig 5, top left): Termites were removed from wood debris.
(Fig 6, top right): The termites were crushed using a mortar and pester with PBS as a solvent.
(Fig 7, bottom left): The supernatant was placed in eppendorf tubes and centrifuged at 1300 rpm
for 10 minutes.
(Fig 8, bottom right): The supernatant was collected and passed through micro-filter tubes to
obtain the sterile-filtered body fluids of termites.
6.2.1 Different pH of PBS
Separate experiments were carried out using different pH of PBS (pH 7.3 and pH 10.0).
PBS of pH 7.3 simulates the neutral condition in the hemolymph of termites, while pH
10.0 simulates the alkaline condition in the gut of termites. By simulating the different
pH conditions of the hemolymph and gut, we can determine whether the main source of
anti-fungal properties is from the hemolymph or gut.
9
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
6.2.2 Different micro-filter pore sizes
By passing the body fluids or termites through micro-filter (0.8 um pore size), protozoa
are removed. By passing the body fluids through micro-filter pore size (0.45 um pore
size), both protozoa and bacteria are removed. By comparing the results of 0.8um and
0.45um micro-filter pore-size, we can determine whether it is the protozoa or bacteria
that is responsible for the anti-fungal properties.
6.3 Anti-fungal assay
We used 2 different methods for anti-fungal assay. Method 1 tests the ability of body
fluids of termites in inhibiting mould growth, while method 2 tests the ability of body
fluids in killing mould spores.
6.3.1 Anti-fungal assay: Method #1
First, the laminar hood was sterilized with UV light and ethanol. Safety measures
such as lab-coats and gloves were worn at all times as mould is a biohazard. 2 ml of
body fluids of termites which were collected from the extraction process was mixed with
10ml of sterile molten PDA. The molten PDA was poured onto a petri-dish and left to
harden. Sterile forceps were used to cut out cubes of the cultures of A. niger and
placed face down. Triplicates were conducted and the steps were repeated at least
twice for both strains of mould, namely, Aspergillus niger and Trichophyton kanei
summerbell.
Quantification
A grid transparency was laid over the agar plates and the number of squares of
mould growth was counted. A percentage of mould growth on the plate can then be
counted. A square is counted only when at least 50% of the square is occupied by the
mould.
10
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
6.3.2 Anti-fungal assay: Method #2
The laminar hood was sterilized with UV light and ethanol. 3 sterile filter papers
were immersed in the body fluids of termites, PBS and bleach separately, and placed
well spaced out on a PDA plate. The plate was overlaid with soft agar containing mould
and incubated for 48 hours. Zones of inhibition were recorded and triplicates as well as
at least 2 repeats were conducted for both strains of mould.
7. Results
Results for anti-fungal assay method #1 against A. niger, using the body fluids of
respective termites
Fig 9: Effect of the first supplied batch of termites (Rhinotermes spp.) and the harvested batch of
termites (C. gestroi) against A. niger. T-test p value: 0.045 (Significant)
11
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
Results for anti-fungal assay method #1 against T. kanei, using the body fluids of
respective termites
Fig 10: Effect of the first supplied batch of termites (Rhinotermes spp.) and the harvested batch
of termites (C. gestroi) against T. kanei. T-test p value: 0.139 (Insignificant)
12
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
Results for anti-fungal assay method #2 against A. niger, using the body fluids of
respective termites
Fig 11: Effect of the second supplied batch of termites (Macrotermes) against A. niger. T-test p
value: 0.0163 (Significant)
13
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
Results for anti-fungal assay method #2 against T. kanei, using the body fluids of
respective termites
Fig 12: Effect of the second supplied batch of termites (Macrotermes) against T.kanei. T-test p
value: 0.250 (Insignificant)
14
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
Results for anti-fungal assay method #1 on A.niger, using body extracts of termites mixed in
PBS (pH 10)
Control (PBS)
w/ extract (0.8um)
w/ extract (Not filtered)
w/ extract (0.45um)
Fig 13: Effect of body fluids of termites (in pH 10 PBS) against A.niger.
T-test p-value for extract (not filtered): 0.169 (Not significant)
(Note: Extract (not filtered, and 0.8um) is contaminated with other strains of mould.
It is inevitable for extract (not filtered, and 0.8um) to be contaminated with other strains of
mould or bacteria, since the micro-filter pore size is too huge and allows mould and bacteria to
pass through.)
15
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
Results for anti-fungal assay #1 on T.kanei, using body extracts of termites mixed in PBS (pH
10)
Control (PBS)
w/ extract (0.8um)
w/ extract (Not filtered)
w/ extract (0.45um)
Fig 14: Effect of body fluids of termites (in pH 10 PBS) against T.kanei.
T-test p-value for extract (not filtered): 0.0398 (Significant), extract (0.8um): 0.0457
(Significant)
Note:
Extract (not filtered, and 0.8um) is contaminated with other strains of mould.
It is inevitable for extract (not filtered, and 0.8um) to be contaminated with other strains of
mould or bacteria, since the micro-filter pore size is too huge and allows mould and bacteria to
pass through.
16
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
Results for anti-fungal assay method #1 on A.niger, using body extracts of termites mixed in
PBS (pH 7.3)
Control (PBS)
w/ extract (Not filtered)
w/ extract (0.45um)
w/ extract (0.8um)
Fig 15: Effect of body fluids of termites (in pH 7.3 PBS) against A.niger.
T-test p-value for extract (not filtered): 0.193 (Insignificant), extract (0.8um): 0.193
(Insignificant), extract (0.45um): 0.0359 (Significant)
Note:
Extract (0.45 um) is contaminated with bacteria.
17
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
Results for anti-fungal assay #1 on T.kanei, using body extracts of termites mixed in PBS (pH
7.5)
Control (PBS)
w/ extract (0.45um)
w/ extract (Not filtered)
w/ extract (0.8um)
Fig 16: Effect of body fluids of termites (in pH 10 PBS) against T.kanei.
T-test p-value for extract (not filtered): 0.0279 (Significant), extract (0.8um): 0.0367
(Significant)
Note:
Extract (not filtered, and 0.8um) is contaminated with other strains of mould, extract (0.45um) is
contaminated with bacteria.
It is inevitable for extract (not filtered, and 0.8um) to be contaminated with other strains of
mould or bacteria, since the micro-filter pore size is too huge and allows mould and bacteria to
pass through.
18
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
8. Analysis
The results from the anti-fungal assay have shown that the body fluids of
termites are effective in reducing fungal growth of Aspergillus niger. The t-test p-value
of the results recorded is 0.0450 (Fig. 9), showing that the results are significant. The
body fluids of termites are also effective against Trichophyton kanei summerbell, as
seen from the recorded t-test value of 0.0279 (Fig. 16) However, A. niger is ubiquitous
in soil and is commonly reported from indoor environments, whereas T. kanei is a
species of fungi parasitic in humans. A. niger would therefore be a closer substitute for
mould in AC systems, and the results would be more relevant than that of T. kanei to
be used as a potential mould inhibitor.
The results (Fig.13 & Fig. 14 as compared to Fig. 15 & Fig. 16) also show that
body fluids of termites in pH 7.3 PBS have greater anti-fungal properties compared to
body fluids of termites in pH 10 PBS. Hence, the source of anti-fungal properties is
mainly from the hemolymph of termites (since pH 7.3 simulates neutral condition in
hemolymph and pH 10 simulates alkaline condition in gut)
Also, the results (Fig. 14 & 16) shows that extract (not filtered) has the greatest
anti-fungal properties against T.kanei, followed by extract (0.8um). Hence, we can
conclude that the both the protozoa and bacteria possess anti-fungal properties, but
bacteria has relatively greater anti-fungal properties compared to protozoa. (since
0.8um micro-filter pore size filters out the protozoa from the body fluids). A.niger
19
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
9. Relevance to practical application
As A.niger is commonly reported from indoor environments, and experiments
have shown that A.niger is found in air-conditioners, we will be able to apply body fluids
of termites on air-conditioners as a potential mould growth inhibitor, decreasing the
amount of mould which are health hazards.
10. Conclusion
Results have shown that termites can indeed be harvested from trees, and the
body fluids of termites contain anti-fungal properties. Its introduction has shown
significant reduced fungal growth of Aspergillus niger and Trichophyton kanei
summerbell. PBS and bleach are negative controls and did not contribute to the
significant results of reduced fungal growth of certain species. A. niger is ubiquitous in
soil and is commonly reported from indoor environments, whereas T. kanei is a species
of fungi parasitic in humans. Therefore, A.niger is a closer substitute for mould in AC
systems, hence the results from A.niger would be more relevant in addressing the
problem of mould accumulation in air-conditioners.
Results have also shown that the body fluids termites in PBS pH 7.3 have
greater anti-fungal properties as compared to body fluids in PBS pH 10. Hence, we can
conclude the hemolymph has greater anti-fungal properties than gut. Also, extract (not
filtered) has the greatest anti-fungal properties against T.kanei, followed by extract
20
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
(0.8um). Hence, we can conclude that the both the protozoa and bacteria possess antifungal properties, but bacteria has relatively greater anti-fungal properties compared to
protozoa. To sum up, the bacteria in hemolymph is the main source of anti-fungal
properties in termites. A. niger
21
Samuel Lau Yu Yi, Wong Hong Jie, Tan Wei Jie
11. References
Wong M.L.(2009) Air-conditioning can spread germs The Straits Times article, February 26.
Lankarge, V. (2003) What Every Home Owner Needs to Know about Mold and What to Do
about It. Blacklick, OH, USA: McGraw-Hill Trade. p 14.
Yupaporn, S. (1996) Studies on Damage of Constructions Caused by Subterranean Termites
and Its Control in Thailand. Bangkok: Forest Products Research Division, Royal Forest
Department.
Bultman, D. and Southwell, C.R. (1976) Natural resistance of tropical American woods to
terrestrial wood-destroying organism. Biotropica. V.8, p.71-95.
Scheffrahn, R. and Nan-Yao, S. (2000) Asian Subterranean Termite, Coptotermes gestroi.
Document EENY-128, one of a series of Featured Creatures from the Entomology and
Nematology Department, Florida Cooperative Extension Service, Institute of Food and
Agricultural Sciences, University of Florida.
US Patent 5866317 (1999) Method for collecting hemolymph of insects
Chapman, R.F. (1998) The insects: structure and function, University of Cambridge, p.101 &
106
Pasanen, A.L. et al. (1991) Laboratory studies on the relationship between fungal growth and
atmospheric temperature and humidity. Environ Int 1991;17: 225–228.
Araújo, R.L. (1958) Contribuição à biogeografia dos térmitas de São Paulo, Brasil. Insecta –
Isoptera. Arquivos do Instituto Biológico de São Paulo 25: 185-217.
Radek, R. (1999). Flagellates, bacteria, and fungi associated with termites: diversity and
function of nutrition- a review. European Journal of Protistology: Ecotropica, 5, 183-196.
12. Acknowledgements
Mrs Koh-Oon Hsiu Leng, Project mentor.
Mr Carl Baptista, Director, ORIGIN Exterminators Pte Ltd
Mr Choy Chen Yep, R&D Executive, ORIGIN Exterminators Pte Ltd.
Mdm Lim Cheng Fui, Science Research Center-in-charge.
Mr Patrick Chong, Aardvark Pestkare Pte Ltd
22