Tushara's Capstone Presentation

1. Interleukin-17 expression in
Crassostrea gigas following
Escherichia coli exposure
Tushara Williamson (Saint Vitus)
Steven Roberts

2. Proposal / Experiment
Question: How do Crassostrea gigas
oysters respond to a bacterial stressor?
The purpose of this experiment is to
compare immune response in C. gigas
oysters following exposure to heat-killed E.
coli bacteria.

3. Where we’re going
• The role of cytokines
• The role of interleukin-17
• E. coli bacteria information
• Methods and Materials
• Results
• Discussion / Implications to the scientific
community

4. What are cytokines?
• Protein
• Functions in the vertebrate immune
system
• Acts as a messenger between cells to
inhibit or stimulate immune functions
• Three types: interleukins, interferons,
chemokines

5. Interleukin-17
• T-cell derived cytokine
• Implicated in inflammatory diseases such
as arthritis, cystic fibrosis and asthma in
mammals
• Nothing about interleukin-17 expression in
invertebrates
• Six types: A-F
• IL-17B of interest for this experiment

6. E. coli
• Bacteria commonly found
in gastrointestinal tract of
endotherms
• Ferment lactose into gas
and acids
• Most strains harmless but
some can cause
infections of the bladder
and fatal diarrhea in
humans

7. Methods and Materials
• Interleukin-17B primers designed
• PCR and gel electrophoresis of C. gigas and O.
Conchaphila gill, mantle and muscle tissue
carried out to determine where interleukin-17 is
expressed in organism
• Oyster exposure to heat-killed E.coli trial
• RNA extracted from trial oysters; reverse
transcribed into cDNA
• Quantitative PCR of control and experimental C.
gigas gill cDNA

8. Interleukin-17 primer design
• Primers designed using fragment of gene sequence that
appeared to be interleukin-17 based on sequence
similarity
• Primers designed using Geneious, bioinformatics
software for sequence alignment (Biomatters, Ltd.)
http://www.geneious.com

9. PCR and Gel Electrophoresis
• Polymerase Chain Reaction (PCR) done
to show interleukin-17 expression in
C.gigas gill, muscle and mantle with
Ostrea conchaphila (Olympia oyster)
control
• Gel electrophoresis done as an 0.8%
agarose mix stained with ethidium bromide

10. C. gigas exposure trial
• Two tanks set up (one experimental, one
control)
• Four adult C. gigas oysters each tank in
seawater
• Temperature controlled room (15.5˚ C / 60˚F)
• Experimental tank exposed to 6.84 x 1011 heat
killed bacteria suspended in 2L seawater for
three hours

11. Pictures

12. Pictures

13. Pictures

14. http://video.google.com/videoplay?docid=6469897211658098497&hl=en

15. RNA Reverse Transcription
• Process where single-stranded RNA is
transcribed with reverse transcriptase into
cDNA

16. Quantitative PCR
• Modification of traditional
PCR method
• Used to find quantity of
DNA, cDNA or RNA
present in a sample
• Uses fluorescent markers
to find DNA (SYBR
green) by monitoring
fluorescence emitted after
each detection cycle

17. Results
• Initial IL-17 C. gigas cDNA gel
electrophoresis (gill, muscle, mantle)
• Quantitative PCR results from oyster
bacterial experimental and control cDNA

18. IL-17 gel electrophoresis
1. O. conchaphila gill
2. C. gigas gill
3. O. conchaphila
mantle
4. C. gigas mantle
5. O. conchaphila
muscle
1 2 3 4 5 6
6. C. gigas muscle

19. Quantitative PCR results
Figure 1. Comparison of control and experimental relative expression levels
of interleukin-17b in C. gigas gill samples
300
Relative Expression Level
250 1 - 4 Control
6 - 8 Experimental
200
150
100
50
0
1 2 3 4 5 6 7 8

20. Quantitative PCR results
Figure 1. Control cDNA relative expression levels of C. gigas gill tissue samples
1.7
1.8
Relative Expression Level
1.6
1.4
1.2
1
0.8
0.5
0.6
0.4
0.1
0.2 0
0
1 2 3 4
Sample Number

21. Quantitative PCR results
Figure 1. Experimental cDNA relative expression levels of C. gigas gill tissue samples
1000 926.1
900
Relative Expression Level
800
700
600
500
400
300
183.4
200
39.8
100 0.1
0
1 2 3 4
Sample Number

22. Quantitative PCR results
• On average, relative expression level in
experimental treatments was 500 times
higher than in control treatments

23. Implications to the scientific
community
1. Gain knowledge on IL-17 in
invertebrates; provide inspiration and
basis for future research
a. Majority of information about IL-17 is about
mammals, humans more specifically
2. Use IL-17 expression in C. gigas as an
indicator of water quality
a. IL-17 expression can imply high levels of E.
coli, making surrounding waters unsafe for
shellfish harvest, swimming and fishing

24. Implications to the scientific
community
3. Marker assisted selection
a. Broodstock in aquaculture can be
possibly selected based on expression
4. Can give implications as to the effects of
other pathogens that can affect C. gigas
such as Vibrio sp.

25. Future directions
• Increase sample size
• Look at different exposure times
• Look at other genes
• Look at other pathogens