2. What is a Crinoid?
• Marine animals inphylum Echinodermata
• Common as fossils – typically stalked
• Davidaster rubiginosa, Davidaster discoidea –
Comatulidae – extant stalklesscrinoids
• These species may be found on shallow reefs
3. Objective
• To test, through genetic bar-coding, the
hypothesis that a cryptic species of crinoid
exists within Davidaster.
• Why is this important? – Identifying full
genetic diversity allows better
understanding of fragile tropical reef
ecosystems.
8. Sample acquisition led us to San
Salvador, Bahamas where we could find two of
the three crinoids in question right off the
coast.
9. Lab Work
This particular crinoid (D. discoidea) was
extracted from a reef, brought to the research
facility for observation in a water table, and
inspected under a microscope. The next day it
was returned to its reef with no harm done.
17. Back at Home – Cincinnati Museum
Center Molecular Genetics Lab
18. Immediately upon return from the
Bahamas, Polymerase Chain Reactions (PCR)
began. After several unsuccessful attempts to
amplify the DNA, it was decided that another
round of extractions should be done in the
comfort of our own lab.
19. Polymerase Chain Reaction
- Amplifying DNA
• Literature search to find Primers
• Record list of Primers for purchase
• Order
• Wait/Receive
• Set up PCR
20. Performing PCR
• Requires exact measurements of
– Water
– Taq (thermostableT. aquaticus
DNA polymerase)
– Forward and reverse primers
– DNA
22. After PCR, one must check to see if
amplification was successful by
inserting PCR’d sample and
loading dye into a Gel
Electrophoresis apparatus.
23. Gel is ‘run’ and then placed over
ultraviolet light. This is an example of
what you should see.
24. Sequencing
• Used PRC’d sample for sequencing prep
• Ran a different program in PCR machine to
complete sequencing reaction
• Once sequenced, collected sample sequence
was used to create new primers for increased
precision in amplification results
• Amplified DNA with crinoid-specific primers
25. The catch…
•Preliminary sequences are not adequate –
sampled tissues did not yield full genetic code
Originally, to spare crinoids, we only sampled
their arms (able to regenerate).
Unfortunately, DNA amplification has led to
mixed results. There appears to be little tissue
in crinoid arms and we may need to collect
larger crinoid samples for better results.
26. What’s Next?
• Obtain better samples (Jamaican expedition
planned)
• Repeat amplification process
• Sequence and read genetic code
• Compare with knownDavidastercrinoids
• Test hypothesis of cryptic species
27. Thank You!
I would like to give a big thank you to the Cincinnati
Museum Center, The National Geographic Society
for travel funds, Professor David Meyer, his wife
Kani, Dr. Herman Mays, and my father.
