Jia Wern Pan
Internship Report for Summer 2009
Sewanee, TN
August 29, 2009
The evolutionary history of local land snails (Anguispira sp.) and the population
structure of the eastern mud-snail (Ilyanassa obsoleta).
This project examines the evolutionary history and genetics of two kinds of
snails, “snake-coiled forest snails” (Anguispira sp.) and the eastern mud-snail
(Ilyanassa obsoleta). Snails from the genus Anguispira are endemic to southeastern
Tennessee and I examined the evolutionary history of this genus by using DNA
analysis to determine the phylogenetics of the genus leading to current snail diversity.
I also used similar DNA analysis techniques to determine the population structure and
genetic diversity of Ilyanassa obsoleta individuals collected from different parts of
coastal America.
In the project involving Anguispira sp., I was primarily involved in refining
methods of DNA extraction to extract DNA from individuals without harming them,
as required in working with endangered species such as A. picta. To do so, I
established a protocol using chemical techniques and kits from plant genetics to bind
the polysaccharides in snail “slime” so that surface epithelial cells can be used for
DNA analysis. With that done, Dr Haskell and I tried various different ways of
obtaining DNA from Anguispira snails that did not involve killing them. Our test
population consisted 24 Anguispira cumberlandiana snails that we collected from the
bottom of Shakerag Hollow, as well as a few Mesodon thyroidus snails that Dr
Haskell collected from his garden, that we reared in the lab. We first collected fecal
matter from the snails over two weeks and attempted to extract snail DNA from that.
We were able to obtain some DNA from the fecal matter, however to date we have
not been able to amplify or sequence that DNA for identification. We then tried more
invasive methods: scraping the foot of the snail with a file, and cutting off a small part
of the tip of the snail’s foot after it had fully extended. The former method failed to
Jia Wern Pan
Internship Report for Summer 2009
Sewanee, TN
August 29, 2009
obtain DNA (the snails produced too much defensive slime) but we were able to
harvest DNA from the tissue collected from the latter; as of now this DNA has been
amplified and is in the process of being sequenced. We also tracked the survivorship
of the snails after the invasive method of cutting the tip of their feet, and preliminary
results indicate that the method does not affect their survivorship to any significant
degree.
My daily duties for this project include caring for the snails kept in the lab
(feeding them, changing and cleaning the jars in which they were kept, etc.) as well as
carrying out the different methods for obtaining DNA and the various different lab
procedures to extract and amplify the DNA obtained.
As for the project involving the eastern mud-snail (Ilyanassa obsoleta), snails
of that species were mailed to Sewanee by contacts of Dr Zigler from various
different localities on both the East and West coast of America, including Maine, New
York, Washington, San Francisco, and Nova Scotia (Canada). Once the snails arrived,
I extracted the snails from their shells and stored them in a freezer. Over the summer,
I extracted DNA from these specimens and attempted to amplify and sequence a
specific part of the DNA (the mitochondrial CO1 gene). These sequences will be
compared statistically to determine the population and genetic structure of the
population. Over the summer I have managed to obtain sequences from roughly 40
different individuals, and I also have been able to refine the DNA extraction and
amplification procedures (determining optimum amplification temperatures, finding
the best combination of primers used for DNA amplification, etc.).
My daily duties for this project include processing the snails that arrive from
the various different localities (labeling, extracting, and storing specimens), as well as
Jia Wern Pan
Internship Report for Summer 2009
Sewanee, TN
August 29, 2009
carrying out various different lab procedures to extract, amplify, and sequence the
DNA of the specimens (DNA extraction, PCR, purification of PCR product, etc.).
It should be noted that both of these projects are still ongoing. I will continue
to work on both of these projects through the school year, in the hope that they will
eventually be able to be published in scientific literature. As such, the Yeatman fund
provided an invaluable opportunity for me to get started on these projects and get
them into full swing. Being able to work on these projects over the summer has
enabled me to iron out many of the problems that inevitably plague such projects, and
will make future work on these projects much easier.
Professionally, this internship has provided me with valuable experience that
will be very useful in future endeavors. As my current goal is to become a research
scientist in the field of evolutionary biology, this internship has allowed me to gain
experience in many of the basic techniques and procedures used in that discipline. For
example, as my internship consisted of both field and lab work, I was able to learn
important field methods (data collection, describing the biogeography of the studied
organism), as well as important lab techniques such as primer design, genetic data
analysis and DNA barcoding. Also, if all goes well, I will have been able to contribute
towards the publication of one or two scientific articles on my research subject (snail
genetics and evolution), which will both improve my scientific credentials and allow
me to learn the processes that lead towards the publication of such articles.
Furthermore, during the course of my internship this summer I was able to participate
in an informal malacology conference regarding the current status and future of the
endangered Anguispira picta, which was an eye-opening experience as to the daily
life and work of conservation biologists. Last but not least, I had to carry out these
Jia Wern Pan
Internship Report for Summer 2009
Sewanee, TN
August 29, 2009
projects mostly on my own, and the experience has taught me a lot about the
importance of personal initiative and motivation and how to be independent.