Wow, what an amazing last few weeks here at OIMB! I am surprised by what we have already accomplished and learned in such a short period of time. We have really hit the ground running to get started on the biodiversity project for nemerteans that are found on the Oregon coast. As I have now learned there are many parts to properly completing a modern biodiversity survey and creating an identification guide. Some of this process has been unchanged for hundreds of years, such as the observation and classification of different types of species based on their morphological features. Morphology is the study of the forms of things and has historically been the primary tool for identifying different species of organisms. The problem being that if you ask a group of ten people to observe something and then describe it, chances are there will be variations in each persons description. So as you might assume, this was never an exact science and there is lots of room for error and no way to “clean it up” with any amount of certainty, But now with DNA sequencing there is a means to more objectively identify and group different species. This not only helps to confirm specific features belonging to a specific species but can also alert us to cryptic species. Cryptic species are organisms that look alike. DNA barcoding can reveal that although these species can look very similar their DNA is, in fact, different enough for them to belong to different species, tells us they are genetically distinct . The opposite can also be true; some animals that appear to be very different, are in fact the same species but with varying color patterns. A Glimpse into Molecular Biology:)The first week here at OIMB we went through the steps for processing the DNA. Christina Ellison, a graduate student working with Dr. Svetlana Maslakova, our research mentor, guided us step by step through this fascinating process. The first step in this process was to extract and purify the DNA from the nemertean tissue samples. These first samples were collected prior to our arrival and placed in individual vials of 95% ethanol, which where then stored in a freezer at -80 ℃ to prevent tissue degradation. The first step was to extract the DNA from the chunks of tissue. This is accomplished using a miniature pestle to manually grind the tissue and by adding a lysis buffer solution. The lysis buffer helps to break open the microscopic cell and nuclear membranes, essentially freeing the DNA strands from inside their protective nucleus walls. A small amount of proteinase K is also added to the mixture to help digest the proteins and enzymes in the tissue. The Proteinase K keeps the naturally occurring enzymes, DNAse and RNAse, from doing their job of breaking down the DNA and RNA, thereby keeping the DNA intact for us to use for DNA-barcoding. The DNA then needs to be separated from the other cell debris. One way this is accomplished is by running the mixture through a special “DNeasy” column that has an artificial filter membrane. This process includes a variety of steps but essentially the DNA binds to the membrane in the vial so that the debris can be washed through it, leaving clean DNA strands attached to the membrane. The DNA is then released from the membrane into nuclease-free water, to be collected and used in the next step. Here comes the really fascinating part. In 2003, the Cytochrome C oxidase subunit I gene (commonly abbreviated as cox1 or COI) was proposed to be used as a universal gene region for DNA-barcoding of animals. This sequence changes fast enough over evolutionary time to differentiate between even closely species, but is also flanked by two regions of DNA that have not changed much across the animal kingdom. Therefore the markers on each end are very similar in most animals, and can be targeted to help amplify the highly variable region of DNA in between, regardless of wether you are sampling tissue from a rabbit or a nemertean worm. The key here is that you need to have the same region of DNA from a large number of organisms in order to be able to compare and contrast sequences across the animal kingdom. The DNA processing is of course not the first step in completing a biodiversity survey but by running through this procedure first it helped me to see the value of DNA barcoding in species identification, and cataloguing species diversity. It also made me aware of the importance of properly handling and labeling the tissue during field work and the importance of not contaminating the tissue samples with DNA from other sources. The Journey Begins!So, last week we officially began our first biodiversity survey. The first step of a survey is actually preparation and includes things like getting a collection permit, scheduling and preparing for the field work, planning for specimen storage and tracking. This step was already completed by our mentor, Prof. Svetlana Maslakova, who has been collecting and studying nemerteans in various parts of the world for the past 20-some years. The beginning of last week was spent getting familiar with the taxonomy and differentiating features of the various types of nemerteans. I admit the taxonomic names and pronunciations have been a bit of a challenge for me, but I am persistent and determined to keep trying!
The next step is field work, which for us meant a lot of early morning tide pooling and collecting nemerteans from various habitats. Enjoying the views and getting to know Southern Oregon’s diverse marine life was a fantastic way to spend the mornings and explore the coastline. It was important to collect as much information as possible for each individual worm, and it helped that our mentor has an established system of recording and cross referencing data from each trip. The tide pools provided a variety of habitats to sample from such as underneath rocks, in mussel beds, in the sand, on surface of rocks or among kelp holdfasts or surfgrass roots. Each location and habitat is recorded and given a reference number. Once back at the lab the worms are put into beakers and bowls and placed in the sea table as quickly as possible to stay cool. Then, they are grouped by morphology, identified to the closest known taxon, and labeled with location and habitat.. This made for a few busy but exciting days, and it was really fun getting to know the worms I have heard so much about. They were surprising and definitely have more personality then I would have ever thought. We have begun processing the worms this week. Every worm that is being processed gets assigned an individual number, and is documented with color photographs of its features. Some worms are large enough for a standard macrophotography (using an SLR camera with a macro lens), while others need to be photographed through a microscope. Learning how to work with the different camera equipment and microscopes has been a really cool bonus that I was not expecting and have thoroughly enjoyed. Once the specimens have been properly documented, they are preserved in two separate vials. Some of the tissue (usually the posterior end) is placed in 95% ethanol for DNA-barcoding and some (usually the anterior end, which is more feature rich in these worms) into 10% formalin to be preserved and later deposited into a natural history museum as a morphological voucher. The morphological voucher is a way to provide a reference specimen for other scholars to see the features first hand.
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Rebecca OrrHi, my name is Rebecca I’m from Northern California. My major is in biological sciences. I am so excited to be working in Dr. Svetlana Maslakova’s lab and learning the protocol and procedures for completing a modern biodiversity survey. Archives
August 2021
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