Or, A diſcourſe on the abſence of monotony, being a time of inopportune distreſs, initiated by forces outſide the author's demeſne "Who are you, who are so wise in the ways of science?" -Sir Bedivere, Monty Python and the Holy Grail Saturday was the coolest day -- we went dredging on OIMB's shiny new research vessel, the R/V Megalopa. Dr. Emlet was an ace at identifying the many organisms we pulled up from 150 to 300 ft deep, and the incredible crew (Captain Bradd Beckett and James Johnson) made the trip smooth and enjoyable. The boat is equipped with an A-frame to support the small trawl net, and I operated the winch for a bit. Several people got a bit queasy, including me, but no one lost their lunch, since it was a fairly calm day. What helped me most were ginger chews, saltines, and sitting in the cold wind, so I'll come prepared in the future! The basket stars we pulled up were magnificent, but our table was overflowing with over a dozen phyla: sea cucumbers and brittle stars, corals, a Cerebratulus nemertean, sponges, jellyfish, sipunculids, fish, Dungeness crabs, nudibranchs and chitons, serpulid tubeworms, phoronids, bryozoans, brachiopods, scallops, and several more I must have forgotten. We presented about our projects to the other REU students on Wednesday, and it was really cool to see what everyone's been up to! It was revealing that everyone thinks their own project is monotonous, but everyone else sees how cool the research is. "Thus took form by degrees those hundred thousand diverse races, the classification of which so cruelly embarrasses the unfortunate race that habit has changed into naturalists." -Georges Cuvier, Recherches sur les ossemens fossiles de quadrupèdes As for my project, it has been anything but monotonous. It's hugely exciting to see how many ways research can go wrong. Since last Friday, we have passed 670 amplification reactions, but there are still 24 samples (out of 180 that are new this summer) which we can't amplify. We have a few last tricks up our sleeve, but many of these will remain unsequenced. This is disappointing, but today Dr. Maslakova reassured us that there is always a percentage that cannot be sequenced, and moreover, with or without these samples, our project is groundbreaking in documenting nemerteans from the Arabian Peninsula. Her outlook is both pragmatic and optimistic, for when we have made every reasonable attempt, we must be able to cut our losses and focus on the many other tasks and endeavors that are always begging for our attention. Banging your head against a wall over and over won't break the wall -- just your head. However, we were enormously excited this morning to have 96 samples loaded into two plates to send for sequencing -- when we discovered that the sequencing company the Maslakova lab has been using for years permanently shut down last week. This caused a real scramble. The University of Oregon Core Facility doesn't offer Sanger sequencing service, but the Oregon State University Core Facility doesn't measure and adjust the DNA concentration, which is necessary for a high quality read. In the past, the sequencing company would quantify samples for us, so we weren't prepared for the procedure. We tried a Qubit fluorometer, but the standards wouldn't read properly. We also tried the gel-quantification method, which uses a ladder with DNA fragments of known lengths and concentrations. The ladder we borrowed from Dr. Emlet did not give a very clear image, but it was enough to roughly determine the concentration of test samples, by guesstimating how much brighter or dimmer each sample seemed compared to the ladder. We now have to go through our gel images, decide how bright each successful band was compared to our test samples, remove our products from the loaded plates, dilute them by a factor between 1 and 12, and load them into a different layout, plus primers. This promises to be a time-consuming and monotonous process. Alas, we have no other option, and we desperately want to get these 96 specimens sequenced, so we're going to go through with it. I am experiencing firsthand that science can be tortuous and arduous, but with luck, we will send our samples for sequencing next week, and soon crest back atop the emotional high of species discovery! "I must have been distracted when I left my home because P.S. I wrote this blog just after our dies horribilis, but since Friday we have finished loading up the new plates. It was a lot of work, but we are prepared to send them for sequencing on Monday, and I am very excited to see what we will discover next week!
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...which must in time find the sea I look back and can't believe it's already been a month here. We submitted our research proposals to the faculty organizers of the REU program, Drs. Maya Watts and Richard Emlet, for feedback this week. Next week we'll present our projects to the other REU students, so we're really in the thick of it. Also great news, I have finally met Dr. Emlet in person! He has returned to OIMB after teaching a course at Friday Harbor Laboratories up in Washington, and at lunch today he had some very insightful advice about career paths and the state of academia. Last weekend we hit up a string of excellent thrift and vintage stores in Coos Bay, where I picked up a flannel and a book of Ansel Adams prints. That evening we went to the 7 Devils Pub and Brewery for a "Three for Silver" concert recommended by my mentor, Dr. Maslakova, and her husband, Dr. von Dassow. He knows the band particularly well, since he once hosted them as artists-in-residence for two weeks in his lab. They were real crowd pleasers, and I had a great time dancing by the fire. Audrey and I have been emerging from our lab occasionally to poke around in the inter- and subtidal. We joined Jay and Adam to count the daily catch of the Dungeness crab megalopae (spiky larval forms), and it was fun to see the long-term monitoring that can happen at a marine station. We also collected a plankton tow with Chloe last Tuesday, which is a simple but useful technique. Later in the week, Chloe came into our lab for a little research exchange, pipetting a ladder in our agarose gel. Thursday morning, we went with Randi, Madison, and Chloe on the mud flats to muck around with the phoronid worms, clams, and polychaetes. As I write this, we just got back from a new hike with El -- literally a new trail that a couple students hacked up the coast. It's still rough in spots, but the mist was beautiful today, and I love investigating these small pockets of wilderness. Audrey and I are, however, still doing plenty of lab work. We received our second plate of sequences, and after hours of cleaning data and sorting spreadsheets, we have a grand total of seventeen tentative species newly sequenced this summer (and likely new to science!). This is a pretty incredible haul, and we still have at least one more plate of samples. We compare each sequence against the several hundred samples Dr. Maslakova has collected and the GenBank database. Assuming the attached identifications are correct (which is not always safe to do), the matches suggest what taxon we have sampled. The worst mismatches to decipher are the subtle ones, which sort to the wrong genus or clump visually disparate specimens together. This could be because of mislabeling in the lab or very surprising phylogenetic similarities. Assuming our DNA extracts are uncontaminated, we can re-run amplification to get more accurate results. However, some of these samples are likely irretrievable, which is always a risk. We'll keep chugging along, testing different tips and tricks, and I hope I'll have good news about them next week. I know everyone is eager to learn the worm poll results. This highly scientific survey shows that one third of my audience is Tetrastemma melanocephalum (shadow face). There are also quite a few T. vermiculus (crying) and T. longissimum (aaahhh). Also represented are T. peltatum (smiley face), T. laminariae (choker with large dots), and juvenile T. coronatum (sad baby). Species richness: 6. Average population size: 3.8 ± 1.8. Simpson's Inverse D: 6.17. The species discovery curve shows that I can expect to discover about 10 species of Tetrastemma in my audience if I sample 184 respondents.
No matter what I say,
Saturday night, we gathered around the fire, ate some innovative cookie-s'mores proselytized by Chloe and Audrey, and tried to tell ghost stories. El opened with a fabulously spine-chilling campfire tale, but no one else knew any good ones, so I told a very silly joke about a village of elves, an old wise wizard, and the frightful Medikin. If you're curious and have 15 minutes to spare, I'll tell it to you someday... I'm working on my research proposal, which includes seeking out original species descriptions, and there are some crazy illustrations! These are serious business. Wormy squirm. Lab work this week has been super exciting, because we received our first 48 sequences on Monday! With a nifty program called Geneious Prime, we first trimmed off the ends where we saw low quality reads or primers, which don't reflect the nemertean's actual genetic sequence. Since we sequenced from forward and reverse primers (one on each DNA strand), we had two reads of each region, which Geneious Prime aligned and searched for discrepancies. We then BLASTed the sequence (a great opportunity for fun sound effects *whoosh* *blaaaast*), meaning a search against a national database called GenBank for similar genetic sequences. After checking for mismatches with initial field identifications, which would suggest contamination or mislabeling, we constructed a phylogenetic tree to see how our sequences grouped, and it was awesome! Some species overlapped with previous specimens, which was both expected and good confirmation, but we also found 5 entirely new, never-before-sequenced “operational taxonomic units” (OTUs) in just the past three weeks! These OTUs are genetically distinct enough that we believe they are reproductively isolated populations, a.k.a. separate species. Here's a sneak peak at our results, just using the couple that I have images for: One specimen groups genetically with an OTU identified to the suborder Reptantia, and visually they are also quite similar. On the other hand, this one represents an entirely new species of Cratenemertidae. Here is another specimen matching a Lineidae OTU. But it's important to remember that a big reason we are using genetic techniques is because of cryptic species. Here are three OTUs within the genus Carinoma that are visually indistinguishable but genetically distinct. Here's the branch of our phylogenetic tree for Carinoma. Purple sequences are ones Audrey and I just added. Most of them group closely with one of the three OTUs documented from Oman (pictures above), but the ones boxed in red are completely separated -- a new OTU! I don't have pictures for it yet, but those worms probably look pretty similar to the other three tentative species. So hopefully this gives you a better idea of what we're trying to identify, why we're doing it, and what we're discovering!
You can really have no notion how delightful it will be My highlight this week was tidepooling during the spring tide, when the sun and moon align to create dramatic changes in water level, which gave us a strong -2.2 ft low tide. I went with Riley (our awesome REU coordinator), Randi, Naia, and El to Sunset Bay to poke around in the eelgrass beds and rocks. There was lots of scrambling, down the cliff and then across algae-covered rocks, sea urchins, and eelgrass. I slipped a couple of times, but luckily I got off with only a few scratches. Besides the abundant purple sea urchins and ochre sea stars, we spotted red sea urchins, gumboot and leather chitons, six-rayed sea stars, sea slaters and kelp isopods, some very pretty nudibranchs, and a bright orange Tubulanus ruber nemertean worm. I got pretty cold after a couple hours, but it was an incredible experience, and I'm very glad I went! Tuesday was the Fourth of July, so us REUs went to the city of Coos Bay to watch the fireworks. We were also planning to wander around and find something to eat, but almost everything was closed, so we ended up at McDonald's, which was oddly apropos. On the way back, we had a lot of trouble wrangling the van, first with the parking brake (who knew you needed to pull the release handle?), then with turning on the headlights, then with turning off the roof light I had accidentally turned on while trying to find the headlights, but we made it back okay. On the research side, we have finished the first pass on all of our nemertean genetic samples currently on-hand, which is great news! Unfortunately, we are now in the dreaded troubleshooting phase, with finicky samples that did not work initially, for several possible reasons. Some nemerteans had a delicious meal of polychaete worms right before they were collected, so we ended up with a sequence matching the wrong phylum. Others might have enzymes bound up in their DNA which inhibit replication. Earlier this week, we used a 10% dilution of the DNA, which counterintuitively can improve results by diluting these enzymes, and in fact that technique solved about half of our problematic samples. For a third group, we may need to test different primers because of genetic variation in the target gene. Primers are short DNA sequences that provide the starting site for the replication enzyme. We usually start with the so-called 'universal' Folmer primers, but we moved into testing nemertean-specific primers in various combinations, which might bind better to nemertean DNA and improve the signal. Next week, we'll continue testing combinations of primers, a 1% dilution, or lowering the temperature during polymerase chain reaction to encourage binding even when there are slight mismatches to the DNA. In an exciting development, we mailed our first plate of samples to an outside company for sequencing today, and we should have that data back by Monday, which will allow us to start identifying tentative species. I'm looking forward to our first results! And there came of a sudden creatures with backs as hard as anvils, bent of claw, walking aslant, squinting, scissor-mouthed, shell-skinned, bony-natured, flat-backed, gleaming-shouldered, bandy-legged, with tendons for hands, peering from their chests, eight-legged, twin-feelered, unwearying: those known as crabs. -Batrachomyomachia (The Battle of Frogs and Mice) My first week at OIMB has been a whirlwind! It's a wonderful experience to live and work on a campus with a small community where everyone is captivated by the marine world. My first day on campus, a big group of students went down to the beach to poke around, and everyone was exclaiming in delight at the ochre sea stars, picking up crabs, or staring intensely at the little tidepool sculpins. Everything I need is nearby, from the dining hall and 24-hour library to the OIMBeach and everyone's favorite convenience store, Davy Jones' Locker. I visited the Charleston Marine Life Center yesterday, where the collections of living and preserved organisms are fascinating! Highlights include the Humboldt squid and the dogfish. There are also a series of trails running up the mountain behind campus, and it's impossible to convey how stunning the Pacific Northwest conifer forest is. My research is off to a great start -- from samples of ribbon worm (aka nemertean) tissue preserved in ethanol, I have extracted DNA, run it through a polymerase chain reaction (a pivotal method to amplify the specific regions of DNA), checked product quality in an agarose gel, and purified the PCR products. Early next week, when we have a few more samples to fill a plate, we'll send them off to an outside company and get our first sequences! With these genetic sequences, I'm going to look for which specimens belong to the same species and which have enough divergence to be separate species.
This is the crux of the project: by comparing genetic barcoding sequences to images which were taken in the field, we can look for ribbon worm species that are visually similar, but don't interact as one interbreeding species. Nemerteans desperately need this descriptive work because they have few notable external features except for color, which fades in preserved specimens. Also, these smooth, sliding, predatory worms with proboscides which pop out of their body cavities simply haven't received much attention. One of the best (and only) resources I've found so far about nemerteans in the Red Sea is from 1831, out-of-date in its descriptions and taxonomy, and written entirely in Latin. I never thought those four years of Latin classes could be so useful, so special thanks to Drs. Dombrowski, Beckelhymer, Martelli, and Goldberg! Next week, I'm looking forward to even more jigsaw puzzles, starting up the OIMBand -- I hear we have two violins, a ukulele, a guitar, and a banjo, so it's going to be awesome -- more lifer birds (I've seen Golden-Crowned Kinglets and Pigeon Guillemots for the first time), and learning more about the fascinating biology of nemerteans! |
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