Sticks in the Mud

As mentioned previously, one of the aims for this project was to compare allele frequencies between life stages of the Olympia oyster to see if there are any significant shifts and at which loci. These shifts could be due to stochastic processes, such as Sweepstakes Reproductive Success, or strong selection during a life stage that causes high mortality for individuals with certain alleles. Discriminating between the two is veryyyy difficult. As this field season is exploratory, I designed an experiment to determine if allele frequencies might indeed be shifting between life stages and whether this varies by geographic location and environmental conditions.

Larval Olympia oysters preferentially settle on oyster shells, so I made sticks with shells on them to place in various locations at the beginning of my field season. Olys (the affectionate term for Olympia oysters) start spawning as early as mid June and can go until September. After settling, the spat is about 0.5mm in size and under the microscope faintly resembles a contact lens. At the end of my field season I planned to gather some of the oyster sticks and carefully scrape off the spat for subsequent genetic analysis. The remaining oyster sticks would be left to allow the spat to mature to juveniles and then sampled at a later date.

Drilling shells

Drilling holes into Pacific oyster shells…somehow I finished with all 10 fingers still intact…

 

 

Shell sticks after being left in the water for almost a year

Shell sticks after being left in the water for almost a year.

Due to Jennifer Ruesink’s longterm monitoring of larval settlement in the area and recent characterization of pH variability, I chose Willapa Bay as one of the geographic regions for this project. The pH in the southeast part of the bay remains relatively stable at 8 during the summer, while the pH in the north part of the bay can range between 7.6 to 8.6 (a huge difference when you consider the pH scale only goes to 14!!). This difference in variability between the sites is likely due to the Willapa River input in the north and other oceanographic features of the bay. To see if these environmental differences affect allele frequency shifts, I decided to place shell substrates in both areas. While setting out oyster sticks in the southeast was easy, the olys in the north part tend to settle on substrate 30 feet underwater. To address this, Alan (introduced here) helped me construct the concrete version of shell sticks, because apparently oly larvae are not very discriminating between shell and cinder blocks. The blocks were tied to buoys and then deployed at a later date by Alan to be picked up again sometime in October.

The other region I chose was Puget Sound, WA, which also demonstrates environmental heterogeneity between different subregions. Dr. Brent Vadopolas and others at the University of Washington are doing some really interesting work looking for signals of local adaptation in 3 populations of Olympia oysters by outplanting oysters from each population to 4 locations around Puget Sound. During the course of this study, they observed extreme mortality in one location (Dabob Bay) for all transplanted populations. Additional observations from the region indicate that natural populations of olys in south Puget Sound are doing well, while those in the north are starting to grow despite previously low numbers. Some questions these observations raise: was the mortality event stochastic, or was it due to consistent ecological factors in the area? If oysters are locally adapted to their specific subregion of Puget Sound, where at the northern oysters recruiting from? To tie in my project with theirs, I placed two shell sticks next to their experiment in the North Sound and two near the South Sound location. Stephanie Valdez, a recent graduate of UW, is monitoring Olympia and Pacific oyster settlement in Hood Canal (body of water that offshoots Puget Sound) and agreed to give me the oly recruits from her Dabob Bay shell sticks for genetic analysis.

Locations of Puget Sound shell sticks

Locations of Puget Sound shell sticks

Sounds like a great plan, right? As I’m writing this post facto, I can say that this aspect of my field season was essentially a failure from the get go, at least with the Puget Sound shell sticks. First, I was unable to find the oyster transplant site in South Sound due to a miscommunication about the GPS coordinates and my own stubbornness about asking for clarification. Instead, I decided to find my own spot in the vicinity to put my shell sticks. The area of the sound I was in, Oyster Bay, is surrounded by private homes with “No Trespassing” signs and actual commercial oyster farms. After an unsuccessful attempt to ask a homeowner for permission to access the bay from their backyard, I gave up on that idea and decided to park at a wildlife viewing area near the tip of the estuary and cross the (deceptively) short distance to the water’s edge. It took an hour of slogging through the mud, losing my boots, being laughed at by raccoons, and eventually breaking one of my shell sticks for me to accept defeat.

10463887_10152988725794128_5124704887406444502_n

That night, after a campground shower and a couple IPAs, I found the resolve to try again the next day. This time, instead of directly cutting across the soul-crushing mud to the water, I decided to walk along the rocky edge of the bay until I reached an area that would remain underwater even during minus tides.

Oyster Bay

Green: path to stick placement
Red: path back to car

This Google Earth image was obviously taken during a normal tide, because when I trekked out there with a 1.3 ft low tide the water didn’t reach nearly as far inland. From the car to the shell stick placement site was about 1.2 miles of sweating, slipping, cursing, and  overall misery. To avoid such a return trek, I cut through someone’s backyard and walked 2 miles on the backroads- no doubt looking like the creature from the black lagoon. The placement in North Puget Sound was considerably easier. I quickly found the UW oyster transplant site and wedged my shell sticks about 50 ft away.

Fast forward 5 weeks to the beginning of August. By that time, I had finally internalized the value of asking for help and simply drove up to the house in Oyster Bay I had previously trespassed by and asked if I could walk through their backyard to the bay. They found the request strange, but mildly amusing and let me go by. When I get to the coordinates for my shell sticks, however, they were gone. Both of them. I frantically waded around the entire area to no avail. Talking to Stephanie later, she said that in her work shell sticks do occasionally go missing. Maybe a nearby oyster farmer saw the pink flag tape sticking out of the water and thought it was trash. Maybe I didn’t hammer them in deep enough and they washed out with the tides. Maybe otters came and made a shell necklace out of them. Whatever the cause, by not having one of the sites it took away any interesting comparisons I could do with the others in the region so I decided to focus on my phylogeographic collections for the remainder of my field season. Also, I still have hope to get something interesting out of the recruits I’ve collected from Willapa Bay.

While totally a bummer, the entire experience taught me a lot about field work, experimental design, and myself.

  1. When setting out an experiment, talk to all of the nearby landowners and workers so they know to keep an eye out and not disturb anything. They may even be interested in the science!
  2. Don’t put all of your eggs in one basket, i.e. put your shell sticks in multiple sites for a sampling area.
  3. Ask for help! You don’t get any points for being stubborn and trying to work something out alone. In fact, you’re more likely to get covered in mud.

 

See You in Seiku

I’m going to be a little anachronistic for this next post, because I’m waiting on GPS coordinates to make a pretty map for my last couple of days in Willapa. So fast forward to the evening of June 29….

WBtoSekui

 

Off Highway 101 near Kalaloch, WA

Off Highway 101 near Kalaloch, WA

After a beautiful 3 hour drive along the Olympic peninsula, I arrived late at the house of my advisor Cathy Pfister and her husband Tim Wootton, another ecologist and faculty at UChicago. For most of the year they live in Chicago, but during the summer stay out in Seiku, WA so that they are much closer to their primary field site, Tatoosh Island.

tatoosh_aerial

A tiny seaside community of 27 year-round residents, where cell service was negligible and kids were more likely to be running around outside than sitting in front of a TV, Seiku had the feeling of quintessential Small Town America without all the commercial trappings. Being a 5 minute walk from the Pacific wasn’t too bad either. My purpose for this visit was not particularly oyster related, but instead to catch up with Cathy and explore some of her field sites around the area to see if a particular organism, ecosystem, or potential research question tickled my fancy. While oysters are certainly my species du jour, it’s a good idea at this stage of my PhD to keep an open mind. Choosing a thesis project requires discovering a relatively unfilled niche so that other researchers aren’t scooping your findings and publishing them before you, making all the hard work seem for naught!

The next morning, Cathy drew me out directions to Slip Point, a rocky intertidal habitat a few miles away.

Slip Point

Armed with pH and dissolved oxygen sensors, a waterproof notebook, and a rain bib I spent a few hours poking around in tide pools and climbing over slippery rocks. Having previously lived in Texas and Florida, I had never actually seen a Pacific Northwest rocky intertidal habitat in person before. Similar to those I was familiar with on the east coast, there was a very visible gradient in different species leading away from the water. This follows the gradient of stresses an inhabiting organism might be subjected to, from being constantly submerged by water in the subtidal zone to experiencing huge, daily fluctuations in salinity, temperature, and desiccation in the upper intertidal. Tide pools dotting the shore offer respite from drying out, but even adjacent tide pools can vary drastically in species composition and environmental factors.

http://sky.scnu.edu.cn/life/class/ecology/chapter/Chapter3.htm

Intertidal zonation

What struck me the most was the diversity of seaweeds covering the rocks (struck being the operative word as I slipped and fell on my ass because of them quite a few times).

seaweed

While I may know more at this point about oysters than my adviser, she is much more familiar with the ecology of kelp in the PNW than me. I can’t even tell my reds from my greens! Despite that I was intrigued about the possibility of local adaptation in these sessile species, for in many ways oysters are similar to some plants in the way they produce large numbers of offspring and release them into the water/air with a small chance of finding suitable habitat to settle. Another PhD student in my lab, Courtney Stepien, is doing a lot of work in this area and I resolved to talk to her more about her research once I returned to Chicago.

Blood, Sweat, Mud, and Oyster Guts

WillapaMap

Thursday, July 26, 2014

Breakfast was at 4:30am, followed by gearing up for work in the tidal flats. This involves hip boots, a bright orange Pvc rain bib, my matching orange Carhartt rain jacket, and lots of layers. Unfortunately I don’t have the documentation of this fashionable outfit, but imagine a cross between Deadliest Catch and the Stay Puff Marshmallow Man and you’re close.
Jennifer Ruesink is part of a project called the Zostera Experimental Network (ZEN) , which is a collaboration of scientists around the world studying eelgrass (Zostera marina). I got to help out with one of their predation intensity experiments this morning, which involves super gluing live amphipods (tiny crustaceans about 6-10 mm long) to fishing line attached to poles and sticking them in an eelgrass bed to then check the next day. The number of creatures missing is a rough indicator of the level of predation, which can be compared to the level of herbivory (tested by, I’m not kidding, gluing organic kale to similar poles). While not always my first choice of an activity at 6:30a on a boat in the drizzly cold, the company was good and I got to pick Jennifer’s brain about feasible locations to sample from and the general ecology of the area. I also had my first taste of walking in tidal flat mud…
That afternoon I had an in depth conversation with Alan about his natural history observations of the bay and how the oceanography, such as river inputs and currents, may affect population structure. He also seems to know everyone who’s anyone that deals with oyster growing and management at the local and state levels. We mapped out a plan of where to put settlement substrates and where to collect adult oysters from so both the north and south parts of the bay are represented.
For the north part of the bay, the native oysters primarily live 30 feet underwater, and therefore can only be collected from trawling. Luckily for me, a trawl had been done that day to collect Pacific oysters to sell, and they were able to set aside a couple dozen native oysters that had also been picked up. These oysters had been cleaned on the outside and separated from each other, so excellent subjects for my first dissections! Up until then, I had never shucked an oyster, nor even seen an Olympia oyster myself, so this first group was slow going. For each oyster, I shucked off the top shell, rinsed the body with special purified water, and then dissected a small section of the adductor muscle to preserve in a buffer called RNALater (which keeps the DNA from degrading). I then noted the length/width of the shell, whether gonads were present, and the gender when I could tell.
oysterdiagram
For most native oysters, you need to look at the gametes (egg/sperm cells) under a microscope to tell if it is a boy or girl. Olympia oysters are different compared to most oysters in another interesting way. When the water temperature is just right, the males release their millions of sperm into the water for the females to filter out and use to fertilize their eggs. The young larvae are then brooded in the female’s mantle cavity for about 12 days until released into the water. This theoretically gives each larvae a much better chance of surviving the harsh ocean than the Pacific oyster larvae, which are floaters from the second a sperm fertilizes an egg.
OlyLife

PacOysterLife

  Friday, June 27, 2014

ParcelA
This day will forever remain in my memory as the day I almost died 500 meters from dry land…that’s obviously an exaggeration, but at the time it certainly felt that way. My goal was to collect adult Pacific oysters from a site that Jennifer and Alan termed “Parcel A”, look to see if there happened to be any natives hanging around, as well as pick some shell sticks they had set out earlier to look for Olympia oyster spat. Jennifer dropped me off on an oyster hummock with the boat around 7:30am, geared up in my orange Stay Puff outfit with my gloves and little collecting bag. The plan was for me to wait for the tide to go down so I could find the oyster stick, collect about 2 dozen Pacifics, and then walk back to shore and walk the little ways back to their house. I found a comfy spot amongst the razor sharp shells to sit, and then used an incredibly rough method to sample Pacific oysters at random from the surrounding area.
http://depts.washington.edu/jlrlab/oysters.phpLarge oyster hummocks
Oysters grow best on the shells of other oysters, and so in relatively undisturbed areas like this all of the oysters are in clumps, with the younger ones crowding on the older, larger ones. Some of these ladies were 16 cm long, and surprisingly heavy! An hour of traipsing around didn’t yield any native oysters, so I started back towards shore across what seemed to be a flat expanse of easily traversed damp brown. The second I stepped off the oyster reef, however, I sunk down to my knees. As thick mud is one of those non-Newtonian fluids where it actually solidifies under a shearing force, I tried to “sprint” forwards and tired quickly. This is where things got bad, like crying and calling for my momma bad. I made the mistake of sitting down and immediately sank so that mud and water went over my chest high rain bib and filled up my boots, making me even heavier. The Neverending Story came to mind more than a few times…

Admitting defeat, I trudged back to the safety of my oyster hummock, and decided the path of least resistance was to walk through the rising water around the rocks, climb over to the pier, and walk back to the house from there. Numerous people working around the docks must’ve seen this entire scene and I can only guess what they were thinking. Upon arriving at the house, Alan greeted me with a spray hose and congratulations- apparently plenty of people they’ve taken out to the tidal flats end up needing some kind of “rescuing”. He also recommended a bucket next time to help me push myself out when I get stuck (advice I have kept to avidly since).
The afternoon involved dissecting and measuring the Pacifics I had collected, and I discovered the joy of opening a “mudder”- a oyster shell that’s actually full of large polychaete worms and the same thick black mud that irked me so earlier that day. I also practiced collecting the tiny 0.5 mm spat off of the shell sticks. How I’m going to extract DNA from each of these tiny oyster babies is a challenge for back in Chicago.

Arriving in Willapa Bay

My field season started with a midnight arrival into LA on Monday, June 23 followed by a 10 hour drive to Medford, OR the next day. These back to back traveling days were good preparation, as they became more the norm than the exception over the next few weeks. I arrived into Willapa Bay, WA the afternoon of Wednesday, June 25 and met up with Jennifer Ruesink and Alan Trimble from University of Washington, my hosts and guides for the next few days. They’ve been working in Willapa Bay for almost 20 years, and are fountains of information on the natural history, ecology, and local recruitment patterns of both the Pacific and Olympia oysters.

Willapa Bay is arguably the most involved site on my list, as I plan to address elements from all three of my stated aims here. My goals during this tide series were to:
1) Collect Olympia oysters from around the bay for the phylogeographic study
2) Collect adult Pacific oysters
3) Set out settlement substrates in areas of the bay that experience different variability in pH. I would then return in August to collect the newly settled oyster babies (hereon referred to by the slightly more scientific term “spat”).

Before continuing, it’s worth mentioning why my sample collection schedule is structured the way it is, with a stretch of very busy days and then “down” time in between. While low tides usually remain at least a couple feet above the sea level mark (referenced as zero for describing tidal heights), there are certain periods that have a minus low tide where the tide falls below the zero mark. This is due to the sun and moon either being on exact opposite sides of the earth or on the same side. For the west coast of North America, the best minus tides are during the summer. Here are the estimated tides for Willapa Bay. Note how the minus tides start early in the morning and then gradually become later in the day.

June Tides for Willapa Bay, WA Willapa Tides June

Jennifer and Alan had two other people staying and working with them this tidal cycle: Alex Lowe, also a first year PhD student studying at UWash; and an intern, a recent high school graduate starting at UWash in the fall who was from a third-generation oyster farm. Jennifer is an excellent cook, and dinner that first night more than compensated for the fast food I’d been dining on the past few days. The conversation thoroughly tested my abilities to convey my knowledge of population genetics (most of which I had just learned a few months prior!) and what I intended to learn from these oyster species that wasn’t already known.

As proof that the best ideas come out of lively discussion, an additional potential project emerged from the chat. For as long as they’ve been working in Willapa Bay, Alan said that there was always an early set in July and a late set in August for the Pacific oysters. Set here refers to when the oyster larvae leave the water column and settle on their preferred substrate (usually other oyster shells), where they then stay for the rest of their lives. While there are always fewer larvae in the water before the early set, the proportion that survive to be juveniles is much higher than those in the late set. This may be because the early set are able to reach a bigger size before winter storms come and are better able to weather the rough conditions. From a genetics perspective, this poses a few interesting questions. Are the early spawning oysters more related to each other than you’d expect by chance? Is there a heritable trait that the early spawning adults have that confer them this advantage? What genes are responsible for this apparent shift in phenology that’s occurred for only some oysters in the population?

Oysters on the Brain

This summer I’ve embarked on my first official “field season”, collecting oyster tissue from two species on the West Coast of North America for an exploratory project to help me determine which species will be favorable for my thesis research. If you’re feeling up to a potential snoozefest, here is a proposal I wrote for the Hinds fund, an award for evolutionary biology students at UChicago primarily in their 1st or 2nd years to help support preliminary data collection and develop grant writing skills. It outlines some of the where/what/why/how of this project. Hinds Proposal 2014

Meet The Players

THE NATIVE

Olympia oyster
Olympia oyster (Ostrea lurida)

THE INTRODUCED

Pacific oyster
Pacific or Japanese oyster (Crassostrea gigas)

Project Aims

Starting out, I had three (overly ambitious) aims for this project:

  1. Analyze the fine scale phylogeographic structure of Olympia oysters from San Diego, California to Ladysmith Harbor, British Columbia; incorporate records of local extinctions/introductions into the analysis, estimate parameters of gene flow and dispersal, and correlate with environmental parameters.
  2. Use larvae and adults from both naturalized (producing successful offspring without human aid) and commercially reared populations of Pacific oysters to help elucidate how commercial practices either homogenize or diversify the species (or if there even are any detectable patterns)
  3. Sample newly settled oyster recruits, juveniles, and breeding adults from both species in Washington that experience different means and variances in pH and upwelling. These will be sequenced to identify the areas of the genome that experience allele frequency shifts between life stages.

As of writing, I’m about halfway through my field season and already there have been some reality checks about what is and isn’t feasible, though I’ve picked up some new ideas, too! Stay tuned, as I’ll try to retroactively write up some of my coastal adventures so that I can update the second half of my season in real time.