Tuesday, May 21, 2019

Spawn city

A Crepidula fornicata larva. You can see its spiral shell, and
the lob hanging down is its velum.
After two weeks of successfully keeping Crepidula fornicata adults alive in the lab, I am happy to announce they have given me larvae! Now my experiment really takes off.

I'm investigating whether the conditions brooding mothers are kept in have carryover effects on the larvae. My mothers are in two different temperatures and only half of them are getting fed, so I can test those two crossed factors (high/low temperature and food/no food). To tell if there are any effects on the larvae, I'm measuring several variables - how big the larvae are when they're spawned, how quickly they grow, and how long it takes them to become competent to settle.

Crepidula larvae swimming in a dish under the microscope.
The clear lobes on each individual are the velum.
Measuring larval size is a tricky game. I collect a random sub-sample of individuals by sucking up some water from the larval culture jar, and then view them individually under the microscope. Crepidula larvae love to swim and move around almost constantly with two fleshy lobes called the velum. The edge of the velum is lined with cilia, so the rapidly beating micro-hairs create waves that propel the larvae through the water.

I'm able to make them stop swimming by cooling them down - basically sticking them in the freezer for a few minutes. Then I measure them using an ocular micrometer, which is a type of ruler built into the eyepiece of a microscope. I record the magnification and how many tick marks long the larva is so I can calculate their true size later. Voila - data!

It is very exciting for me to be collecting data for my experiment, and I hope I have good results!

Thursday, May 16, 2019

Balance

"What's a work-life balance, and should we buy one for the lab?"
- online meme

I walked out of my office at 4 pm, and it felt weird.

Wet and happy after my dive in Hathaway Pond
I've been going full-steam-ahead for the last several months, pulling 10-hour days, coming in on weekends, and doing whatever necessary to complete my self-assigned work. It was time for a break. So on the first sunny afternoon in weeks, I loaded my dive gear into the car and headed out to Hathaway Pond.

I hadn't been in the water in months. After diving for 21 days straight in the tropics last December with Carl, I stayed on land for the New England winter. I had one dive in March this year, and that was it. It was high time to get back in the water.

It was an amazing dive. Hathaway Pond is not the most exciting dive site by any measure - it's just a little freshwater pond - but whatever aquatic life the pond had to offer came out for me in full force yesterday. As I entered the pond from the beach, a large fish swam around my legs. I knelt on the sand with my face just under the surface, and he faced me head-on. My freshwater fish taxonomic skills are a decade old and rusty, but maybe he was a crappie? Either way, I appreciated the welcome to the pond.

Most of the limnetic zone was covered in long, fluffy macroalgae, like green feathers covering the pond floor. As I went deeper, the algae disappeared, but I noticed something even more interesting: a green encrusting species that covered rocks, cinder blocks, and logs. It looked like a sponge, so I drew closer for a better look (freshwater sponges are pretty rare). I looked it up, and what I saw looks exactly like Spongilla lacustris, which is indeed a freshwater sponge! The sponge has a bright lime-green color from symbiotic algae living within it, and it's been documented in a few places in Massachusetts. As I emerged from the depths, I started noticing the sponge growing on algal fronds as well - it was actually pretty common in the pond. Very cool!

It was evening, so by the time I reached the surface again, the sun was at an angle. Instead of white light, the sun shone yellow through the turbid water column. There was one moment when I rolled onto my left side, facing the sunlight, and caught a magnificent view of small fish (maybe salmonid parr, based on their vertical stripes) back-lit by the angled yellow sun. It was the most fish I've ever seen in the pond, and it was a beautiful, serene moment.

By the time I got back to my car, I was cold but invigorated. Diving is my absolute favorite thing, and I was glad to get back in the water. Here's to a long and productive diving season ahead!

Tuesday, May 14, 2019

Tube city: part 2

Well, friends, I wish I could tell you everything in science went as planned, but I can't. Gear implodes; weather turns bad; organisms die; and every once in a while, the Navy gets in your way.

Tube City indeed!
I wish I could tell you the experiment I had set up two weeks ago worked perfectly, but that's not true. Over half my organisms died. Crepidula fornicata is an incredibly resilient organism (I've even sanded their shells before and they've lived!), but I figured out the one thing they can't handle: having their feet exposed. Crepidula have to have a substratum to grip onto, or they keel over.

So I started over. I collected more individuals and re-vamped the design of my experiment. Now each individual has its own jar, and they're all happily attached to the rock or shell I found them on. Dividing the individuals into their own jars had one main effect: the number of tubes increased by a factor of 3! My experiment really is Tube City!

So far, all the individuals are still alive, and it's been over a week. It should be a good experiment!

Friday, April 26, 2019

Tube city

Inside ESL. This place is Tube City!
Ladies and gentlemen, boys and girls, I'd like to introduce you to the Woods Hole Oceanographic Institution's Environmental Systems Laboratory. This building, located just a few hundred feet from the beach on the south shore of Cape Cod, is home to numerous experiments maintained by WHOI scientists, all the plumbing they require, and the dull roar you would expect from an uninsulated wooden building full of running seawater lines. I'm using the ESL facilities for an experiment using the slipper limpet, Crepidula fornicata, and in short, I will be spending a lot of time here this summer.

Crepidula females in one of my experimental treatments.
I set up my experiment in one corner of ESL, on a metallic rack with two seawater tables (in the foreground of the photo above). Thankfully, a technician was able to set up the rack and the seawater lines for me. My experimental design involves keeping female Crepidula fornicata at two different temperatures, so I rigged up one side with water from a cooled seawater line (about 15° C) and the other with heated water (about 22° C). I also want to see how the Crepidula are affected by food supply, so I separated my specimens into three bins on each side, one for no food, one for low food, and one for high food.

Over the next couple months, I will be maintaining Crepidula females under these conditions (different temperature and food supply), and then when they spawn, I will see if there are any carryover effects of the mother's environment on the larvae. This experiment is part of a new research direction I'm exploring, about variation in larval dispersal within a single species. If some individuals are born larger, stay in the larval form longer, or have different behaviors, they could disperse farther and colonize island-like habitats like shipwrecks or dropstones or experimental panels or fjords (as you can tell, this work builds on my previous analyses of island-like habitats). I'm starting with an easy species for my first experiment, and then we'll see where the science takes me.

Stay tuned over the next couple months for updates on this experiment. Fingers crossed that it works!

Wednesday, April 24, 2019

Little Harbor

I was calf-deep in water, wearing jeans and thick rubber boots. The sunlight draped over my shoulders and reflected back up at my face from the shimmering surface below. To my left and to my right, I could see the faint black shadows of land surrounding Buzzards Bay, but in front of me was only the sea. It started out clear near my feet, then took the color of the sand in front of me, and finally deepened in shades of blue toward the horizon. I waded through the water, careful not to splash over the top of my boots. Beside me, I half-dragged, half-carried a 5-gallon bucket on the surface of the water. The mess of green algae in the bottom was already home to ~150 limpets, my catch for the day. Not a bad day at work!   

My trip to the idyllic sand flat known as Little Harbor was motivated by a singular need for Crepidula fornicata. The little slipper limpet is common in New England and a great model species for experiments. I'm starting a new study now that it's spring (and most of my coral DNA work is finished), so over the next few months, I plan to become intimately acquainted with Crepidula.

Codium algae on top of Crepidula in Little Harbor
The limpets like to live in shallow water, either in the intertidal zone or in the subtidal, just below the water line. Where there are rocks and shells available, they adhere themselves to the substrata, but they can also live in sandy and muddy environments by settling on the empty shells of limpets past. Crepidula like to live in stacks, so when you find one, you'll find many. In Little Harbor, most of the stacks are covered in the alga Codium, which settles on Crepidula and uses the shells as its anchor. We went at low tide, when the sand flat is covered by a foot of water or less, and scanned the white-yellow sand for clumps of green Codium. Sure enough, at the base of each one was a stack of Crepidula fornicata.

In just 20 minutes, I managed to fill my bucket. We brought the limpets back to the lab and set them in flowing seawater overnight. It was a successful collection trip, and I look forward to the coming experiment!

Biology megablaster

Scientists love acronyms. Every project, every technique, every fancy new invention needs a good acronym. In fact, I once saw a presentation by a fellow researcher who openly admitted that she had named her project what she did just to have a cool acronym for it.

Today, I got to use a technique with one of the most powerful-sounding acronyms in all of science: BLAST. It stands for "Basic Local Alignment Search Tool," and it's used for matching DNA sequences. You enter your sequence into the website's search bar, select your parameters, and compare it to sequences in a database called GenBank. (I know, it's anti-climatic, but if it makes you feel any better, one of the parameter options is called "megablast.")

This story is going to sound a lot more exciting if I add "blast" sounds to each sentence. Here we go: I sat down at my laptop with the DNA sequences from coral larvae and spat I had collected with Hanny in Palau (blast!). I copied the first one into the search field on the website (copy-blast!). I pressed "search" and watched the page refresh every 5 seconds (waiting-blast!). The website returned a list of other sequences that matched mine, along with the percent match (results-blast!).

Hanny looking at some adult Montipora corals (white arrows)
in German Channel, Palau
The top result was Montipora peltiformis, which is an Indo-Pacific coral (happy-blast!). The procedure worked, and I even got a reasonable result! The match wasn't perfect - only about 90% - so the coral larvae I had just BLAST-ed might not be exactly the same species but rather something related. I can conclude with a reasonable level of certainty that my larva is a plating coral in the genus Montipora.

Just to give you a reality check, I should mention that not every sequence I BLAST-ed had such a promising result. A number of them didn't match anything in the database, and for one of them, the closest match was a Canadian bighorn sheep (um, no). The usefulness of a database depends entirely on what's in it, so the most likely case is that no other researcher had sequenced the DNA of the species I was BLASTing. Coral larvae and spat are super small and difficult to identify by eye, so without a genetic reference, we may never know what species they are.

This story has four morals:
1) There is a lot of research left to be done, especially in remote parts of the world.
2) Acronyms rock.
3) Canadian bighorn sheep do not live on coral reefs in Palau.
4) Sometimes, hard work pays off in reasonable, exciting species identifications!

Friday, April 5, 2019

Banana test

"We shall conquer the larvae. They may be small but shall not escape!"
- an email from Hanny

Alright, friends, so I got all the DNA that Hanny and I collected from young corals in Palau sent off for sequencing. However, there are still a few samples left. In addition to tissue chips we collected from coral colonies (the DNA I just sent off), we had collected a few coral larvae and young settlers called spat.

A coral spat on a terra cotta panel. The scale bar is in the
bottom left, and as you can see, this settler is < 1 mm across.
We don't have enough larvae and spat for a population-level genetic analysis, but we still want to get as much information as we can from the samples. By extracting the DNA from our larvae and spat, Hanny and I can identify them and see what species are dispersing and settling at each of our study sites. But that's just the thing - extracting DNA from larvae and spat is hard. There's so little tissue in a single individual that conventional methods don't work. I tried three different methods with no success, and I was wasting samples along the way. Time for a different strategy.

Time...for a banana test.

Fresh fruit has some of the highest DNA concentrations of any biological tissue. In fact, extracting DNA from strawberries and bananas is a great science lab to do with children. I needed an idiot-proof way to develop my own DNA extraction method, so I brought my breakfast banana into the lab and scraped off tiny flecks of the fruit with a sterile pipet tip.

I tried one standard DNA extraction kit and three variations on a less-established method. After searching in the scientific literature, I decided to throw in my own method as well, by combining reagents from two different kits. I added all the reagents to the tubes, went through the proper incubation steps, and then tested the results using a NanoDrop. The machine showed ambiguous results - could be DNA, could be protein, could be DNA with high concentrations of protein contamination. Interestingly, my pieced-together method showed the most promising result.

I decided to try with some larvae. Another WHOI scientist was kind enough to give me some anemone larvae for the test - they're similar to coral larvae and served as a good analogue. I tried my pieced-together method, plus the two other variations, but the NanoDrop again showed ambiguous results. I needed another way to test for the presence of DNA.

Here's where the story takes an ironic twist: the best way to test for DNA in my samples was to try and make copies of it using PCR - a procedure that until just a few weeks ago was driving me nuts. I've gotten the hang of it now, thanks to Hanny, and been able to use it to answer important questions. In this case, PCR was my friend.

My electrophoresis gel
I got a tried-and-true PCR recipe to use on the anemone DNA from Hanny. I combined the reagents and subjected them to the prescribed thermal cycles. I ran the samples out on an electrophoresis gel and used UV light to visualize the DNA.

Drumroll please....

It worked! The gel you see to the right here shows bright bands of DNA that was amplified during the PCR procedure. This could only have happened if there was DNA in the sample originally - meaning that my extraction worked!

I was very proud of myself for working out the DNA extraction procedure. I can't wait to use it on the coral larvae and spat!