My Thankful List :)

22 11 2012

On this wonderful holiday, full of stuffing my face with turkey and pumpkin pie (oh, how I love you, pumpkin pie), I thought I would take a second to reflect over my last year and tell you all about what I’m really thankful for.

The beginning of the hike down Heartbreak Hill (B. Blaud)

 

Last year, on Thanksgiving, around this time of 11:14am, I was preparing to go out in the field on San Nicolas Island.  We were headed to Site 8, which involves a 2-mile hike to get to our chosen tide pool.  The hike to the site isn’t so bad, all down hill and passed hauled out California sea lions and elephant seals, a bit brutal on the knees but nothing to stress about.  The run itself went smoothly, which was a blessing following the devastating failure the day before.  The sperm-sized particles were clogging the tube and not flowing consistently, so we tried removing the drip trap and connecting the tube directly to the bag the night before.  It worked, and I had my second complete, successful run.

 

After the run was completed, all the samples collected and everything packed back into my backpack, we took turns carrying the burden back up the hill.  The first 1 ½ mile back isn’t back, but the last ½ mile involves a 600-foot climb, which is, in a word, BRUTAL!  To save my pride, I insist on looking for whales in the ocean every 100-feet or so (ok, sometimes every 50-feet.  Don’t judge!).

 

E.O.D.B. (B. Blaud)

Once we reach the top of the mountain, we celebrate our success and survival summiting Heartbreak Hill (as the hill was christened decades ago) with an End Of the Day Beer (EODB), kept chilled in a cooler of ice.  Nothing tastes as sweet as a cold beer at the end of a long day of fieldwork and good hike.  When we get back to Nick Town, I cleaned off my equipment and stored my samples for safe travel back to Seattle while the rest of the team started on dinner, and what a dinner it was! Turkey, potatoes, stuffing, cranberries, green bean casserole, warm rolls, apple pie, pumpkin pie, and lots of wine.  Yum!!!

 

 

Mike Kenner’s improvised turkey baster (B. Blaud)

This trip and the following trips were great opportunities following the loss of funding.  March and April were spent measuring particles in my numerous water samples and analyzing the results.  During the summer though, I hit a bit of a slump.  I was no longer doing fieldwork, all my samples were processed and analyzed, and my future fieldtrips were months away.  I became unmotivated and uninspired.  I was still faced with the same lack of funding, so circumstances looked pretty bleak.  Everything always works out though, and I know this!  I just had to hold on, keep working and doing my best, but sometimes, you lose sight.  To promote my project and let you all know what I’ve been up to, I started this blog.  In writing about what got me into researching abalone and what an amazing species they are, I started to get excited again.  I fell in love with my research all over again.  I describe this in my interview with Anthony in his Notebook.

 

 

SciFund – Black Abalone Dating Service

At a conference at the end of September, when discussing the situation I was in with a fellow researcher, they suggested I try crowfunding on RocketHub.  It was less than a week later that I received an email from SAFS about fundraising through SciFund, which is promoted on RocketHub.  Everything is worth a try, so I went for it.  This bring us up to present, Thanksgiving Day of 2012, and my thankful list:

 

Mainly, I’m thankful for my friends and family, who have unconditionally supported me.  You have cheered my successes; been my shoulder to cry on; offered advice, ideas, and support; read my writings and drafts; and most importantly, are there.

I’m thankful for all my fuelers generous support.  On RocketHub, I have raised $1,150 and am at 46% of my goal with 22 days left.  I’m confident and optimistic that we will get there!

I am in awe of the wonderful opportunity I have to work with an incredibly species in a truly relevant field of conservation biology and ecology.  I’m doing what I love, and someday (hopefully sooner rather than later) will have a piece of paper that says all of you must call me Master.  That’s what a Master’s degree means, right?

I should also mention that I’m thankful for this blog, for reminding me what I do and why, and why I love it.  Sometimes, we just need to be reminded.  It’s actually been a busy month so far with my project and the fundraising; so much so that I have struggled keeping up with my promised 3 posts a week, but will work on it and do better!

 

So in closing, I wish you all a safe and happy Thanksgiving!  May you all eat too much and spend it in wonderful company!

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Ew! There’s Sperm on my Shoe!!!

16 11 2012

Recap: I now have 14 red abalone adjusting to life in Seattle in the basement at UW (unfortunately, two didn’t survive the adjustment – Seattlites have to be a little tougher than those Californians!).  Once they are comfortable with their new home, I’m going to practice spawning them so I’m able to use live gametes in my field experiments.  But before I can spawn them, I need to learn how. 

Pinto abalone sperm under 80x magnification (B. Blaud)

Apparently there’s more to spawning abalone than putting on soft music and chilling a bottle of Riesling in front of a romantic fireplace.  Although their tastes are a little different from ours on what gets them in the mood to make babies, they are still very particular and can be temperamental.

 

I was invited to participate in spawning pinto abalone up at the hatchery at the NOAA Research Facility in Mukilteo, located 20 miles north of Seattle.  Pinto abalone are declining at a rapid rate in Washington, and are functionally extinct in Puget Sound.  In efforts to reinstate this amazing species, ongoing restoration projects focus on spawning, raising, and outplanting young abalone, and monitoring sites for population growth and recruitment.

 

Pinto abalone (J. Bouma)

     Some basic facts about pinto abalone:

Range from Sitka, Alaska to California

Are relatively small (4 to 6 inches)

Live in waters up to 35 feet deep (10m)

Spawn between May and October.

 

I mentioned before that it can be rather difficult to get abalone to spawn, but I didn’t mention how hard.  It is a little easier to get males to spawn (big surprise there), so a lot of focus goes towards getting the females ready.  Although the typical spawning season is between May and October and it’s already November, the pinto in the hatchery hadn’t spawned yet.  Researchers have been trying for months with no success, so no one anticipated big results on this spawning attempt either, but it’s worth a try.

 

Set up for spawning pinto abalone (B. Blaud)

Several methods are used to set the mood and induce spawning, which affect each species differently and vary in success.  Temperature increases and exposing abalone to air naturally stress the animals, making them more inclined to spawn, but curiously, the exposure to UV-light irradiated water and diluted hydrogen also induce spawning.  For successful fertilization, one of the key requirements is synchronicity in spawning, where eggs and sperm must be released in the same location and same time.  To cue neighboring abalone that someone is spawning, a chemical called prostaglandin, aptly named after the location of its discovery – in the human prostate, is released into the external environment whenever an abalone spawns.  Both the UV-irradiated water and hydrogen peroxide stimulate the production and release of prostaglandin, which in turn excites abalone, effectively setting the mood and getting them to spawn.  It’s not really my preferred romantic setting.  I prefer more wine and a little less hydrogen peroxide, myself.

 

For spawning pinto abalone, we used a combination of stimulants: we increased the temperature of their water by 3 degrees Celsius and added diluted hydrogen peroxide to their water.  Ideally, we let them sit in the water for 3 hours and when they are put into clean seawater, they spawn shortly after.  During those 3 hours, we check periodically for early spawners and get them into clean water ASAP if they start spawning – hydrogen peroxide damages the eggs and sperm.

 

Spawned female pinto abalone (B. Blaud)

Until spawning begins, it’s a waiting game and a good time to get coffee and hang out.  Once spawning begins, things get busy!  All the abalone have their own buckets to spawn in so all fertilization crosses can be controlled to preserve genetic diversity in later crosses.  Sperm is collected and examined under a microscope to measure sperm count.  Similarly, eggs are put in clean seawater and counted for density.  Overall, we had two males and four females spawn, producing over 3 million eggs.

 

When all counts are known, fancy arithmetic with large numbers is done to determine how much sperm is required to fertilize a bucket full of eggs.  The desired concentration is approximately 500 sperm to each egg.  Surprisingly, this included adding less than 10mL of sperm to a bucket containing ~500,000 eggs.  After letting them marinate for 15 minutes, the eggs are washed and put into a tank where they will develop for 12 to 24 hours before hatching.

 

And that is the miracle leading to abalone babies!!!

 

Morse, D.E., H. Duncan, N. Hooker, and A. Morse.  1977.  Hydrogen peroxide induces spawning in mollusks, with activation of prostaglandin endoperoxide synthetase.  Science, 196(4287): 298-300





RocketHub Launches my Project

14 11 2012

Black abalone on San Nicolas Island (B. Blaud)

I have definitely over-used this word lately, but in my overwhelming job to be a part of such a fun way to raise money, I can’t think of any more appropriate vocabulary, so I will keep using the word.  I am EXCITED!!!  SciFund has helped me become organized in the crowfunding world, and through RocketHub, I have launched a fundraising campaign, the Black Abalone Dating Service.  My goal is to raise $2500, and to help people understand what I’m trying to accomplish and what the money will be used for, I put together a short description and video.

 

Black Abalone Dating Service

Are you a lonely black abalone, seeking a nearby abalone of the opposite sex to spawn near and reproduce with?  If that’s the case, you’re not alone!

There were once so many black abalones in California that this intertidal sea snail was stacked 6-deep in the open, so finding a mate wasn’t hard.  After a disease culled them to a fraction of their original numbers, they are now more cryptic and located much further from potential mates.

Black abalones are broadcast spawners, meaning they release their eggs or sperm directly into the water in a Hail Mary attempt to reproduce, hoping they are close enough for their gametes to mix and the eggs to fertilize.  But the question is: how close is close enough?

 Fundraising Target: $2,500

Because black abalones are an endangered species and we have not been able to successfully spawn them in laboratory settings, we use small particles as surrogates for black abalone sperm and eggs in our experiments.  We release these particles in the intertidal and collect water samples to measure the concentration of sperm and eggs over distance and time.  The cost of the particles for each experimental run is approximately $2,500.  We aim to raise enough to fund one run in the hopes of finding out how close two abalones have to be in order to successfully reproduce.   Any money we receive over our fundraising target will go to funding further experimental runs to see how different variables affect the necessary proximity two abalone must be in to successfully reproduce.

 

You can view the video through the SciFund link at: http://www.rockethub.com/projects/11912-black-abalone-dating-service

 

I literally check the website 10 times a day to see if anyone has contributed, and to show that I believe in myself and my project, I was the first to fuel it with a donation.  Any help is appreciated, seriously appreciated.  So go check out the link, and open your wallets!





Results, Revisions, and Revamping

7 11 2012

Recap: So I set up the experiments, collected the samples, counted all of the different sized particles, and entered them into a spreadsheet.  Now what?

An example of R code I used to generate a figure (B. Blaud)

 

I have a secret… I hate R.  Ok, I don’t mean that; I just don’t like it as much as I should, as a scientist.  R  is an amazing free data analysis tool that you can do anything on, as long as you speak computer (specifically, S Language).  I, unfortunately, don’t.  Learning to speak computer is like learning a whole new language, which takes time, dedication, and motivation to practice.  To learn a language, you sing the alphabet song, study vocabulary on flashcards, and practice grammar by writing meaningless sentences in four different tenses over and over.  Learning a computer language is similar, and so I cheat.  I use Excel.  Instead of fourteen lines of code that may be necessary to generate a figure of the average volume of particles in each run, I just hi-lite the desired cells, and type “=AVERAGE(B2:B115)” to compute my run average, and fill in the blanks under the “Charts” tab to make a pretty picture.

 

Why is this a dirty secret?  Several great blogs and articles out there explain in great detail the difference between the two types of software, mainly focusing on user-friendliness and statistical power.  Overall, though, in the professional world, it’s frowned upon to use something like Excel.  So for now, in a very public blog that I share with everyone I know and a lot of people I don’t know, I will just ask that you keep my secret until I become more proficient in the more professional software – which I am working on, slowly but surely. 

This is from my PowerPoint presentation on my project showing figures generated on Excel.  The legend in the top right shows how the colors of the bars change with the increase in probability of successful fertilization in sperm concentration. (B. Blaud)

Now, I get back to the actual results.  It was incredibly exciting counting egg sized particles.  I was finding them in samples as far as 8m away from where they were released, yet they were still hanging out at each distance as long as 5 minutes after the initial release.  Pretty cool!  This means that in the turbulent intertidal, where water is constantly coming and going, the eggs still remain in the immediate area, but just disperse more depending on how the strength of the water activity.

Abalone egg
(oceanlink.info/Conservation/
abalone/BHCAP/
development.html)

 

The sperm sized particles were what I was initially more invested in, seeing as how it’s the guys that really get the girls going, and it’s the concentration of sperm that determine fertilization success.  You see, for an egg to be fertilized, there just needs to be at least one egg present, but it requires 10,000 to 1,000,000 sperm per milliliter (mL) of seawater for a 60 to 100% probability of fertilization (according to laboratory studies).  Fertilization can occur with as few as 100 to 1,000 sperm/mL of seawater, but the probability drops below 10%.  In my results, the concentrations of sperm rarely went above 500 sperm/mL, and only twice were above 1,000 sperm/mL.

 

At first, I was disheartened by these results, thinking this means that my experiment failed or spawning success was extremely unlikely in the intertidal.  After putting this into more realistic terms, however, I realized that my experiment only represents a single male spawning for a period of 10-minutes, whereas actual spawning events may take as long as an hour, and gametes continue to remain viable (alive and able to fertilize) for an additional hour after being spawned.

 

So I revised my project.  Instead of a single male spawning for 10 minutes, I’m going to compare results from a single male spawning for 30 minutes (with samples collected 30 minutes after spawning commences) with three simultaneous males spawning for 10 minutes, and see how those results differ from my original experimental runs.  And in the future, I will avoid coulter counters at all costs!!!  (That’s the revamp).





MY ABALONES ARE HERE!!!

6 11 2012

My 12 female red abalone in their new tank (B. Blaud)

I am so EXCITED!!!  My 16 red abalones (4 males and 12 females) arrived today.  The abalone stork (also called FedEx) just dropped them off.  They are now settling in nicely down in the basement, twisting and stretching as they get used to their new home…

 

But I know you are probably thinking, “Why do you need abalones?  Wasn’t the point of the fake eggs and sperm to bypass the need for actual abalones?”  Well, yes, but that’s no reason to rain on my parade.  Geesh.  Can’t a girl be excited about her new abalone for five minutes?  In all seriousness, there is a very good and valid reason to me getting my own abalone.

 

My experiments in tide pools are fairly simple: I release fake sperm, allow them to build up for 5 minutes, then release the fake eggs (allowing fake sperm to release for another 5 minutes), and collect water samples to count each of the sizes of particles later in a lab (or kitchen-lab).   This worked well, but the question that was brought to my attention was how real is the experiment?  Are the fake egg and sperm realistic representations of live egg and sperm?  That’s where my shiny new red abalones come in.

 

The four male red abalone, with decorative marks to indicate their masculinity (B. Blaud)

To provide some plausibility to my experiment, I will get red abalone to spawn and will collect their eggs and sperm.  Red abalone not only are easier to spawn (less shy, I guess), but are not endangered and are raised in farms, so no permit is required to work with them.  In my field experiments, the eggs will be put in plastic containers surrounded with a small mesh (~80um) that keeps the eggs enclosed, but still allows water and sperm to pass through.  I will anchor each of the containers at various distances from where I will be releasing the sperm.  The live sperm will be released in a similar manner as my fake sperm: through an IV bag that controls the rate of release to simulate an actual male spawning.  No actual abalone will be released into the environment.  I will also collect water samples to measure the concentration of sperm at different distances and times to compare with my simulated spawning events using fake gametes.

 

When the experimental run is complete, we will collect containers containing the eggs and count how many of the eggs were fertilized back at a lab (or kitchen-lab) under a dissecting scope.  No larvae will be released into the environment.  The results I get from this experiment will be used to validate future releases with fake gametes.  I’m anxious to learn more and start spawning my new abalones, and can’t wait for them to adjust!

 

But for right now, I’m content to just be a proud mama of 16 new abalones!





Going Gangsta on a Coulter Counter

4 11 2012

Recap: In the field, I went through a series of trial and error to develop a realistic simulation of black abalone spawning at different distances apart.  In this process, I collected almost 700 water samples that (hopefully) contained an abundance of egg (212 to 250um) and sperm (10 to 45um) sized particles.  Now, it’s time to count the two different sized particles in each of the water samples (fun, fun)!

From Office Space (1999)

 

Have you seen that scene in the Office Space where they take the dysfunctional copier into a field and beat the shit out of it with a baseball bat while rap music fuels their rage in the background.  I dreamed, day and night, of being able to do that to a coulter counter.

Coulter Counter

 

A coulter counter is a piece of equipment that was supposed to make my life easier.  It is used to count small particles by pulling them through a micro-channel that causes the change in the electric current, which is registered on the machine.  I don’t fully understand the process; mostly what I know is that I put my water sample in a tube that goes in the apparatus, turn a knob, and, VOILA!  A number appears on the left indicating the amount of particles per 0.5mL in my sample.  Or that was how it was supposed to work in a perfect world…

 

But alas, my world is far from perfect.  The machine only counted the smaller particles, it often clogged, it gave me unrealistic numbers that didn’t always match my quality checks where I compared the coulter counter numbers to hand counts under a dissecting scope, and eventually (with only 67 samples left) broke, forcing me to finish the counts by hand.

The lab space in my parents kitchen (B. Blaud)

 

I started off by filtering out the larger size particles to count them separately.  I turned my parents kitchen into a lab space.  It had the required sink next to counter space, and an extra computer for watching movies on while going through the tedious counting process.  To filter the larger, egg-sized particles, I built a screen with 180um mesh that would catch the eggs, but allow the sperm to pass through, and used a microscope to count these by hand.  This process was fairly simple and smooth.

 

It was the sperm-sized particles that caused me to lose hair.  It’s always the boys that complicate things, isn’t it.  At first, I had the coulter counter set up in my lab space at UW in Seattle, but after it broke a couple times and I wasn’t able to fix it quickly, it went back to the Taylor Hatchery in Quilcene, which is a 30 minute ferry ride and hour drive away.  So I made the daily trek out to Quilcene every day for a week to count samples, fighting the coulter counter tooth and nail for every tally of sperm sized particles, living off diet Coke to survive my grueling 18 hour days (mornings making coffee, afternoons counting particles, and evenings entering data).

 

And my reward for all my efforts?  Data!

Next up, my results and what they mean…





MacGyver-ed Experiments

1 11 2012

If MacGyver were a marine biologist, I try to imagine what complicated experiments he could create using a roll of duct-tape, a shoelace, and an empty Coca-Cola bottle he found on the beach.  I have a strange imagination.

I tried to channel some inner-MacGyver while working on setting up my experiment.  Remember, I’m trying to figure out how close together two abalones must be in order to successfully reproduce.  I had a rough idea of how the experiment should go in my head, but sometimes it’s hard to connect what I envision with reality.

Tide pool used in experiments on San Nicolas Island (B. Blaud)

So here’s what I initially pictured: I would let a sperm cloud build up for five minutes using a controlled release from an IV bag, commandeered from a local hospital by sources unnamed (love you big sis!!!), then I would release the eggs all at once and collect water samples.  This seemed pretty simple and straightforward in my head until my advisor, Glenn VanBlaricom, asked how I would collect the samples.  We brainstormed different systems, one involving placing 10 people in the tide pool with bottles, scooping up water samples, and another with sampling tubes rigged onto a frame that would be anchored to the bottom of the tide pool.

Sampling bottles (B. Blaud)

What we ended up doing was something in between the two ideas.  I thought it would be useful if the sampling bottles functioned similar to a Niskin bottle, which is used in oceanography to take water samples from different depths.  It is hollow and open on both ends, which allows water to flow through.  Water samples are collected at a desired depth by triggering a mechanism that allows the stoppers on either end of the tube to close.  These bottles range from a couple hundred to several thousand dollars, are much larger than I need for my samples, and are entirely out of my budget of $52.37 (all that I had in my checking account at the time).

With the invaluable help from Julia Eggers, I was able to create unique sampling bottles using PVC pipes, racquetballs, surgical tubing, and zip-ties.  Eggers and I spent a couple afternoons scouring the local hardware stores and harassing poor Home Depot employees to figure out how we could make this contraption hold water.  After collecting supplies, we would run back to the shop at school and experiment with different designs.  Through trial and error, we came up with something pretty darn cool and simple.  To make them in bulk (I needed 40 of them), I got all the supplies together, bought pizza and beer, and invited friends over for an assembly party.  Ah, what my friends won’t do for a free beer…

Chris Yates with a water sample that has visible egg- and sperm-size particles (B. Blaud

To get the whole thing to work, the simplest plan was to place actual people into the tide pool at the time of the experiment, and run it like I had envisioned.  We had a couple other experiences in the trial and error process that were fairly painful.  To get an idea of how the experiment would go, VanBlaricom and I headed out to practice in a tide pool.  We set up an IV bag with the fake-abalone sperm and put the fake-abalone eggs into the water using a turkey baster.  One problem we didn’t anticipate: the particles floated – and so while we watched, literally $1,000 floated away into the sunset!  I could have cried, and maybe did a little.  All my research online indicated they would be neutrally buoyant in the water, but we totally discounted needing a dispersant, something that would break the surface tension of water and allow the particles to enter the water column.  Dawn detergent to the rescue!  The next day, we headed back to the same tide pool with Dawn and another batch of particles, and this time we were successful.

IV bags, the right one containing sperm-size particles (B. Blaud)

For the short experiment practicing to see how the particles would react and brainstorming how we would choreograph the entire experimental run, the IV bag worked great!  Unfortunately, in the extended, real runs, it clogged (another $1,000 floats off into the sunset with no data collected – more tears shed, and not the last).  I used swiss army knife to remove the drip chamber where the clog was occurring and attached the tube directly into the bag with duct tape (see, I AM like MacGyver, solving the world’s problems with a piece of duct-tape!).

I set up a tripod to hold the IV bag, filled it with fake-abalone sperm mixed with seawater and Dawn detergent, and let it release for 5-minutes before releasing the eggs.  Everyone had his or her own location, a set distance from the egg release location and collected water samples at precisely 0, 15, 30, 60, and 300 seconds from the time the eggs were released.  The choreography was something else that was worked out through trial and error, but after several runs (some successes, some failures), we got a good system worked out.

And thus, we completed six successful runs of simulated spawning experiments, providing me with roughly 684 samples to run process.

Next up, from the field to the lab…