Invincible Black Abalone

29 12 2012
Pre-disease black abalone population on San Nicolas Island (G. VanBlaricom)

Pre-disease black abalone population on San Nicolas Island (G. VanBlaricom)

When I first began this blog, I introduced withering syndrome and it’s devastating effect on black abalone populations in Southern California.  The effect of the disease on the populations on San Nicolas Island is the equivalent of wiping out all the people in the world except for the populations in the US, Indonesia, and Brazil.  Only three of the most densely populated countries out of the total 242 countries in the world manage to survive.  It’s a little sobering when you think of it that way.  If you break it down so populations are evenly lost among each country, only the populations in Washington and Idaho would survive in the United States (I’m in Washington, so I’m happy I survive the fake apocalypse).  I’m not stating the statistics in the manner to convince everyone to move to the northwest, even though it’s the best place to live, I’m just trying to paint a picture of how the US would look if only 1% of the population survived.  Out of the 315,077,987 people in the US, only approximately 3 million make it.

 

Then, imagine among the people dodging the zombies in the apocalypse (I’ve been watching a lot of Walking Dead, so that’s the only apocalypse scenario I can picture right now), there is a group of individuals resistant to the brain-eating bacteria!  And they’re found on San Nicolas Island.

 

Disease-resistant black abalone discovered on US island

Biologists have discovered that black abalone on San Nicolas Island in the Santa Barbara Channel are more resistant to the deadly bacterial disease known as withering syndrome than abalone on the mainland. The discovery may help save these now rare intertidal molluscs from extinction, as scientists hope to soon breed these animals in captivity for release in the wild.

According to Carolyn Friedman, from the School of Aquatic and Fishery Sciences at the University of Washington in Seattle, San Nicolas Island has been the site of several severe outbreaks of withering syndrome. The present population appears to be the descendants of that 1% of the population that survived the onslaught.

Friedman and her colleague on the California Sea Grant project, Steven Roberts, also at the University of Washington, are now trying to identify which genes are responsible for resistance and the mechanisms by which this resistance is conferred. This work includes studying differences in gene expression between island black abalone and those from Carmel in Monterey, as the animals are subjected to high loads of the withering syndrome pathogen.

Withering syndrome, which causes severe atrophy of the animal’s foot muscle and is caused by a water-borne pathogen excreted in abalone faeces, occurs in relatively warm water, such as those found in the Santa Barbara Channel. Until recently, waters off the more northerly Carmel have been too cold to trigger outbreaks. As a result, abalone in Carmel have little natural protection against the disease.

Source: California Sea Grant

Anonymous.  2008.  Disease-resistant black abalone discovered on US island.  Marine Pollution Bulletin, 56(1): 7

 

Super Black Abalone (B. Blaud)

Super Black Abalone (B. Blaud)

Beginning in 2001, isolated populations on San Nicolas Island began to increase, so Dr. Friedman hypothesized that there may be disease resistant animals.  My very first trip to the island in 2004 was to collect animals and bring them back to UW to test this hypothesis.  The exposure of naïve (uncontaminated, un-diseased) black abalone from Carmel and comparing the rate of mortality to infected animals from San Nicolas Island indicated that San Nicolas Island black abalone are, in fact, resistant to the disease.  It’s a snail…it’s a rock…it’s SUPER BLACK ABALONE!  Yeah, I’m a dork.

 

But the story doesn’t end here.  Continuing this work, Dr. Friedman and PhD student Lisa Crosson investigated which genes were potentially responsible for the disease resistance in black abalone populations, and in doing so, made a surprising discovery.  But that is a story for another time…

 





Run #7

24 12 2012

Recap: My preliminary runs were exciting, although it was difficult to get used to releasing a thousand dollars in the form of small particles and watching it drift away in the ocean.  In my experiment, as designed, a single male abalone was incapable of fertilizing an egg, even if a female was spawning right next to him.  I wasn’t convince that my design was perfectly awesome since it only showed the results of a single male spawning for 10-minutes when often a spawning event takes place for up to an hour.  After revising my project design, I was ready for a second take…

My tide pool at Site 8 (B. Blaud)

My tide pool at Site 8 (B. Blaud

 

It was the perfect day for a tide-pool experiment.  The tide was low, but not too low, the sky was clear, the temperature a comfortable 72 degrees, and the waves were not too high.  By all reasoning, I should have been in a great mood, but I couldn’t relax.  The experiment has tripled in size, and we are now releasing three times as many particles into the water, which means that over $2500 is now being released into the tide pool to float away.  I couldn’t deal with any thought of failure and wanted this run to be successful so bad, I could taste it.  It tastes salty, like a combination of ocean, sweat, and tears. 

 

 

I scrambled to get everything ready: all the sample bottles clean and present, all the tubes labeled and secured, triple checked that the particles were packed (there’s nothing like hiking for an hour to realize the guest of honor is back in the room), and hoped I wasn’t forgetting anything major – I knew I would forget something, I could feel it, but was hoping it was something that could be fixed in the field.  While setting up for the run down at the site, I realized I forgot the snap-shackles, which are used to firmly anchor the nose of the IV tube in the water.  I knew I forgot something!!!  Annoying, but not major, and something that can be improvised with some well-placed rocks.  Catastrophe averted, but I still couldn’t relax.  There were too many possibilities for things to go wrong along the way.

 

Black abalone on San Nicolas Island (B. Blaud)

Black abalone on San Nicolas Island (B. Blaud)

I went over the choreography for the experiment, because in my mind, the whole thing is like one big dance.  It needs to be timed, everyone’s movements synchronized, and when it goes well, it’s quite pretty to watch.  Dave, to clarify after I described the process, asks if he is releasing his sperm at 0, 1, 5, 10, 20, 30, 45, and 60 minutes after the initial release.  Incredulous, and rather impressed that anyone would be able to release their sperm in timely intervals like that, I suggested that I be the only one to release sperm, as mine is in the form of surrogate, sperm-sized particles and not real sperm.  Everyone agreed that that would be best, and would collect samples at those intervals instead.

 

Everyone took their place, and I had enough amazing volunteers that each person was responsible for only one distance.  I was the bucket lady, running around rinsing sample bottles between collections to avoid left-over particles, making sure the sperm supply didn’t run out, keeping track of the time and orchestrating the sample collections.  Someone later suggested I should teach an aerobics class, because my voice barked orders in a no-nonsense way.  I took it as a compliment.

 

It all went rather smoothly.  I glared at Glenn when he suggested as much halfway through the experiment.  “Don’t jinx it!”  I know science and superstition don’t exactly go hand in hand, but science is about exploring the unknown.  I’m not about to tempt fate and will do anything to ensure a successful run, including not mentioning how well it’s going until I’m sure nothing can go wrong!  It’s along the same line as not saying the number seven at the craps table when the button is on.  If someone had suggested that I can also improve my odds of success by clicking my shiny, red heels together while exclaiming, “this WILL work” three times, I would have done it without a second thought, totally comfortable looking like an idiot as long as all is right in my world.  Essentially, even though it was going amazingly well, I still couldn’t relax until the last sample was collected and safely packed away.

Mel, Eric, and Glenn enjoying an E.O.D.B. at the top of Heartbreak Hill (B. Blaud)

Mel, Eric, and Glenn enjoying an E.O.D.B. at the top of Heartbreak Hill (B. Blaud)

 

Despite my fears and insecurities, it was perfect.  There are always things that can be corrected and perfected, but nothing major went wrong.  Once all my samples were stowed, I could finally smile and laugh at the sperm-related jokes.  At the end of the day, for the first time ever, I was able to hike up Heart Break Hill without stopping to “look for whales,” while carrying a 50lb backpack.  Although my backpack was heavier, weighed down with water samples, my burden was lighter now that the run was successfully completed. 

 

Since we were only able to find funds to pay for one run, the rest of the week was spent in the kitchen counting particles.  Planning for this, I brought a dissecting scope (I’m no longer going to even try to deal with a coulter counter) and all the supplies needed for counting the egg and sperm-sized particles.  With few distractions that life on SNI offers (mainly, no cell reception and limited internet access), I was able to buckle down and power through the counting, completing over half my samples.  It’s a good start!





Spawning By Myself…

20 12 2012

Recap:  To bring more credibility to my project and determine how realistic my experimental design with surrogate particles is, I will release live gametes and measure the amount of fertilized eggs in a mesh container as a function of distance from live sperm, released from a fixed distance using an IV bag.  To learn how to spawn abalone, I participated in a pinto spawning up in Mukilteo several weeks ago.  I received 16 red abalone from the Cayucos Hatchery, and have been holding them for six weeks with the intention of further honing my spawning skills…

 

My red abalone (B. Blaud)

My red abalone (B. Blaud)

Spawning by myself sounds like something completely dirty, but I assure you, it’s not.  Since November 6th, I have been holding red abalone in the basement at the University of Washington with the intention of practicing the spawning protocol I learned with pinto abalone up in Mukilteo and honing the methods to something I can use in the field as part of my experimentBy the time I was ready to practice spawning them, I had 13 red abalone, 9 females and 4 males.  I was preparing to go to San Nicolas Island (SNI) for one of my experimental runs with surrogate particles, but wanted to have one solo spawning under my belt beforehand.

 

I headed down to the basement fairly early on the Friday morning to set up for the spawning experience.  The first steps are measuring up the participants, their gonads to be exact.  I measured the gamete index for each abalone, to identify which abalone was ripe, and separated them into buckets based on gender – boys in one bucket, girls in another.  Determining ripeness was a little more difficult than I anticipated, as none of my abalone were all that ready to spawn.  I looked under each of their skirts, but had a hard time identifying males from females in many cases.  It’s supposed to be fairly straightforward.  I hold the abalone, shell down in my right hand with the base of the swirl in the shell, their head near my fingertips and the apex in my palm.  After the abalone calms down for a second, I’m able to move the foot to the side to see the gonad at the base of the shell.  The gonad index identifies ripeness based on color and the amount of swelling in the gonad.  Yellowish-creamy white means male (makes sense – sperm is white), and a greenish-purple indicates female (the eggs come out an olive green).  An index of 0 is unripe, where the gonad is indistinguishable as either male or female; an index of 1 indicates when there is slight coloration and swelling of the gonad; an index of 2 has swelling of the gonad up to the mantle, the edge of the shell; and the highest index of 3 is visibly swelled over the edge of the shell, where you can see it with barely moving the foot to the side.  All of my abalone were at my un-expert opinion a gonad index of 0 or 1. 

 

Diagram of abalone gonad (Rogers-Bennett et al. 2004 J Shellf Res 23: 553).

Diagram of abalone gonad (Rogers-Bennett et al. 2004 J Shellf Res 23: 553).

I separated the abalone I rated as a gonad index of 1 into separate buckets, then put a couple abalone rated as 0 into their own buckets, since I couldn’t conclusively determine their gender, but wanted to spawn more than a handful of my abalone.  I added the appropriate amount of Tris(hydroxymethyl) aminomethane (or Tris, for short) and 6% hydrogen peroxide, covered them in a tarp to provide darkness (and a little privacy), and then left them alone with only short, periodic checks for spawners.  I mentioned before that these are all unripe abalone with a gonad index of 0 or 1, so it goes without saying that I wasn’t overly optimistic about the possibility of getting any of them to spawn.  So, imagine my surprise when I went down after only 90 minutes of them sitting in the dark to find one male and one female spawning in separate buckets!  I was so excited, I barely remembered that I was supposed to rinse them off and put them in a bucket of clean seawater (with no chemical additions), so they could finish spawning and have usable gametes.  As soon as that was done, I ran up a couple flights of stairs to find Glenn, who would share my astonishment and simple joy at these unripe, unready abalone suddenly releasing their gametes.

 

Hatched abalone larvae (http://www.lib.noaa.gov/)

Hatched abalone larvae (http://www.lib.noaa.gov/)

Eventually, after about 3 hours, all the red abalone had been rinsed and placed in buckets of clean seawater.  Due to the unripe nature of the abalone, they didn’t spawn a significant amount of gametes.  It was a great exercise going through the protocol, and learned a lot about what would work in the field, what needs to be modified, and ponder what it would be like to be an abalone parent, with millions of children, somewhere out there… but I’m weird.

 

After spawning my red abalone, a good practice run, I felt oddly optimistic about future experiments.  I should say, cautiously optimistic, because when you say the word “experiment,” I get an ominous chill and view of everything that could potentially go wrong.  I am starting to get more and more excited about the results that experiments with live gametes will yield.  But that, my faithful readers, is a story for another day.

 

And next, I will tell you all about Run 7, and my latest stay on the island…I can’t wait to go back!





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





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).





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…