Take Two

1 02 2014

Recap: I am a Master of Science (and the Universe, according to my nephew when he wants the tickle attacks to stop), and my experiments in the intertidal were mostly successful, except for the live spawning attempt.  Again, to validate my project design and strengthen my simulated spawning results, we tried a second live spawning experiment at the end of last year before the permits expired… 

Me showing some abalone love (B. Blaud)

Me showing some abalone love (B. Blaud)

San Nicolas Island is one of my favorite places on earth, if you haven’t picked that up from my previous posts.  I feel incredibly blessed to have the unique opportunity to venture back, even after my schooling is complete.  I was plotting ways to get back to SNI, thinking of leveraging my experience on the island and in the intertidal to get a volunteer position with another group, when I got an email from Glenn that we were on for another live spawning experiment, and they would like me to be involved.  I didn’t even need to think about it before quickly replying, a hearty and enthusiastic “YES!  When do we leave?”

 

Withering syndrome is still a concern, even though it doesn’t appear to be as virulent as it was in the 1990s during its initial appearance on the island.  Although the disease presence has been documented on the island, the conservative approach to preventing further spread of withering syndrome dictates using only disease free animals.  The last spawning experiment involved naïve animals that were never exposed to the disease.  However, the difficulty we experienced spawning the larger animals and the learned knowledge that smaller animals are more gravid and easily spawned encouraged us to get younger red abalone.  To stay within the permit guidelines and only bring disease free animals to the island, the thirty red abalone we got for the experiment had to be treated with oxytetracycline (OTC), a broad spectrum antibiotic active against many types of bacteria, including rickettsiales.

 

The most ripe red abalone with bulging gonads were hand-picked by an abalone expert, Jim Moore, to improve our odds of a successful experiment.  Unfortunately, the OTC treatment is rather stressful, and involves the abalone sitting in a series of eight baths for 24 hours each over the course of three weeks.  During this time, the horny little abalone reabsorb their gametes, or may spawn in response to the stress.  To reduce their stress and hopefully maintain fuller gonads, the abalone were fed during the treatment.  They were allowed to recover for an additional three weeks after treatment concluded, and we hoped it would be enough.

 

Armed with a greater number of smaller abalone and knowledge from what led to failed experiments previously, we headed out to the island.  I think I inadvertently cursed myself after my last blog, Failure in the Field:

It is common for the first, and even the second field experiment to not work.  Unfortunately, I had everything riding on this one spawning release. There were many things I learned from this venture, the most obvious being to mix the iodine into the Lugol’s solution before adding it as a preservative to the eggs (duh).  I also later learned that the larger abalone are more difficult to spawn, and more successes are achieved with younger, smaller abalone.

In this second field experiment, I was in no way about to be in charge of the Lugol’s solution, and we brought formalin as a back-up preservative, just in case something else went wrong.  It’s science, and “things going wrong” is the name of the game. 

 

Red abalone in 3L of sexy spawning cocktail (Tris and hydrogen peroxide) (B. Blaud)

Red abalone in 3L of sexy spawning cocktail (Tris and hydrogen peroxide) (B. Blaud)

We set up the abalone in the temporary housing outside the archeological labs and storage, and less than a mile from a good release location at Site 2.  The plan was to spawn the abalone in the comfort of civilization, near electricity and running water, then take the gametes down to the experimental site, run the experiment at Site 2, and transport the canisters back to the lab where we would distribute eggs among the transportable tubes and add preservative.  That was the plan.

 

Now we just need the little buggers to spawn!  We added the sexy cocktail of Tris and hydrogen peroxide, ran down to quickly set everything up, and then waited… and waited… and waited…  When the tide started coming back in, and only one female had spawned a small amount of eggs in 6 hours, we decided to call it a day.  We put them all back in their temporary tank, and prepared to try again the next day.

 

Unfortunately, we were out of 6% hydrogen peroxide.  Luckily, someone thought ahead and brought back-up 3% hydrogen peroxide, purchased for $0.89 a bottle at the local pharmacy.  We doubled up the dose to try, try again.  Feeling slightly less optimistic, but still hopeful, we tried all the tricks we had learned in our combined spawning experiences and reading: temperature stress (exposure to sunlight to increase water temperature to 20°C in their 3L buckets, then water changes to bring the temperature back to 14°C); mechanical stress (EARTHQUAKE!  Shaking the buckets, tapping the buckets, mainly just agitating the poor abalone); musical stimulation (Barry Manilow, Madonna, Bruno Mars, Rhianna and Brittany – don’t judge!  It worked last time, and I was willing to try anything).  Our efforts were rewarded with some stressed out abalone that released several mucus plugs and poop, and appeared to want to spawn but had nothing to give.

 

Our conclusion: they hadn’t had enough recovery time after the OTC treatment.  Next steps include renewing the permits, applying for grants, and praying that the third try does the trick.  On the bright side, I get to go to the island at least one more time.  Can’t complain about that!

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Failure in the Field

3 11 2013

Recap: I have completed three successful experimental releases of surrogate egg and sperm in the tide pool habitat, and three successful experimental releases in the crevice habitat.  To support my project design and give plausibility to the experiments, I wanted to replicate the experimental release using live gametes from red abalone.  Last November, I received red abalone with which to practice the spawning protocols, and learned how to spawn pinto abalone up at the Mukilteo hatchery.  Now it’s time to do it in the field…

 

I realize that my last post alluded to the devastation I have felt with some of my experiments, and yeah, this is going to be the post that goes more in depth about that topic.  In truth, science makes me feel bipolar.  I reach such highs, such levels of excitement, triumph, and pure, unadulterated joy when things that shouldn’t possibly work do; but the higher the climb, the steeper the fall.

 

Camp at Site 8 (B. Blaud)

Camp at Site 8 (B. Blaud)

Once my 12 abalone (4 males and 8 females) arrived following the last experimental particle release in the crevice habitat, we set the abalone up in their temporary San Nicolas Site 8 hotel, and made our own camp.  Glenn and I would spend the next two nights camping at Site 8.  Camping was an unforgettable experience, both exciting and exhausting.  The abalone were set up in their temporary home mere feet from high tide (which happened in the middle of the night), and our tents were a short distance up the hill.  The wind was extremely high, reaching speeds of almost 35 mph, and made sleeping both uncomfortable and difficult.  That, combined with the required call-in checks every 4 hours to the Navy, and the water changes to keep the abalone clean, made solid sleep an unachievable luxury.  Did I forget to mention the sea lion serenades – it’s a thing of beauty.  These are not complaints though!  I loved every second of this – ok, I could have done without the cold, wet socks at 4am – but I loved everything else!

 

Finally, spawning day arrived.  It was a gorgeous day, like many days on San Nicolas Island.  No clouds, uncharacteristically low winds, air temperatures in the low-70s (in March – love it!!!), and I was ready to do this.  When everyone arrived, we scoped out the two areas for potential spawning (the crevice habitat and the tidepool habitat) and decided to aim for one spawning release in the tide pool habitat.  Each abalone was placed in a separate bucket, Tris and hydrogen peroxide cocktail, and then we waited.  To get the abalone in the reproducing mood, John played some Hot Chocolate, Barry Manilow, Madonna, Salt N’ Pepa, and other sexy tunes.

Spawning red abalone, marinating in a Tris and hydrogen peroxide cocktail (B. Blaud)

Spawning red abalone, marinating in a Tris and hydrogen peroxide cocktail (B. Blaud)

The spicy cocktail and sexy music worked for two abalone.  Of the 12 we brought to the island, only one male and one female released their gametes, but that’s all we needed!  Once egg and sperm were released, there was so much activity and we needed all sets of hands.  While someone attended to the abalone, getting them into clean, cool water, the gametes were taken to be washed of any residual hydrogen peroxide and be distributed between 21 canisters.  No water was added to the sperm to keep it as concentrated as possible.

 

Anchored canisters located  at -1 m, 0 m, 1 m, 2 m, 4 m, 8 m, and 12 m from the sperm release location (B. Blaud)

Anchored canisters located at -1 m, 0 m, 1 m, 2 m, 4 m, 8 m, and 12 m from the sperm release location (B. Blaud)

The live-gamete release experiment is set up much similar to the previous simulated spawning experiments.  Three canisters containing live eggs are anchored with fishing weights or sandbags at -1 m, 0 m, 1 m, 2 m, 4 m, 8 m, and 12 m from the live sperm release location.  The live sperm is released through an IV bag in the same method as the surrogate sperm in previous experiments.  There was only enough sperm for 13 minutes of release, and the canisters remained in the tide pool for an additional 30 minutes.  Once removed from the water, the eggs were preserved in falcon tubes with Lugol’s solution, and taped up for transport.  The rest of the short day was spent packing and hauling everything up the hill, everyone elated with the apparent success of the venture.

 

From my foreshadowing early on, you, my intelligent reader, may have surmised that something went horribly wrong.  Once the eggs made it back to Seattle, I opened the tubes to discover they have all degraded beyond identification.  I had not properly mixed the Lugol’s solution with the iodine, and therefore no preservative was added to the eggs.  It’s still difficult for me to think back on these moments when I discovered that all the work that everyone put in to getting abalone to the island, the nights camping on location, all the gear carried to the location, the sweat, the tears, the highs of perceived success on spawning day were all for nothing when the eggs became unidentifiable.  I won’t go into detail about the tears shed over this loss.  I had amazing support, and was heartened as several other researchers shared their tales of woe and failures in the field.  It is common for the first, and even the second field experiment to not work.  Unfortunately, I had everything riding on this one spawning release.

 

 

There were many things I learned from this venture, the most obvious being to mix the iodine into the Lugol’s solution before adding it as a preservative to the eggs (duh).  I also later learned that the larger abalone are more difficult to spawn, and more successes are achieved with younger, smaller abalone.  While this experiment would have strengthened my project and supported my results, it is not my only experiment.  I still have a Master’s project without it.  I would like to try again.  It can still be done and may support my project once published.  I promise, I will keep you updated on any more ventures.  This part of the story is not over…





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!





Project: Funded!

17 12 2012

After 33 days of nail biting, annoying spamming all my dear friends and family, and cajoling strangers into coughing up their hard-earned dough, my crowfunding experience has come to a close.  Although I didn’t quite reach my goal of $2,500, making $1,990 is nothing to cry about.  I am in awe of everyone’s generosity!  I couldn’t have asked for a better experience.

 

Black abalone on San Nicolas Island (B. Blaud)

Black abalone on San Nicolas Island (B. Blaud)

What next, you ask?  First off, I’m going to stop slacking on my blogging responsibilities.  I apologize.  Sometimes, when I have a lot going on in my life, I have to drop one or two balls that I’m juggling or else I risk them all falling and hitting me on the head.  I have had enough bruises from past experiences to learn that lesson the hard way.  So while I haven’t been updating you as diligently as promised, I have been busy, getting new material for posts and always, always, always working on my project.

 

Second, I’m going to spend my money!  I love shopping!!!  But my RocketHub funds will be responsibly spent on small, white particles the size of abalone sperm.  I just got back from SNI, where I had an amazing trip and completed a successful extended run, and hope to repeat that experience with particles I purchase using what you all generously donated.

 

Third, I’m going to put my spawning practices to use by releasing live gametes in tide pools and find out how realistic my project design is.  And then… a lot of data analysis and writing.  That’s when all the free coffee I get at work at Starbucks will really come in handy, because sleep will become an expensive luxury.

 

So, while this quick update has been short and sweet, I wanted to get a HUGE THANK YOU out there to all my incredible funders.  Seriously, thank you.thankyou





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!