Post #20 - Camp SeaFET and our last day

Another crazy busy day today doing everything from cleaning out tanks to taking time-lapse photos of COT fertilization and early development to.....well, more on today later.  First, let's go back two days to Wednesday which we dubbed "Camp SeaFET Day."  Emily decided it would be interesting to take water samples at the SeaFET every hour and do water chemistry (pH and TA) to correlate these data with the information the SeaFET was collecting.  So, we got up at 5 a.m. and were out in two boats at the SeaFET site by 6 a.m., just as the sun was peeking over the horizon.  Emily snorkeled down to collect a water sample with a Niskin bottle.  (This picture here we took today when scuba diving over there to retrieve the SeaFET.  On Wednesday, we were too busy to take underwater pictures.)

Emily's right hand is about to squeeze the "trigger" that would cause the stoppers at the each end of the tubular Niskin to close.  She collected samples this way every hour on the hour from 6 a.m. until 6 p.m.   Back in the boat she attached a tube to the white nipple at the right end of the Niskin and filled two glass bottles.  I came out in a separate boat every other hour to take the samples back to the lab to begin the water chemistry measurements.  Emily prepared well for staying out there, with life jackets for cushions, an umbrella for the sun or rain, towels and a book to read.  So when I returned to Camp SeaFET, this is what I found:

The weather turned really nasty in the morning, with rain and strong winds.  Emily hunkered down under her umbrella, peering out at the world.

Although to those of us from upstate New York this may look cold, the temperature wasn't too unpleasant.  Our final sample was taken just before the sun set, so we were back at the dock before dark.  However, running the TA samples continued late into the evening and all the next morning, when it was really POURING outside.  We had thought the weather on Wednesday was bad....

This morning (Friday) we took our Crown-of-Thorns back to where we collected them.  It was good to see them back in their natural environment.

What was really interesting is that as soon as they were on the bottom, several of the males started spawning, releasing strings of white sperm that dissipated into clouds of gametes!

I don't know if they were spawning because they were so excited to be home again, or because of the stress of being crammed into a cooler for the half an hour trip out from the lab.

On the way back, we just had to jump in the water at the spot where tour operators feed the sting rays and sharks.

After stopping briefly at the lab, we went back out to retrieve the SeaFET, loosening the clamps that held it to the sunken cement piling.

Emily carried this valuable piece of equipment back to the lab eager to see whether it had worked, collecting data on pH, temperature and salinity every hour since we deployed it back in January.

The SeaFET was opened, and indeed had a data file that was downloaded onto the laptop.
Here is what the insides look like:

Emily was super excited about this, for among other things it meant that all the data we collected Wednesday on water chemistry can be united with the SeaFET data.

Our days aren't always filled with exciting science, especially as we prepare to leave.   I had to disassemble an air compressor in preparation for storage, organize gear for packing, etc.   Later in the afternoon we donned scuba tanks again, but this time to scrub the undersides of the boat to remove the algae and invertebrates that had formed a thick layer over the hull.  Still, I made sure to periodically look around at the corals and the beautifully colorful fish.

When we finally went up the hill to clean up, we had to stop and watch the show that the final Moorean sunset we may ever see put on for us.

Post #19 Biocode and other projects at the Gump Station

One of the rewarding aspects of doing a sabbatic at a marine lab (or any other busy place) is the interesting people you meet doing fascinating work.  For example, in for the first several weeks while I was here, the BIOCODE folks occupied the entire lab next door.

This is a multiyear project that brings in some of the world's best specialists in different marine plant and animal groups in an attempt to identify every living plant and animal here in Moorea.  It's a prodigious effort that is well described at their web site, which can be found by clicking on BIOCODE here.  One of the ring-leaders is Gustav Pauley, Curator of Marine Malacology at the Florida Museum of Natural History:

It was a surprise to see him here and a great pleasure because we were graduate students together at the University of Washington.  Gustav knows just about every marine invertebrate here, he has an amazing encyclopedic mind.  Another member of the team this year was Jeff Cordell, also from UW.

Jeff specializes is tiny crustaceans.  These pictures of Gustav and Jeff accurately show how they spent 10-14 hours a day working through samples either they had collected, or possibly specimens collected by special divers (see below).

Every organism has its picture taken to be put in a reference collection of photos.  Here are just a couple of their wonderful photographs:

Every species also has a DNA sample taken to be sequenced.  The growing data bank of DNA sequence information from various species is incredibly useful in so many different fields of study, including taxonomy, ecology, population dynamics, etc.

This year, there was an effort to collect specimens from water at depths below where most scuba divers venture.  For this they brought in some specialists that use carefully engineered mixed gas rigs that enabled them to get to over 350 feet.  They used what is called a "rebreather," for the diver rebreathes some of the same gas over and over while the equipment adds oxygen as needed and scrubs the CO2 that is exhaled.

Here is David Pence from the University of Hawaii Diving Safety Office with his rebreather:

When diving very deep, he may actually stay only about 30 minutes at the deepest level, but then spend several HOURS gradually rising to the surface so that gases such as nitrogen dissolved in his blood can slowly come out.  However, the rewards of such efforts can be great.  Here is world-renowned photographer David Liitschwager getting ready to photograph a peppermint butterfly fish collected at 360 feet.  (David was working at the station on some of this own projects.  He has had many articles in National Geographic Magazine, among others.  You can check out some of his work here.)

Here is my close-up of this amazingly colorful fish:

David's photograph would be far better.  If for a publication it would show nothing but the fish against a white background.  He might take 600 to several thousand shots to get that one "perfect" picture.

Post #18 - Experiments are over!!!!

Time to celebrate!! Yesterday (Sunday) morning we started what turned out to be our last fertilization experiment.  After doing the water chemistry from 7 until 8, I selected my last female COT,

dissected out my last COT ovaries,

(Notice how the spines have been trimmed both for my protection and to mark the individual.  This is female #4 because she has spines trimmed in all four quadrants.)

...and put the ovaries in a convenient food storage container used to transport the ovaries across the street to the lab:

After the lid was put on the container, I found the right male (each cross is between a specific female and a specific male so that we can investigate if different parents make a difference in fertilization success),  removed some testes....

...and put them in their own container where they immediately began leaking milky clouds of sperm.

Eggs and sperm were put in the experimental tubes by mid-morning.  I let them develop until around 6 p.m. when I preserved them.  After dinner I went back to the lab to start analyzing not only these tubes but also the many others that I had been accumulating from the six new experiments we conducted over the past several days.  It was important to examine them quickly as we didn't want to turn off the CO2 system until we knew all the experiments produced satisfactory results.  So after an evening in the lab, I returned by 6:30 a.m. this morning and continued processing.  It wasn't until 6 hours later that I finally finished looking all the tubes:

I categorize 200-300 embryos (or unfertilized eggs) from each tube under the microscope.  Although a bit arduous, it is never tedious because it is so fascinating to see the process by which sea stars develop, even though some of the embryos are abnormal.

All three embryos seen here have a clear fertilization membrane around them.  The embryo with the large cells at the upper left is abnormal, whereas the other two are a normal "late blastula" which is basically a hollow ball of cells.

Having run 24 fertilization experiments, with 16 of them giving us good enough results to further analyze statistically after we get back, we were happy to start the process of shutting down the CO2 system and cleaning out the aquaria.   It will be a huge relief not having to oversee and maintain all the equipment everyday, but a bit sad to think that our days of working with live Acanthaster are nearing an end.

Still, we wanted to celebrate the end of our COT fertilization experiments, so we choose an interesting roadside pizza restaurant for a delightful dinner...

...culminating in a silky smooth homemade Belgian chocolate mousse for dessert.   (Sorry, no pictures of this sublime creation were taken for we were too busy indulging!)

Post #17 - One doesn't use litmus paper anymore....

In science labs in school when growing up, we used colorful litmus paper to measure pH. When dipped in a solution, the paper turned a color based on the solution's pH.  Things are a LOT different these days.  In order to publish work on ocean acidification, you need to use techniques that give you both the pH and what is called the pCO2 (basically the amount of CO2 dissolved in the water) with great precision.  The techniques can be very time consuming, and thus I have spent a great deal of my time here doing the water chemistry necessary to support both Emily's and my research.

The pH is measured with a spectrophotometer.  Here, the sliding top door is open so that you can see the white cap of a quartz glass cuvette.   The cuvette is filled with 3 mL of the water we are testing.  With the door shut, the machine shines light of very specific wavelengths through the cuvette and measures how much makes it through the solution.  Then I add a special dye and the process is  repeated.  A computer (out of view to the right) records the before-dye and after-dye numbers.  We put these numbers into our laptop where they are fed into calculations that give us a tentative pH.

The water bath on the floor does two things.  It is set at 25oC and sends water through the hoses to the cuvette holder, because this process is very temperature sensitive so we try to keep the sample as close to 25oC as possible.  This is why we place the samples in the water bath reservoir prior to measuring their pH.  On top of the spectrophotometer is a digital thermometer that has a thermocouple on the end of a thin wire.  The last thing we do after the sample is read by the spec is to take the temperature of the sample as it sits in the cuvette inside the spec.  This temperature is used in the calculation of the pH.

This is the prep area for our samples.  In the dish at right are white-capped glass vials in which we collect our water samples.  During Emily's experiments, I was collecting two vials of water from each of eight aquaria.  When the samples are collected, we must make sure there are no air bubbles, because that could change the CO2 levels in the water and hence the pH.  The dye takes a full morning to prepare if everything goes right, and is stored in an aluminum foil-wrapped bottle (at left) because it is light sensitive.  The dye can go bad, as ours did about several days ago.  The new batches were not checking out with the standards we were using, and we spent a good two and a half days figuring out how to adjust things to get it to work.  It disrupted my fertilization experiment schedule in a big way, so now I'm running two experiments per day for the next several days.

When the dye is working well, even your naked eye can see the color difference between normal sea water in the cuvette to the left (it is more purplish) and the water acidified by CO2 at right.

Using the spectrophotometer is only one part of the process.  A titrator is used to determine what is called the "total alkalinity" or TA of each water sample.  This is what the $50k machine looks like:

The red cylinder on top has a piston that sucks an acid from the bottle to the left of the titrator and pumps it through the red hoses into the sample cup suspended at the right.  Here's a closeup of the sample cup:

The cup holds a very carefully weighed sample of sea water.  Projecting down into the sample are a pH probe, a tube for delivering the acid, a temperature probe, a white stirrer, and for our samples an air bubbler.  Over a period of about 20 minutes, very precise volumes of acid are periodically pumped into the sample as it is being stirred, and the pH and temperature are measured.   All the data are fed into a computer program that  calculates the TA.  The TA numbers are then added to another program along with the pH measurements from the spectrophotometer to calculate the pCO2 levels in the samples.

All this is necessary in order to do OA research, and with eight different experimental aquaria running, you can see why, when I was running samples during Emily's experiments, it could take from 7 in the morning (when the samples are carefully collected) to early- to mid-afternoon (if there are no major interruptions) to complete that day's water chemistry.  And this went on day after day as long as Emily's corals were producing larvae...

Now with Emily's coral experiments over and our COT fertilization experiments going full speed, she's doing most of the water chemistry while I'm focussing on the rest of the experiments.  It's a lot of lab work for both of us, but worth it.  Still, getting out in the late afternoon to snorkel, even if it is a quick trip to check the SeaFET, is a wonderful break when you can see so many colorful fish hovering around the coral........

Post #16 - Emily's research on coral larvae

I think it's time I explain what Emily is up to here in a bit more detail.  I have briefly described her dissertation research earlier, but just to recap, she is looking at what effects ocean acidification (due to increased CO2 in the atmosphere from burning fossil fuels) and temperature have on the larvae of a Pacific coral whose common name is "cauliflower coral" and whose Latin name is Pocillopora damicornis.   There is a picture of  a "P-dam" colony in Post #12.  Interestingly, this species of coral is a brooder as opposed to a broadcast spawner, so that it releases fully formed larvae instead of gametes.  Each month for about 7-10 days after the new moon, P-dam colonies release larvae during the hours of darkness.

Since P-dam lives in shallow water, it can be collected by wading (see Post #3)  or snorkeling. Below, Emily is using a small chisel and a hammer to break free selected colonies.  I hold open a plastic bag into which Emily carefully places a coral colony.  I then swim it back to the boat which is anchored nearby and place it in a mesh bag suspended in the water to minimize how much the coral gets jostled.

When eight colonies are collected, we gently lift the plastic bags into the boat and place them into a sea water-filled cooler.  Back at the lab, Emily has made an impressive array of buckets for her coral colonies.

One colony is placed in each bucket.  There is a slow trickle of filtered sea water delivered to each bucket, with excess water flowing out the hose on the side. 

Since the larvae float when released, they are swept out of the bucket and down the hose, right into Emily's larval trap.  Each morning before the sun is up, Emily is down collecting her larvae.  At the beginning and end of a larval-release-cycle, she may have just a few dozen to deal with, but in the middle she may have thousands!   She takes the larvae into the aquarium room where they are counted and if there are enough, allocated into different groups.  

This is what the larvae look like in her beaker.

The larvae are cigar-shaped and only about one mm long.  They are brownish in color because they have the same symbiotic algae inside of them that the adult colonies do.

To test the effects of ocean acidification and temperature on the larvae, some are placed in home-made incubation containers with holes cut in the sides that are covered with a fine mesh.  These containers are floated in aquaria that have different conditions of temperature and CO2 levels.  

The tape label at the lower left tells us that this tank has ambient CO2 levels and an ambient temperature.

Some larvae are taken into the lab where they are digitally photographed under a microscope so that the size their size can be easily measured.

This is one of Emily's pictures of a P-dam larva:

  

When the larvae are abundant, each morning Emily is dealing with both the new larvae produced overnight and the larvae that have been incubating in the aquaria since the previous morning.  This is Emily's third (and probably last) visit to Moorea; in previous field seasons she has looked at other aspects of the larval physiology, but this year's experiments are designed to determine if the energy reserves in the larvae are impacted by ocean acidification or increases in water temperature.  Prior to analysis, the larvae are put in microcentrifuge tubes and placed in the -80 degree Centigrade freezer.

Each good day produces dozens of tubes, and each tube may involve one or more hours to process later!

The success of her experiments hinges on so many things going right.  One of the key elements involves monitoring the water chemistry in the aquaria to make sure the pH levels are where they should be.   This monitoring takes hours and hours every day.  How this is done will be described soon.....

Post #15 - How one counts COT sperm

It's been another long but productive day in the lab, doing a lot of water chemistry for Emily's experiments (more on that soon).  It's late and we need to get up early, but I want to get this out to you.

I showed you in Post #13 a test tube with a stock suspension of COT sperm and described how a serial dilution is made so that eggs in different culture tubes are exposed to vastly different concentrations of sperm.  As I've described previously, I count in each tube how many eggs develop normally, how many develop abnormally, and how many are unfertilized.  In order to analyze the results though, I need to know the concentration of sperm to which the eggs are exposed.  I do this by preserving three samples of each sperm stock suspension in tiny plastic microcentrifuge tubes.  Here is a picture similar to one in Post #12 that shows the tubes and the pipetter with which a tiny drop of a sperm sample is placed on the counting slide seen at the bottom.

The counting slide has a tiny grid etched into it and is designed so that a precisely determined volume of your sample is held between the cover slip and the grid.  This allows you to count the number of things (sperm in this case) in a known but tiny volume of fluid, and from this the concentration in your suspension can be calculated.  Here is what I see when counting sperm:

The picture is not real clear because I had to jury-rig a camera onto the microscope, but at least you can see the dots that are the sperm.  I count the sperm in 25 of those squares for each of 6 samples for each sperm suspension, with the data immediately going into a spreadsheet on my laptop.  Tedious but necessary, and one can't help but smile when done!

Post #14 - What is it?

It's been a busy day in the lab, but the afternoon ended with a quick snorkeling trip.  Saw some things I thought you'd like to see, though they are presented here through a serious of questions.....

(It was a late afternoon snorkel, with threatening clouds.  If fact, it was raining by the time we were getting out, and pouring later.  Because of the time of day and weather, the colors in these pictures are rather muted.)


First question: This is a close-up of what?

The top side of a cushion star, which like the COT eats corals.  This one is about 6 inches across:

Second question: What kind of animal produces all this mucous coming out of a hole (at least 4 of which you see here) in the coral?

It's hard to believe it's a snail, but not at all like this cowry which crawls about freely:

The mucous is a sticky trap for organic rich particles that are reeled in and eaten (along with the mucous) by vermetid snails.  The shells of these animals look more like the calcareous tube of some fan worms, and are usually buried down in the coral skeleton but not here:

The round dark "trap door" is the operculum, just like that of most free-crawling snails that block the opening to the shell should a predator arrive.  In this full-sized animal, the operculum is just less than an inch across.

Third question: What did Emily take a picture of here?  An eel?  A snake?  A worm?

Nope - it's a sea cucumber, at least the back 12 inches of it.  If you touch it it feels "sticky" because tiny barbed ossicles (little bones) protrude from the skin.

Lastly, see if you can find Waldo, the 3 inch baby flounder: