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Imagine spending your entire life up in the water column as a creature of the plankton. You use cilia to swim but are more or less blown about by the currents, never (hopefully!) encountering a hard surface, and feeding on phytoplankton and other particulate matter suspended in the water. Then, several weeks into your life's adventure, you fall out of the plankton, dismantle your body while simultaneously building a new one, and about a day later have to begin walking using anatomical structures that you didn't have 24 hours earlier. Not only that, but the food that you've been eating your entire life is no longer available to you, for you no longer possess the apparatus that can capture it. And, finally, your body symmetry makes a wholescale change from bilateral to pentaradial--just think of what that means in terms of how your body is oriented and moves through three-dimensional space. That's what metamorphosis is like for sea urchins and many other echinoderms.

The objects of my complete and utter obsession for the past month and a half have started metamorphosing from small larvae into tiny urchins. When I did my daily check yesterday I had two that had completed metamorphosis since the previous day. One of them still had a bit of puffiness on the aboral surface, which I think may be the very last remnants of the larval body. This little guy has only its first five tube feet, from the juvenile rudiment of the competent larva.

Newly metamorphosed juvenile urchin (Strongylocentrotus purpuratus), 11 March 2015. Age = 50 days. ©Allison J. Gong
Newly metamorphosed juvenile urchin (Strongylocentrotus purpuratus), age 50 days, 11 March 2015. 
© Allison J. Gong

Its companion in metamorphosis was a bit farther along in terms of development; while it still had only the first five tube feet, it has more spines:

Juvenile sea urchin (Strongylocentrotus purpuratus), 11 March 2015. ©Allison J. Gong
Juvenile sea urchin (Strongylocentrotus purpuratus), age 50 days, 11 March 2015.
© Allison J. Gong

But just having feet doesn't mean you automatically know how to walk with them, and it's no easier for these guys than it is for humans. It's probably more difficult, actually, because the urchins have to coordinate movement of five appendages simultaneously. They typically pick up one or two tube feet from the same side of the body and wave them around until one of them randomly sticks to something. Then they remain stretched out until the tube feet on the opposite side of the body let go. Well, you can watch for yourself; this is the same individual that is in the top photo above:

Being a bit farther along in the developmental process means having more spines, but not necessarily any more coordination. I watched the second urchin for several minutes, and while it repeatedly detached and re-attached tube feet, it didn't actually go anywhere. Here's a short clip:

It's amazing how quickly they learn, though. When I go to the lab to look at them tomorrow, they'll be running around as though they've been ambulatory their entire lives. Which, in a peculiar sense, depending on when you start counting, maybe they have.

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After much teasing and titillation, my urchin larvae have finally gotten down to the serious business of metamorphosis. It seems that I had jumped the gun on proclaiming them competent about a week ago, or maybe they were indeed competent and just needed to wait for some exogenous cue to commit to leaving the plankton for good. In any case, I've spent much of the last five days or so watching and photographing the larvae to document the progress of metamorphosis as it occurs. While I was unable to follow any individual larva through the entire process of metamorphosis, I did manage to put together a series of photographs that document the sequence of events.

To recap: A competent larva is anatomically and physiologically prepared to undergo metamorphosis. This batch of larvae reached competence at the age of about 45 days. The larva below is very dense and opaque in the main body. It can still swim, but has become "sticky" and tends to sit on the bottom of the dish.

Competent pluteus larva of Strongylocentrotus purpuratus, 6 March 2015. ©Allison J. Gong
Competent pluteus larva of Strongylocentrotus purpuratus, age 45 days, 6 March 2015.
© Allison J. Gong

Sometimes the first tube feet emerge from the larva while it is still planktonic. Other times the larva falls to the benthos and lands on its (usually) left side, where the rudiment is located.

This larva is lying on its right side, so the tube feet are sticking straight up out of the plane of view. You can clearly see two of them, though.

Metamorphosing larva of Strongylocentrotus purpuratus, 8 March 2015. Age = 47 days. ©Allison J. Gong
Metamorphosing larva of Strongylocentrotus purpuratus, age 47 days, 8 March 2015.
© Allison J. Gong

Just for kicks, here's the same larva, photographed with dark-field lighting. This kind of light illuminates the surface of the object being viewed, which is very helpful when the subject is opaque, making it possible to see four tube feet in this picture.

Metamorphosing larva of Strongylocentrotus purpuratus, photographed with dark-field lighting, 8 March 2015.  ©Allison J. Gong
Metamorphosing larva of Strongylocentrotus purpuratus, photographed with dark-field lighting, age 47 days. 8 March 2015.
© Allison J. Gong

As the tube feet are emerging from the juvenile rudiment, the larval body contracts and gets denser. The arms shrink and the internal skeletal rods that supported them are discarded. At this stage the larval juvenile (larvenile? juvenal?) begins to crawl around on the bottom. The ciliated band that used to propel it through the water and create the feeding current may still be beating, but eventually will stop, as the larvenile will no longer need it. This is usually the time that I see the first spines waving around; it's interesting to note that tube feet, which originate from the inside of the animal, come first, then are followed by spines. Then again, the spines are part of the animal's endoskeleton, so maybe it's not so noteworthy after all.

Metamorphosing larva of Strongylocentrotus purpuratus, showing spines and tube feet, 9 March 2015. Age = 48 days. ©Allison J. Gong
Metamorphosing larva of Strongylocentrotus purpuratus, showing spines and tube feet, age 48 days. 9 March 2015.
© Allison J. Gong

So they're getting close to becoming real urchins! Next up: Learning to walk.

Did you notice that I invented a new word? I'm going to start using it regularly.

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In the parlance of invertebrate zoologists, competence is the state of development when a larva has all of the structures and energy reserves it needs to undergo metamorphosis into the juvenile form. In the case of my sea urchins, this means that they have four complete pairs of arms, each with its own skeletal rod, and a fully formed juvenile rudiment, which contains the first five tube feet of the water vascular system. A continuous ciliated band runs up and down all eight arms and provides the water current used both for swimming and feeding. The larva will have been eating well and its gut will be full of food. It will have lost the transparency it had when it was younger and will appear to be more solid-looking in the central area.

The first batch of larvae that I began culturing this season are now 42 days old. Some of these are competent, or very nearly so. Last week I isolated about a dozen of these big guys into a small dish, making it easier for me to observe them closely every day. Today they looked decidedly opaque and dumpy, and although some of them were still swimming others were heavy and tended to rest on the bottom of the dish.

Here's a photo that I took yesterday:

41-day-old pluteus larva of Strongylocentrotus purpuratus, 2 March 2015. ©Allison J. Gong
41-day-old pluteus larva of Strongylocentrotus purpuratus, 2 March 2015.
©Allison J. Gong

General orientation: This is a ventral view. The animal swims with its arms forward, which defines the anterior portion. Thus the bottom of the cup-shaped body is the posterior. This larva measures ~900 microns along the anterior-posterior axis. Plutei have bilateral symmetry that goes all to hell during metamorphosis, from which the urchin crawls away with typical echinoderm pentaradial symmetry. This wholescale change in body organization is one of the truly amazing things about metamorphosis in these animals. It boggles my mind every time I think about it.

You can see that this pluteus has eight arms. The oblong reddish structure in the middle is the stomach, which has taken on the color of the food the animal has been eating. The strange mixed-up looking structure adjacent to the stomach on the animal's left side is the juvenile rudiment. Focusing up and down through the rudiment shows that it contains five tube feet. After metamorphosis, the juvenile urchin will use those first five tube feet to walk around as a benthic creature, having spent all of its life up to this point as a member of the plankton.

Today I captured about 20 seconds of a larva feeding. This individual is a day older than the one in the photo above and has more of that opacity that I associate with competence.

This is a dorsal view; if you imagine that you're looking at the animal's back, you see that the rudiment is indeed on its left side. The larva's ciliated band is moving a lot of water, and the little specks that you can see flying around are food cells. There wasn't enough water in this drop for the pluteus to do any actual swimming, but at this point it's pretty heavy and would tend to sink to the bottom.

Some time in the next several days these guys are going to start metamorphosing. I will be examining them every day; keep your fingers crossed that I catch them in the act!

 

 

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Thirty-one days ago, on 20 January 2015, I spawned purple sea urchins (Strongylocentrotus purpuratus) and generated six jars of larvae. I've been examining the larvae twice a week ever since. At first they were doing great, developing on schedule with no appreciable abnormalities or warning flags. Then, at about Day 24, the cultures began crashing for no apparent reason. At first I expected to see lots of malformed, shriveled, or underdeveloped larvae, but the thing that I don't understand is that for the most part they look great. They're eating, pooping, growing, and (apparently) doing everything that they should be doing.

Case in point:

31-day-old pluteus larva of Strongylocentrotus purpuratus, 20 February 2015. © Allison J. Gong
31-day-old pluteus larva of Strongylocentrotus purpuratus, 20 February 2015.
© Allison J. Gong

This larva is PERFECT. It has all four pairs of arms now, and they are growing symmetrically. The stomach (the inverted-pear-shaped structure in the middle of the cup-shaped region) is pigmented with the red food it has been eating, and there are no skeletal rods protruding beyond the tips of the arms. This individual doesn't give me any clues as to why the culture it came from took a nosedive this past week. The other larvae that I sampled from this jar today also look good. There aren't many left in the jars from this spawning, but if they all look as promising as this one then I still have hope that some will be able to metamorphose successfully.

So what gives? I suspect that Day 24 has something to do with it. I'm working on a hypothesis and need to let it percolate inside my brain a bit more. When it's ready I want to test it, although that will have to wait until next year, as we've reached the end of this year's spawning season.

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On another glorious afternoon low tide the other day, with the help of a former student I collected six purple urchins, Strongylocentrotus purpuratus. Given that we're in about the middle of this species' spawning season, I reasoned that collecting six gave me a decent chance of ending up with at least one male and one female that hadn't spawned yet.

Yesterday, after the urchins had been in the lab for somewhat less than a whole day, I shot them up and waited. Three females began spawning almost immediately (yes!) and one male started a few minutes later. When all was said and done I ended up with four females and two males. It turns out that the largest individual, with a test diameter of almost 10 cm, was a male but didn't spawn very much at all. I infer from this that he had already spawned in the field before I collected him.

Female (left) and male (right) spawning purple sea urchins (Strongylocentrotus purpuratus). 20 January 2015. Photo credit:  Allison J. Gong
Female (left) and male (right) spawning purple sea urchins (Strongylocentrotus purpuratus), 20 January 2015.
© Allison J. Gong

At the current ambient sea water temperature of 14°C, hatching begins around 24 hours post-fertilization. Early this afternoon I checked on the beakers and they had indeed begun hatching. Sea urchins hatch at the blastula stage of development, when they are essentially a ciliated hollow ball of cells. The cilia allow the larvae to swim, but at this size they are at the mercy of even the weakest current. Thus, for the most part they act as particles, getting carried wherever the current takes them.

1-day-old embryos of S. purpuratus. The empty space inside each embryo is called the blastocoel. 20 January 2015. Photo credit:  Allison J. Gong
1-day-old embryos of S. purpuratus. The empty space inside each embryo is called the blastocoel. 20 January 2015.
© Allison J. Gong

As the embryos hatch, they swim up to the top of the beaker, then move down towards the bottom. I call this "streaming." At this point in our artificial culturing system the embryos are living in still water without any current, so this behavior is due primarily to their ability to swim. There is probably some interesting physics involved, but I'm not enough of a physicist to figure out what's going on at that level. But whatever it is, it's a really cool behavior to watch:

Rather mesmerizing, isn't it? Each of those tiny orange dots is an individual embryo. Once the embryos hit the water column I pour them off into larger jars and begin stirring them. Right now they're small enough to swim on their own, but once they start feeding and growing they get heavier and would sink to the bottom without some current to keep them suspended. The contraption we use to stir jars of larvae is a manifold of paddles connected to a motor that moves the paddles back and forth, creating the right amount of current to keep the larvae from settling on the bottom without getting beat up by the turbulence.

Here's the paddle table in action. It's a noisy SOB.

For now the embryos just hang out in the jars and get stirred. Their first gut, the archenteron, will be visible tomorrow and the larvae will be able to eat on Friday. Stay tuned!

I've shown you how sea urchin eggs are fertilized in the lab, and you've watched the fertilization membrane develop in real-time.

One day a few years ago, my colleague, Betsy, and I set up shop to spawn urchins.  We do this just about every year because it is super fun and we both enjoy watching larval development; plus, if all goes well we end up with a cohort of urchins whose genetic lineage is known to do growth experiments.

Anyway, after we shot up the urchins and they began spawning we took a sample of eggs to check on their shape.  They should be uniformly round and about 80 microns in diameter.  The first slide that we set up looked like this:

How did this egg get fertilized?

See that egg in the center, with the fertilization membrane?  Somehow that egg got fertilized.  This sample of eggs had not been in contact with sperm or any tools that might have been in contact with sperm, so how did this single egg get fertilized?  None of the other eggs on the slide had been fertilized, nor was there any visible sperm swimming around.

Betsy and I never did figure out what was going on here.  We decided it was one of the Mysteries of Life, and continue to marvel at all the complexities of life that we don't understand.  That's what makes being a biologist so cool--it wouldn't be nearly as much fun if we already understood everything.

In my next post I'll show you pictures of sea urchin larval development.

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One of the all-around coolest things I do with my students is spawn sea urchins to show them fertilization.  We can actually watch fertilization occur under the microscope.  And since the early stages of development are the same in sea urchins and humans the students get to see how their own lives started--not in dishes of seawater and probably not on a microscope slide, but you get the drift.  I've probably spawned and fertilized sea urchins dozens of times, and I never get tired of it.

Part of the reason we can spawn urchins on demand (sort of) is that they are broadcast spawners.  In nature, urchins of both sexes shed their gametes to the outside and fertilization and all ensuing development occur in the water column.  This is convenient for us because it means we can culture the larvae and observe them at various stages of development.

Gametogenesis is seasonal in urchins, with the local purple urchin (Strongylocentrotus purpuratus) generally ripe from December through March-ish.  In the lab we can manipulate the timing of gametogenesis by subjecting the urchins to artificial photoperiod, tricking them into "thinking" that they are experiencing winter when the calendar says otherwise.

Fertilization is a complex series of events, some of which happen very quickly and some of which are a bit slower.  Here's a brief rundown:

  1. Sperm fuses with the outer layer (called the vitelline layer) of the egg.
  2. Sperm nucleus begins to enter the cytoplasm of the egg.  This causes the egg membrane to become impenetrable to other sperm and is called the fast block to polyspermy.  The egg is impenetrable about 1 second after the sperm and egg membranes begin to fuse.
  3. Once an egg has been penetrated by a sperm, vesicles in the outer layer of the egg fuse with the egg's plasma membrane and release cortical granules into the space between the plasma membrane and the vitelline layer.
  4. The granules trigger a cortical reaction that results in the lifting of the vitelline layer off the egg surface.  The vitelline layer hardens and is now called a fertilization envelope.  The hardened envelope keeps other sperm from penetrating the egg and is referred to as the slow block to polyspermy.

Why are there two blocks to polyspermy?  Everyone knows that it takes only one sperm to fertilize an egg.  It turns out that if multiple sperm enter an egg at the same time, development goes awry.  I've had cultures that I fertilized with too high a concentration of sperm that get through the early stages fine but crash soon afterwards.  So polyspermy is bad and it definitely makes sense that natural selection would come up with redundant ways to prevent it.

All this is to set up the following video clip.  These eggs were spawned at the end of February 2012 for my zoology class, and after all these years I was finally to record fertilization as it occurred in real time.  Actually, I can't take credit for the recording; Sid and Moriah were the ones who figured out how to make the camera play nice with the microscope and actually record video to a computer.

What you will see at the beginning is several large dark eggs on a yellow background, with lots of little sperms wiggling around.  There are way too many sperm for this particular set of eggs NOT to be dealing with polyspermy, by the way.  A few seconds into the video you will see what looks like a bubble forming around some of the eggs. The bubble is the fertilization envelope rising off the egg surface.  There's one egg that seems to be holding out, but by the end of the 1-minute-long video all the eggs will be fertilized.

Pretty cool, eh?

 

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