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A recent college graduate and fellow marine lab denizen (Scott) and I are collaborating on a project to quantify growth rates in juvenile Pisaster orchraceus stars. This is one of the intertidal species whose populations in the field and in the lab were decimated by the most recent outbreak of sea star wasting syndrome (SSWS). We are interested in seeing how quickly the stars grow once they metamorphose and recruit to the benthos, and hope that the information will help researchers guesstimate the age of the little stars that are now being seen in the field. This would in turn tell us whether the little stars are survivors of SSWS or post-plague recruits. I keep seeing people refer to them as "babies," but they could very well be several years old. We just don't know, hence this study.

Large, healthy specimen of Pisaster ochraceus at Davenport Landing. 20 May 2015. © Allison J. Gong
Large, healthy specimen of Pisaster ochraceus at Davenport Landing. 20 May 2015.
© Allison J. Gong

But before we get to measure juvenile growth we have to get through larval development, which is perfectly fine by me because I'm always up for observing marine invertebrate larvae. Two weeks ago Scott and I ventured into the field in search of prospective parents. We brought back eight individuals from two different sites, making sure to leave many more in place than we took away. It was actually rather gratifying to see how many hand-sized-or-larger P. ochraceus there were. This morning we met at 07:30 to shoot up the stars with magic juice and then wait for them to spawn.

We have injected the stars (Pisaster ochraceus) and are waiting for them to spawn. 2 June 2015 © Allison J. Gong
We have injected the stars (Pisaster ochraceus) and are waiting for them to spawn. 2 June 2015.
© Allison J. Gong

It has been a while since I tried to induce spawning in Pisaster, and I had forgotten how much longer everything takes compared to the urchins. For one thing, the magic juice itself isn't the same stuff that we use on the urchins, and works by an entirely different mechanism. The stars' response to the magic juice takes 1.5-2 hours, whereas if the urchins aren't doing anything 30 minutes after getting shot up they either need another injection or simply don't have gametes to share.

However, despite my misgivings the animals spawned. Two large females gave us enormous quantities of eggs, and three more donated trivial amounts that we didn't end up using.

This purple individual is the one we designated Female 1. See the huge piles of salmon-pink eggs?

Large purple female Pisaster ochraceus, spawning. 2 June 2015 © Allison J. Gong
Large purple female Pisaster ochraceus, spawning. 2 June 2015.
© Allison J. Gong

and

Large orange female Pisaster ochraceus, spawning. 2 June 2015 © Allison J. Gong
Female 2, a large Pisaster ochraceus, spawning. 2 June 2015.
© Allison J. Gong

Although we had to wait for a male to spawn, we finally did get some sperm and fertilized the eggs at about 12:30. Another thing I had forgotten was that Pisaster eggs, when shed, are lumpy and strange. I was used to the urchin eggs, which are usually almost all beautifully spherical and small. The stars' eggs are about twice as big, at ~160 µm in diameter. The lumpiness doesn't seem to hamper the fertilization process, as you can see below.

Fertilized eggs of Pisaster ochraceus, 2 June 2015 © Allison J. Gong
Fertilized eggs of Pisaster ochraceus, 2 June 2015.
© Allison J. Gong

In this photo you can see the fertilization envelope surrounding most of the eggs. In stars the perivitelline space (the space between the egg surface and the fertilization envelope) is very narrow, which makes it difficult to see the envelope; in urchins the space is much larger, and as a result the envelope quite conspicuous. The rising of the fertilization envelope off the surface of the egg is referred to as the slow block to polyspermy, a mechanical barrier that keeps multiple sperms from penetrating any individual egg. There's also a fast block to polyspermy, but it happens on a molecular level milliseconds after a sperm makes contact with the egg surface; you can't see it happen in real time.

Cleavage in stars proceeds much more slowly than it does in urchins, too. In embryological terms, "cleavage" refers to the first several divisions of the zygote, during which the cell number increases as the cell size decreases. This inverse relationship between cell size and number logically has to occur because the embryo can't get any larger until it has a mouth and begins to feed, which won't happen for at least a couple of days. It took our zygotes about four hours to undergo the first cleavage division.

2-cell embryo of Pisaster ochraceus, 2 June 2015 © Allison J. Gong
2-cell embryo of Pisaster ochraceus, 2 June 2015.
© Allison J. Gong

I left the slide on the microscope to warm up and speed development a bit, and about 45 minutes later was rewarded with this mishmash of embryos at different stages. Nine hours after we started this whole process, there were 2-cell, 4-cell, and 8-cell embryos, as well as eggs that had not divided yet.

Embryos of Pisaster ochraceus, about four hours post-fertilization. 2 June 2015 © Allison J. Gong
Embryos of Pisaster ochraceus, about four hours post-fertilization, 2 June 2015.
© Allison J. Gong

This asynchrony in early development is another way that stars differ from urchins, and it takes some getting used to. I expect that development will become more synchronized as the embryos continue to cleave, and that hatching will occur for all of them at about the same time, probably before Thursday. At least it won't take another 9-hour day to see how far they've come.

 

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?

 

The Dendronotus veligers are still alive.  I've been running into the same difficulties I've always had when trying to rear nudibranch larvae:  hydrophobic shells that tend to get stuck in the surface tension of the water.  Larvae that are trapped at the surface can neither swim nor feed.

We can pretty easily rear sea urchin larvae in culture by stirring jars on a paddle table.  The stirring keeps the larvae and their food in suspension; without stirring the larvae would settle on the bottom and die.  Nudibranch veligers are stronger and faster swimmers than sea urchin larvae and I thought I could get away with not stirring them, as I worried that the paddles might break the larval shells.

Two jars are being stirred on the paddle table.

Two jars of larvae are being stirred on the paddle table, along with several jars of sea urchin juveniles that resulted from a spawning I did back in late February.  The paddles move back and forth and keep the water moving, ensuring that the larvae have pretty consistent access to food.

It's a little early to tell, but it seems that there may be fewer larvae trapped at the surface in these jars.  And I didn't see more smashed or broken larvae in these jars compared to the others.  I'll look at them again tomorrow to reassess.

One jar being bubbled and two beakers with no water movement

One jar of larvae is being gently bubbled, to see if this helps break the surface tension.  I started with bubbling that was too gentle, and the other day upped the airflow a bit.  There is a slow circular current in the jar that might be helping.

The two beakers in the front of this table have no agitation at all.  These larvae are dependent on oxygen dissolving into the water from the surface, and I'm a little worried that they might be a little oxygen-stressed.  They are definitely getting stuck at the surface, so I doubt this will be a long-term solution to that particular problem.

Tomorrow I will change the water in all the jars and beakers, and try to assess the amount of stuckage in each.  Hopefully either stirring or bubbling will be the way to maximize survival of my larvae.

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Today a lot of my Dendronotus eggs had hatched on their own, swimming through the water as bona fide veliger larvae.  Nudibranch larval culture has officially started!

These bad boys are much more spherical now--whew!-- which makes me think that pointy-shell thing I saw last week was an artifact of their premature hatching.  Now they look like little swimming bubbles.  Interestingly, their shells are mostly empty.  My invertebrate larval culture guide says that planktotrophic larvae (those that feed while in the plankton) such as these hatch with relatively tiny bodies that grow as the larvae feed. We'll see if that holds for these guys.

I captured some video of the little veligers zooming around.  Here they are at 10X magnification:

Here's another short video clip of some veligers that were conveniently squished under the coverslip.  This kept them from swimming away and I was able to film them at higher magnification.  You can see the little velum whirling away and then being retracted.  See also how most of the shell space is empty?

So, now that these guys have hatched and have all that empty space inside their shells to fill up, they need to eat.  What do I feed them, you ask?  Well, because I was in a hurry to get them something, anything, to eat this morning, I fed them a bit of Isochrysis galbana, which is a haptophyte.  Algal taxonomy is not well established yet, and there are many ways of classifying both micro- and macroalgae.  I hesitate to wade into those murky waters, so suffice it to say that Isochrysis is a unicellular alga, golden-brown in color but neither a diatom nor a dinoflagellate.

This is what Isochrysis galbana looks like in culture.  We grow it in 1000-mL flasks of sterilized seawater and nutrients.

Isochrysis galbana in culture

According to the literature, veligers of Dendronotus frondosus can be raised on a mixture of Isochrysis galbana and a red alga called Rhodomonas salina.  And it just so happens that we also have R. salina in culture, so starting tomorrow the veligers will get a mixture of algae for their breakfast.

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The marine gastropods and bivalves go through a larval stage called a veliger.  This larva gets its name from the ciliated structure, called a velum, that the animal uses for swimming.  Veligers have shells--1 for gastropods and 2 for bivalves--and can withdraw the velum into the shell.  Even gastropods that lack shells as adults, such as nudibranchs, have shells as larvae.

The egg mass from Dendronotus is still intact and the embryos are developing nicely.  This morning when I looked at it through the microscope I could see the little larvae swimming around inside their egg capsules.  I wanted to take a closer look under the compound scope, and when I teased apart the egg mass some of the larvae were forced to "hatch" prematurely.  They're not yet ready for life on their own but now they're out in the real world swimming, for better or for worse.

Not being one to let an opportunity like this go to waste, I took some video of the almost-veligers.

You can see the cilia on their little velums whirling around.  The larvae aren't as spherical as I had expected, based on what I've seen in other nudibranchs, and I think it'll be fun seeing how they develop.  More as things unfold!

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