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Well, it looks like the end is indeed nigh. That last Pisaster, for whom I held out unreasonable hope for so long, seems to be on its way out. Today it has lost its last two arms, leaving a central disc attached to a single arm:

Remains of Pisaster ochraceus that has lost four arms. ©2013 Allison J. Gong
Remains of Pisaster ochraceus that has lost four arms.
© 2013 Allison J. Gong

As bad as it looks, it could be a lot worse. The other stars that disintegrated to this degree were essentially amorphous piles of goo, and this one is still somewhat intact. It also hasn't gone entirely mushy, so it is somehow maintaining its internal pressure. I'm going to keep it for another day and see how it looks tomorrow.

The other two arms, on the other hand (ha!), were a mess. When I got to the table this afternoon they were both semi-attached and semi-upside down behind one of the quarantine tanks. And they were very mushy; when I picked them up they just collapsed the way sea cucumbers do before they start firming up. Gross.

Autotomized arms of Pisaster ochraceus ©2013 Allison J. Gong
Autotomized arms of Pisaster ochraceus
© 2013 Allison J. Gong

This has to be the end, if only because I don't have any more Pisaster stars to die. Unless the Patiria and Dermasterias stars that I quarantined start getting sick, the outbreak in my seawater table is over, simply because there are no more victims to be infected. From a pathogen's perspective a 100% mortality rate is a bad thing--if all hosts of a population are killed then the pathogen will die with them. However, my table is connected by water supply to other tables and labs, and I have a sneaking suspicion that the pathogen is out there in Monterey Bay (the source of our seawater), in which case there's nothing I can do about it. Actually, I can do something. I can cross my fingers and hope for the best.

 

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Against all odds, my last Pisaster star is (literally) hanging in there. It hasn't lost any more arms in the past 24 hours, and by the standards of the past two weeks that's a rousing success.

Pisaster star that lost two arms yesterday but no more since. ©2013 Allison J. Gong
Pisaster ochraceus star that lost two arms yesterday but no more since.
© 2013 Allison J. Gong

And it hasn't lost the turgor pressure of its body, so it isn't as limp as the others were before they died. I didn't want to mess with the animal too much, but it was pretty strongly attached to the table, indicating that the water vascular system hasn't lost all of its integrity. If that inter-radial area towards the top of the photograph is one of the areas where an arm was autotomized, the wound has healed surprisingly well. I will have to see what happens tomorrow.

On the other hand, the disease has spread to the lab next door, where a Pisaster giganteus started melting away two days ago. It was discovered with a small P. ochraceus feeding on the sick star, and the two stars have been since isolated. Today the P. giganteus looked horrifying:

Pisaster giganteus star melting from wasting disease. ©2013 Allison J. Gong
Pisaster giganteus star melting from wasting disease.
© 2013 Allison J. Gong

This is a really sick animal. There's a large wound on the bottom edge where an arm had been autotomized; it looks like the wound hasn't started healing at all. One of the remaining arms has twisted so that it is upside-down with the ambulacral groove--where the tube feet are visible--is facing upwards; that arm is probably going to be cast off soon. The beige-ish fluffy bits in the top of the photo are pieces of gut and water vascular system that are protruding through wounds in the body wall. I would be very surprised if this poor animal is still alive tomorrow. So far, the one that was feeding on this creature doesn't look diseased, so perhaps it will escape the pestilence.

19

The last of my Pisaster ochraceus stars waited until today, three whole days after all of its conspecifics had died, to start ripping itself into pieces. This is the sight that greeted me when I checked on my animals this morning:

My last Pisaster and its autotomized arm ©2013 Allison J. Gong
My last Pisaster and its autotomized arm
© 2013 Allison J. Gong

I spent some time examining the severed arm because it is freakishly fascinating to watch autotomized parts continue on as though they were still attached to the main body. They literally don't know that they're dead.  I've seen almost completely eviscerated sea urchins lumber around a seawater table on about 10 tube feet for days before finally giving up the ghost. This arm remained very active for quite a while--at least an hour--before I gave up and threw it away.

While I had this severed arm in a bowl under the dissecting scope I thought I'd take a few photos of the surface. Beautifully complex animals, sea stars are, when you look at them up close.

View through dissecting microscope of aboral surface of arm of Pisaster ochraceus. ©2013 Allison J. Gong
View through dissecting microscope of aboral surface of arm of Pisaster ochraceus.
© 2013 Allison J. Gong
Oral surface of arm of Pisaster ochraceus, showing tube feet. ©2013 Allison J. Gong
Oral surface of arm of Pisaster ochraceus, showing tube feet.
© 2013 Allison J. Gong

Meanwhile, the remaining 4/5 of the star continued to walk around the table. It ended up behind one of the quarantine tanks in which I had sequestered the bat stars, where over the course of the next couple of hours it dropped another arm. Because of its location I wasn't able to get a decent photo of it, but here is a shot of the wound from the first autotomization:

Wound caused by autotomy of an arm in Pisaster ochraceus. ©2013 Allison J. Gong
Wound caused by autotomy of an arm in Pisaster ochraceus.
©2013 Allison J. Gong

And I'm not the only one at the lab dealing with this disease outbreak. The lab next door is losing a couple of stars, and the Seymour Center lost one of their Pycnopodia helianthoides (sunflower star) yesterday. And, I heard second-hand that a student in the Santa Cruz area saw some dying stars on a dive in the past few days. What happened in my seawater table over the past few weeks may be just the beginning of something really, really bad.

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And I don't mean plague as in "too many stars to know what to do with," but as in "disastrous sickness that you don't want to catch." Some of the stars in my seawater table have been succumbing to some awful disease lately. A week ago today I noticed that many stars had been busy cannibalizing one of their compadres. Sometimes this just happens, and it doesn't necessarily indicate that things are about to go south. But when I looked more closely I noticed that the victim, instead of just being eaten, had autotomized its arms. Autotomy occurs in most sea stars and other invertebrates, and in fact is used as a method of clonal replication in some stars and many cnidarians. The species of star that is being affected by this plague (Pisaster ochraceus, the common ochre star) isn't one that readily autotomizes except in response to some external stress, such as a predator pulling on an arm.

So something was going on in this table. On Monday (Labor Day) I popped in for a quick check and although nobody had lost any arms I couldn't be absolutely sure that everything was okay. Some of the Pisasters were a little squishy and had arms that were a little twisted. On Tuesday morning there was no autotomy but in the afternoon a star had lost an arm, greatly disturbing the student lab assistant who discovered it. On Wednesday the table looked like an asteroid battlefield:

Large Patiria miniata (bat star) scavenging on dead Pisaster ochraceus (ochre star)
Large Patiria miniata (bat star) scavenging on dead Pisaster ochraceus (ochre star).
© 2013 Allison J. Gong

Many of the other Pisasters were also showing signs of sickness: curly arms (visible in the yellow star in the lower right corner of the photo above. Another ominous sign is that some of the apparently sickly stars were kind of squishy, indicating that the water vascular systems were somehow compromised.

Severed arms littered the table. The autotomized arms retain mobility for quite a while after being cast off--they literally don't know that they're dead.

IMG_2075
Autotomized arm of a sick Pisaster ochraceus. The other, intact, star is Orthasterias koehleri, the rainbow star.
© 2013 Allison J. Gong 

After removing the corpses and cleaning the table as best I could I was able to take a closer look at the survivors. I noticed that most of the remaining Pisasters had twisty or crossed arms, and some showed pretty severe stretching in an interambulacral area ("armpit" area between adjacent arms), which I think is the first stage of autotomy.

IMG_2079
Pisaster ochraceus stretched interambulacral area, pulling its own arm off.
© 2013 Allison J. Gong

The disease progresses very rapidly, and within an hour a star in this condition had pulled off one arm and was working on another.

IMG_2083
Pisaster ochraceus that has autotomized an arm. Injury site is visible as a white area in lower edge of central disc. The autotomized arm is located at the top of the photo.
© 2013 Allison J. Gong

Unfortunately, this disease also affects other species. My Orthasterias koehleri (rainbow star) decided to join the fun. When I arrived Wednesday morning it was intact. It dropped an arm, I went away for about 40 minutes to take care of tasks in a different building, and when I returned it had lost two more arms:

IMG_2088
Orthasterias koehleri that dropped three arms in about an hour. The autotomized arms are indicated by yellow arrows. The remaining 2/5 of the star are attached to the outside of my urchin tank.
© 2013 Allison J. Gong

Alas, my one and only Orthasterias succumbed later in the day and was dead on Thursday. Interestingly, the disease does not seem to affect either Patiria miniata (bat stars) or Dermasterias imbricata (leather stars). In fact, the Patiria have been eating pretty well over the past week, scavenging on the carcasses of the plague victims. I don't know if eating the diseased tissue will cause problems later on.

On Friday I lost two more Pisasters and isolated the Patiria and Dermasterias into tanks. A colleague of mine calls this the Molokai treatment, and I probably should have done it sooner, but I figured that at this point all the stars in the table were exposed to whatever pathogen is causing this disease so at that point why bother? However, I will need to sequester the healthy stars in order to disinfect the table once the disease has run its course, so into tanks they went.

After checking on the stars Saturday morning I am cautiously optimistic that the plague may have run its course. One more Pisaster, that was looking sickly the day before, had died, but my last two appeared healthy. Their arms were not curly, I didn't see any interambulacral stretching, and they felt nice and hard when I poked at them. All of these are good signs, but I will continue to keep close watch on them. If they make it to Monday we just might be out of the woods.

As of today, one week after I noticed the first severe symptoms, I have lost 80% of my Pisaster collection. To put that in to context, this mortality rate is every bit as bad as some villages that were virtually wiped out by the medieval Black Death.

It has been almost a month since my big female whelk started laying her eggs, and the embryos seem to be developing nicely. The first time I witnessed this phenomenon I saw the egg capsules begin to turn black, and worried that the eggs inside were dead and decomposing. But the cool thing about Kelletia development is that the larvae themselves become darkly pigmented as they develop, which we see as an overall dingy grayness of the egg capsules:

Kellettia eggs

 

Nosy as ever, I pulled one of the egg capsules off the side of the bin and took it back to my desk for closer examination under my dissecting scope. At the "top" of the capsule (the end that is attached to the bin), the material was quite thin, and I could some vague dark lumps inside. They were slowly moving around, so I knew they were alive.

Individual larvae resemble bubbles with dark stuff inside.
Individual larvae resemble bubbles with dark stuff inside.

 

Viability! This makes me happy and encourages me to "liberate" a few larvae to look at under higher magnification. I squeezed out a few veligers and put them under a coverslip with just enough water to keep their shells from cracking but not enough to let them swim away. Here's a tip for observing small aquatic critters under a microscope:  If you make their universe (i.e., the drop of water you are observing) small, they will be less able to swim away from you. Flattening the drop of water with a judiciously placed coverslip will also help immobilize the creature, as well as taking best advantage of the microscope's optics.

Early veliger of Kelletia kellettiiNot too much to look at while stationary, is it? You can see a coiled shell (this is a snail after all) and some blobby structures inside it. At this stage the larva isn't feeding and relies on yolk reserves provided by the mother when she deposited the eggs. Some of the opaque stuff inside the shell is yolk and other bits are various parts of the digestive system. At about 11:00 just underneath the shell there is an elongated transparent area: the larva's heart; you can see it beating in the video below. The light mohawk-looking structure facing to the right is the larva's velum, a lobed ciliated structure that the animal will use to swim after it hatches. The last structure of note is the wedge-shaped thing that points to about 5:00; this is the larva's foot, on the back of which sits the operculum that is used to close up the shell.

After a bit of trial and error I was able to catch some decent video footage through the microscope of a trapped larva:

Kellettia larva under compound scope

The larva rhythmically extends and retracts its velum. Because of the coverslip the larva can't go anywhere, but if unencumbered it would be able to use that velum to zip around really fast. It is very difficult to keep up with swimming veligers under a microscope!

My guess is that the larvae will begin hatching on their own in the next couple of weeks. They will be washed out of their tub and down the drain of the seawater table, to take their chances in the big ol' Pacific Ocean.

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This week my female Kellet's whelk (Kelletia kelletii) started laying eggs. She's been doing this every summer for the past several years. She lives with one other whelk, presumably the father of her brood, as the eggs are both fertilized and viable even though I've never seen the snails copulating.

That's right, copulating. Whelks are predatory marine snails, some of which get quite large. My big female's shell is a heavily calcified 12 cm or so; she's a beefy mother! Her mate is smaller, but other than the size difference I wouldn't be able to tell them apart. Anyway, whelks copulate, with the male using a penis to transfer sperm into the female's body. Not very different from the way we humans do things, actually.

So at some point in the recent past my whelks copulated, and this week the female began depositing egg cases on the walls of their shared tub. I first noticed them on Monday, but she may have started over the weekend.

Female whelk (right) laying eggs. ©Allison J. Gong
Female whelk (right) laying eggs.
© 2013 Allison J. Gong

Those pumpkin seed-shaped objects are the egg capsules. Each is actually about the size and shape of a pumpkin seed and has a tough outer covering that contains 20-50 developing embryos. After the entire clutch is lain, which usually takes this particular female a week or so, the mom will leave the eggs to develop on their own.

I'll keep an eye on these eggs for the next week or so, and might be able to get some photos of the embryos and larvae as they begin developing. Keep your fingers crossed!

2

Over the Memorial Day weekend I took my students out on the early morning low tides at Natural Bridges State Beach.  While they were ooh-ing and ahh-ing and filling out their assignment worksheet, I was playing around with my new camera, taking pictures in the water.  Because I am not a photographer and sea anemones just sit there, they quickly became my favorite subjects.  Not to mention the fact that they are simply  beautiful and photogenic creatures.

At Natural Bridges we have four species of anemones in the genus Anthopleura:

  • A. xanthogrammica - giant green anemone
  • A. sola - sunburst anemone
  • A. elegantissima - aggregating anemone
  • A. artemisia - moonglow anemone

Of these species, the first two are notable for their large size.  At Natural Bridges they can get to be the size of a dinner plate.  They live side-by-side in tidepools, and since there are many deep-ish pools at Natural Bridges they are among the most conspicuous animals in the intertidal along the northern California coast.

Anthopleura sola (left) and A. xanthogrammica (right) in a shallow pool at Franklin Point.
Anthopleura sola (left) and A. xanthogrammica (right) in a shallow pool at Franklin Point.

It's easy to identify these animals when they're sitting right next to each other.  The difficulty comes when you see only one in a pool by itself with nothing to compare it to.  In a nutshell, here are some things you can use as clues to determine which species you have in front of you.

Let's start with Anthopleura xanthogrammica, the giant green anemone.  This animal's oral surface and tentacles are a solid color, varying from bright green to golden brown.  There are no conspicuous stripes on the central disc and the tentacles are relatively short and stubby, without any white patches.

Anthopleura xanthogrammica, photographed at Natural Bridges State Beach
Anthopleura xanthogrammica, photographed at Natural Bridges State Beach

Anthopleura sola, on the other hand, usually has distinctive radiating lines on the oral disc.  Hence the common name of Sunburst Anemone.  Its tentacles are generally longer and more slender than those of A. xanthogrammica, and often have sharp-edged white patches.  Sometimes the tips of the tentacles are tinged a pale purple.  Anthopleura sola are usually brownish-green in color, and I haven't seen any that are as bright green as the A. xanthogrammica anemones.

Anthopleura sola, photographed at Natural Bridges State Beach
Anthopleura sola, photographed at Natural Bridges State Beach

That's all well and good, but sometimes you come across an individual that doesn't completely follow the rules.  Or rather, it looks like it could belong to both species. Such as this fellow (fella?):

Hmmm...sola or xanthogrammica?
Hmmm. . . sola or xanthogrammica?

The animals obviously don't read the descriptions.  This one has xanthogrammica shape and overall color, but those lines on the disc read as sola-ish.  I would call this one a xanthogrammica.  What do you think?

1

I was making my usual feeding and checking rounds at the marine lab last Wednesday, when I saw this:

Pugettia producta, molting.  Time 10:09:12

This crab is a kelp crab, Pugettia producta. It is one of the common crab species on the California coast; you can find them in the low intertidal clinging to algae. Many of them are this golden-brown color, coincidentally(?) the same color of the kelp Macrocystis pyrifera. Juveniles are often reddish or dark brown in color, again matching or blending in with the algae where you see them. This particular crab has always been this color, at least since it has been in my care.

Crustaceans, as all arthropods, periodically molt their entire exoskeleton in one fell swoop. The exoskeleton splits along the transverse seam between the carapace and the abdomen, then the crab sort of slithers out backward. The entire exterior of the body, including legs, antennae, and mouthparts, is left behind as a larger version of the crab scuttles away to hide out for a few days until its new shell hardens.

I've kept lots of crabs and seen lots of molts show up in their tanks, but have never caught one in the act before. From when I started watching, in the photo above, to the final wiggle out of the old exoskeleton took no longer than 5 minutes.  Here's the sequence of photos documenting the molt:

Pugettia producta molting. Time 10:09:41
Pugettia producta molting. Time 10:12:18
Pugettia producta, molting. Time 10:13:57

Pretty nifty, eh?

 

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.

1

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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