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When it comes to the natural world, I have always found myself drawn to things that are unfamiliar and strange. I think that's why I gravitated towards the marine invertebrates: they are the animals most unlike us in just about every way imaginable. Even so, some of them have bodies at least that are recognizable as being both: (1) alive; and (2) animal-ish. Think, for example, of a lobster and a snail. Each has a head and the familiar bilateral symmetry that we have. Obviously they are animals, right? I, of course, am most fascinated not by these easy-to-understand (not really, but you know what I mean) animals, but to the cnidarians and the echinoderms. And for different reasons. The cnidarians astound me because they combine morphological simplicity with life cycle complexities that boggle the mind. I hope to write about that some day. Today's post is about my other favorite phylum, the Echinodermata.

For years now I've been spawning sea urchins, to study their larval development and demonstrate to students how this type of work is done. I have a pretty good idea of what to expect in urchin larvae and can claim a decent track record of raising them through metamorphosis successfully. Urchins are easy. To contrast, I have much less experience working with sea stars. I have found that some species are easy to work with, while others are much more problematic. Bat stars (Patiria miniata), for instance, are easy to spawn and raise through larval development into post-larval life. Ochre stars (Pisaster ochraceus), on the other hand, go through larval development beautifully, but then all die as juveniles because nobody has figured out what to feed them. I've already chronicled my and Scott's attempts in 2015 to raise juvenile ochre stars in a series of posts starting here.

Sea urchins and sea stars have long been model organisms for the study of embryonic development in animals, for a few reasons. First, many species of both kinds of animals are broadcast spawners, which in nature would simply throw their gametes out into the water. This means that development occurs outside the mother's body, so biologists can raise the larvae in the lab and observe what happens. Second, spawning can be induced by subjecting the parents to nonlethal chemical or environment stresses. Third, the larvae themselves are often quite happy to grow in jars and eat what we feed them. Fourth, the larvae of the planktotrophic species are often beautifully transparent, allowing the observer to see details of internal anatomy. Lucky me, I've been able to do this several times. And it never gets old.

All that said, there are differences between urchins and stars that force the biologist to treat them differently if we want them to spawn. For the species I work with, spawning occurs after I inject a certain magic juice into the animals' central body cavity--urchins get a simple salt solution (KCl, or potassium chloride) and stars get a more complex molecule (1-MA, or 1-methyladenine). The fact that you can't use the same magic juice for urchins and stars reflects a fundamental difference in gametogenesis and spawning in these groups of animals.

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

Sea urchins will spawn only if they have fully developed gametes. In other words, gametogenesis must be complete before gametes can be released to the outside. You can inject as much KCl into a sea urchin as you want, but if it's the wrong time of year or the urchin doesn't have mature gonads (due to poor food conditions, perhaps), it won't spawn. I've never investigated the mechanism by which KCl induces spawning in ripe urchins, but here's what I think happens.

When students dissect animals in my invertebrate zoology class, we use magnesium chloride (MgCl2) to narcotize the animals first. A 7.5% solution of this simple salt is remarkably effective at putting many animals gently to sleep, especially molluscs and echinoderms. Placing the animals in a bowl of MgCl2 and seawater causes them to relax and gradually become unresponsive. A longer bath in the MgCl2 puts them to sleep for good.

Given the relaxation effects of MgCl2 on urchins, I suspect that injecting a solution of KCl into the body cavity relaxes the sphincter muscles surrounding the gonopores. This relaxation opens the gonopore, and if the gonads are ripe the mature gametes are released to the outside. As I said above, I don't know for certain if this is how it works, but the hypothesis makes sense to me. It also explains why that I can shoot up a dozen urchins and get none of them to spawn: the KCl might be doing what it normally does (i.e., opening the gonopores) but if the gonads aren't ripe there are no gametes to be released.

For completely different reasons, injecting a star with KCl does absolutely nothing at all except probably make the animal a bit uncomfortable. The KCl may very well open gonopores as it does in urchins, but a star will never have mature gametes, especially eggs, to release in response to this muscle relaxant. This is because at least in female stars, meiosis (the process that produces haploid gametes) isn't complete until the eggs have been spawned to the outside. What, then, is the magic juice used to induce spawning in stars, and what exactly does it do?

The magic juice is 1-methyladenine, a molecule related to the nucleobase adenine, most commonly known as one of the four bases that make up DNA. The nomenclature indicates that the difference between the two molecules is the addition of a methyl group (--CH3) to the #1 position on an adenine molecule:

Chemistry aside, what I'm interested in is the action of 1-MA on the eggs of sea stars. Meiosis, the process that produces gametes, has two divisions called Meiosis I and II. Meiosis I starts with a diploid cell (i.e., containing two sets of chromosomes) and produces two diploid daughter cells; these daughter cells may not be genetically identical to each other because of recombination events such as crossing over. It isn't until Meiosis II, the so-called reduction division, that the ploidy number is halved, so each daughter cell is now haploid (i.e., containing a single set of chromosomes) and can take part in a fertilization event. In a nutshell, the end products of meiosis are haploid cells, all of which ultimately result from a single diploid parent cell.

In female sea urchins, the entire meiotic process is completed before the eggs are spawned, which is why the relaxation effects KCl can induce spawning.

In females of many other animal species, meiosis is arrested for some period of time after the Meiosis I division. For example, this happens in humans: baby girls are born with all of the eggs they will ever produce, maintained in a state of suspended animation after Meiosis I. It isn't until puberty that eggs begin to complete meiosis, one egg becoming mature and being ovulated approximately monthly for the rest of the woman's reproductive life. Sea stars are sort of like this, with the notable exception that a female star will ripen and produce thousands of eggs in any spawning event rather than doling them out one at a time.

One of the really cool things about working with sea star embryology is that I get to see the completion of meiosis after the eggs have been spawned. I know that the gonads have to reach a certain level of ripeness before 1-MA will induce spawning. Reviewing my notes from a course I took in comparative invertebrate embryology when I was in graduate school, I came across the mention of 'polar bodies,' tiny blobs that I remember seeing in just-fertilized sea star eggs but which I have never seen in sea urchin embryos. Then I needed to remind myself what polar bodies are all about.

Remember how there are two cell divisions in meiosis? Well, despite what's shown in the diagram above, each of the divisions is asymmetrical. In other words, each division of meiosis produces one big cell and one tiny cell. The tiny cells are the polar bodies. They are too small to either divide or be fertilized, and generally die on their own. Here's a chronology of what happens. First, a cell divides, producing a large cell and a tiny polar body:

I've x'd out the polar body in red because it cannot divide or be fertilized and will soon die. Then the large cell divides to produce the final egg and a second polar body:

It turns out that in sea stars things get even more complicated. 1-MA acts as a maturation-inducing substance in these animals, effectively jump-starting the eggs that have been sitting around in an arrested state after undergoing Meiosis I. This initiates the continued maturation of the eggs to the stage when they can be spawned. Even now, though, meiosis doesn't complete until an egg has been fertilized, at which point the second polar body is produced. The production of that second polar body is the signal that Meiosis II has occurred, and the now-fertilized egg can begin its embryonic development.

Here's a freshly fertilized egg of Pisaster ochraceus, with the two polar bodies smushed into the narrow perivitelline space between the surface of the zygote and the fertilization envelope:

Zygotes of the ochre star Pisaster ochraceus, showing two polar bodies
25 April 2017
© Allison J. Gong

Sea urchins, remember, do not have polar bodies when I spawn them. That's because meiosis is complete by the time the eggs can be spawned, so the polar bodies have already died or been resorbed by the final mature egg. The photo of the P. ochraceus zygotes was taken within a few minutes of fertilization. Let's contrast that with a photo of a brand new urchin zygote:

Egg of purple sea urchin (Strongylocentrotus purpuratus) fertilized by sperm from a red urchin (Mesocentrotus franciscanus)
30 December 2016
© Allison J. Gong

See? No polar bodies!

All of this is to explain why we can't use the same magic juice to spawn both urchins and stars. Kinda cool when the madness in our method has a biological context, isn't it?

I seem to have a need to keep investigating seastar wasting syndrome (SSWS) and trying to make sense of what I and others see in the field. I think it parallels my morbid fascination with the medieval Black Death. In any case, I've devised a plan to continue experimenting with one aspect of the potential recovery of one species, the ochre star, Pisaster ochraceus.

The first step of this plan was to collect a few more stars, which I did back in early March. For the past year or so the stars had been becoming more abundant at certain sites, leading to hope that the populations were beginning to recover and speculation as to whether these individuals were pre-SSWS survivors or post-SSWS recruits. I think they are survivors, because it seems highly unlikely that a star can grow from teensy (a few millimeters in diameter) to hand-sized on a few years. This is what I want to address experimentally in the lab.

The three stars that I collected seemed to adjust well to life in the lab. They all ate well and were crawling around in their tank. Then, last Friday (31 March 2017, to be exact) I checked on the stars as I usually do and was horrified to see this:

Dismembered bits of an ochre star (Pisaster ochraceus)
31 March 2017
© Allison J. Gong

Knowing from experience how quickly this can happen, I'd guess this star had begun ripping itself into pieces in the previous 24 hours. And meantime, its tankmates had stuck themselves to the underside of the cover of the tank. This is not unusual behavior and once I poked them both to make sure they weren't getting mush I decided not to worry about them for the time being. The important thing was to remove the not-dead-yet pieces of the exploded star and bleach the tank before returning the apparently healthy stars to it.

One of the most horrific aspects of SSWS is that it is both blindingly fast and agonizingly slow. It appears to strike out of the blue, by which I mean that stars can look absolutely fine one afternoon and be torn to bits the next morning. And it's slow because the individual pieces can live for hours or even days before finally dying.

31 March 2017
© Allison J. Gong

This star broke itself into five pieces. The three pieces of arm had started getting mushy but still responded by sticking harder when I picked them up. That larger section with two arms and the madreporite was actually walking around the bowl. The torn-off pieces were all oozing sperm into the water, so at least I know this individual was a male. Small comfort, that, when I had to bag up the pieces and throw them in the trash.

Being confronted with the specter of SSWS, I wondered exactly what it meant. I've never been under the illusion that SSWS goes away entirely. I suspect that it is always present in the wild, possibly at low enough levels that we don't notice it for decades at a time. Seeing one dead star, which presumably was infected in the field before I brought it into the lab. . . does it mean the plague is rearing its ugly head again? Or is this a one-off that I just happened to catch? There's only one way to find out, and that is to see if there are more sick stars in the field. So that's what I did the following two days. I had planned to visit three intertidal sites where I expect Pisaster ochraceus to live, but my concussed brain allowed me to drive to only the two nearest sites.

I went to Natural Bridges on Saturday, where I'd been seeing lots of ochre stars over the past several months. I hadn't seen a sick star there for years, although at the outbreak of the plague in 2013 the ochre stars disappeared suddenly. In the past couple of years I'd been happy to see lots of healthy hand-sized stars there. Last weekend it seemed I saw fewer stars than I had gotten used to seeing, but none of them were sick. Whew!

Pair of healthy Pisaster ochraceus at Natural Bridges
1 April 2017
© Allison J. Gong

The next day I went to Mitchell's Cove, where I'd collected those three stars back in March. I did see lots of great-looking stars, some as small as ~6 cm in diameter and others bigger than my outstretched hand.

Trio of healthy Pisaster ochraceus at Mitchell's Cove
2 April 2017
© Allison J. Gong

But I also saw this:

Arm of a P. ochraceus that was killed by SSWS at Mitchell's Cove
2 April 2017
© Allison J. Gong

This is all that remains of an ochre star that apparently succumbed to SSWS. No other body parts are visible in the vicinity, and this arm bit was barely hanging on to the rock. Given how quickly stars can disintegrate when SSWS hits, this one probably began showing symptoms the previous day, while the tide was in and nobody would have seen it. And who knows how many other stars got sick and died without anybody noticing.

The take-home message is that I need to not let SSWS fall off my mental radar. I hope to god that my six remaining P. ochraceus in the lab remain healthy and that I can spawn them in a couple of weeks. I've obtained from a friend some small dishes seeded with food that tiny juvenile stars may be able to eat. I'm not too worried about getting through the larval development stage, although I probably shouldn't get too cocky about that. In any case, it's the post-larval juvenile survivorship that I'm really interested in. This year I don't have Scott to help me with the husbandry and data collection. I will instead be working with another colleague, Betsy. We have a spawning date at the end of April, when the next phase of my ongoing SSWS investigation will begin.

It has been almost three and a half years since I first documented seastar wasting syndrome (SSWS) in the lab. Since then many stars have died, in the field and in the lab, and more recently some species seem to be making a comeback in the intertidal. This circumstantial evidence may not be reason enough to conclude that the epidemic is over, but I think there is reason to be hopeful. Any disease outbreak eventually runs its course, and despite its death toll there are always at least some survivors. And I have an individual star that was very sick but seems to be recovering.

In September of 2015 one of my bat stars (Patiria miniata) developed the first tell-tale lesion of SSWS on its aboral surface. At the time the lesion was small (less than 10 mm in diameter) and superficial. Knowing that SSWS starts with minor symptoms and rapidly progresses to something horrific within a day or so, I wanted to keep an eye on this star. It held the same morbid fascination as a car accident or any other impending catastrophe.

5 September 2015

Bat star (Patiria miniata) with small aboral lesions.
4 September 2015
© Allison J. Gong
Dermal lesion on the aboral surface of a bat star (P. miniata).
5 September 2015
© Allison J. Gong

24 November 2015

By November 2015 the main lesion hadn't grown much but a few others had developed. The star still wasn't acting sick and was eating every once in a while, although it occasionally ignored the food that I offered.

Bat star (P. miniata) with aboral lesions.
24 November 2015
© Allison J. Gong

So far, so good. I was thinking that the star doesn't look too much worse, so maybe it wouldn't keep getting sicker. I checked on it regularly, offered food a few times a week, and left it alone.


4 May 2016

Several months later I noticed that the first lesion had gotten much deeper. The outer dermal layers had been completely compromised, exposing the animal's internal organs (gonad and digestive caecum) to the external environment. This was bad, very bad. Even in stars, internal organs are supposed to be internal, except when stars extrude their stomachs to feed.

Bat star (P. miniata) with deep aboral lesion.
4 May 2016
© Allison J. Gong
Lesion on aboral surface of a bat star (P. miniata). Note the internal structures that are exposed to the surrounding seawater.
4 May 2016
© Allison J. Gong

This was the point in time when things started going south. The star lost the ability to maintain its internal turgor pressure and became lethargic and floppy. It stopped eating, or even responding to food. It spent most of its time in a corner of the seawater table where it lives, although a few times I saw it wrapped around one of the hoses that feeds the table. However, its body never started disintegrating the way I'd seen with other SSWS victims.


19 January 2017

Fast-forward another several months. About a month ago the sick bat star began perking up a bit when I placed food near the tip of one of the arms. A week later it actually wrapped its arm around the food, and I assume ate it. It has since been eating about once a week, after fasting for at least eight months. I began to think it would recover.

Today I had some time to photograph the star again, and it really appears to be doing much better!

Bat star (P. miniata) with healing lesions.
19 January 2017
© Allison J. Gong

The lesions are apparently healing over; at any rate, the internal organs are no longer exposed to the outside. The body margin between the arms has a few small divots, but they look superficial. Lately the star has been more active, too, cruising around the table instead of hunkering down in a corner. I'm going to keep feeding it to see if it continues to improve.

One of the most remarkable things about many animals with a decentralized nervous system, such as echinoderms and cnidarians, is their ability to regenerate lost parts and repair damage to their bodies. This bat star is a prime example. It has been sick for almost a year and a half now, and for at least half that time it hasn't eaten. Yet it somehow had the metabolic reserves to heal a major wound to its body wall. That's some astounding resilience there. I am very impressed, and you should be, too.

1

When the most recent epidemic of seastar wasting syndrome (SSWS) began back in 2013, the forcipulate stars were the first to succumb. This group includes conspicuous members of intertidal and subtidal habitats, such as:

  • Pisaster ochraceus -- the intertidal ochre star
  • Pisaster giganteus -- the giant spined star, which lives in the low intertidal and subtidal
  • Pycnopodia helianthoides -- the sunflower star, a huge monster of the low intertidal and subtidal.

In the past year or so, I've noticed P. ochraceus making a comeback at local intertidal sites. At first I was seeing stars in the 2-3 cm size range, and now I'm regularly seeing hand-sized ones clinging to the rocks.

4 mm juvenile Pisaster ochraceus star at Pescadero State Beach.
11 May 2016
© Allison J. Gong

You read that right. 4 mm in diameter. This is the tiniest forcipulate star that I've ever been able to ID in the field with any certainty.

Pair of Pisaster ochraceus stars in the low-mid intertidal at Natural Bridges.
22 July 2016
© Allison J. Gong
A hand-sized (dark orange) and much smaller (dark purple, tucked far back in the little cave) Pisaster ochraceus at Mitchell's Cove.
28 November 2016
© Allison J. Gong

It seems pretty clear that the ochre stars, at least, are making a comeback. It's likely that the larger ones are survivors of the SSWS plague. That little tiny one, though, may well be a post-SSWS recruit. Unfortunately we don't know how fast they grow once they recruit to the benthos. We do know that when they recruit they're about 500 µm in diameter, so even that little guy has grown a lot in however long it has been since it settled.

The really exciting news is that yesterday I saw my first P. giganteus since the SSWS outbreak began! I was up at Davenport Landing collecting sea urchins and saw this star in an urchin hole. The rock around here is a soft mudstone that is easily eroded. Urchins excavate holes by twisting their spines against the rock, and then live in them. Holes that are urchinless, for whatever reason, are quickly colonized by other organisms (including baby urchins).

A not-so-gigantic Pisaster giganteus star in an urchin hole at Davenport Landing.
13 December 2016
© Allison J. Gong

For a sense of size, this urchin hole is about 8 cm in diameter. The star is sharing it with a small anemone, most likely Anthopleura elegantissima.

Pisaster giganteus generally occurs lower in the intertidal than P. ochraceus, and I wouldn't expect to see it on a tide that isn't at least as low as -0.8 ft. It isn't as closely associated with mussel beds as P. ochraceus, either, because it lives lower in the intertidal. Fortunately, this week's low tide series includes a few days with tides below -1.0 ft, and I'm going back out today. I'll be keeping my eyes open for not only Pisaster stars, but also the Pycnopodia that disappeared a few years ago. Although Pycnopodia gets very large, I don't expect to see any really big ones running across the intertidal. However, Pycnopodia juveniles would indicate at least the beginning of a possible population recovery  from the SSWS plague.

So, wish me luck and keep your fingers crossed!

Seeing as today is the third anniversary of the first blog post I wrote about sea star wasting syndrome (SSWS), I thought it would be appropriate to take inventory of my remaining stars and see how they're doing. Right now I have custody of ~10 bat stars (Patiria miniata), 7 ochre stars (Pisaster ochraceus--collected last year for the juvenile survival experiment I did with Scott), and 1 Mediaster aequalis. For whatever reason the M. aequalis hasn't been affected by SSWS so I'm going to disregard it for now. Of the 10 or so bat stars, four live in one of my seawater tables, roaming free-range in quite a large volume of water. The other half-dozen or so live in a tank in a different building. The Pisasters live in 1s and 2s in tanks distributed in two rooms in the same lab.

After the initial horror and shock of the spectacular onset of SSWS, in which we watched stars rip themselves into pieces right before our eyes, what we've seen has followed the standard epidemiology pattern. Any time a novel pathogen enters a population, the individuals that have no immunity or resistance are the first to die. The disease spreads rapidly through the population, wiping out all of these weaker individuals. However, not everyone dies. Even during the Black Death of the 14th century, the very fact that 1/3-1/2 of the human population died of bubonic plague means that 1/2-2/3 survived. Those survivors presumably had some degree of resistance to the disease.

At the same time three years ago that all of my forcipulate stars died, divers were noticing similar phenomena happening subtidally. It didn't take long for us to realize that Something Big was going on, which was eventually dubbed SSWS. Fast-forward three years and now I'm seeing healthy, hand-sized P. ochraceus in the intertidal again. These individuals are certainly survivors from the SSWS outbreak; they were likely small juveniles during the plague, and were able to come out of hiding and expand into open niches after so many of the adults died. Whether or not natural populations will recover completely remains to be seen, but as of right now things look promising.

About a year ago, having gone two years without showing any signs of being sick, one of my bat stars developed lesions on its aboral surface. It's the red star in the middle of that blog post. This star is one of the four that live in my shallow table. It has now been sick for a year. See how it has changed since then:

Patiria miniata (bat star) with small lesion. 4 September 2015 © Allison J. Gong
Patiria miniata (bat star) with small lesion.
4 September 2015
© Allison J. Gong

and

Patiria miniata with aboral lesions. 7 September 2016 © Allison J. Gong
Patiria miniata with aboral lesions.
7 September 2016
© Allison J. Gong

The lesions have all gotten worse--the largest is about 2 cm long now--and the body margin has some ripples that it didn't have before, but the star is still alive. For a while it wasn't eating, as far as I could tell, but two days ago I watched it eat a piece of fish. Perhaps the return of cooler water is helping this animal survive.

One of its tablemates, however, hasn't been so lucky. I first noticed apparent SSWS damage in a second star several months ago. Today was the first chance I had to look closely at it.

Aboral view of Patiria miniata with damage to body wall. 7 September 2016 © Allison J. Gong
Aboral view of Patiria miniata with damage to body wall.
7 September 2016
© Allison J. Gong
Oral view of Patiria miniata with damage to body wall. 7 September 2016 © Allison J. Gong
Oral view of Patiria miniata with damage to body wall.
7 September 2016
© Allison J. Gong

The most noticeable injury to this star is that big interradial divot. It looks like someone took a bite out of the body at that spot. The margins of the wound are white and fluffy, similar in appearance to the lesions caused by SSWS.

For years now this star has had an abnormal spot on its aboral surface. I've been calling it a bubble, for lack of a better word. The bubble may be an over-inflated papulla (skin gill) and it didn't seem to be causing any problems for the star. I'd touch it and it would deflate, then re-inflate almost immediately. When I touched it today, it shrank back a little but didn't really deflate.

Strange "bubble" on aboral surface of P. miniata. 7 September 2016 © Allison J. Gong
Strange "bubble" on aboral surface of P. miniata.
7 September 2016
© Allison J. Gong

If you look really closely at the above photo, you can make out clusters of small, clear, clublike projections. These are papullae, extensions of the internal body lining that project through the skeletal ossicles to the outside and act as gas exchange surfaces. The bubble is many times larger than the normal papullae. Because it has been there for so long, years before the divot in the interradial margin, I don't think the bubble is due to SSWS. I don't even know if it's a wound, or merely an overinflated papulla. The largest star in this table has also had a bubble for many years, but no lesions or wounds indicating SSWS or other disease.

So. Three years after the outbreak of SSWS I still have stars that are sick. They've been sick for a long time and aren't getting worse very quickly, from which I conclude they may eventually recover. At the very least they must have some resistance to the SSWS pathogen because they've managed to survive so far. One more thing. Way back in 2013 when all of the forcipulates were tearing themselves into pieces and melting into piles of goo, these bat stars were among them, scavenging on the dead and decaying tissue. For a while I feared that eating contaminated tissue might cause the disease, but that doesn't seem to be the case, as these two didn't get sick until two years after the initial exposure.

I hope these two stars make it. Cooler water temperatures should help. When they're really sick they stop eating (they haven't eaten much in the past year) but if they're going to eat now I'll keep feeding them. Fingers crossed!

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