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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.

A few days ago I was in the intertidal with my friend Brenna. This most recent low tide series followed on the heels of some magnificently large swells and it was iffy whether or not we'd be able to get out to where we wanted to do some collecting. Our first day we went up to Pistachio Beach, just north of Pigeon Point, where the rocky intertidal is bouldery and protected by some large rock outcrops.

Pigeon Point lighthouse, viewed from Pistachio Beach.
27 January 2017
© Allison J. Gong
27 January 2017
© Allison J. Gong

So while the swell was indeed really big, we were pretty well protected in the intertidal. The Seymour Center has a standing order for slugs, hermit crabs, and algae. I was easily able to grab my limit (35) of hermit crabs over the course of the afternoon, and while it's too early in the season for the algae to do much I had my sluggy friend with me to take care of finding nudibranchs, which left me free to let my attention wander as it would.

Codium setchellii at Pistachio Beach.
27 January 2017
© Allison J. Gong

The very first thing to catch my eye as we go out there was the coenocytic green alga Codium setchellii, which I wrote about last time. I've seen and collected C. setchellii from this site before, but don't remember seeing it in such large conspicuous patches. I need to review what I learned about the phenology of various intertidal algae, but here's a thought. Maybe Codium is an early-season species that gets outcompeted by the plethora of fast-growing red algae later in the spring. Red algae were present at Pistachio Beach but not in the lush (and slippery!) abundance that I'll see in, say, June. I'm willing to bet that Codium will be less abundant in the next few months.

Leptasterias sp. at Pigeon Point.
24 April 2016
© Allison J. Gong

In my experience, the six-armed stars of the genus Leptasterias have always been the most abundant sea stars on the stretch of coastline between Franklin Point and Pescadero. Even though they are small--a monstrously ginormous one would be as large as the palm of my hand--they are very numerous in the low-mid intertidal. I've seen them in all sorts of pinks and grays with varying amounts of mottling. Alas, I don't know of any really reliable marks for identifying them to species in the field.

Unlike other familiar stars, such as the various Pisaster species and the common Patiria miniata (bat stars), which reproduce by broadcast spawning their gametes into the water, Leptasterias is a brooder. Males release sperm that is somehow acquired by neighboring females and used to fertilize their eggs. There isn't any space inside a star's body to brood developing embryos, so a Leptasterias female tucks her babies underneath her oral surface and then humps up over them. Leptasterias also humps up when preying on small snails and such, so that particular posture could indicate either feeding or brooding.

Here's a Leptasterias humped up on a rock, photographed last spring:

Leptasterias sp. at Pigeon Point.
5 May 2016
© Allison J. Gong

The only way to tell if a Leptasterias star is feeding or brooding is to pick it up and look at the underside. I did that the other day and saw this:

Brooding Leptasterias sp. star at Pistachio Beach.
27 January 2017
© Allison J. Gong

Those little orange roundish things are developing embryos. While the mother is brooding she cannot feed, and can use only the tips of her arms to hang onto rocks. Don't worry, I replaced this star where I found her and made sure she had attached herself as firmly as possible before I left her. In a few weeks her babies will be big enough to crawl away and she'll be able to feed again.

Looks like the reproductive season for Leptasterias has begun.


The next day Brenna and I went to Davenport, again hoping to get lucky despite another not-so-low tide and big swell.

Davenport Landing Beach and adjacent rocky areas.
© Google Earth

Davenport Landing Beach is a popular sandy beach, with rocky areas to the north and south. The topography of the north end is quite variable, with some large shallow pools and lots of vertical real estate to make the biota very diverse and interesting. The big rocks also provide shelter from the wind, a big plus for the intrepid marine biologist who insists on going out even when it's crazy windy. The southern rocky area is very different, consisting of flat benches that slope gently towards the ocean, with comparatively little vertical terrain. The southern end of the beach is always more easily accessible, which is why I almost always go to the north. But this day the north wasn't going to happen. The winter storms had washed away at least a vertical meter of sand between the rock outcrops. That and the not-so-low tide combined for conditions that made even getting out to the intended collecting site a pretty dodgy affair. So Brenna and I trudged across the beach to the south.

28 January 2017
© Allison J. Gong

Along the way we saw lots of these thumb-sized objects on the beach. At first glance they look like pieces of plastic, but after you see a few of them you realize that they are clearly (ha!) gelatinous things of biological origin. They are slipper-shaped and you can stick them over the ends of your fingers. They have a bumpy texture on the outside and are smooth on the inside.

Any guesses as to what they are?

Pseudoconch of Corolla spectabilis, washed up on Davenport Landing Beach.
28 January 2017
© Allison J. Gong

These funny little things are the pseudoconchs of a pelagic gastropod named Corolla spectabilis. What is a pseudoconch, you ask? If we break down the word into its Greek roots we have 'pseudo-' which means 'false' and 'conch' which means shell. Thus a pseudoconch is a false shell. In this case, 'false' refers to the fact that this shell is both internal (as opposed to external) and uncalcified.

The animal that made these pseudoconchs, Corolla spectabilis, is a type of gastropod called a pteropod (Gk: 'wing-foot'). Pteropods are pelagic relatives of nudibranchs, sea hares, and other marine slugs. They are indeed entirely pelagic, swimming with the elongated lateral edges of their foot. Like almost all pelagic animals, Corolla has a transparent gelatinous body. Even their shell is gelatinous, rather flimsier than most shells, but it serves to provide support for the animal's body as it swims.

You can read more about Corolla spectabilis and see pictures and video here.

Why, you may be wondering, do the pseudoconchs of C. spectabilis end up on the beach, and where is the rest of the animal? The body of Corolla and other pteropods is soft and fragile. When strong storms and heavy swells seep through the area, the water gets churned up and pteropods (and other pelagic animals) get tossed about and shredded. This leaves their pseudoconchs to float on currents until they are either themselves demolished by turbulence or cast upon the beach. Corolla is commonly seen in Monterey Bay, and it is not unusual to find their pseudoconchs on the beaches after a series of severe storms.

Brenna and I were wondering if we could preserve the pseudoconchs somehow. I took several back to the lab and tried to dry them, thinking that they might behave like Velella velella does when dried. Unfortunately, the next day they had shriveled into unrecognizable little blobs of dried snot, and the day after that they had disintegrated completely into piles of dust. Maybe drying them more slowly would work. Something to consider the next time I run across pseudoconchs in the sand.

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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!

So. I have a batch of larvae from a spontaneous spawning of the leather star, Dermasterias imbricata, that occurred four weeks ago tonight. Until now I've never had an opportunity to work with this species, even though we have quite a few of them at the marine lab. I had my own for several years, until they became casualties of the plague about a year into the current sea star wasting syndrome event. In any case, this is the first time I've been able to spend time with larvae of this species. At the very least I wanted to see how big they would get and how quickly they would develop, compared to the species I'm more familiar with, Patiria miniata (bat star) and Pisaster ochraceus (ochre star).

When the Dermasterias spawned, the first thing I noticed was that the eggs are huge. I measured them at 220 µm in diameter, which is big even compared to what I've seen in other stars. Hatch rates were pretty good, and four days later the larvae were already in the 400-430 µm range. Since I have no experience culturing this species, I thought I'd divvy up my larvae and put them into three feeding treatments to see which larval diet resulted in the best overall success. According to the literature, Dermasterias larvae can be raised on a mixture of the unicellular algae Dunaliella tertiolecta (green) and Isochrysis galbana (golden). My three feeding treatments are: Dun only, a Dun/Iso mix, and Iso only.

A week into the experiment there was a clear difference between the larvae eating only the green food, and those eating either a mixture of green and golden or only the golden. Larvae from all food treatments were about the same size, but the ones eating only Dunaliella had noticeably green guts.

Bipinnaria larva of Dermasterias imbricata. 29 February 2016 © Allison J. Gong
Bipinnaria larva of Dermasterias imbricata, fed on Dunaliella tertiolecta, age 7 days.
29 February 2016
© Allison J. Gong
Bipinnaria larva of Dermasterias imbricata, fed on a mixture of D. tertiolecta and I. galbana. 29 February 2016 © Allison J. Gong
Bipinnaria larva of Dermasterias imbricata, fed on a mixture of Dunaliella tertiolecta and Isochrysis galbana, age 7 days.
29 February 2016
© Allison J. Gong
Bipinnaria larva of Dermasterias imbricata, fed on I. galbana. 29 February 2016 © Allison J. Gong
Bipinnaria larva of Dermasterias imbricata, fed on Isochrysis galbana, age 7 days.
29 February 2016
© Allison J. Gong

Fast forward two weeks, and the larvae were 20 days old. By this time they had progressed from the bipinnaria stage to the brachiolaria stage. The interesting thing was the absence of green pigment in any of the guts, even those that were eating only green food. The D. tertiolecta larvae looked good, actually. They were a little smaller than the other larvae but were perfectly formed.

Brachiolaria larva of Dermasterias imbricata, fed D. tertiolecta, age 20 days. 14 March 2016 © Allison J. Gong
Brachiolaria larva of Dermasterias imbricata, fed D. tertiolecta, age 20 days.
14 March 2016
© Allison J. Gong
Brachiolaria larva of Dermasterias imbricata, fed D. tertiolecta and I. galbana, age 20 days. 14 March 2016 © Allison J. Gong
Brachiolaria larva of Dermasterias imbricata, fed D. tertiolecta and I. galbana, age 20 days.
14 March 2016
© Allison J. Gong
Brachiolaria larvae of Dermasterias imbricata, fed I. galbana, age 20 days. 14 March 2016 © Allison J. Gong
Brachiolaria larvae of Dermasterias imbricata, fed I. galbana, age 20 days.
14 March 2016
© Allison J. Gong

Obviously all of the larvae are assimilating enough of their food to grow and develop normally. I looked at them today but didn't have time to take pictures. Qualitatively there is no difference between the Dun larvae and the Dun/Iso larvae. In the Iso jars, however, there are many larvae at earlier stages; some are still at the "jellybean" stage. I don't know if this is because these larvae are developing more slowly, or because of some nonrandom distribution of earlier stages into those jars when I was setting up the feeding treatments.

Next week I'll measure the larvae again, and will have three data points to track growth trajectories.

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When serendipity strikes, I try to go with the flow and ride it as long as I can. The latest wave is my batch of Dermasterias larvae, which are developing nicely for the first four days of life. And now they look just like jellybeans!

They have complete guts now and have already grown a bit, measuring 400-430 µm long. It's not always easy to catch these guys in the right orientation to take a photo, as they are spinning and swimming through three-dimensional space, but I got lucky:

4-day-old dipleurula larva of Dermasterias imbricata. 26 February 2016 © Allison J. Gong
4-day-old dipleurula larva of Dermasterias imbricata.
26 February 2016
© Allison J. Gong

I did try to follow an individual larva as it swam around on a microscope slide. I confined the larvae to a single drop of water under a cover slip so their movements are a bit constrained, but they manage to swim along fairly quickly. The resulting video might be a little nausea-inducing, so don't click on it if you're susceptible to motion sickness.

For now I've got the larvae divvied up into different feeding treatments. More on that later.

Actually, it was a fortunately placed phone call from an aquarium curator that struck the other night. I was at home, having eaten dinner and reviewed my lecture for the following morning, when my phone rang. It was the curator, saying that he was making his last rounds of the evening and had noticed that some of his sea stars were spawning. Echinoderm sex--more specifically, the opportunity to collect gametes and observe larval development--always grabs my attention, so I told him I'd throw on some shoes and meet him at the marine lab in five minutes.

Lo and behold, there were leather stars (Dermasterias imbricata) spawning in several of the tanks and seawater tables. Many of the tables were cloudy with sperm, but I found only one female, which seems strange but isn't so unusual. These spawning events occur in response to some environmental cue, such as day-length, a chemical of some sort, or the phase of the moon. When a sea star (or sea urchin) spawns it also releases chemicals that trigger spawning in nearby conspecifics, as to spawn by oneself is an enormous waste of energy. A single spawning animal can result in all the others of its kind spewing out huge numbers of gametes in an orgy of passive sex. However, an animal can be induced to spawn only if its gonads are ripe. Ripeness depends on the overall health of the animal and requires adequate food; animals that don't receive enough food don't have energy to allocate towards gamete production. As eggs are energetically expensive to produce, compared to sperm, it is not unusual for males of a species to mature earlier in the reproductive season than the females. In Washington the spawning season for D. imbricata is April-August. Here in California the reproductive season hasn't been clearly defined, but I do remember a springtime spontaneous spawning event in the lab several years ago.

Spawning female leather star (Dermasterias imbricata) at Long Marine Lab. 22 February 2016 © Allison J. Gong
Spawning female leather star (Dermasterias imbricata) at Long Marine Lab.
22 February 2016
© Allison J. Gong

That creamy looking mass of goo on the star's aboral surface is a pile of eggs. Sea star eggs are fairly large, compared to the urchin eggs I'm used to, and sticky. They tend to clump together in stringy globs until they are dispersed by water currents. The star whose arm is photobombing in the lower right corner is a male. He was also spawning copiously and is probably the individual who fertilized most of this female's eggs.

Given the lateness of the hour and the fact that I had to get up early the next morning I didn't take many pictures of the eggs, although I did look at them to make sure they were fertilized. They were, so I put them into a 1000-mL beaker of seawater and let them do their thing.


Fast forward to today, about a day and a half after fertilization. About two-thirds of the embryos had hatched and were swimming in the water column. Here's what they look like under the dissecting scope:

I poured off the swimmers into jars and set them up on the paddle table. I gave them a little bit of food, in case their mouths break through before I can get back to the lab tomorrow afternoon. In the meantime, I took a sample of embryos and examined them under the microscope. They look really cool!

Almost-two-day-old embryo of Dermasterias imbricata. 24 February 2016 © Allison J. Gong
Almost-two-day-old embryo of Dermasterias imbricata.
24 February 2016
© Allison J. Gong

The embryos are almost spherical, measuring 290 µm long and 270 µm wide. They are ciliated all over and swim with the rounded end forward. The flattened end is where the process of gastrulation started. That visible invagination begins at a section of the embryo called the blastopore; the channel is the archenteron, the first gut of the larva. In echinoderms, as in chordates (including us humans), the blastopore will end up being the larva's anus; the mouth breaks through later at the other end of the archenteron. This is why I don't need to start feeding the larvae right away even though their gut has begun forming.

Tomorrow afternoon I'll have a brief window of time when I can check on the larvae and see how they're doing. I think they may have complete guts by then!

1

. . . must come to an end, so they say. And Scott's and my little experiment growing Pisaster ochraceus came to its end when the last of our teensy stars gave up the ghost a week ago. We aren't entirely surprised, as nobody before us had succeeded in growing these guys in the post-larval stage, but it's still sad to see the empty paddle table and disappointing to know that we haven't really added to the body of knowledge about how to grow them.

But we did make a small bit of progress, at least to further our own understanding of exactly how difficult it is to do what we attempted. To summarize, here's a timeline of what we did and what happened:

  • 18 and 20 May 2015 -- Collected adult stars from local intertidal sites. Made up the solution of "magic juice" (100 µM 1-methyladenine).
  • 2 June 2015 -- Shot up stars with 1-MA. Got usable amounts of gametes from a total of three stars: 2 Purple (1 female + 1 male) and 1 Orange (female). After examining gametes to make sure they were okay, set up two matings: Purple x Purple; and Orange x Purple. The Purple x Purple embryos went through the earliest developmental stages just fine. The Orange x Purple embryos got off on the wrong foot and never recovered.
  • 5 June 2015 -- Purple x Purple embryos began undergoing gastrulation. Began feeding them. Orange x Purple embryos all dead.
  • 20 July 2015 (age 48 days) -- Larvae began settling.
  • 27 July 2015 (age 55 days) -- Counted a total of ~22 tiny stars in the jars. Removed a few to measure, and they were all 500 µm or smaller in diameter. It was very difficult keeping track of things this tiny in our 1-gallon jars.
  • 13 August 2015 (age 73 days) -- Paintbrushed out all of the little stars into a bowl and divvied them up into six food treatments. Replaced bowls in the paddle table to provide very gentle stirring.
  • 21 August - 7 September 2015 -- Stars died off in all but one of the food treatment bowls. By 7 September (age 96 days) the only surviving stars (N=4) were the ones we kept in a bowl with a small piece of mussel shell.
  • 11 September 2015 (age 100 days) -- And then there were three.
  • 28 September 2015 (age 117 days) -- Two survivors + 1 corpse.
  • 2 October 2015 (age 121 days) -- And then there were none.

In a nutshell, the larval development went fairly well, as we expected, and the post-larval survival sucked, also as we expected. We did manage to get those last two stars to survive 48 days post-metamorphosis, which is something. I'm not sure how much credit we can take for that, though, as I suspect that the reason the other juveniles died had to do with poor water quality as much as lack of food.

Here's what I think might have been going on: We are in our second consecutive year of elevated seawater temperature, and coupled with the massive El Nino that yesterday was proclaimed to be among the strongest ever this means that coastal animals are being subject to higher-than-normal temperatures. In ectothermic poikilotherms such as marine invertebrates, metabolic rate is directly related to environmental temperature. Thus, higher ambient seawater temperature should result, all else being equal, in a faster growth rate.

This sounds like it might be a good thing for the Pisaster larvae, especially if predation and other risks are higher in the planktonic larval stages than as benthic juveniles. However, I think there's more to the problem than simple growth rate. What if success as a juvenile depends not only on how quickly an animal progresses through all of its developmental stages, but also on how much time it spends in the different stages? For some larvae, notably the nauplius larva of barnacles, the primary job is to eat as much as possible and deposit energy reserves in the form of oil droplets; these food reserves will be utilized by the second larval stage, the non-feeding cyprid, as it hunts around for a place to establish a permanent home in the benthos. Perhaps part of the job of the developing Pisaster brachiolaria larvae is also to sequester energy reserves. Although no oil droplets were visible in any of the larvae that Scott and I observed this summer, energy could have been stored in other tissues of the larval body.

Back to the problem of post-larval survival. Our larvae began metamorphosing after only 48 days in the plankton. One of our sources has Pisaster ochraceus undergoing metamorphosis at 76-228 days in culture, at temperatures of about 12°C (for the duration of our experiment this summer ambient seawater temps were 15-18.5°C). So, if the warmer temperatures caused the larvae to develop more quickly than normal, and the larvae spent ~25 fewer days in the plankton than they "should" have, they may simply not have had time to accumulate whatever energy reserves they'd need to draw on once they metamorphosed.

That's just a guess on my part. I also imagine that poor water quality played a part in our juvenile stars' demise. It proved to be impossible to make potential food available to such tiny animals while keeping their water clean. We thought that stirring on the paddle table might help, and who knows, maybe it did.

In any case, RIP, little guys. Thanks for what you taught us, and I'm sorry we weren't able to help you succeed.

Juvenile Pisaster ochraceus, age 75 days. 16 August 2015 © Allison J. Gong
Juvenile Pisaster ochraceus, age 75 days.
16 August 2015
© Allison J. Gong

 

1

I did a quick search, and there doesn't seem to be a collective noun for sea stars. I'm going to remedy that by declaring "constellation" to be the official term for a group of sea stars. And by "official" I mean that's the term I'm going to use. Who knows, maybe it'll take.

In any case, I certainly have a constellation of sea star larvae in each of my jars. Today I pipetted a lot of them into a bowl, and they look pretty cool all swimming together, like strange alien spaceships. What do you think?

The largest of the larvae are over 2 mm long now, and the brachiolar arms have grown much longer. They have three adhesive papillae on the ventral side of the anterior projection and well-formed juvenile rudiments, where the water vascular system is forming. They're much too big to fit under the compound scope, so the only way to get pictures of the entire body is through the dissecting scope:

Brachiolaria larva of Pisaster ochraceus, age 31 days. 3 July 2015. © Allison J. Gong
Brachiolaria larva of Pisaster ochraceus, age 31 days. 3 July 2015.
© Allison J. Gong

In the above photo you are looking at the larva's ventral surface, so the animal's left side on the right side of the photo, and vice versa. If you squint you might be able to convince yourself that you see a small whitish bleb on the left side of the stomach; that's the rudiment. Since it doesn't make much sense under this magnification, I removed this individual to a slide and put it under the compound scope. It doesn't fit in the field of view, so I took pictures of each half of the body. If I were clever with photo editing software I'd be able to mesh these photos into a single image. Alas....

Ventral view of the anterior end of a brachiolaria larva of Pisaster ochraceus, age 31 days. 3 July 2015. © Allison J. Gong
Ventral view of the anterior end of a brachiolaria larva of Pisaster ochraceus, age 31 days. 3 July 2015.
© Allison J. Gong
Ventral view of the posterior end of a brachiolaria larva of Pisaster ochraceus, age 31 days. 3 July 2015. © Allison J. Gong
Ventral view of the posterior end of a brachiolaria larva of Pisaster ochraceus, age 31 days. 3 July 2015.
© Allison J. Gong

This gives you a better view of the juvenile rudiment on the animal's left. Those three roundish blobs are tube feet! I think it's likely that at some point in the not-too-distant future the larvae will be competent, which means they'd be physiologically and anatomically capable of metamorphosis. It seems to me that they are still developing very quickly, and with seawater temperatures consistent at 15-16°C I don't expect that to change. So far, so good!

Edit 4 July 2015:  Look at what my online friend Becca can do! She was able to merge my photos into a single image. Now you can see the entire body! Thanks, Becca!

Composite image of brachiolaria larva of Pisaster ochraceus, age 31 days. 3 July 2015.
Composite image of brachiolaria larva of Pisaster ochraceus, age 31 days. 3 July 2015.

 

1

Today my Pisaster ochraceus larvae are 10 days old. Although they seemed to be developing slowly, compared to the urchins that I'm more used to, in the past several days they have changed quite a bit. They've also been growing quickly, which makes me think that they're off to a strong start. Of course, there's still a lot of time for things to go wrong, as they have another couple of months in the plankton. However, at this point in time I feel optimistic about their chances.

In the dish under the dissecting scope they swim around like bizarre space ships. All the bits of detritus in the water add to the effect. The only thing missing is the sound effects.

The magnification of my dissecting scope goes up to 40X. To see any details of anatomy I have to use the compound microscope, through which I can see this, under 100X magnification:

10-day-old bipinnaria larva of Pisaster ochraceus, 12 June 2015. © Allison J. Gong
Ventral view of 10-day-old bipinnaria larva of Pisaster ochraceus, 12 June 2015.
© Allison J. Gong

Aside from the dramatic increase in overall size (almost 1 mm long now!), the larva's body has gotten a lot more complicated. For one thing, the animal's marginal ciliated band, which propels the larva through the water, has started becoming more elongate and elaborate. In this view the larva is lying on its back, and I have focused on the plane of its ventral surface. The left and right coeloms are in the plane of the dorsal surface, and thus are not really in focus. You should still be able to see how long they have gotten, though. Eventually they will fuse anteriorly to form a single cavity. The stomach of the larva has a nice green-golden color due to the food it has been eating. It makes perfect sense, as we are feeding them a cocktail of green algae and a diatom-like golden alga.

The larvae are very flexible and can be quite animated when they're swimming around. They bend, scrunch up, and swallow food cells. They have already gotten so big that they take up much of the field of view under the microscope, even at the lowest magnification. Watch some larval gymnastics:


Part of the reason that I wanted to spawn Pisaster and raise the larvae this summer is that I want to put together a series of pen-and-ink drawings of the developmental stages. I did the same for the bat star Patiria miniata several years ago, but the Pisaster larvae will have longer and more elaborate arms when they mature; capturing these in drawings will be a challenge for me. I also hope to include the juveniles in this set of drawings. With that goal in mind, I've been sketching the larvae every few days, just to get some practice under my hand and remind myself what it feels like to draw. I've missed it!

10-day-old bipinnaria larva of Pisaster ochraceus, drawn from life. 12 June 2015. © Allison J. Gong
10-day-old bipinnaria larva of Pisaster ochraceus, drawn from life. 12 June 2015.
© Allison J. Gong

For whatever reason, I really like how this sketch turned out. It's not pretty, but it does truly represent what I saw under the microscope. I'm going to have to work on depicting three-dimensional structures on a two-dimensional page, which will take some practice. Fortunately I have several weeks to brush up on my skills!

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