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It has been a few weeks since I posted about my most recent batches of urchin larvae. Some strange things have been happening, and I'm not yet sure what to make of them. It would be great if animals cooperated and did what I expect; somehow that never seems to be the case. The upshot of all this uncertainty is that there is always something new to learn. I, for one, am not going to complain about that.

One noteworthy thing to report is that my hybrids all died, very quickly and unexpectedly. They had been racing through development and on the dreaded Day 24 they looked great.

Hybrid larvae of purple urchin (Strongylocentrotus purpuratus) eggs fertilized by red urchin (Mesocentrotus franciscanus) sperm, age 24 days.
23 January 2017
© Allison J. Gong

And the next time I changed their water, they were all dead. So much for the hybrid vigor I had written about earlier. Teach me to get cocky and think I know what's going on.


Fast forward to Day 52, and some of the cultures are still going strong. I originally set up four matings, and at least some individuals from each are alive. One thing that seems to happen when I start multiple batches of larvae at the same time is that the batch with the fewest numbers does the best. This time my F3xM1 mating was always the least dense culture, but some of them have already begun and completed metamorphosis. And the ones that are metamorphosing are the ones being fed what I expected to be the less desirable food source. As I said, not much of this whole experience is making sense.

The good thing is that I have an opportunity to observe these larveniles in action. As long as they don't get arrested in this neither-here-nor-there stage, they should soon join their siblings as permanent inhabitants of the benthos.

This video contains short clips of three different larveniles. I've arranged the clips from earlier to later stages of metamorphosis. Although these are three separate individuals, you can imagine that each one goes all of these stages.

Having both tube feet (for crawling around the benthos) and ciliated bands (for swimming in the plankton) make these animals unsuited for either habitat. They have gotten very heavy and sink to the bottom, but it doesn't take much water movement to knock them off their five little tube feet. It always amazes me that teensy critters like this, so fragile and easily killed, manage somehow to stick in the intertidal and survive long enough to be grown-up urchins on their own. And yet some of them will. I've seen it happen.

2

Although at this stage it's a close race. Two and a half weeks ago I spawned sea urchins in the lab, setting up several purple urchin crosses with the hope of re-doing the feeding experiment that I lost this past summer when I was on the DL (that's Disabled List, for those of you who don't speak baseball). I was also fortunate enough to set up a hybrid cross, fertilizing purple urchin (Strongylocentrotus purpuratus, or "Purp") eggs with red urchin (Mesocentrotus franciscanus, or "Red") sperm. I would have done the reciprocal hybrid cross (red eggs by purp sperm) as well if I'd gotten any of female red urchins to spawn. However it wasn't really spawning season for the reds, and I consider myself lucky to have persuaded that one male to release some sperm for me.

This is the first time that I've tried to raise the hybrid larvae, although I know it can be done because my colleagues Betsy and John did it many years ago, before I came to the marine lab. All of my larvae are the exact same age and are being raised side-by-side, so I can make direct comparisons between the Purp by Purp crosses and the Purp by Red hybrids. Incidentally, when speaking or writing about a hybrid cross the convention I've adopted is to reference the female parent first, so when I say Purp by Red I mean a Purple eggs fertilized by Red sperm. A Red by Purp hybrid would logically result from red urchin eggs fertilized by purple urchin sperm.

My experience raising sea urchin larvae is that things almost always go well for the first 48 hours or so; most (but not all) of the fertilized eggs develop into embryos and undergo the crucial processes of gastrulation and hatching. In some cultures the hatching rate is close to 100%. After that there's a window of 3-4 days when cultures can crash for no apparent reason, although food availability or quality may be a factor. If the larvae make it past their first week of post-hatching life they generally cruise along until the next danger period which occurs at about 24 days. I change the water in the culture jars and observe the larvae under the microscope twice a week.

Today the larvae are 18 days old. It's a little early for that second mortality period, but some of the Purp by Purp cultures never really took off. The larvae don't seem to be growing or developing as quickly as I'm used to. Perhaps this has to do with lower water temperatures, especially after the prolonged period of high temps in 2014-2015. In any case, two of the four Purp by Purp crosses are doing well and the other two are just hanging in there.

There are two things I can see with the naked eye that give me a heads-up when cultures are crashing: the first sign is an accumulation of debris at the bottom of the jar and the second is an absence of larvae in the water column. The debris can be due to excess food, a build-up of fecal matter (not usually the case, as I'm pretty good at doing the water changes on time), the disintegration of larval bodies, or some combination thereof. If the water column is clear then the culture has already crashed and everybody is dead.

Today one of my jars had crashed. The water column was very clear and there was a lot of fluff at the bottom of the jar. I'd been wondering if I could figure out what the fluff was made of, so I sucked up a bit in a pipet and examined it under the microscope. I thought I'd see dead algal cells or pieces that look like defecated algal cells. This is what I saw:

18 January 2017
© Allison J. Gong

Silly me. I had forgotten that the corpses of pluteus larvae would disintegrate pretty quickly, leaving behind only the skeletal rods. The rods get all tangled together and trap the organic stuff, which is probably a mixture of uneaten and defecated algal cells and the soft tissues of the larval bodies. This explains the clear water column in the jar.

While the Purp by Purp larvae have had mixed success so far, the Purp by Red hybrids have been doing well. From the outset they appeared to be more robust than the Purps, and even though the fertilization rate was only about 50% the post-hatching mortality seems low. The hybrid larvae are also larger than the Purps, and are developing more quickly. In the two photos below the scale bar indicates 100 µm.

Pluteus larva of Strongylocentrotus purpuratus, age 18 days.
17 January 2017
© Allison J. Gong
Pluteus larva of a hybrid cross between S. purpuratus and Mesocentrotus franciscanus, age 18 days.
17 January 2017
© Allison J. Gong

The hybrid larva is about 10% larger than the Purp larva. Other than that they look similar, but to me the hybrid larva seems farther along the developmental process: its arms are proportionally longer and have a more mature look (although I don't have any way to describe that to a naive observer). There's something about the gestalt of the animal that makes me think it's more robust than the Purp individual.

We'll see how the pure Purps and the hybrids do from here on. I actually have the Purp larvae divided up into different feeding treatments, which I may discuss in a future blog post. In the meantime I'm trying to baby the hybrid larvae as much as possible, to maximize their probability of successful metamorphosis in six weeks or so.

1

Sea urchins have long been among my favorite animals. From a purely aesthetic perspective I love them for their spiky exterior that hides a soft squishy interior. I also admire their uncanny and exasperating knack for getting into trouble despite the absence of a brain or centralized nervous system. Have you ever been outsmarted by an animal without a brain? I have. It's rather humbling.

Red sea urchins (Mesocentrotus franciscanus) and purple sea urchins (Strongylocentrotus purpuratus) share a common geographic range along the northeastern Pacific but generally live in different habitats. S. purpuratus is the common urchin in tidepools, while reds are almost always subtidal (although I have seen them in the intertidal on very low minus tides). The two species' habitats do overlap a bit, as the purple urchin can live in subtidal kelp forests alongside the reds. There is a commercial fishery for the gonads of red urchins, which are prized as uni by sushi aficionados. I've tried uni once, and it tasted exactly the way I imagined the gonads of a sea urchin would taste. Not a fan. I'd much rather make a different use of urchin gonads.

The other week I collected some urchins from the field, hoping that they'd have nice full gonads. Gametogenesis in many marine invertebrates, including sea urchins, is governed at least partly by annual light cycles. Provided they have sufficient food, purple urchins have ripe gonads and spawn in the winter, from December through March. Reds spawn in the spring, from March through June. In my experience the best time to induce spawning of purps in the lab is December or January, when the urchins have developed gonads but likely haven't spawned yet. There is no way of knowing the sex of any given urchin or the condition of its gonads, so this exercise is somewhat of a crap shoot even with the best of planning.

Ready to induce spawning!
30 December 2016
© Allison J. Gong

Today I shot up my eight field-collected purps, hoping to get at least one male and one female out of the deal. I got lucky with the timing, as one of the smallest urchins was a female and began spewing out eggs. This little female gave a lot of eggs! She was followed by three males and two more females. So out of my eight purps I ended up with three of each sex, and a spawning rate of 75% ain't bad.

I set up some mating crosses and fertilized all of the eggs. I divided the little female's eggs into two batches and fertilized them with the sperm of two different males (M1 and M2). Each of the other females' eggs was fertilized by M1, who gave huge amounts of sperm. When I checked on the eggs about two hours post-fertilization most of them had gone through the first cleavage division and seemed to be developing normally and on schedule.

2-cell embryos of Strongylocentrotus purpuratus
30 December 2016
© Allison J. Gong

Just for the hell of it I decided to shoot up some of the red urchins we have in the lab. I didn't really think they'd spawn, as it's not the season for them to be gravid. Red urchins are large, heavy animals with long and sharp spines and they are much more difficult to handle. Four of the five that I shot up did nothing, as expected. It took a long time, but just as I was about to give up on them the biggest red began dribbling out a couple thin streams of sperm. I examined the sperm under the microscope and they were very active and healthy. Fortunately I hadn't returned the purps to their tanks, and two of the female were still putting out some eggs. I rinsed the purp eggs into a clean beaker, pipetted up some of the red sperm, and added it to the eggs.

Sea urchin eggs are covered by a thick jelly coat. In the video you can see many of the red urchin sperm embedded in the jelly coat of the egg. Despite the frantic activity of the sperm, fertilization (as evidenced by the rising of the fertilization envelope off the surface of the egg) took much longer than it does when eggs and sperm come from the same species.

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

Look at that beautiful zygote! Fertilization success in this hybrid cross was low, only about 50%. The eggs that did get fertilized went through the first cleavage division after about two hours later, which is right on time.

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

It remains to be seen whether or not the few hybrid embryos I have continue to develop. I have a colleague who has hybridized red and purple urchins successfully in the past, and has raised the offspring to adulthood. I don't have any expectations of great success with this little experiment, but it would be very informative to raise known hybrid urchins. I've seen animals in the field that look like hybrids and there's no reason to assume that hybridization between these two free-spawning species never occurs. The adults can be found living side-by-side subtidally, and there's enough overlap in their reproductive seasons that some individuals of each species could very well spawn at the same time. On the other hand, hybridization that can be forced in the lab doesn't necessarily occur in the field. I dumped a lot of red urchin sperm on those purple urchin eggs, and such high sperm concentration may overcome any mechanisms of reproductive isolation that exist under real-life conditions.

I'll know more when I check on things tomorrow.

My most recent batch of sea urchin larvae continues to do well, having gotten through the dreaded Day 24. I haven't written about them lately because they're not doing very differently from the group that I followed last winter/spring. However, I've been taking photos of the larvae twice a week and it seems a shame to let them go to waste, so I've put together a progression of larval development. As a reminder, the last time I wrote about these larvae they were six days old.


Age 9 days: The larvae had four arms and were growing their skeletal arm rods. Their stomachs, which we keep an eye on because their size can tell us whether or not we're feeding them enough, were a bit small but not so much so that I worried.

9-day-old pluteus larva of the purple sea urchin, Strongylocentrotus purpuratus. 13 November 2015 © Allison J. Gong
9-day-old pluteus larva of the purple sea urchin, Strongylocentrotus purpuratus.
13 November 2015
© Allison J. Gong

Age 12 days: The larvae were growing their third pair of arms. Some had just begun growing the fourth pair of arms. Red pigment spots also start appearing all over the body. Some larvae develop lots of red spots, others have very few. Notice that the stomach is slightly pear-shaped; this is normal.

12-day-old pluteus larva of the purple sea urchin, Strongylocentrotus purpuratus. 16 November 2015 © Allison J. Gong
Ventral view of a 12-day-old pluteus larva of the purple sea urchin, Strongylocentrotus purpuratus.
16 November 2015
© Allison J. Gong

Age 17 days:  This larva doesn't look appreciably different from the previous one. This photograph, though, is a bit clearer. The stomach has taken on a pink tinge, due to the red color of the food the animal is eating, and the mouth is the large rounded triangular in the in-focus plane. The pair of skeletal arm rods that are in focus are protruding from the ends of the arms, which raises is something to be concerned about. Sometimes the first sign of imminent doom is the shriveling of the arms, so seeing the rods sticking out makes me think "Uh-oh. . ."

Dorsal view of a pluteus larvae of the purple sea urchin, Strongylocentrotus purpuratus. 21 November 2015 © Allison J. Gong
Dorsal view of a pluteus larvae of the purple sea urchin, Strongylocentrotus purpuratus.
21 November 2015
© Allison J. Gong

Age 24 days: This is about the time in larval development when things often start to go wonky. I've looked back at my notes from previous spawnings of S. purpuratus, and seven of the 20 cultures that crashed did so in the week between days 20-28 of development. Some of these cultures were doing well right up to the point that they all died. They were literally there one day and gone the next.

Nonetheless, the current batch of larvae continued to do well. The fourth pair of arms were slow to grow but otherwise the larvae look fine. The top larva in the picture below is lying on its back, so you are looking onto the ventral surface. On the left side of the stomach there's a little upward-facing invagination; this is part of the initial water vascular system forming. Note also that the overall shape of the larvae is changing a bit. They are becoming less pointy and a bit rounder.

Pair of 24-day-old pluteus larvae of the purple sea urchin, Strongylocentrotus purpuratus. 28 November 2015 © Allison J. Gong
Pair of 24-day-old pluteus larvae of the purple sea urchin, Strongylocentrotus purpuratus.
28 November 2015
© Allison J. Gong

Age 30 days:  At this stage the juvenile rudiment is clearly visible. You can see it as a rather nondescript blob of stuff to the left of the gut. The fourth pair of arms have also grown quite a bit but are still considerably shorter than the others. This individual has two bands of cilia, called epaulettes, that encircle the body. These epaulettes will become more conspicuous as the larva approaches competency.

Ventral view of a 30-day-old pluteus larva of the purple sea urchin, Strongylocentrotus purpuratus. 4 December 2015 © Allison J. Gong
Ventral view of a 30-day-old pluteus larva of the purple sea urchin, Strongylocentrotus purpuratus.
4 December 2015
© Allison J. Gong

Age 33 days: Today I got lucky! The larvae looked good when I changed their water this morning <knock on wood> and although I'm keeping my fingers crossed I have high hopes for these guys. They're about as big as they're going to get, measuring 760-800 µm in length. They will get heavier and more opaque as the juvenile rudiment continues to develop.

33-day-old pluteus larvae of the purple sea urchin, Strongylocentrotus purpuratus. 7 December 2015 © Allison J. Gong
33-day-old pluteus larvae of the purple sea urchin, Strongylocentrotus purpuratus.
7 December 2015
© Allison J. Gong

The really cool thing is that one of the larvae landed on the slide exactly as I wanted it to. It happened to fall onto its left side and stayed there, so I was able to focus up and down through the body to get the rudiment into focus.

Left-side view of a 33-day-old pluteus larva of the purple sea urchin, Strongylocentrotus purpuratus. 7 December 2015 © Allison J. Gong
Right-side view of a 33-day-old pluteus larva of the purple sea urchin, Strongylocentrotus purpuratus.
7 December 2015
© Allison J. Gong

Do you see five small roundish blobs that are evenly spaced around the larger golden circular blob? The large blob is the stomach, seen in side view. Those smaller blobs are tube feet! Don't believe me? Then take a look at this close-up:

Juvenile rudiment of 33-day-old pluteus larva of the purple sea urchin, Strongylocentrotus purpuratus. 7 December 2015 © Allison J. Gong
Juvenile rudiment of 33-day-old pluteus larva of the purple sea urchin, Strongylocentrotus purpuratus.
7 December 2015
© Allison J. Gong

Now if those don't look like tube feet, then I'll eat my hat. What's also noteworthy about this larva is that its epaulette bands are both visible, especially the posterior-most one.

So far, so good. I won't know how successful larval development is for these guys until they either make it through metamorphosis, or not. In a very real sense, I won't be able to draw any conclusions about the success of larval development until they either become established as juvenile urchins, or not. One of my graduate advisors inherited a couple of sayings that he passed on to me, as well as to a whole generation of aspiring invertebrate zoologists:

The animal is always right.

and

The life cycle is the organism.

The first is a given, right? The animal knows what it is and what it's doing, even if we humans have no clue about what's going on and can't decide what its name should be.

The second saying might be a little less intuitive. What it means is that, for organisms with a multi-stage life cycle, you have to consider all of the stages if you want to understand them. This is a much more holistic view of biology, and it's the one that appeals most strongly to me. When I'm thinking as a naturalist, I find my thought process constantly switching between "forest" and "trees" as I seek to understand even a teensy bit of the world around me. While it's easy to get distracted by all the cool details of organisms, it's important to step back and ask myself, "What does it all mean? What is the big picture here?" So yeah. Perhaps when (if!) these larvae turn into urchins and I've got them feeding on macroalgae in a few months, I'll be able to say whether or not larval development was successful. If all goes well this larval phase, as all-consuming and fascinating as it is to me, will be only a small part of these animals' lives.

Today my most recent batches of urchin larvae are six days old. Yesterday being Monday, I changed their water and looked at them under the scopes. I was pleased to be able to split each batch into two jars, as the larvae have already grown quite a bit; I now have a total of four jars to take care of. This makes me inordinately happy. Having only two jars is risky, as it wouldn't take much for both of them to crash, but for some reason I feel more confident of success with four jars. It's probably one of those all-your-eggs-in-one-basket things.

In any case, this is what they look like now:

Pluteus larvae of the sea urchin Strongylocentrotus purpuratus, age 5 days. 9 November 2015 © Allison J. Gong
Pluteus larvae of the sea urchin Strongylocentrotus purpuratus, age 5 days.
9 November 2015
© Allison J. Gong

These larvae are perfectly formed. At this point they are shaped essentially like squared-off goblets, with four arms sticking up at the corners of the goblet. They will continue to grow arms in pairs until they have a total of eight (four pairs). The stomachs (the round-ish pale red structures in the middle of the body) are big and round; the color of the stomachs is due to the food that the larvae are eating. And can you see the skeletal rods extending into each of the arms? Each of the eventual larval arms will be supported by one of these rods, and additional rods will serve as cross-braces going horizontally across the body.

Ever wondered what these animals eat? In the wild they would be feeding on whatever phytoplankton they can catch. In the lab we have several types of phytoplankton growing in pure culture, but trial and error has taught us that urchin larvae do best on a diet of the cryptophyte Rhodomonas sp.

The cryptophyte Rhodomonas sp., growing in pure culture. 9 November 2015 © Allison J. Gong
The cryptophyte Rhodomonas sp., growing in pure culture.
9 November 2015
© Allison J. Gong

The red color of the cultures is due to the color of the cells. When the larvae eat this food their stomachs turn pinkish. Rhodomonas cells are about 25 µm long and have two flagella that they use to zip around. Here's a short video of a drop of Rhodomonas culture on a slide:

They sort of look like sperms, but the cells are much larger than sperms, the flagella are much shorter than the single flagellum of a sperm, and their swimming isn't quite right to be sperms, either.

The larvae themselves live in glass jars in one of the seawater tables that I converted into a paddle table. The larvae are negatively buoyant and would sink to the bottoms of the jars if left unstirred, and the gentle back-and-forth motion of the paddles keeps them, and their food, suspended in the water column.

See my four jars? They are a sign of short-term success. There's still a lot of time for things to go south with these larvae, and I certainly don't take for granted that I'll be able to keep them alive for the duration. But today, as my students were dissecting urchins in lab, I was able to show them the offspring of said urchins. I hope to keep the larvae alive through the end of the semester, to show the students as much as I can of larval development in one of my favorite animals.

Having obtained decent-ish amounts of gametes from sea urchins, the next step is to get eggs and sperm together. The first thing I did was examine the spawned eggs to make sure they were round and all the same size. Lumpy eggs or a variety of sizes of eggs indicates that they are probably not fertilizable. These eggs from F1 looked just about perfect:

Freshly spawned eggs of Strongylocentrotus purpuratus. 4 November 2015 © Allison J. Gong
Freshly spawned eggs of Strongylocentrotus purpuratus.
4 November 2015
© Allison J. Gong

Note that the eggs are all similarly sized (80 µm in diameter) and round. These look good to go.

The next step is to dilute the sperm in filtered seawater and introduce a small amount to the eggs. The sperm need to be diluted because, believe it or not, in this case too much of a good thing is bad. There's a phenomenon called "polyspermy" which is pretty much exactly what it sounds like: an egg being penetrated by more than one sperm. Polyspermy leads to wonky development down the road, and while it probably rarely happens in the field, where sperm would be diluted immediately upon being spawned, it definitely does occur in the lab. However, eggs are smart and have evolved a couple of mechanisms to prevent polyspermy.

The fast block to polyspermy occurs within a few seconds of the fusion of the sperm and egg plasma membranes. As the sperm nucleus begins to enter the cytoplasm of the egg, Na+ ion channels in the egg membrane open and cause a depolarization of the egg membrane; this depolarization makes the egg impenetrable to other sperm. However, the egg membrane cannot remain depolarized indefinitely, so after about a minute the slow block to polyspermy takes effect.

The slow block is the rising of the egg's vitelline layer above the surface of the egg, creating what we call the fertilization membrane. This envelope acts as a physical barrier against additional sperm. The really cool thing about studying fertilization in sea urchins is that you can watch it happen in real time. I mean, how often do you get to observe the formation of a brand new life at the moment that is is being formed?

In this video there are 2.5 eggs in the field of view. Concentrate on the two whole eggs. The one on the top has already been fertilized, which you know because you can see the fertilization membrane surrounding it. You can also see a lot of sperm zooming around. Keep an eye on the lower of the whole eggs; can you see the rising of its fertilization membrane?

Of the two female urchins that spawned for me this morning, F2 had only a few eggs to give but her fertilization rate was 100%. F1, on the other hand, spawned a lot of eggs but only about 50% of them were fertilized. I have no explanation for this. Sometimes (quite a lot of times, actually) things simply don't work.

That said, at our local ambient temperature the first cleavage division occurs about two hours post-fertilization. That's when I saw this:

Two-cell embryo of Strongylocentrotus purpuratus, approx. two hours post-fertilization. 4 November 2015 © Allison J. Gong
Two-cell embryo of the sea urchin Strongylocentrotus purpuratus, approx. two hours post-fertilization.
4 November 2015
© Allison J. Gong

A few hours later the embryos had progressed to what I think is the 16-cell stage. At this point it starts getting difficult to distinguish the different cells without focusing up and down through the embryo. But if you know what you're looking at, the three-dimensional structure does make some sense. In the embryo below I can talk myself into seeing two rings of eight cells each, one ring lying on top of the other.

16-cell embryo of the sea urchin Strongylocentrotus purpuratus. 4 November 2015 © Allison J. Gong
16-cell embryo of the sea urchin Strongylocentrotus purpuratus, approx. five hours post-fertilization.
4 November 2015
© Allison J. Gong

If the embryo is at the 16-cell stage, then it has undergone four cleavage divisions. The early divisions of an embryo are called "cleavages" because the cells divide in half to form equal-sized daughter cells. In other words, the cell cleaves. During cleavage the embryo doesn't grow, which means that the average cell size necessarily decreases. Cleavage divisions will continue for a total of about 24 hours, resulting in a stage called a blastula.

UP NEXT (hopefully): hatching and swimming

We are finally heading into the time of the year that our local intertidal sea urchin, Strongylocentrotus purpuratus, spawns. Usually I would wait until December or January to try to spawn urchins in the lab, but next week my students will be dissecting urchins in lab and I thought I might as well evaluate gonad development in the animals that are going to be sacrificed anyway. In early December I'm going to loan several urchins to a colleague who will be spawning them to show the earliest stages of development to students in one of the lower-division classes at the end of the semester. If I have any luck today, I'll be able to: (1) start my own cultures of urchin larvae so that I can show the later larval stages to students in my upper-division class; and (2) let my colleague know how likely it is that the urchins I loan to her will be spawnable.

4 November 2015 © Allison J. Gong
4 November 2015
© Allison J. Gong

I know, it ain't as romantic as the Ritz-Carlton but this is where I hope to make the sea urchins have sex. We have our victims lucky individuals in their "live only" tub, two beakers for eggs, two sperm dishes on ice, a box of glass pipets, a bottle of magic juice, and a syringe with needle to get the magic juice into the animals. Ready to go!

What is the magic juice, you ask? It's a solution of KCl in filtered seawater. I'm not sure exactly how it works, but here's what I think happens. We use a solution of MgCl2, a similar salt, to narcotize animals before dissecting them. Sea urchins sitting in a bath of  MgCl2 isotonic with seawater get sleepy pretty quickly, becoming entirely nonresponsive after about 30 minutes. I suspect that KCl has a similar effect. We inject KCl into the main body cavity of the urchin (I call this "shooting them up") and I think it relaxes the muscles surrounding the gonopores. If the gonads are ripe, then gametes are released as the gonopores open. If gonads are immature, then nothing happens.

A sea urchin is a well-armored beast. Its endoskeleton, or test, is a solid structure composed of calcareous ossicles that are perforated only where tube feet extend. Getting a needle through the test without damaging the animal is pretty much impossible, so we go through the peristomial membrane instead. This membrane surrounds the mouth on the oral (bottom) side of the urchin. It's the only way to get into an urchin without breaking the test.

The urchins don't seem to like being injected with KCl--they wave their tube feet and spines all around and generally appear somewhat agitated--but they don't suffer any lasting effects.

If the urchins are ripe, they should start spawning shortly after being injected with KCl. Sometimes the response is immediate, with urchins pouring out gametes through all five gonopores at an astounding rate. Today it was much slower. It took about 5 minutes for the first female to spawn:

Spawning female sea urchin (Strongylocentrotus purpuratus). 4 November 2015 © Allison J. Gong
Spawning female sea urchin (Strongylocentrotus purpuratus).
4 November 2015
© Allison J. Gong

That little blotch of pale orange is is the mass of eggs that she is spawning. At this point you can pipet off the eggs into a beaker of filtered seawater, but I decided to go the less-invasive route and simply invert the spawning animal onto a beaker filled with water and let the eggs drop to the bottom as they flowed out of her.

The only difficulty with this method is that the animal doesn't like being upside down and immediately tries to right herself. I kept having to remove her from the beaker and replace her in the orientation we wanted. I designated this urchin as F1. She gave us a decent number of eggs. A second, smaller female (F2) spawned just a few eggs but we kept them all.

Sperm get a different treatment. I had only one male spawn this morning and he wasn't exactly a gusher. I pipetted off the concentrated sperm into a cold dish on ice, and didn't dilute the sperm until the eggs were ready for fertilization.

UP NEXT: Fertilization and subsequent events.

The juvenile sea urchins I've been raising this year are now nine months old. Back in June I put them on three different macroalgal diets and have been measuring their test diameters monthly. I do the measuring in the first week of every month, and today was the day for November. Over the past few weeks I lost a lot of my Ulva urchins, for no reason that I could discern. Judging from the poop production they were definitely eating, but on some days there would be a handful of corpses in the bowl when I changed the water. They all seemed healthy and happy today, including this beautiful creature:

Juvenile sea urchin (Strongylocentrotus purpuratus) that has been eating Ulva, age 9 months. 2 November 2015 © Allison J. Gong
Juvenile sea urchin (Strongylocentrotus purpuratus) that has been eating the green alga Ulva sp., age 9 months.
2 November 2015
© Allison J. Gong

Seriously, this has to be the most gorgeous photo of a sea urchin I've ever taken. This individual is the largest of my Ulva urchins, with a test diameter of 12.7 mm. I love the coloration of this animal: the younger spines are green, the older spines are pale purple, and the tube feet are beautifully transparent and tipped with purple suckers.

By contrast, the urchins eating Macrocystis continue to be a more uniformly golden color:

Juvenile sea urchin (Strongylocentrotus purpuratus) that has been eating the kelp Macrocystis pyrifera, age 9 months. 2 November 2015 © Allison J. Gong
Juvenile sea urchin (Strongylocentrotus purpuratus) that has been eating the kelp Macrocystis pyrifera, age 9 months.
2 November 2015
© Allison J. Gong

This Macrocystis urchin is actually a tad bigger than the Ulva urchin and has a test diameter of 13.0 mm. It looks smaller because its tube feet are fully extended, so I had to zoom out a bit to get the entire body in the frame. It was also crawling around very fast and I had to hold it down to get it centered, then remove the forceps and take the picture quickly before it walked out of the picture. Every photo of this individual that I managed to get is a little blurry because of the movement.

Last but not least, the urchins eating coralline algae are hanging in there. None of them died in the past month and they are growing. Their color patterns are qualitatively different from the those of urchins eating Ulva or Macrocystis. To my eye there is more contrast in the coralline urchins; they all seem to have prominent dark coloration in the lines that radiate outward from the apical region. The other urchins have it too, but in the coralline urchins this dark pigmentation is concentrated into more clearly defined streaks and contrasts more strongly with the paler background color.

Juvenile sea urchin (Strongylocentrotus purpuratus) that has been eating coralline algae, age 9 months. 2 November 2015 © Allison J. Gong
Juvenile sea urchin (Strongylocentrotus purpuratus) that has been eating coralline algae, age 9 months.
2 November 2015
© Allison J. Gong

This animal, with a test diameter of 6.08 mm, is about half the diameter of the largest of its full siblings in each of the other food treatments. Food quality definitely has an effect on size, as these data indicate:

Test diameters of juvenile sea urchins (Strongylocentrotus purpuratus) on three food treatments. 2 November 2015 © Allison J. Gong
Test diameters of juvenile sea urchins (Strongylocentrotus purpuratus) on three food treatments.
2 November 2015
© Allison J. Gong

It remains to be seen whether or not I'll be able to provide Ulva and Macrocystis to these animals throughout the winter. If we get the strong El Niño storms that are predicted, the nearshore algae could be wiped out for a while. I'll make sure that if I run out of one food then urchins in the other treatment will also fast until I can feed both of them again. In the meantime, because the coralline urchins are so far behind in their growth, I'll continue to give them access to food. I don't want any of them to die of starvation, and the coralline eaters are the most vulnerable, I think.

2

Two months ago now I gave my juvenile sea urchins a job. It's the kind of job they're perfectly suited for:  eating algae. I measured them all and randomly divvied them up into three food treatments. One group remains on the pink coralline alga they'd all been eating once they graduated from a diet of scum, one group gets to eat the soft green alga Ulva sp., and the third group is eating the kelp Macrocystis pyrifera. I fully expected that the urchins on coralline algae would grow much more slowly and experience higher mortality than the other groups. And now I have data to validate my intuition!

Test diameter of juvenile sea urchins (Strongylocentrotus purpuratus) as a function of diet. 3 August 2015. © Allison J. Gong
Test diameter of juvenile sea urchins (Strongylocentrotus purpuratus) as a function of diet. 3 August 2015.
© Allison J. Gong

It has been clear from the get-go that the Ulva and Macrocystis urchins are growing faster than the poor guys relegated to coralline algae. The coralline urchins are hanging in there, though, and are even growing a bit. They are also dying, a lot.

Population sizes of juvenile sea urchins (Strongylocentrotus purpuratus) as a function of diet. 3 August 2015. © Allison J. Gong
Population sizes of juvenile sea urchins (Strongylocentrotus purpuratus) as a function of diet. 3 August 2015.
© Allison J. Gong

During the first month of the experiment I was surprised to see the high attrition rate of urchins eating Macrocystis. I think these early deaths were due to the fact that Macrocystis, once it starts to go bad, goes bad fast. Even with daily water changes to rinse out the poop, the Macrocystis bowl tended to get dirty faster than the others, so poor water quality may have killed the urchins. The copious slime from the Macrocystis itself doesn't help, either. Eventually I will be able to graduate the urchins to containers that will allow flow-through water, but for now most of them are too small to be kept in screened containers because they would escape through the mesh.

Overall, the Ulva urchins seem to be the happiest. I haven't lost any this past month and they eat and poop a lot. These individuals have the good fortune that Ulva doesn't foul the water as quickly as Macrocystis does. They are extraordinarily beautiful, too, and are becoming much more colorful:

Juvenile sea urchin (Strongylocentrotus purpuratus), age 196 days. 3 August 2015. © Allison J. Gong
Juvenile sea urchin (Strongylocentrotus purpuratus), age 196 days. 3 August 2015.
© Allison J. Gong

I've always wondered about the biochemical magic that allows this species of sea urchin to eat algae (primarily kelps, but also some red and green algae) and end up so unabashedly purple as they grow to adulthood. I know from experience in the intertidal that juveniles of S. purpuratus usually go through a green stage when they're in the 1-2 cm size range, before they become purple. And once they're purple, they stay purple. Part of the reason I wanted to do this feeding experiment is to see how the juvenile diet affects color of the animal. These urchins are all from the same mating, so they are full siblings. Presumably there would be some color variation even among a cohort of full-sibs, but if I can distinguish differences between urchins eating Ulva and urchins eating Macrocystis, then perhaps these would be at least partly due to diet?

The difficulty is in photographing individual urchins under the same lighting and background conditions so that color can be somewhat objectively registered. I'm going to have to become a much better photographer, and the urchins are going to have to be more willing to sit still and pose for me. In the meantime, it is easier to compare overall color between the two groups, rather than individual urchins. Looking at the two bowls side-by-side, I get a better feel for the gestalt of each group; can you see the difference? Before you read the caption, can you guess which is the Ulva group and which is the Macrocystis group?

Juvenile sea urchins (Strongylocentrotus purpuratus), age 196 days. Urchins on the left are eating the green alga Ulva; urchins on the right are eating the kelp Macrocystis. 3 August 2015. © Allison J. Gong
Juvenile sea urchins (Strongylocentrotus purpuratus), age 196 days. Urchins on the left are eating the green alga Ulva; urchins on the right are eating the kelp Macrocystis. 3 August 2015.
© Allison J. Gong

To my admittedly very subjective eye, the urchins on the left have more dark pigment and the ones on the right have a more overall golden color. The golden color makes sense because Macrocystis is golden in color (even though taxonomically it is considered a brown alga). But the darker purple in the urchins eating green algae? That makes less sense to me. In any case, I'll have to wait and see how the color develops in both groups of urchins. I suspect that in the long run they'll all end up purple, because that's what they do in the field, but they may take different routes getting there. Stay tuned!

1

I'm sorry. I had to go there. You didn't really expect me not to, did you?

The reason, of course, is that today we got our first settled and metamorphosed Pisaster stars! We were doing our normal Monday water change when I noticed a teensy orange speck on the bottom of one of the jars. I used my beat-up old paintbrush to remove the tiny dot to a dish, put it under the dissecting scope, and saw this:

Metamorphosing ochre star (Pisaster ochraceus), age 48 days. 20 July 2015. © Allison J. Gong
Metamorphosing ochre star (Pisaster ochraceus), age 48 days. 20 July 2015.
© Allison J. Gong

From this picture it's a little hard to see what's going on. The entire body has contracted a lot, from a 2.5-mm larva to about 1/4 of the original size as a 600-µm juvenile, and become much more opaque. There are tube feet and spines as well as some remnants of larval body (the soft bits at the bottom of the animal) at this in-between larvenile stage.

Here's a picture of a fully metamorphosed little star:

Newly metamorphosed ochre star (Pisaster ochraceus), age 48 days. 20 July 2015. © Allison J. Gong
Newly metamorphosed ochre star (Pisaster ochraceus), age 48 days. 20 July 2015.
© Allison J. Gong

I expect we'll be seeing more tiny orange dots on the bottoms and sides of the jars in the next several weeks. At some point we will have to figure out what they eat and provide it for them. But at least we know we're able to get them through the larval phase.

Just for kicks, here are some pictures of where we grow the larvae and how we do the twice-weekly water changes.

Larval culturing paddle table. © Allison J. Gong
Larval culturing paddle table.
© Allison J. Gong
Step 1:  We pour the larvae into a filter to concentrate them into a smaller volume of water. Then we can wash or rinse the jar. © Allison J. Gong
Step 1: We pour the larvae into a filter to concentrate them into a smaller volume of water. Then we can wash or rinse the jar.
© Allison J. Gong
Steps 2 and 3:  We use a turkey baster to transfer most of the larvae from the filter into a jar of clean water. The final step is to turn the filter over and wash the last larvae into the jar. © Allison J. Gong
Steps 2 and 3: We use a turkey baster to transfer most of the larvae from the filter into a jar of clean water. The final step is to turn the filter over and wash the last larvae into the jar. Then we fill up the jar and resume the stirring.
© Allison J. Gong

An update on other matters:

Today is the six-month birthday of my baby urchins! Six months ago to the day these little guys were zygotes, and six-months-plus-one-day ago their parents were roaming the intertidal. They grow up so fast!

Juvenile sea urchin (Strongylocentrotus purpuratus), age 6 months. 20 July 2015. © Allison J. Gong
Juvenile sea urchin (Strongylocentrotus purpuratus), age 6 months. 20 July 2015.
© Allison J. Gong

And lastly, that little shmoo-type thing that I found in the plankton yesterday has revealed itself to be. . . an anemone!

One of the things I like best about cnidarians is the beautiful transparency of their bodies. I love how you can see fluid circulating through the tentacles. Gorgeous, isn't it?

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