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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.

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