Tag: larvae

Progress report

Posted on by Allison J. Gong

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.

Some ins and outs of raising larvae

Posted on by Allison J. Gong

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.

Boy meets girl, urchin style

Posted on by Allison J. Gong

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

Playing matchmaker

Posted on by Allison J. Gong

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.

A star is born!

Posted on by Allison J. Gong

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?

All in the family

Posted on by Allison J. Gong

Earlier this week an acquaintance asked me about the development of sand dollars, specifically if it is anything like that of sea urchins. It just so happens that sea urchins and sand dollars, while not in the same taxonomic family, are in the same class, the Echinoidea. As close kin, they share a similar larval form, the pluteus larva, and undergo more or less the same development. If you’re satisfied with the short answer, you can stop reading here.

Interested in the details? Then read on!

In my first year of graduate school I took a course in comparative invertebrate embryology through the University of Washington up at the Friday Harbor Lab in Puget Sound. It was a blast! We spent mornings in lecture and afternoons in the field and/or in the lab observing and drawing embryos and larvae. At night we would lie on our bellies on the dock and shine a light over the water, scooping up the critters that rose to the surface. It was in this class that I did my first studies of larval development and fell in love with the echinopluteus. In that class we studied several echinoid species, but the only one I was able to follow all the way through to metamorphosis was the sand dollar Dendraster excentricus.

Even a casual beachcomber has likely encountered the naked tests of dead sand dollars, but I’d bet that most people haven’t seen them alive. The bare test is white, while in life the animal is a fuzzy grayish-purple color. As their name indicates, they live on sandy bottoms in the shallow subtidal, where they prop themselves in the sand and catch particles of food that fall on them. This photo below is of the sand dollar exhibit at the Monterey Bay Aquarium. My friend, Chris Mah, is the owner of the most excellent Echinoblog and has explained how sand dollars really are sea urchins. Check it out for some good information from a real-life echinodermologist (yes, I just made up that word).

Dendraster excentricus, the eccentric sand dollar. © Monterey Bay Aquarium
Dendraster excentricus, the eccentric sand dollar.
© Monterey Bay Aquarium

But the original question I was asked to address is about the development of sand dollars as it compares to that of sea urchins. Given that these animals share the same larval form, you would probably not be surprised to learn that their overall development is very similar. Just to remind myself of exactly how similar, I dug through my old embryology notebook and took pictures of some of my drawings. Keep in mind that these drawings were intended to document my observations, not to be pretty or artistic. However, they may be useful as comparisons with the photos of sea urchin larvae that I’ve been taking over the years.

Early pluteus larva of Dendraster excentricus (eccentric sand dollar), drawn from life. © Allison J. Gong
2-day-old early pluteus larva of Dendraster excentricus (eccentric sand dollar), drawn from life.
© Allison J. Gong

According to my notes, to speed up development we cultured these larvae at room temperature so we could get through the entire larval period in the 5-week course. I didn’t record the temperature, but would guess it to be about 17ºC. At this temperature it took the Dendraster larvae only two days to get to the early pluteus stage, complete with functioning gut and skeletal rods (see drawing on right).

In contrast, the Strongylocentrotus larvae that I started this past January were still forming their guts at the ripe old age of 2 days. You’ll have to take my word for that, as I didn’t take pictures. I do have a photo of the embryos when they were 1 day old and had just hatched:

1-day-old embryos of S. purpuratus. The empty space inside each embryo is called the blastocoel. 20 January 2015. Photo credit: Allison J. Gong
1-day-old embryos of S. purpuratus
21 January 2015
Photo credit: Allison J. Gong

By the age of 7 days, the Dendraster larvae already had three pairs of fully developed arms (the anteriolateral, postoral, and posterodorsal arms), with the fourth and final pair (the preoral arms) just beginning to form:

7-day-old pluteus larva of Dendraster excentricus. © Allison J. Gong
7-day-old pluteus larva of Dendraster excentricus, drawn from life.
© Allison J. Gong

The Strongylocentrotus larvae, on the other hand, had barely started growing their first arms at 6 days of age:

6-day-old pluteus larva of Strongylocentrotus purpuratus. © Allison J. Gong
6-day-old pluteus larva of Strongylocentrotus purpuratus
26 January 2015
© Allison J. Gong

After a few weeks the Dendraster larvae had grown all four pairs of their arms as well as their juvenile rudiment on the left side of the gut. The individual I drew has a fully formed rudiment with five tube feet (labelled ‘podia’ in the drawing) and an additional waviness to the ciliated band (shaded in the drawing). My guess at the time was that this individual was developmentally competent, or ready to settle out of the plankton and metamorphose.

27-day-old pluteus larva of Dendraster excentricus. © Allison J. Gong
27-day-old pluteus larva of Dendraster excentricus, drawn from life.
© Allison J. Gong

Most of the sea urchin larvae had not even started forming rudiments by the age of 31 days:

31-day-old pluteus larva of Strongylocentrotus purpuratus, 20 February 2015. © Allison J. Gong
31-day-old pluteus larva of Strongylocentrotus purpuratus
20 February 2015
© Allison J. Gong

When I was in Friday Harbor I was lucky to see one of my Dendraster larvae undergo metamorphosis more or less as I was watching under a microscope. I wish I’d had the set-up to take microscope pictures, because it was an amazing phenomenon to observe. I did make one last drawing of the newly metamorphosed and benthic tiny sand dollar and its discarded larval skeletal rods:

Newly metamorphosed Dendraster excentricus, drawn from life. © Allison J. Gong
Newly metamorphosed Dendraster excentricus, age 29 days, drawn from life.
© Allison J. Gong

I’m sure that I drew what I saw, but looking at this drawing with more experience I wonder if the tube feet really looked like that. Oh well. One of the last things we did as a class was “graduate” our remaining larvae off the dock and wish them luck as we released them into the real world.

With my most recent batch of S. purpuratus larvae, I began seeing competence at 45 days post-fertilization. The first bona fide juvenile urchin didn’t begin crawling around on tube feet until 50 days:

Newly metamorphosed Strongylocentrotus purpuratus, age 50 days. 11 March 2015. © Allison J. Gong
Newly metamorphosed Strongylocentrotus purpuratus, age 50 days
11 March 2015
© Allison J. Gong

The next logical question would be: Why do the sand dollars develop so much more quickly than the urchins? I don’t have a definitive answer for that. Since that class in Friday Harbor I haven’t had another chance to study sand dollars, but in my experience my most recent cohort of sea urchins progressed through development at the normal pace for the species in this location. Some species just take longer than others, and the differences could be due to any number or combination of factors: water temperature, genetics, presence or absence of settling cues, water chemistry, and so on. The take-home message, if you’ve managed to read this far, is that yes, sand dollars and sea urchins undergo pretty much the same development. It’s the same as the short answer at the top of the post, but wasn’t it fun getting there the long way?

Constellations

Posted on by Allison J. Gong

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

17 days old

Posted on by Allison J. Gong

What a difference a week makes! The Pisaster larvae have grown and developed quite a bit since I looked at them a week ago. Here they are as little space ships again.

Early brachiolaria larvae of Pisaster ochraceus, age 17 days. 19 June 2015. © Allison J. Gong
Early brachiolaria larvae of Pisaster ochraceus, age 17 days
19 June 2015
© Allison J. Gong

Since they are getting so big, Scott and I decided to redistribute the larvae from four jars into six. This will give them room to grow and ensure that they aren’t overcrowded. To do this we first concentrated them all into a single beaker, then divided the entire population into two jars, then subdivided each jar into three jars, for a total of six. See all the larvae in the beaker?

Brachiolaria larvae of Pisaster ochraceus, aged 17 days. 19 June 2015. © Allison J. Gong
Brachiolaria larvae of Pisaster ochraceus, aged 17 days
19 June 2015
© Allison J. Gong

The largest larvae are ~1200 µm long, getting big enough to fill up the field of view under the lowest magnification of the compound microscope. The most noticeable difference from last time, aside from the overall increase in size, is that the ciliated band is becoming more lobed. These lobes will eventually be elaborated into the long arms of the mature brachiolaria larva (‘brach-‘ is Greek for ‘arm’). See below:

Brachiolaria larva of Pisaster ochraceus, age 17 days. 19 June 2015. © Allison J. Gong
Brachiolaria larva of Pisaster ochraceus, age 17 days
19 June 2015
© Allison J. Gong

The other rather obvious development is that the left and right coeloms from the previous observation a week ago have fused together in the anterior (top of the picture) and posterior (bottom of the picture) region of the body.

From here on out the larvae won’t get too much bigger; if I remember correctly they’ll grow until they’re about 1500 µm long. Their brachiolar arms will get really long and pretty, though, greatly increasing the length of the ciliated band. Eventually their juvenile rudiments will form . . . but that’s a post for another day. More on that when it happens.

Growing fast

Posted on by Allison J. Gong

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!