These sand dollar (Dendraster excentricus) larvae that I've been raising will be 21 days old tomorrow, and they are still on the fast track. They're developing much more quickly than any of the sea urchin cohorts I have raised. Some of them already have juvenile rudiments with tube feet visible. With the urchins (Strongylocentrotus purpuratus) this is the age when I worry about the cultures crashing for no apparent reason, and so far these sand dollar plutei look great. I hope I didn't jinx them by writing that. In any case, the sand dollars are known to go through larval development more quickly than their sea urchin cousins, so my larvae appear to be playing by the book, at least as far as timelines go.
Just for kicks I took the largest full-sib cohort I had and split it into two batches. One batch I'm feeding the recommended combination of Rhodomonas sp. (red) and Dunaliella tertiolecta (green), and the other I'm feeding Rhodomonas sp. only. I've been able to raise urchin larvae through metamorphosis on a diet of Rhodomonas so I assumed that this food might work for the sand dollars as well. It turns out, however, that the Rhodomonas-fed larvae look a little strange now.
Their bodies have become more opaque and compact; they've shrunk to a length of 450-500 µm. I wonder if this is the first stage in metamorphosis. I didn't see a well-defined juvenile rudiment in any of the larvae I examined but that doesn't mean it isn't there. And although they look weird and deformed, they don't necessarily look bad. They just don't look . . . right.
On the other hand, there may indeed be something wonky going on. I have a jar of siblings of these larvae being fed a red/green diet, and they look totally different.
This is a beautiful 8-armed pluteus larva. It looks great! The arms are nice and long but none of the arm spines are poking through the ends. They appear to be eating well and have grown to a length of 700-800 µm. This is a ventral view, and that oblong blob on the left side of the pigmented stomach is the juvenile rudiment.
Here's a close-up view of the rudiment:
See how the rudiment is crowding into the stomach? And if you squint you might be able to talk yourself into seeing a couple of round blobs in the rudiment. These would be tube feet, which I can see as I focus the microscope up and down through the animal's body but which don't show up very well in a photograph.
The next day that I change the water and have a chance to look at these guys under the microscope is Friday. It's only three days from now, but given how quickly the larvae are developing, a lot could happen between now and then. I'm a little nervous.
Friday 1 April was the last day of my spring break, and tomorrow I go back to teaching. Spring break felt very short this year, and I was busy the entire week. I decided to spend my last day of freedom doing my favorite lab-related things: looking through microscopes at tiny organisms. I had already planned on spending a few hours dealing with my two batches of larvae, and figured I might as well make a day of it and collect a plankton sample on my way in.
It was a beautiful morning out on the bay.
Alas, as gorgeous as the outdoor scenery was, I couldn't linger long once I'd collected the plankton sample so I headed to the lab. If you've ever wondered what a marine biologist's desk looks like, here's mine:
The dissecting scope on the left belongs to me, as it was a graduation gift I bought for myself when I finished graduate school. The compound scope on the right belongs to the lab, but I'm the person who uses it most frequently. I find that, when looking at something like plankton, it's easiest to start by looking at a bit of the sample in a small dish under the dissecting scope; then, when I find interesting critters I can pipet them out and put them on a microscope slide for observation under the compound scope. It may seem a little awkward, but this switching back and forth between "forest" and "tree" views works for me. And honestly, any field biologist worth her salt should be able to switch focus from "big picture" to "small detail" fairly easily. How else would she be able to develop a solid understanding of the system(s) she studies?
Now back to the plankton. Right off the bat I could see with the naked eye some big (by plankton standards) crustaceans zooming around. It wasn't easy chasing them down with the pipet, but after a while I caught one and dumped it on a depression slide. It was a mysid shrimp.
Those big compound eyes are stereotypical of many crustaceans--think crabs, lobsters, large shrimps, etc. Looking carefully at the tail of this particular individual, can you see two small circular structures? Those are statocysts, the organs that give the animal information about its orientation with respect to gravity. The presence of two statocysts in the uropods (the appendages on the most posterior segment of the body) tell me that this animal is a mysid, rather than one of the gazillion other shrimplike crustaceans living in the sea. I saw at least half a dozen mysids in this plankton sample.
Pelagic crustaceans tend to be quite spastic, and mysids are no exception. Their thoracic appendages beat almost constantly to generate a current that brings particles close to the ventral midline, where they are passed forward to the head and sorted as either "food" or "not-food" and disposed of accordingly. The action of the thoracic appendages also moves the animal slowly through the water, but for quick swimming the mysid rapidly flexes its abdomen and moves away in short bursts.
Overall, this wasn't the most interesting plankton sample I've ever collected. When my students and I collected and examined a sample a week earlier, we saw much more animal diversity than I saw the other day. We had some strong winds on Monday-Thursday of last week (I'm writing this on Sunday) and the surface water temperature dropped to 12°C; I thought this would be the start of the spring upwelling season. If it was, then the phytoplankers hadn't responded when I collected this plankton sample on Friday. In any case, it appears that the spring phytoplankton bloom hadn't yet begun. I expect that in another week or two I'll find more diatoms in the plankton.
After lunch it was time to tend and observe my larvae. There's not much to report about the Dermasterias (leather star) larvae. If you remember, I've split these larvae into three different food treatments: (1) Dunaliella only; (2) a combination of Dunaliella and Isochrysis; and (3) Isochrysis only. At this point, 38 days into development, there is no discernable difference between treatments 1 and 2. The larvae in treatment 3, however, don't look so good. They are stunted and appear to be regressing to earlier developmental stages.
On the other hand, the Dendraster (sand dollar) plutei continue to astound and fascinate me. They are stunning!
They are happy and healthy and seem to be doing well. Their posterodorsal arms have grown and their pre-oral arms (the fourth and last pair to form) are poking out. The larvae are eating all the food I'm giving them and are putting it to good use. At this rate I expect to see their rudiments developing soon.
Because I was so surprised at how quickly my sand dollar larvae (Dendraster excentricus) were developing, I checked my notebook from the invertebrate embryology course I took while in grad school to see if what I'm observing now is normal for these animals. It turns out that yes, Dendraster does develop at a much quicker rate than its cousin the sea urchin. And now that I think of it, when I took that 5-week embryology course the sand dollars were the only echinoids that we followed all the way to competence; we spawned and observed urchins as well, but none of them were as far along as the sand dollars by the time the class ended and we "graduated" our larvae off the dock.
Yesterday my Dendraster larvae were five days old. They already had two well-developed pairs of arms and were working on the third pair.
These larvae are big, too--500 µm long. Of course, they started from eggs that were over twice the size of urchin eggs, but they've still grown a lot in only five days. The fourth pair of arms will be the preoral arms. At the rate these larvae are developing, I wouldn't be surprised if these arms show up in the next few days.
As beautiful as those long arms are, they may be a little too long. The larvae swim and gather food using a band of cilia that runs up and down all the arms; the entire body is ciliated, but the ciliated band is the primary locomotory system. I remember the instructor of my embryology course telling us that echinoid plutei will respond to lack of food by growing longer arms, which increases the length of the ciliated band and thus (presumably) the animal's ability to capture the food that is available. There are two pieces of circumstantial evidence that my larvae may be a little food-deprived: (1) the really long arms; and (2) the lack of visible food cells in the stomachs. In urchin plutei that are feeding well I can see food cells churning away in the stomachs. These Dendraster plutei have beautifully transparent bodies, but I don't see food in the guts. On they other hand, they are growing, so obviously they are eating. Just in case they are short of food, though, I'll increase their food ration for the next few days and see how the animals respond.
In the meantime, I continue to be fascinated by the intricacy of the larval skeleton and the complexity of the skeletal rods themselves. Next time I'll try to take photos of these.
This afternoon I met up with Joanna and Amy, who had come to the marine lab with some sand dollars (Dendraster excentricus) to try to spawn. Since sand dollars are in the same taxonomic group (the Echinoidea) as sea urchins, I'd try the same techniques on these animals I'd never spawned before. I did have to modify some things a bit, mostly to account for the difference in body shape between sand dollars and urchins. Urchins are globular, with quite a large internal body volume, while sand dollars are flat. There's much less space inside a sand dollar for gonads and guts.
Gravid echinoids such as urchins and sand dollars can be pretty easily induced to spawn by injecting their internal body cavity with a solution of KCl. We shot up all eight sand dollars and five of them spawned, two males and three females. One of the males didn't give enough sperm to be collected, so we didn't use his gametes. The other male, though, gave us lots of sperm. And they were good sperm, too.
If you've never had a chance to see swimming sperm under a microscope, today is your lucky day!
And the eggs. Wow, sand dollar eggs are freakin' cool! For one thing, they're big, ~130 µm in diameter, compared to the 80 µm eggs of the purple urchin Strongylocentrotus purpuratus. Plus, they have a really thick jelly coat that contains red pigment cells; urchin eggs don't have the pigment cells, either.
The eggs themselves were a little lumpy, not as perfectly round as I'm used to seeing with the urchins, but they fertilized just fine. In all three of the crosses, the fertilization rate was 90-95%. Apparently the sperm have no problem digging through the jelly coat to get to the egg surface.
In this photo you can see the familiar fertilization envelope raised off the surface of the egg, as well as the red pigment cells in the jelly coat. This may very well be the most beautiful zygote I've ever seen. How many people can say things like that?
After an hour and 20 minutes sitting on my desk at room temperature the zygotes started to cleave:
The blastomeres are still a little wrinkled and lumpy, but I think they'll be okay. I've poured them into 1000-mL beakers and they're sitting in one of my seawater tables. Tomorrow afternoon I hope to see them swimming up in the water column. Fingers crossed!
So. I have a batch of larvae from a spontaneous spawning of the leather star, Dermasterias imbricata, that occurred four weeks ago tonight. Until now I've never had an opportunity to work with this species, even though we have quite a few of them at the marine lab. I had my own for several years, until they became casualties of the plague about a year into the current sea star wasting syndrome event. In any case, this is the first time I've been able to spend time with larvae of this species. At the very least I wanted to see how big they would get and how quickly they would develop, compared to the species I'm more familiar with, Patiria miniata (bat star) and Pisaster ochraceus (ochre star).
When the Dermasterias spawned, the first thing I noticed was that the eggs are huge. I measured them at 220 µm in diameter, which is big even compared to what I've seen in other stars. Hatch rates were pretty good, and four days later the larvae were already in the 400-430 µm range. Since I have no experience culturing this species, I thought I'd divvy up my larvae and put them into three feeding treatments to see which larval diet resulted in the best overall success. According to the literature, Dermasterias larvae can be raised on a mixture of the unicellular algae Dunaliella tertiolecta (green) and Isochrysis galbana (golden). My three feeding treatments are: Dun only, a Dun/Iso mix, and Iso only.
A week into the experiment there was a clear difference between the larvae eating only the green food, and those eating either a mixture of green and golden or only the golden. Larvae from all food treatments were about the same size, but the ones eating only Dunaliella had noticeably green guts.
Fast forward two weeks, and the larvae were 20 days old. By this time they had progressed from the bipinnaria stage to the brachiolaria stage. The interesting thing was the absence of green pigment in any of the guts, even those that were eating only green food. The D. tertiolecta larvae looked good, actually. They were a little smaller than the other larvae but were perfectly formed.
Obviously all of the larvae are assimilating enough of their food to grow and develop normally. I looked at them today but didn't have time to take pictures. Qualitatively there is no difference between the Dun larvae and the Dun/Iso larvae. In the Iso jars, however, there are many larvae at earlier stages; some are still at the "jellybean" stage. I don't know if this is because these larvae are developing more slowly, or because of some nonrandom distribution of earlier stages into those jars when I was setting up the feeding treatments.
Next week I'll measure the larvae again, and will have three data points to track growth trajectories.
When serendipity strikes, I try to go with the flow and ride it as long as I can. The latest wave is my batch of Dermasterias larvae, which are developing nicely for the first four days of life. And now they look just like jellybeans!
They have complete guts now and have already grown a bit, measuring 400-430 µm long. It's not always easy to catch these guys in the right orientation to take a photo, as they are spinning and swimming through three-dimensional space, but I got lucky:
I did try to follow an individual larva as it swam around on a microscope slide. I confined the larvae to a single drop of water under a cover slip so their movements are a bit constrained, but they manage to swim along fairly quickly. The resulting video might be a little nausea-inducing, so don't click on it if you're susceptible to motion sickness.
For now I've got the larvae divvied up into different feeding treatments. More on that later.
Actually, it was a fortunately placed phone call from an aquarium curator that struck the other night. I was at home, having eaten dinner and reviewed my lecture for the following morning, when my phone rang. It was the curator, saying that he was making his last rounds of the evening and had noticed that some of his sea stars were spawning. Echinoderm sex--more specifically, the opportunity to collect gametes and observe larval development--always grabs my attention, so I told him I'd throw on some shoes and meet him at the marine lab in five minutes.
Lo and behold, there were leather stars (Dermasterias imbricata) spawning in several of the tanks and seawater tables. Many of the tables were cloudy with sperm, but I found only one female, which seems strange but isn't so unusual. These spawning events occur in response to some environmental cue, such as day-length, a chemical of some sort, or the phase of the moon. When a sea star (or sea urchin) spawns it also releases chemicals that trigger spawning in nearby conspecifics, as to spawn by oneself is an enormous waste of energy. A single spawning animal can result in all the others of its kind spewing out huge numbers of gametes in an orgy of passive sex. However, an animal can be induced to spawn only if its gonads are ripe. Ripeness depends on the overall health of the animal and requires adequate food; animals that don't receive enough food don't have energy to allocate towards gamete production. As eggs are energetically expensive to produce, compared to sperm, it is not unusual for males of a species to mature earlier in the reproductive season than the females. In Washington the spawning season for D. imbricata is April-August. Here in California the reproductive season hasn't been clearly defined, but I do remember a springtime spontaneous spawning event in the lab several years ago.
That creamy looking mass of goo on the star's aboral surface is a pile of eggs. Sea star eggs are fairly large, compared to the urchin eggs I'm used to, and sticky. They tend to clump together in stringy globs until they are dispersed by water currents. The star whose arm is photobombing in the lower right corner is a male. He was also spawning copiously and is probably the individual who fertilized most of this female's eggs.
Given the lateness of the hour and the fact that I had to get up early the next morning I didn't take many pictures of the eggs, although I did look at them to make sure they were fertilized. They were, so I put them into a 1000-mL beaker of seawater and let them do their thing.
Fast forward to today, about a day and a half after fertilization. About two-thirds of the embryos had hatched and were swimming in the water column. Here's what they look like under the dissecting scope:
I poured off the swimmers into jars and set them up on the paddle table. I gave them a little bit of food, in case their mouths break through before I can get back to the lab tomorrow afternoon. In the meantime, I took a sample of embryos and examined them under the microscope. They look really cool!
The embryos are almost spherical, measuring 290 µm long and 270 µm wide. They are ciliated all over and swim with the rounded end forward. The flattened end is where the process of gastrulation started. That visible invagination begins at a section of the embryo called the blastopore; the channel is the archenteron, the first gut of the larva. In echinoderms, as in chordates (including us humans), the blastopore will end up being the larva's anus; the mouth breaks through later at the other end of the archenteron. This is why I don't need to start feeding the larvae right away even though their gut has begun forming.
Tomorrow afternoon I'll have a brief window of time when I can check on the larvae and see how they're doing. I think they may have complete guts by then!
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.
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.
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. . ."
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.
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.
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.
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.
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:
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.
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:
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 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:
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:
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.
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.