This morning as I was doing my rounds at the marine lab I noticed a pile of eggs next to one of the bat stars (Patiria miniata) in a large table. Somebody, or more likely, multiple somebodies, had spawned overnight. I have absolutely zero time to deal with another ongoing project right now, but I have even less self-control when it comes to culturing invertebrate larvae. So I sucked up as many of the eggs as I could, along with a fair amount of scuzz from the bottom of the table, and took a look.
As I've come to expect with stars, the early embryonic stages are developing asynchronously. There were unfertilized eggs (obviously not going to develop at all), zygotes that hadn't divided yet, and other stages.
The coolest thing, though, will take some explaining. Animals begin life as a zygote, or fertilized egg. The zygote undergoes a number of what are called cleavage divisions, in which the cell divides but the embryo doesn't grow. A logical necessity of these two facts is that the cells get smaller and smaller as cleavage continues.
Now let's go back to the earliest cleavage divisions. One cell divides into two, each of those divides into two, and so on. The cell number starts with 1 and goes to 2, then 4, then 8, then 16, and so on. The process is more or less the same for all animals, but in only a few can these divisions be easily seen. Many echinoderms have nice distinct cleavage divisions and transparent-ish embryos, which is why the old-school embryologists in the early 1900s studied them.
Echinoderms are the major phylum in a group of animals called the deuterostomes. Incidentally, chordates (ahem--us) are also deuterostomes. The word "deuterostome" refers to the fact that during development in these animals the anus forms before the mouth does. That's right, folks, you had an anus before you had a mouth.
Another feature that is generally associated with the deuterostomes occurs in early cleavage. Picture this: A cell divides into two cells. Then each of those divides, resulting in four cells. Geometry dictates that the four cells form a plane. That makes sense, right? When the four cells divide again to make the 8-cell embryo, a second plane of cells is formed on top of the first. The second tier can either sit directly on top of the cells of the first tier (radial cleavage) or be twisted 45º so that the cells sit in the grooves between cells in the first tier (spiral cleavage).
Take a look at this embryo. Do you think it has undergone spiral cleavage or radial cleavage?
This is a textbook example of radial cleavage. In all the sea urchin embryos I've watched over the years, I've never seen radial cleavage as clear and unambiguous as this. It was one of those moments when you actually get to see something that you've known (and taught) about forever.
So yes, echinoderms and other deuterostomes generally undergo radial cleavage. And I will hopefully have larvae to look after again! They will probably hatch over the weekend. On top of everything else that's going on now, additional mouths to feed are the last thing I need. But fate dropped them into my lap and who am I to argue with fate?
Every once in a while some random person drops off a creature at the marine lab. Sometimes the creature is a goldfish that had been a take-home prize at a wedding over the weekend (now weddings taking place at the Seymour Center are not allowed to include live animals in centerpieces). Once it was a spiny lobster that spent the long drive up from the Channel Islands in a cooler, and became the Exhibit Hall favorite, Fluffy. This time the objects had been collected off the beach and brought in by somebody who thought they might still be alive.
These white objects are egg masses of the California market squid, Doryteuthis opalescens, that had been cast onto the beach at Davenport. Sometimes the masses are called fingers or candles, because they're about finger-sized. Each contains dozens of large eggs. Squids, like all cephalopods, are copulators, and after mating the female deposits a few of these fingers onto the sea floor. Many females will lay their eggs in the same spot, so the eggs in this photo represent the reproductive output of several individuals. The cephalopods as a group are semelparous, meaning that they reproduce only once at the end of their natural life; salmons are also semelparous. After mating, the squids die. Not coincidentally, the squid fishing season is open right now, the idea being that as long as the squids have reproduced before being caught in seines, little harm is done to the population. Most of the time the squids are dispersed throughout the ocean, and the only time it is feasible to catch them in large numbers is when they gather to mate.
These egg masses look vulnerable, but they're very well protected. The outer coating is tough and leathery, and the eggs must taste bad because nothing eats them. I've fed them to anemones, which will eat just about anything, and they were spat out immediately.
The eggs were brought to the Seymour Center because the person who brought them in thought they might make a good exhibit. I happened to be there that day and got permission to take a small subset of the bunch so I could keep an eye on them. And they did and still do make a good exhibit.
16 April 2018: I obtain squid eggs!
At this stage it is impossible to tell whether or not the eggs are alive. The only thing to do was wait and see.
30 April 2018: After waiting two weeks with apparently no change, I decided it was time to look at the egg fingers more closely again. Lo and behold, they are indeed alive! Look at the pink spots in the individual eggs--those are eyes. And if you can see the smaller pink spots, those are chromatophores, the 'color bodies' in the squids' skin that allow them to perform their remarkable color changes.
9 May 2018: A week and a half later, the embryos definitely look more like squids! Their eyes and chromatophores have darkened to black now. The embryos are also more active, swimming around inside their egg capsules. You can see the alternating contraction and relaxation of the mantle, which irrigates the gills. Squids have two gills. More on that below.
At this point the squid fingers began to disintegrate and look ragged. They became flaccid and lightly fouled with sediment.
14 May 2018 (today): Almost a month after they arrived, my squid eggs look like they're going to hatch soon! I didn't see any chromatophore flashing, though.
In the meantime, some of the eggs on exhibit in the Seymour Center have already started hatching. The first hatchlings appeared on Friday 11 May 2018. The hatchlings of cephalopods are called paralarvae; they aren't true larvae in the sense that instead of having to metamorphose into the adult form, they are miniature versions of their parents.
Peter, the aquarium curator at the Seymour Center, allowed me to take a few of the paralarvae in his exhibit and look at them under the scope. The squidlets are about 3mm long and swim around quite vigorously. Trying to suck them up in a turkey baster was more difficult than I anticipated. But I prevailed!
You can actually see more of what's going on in a video:
The cup-shaped layer of muscular tissue that surrounds the squid's innards is the mantle. When you eat a calamari steak, you are eating the mantle of a large squid.The space enclosed by the mantle is called the mantle cavity. Because the paralarvae are transparent you can see the internal organs. Each of those featherlike structures is a ctenidium, which is the term for a mollusk's gill. The ventilating motions of the mantle flush water in and out of the mantle cavity, ensuring that the gill is always surrounded by clean water.
And now we get to the hearts of the matter. At the base of each gill is a small pulsating structure called a branchial heart ('branch' = Gk: 'gill'). It performs the same function as the right atrium of our own four-chambered heart; that is, boosting the flow of blood to the gas-exchange structure. So that's two hearts. Between the pair of branchial hearts is the systemic heart, which pumps the oxygenated blood from the gills to the rest of the squid's body. This arrangement of multiple hearts, combined with a closed circulatory system, allows cephalopods to be much more active swimmers and hunters than the rest of their molluscan kin.
I expect that my fingers will hatch very soon. If and when they do, it will be a challenge getting them to eat. I've never tried it myself, and cephalopods are known to be difficult to rear in captivity. But I'm willing to give it a shot!
Five days ago I collected the phoronid worms that I wrote about earlier this week, and today I'm really glad I did. I noticed when I first looked at them under the scope that several of them were brooding eggs among the tentacles of the lophophore. My attempts to photograph this phenomenon were not entirely successful, but see that clump of white stuff in the center of the lophophore? Those are eggs! Oh, and in case you're wondering what that tannish brown tube is, it's a fecal pellet. Everyone poops, even worms!
Based on species records where I found these adult worms, I think they are Phoronis ijimai, which I originally learned as Phoronis vancouverensis. The location fits and the lophophore is the right shape. Besides, there are only two genera and fewer than 15 described species of phoronids worldwide.
Two days after I first collected the worms, I was watching them feed when I noticed some tiny approximately spherical white ciliated blobs swimming around. Closer examination under the compound scope showed them to be the phoronids' larvae--actinotrochs! Actinotrochs have been my favorite marine invertebrate larvae--and that's saying quite a lot, given my overall infatuation with such life forms--since I first encountered them in a course in comparative invertebrate embryology at the Friday Harbor Labs when I was in graduate school.
The above is a mostly top-down view on an actinotroch, which measured about 70 µm long. They swim incredibly fast, and trying to photograph them was an exercise in futility. They are small enough to swim freely in a drop of water on a depression slide, so I tried observing them in a big drop of water under a coverslip on a flat glass slide. At first they were a bit squashed, but as soon as I gave them enough water to wiggle themselves back into shape they took off swimming out of view.
Here's the same photo, with parts of the body labelled:
The hood indicates the anterior end of the larva and the telotroch is the band of cilia around the posterior end. The hood hangs down in front of the mouth and is very flexible. At this stage the larva possesses four tentacles, which are ciliated and will get longer as the larva grows. These are not the same as the tentacles of the adult worm's lophophore, which will be formed from a different structure when the larva undergoes metamorphosis.
As usual, a photograph doesn't give a very satisfactory impression of the larva's three-dimensional structure. There's a lot going on in this little body! The entire surface is ciliated, and this actinotroch's gut is full of phytoplankton cells. You can see a lot more in the video, although this larva is also a little squished.
I've been offering a cocktail of Dunaliella tertiolecta and Isochrysis galbana to the adult phoronids, and these are the green and golden cells churning around in the larva's gut. However, good eaten is not necessarily food digested, and the poops that I saw the larvae excrete looked a lot like the food cells themselves. Today I collected more larvae from the parents' bowl and offered them a few drops of Rhodomonas sp., a cryptonomad with red cells. This is the food that we fed actinotrochs in my class at Friday Harbor. We didn't have enough time then to observe their long-term success or failure, but I did note that they appeared to eat the red cells.
I don't know if phoronids reproduce year-round. It would be a simple task to run down and collect a few every month or so and see if any worms are brooding. Now that I know where they are, it would also be a good idea to keep an eye on the size of the patch. Some species of phoronid can clone themselves, although I don't know if P. ijimai is one of them. In any case, even allowing for the possibility of clonal division, an increase in the size of the adult population would be at least partially due to recruitment of new individuals. If recruitment happens throughout the year, it follows logically that sexual reproduction is likewise a year-round activity. Doesn't that sound like a nifty little project?
Besides, it's never a bad idea to spend time at the harbor!
When it comes to the natural world, I have always found myself drawn to things that are unfamiliar and strange. I think that's why I gravitated towards the marine invertebrates: they are the animals most unlike us in just about every way imaginable. Even so, some of them have bodies at least that are recognizable as being both: (1) alive; and (2) animal-ish. Think, for example, of a lobster and a snail. Each has a head and the familiar bilateral symmetry that we have. Obviously they are animals, right? I, of course, am most fascinated not by these easy-to-understand (not really, but you know what I mean) animals, but to the cnidarians and the echinoderms. And for different reasons. The cnidarians astound me because they combine morphological simplicity with life cycle complexities that boggle the mind. I hope to write about that some day. Today's post is about my other favorite phylum, the Echinodermata.
For years now I've been spawning sea urchins, to study their larval development and demonstrate to students how this type of work is done. I have a pretty good idea of what to expect in urchin larvae and can claim a decent track record of raising them through metamorphosis successfully. Urchins are easy. To contrast, I have much less experience working with sea stars. I have found that some species are easy to work with, while others are much more problematic. Bat stars (Patiria miniata), for instance, are easy to spawn and raise through larval development into post-larval life. Ochre stars (Pisaster ochraceus), on the other hand, go through larval development beautifully, but then all die as juveniles because nobody has figured out what to feed them. I've already chronicled my and Scott's attempts in 2015 to raise juvenile ochre stars in a series of posts starting here.
Sea urchins and sea stars have long been model organisms for the study of embryonic development in animals, for a few reasons. First, many species of both kinds of animals are broadcast spawners, which in nature would simply throw their gametes out into the water. This means that development occurs outside the mother's body, so biologists can raise the larvae in the lab and observe what happens. Second, spawning can be induced by subjecting the parents to nonlethal chemical or environment stresses. Third, the larvae themselves are often quite happy to grow in jars and eat what we feed them. Fourth, the larvae of the planktotrophic species are often beautifully transparent, allowing the observer to see details of internal anatomy. Lucky me, I've been able to do this several times. And it never gets old.
All that said, there are differences between urchins and stars that force the biologist to treat them differently if we want them to spawn. For the species I work with, spawning occurs after I inject a certain magic juice into the animals' central body cavity--urchins get a simple salt solution (KCl, or potassium chloride) and stars get a more complex molecule (1-MA, or 1-methyladenine). The fact that you can't use the same magic juice for urchins and stars reflects a fundamental difference in gametogenesis and spawning in these groups of animals.
Sea urchins will spawn only if they have fully developed gametes. In other words, gametogenesis must be complete before gametes can be released to the outside. You can inject as much KCl into a sea urchin as you want, but if it's the wrong time of year or the urchin doesn't have mature gonads (due to poor food conditions, perhaps), it won't spawn. I've never investigated the mechanism by which KCl induces spawning in ripe urchins, but here's what I think happens.
When students dissect animals in my invertebrate zoology class, we use magnesium chloride (MgCl2) to narcotize the animals first. A 7.5% solution of this simple salt is remarkably effective at putting many animals gently to sleep, especially molluscs and echinoderms. Placing the animals in a bowl of MgCl2 and seawater causes them to relax and gradually become unresponsive. A longer bath in the MgCl2 puts them to sleep for good.
Given the relaxation effects of MgCl2 on urchins, I suspect that injecting a solution of KCl into the body cavity relaxes the sphincter muscles surrounding the gonopores. This relaxation opens the gonopore, and if the gonads are ripe the mature gametes are released to the outside. As I said above, I don't know for certain if this is how it works, but the hypothesis makes sense to me. It also explains why that I can shoot up a dozen urchins and get none of them to spawn: the KCl might be doing what it normally does (i.e., opening the gonopores) but if the gonads aren't ripe there are no gametes to be released.
For completely different reasons, injecting a star with KCl does absolutely nothing at all except probably make the animal a bit uncomfortable. The KCl may very well open gonopores as it does in urchins, but a star will never have mature gametes, especially eggs, to release in response to this muscle relaxant. This is because at least in female stars, meiosis (the process that produces haploid gametes) isn't complete until the eggs have been spawned to the outside. What, then, is the magic juice used to induce spawning in stars, and what exactly does it do?
The magic juice is 1-methyladenine, a molecule related to the nucleobase adenine, most commonly known as one of the four bases that make up DNA. The nomenclature indicates that the difference between the two molecules is the addition of a methyl group (--CH3) to the #1 position on an adenine molecule:
Chemistry aside, what I'm interested in is the action of 1-MA on the eggs of sea stars. Meiosis, the process that produces gametes, has two divisions called Meiosis I and II. Meiosis I starts with a diploid cell (i.e., containing two sets of chromosomes) and produces two diploid daughter cells; these daughter cells may not be genetically identical to each other because of recombination events such as crossing over. It isn't until Meiosis II, the so-called reduction division, that the ploidy number is halved, so each daughter cell is now haploid (i.e., containing a single set of chromosomes) and can take part in a fertilization event. In a nutshell, the end products of meiosis are haploid cells, all of which ultimately result from a single diploid parent cell.
In female sea urchins, the entire meiotic process is completed before the eggs are spawned, which is why the relaxation effects KCl can induce spawning.
In females of many other animal species, meiosis is arrested for some period of time after the Meiosis I division. For example, this happens in humans: baby girls are born with all of the eggs they will ever produce, maintained in a state of suspended animation after Meiosis I. It isn't until puberty that eggs begin to complete meiosis, one egg becoming mature and being ovulated approximately monthly for the rest of the woman's reproductive life. Sea stars are sort of like this, with the notable exception that a female star will ripen and produce thousands of eggs in any spawning event rather than doling them out one at a time.
One of the really cool things about working with sea star embryology is that I get to see the completion of meiosis after the eggs have been spawned. I know that the gonads have to reach a certain level of ripeness before 1-MA will induce spawning. Reviewing my notes from a course I took in comparative invertebrate embryology when I was in graduate school, I came across the mention of 'polar bodies,' tiny blobs that I remember seeing in just-fertilized sea star eggs but which I have never seen in sea urchin embryos. Then I needed to remind myself what polar bodies are all about.
Remember how there are two cell divisions in meiosis? Well, despite what's shown in the diagram above, each of the divisions is asymmetrical. In other words, each division of meiosis produces one big cell and one tiny cell. The tiny cells are the polar bodies. They are too small to either divide or be fertilized, and generally die on their own. Here's a chronology of what happens. First, a cell divides, producing a large cell and a tiny polar body:
I've x'd out the polar body in red because it cannot divide or be fertilized and will soon die. Then the large cell divides to produce the final egg and a second polar body:
It turns out that in sea stars things get even more complicated. 1-MA acts as a maturation-inducing substance in these animals, effectively jump-starting the eggs that have been sitting around in an arrested state after undergoing Meiosis I. This initiates the continued maturation of the eggs to the stage when they can be spawned. Even now, though, meiosis doesn't complete until an egg has been fertilized, at which point the second polar body is produced. The production of that second polar body is the signal that Meiosis II has occurred, and the now-fertilized egg can begin its embryonic development.
Here's a freshly fertilized egg of Pisaster ochraceus, with the two polar bodies smushed into the narrow perivitelline space between the surface of the zygote and the fertilization envelope:
Sea urchins, remember, do not have polar bodies when I spawn them. That's because meiosis is complete by the time the eggs can be spawned, so the polar bodies have already died or been resorbed by the final mature egg. The photo of the P. ochraceus zygotes was taken within a few minutes of fertilization. Let's contrast that with a photo of a brand new urchin zygote:
See? No polar bodies!
All of this is to explain why we can't use the same magic juice to spawn both urchins and stars. Kinda cool when the madness in our method has a biological context, isn't it?
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.
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.
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:
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.
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.
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.
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.
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.
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.
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.
So. Last week when I looked at my sand dollar larvae I wasn't at all sure what to make of them. I thought that all of the offspring from one of the matings (F2xM1) were going south and didn't know how much longer they would survive. The offspring from the other two matings seemed to be doing much better.
Fast forward a week and a half and my, how things have changed. I have some juvenile sand dollars now! And so far they are all from the F2xM1 mating, the ones that had started looking strange and that I thought might die. I'm surprised that any of the larvae metamorphosed, as my general understanding of sand dollars was that competent larvae settle among adults of their species, so that when they finish metamorphosis they would be in a suitable location to grow up. However, the animals is always right, and in this case I was happy to learn that my understanding was wrong.
This larva is almost competent. The main part of its body is almost completely filled by the juvenile rudiment (the tannish structure on the left side of the more reddish stomach) and the arms are shorter.
And here is a video of a trio of competent larvae.
Their bodies are almost entirely opaque now but they are unquestionably pluteus larvae.
As metamorphosis begins, the tube feet in the juvenile rudiment rupture through the body wall and the animal starts sticking to a hard surface, in this case a glass slide. For a while the animal is suspended between the larval and juvenile forms, in a state I call a larvenile. Hopefully the time spent in the larvenile stage is short, as to be neither larva nor juvenile is a bad thing. I've seen both sea urchins and sea stars get stuck in the larvenile stage, and they all died.
Larveniles are strange things. See for yourself.
In this video the right side of the animal (not the anatomical right but the right side of the image as it is presented on the screen) is the juvenile, and the left side is the larva. The larva half still has its fenestrated arm rods, which will eventually be dropped and left behind. It also retains for the time being the ciliated band which it used both to swim and to capture food. Another weird feature of the larvenile is the transition between the bilateral symmetry of the larva and the pentaradial symmetry of the juvenile. The bilateral symmetry has been more or less obliterated by the process of metamorphosis, but there isn't enough of the juvenile to have complete pentaradial symmetry yet.
And, finally, metamorphosis is complete and a little sand dollar walks around on tube feet.
Yesterday this animal was a larva, and today it's a juvenile. The sea urchins do the same thing. But these sand dollars have done everything faster than the urchins, and that includes development immediately after metamorphosis. You may recall that the purple urchins have only five tube feet when they metamorphose, and they struggle to coordinate them to walk. From what I can see these sand dollars have at least twice that many tube feet very shortly after metamorphosis, and they can walk much more quickly.
The tube feet themselves are different, too. Urchins' tube feet are suckered and look like little plungers. Sand dollars' tube feet have those pincher-looking tips (although I haven't seen them open up and grab things yet). Adult sand dollars live partly buried in sand and don't use their tube feet to cling to surfaces; they do use their tube feet to grab food, though.
Speaking of food, I don't know what these juvenile sand dollars will be able to eat. Fortunately I have a while to figure out what to try feeding them, as their mouths won't open up for at least a week (I hope). While it's easy to observe what happens on the surface of the animal as it metamorphoses, it's impossible to see what's going on with the internal reorganization of the body. I do know that an entire new gut will have to be formed before the animal can eat. In the meantime it will have to survive on energy stores stashed in all that opaque part of the body.
Remember that one batch of sand dollar larvae that were looking weird on Monday? Well, they still look weird. In fact, all of the larvae looked the same yesterday as they did on Monday, which seems strange, considering how quickly they galloped through development for the first three weeks of larval life. It's as though they've entered some stasis period during which developmental progress slows way down. Or maybe I just can't see the signs of change.
If I had seen these larvae for the very first time yesterday, I might not suspect that anything was strange. But having watched them twice weekly since fertilization and knowing how different they looked a week ago, my Potential Weirdness-o-Meter™ is redlining. These larvae have definitely changed in a week, and not in the way that I'm used to echinoid larvae developing. With their much shorter arms and overall stunted appearance, these guys appear to be regressing. However, they aren't dying and they don't really look bad. As I said on Monday, they just look . . . weird.
Remember how I said I'd split this cohort of larvae into two batches and fed them different things? At first I thought this strange appearance was due to the change in diet from a Rhodomonas/Dunaliella mixture to Rhodomonas only. The larva in the photo above was from the Rhodomonas-only jar, and perhaps its odd appearance could be explained by some deficiency in the monoculture diet. Then I continued on my rounds and looked at the larvae from the same mating that were still on the Rhodo/Dun diet.
All the larvae in these photos remained on the mixed diet, and they look pretty much the same as their siblings eating the monoculture diet. So I don't think the change in diet explains the appearance of the larvae.
Okay, then. If it's not the food that accounts for what these larvae look like, maybe it's something about the mating itself. These larvae, from both food treatments, are all full siblings from one mother mated with one father. As full sibs they share, on average, 1/4 of their DNA with each other, which could account for the similarity in their appearances. Perhaps this "strange" look is due more to genetics than to the environment (i.e., food).
I can test this hypothesis by examining larvae from the other crosses. Rather fortuitously, as it turns out, when I spawned the adult sand dollars a little over three weeks ago now, only one male contributed enough sperm for me to use. Three females spawned usable amounts of eggs, so I set up three matings:
The female designated F2 gave the most eggs, and her offspring are the ones that I split into the Rhodo-only and Rhodo/Dun diets. Note that all of the larvae in this little experiment have the same father. This gives me the opportunity to test for maternal effects on development; in other words, having controlled for the effects of different fathers--ha! I make it sound as though I did that on purpose--I can now assume that differences (in growth rate, survivability, and successful metamorphosis if we get that far) between the different matings are at least partially due to differences in egg quality among the three mothers. Or to differing gamete compatibilities between each female and the one male.
So now let's take a look at the larvae from other matings. We'll start with F1xM1:
This larva looks normal to me, or at least what I've come to assume is normal. And wow, that was one filthy cover slip,wasn't it?
The offspring of the F3xM1 mating look very much the same:
And here's a short video of that same pair of larvae. They look like they're singing a duet. If I were the clever sort I'd dub in some music; alas, I'm not that clever. Does somebody want to do this for me?
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.