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:
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:
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.
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!
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?
Remember those little urchins I brought into the world back in January? Well, they're doing well, for the most part. About a month ago I took about 250 of them, measured them, and divided them into three feeding treatments: one group I left on the coralline rocks they all cut their teeth on, one group is eating the green alga Ulva, and the third group is eating the kelp Macrocystis pyrifera. My plan is to keep the groups on these foods and monitor growth and survival.
After one month it appears that mortality and growth are not related. I have lost more urchins from the Macrocystis treatment than from the other two, and yet those that have survived this far have grown quite a bit. A month of the experiment gives me exactly two data points, which may over time indicate the beginning of a trend but for now are entirely meaningless. I'll have to wait at least another month to see if what's happening now continues.
However, I also took pictures of the urchins, and some of them are getting so pretty! I'm curious to see if the two macroalgal diets (Macrocystis and Ulva) affect the color of the urchins as they grow. Of course, color is very subjective and I can't duplicate the exact lighting conditions when I take microscope pictures of different subjects, so at this point they all look the same no matter which food they've been eating.
My most colorful urchin at the moment is a little guy from the Ulva food treatment. Its test diameter is only about 4 mm, but its color is very vibrant:
In addition to the five distinct reddish-purple bands on the body, I like that this urchin has so much color on its spines. This individual looks like it may skip the green stage that urchins of this species go through and go straight to purple.
On Monday of this week (today is Thursday) I was transferring my baby urchins into clean bowls as I always do on Mondays, and for some crazy reason decided that I needed to measure all 300+ of them. I don't remember how the details of how this decision came about, but it probably went something like this:
Me #1: You know, we should probably measure these guys. We do want to see how fast they're growing, after all.
Me #2: Are you kidding? Do you know how long it's going to take to measure 300 urchins under the microscope? We don't have that kind of time today!
Me #1: Oh, come on, don't be so lazy. How long can it take, really? Let's do it for science!
Me #2: These things always take twice as long as you think they will.
Me #1: It's not as though you have anything better to do this afternoon. I mean, aside from writing a final exam and grading all those research papers you assigned.
Three-and-a-half hours later, Me #2 was soundly kicking Me #1 in the butt and we were all tired. But the urchins got measured and now I have some baseline data so I can track further growth. And, no, I don't have the urchins separated into individual containers so I won't be following individual growth, but will be able to calculate average growth rates across the cohort.
Having to look at each urchin long enough to get it lined up with the ocular micrometer in the dissecting scope gave me a chance to observe how their colors are developing. In the field, urchins of this species (Strongylocentrotus purpuratus) in this size range (mm-3 cm) are usually greenish in color; when these individuals are brought into the lab they turn purple as they continue to grow. I seem to recall that my last batch of lab-grown urchins (in Spring 2012) didn't go through that green phase as juveniles, at least not as vibrantly as what we see in the field. So while I was holding down the current batch of urchins to measure them, I noted their color.
Some of them have a definite green tinge at the base of the spines, which then fades to a mauve-y purple towards the tips. The green coloration is most evident on the younger spines:
In addition to giving the urchins something more substantial than scum to eat, having them on coralline rocks gives me a chance to see some of the other critters that live on the rocks. This particular rock is inhabited by a number of spirorbid polychaete worms that build tiny circular tubes made of calcium carbonate, as well as assorted small barnacles cemented to the rock and other crustaceans crawling around.
This is a close-up shot of one of the spirorbid worms. The tube is entirely covered by pink coralline alga, but the worm's orange tentacular crown and trumpet-shaped operculum (used to close the tube when the worm withdraws) are extended as the worm filter-feeds:
Another photogenic animal that I happened to find was a very small chiton. By the time I found it after measuring all the urchins I didn't have the brain energy to try and key it out; if I can find it again once I've finished grading final exams I'll give it a shot. It is extremely cute, with its bright blue spots, and was very slowly creeping around on the rock when one of the urchins barged in and ran right over it:
The chiton is probably about 4 mm long, just a bit longer than the urchin's test diameter. To the urchin, walking over a chiton isn't much different from walking over a rock; and while the chiton probably doesn't like being walked on it isn't significantly affected by the incident unless the urchin starts gnawing on it. Chitons are the masters of just hunkering down and waiting for things to get better, whether that means the tide coming back or an uncouth urchin moving along and minding its own business.
Today was a big day for me. I got to graduate some of my baby urchins from glass slides onto coralline rocks. They were growing very quickly on the slides, chowing down on scum faster than I can grow it, so now it's time for the biggest ones to really put their Aristotle's lanterns to the test and chew up some rocks.
Coralline algae are red algae that have calcified cell walls, giving them a crunchy texture. They come in two morphs--erect branching forms and as encrusting sheets--and are pink in color. The corallines that I'm using for urchin food are growing as sheets on rocks. In the field it is not uncommon to see little urchins on coralline rocks, and their teeth are more than capable of grinding up the calcified algae.
So today I used my trusty frayed paintbrush to scoop up a total of ~90 urchins from their slides and dropped them onto rocks. I should have taken a picture of this valuable tool of mine, so you can see just how low-tech (and cheap!) my type of marine biology is.
The largest urchin on this rock has a test diameter of ~2800 µm. Almost 3 mm now!
Here's a closer view of three of the urchins in the photo above:
It didn't take long for the little urchins to start crawling around on their new substrate. I think they'll be happy with this more natural surface to explore and food to eat.
In the meantime, the remaining babies will stay in their jars or on their slides, eating scum. I will continue graduating urchins to rocks as they get too big for slides, feeling more nostalgic each time.
Just think, only 97 days ago these urchins were zygotes! It's not often that you can say that you've known an organism for its entire life, from the moment of fertilization. I am grateful for the privilege of having the opportunity to undertake such an intimate study of these animals' lives. Although I try at least once every year, this is my first successful urchin spawning since 2012. Those animals, by the way, are what I call my most perfect urchins because, well, they just are. I had originally thought I could use them for dissection, but after caring for them as larvae and the three years since they've metamorphosed, I just can't bring myself to sacrifice them. They are simply perfect.
We humans use the term "hitting the wall" when we find ourselves in situations in which progress is elusive despite extreme effort. For endurance athletes or anyone doing any serious physical training it can mean not being able to break one's personal best time for a race, or not being able to continue getting measurably stronger. For me, it felt as though much of graduate school involved hitting various hard walls and coming up with a headache. Maybe it's like that for everyone, but from the perspective inside my own head it sure did seem that I was struggling harder than most.
Sometimes the wall is literal rather than figurative. And for small animals, a surface that we might be able to break through without any effort at all (or without even perceiving it as a surface) can be an impenetrable barrier. The biggest of my baby sea urchins has a test diameter of ~2700 µm now; including spines it probably measures a bit bigger than 3 mm across. While tiny urchins can use the surface tension at the air-water interface to crawl, this big guy is too heavy now to stick to the underside and would fall off of it.
However, I thought this urchin might be able to use the surface tension of a water bubble to grab onto and right itself. A hypothesis like this requires empirical data, so I picked up the little urchin and plopped it, oral side up (so, upside-down), in a bubble of water on a depression slide. As I expected, the urchin crawled over to the edge of the bubble and I could see its tube feet attaching to the underside of the surface tension. Watch here:
I watched continuously for about a minute, and the urchin never did figure out how to turn itself over. I think there may be two reasons for this:
The water bubble at the edge of the depression in the slide was very shallow, probably not deep enough to cover the whole animal for the few seconds that it would be positioned on its edge. If, to the animal, the surface tension proved to be impenetrable, then a comparable situation would be for me to pin you, lying on your side sandwiched between a solid wall in front of you and a hard board against your back, then telling you to roll over. You wouldn't be able to do it, either.
The surface tension of the bubble may simply not have been firm enough for the urchin to grab it and pull. Urchins use their tube feet to pull against hard objects, and adults can actually generate enough leverage to push bricks around. Obviously, it's easier to pull (or push) hard against a solid surface (say, a rock or the side of a glass bowl) than a malleable one such as the inner surface of a bubble.
Now, I'm not by any means an expert in biomechanics, but it seems pretty clear to me that the surface tension is either too hard or too soft to be used by an urchin this size to right itself. Smaller urchins would just crawl on the underside of the surface tension until they reached the side or bottom of the container, and larger urchins would push right through it to reach for whatever was on the other side. I may need to do some more experiments with these urchins and bubbles of various sizes.
Just for fun I took another video of the same animal, this time situated upright. It was much happier this way.
See? It has pedicellariae in addition to spines and tube feet. It's also getting easier to distinguish the ambulacral and interambulacral areas. These urchins are already starting to develop some purple coloration. Typically they go through a greenish stage before turning purple; maybe that will come later. We'll have to see.
My baby urchins have become scum-eating machines! They are 88 days old now and I am beginning to wonder if I can generate scum fast enough to keep up with them. I did a head count this morning and have three bowls, each of which holds a population of ~100 urchins, and a bowl that contains another 33. The first three bowls are going through food very quickly, and I change their scum slide every 2-3 days. And, since eating results in pooping I change the water every day.
Hungry urchins looking for food:
After they eat through the food on the upper surface of the slide, the urchins migrate to the lower side and begin munching there. Once most of the food is gone they go on the prowl, and I'll find them on the sides of the bowl looking for something to eat. In the photo, can you tell which urchins are on the underside of the slide? Most of them are, actually. They're the ones where you see a darkish ring around the center; that ring is the peristomial membrane that surrounds the mouth. That's what "peristomial" means, by the way, but you didn't need me to tell you that, did you?
Changing the slide involves using a paintbrush to pick up each urchin and drop it into the new bowl. It's rather tedious but is also the most convenient time to count them. And they do seem happy every time they find themselves in a new bowl with plenty to eat.
Baby urchins with lots of food to eat now:
Using the clue I gave you up the page, can you find the single urchin on the underside of the slide?
Having pentaradial symmetry means that the urchins don't have forward-backward or left-right axes, and they can and do move in any direction on the horizontal plane. They do, however, have a strong oral-aboral axis, and they definitely have a preference for how their bodies should be oriented with respect to gravity. The normal position is to have the mouth (oral surface) facing downward, with the opposite side (aboral surface) facing up. And for this species, at least, even in the field you don't see them sticking upside-down on overhanging surfaces, unless they have a vertical surface to hang onto as well. These little guys can hang onto the underside of the slide because they're not very heavy yet. Once they get bigger, it'll be a lot more difficult for them.
Setting an urchin down on its aboral surface, with its mouth facing up, will keep it from crawling away very quickly, but sooner or later it will right itself and take off. Even my little babies don't like to be flipped upside-down. This guy was pretty stubborn at first and spent a minute waving its tube feet at me while I looked at it through the microscope, but then took another minute or so to get down to the business of turning over. Don't worry, I cut out the boring first minute so this clip shows only the action sequence.
Quite clearly, urchins don't care about forward-backward or left-right, but they do care about up-down. Like most animals that live in essentially two dimensions, adult urchins prioritize knowing the orientation of one's body with respect to gravity. But remember those bilateral larvae? They swim in any direction in their pelagic, three-dimensional world, although the body always moves through the water in a particular orientation (arm tips first). It seems this is another aspect of metamorphosis that gets overlooked: the transition from a bilateral body that swims in both the horizontal and vertical planes (three body axes, weak response to gravity), to a body with pentaradial symmetry that walks only in the horizontal plane (one body axis, strong response to gravity). Hmm. I'm going to have to think about that for a bit.
As a long-time student of invertebrate zoology I have for most of my life appreciated the immense variety and ingenuity of animal body plans. And one of the things I've always found the most intriguing is the pentaradial symmetry of echinoderms. I remember thinking, the first time I encountered a live echinoderm (probably a star at the beach, when I was in elementary school), "Wow. Five arms. That's weird." And now, all these years later, knowing a bit more than I did then, I still find it weird.
Pentaradial symmetry doesn't occur in any animal group except the echinoderms, and even they begin life as bilateral larvae. Remember these guys?
There isn't a more perfect example of bilateral symmetry out there. Although, even at this stage there are developments within the body that are beginning to interrupt the bilateral-ness of the animal. This is a picture of the animal lying on its dorsal surface, so you are looking down on its ventral surface. See how, to the (animal's) left of the stomach there is a darkish squiggle running mostly horizontally between the stomach and the skeletal rod of that arm, that you don't see on the right side? That squiggle indicates where the juvenile rudiment, which contains the first five tube feet of the water vascular system, will form.
As we've seen already, the rudiment grows to the point that it occupies most of the internal space of the pluteus larva. When the larva settles it lands on its left side, where the tube feet erupt during metamorphosis. The end result is (hopefully!) a little urchin walking around on tube feet that it didn't have the day before. Well, I guess technically it had them, but there weren't useful yet. And the body symmetry will have changed from the bilateral larval form to the pentaradial juvenile.
When looking at a live sea urchin it can be difficult making sense of all the stuff that's going on. A sea urchin is a very active animal, with spines and tube feet waving all over the place. It looks like total chaos at first, but examination of a naked sea urchin test (the endoskeleton made up of interlocking calcareous ossicles) lends a lot of insight into the body plan of this animal.
Here's a cleaned intact urchin test:
Now the pentaradial symmetry of this body plan becomes apparent. You can see that there are five regions of doubled rows of plates that have little holes in them. The holes are where the tube feet protrude to the outside, and the plates that bear them represent the animal's ambulacrum, or ambulacral region. The structures of the water vascular system run up along the inside surface of the test in the five ambulacra. The ambulacral regions are separated from each other by five intermabulacral regions, which do not have holes for tube feet because there are no tube feet here. The bumps on the test are called tubercles, and are where the spines attach. The tubercles fit into the base of the spines like a ball-and-socket joint, similar to our shoulder, that allows the spines to rotate 360˚. You can see this for yourself the next time you have a live urchin available: touch one of the spines and observe how the animal reacts.
There is interesting stuff going on at the apex of the urchin, too. The five large-ish holes, one at the point of each interambulacral area, are the gonopores. When I shoot up urchins to make them spawn, the gametes are released from these holes. The arrangement of the gonopores in the interambulacral regions makes sense, once you remember that on the inside of the test the ambulacral areas are where the water vascular system structures (including tube feet) are located. The only space available for the gonads is in the interambulacral areas. I know, it's confusing. And people think invertebrates are simple. Ha!
That may be enough to digest about urchin symmetry for now. I'll have more on this soon, including the implications of pentaradial symmetry. Stay tuned!
Finally! At long last I have evidence that my juvenile urchins have mouths and are feeding. A week ago I put a batch of seven teensy urchins onto a scuzzy glass slide and have been watching them daily ever since. And yesterday, just as I was beginning to worry that they'd never be able to eat, I saw that some of them had eaten little tracks through the scuzz on the slide.
Here's an example:
The little urchin still has a test diameter of about 0.5 mm, so it hasn't really started growing yet. However, see the squiggly dark paths? Those are areas of the slide that have been eaten clean. The scuzz is algal in origin, giving the slide an overall brownish-green color, so the scuzz-free parts of the slide are clear--or dark, actually, given that I took this photograph against a black background--having been munched clean by the urchin's teeth. And the other bit of evidence that I saw? Poop! Yes, there were fecal pellets on the slide, which proves that the little urchin has a complete functional gut.
And those small round golden objects you see on the slide? Those are big centric diatoms of the genus Coscinodiscus. They are the only local diatoms that I know of that are big enough to be seen with the naked eye.
Lastly, because I just can't seem to stop myself, here's a video of the little urchin:
I love the sculpturing of the spines. And do you see that three-pronged structure at about 9:00 on the urchin? That's a pedicellaria. On adults of the genus Strongylocentrotus there are four types of jawed pedicellariae, three of which, in my experience, are easy to distinguish on a living specimen. But in this young an animal I can't yet tell how many types of pedicellariae it has. I suppose that the formation of pedicellariae might be the next event for me to follow as these urchins continue to grow and develop.
Anyone who went to graduate school in the sciences remembers what oral exams are like. I remember not having any fun at all in mine, and by the time I was dismissed I wasn't sure what my own name was. Fortunately, that is all ancient history and now I get to spend my time performing a different kind of oral examination on other creatures.
My oldest urchins are now 17 days post-metamorphosis and I've been watching them to see when their mouths break through. It seems to me that 17 days is a long time, but the time is near. Besides, the animal is always right. In the urchin that I examined closely the five teeth of Aristotle's lantern are very close to breaking through the thin membrane covering the mouth opening. The teeth are also much more active than they were a week earlier, as you can see in this short video clip:
I also checked out another tiny urchin and noticed that this individual has startlingly red buccal tube feet:
Sea urchins have five pairs of large tube feet on the oral surface, surrounding the mouth. As with all tube feet, the buccal tube feet are part of the animal's water vascular system and are situated in the ambulacral region of the test; they are used to manipulate and grab food. In adults of this species, the buccal tube feet are much larger and more robust than the other tube feet. In this little guy the tube feet are noticeably red, but I can't yet tell if they're bigger than the others.
And just for kicks I took another video:
Yesterday I transferred seven urchins onto a glass slide that I've had basking in the sun in an outdoor tank to develop a thin film of algae. As the urchins' mouths become functional they should be able to start munching on the scuzz on the slide. So far they seem happy to be crawling around on the slide but this morning I didn't see any signs that they'd actually eaten anything.
My oldest baby urchins have been actual sea urchins for eight days now. Their total age, counting from the time they were zygotes, is 58 days. When an animal undergoes a life history event as drastic as this metamorphosis, it can be tricky deciding how to determine its age. Do you count from when egg and sperm formed the new zygote, or from when the juvenile (and eventually adult) body form was achieved? For the sake of this discussion I'm going to count from the date of fertilization, simply because I know exactly when that date was and it's the same for all of these larvae, larveniles, and juveniles. This just makes sense to me.
So, at the grand old age of 58 days, which is five days post-metamorphosis for the oldest individuals, the baby urchins have grown a lot more tube feet, spines, and pedicellariae. However, they haven't gotten any bigger. This is because they aren't eating yet. I'll explain why in a bit. The individual in the picture below measures about 490 µm in test diameter--that's the opaque part in the center of the animal. The spines make the apparent size much larger.
In this short video clip you can see how many more tube feet this animal has, compared to the original five it started with. The movements are now much more coordinated, too, and these animals can walk with what appears to be purposeful direction. You can also see the texturing of the spines and the little pincher-like pedicellariae.
To see the surface details of the animal when it's this opaque, I needed to use a different kind of lighting. Instead of using the transmitted light that shines through the object on the stage of the microscope, giving a brightfield view, I used my fiber optic light to create a darkfield effect that shows the surface details of the animal. Then I shot another video clip with this epi-illumination and focused up and down on the oral surface to see what was going on there. Fortunately the baby urchin isn't yet able to right itself very quickly, and it stayed oral-side-up for as long as I needed to take the photos and video.
What this video clip of the urchin's oral surface shows very clearly is that the animal doesn't have a mouth yet. The pinkish star-shaped structure in the center is actually the negative space between the five triangle-shaped white teeth which all point to the middle. Soon, I expect in the next handful of days or so, that thin membrane covering the mouth will rupture, and the teeth will be exposed for the first time to the outside environment. At that point the urchin will begin feeding.
You may well be wondering, How the heck are they living if they haven't eaten in over a week? They're babies, after all, and don't babies have to eat all the time? Well, yes, they are babies. But before they were baby urchins they were larvae, and as larvae were kept well fed by yours truly for their entire larval life. Part of becoming competent as a larva is sequestering enough energy stores to power the process of metamorphosis and keep the juvenile going until it has a mouth and can feed itself. Remember, this new animal has to do everything--locomote, eat, avoid predators--with body parts that it didn't have when it was a larva. Building whole new body parts and learning how to use them takes time. So these newly metamorphosed juveniles have about 10-12 days to fast until their mouths break through and they can begin eating. Any individual that didn't store enough energy to make it through the fast, will die.
I'll check on them again tomorrow (day 59) and see if it's time to transfer the juveniles to their food source, which will be algal scuzz that I've been cultivating on glass slides for a few weeks. They'll grow quickly once they're eating. I hope I have enough scuzz to keep up with them!