What a difference a week makes! The Pisaster larvae have grown and developed quite a bit since I looked at them a week ago. Here they are as little space ships again.
Since they are getting so big, Scott and I decided to redistribute the larvae from four jars into six. This will give them room to grow and ensure that they aren’t overcrowded. To do this we first concentrated them all into a single beaker, then divided the entire population into two jars, then subdivided each jar into three jars, for a total of six. See all the larvae in the beaker?
The largest larvae are ~1200 µm long, getting big enough to fill up the field of view under the lowest magnification of the compound microscope. The most noticeable difference from last time, aside from the overall increase in size, is that the ciliated band is becoming more lobed. These lobes will eventually be elaborated into the long arms of the mature brachiolaria larva (‘brach-‘ is Greek for ‘arm’). See below:
The other rather obvious development is that the left and right coeloms from the previous observation a week ago have fused together in the anterior (top of the picture) and posterior (bottom of the picture) region of the body.
From here on out the larvae won’t get too much bigger; if I remember correctly they’ll grow until they’re about 1500 µm long. Their brachiolar arms will get really long and pretty, though, greatly increasing the length of the ciliated band. Eventually their juvenile rudiments will form . . . but that’s a post for another day. More on that when it happens.
Today my Pisaster ochraceus larvae are 10 days old. Although they seemed to be developing slowly, compared to the urchins that I’m more used to, in the past several days they have changed quite a bit. They’ve also been growing quickly, which makes me think that they’re off to a strong start. Of course, there’s still a lot of time for things to go wrong, as they have another couple of months in the plankton. However, at this point in time I feel optimistic about their chances.
In the dish under the dissecting scope they swim around like bizarre space ships. All the bits of detritus in the water add to the effect. The only thing missing is the sound effects.
The magnification of my dissecting scope goes up to 40X. To see any details of anatomy I have to use the compound microscope, through which I can see this, under 100X magnification:
Aside from the dramatic increase in overall size (almost 1 mm long now!), the larva’s body has gotten a lot more complicated. For one thing, the animal’s marginal ciliated band, which propels the larva through the water, has started becoming more elongate and elaborate. In this view the larva is lying on its back, and I have focused on the plane of its ventral surface. The left and right coeloms are in the plane of the dorsal surface, and thus are not really in focus. You should still be able to see how long they have gotten, though. Eventually they will fuse anteriorly to form a single cavity. The stomach of the larva has a nice green-golden color due to the food it has been eating. It makes perfect sense, as we are feeding them a cocktail of green algae and a diatom-like golden alga.
The larvae are very flexible and can be quite animated when they’re swimming around. They bend, scrunch up, and swallow food cells. They have already gotten so big that they take up much of the field of view under the microscope, even at the lowest magnification. Watch some larval gymnastics:
Part of the reason that I wanted to spawn Pisaster and raise the larvae this summer is that I want to put together a series of pen-and-ink drawings of the developmental stages. I did the same for the bat star Patiria miniata several years ago, but the Pisaster larvae will have longer and more elaborate arms when they mature; capturing these in drawings will be a challenge for me. I also hope to include the juveniles in this set of drawings. With that goal in mind, I’ve been sketching the larvae every few days, just to get some practice under my hand and remind myself what it feels like to draw. I’ve missed it!
For whatever reason, I really like how this sketch turned out. It’s not pretty, but it does truly represent what I saw under the microscope. I’m going to have to work on depicting three-dimensional structures on a two-dimensional page, which will take some practice. Fortunately I have several weeks to brush up on my skills!
This spring and summer the local beaches have at times been covered by what appear to be small, desiccated, blue or white potato chips. They would typically be seen in windrows at and just below the high-tide line, or blown into piles. The most recently washed up ones are a dark blue-violet color, while the ones that have been on the beach for more than a day or two are faded to white.
These animals are Velella velella, commonly called by-the-wind sailors. Taxonomically they are in the Class Hydrozoa of the Phylum Cnidaria. Other members of this class are the colonial hydroids and siphonophores (such as the Portuguese man-o’-war, Physalia) as well as the freshwater hydras that you may have played around with in high school. Technically speaking, Velella isn’t a jellyfish. Actually, if we want to get uber-technical about it, there’s no such thing as a jellyfish at all; or if there is, it’s a vertebrate (i.e., some kind of actual fish) rather than a cnidarian. Most of the gelatinous creatures that people generally refer to as “jellyfish” are in fact the medusae of cnidarians.
That said, Velella is a special kind of hydrozoan. Its body consists of an oblong disc, 3-10 cm long, with tentacles and such hanging down and a sail sticking up. The little sail catches the wind that propels the animal:
How do so many of these animals end up on the beach? The answer is that they float on the surface of the ocean and are at the mercy of the winds, hence their common name. This is an extremely specialized habitat called the neuston. Organisms living here have to be adapted to both aerial and marine factors. In fact, the blue pigment in these animals is thought to act as a sunscreen, reflecting the blue (and probably UV) wavelengths and protecting the underlying cells. We all know that UV radiation damages DNA, right? That’s why we wear sun protection. Other cnidarian inhabitants of the neuston are things like Physalia and Porpita porpita (blue buttons), which are also blue in color. A former boss of mine used to say that for every hydroid there’s a nudibranch that lives on it, eats it, and looks just like it. Porpita isn’t exactly a hydroid, but it does have a predatory nudibranch, Glaucus atlanticus, which is (of course) blue-purple! Glaucus eats Velella, too.
Porpita porpita (left) and its predator, the nudibranch Glaucus atlanticus. Diameter of P. porpita approx. 2 cm.
The Monterey Bay Aquarium Research Institute (MBARI) has, of course, one of the best video explanations of what Velella is all about. I certainly can’t do any better, so you should watch this:
By the way, MBARI’s YouTube channel is like marine biology and oceanography porn. Just sayin’. If you have some time to kill on the Internet, you could certainly do worse than to spend it there!
Today the Pisaster larvae that Scott and I are following are a week old. Happy birthday, little dudes! Yesterday we did the twice-weekly water change and looked at them. They’re getting big fast since we started feeding them on Saturday when their mouths finally broke through. At this stage they are sort of jellybean-shaped and extremely flexible–they don’t have the calcified skeletal rods that sea urchin larvae have so they bend and flex quite a lot. They are also beautifully transparent, which allows us to see their guts in fine detail. We can even watch them swallow food cells!
In the short term (over the next couple of weeks or so) the larvae will continue to get longer. Their guts won’t change much, but their coelomic systems will develop and become more complex. I’ll try to capture that in photos and drawings to share with you.
I’ve been fielding questions about my recent sea star spawning work from people I’ve shared this blog with, which is a lot of fun! To streamline things and make the info available to anybody who might be following, I decided to put together a very brief FAQ-like post to address the most recent questions.
Question: Can you watch the eggs divide in real time?
In a time-lapse sense you can watch cleavage divisions occur, but not in real time. What I can do is set up a slide on the microscope and leave it there for a while. The gradually warming temperature speeds up development to the point that I can sort of see the division in real time. Of course, the danger is that the embryo will cook on the slide. I generally figure that once I’ve pipetted some embryos onto a slide and dropped a cover slip on top of them, they’re goners (it’s not really possible to remove the cover slip without damaging the cells underneath it) so I feel marginally less bad about sacrificing a few to the gods of observation.
Questions: I’m fairly certain that the stars can go back to the sea, but are you able to keep their eggs with them, too? How difficult is that transport?
Actually, my scientific collecting permit specifically states that I’m not allowed to return animals to the wild. If I needed to, I could apply for additional permits but it has never been necessary for the work I do. Surplus eggs and larvae, therefore, are discharged into the seawater outflow at the lab and do return to the ocean but the parents remain in my care.
Question: Are orange and purple stars usually able to cross with each other?
As far as anyone has been able to determine, the color of stars has zero effect on whether two individuals’ gametes are able to do the nasty together. The sea stars that I’m working with–Pisaster ochraceus, the ochre star–are broadcast spawners, meaning that each individual spews his/her gametes into the water, where fertilization and development occur. The stars are also synchronous spawners, meaning that if one individual in an area begins spawning other stars in the immediate vicinity will also spawn. After all, it does take two to tango, and to spawn while nobody else does is a tremendous waste of energy.
So yes, a purple star and an orange star should be able to mate without any problems… at least not any problems due to the parents’ colors.
Question: If so, what color do they end up being, statstically?
This is a very interesting question. Two of my colleagues are going to spawn Patiria miniata (bat stars) next week to address this. Their plans are to cross a Blue female with an Orange male, an Orange female with a Blue male, and both pure-color matings. They did a preliminary version of this experiment a couple of years ago but didn’t end up with enough juveniles at a size that color could be ascertained; thus they couldn’t calculate any statistically meaningful color ratios.
Questions: Do you suppose that the wasting disease could be now in the genetic makeup? Any thoughts (unofficial of course) about this?
My thought is sort of the opposite, actually. The animals that we brought in from the field are all survivors of SSWS; if anything, I’d expect them to be resistant to whatever causes the plague, and to (hopefully) pass on this resistance to their offspring. Of course, there’s no way of knowing if and how exposure to SSWS affects the quality of the gametes. It’s quite possible that these survivors are less fit after the SSWS outbreak than they were before.
Question: Purple Male with Purple Female developed well and purple Male with Orange female didn’t…some sort of incompatibility?
Well, given what I saw today the Orange (female) x Purple (male) cross almost certainly did not work. Fertilization occurred, but almost none of the embryos had any indication of normal development. Since we know the Purple male was able to mate successfully with the Purple female, we can infer that his sperm were fine. It could be that there was something going on with the Orange female’s eggs; there were a lot of them, but maybe their quality just wasn’t very good. Or perhaps we somehow mistreated and wrecked them the other day.
Any other questions? Use the Comments section to ask them, and I’ll address them in a future post.
Wow, they weren’t kidding about “early developmental asynchrony” in sea stars! This morning I looked at the embryos that I had started almost 24 hours earlier, and noticed two things right off the bat:
Thing #1: Within the F1 x M1 (Purple female x Purple male) mating , developmental rates among full siblings were all over the map. Some embryos had progressed to the blastula stage, which is essentially a hollow ball of ciliated cells, while others were still in the early cleavage stages and rather a lot hadn’t divided at all. In fact, with 24 hours of hindsight I can see that several of these eggs had not even been fertilized.
My first reaction upon looking into the microscope and seeing all these assorted blobs was, “Oh, crap.” But then I looked more closely at some of the embryos and realized that they had become blastulae!
Here’s a picture of a blastula. This embryo is freely swimming inside its fertilization envelope, although it doesn’t have a lot of space (remember that narrow perivitelline space from yesterday? that’s all the elbow room it has). The hollow space in the center of the embryo is the blastocoel ‘sprout cavity.’ Given that the embryo hasn’t grown (or even hatched) yet, it’s still ~165 µm in diameter, the size of the original egg.
The stage that precedes the blastula (a hollow ball of cells) is called a morula (a solid ball of cells). The embryo that is partially visible in the bottom of the above photo may be a morula. Imagine the following sequence of events: (1) an egg is fertilized by a sperm, forming a zygote; (2) the zygote undergoes a number of cleavage divisions, with the cells becoming more numerous and smaller in size; (3) at some stage a solid ball of small cells, the morula, is formed; (4) as cell division continues, the cells migrate toward the outside of the sphere, forming a cavity (the blastocoel) in the middle.
The blastula is a ciliated stage, and in this video clip you can see the cilia moving. I shot this video at only 100X magnification to capture as much depth of field as possible, and suggest viewing at full-screen. This should enable you to see the three-dimensional structure of the embryo, and that it is indeed a sphere.
Thing #2: The F2 x M1 mating (Orange female X Purple male) isn’t doing well at all. I looked at several slides and didn’t see any embryos that were developing normally. They had all been fertilized, as I could see the fertilization envelope surrounding each egg, but most had not even divided. The ones that had divided were all strange and just plain wrong. Here, see for yourself:
Many of the eggs are blurry because they’re below the focal plane of the microscope. But see how many of them are undeveloped? And how, in the ones that have started dividing, the cells are disorganized and of different sizes? Typical echinoderm cleavage, as I see in echinoids (our local urchins and sand dollars) and in my other crossing of these ochre stars, results in a blastula made up of cells that are all approximately the same size. Most of these embryos, on the other hand, appear to consist of one large cell and a bunch of tiny ones.
I assume that these abnormal-looking-to-me embryos will not hatch, although I could be pleasantly surprised tomorrow. I don’t yet have much of an intuition about these Pisaster ochraceus embryos, so this is a huge learning experience for me. I do expect to see hatching in the F1 x M1 cross tomorrow. Fingers crossed!
A recent college graduate and fellow marine lab denizen (Scott) and I are collaborating on a project to quantify growth rates in juvenile Pisaster orchraceus stars. This is one of the intertidal species whose populations in the field and in the lab were decimated by the most recent outbreak of sea star wasting syndrome (SSWS). We are interested in seeing how quickly the stars grow once they metamorphose and recruit to the benthos, and hope that the information will help researchers guesstimate the age of the little stars that are now being seen in the field. This would in turn tell us whether the little stars are survivors of SSWS or post-plague recruits. I keep seeing people refer to them as “babies,” but they could very well be several years old. We just don’t know, hence this study.
But before we get to measure juvenile growth we have to get through larval development, which is perfectly fine by me because I’m always up for observing marine invertebrate larvae. Two weeks ago Scott and I ventured into the field in search of prospective parents. We brought back eight individuals from two different sites, making sure to leave many more in place than we took away. It was actually rather gratifying to see how many hand-sized-or-larger P. ochraceus there were. This morning we met at 07:30 to shoot up the stars with magic juice and then wait for them to spawn.
It has been a while since I tried to induce spawning in Pisaster, and I had forgotten how much longer everything takes compared to the urchins. For one thing, the magic juice itself isn’t the same stuff that we use on the urchins, and works by an entirely different mechanism. The stars’ response to the magic juice takes 1.5-2 hours, whereas if the urchins aren’t doing anything 30 minutes after getting shot up they either need another injection or simply don’t have gametes to share.
However, despite my misgivings the animals spawned. Two large females gave us enormous quantities of eggs, and three more donated trivial amounts that we didn’t end up using.
This purple individual is the one we designated Female 1. See the huge piles of salmon-pink eggs?
Although we had to wait for a male to spawn, we finally did get some sperm and fertilized the eggs at about 12:30. Another thing I had forgotten was that Pisaster eggs, when shed, are lumpy and strange. I was used to the urchin eggs, which are usually almost all beautifully spherical and small. The stars’ eggs are about twice as big, at ~160 µm in diameter. The lumpiness doesn’t seem to hamper the fertilization process, as you can see below.
In this photo you can see the fertilization envelope surrounding most of the eggs. In stars the perivitelline space (the space between the egg surface and the fertilization envelope) is very narrow, which makes it difficult to see the envelope; in urchins the space is much larger, and as a result the envelope quite conspicuous. The rising of the fertilization envelope off the surface of the egg is referred to as the slow block to polyspermy, a mechanical barrier that keeps multiple sperms from penetrating any individual egg. There’s also a fast block to polyspermy, but it happens on a molecular level milliseconds after a sperm makes contact with the egg surface; you can’t see it happen in real time.
Cleavage in stars proceeds much more slowly than it does in urchins, too. In embryological terms, “cleavage” refers to the first several divisions of the zygote, during which the cell number increases as the cell size decreases. This inverse relationship between cell size and number logically has to occur because the embryo can’t get any larger until it has a mouth and begins to feed, which won’t happen for at least a couple of days. It took our zygotes about four hours to undergo the first cleavage division.
I left the slide on the microscope to warm up and speed development a bit, and about 45 minutes later was rewarded with this mishmash of embryos at different stages. Nine hours after we started this whole process, there were 2-cell, 4-cell, and 8-cell embryos, as well as eggs that had not divided yet.
This asynchrony in early development is another way that stars differ from urchins, and it takes some getting used to. I expect that development will become more synchronized as the embryos continue to cleave, and that hatching will occur for all of them at about the same time, probably before Thursday. At least it won’t take another 9-hour day to see how far they’ve come.
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.
Answer: When it’s a snail! Yes, there are snails that secrete and live in white calcareous tubes that look very similar to those of serpulid polychaete worms. Here, see for yourself:
The worms secrete calcareous tubes that snake over whatever surface they’re attached to. When the worm is relaxed, it extends its delicate pinnate feeding tentacles and uses them to capture small particles to eat; they are what we call suspension feeders.
But there are gastropods that secrete calcareous tubes, too. They are the vermetid snails, the local species of which is Thylacodes squamigerus. This is one of my favorite animals in the low intertidal, probably because it is so delightfully un-snail-like.
There are three individuals of T. squamigerus in this photo:
Thylacodes is also a suspension feeder, but it gathers food in a very different way. When submerged, it spins out some sticky mucus threads that catch suspended particles, then reels in the threads and eats them.
So how would you tell these animals apart if you see them? Here’s a hint: Look at the tubes themselves.
I invite you to use the comments section to tell me how you’d distinguish between Serpula and Thylacodes.
This morning I took a small group of Seymour Center volunteers on a tidepooling trip to Point Piños (see red arrow in the photo below). Point Piños is a very interesting site. It marks the boundary between Monterey Bay to the right (east) of the point and the mighty Pacific Ocean to the left (west).
Map of Monterey Bay. Red arrow indicates Point Piños.
As is my usual habit, we began our exploration on the Pacific side of the point. Almost immediately, Victoria found an octopus! And a couple of meters away, she found another one!
As we approach the summer solstice, the algae and seagrasses are at their most lush. Point Piños is a fantastic site for algal diversity; every time I come here I want to take some back with me so I can study it at the lab. Alas, collecting at Point Piños is not allowed even for someone (like me) who holds a valid scientific collecting permit.
And yes, that log-like object towards the upper-left corner is a harbor seal (Phoca vitulina). A handful of seals were hauled out on the rocks.
However, I was much more interested in the invertebrates. I wasn’t looking for anything specific, but in the back of my mind I was keeping track of certain nudibranchs and looking for small stars.
We did see many Patiria miniata (bat stars) in the 1-2 cm size range. Most of them were a bright orange-red color, but some were beige, yellow, or blotchy. There was one large (bigger than my outstretched hand) Pisaster ochraceus that was intensely orange. And Point Piños is always a good spot to see many of the six-armed stars in the genus Leptasterias.
In terms of nudibranchs there were many Doriopsilla albopunctata, a yellow dorid with tiny white spots. We saw quite a few of them crawling around on the emersed surf grass, as well as in pools. And of course Okenia rosacea (Hopkins’ rose) was there, although not in the huge numbers I was expecting.
In the low zone I saw a few thalli of the intertidal form of Macrocystis pyrifera, the giant kelp that forms the forests that the California coast is famous for. I’d seen this intertidal form named Macrocystis integrifolia, but it appears that now the two forms (intertidal and subtidal) are both considered to be M. pyrifera. To my eye, the intertidal form differs morphologically by having rounder pneumatocysts (floats) and a holdfast that is less dense than the subtidal form.
Hermit crabs are diverse and abundant at Point Piños. Here’s an example of Pagurus samuelis, the blue-banded hermit crab; even when you can’t see the blue bands on the legs, the bright red antennae are a major clue to this crab’s identity.
When we climbed over the point to the Monterey Bay side, I found two of these little gastropod molluscs, which I didn’t recognize. They are about 1 cm long, with a brown lumpy mantle that can covers the shell, which is pinkish in color. After putting it out on Facebook that I needed help with the ID, a bunch of friends and friends of friends chimed in (thanks John, Rebecca, Barry, and David!) and I was able to determine that these little guys are Hespererato vitellina:
On our way back up the beach we noticed long windrows of Velella velella, the by-the-wind sailors, washed up. While most of them were faded and desiccated, there were enough freshly dead ones that were still blue, which may have washed up on the previous high tide.
All in all, a very satisfactory morning. I saw things I expected to see, some things I didn’t quite expect but wasn’t surprised to see, and some things I’d never seen before. That Hespererato vitellina was completely new to me, which is always exciting.
Next up: What kinds of things live in white calcareous tubes?
