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About two and a half months ago, the ongoing disaster of sea star wasting syndrome raised its ugly head again when one of my bat stars (Patiria miniata) developed lesions on its aboral surface. Here's what it looked like then:

Patiria miniata (bat star) with small lesion. 4 September 2015 © Allison J. Gong
Patiria miniata (bat star) with small lesion.
4 September 2015
© Allison J. Gong

and here's a close-up of the lesion, taken the following day:

Lesion on aboral surface of Patiria miniata (bat star). 4 September 2015. © Allison J. Gong
Lesion on aboral surface of Patiria miniata (bat star).
5 September 2015.
© Allison J. Gong

See how the lesion is sort of fluffy? It looks as though tissue may be sloughing off the surface. Wanting to see how the syndrome would progress, I let it remain in its table and kept an eye on it. Every so often I took it out and examined it, and nothing really seemed to change. The animal continued to eat, retained its internal turgor pressure, and none of its table mates became sick. Eventually I sort of forgot about it.

Until two of my students last week asked if I had any pictures of sick sea stars that they could borrow for their end-of-the-semester project. This question jump-started my brain and I remembered this particular bat star, and told the students they could come to the lab and take their own pictures of it. . . that is, if it were still alive. They were able to visit me this past Monday and together we looked at the animal.

Lo and behold! it's not dead, and actually looks pretty good.

Patricia miniata (bat star) with aboral lesion. 24 November 2015 © Allison J. Gong
Patiria miniata (bat star) with aboral lesion.
24 November 2015
© Allison J. Gong

The star has a few pale areas in addition to the original lesions, but overall doesn't seem sick at all. It's nice and firm, righted itself quickly when we placed it in the bowl with its oral surface up, and crawled around very actively.

Not only that, but take a closer look at the lesion itself:

Lesion on the aboral surface of Patiria miniata (bat star). 24 November 2015 © Allison J. Gong
Lesion on the aboral surface of Patiria miniata (bat star).
24 November 2015
© Allison J. Gong

The lesion appears to be somewhat sealed off, as if the epidermis has recovered. I gently poked the surface of the lesion with my forceps, and it feels a little firm and nothing squirted out of or peeled off the surface of it. I think it's analogous to a scab that forms over a skinned knee. Of course, while a scrape on my knee would heal after a few days, sea stars have a much slower metabolism so I'm not really surprised that it would take over two months for this individual to show signs of a healing lesion.

Of course, I could be entirely wrong about what's going on with this lesion. It's the same size as it was back in September, so I'm not convinced that it's healing. However, it seems that closure of the wound is better than a wide-open gaping sore that leaves the animal's innards exposed to the external environment. If, over the next several weeks the edges of the wound begin to come together, then I'll be more confident that this animal is on the road to recovery. In this season on Thanksgiving, this is something to be grateful for.

You may have heard that earlier this month the California Department of Fish and Wildlife postponed the scheduled opening of the commercial Dungeness crab season. Gasps of dismay were heard all over the state from Californians whose Thanksgiving traditions include cracked crab, as well as from the folks who make a living fishing for them. The closure is due to the detection of domoic acid (DA) in the crabs. DA is a naturally occurring toxin produced by some species of diatoms in the genus Pseudo-nitzschia. DA is ingested by filter-feeding animals such as mussels, and due to the process of bioaccumulation occurs in higher concentrations in the tissues of animals that feed at higher trophic levels. Humans can be affected by DA also, which is why state officials warn people not to collect and eat mussels when DA levels are high enough to be concerning.

Since the crab fishery closure I've been wanting to do my own informal assessment of Pseudo-nitzschia in the water, but with one thing and another I didn't have the time or opportunity until today. This morning I collected a plankton sample and gave myself a few hours to play with it before I had to start grading papers. Pseudo-nitzschia was present but not incredibly abundant, especially compared to what I saw this past August. Today's Pseudos were in chains of 3-4 cells, instead of the 12 cells that were common in the summer.

Chain of Pseudo-nitzschia sp. cells collected from a plankton tow off the Santa Cruz Municipal Wharf. 18 November 2015 © Allison J. Gong
Chain of Pseudo-nitzschia sp. cells collected from a plankton tow off the Santa Cruz Municipal Wharf.
18 November 2015
© Allison J. Gong

But it turns out that Pseudo-nitzschia wasn't the most interesting thing I found in the plankton today. Just about at the time that I was supposed to stop playing and start grading, I saw one of these:

Mystery phytoplankter collected from a plankton tow off the Santa Cruz Municipal Wharf. 18 November 2015 © Allison J. Gong
Mystery phytoplankter collected from a plankton tow off the Santa Cruz Municipal Wharf.
18 November 2015
© Allison J. Gong

This was a big cell, measuring 250 µm long and 80 µm wide. Right away it had a diatom look about it: the visible protoplasm was golden-brown, the color of diatoms; it didn't have any cilia or flagella; and it was scooting along very slowly, the way a pennate diatom does. But it wasn't anything that I recognized, which made it all the more intriguing. I made an executive decision to investigate further, even if it meant not getting my papers graded. Damn the consequences, science was calling!

I did some poking around, searching through photo databases of local diatom species, not having much success. Since this was a new (to me, at least) critter, it warranted not just a photo and video but an entry in my real lab notebook:

18 November 2015 © Allison J. Gong
18 November 2015
© Allison J. Gong

Besides, spending time with a microscope, notebook, and pencil feels more like doing science than when I take pictures. And it has been a while since I've been entirely stumped, so I was having fun.

It turns out that this diatom isn't all that uncommon in Monterey Bay. I happened across a report of a diatom named Tropidoneis antarctica that had been detected in a plankton tow off our very own Santa Cruz Wharf about a week ago. BINGO! I had a name for my mystery critter, learned something new, and got to play for a morning. And notice that I spelled the genus name wrong in my notebook? Oops.

And, by the way, the papers did all get graded. I am (un)fortunately far too responsible to have let them not get graded. I'm working on that, though. Give me another 50 years or so and I'll be as flaky and unreliable as the next guy.

A few weeks ago I made a pilgrimage to the Great Tidepool in Pacific Grove, where Ed Ricketts did much of his collecting in the 1920-40s. Ricketts is a legend among students of the intertidal here in California, but he is known to a much wider audience as the inspiration for the character Doc in John Steinbeck's novels Cannery Row and Sweet Thursday. Steinbeck and Ricketts were good friends, and in the spring of 1940 the two of them hired a seiner out of Monterey and her captain and crew for a six-week trip to collect intertidal invertebrates from the Sea of Cortez. The journal from that trip, published in 1951 as The Log from the Sea of Cortez, is a classic work of biology, philosophy, and adventure--one of my all-time favorite books and a definite recommended read.

Pacific Biological Lab, the home and workspace of Ed Ricketts. 14 November 2015 © Allison J. Gong
Pacific Biological Laboratories, the home and workspace of Ed Ricketts.
14 November 2015
© Allison J. Gong

For my birthday, I was treated to a tour of the Pacific Biological Laboratories on Cannery Row in Monterey. This is where Ricketts lived and worked. The original building on this site was completely destroyed in late 1936 by a fire that began at an adjacent cannery; Ricketts managed to escape with his typewriter but lost almost all of his collections, research notes, and scientific library. Fortunately for posterity, Ricketts' book on intertidal ecology, Between Pacific Tides, had already been sent to the publisher. Ricketts rebuilt his home and lab, which is the building that currently occupies the site. The city of Monterey provides free docent-led tours of the Lab on the second Saturday of every month.

I was primarily interested in Ricketts the scientist, although Ricketts the music-lover, poet, and philosopher was also discussed in the tour. We did get to see the building and back yard, including what the docent referred to as the "holy of holies," Doc's lab itself.

Bottles and jars at the Pacific Biological Laboratories. 14 November 2015 © Allison J. Gong
Bottles and jars at the Pacific Biological Laboratories.
14 November 2015
© Allison J. Gong

I love this old stuff, even though I probably don't want to know what was in any of these jars. Nor do I really want to be able to read the label on this bottle (okay, yeah, I really do):

Bottle with unreadable label. 14 November 2015 © Allison J. Gong
Bottle with unreadable label.
14 November 2015
© Allison J. Gong

I imagine that all the hazardous stuff was removed once the building became a museum, but the romantic in me wants to believe that these bottles still contain some essence of the work that went on in this room. Besides, I've encountered bottles that appear to be of not-much-younger vintage in old labs, and while they're undoubtedly scary they are also fascinating.

Ricketts' card catalog, which held his extensive collection records. 14 November 2015 © Allison J. Gong
Ricketts' card catalog, which held his extensive collection records.
14 November 2015
© Allison J. Gong

The most interesting artifact in the lab was this desk:

Steinbeck and Ricketts' desk. 14 November 2015 © Allison J. Gong
Steinbeck and Ricketts' desk.
14 November 2015
© Allison J. Gong

This is the very desk that Steinbeck and Ricketts purchased to take on their voyage to the Sea of Cortez. Unfortunately, they hadn't measured the berths on the boat they hired, and the desk didn't fit anywhere. It spent the entire voyage lashed down and covered with a tarp.

Ricketts' back yard holds a big rusted boiler that he used to render the livers of basking sharks (the smell must have been ungodly awful), as well as a series of concrete basins that he used as holding tanks for the animals he collected. The Pacific Ocean breaks literally against what would have been his garden wall if he'd had a garden.

Ricketts' back yard. 14 November 2015 © Allison J. Gong
Ricketts' back yard.
14 November 2015
© Allison J. Gong

Visiting this place made me aware that I hold a teensy bit of Ricketts' legacy in my hands whenever I teach about marine invertebrates or marine ecology. I certainly don't have Ricketts' poetic way of writing about these animals, but I hope that my students come away with a glimmer of what I love about them. And that I can be a conduit through which Ricketts' holistic view of the world he observed is transferred to another generation of naturalists. It's a big job, but somebody's gotta do it.

Today my most recent batches of urchin larvae are six days old. Yesterday being Monday, I changed their water and looked at them under the scopes. I was pleased to be able to split each batch into two jars, as the larvae have already grown quite a bit; I now have a total of four jars to take care of. This makes me inordinately happy. Having only two jars is risky, as it wouldn't take much for both of them to crash, but for some reason I feel more confident of success with four jars. It's probably one of those all-your-eggs-in-one-basket things.

In any case, this is what they look like now:

Pluteus larvae of the sea urchin Strongylocentrotus purpuratus, age 5 days. 9 November 2015 © Allison J. Gong
Pluteus larvae of the sea urchin Strongylocentrotus purpuratus, age 5 days.
9 November 2015
© Allison J. Gong

These larvae are perfectly formed. At this point they are shaped essentially like squared-off goblets, with four arms sticking up at the corners of the goblet. They will continue to grow arms in pairs until they have a total of eight (four pairs). The stomachs (the round-ish pale red structures in the middle of the body) are big and round; the color of the stomachs is due to the food that the larvae are eating. And can you see the skeletal rods extending into each of the arms? Each of the eventual larval arms will be supported by one of these rods, and additional rods will serve as cross-braces going horizontally across the body.

Ever wondered what these animals eat? In the wild they would be feeding on whatever phytoplankton they can catch. In the lab we have several types of phytoplankton growing in pure culture, but trial and error has taught us that urchin larvae do best on a diet of the cryptophyte Rhodomonas sp.

The cryptophyte Rhodomonas sp., growing in pure culture. 9 November 2015 © Allison J. Gong
The cryptophyte Rhodomonas sp., growing in pure culture.
9 November 2015
© Allison J. Gong

The red color of the cultures is due to the color of the cells. When the larvae eat this food their stomachs turn pinkish. Rhodomonas cells are about 25 µm long and have two flagella that they use to zip around. Here's a short video of a drop of Rhodomonas culture on a slide:

They sort of look like sperms, but the cells are much larger than sperms, the flagella are much shorter than the single flagellum of a sperm, and their swimming isn't quite right to be sperms, either.

The larvae themselves live in glass jars in one of the seawater tables that I converted into a paddle table. The larvae are negatively buoyant and would sink to the bottoms of the jars if left unstirred, and the gentle back-and-forth motion of the paddles keeps them, and their food, suspended in the water column.

See my four jars? They are a sign of short-term success. There's still a lot of time for things to go south with these larvae, and I certainly don't take for granted that I'll be able to keep them alive for the duration. But today, as my students were dissecting urchins in lab, I was able to show them the offspring of said urchins. I hope to keep the larvae alive through the end of the semester, to show the students as much as I can of larval development in one of my favorite animals.

Having obtained decent-ish amounts of gametes from sea urchins, the next step is to get eggs and sperm together. The first thing I did was examine the spawned eggs to make sure they were round and all the same size. Lumpy eggs or a variety of sizes of eggs indicates that they are probably not fertilizable. These eggs from F1 looked just about perfect:

Freshly spawned eggs of Strongylocentrotus purpuratus. 4 November 2015 © Allison J. Gong
Freshly spawned eggs of Strongylocentrotus purpuratus.
4 November 2015
© Allison J. Gong

Note that the eggs are all similarly sized (80 µm in diameter) and round. These look good to go.

The next step is to dilute the sperm in filtered seawater and introduce a small amount to the eggs. The sperm need to be diluted because, believe it or not, in this case too much of a good thing is bad. There's a phenomenon called "polyspermy" which is pretty much exactly what it sounds like: an egg being penetrated by more than one sperm. Polyspermy leads to wonky development down the road, and while it probably rarely happens in the field, where sperm would be diluted immediately upon being spawned, it definitely does occur in the lab. However, eggs are smart and have evolved a couple of mechanisms to prevent polyspermy.

The fast block to polyspermy occurs within a few seconds of the fusion of the sperm and egg plasma membranes. As the sperm nucleus begins to enter the cytoplasm of the egg, Na+ ion channels in the egg membrane open and cause a depolarization of the egg membrane; this depolarization makes the egg impenetrable to other sperm. However, the egg membrane cannot remain depolarized indefinitely, so after about a minute the slow block to polyspermy takes effect.

The slow block is the rising of the egg's vitelline layer above the surface of the egg, creating what we call the fertilization membrane. This envelope acts as a physical barrier against additional sperm. The really cool thing about studying fertilization in sea urchins is that you can watch it happen in real time. I mean, how often do you get to observe the formation of a brand new life at the moment that is is being formed?

In this video there are 2.5 eggs in the field of view. Concentrate on the two whole eggs. The one on the top has already been fertilized, which you know because you can see the fertilization membrane surrounding it. You can also see a lot of sperm zooming around. Keep an eye on the lower of the whole eggs; can you see the rising of its fertilization membrane?

Of the two female urchins that spawned for me this morning, F2 had only a few eggs to give but her fertilization rate was 100%. F1, on the other hand, spawned a lot of eggs but only about 50% of them were fertilized. I have no explanation for this. Sometimes (quite a lot of times, actually) things simply don't work.

That said, at our local ambient temperature the first cleavage division occurs about two hours post-fertilization. That's when I saw this:

Two-cell embryo of Strongylocentrotus purpuratus, approx. two hours post-fertilization. 4 November 2015 © Allison J. Gong
Two-cell embryo of the sea urchin Strongylocentrotus purpuratus, approx. two hours post-fertilization.
4 November 2015
© Allison J. Gong

A few hours later the embryos had progressed to what I think is the 16-cell stage. At this point it starts getting difficult to distinguish the different cells without focusing up and down through the embryo. But if you know what you're looking at, the three-dimensional structure does make some sense. In the embryo below I can talk myself into seeing two rings of eight cells each, one ring lying on top of the other.

16-cell embryo of the sea urchin Strongylocentrotus purpuratus. 4 November 2015 © Allison J. Gong
16-cell embryo of the sea urchin Strongylocentrotus purpuratus, approx. five hours post-fertilization.
4 November 2015
© Allison J. Gong

If the embryo is at the 16-cell stage, then it has undergone four cleavage divisions. The early divisions of an embryo are called "cleavages" because the cells divide in half to form equal-sized daughter cells. In other words, the cell cleaves. During cleavage the embryo doesn't grow, which means that the average cell size necessarily decreases. Cleavage divisions will continue for a total of about 24 hours, resulting in a stage called a blastula.

UP NEXT (hopefully): hatching and swimming

We are finally heading into the time of the year that our local intertidal sea urchin, Strongylocentrotus purpuratus, spawns. Usually I would wait until December or January to try to spawn urchins in the lab, but next week my students will be dissecting urchins in lab and I thought I might as well evaluate gonad development in the animals that are going to be sacrificed anyway. In early December I'm going to loan several urchins to a colleague who will be spawning them to show the earliest stages of development to students in one of the lower-division classes at the end of the semester. If I have any luck today, I'll be able to: (1) start my own cultures of urchin larvae so that I can show the later larval stages to students in my upper-division class; and (2) let my colleague know how likely it is that the urchins I loan to her will be spawnable.

4 November 2015 © Allison J. Gong
4 November 2015
© Allison J. Gong

I know, it ain't as romantic as the Ritz-Carlton but this is where I hope to make the sea urchins have sex. We have our victims lucky individuals in their "live only" tub, two beakers for eggs, two sperm dishes on ice, a box of glass pipets, a bottle of magic juice, and a syringe with needle to get the magic juice into the animals. Ready to go!

What is the magic juice, you ask? It's a solution of KCl in filtered seawater. I'm not sure exactly how it works, but here's what I think happens. We use a solution of MgCl2, a similar salt, to narcotize animals before dissecting them. Sea urchins sitting in a bath of  MgCl2 isotonic with seawater get sleepy pretty quickly, becoming entirely nonresponsive after about 30 minutes. I suspect that KCl has a similar effect. We inject KCl into the main body cavity of the urchin (I call this "shooting them up") and I think it relaxes the muscles surrounding the gonopores. If the gonads are ripe, then gametes are released as the gonopores open. If gonads are immature, then nothing happens.

A sea urchin is a well-armored beast. Its endoskeleton, or test, is a solid structure composed of calcareous ossicles that are perforated only where tube feet extend. Getting a needle through the test without damaging the animal is pretty much impossible, so we go through the peristomial membrane instead. This membrane surrounds the mouth on the oral (bottom) side of the urchin. It's the only way to get into an urchin without breaking the test.

The urchins don't seem to like being injected with KCl--they wave their tube feet and spines all around and generally appear somewhat agitated--but they don't suffer any lasting effects.

If the urchins are ripe, they should start spawning shortly after being injected with KCl. Sometimes the response is immediate, with urchins pouring out gametes through all five gonopores at an astounding rate. Today it was much slower. It took about 5 minutes for the first female to spawn:

Spawning female sea urchin (Strongylocentrotus purpuratus). 4 November 2015 © Allison J. Gong
Spawning female sea urchin (Strongylocentrotus purpuratus).
4 November 2015
© Allison J. Gong

That little blotch of pale orange is is the mass of eggs that she is spawning. At this point you can pipet off the eggs into a beaker of filtered seawater, but I decided to go the less-invasive route and simply invert the spawning animal onto a beaker filled with water and let the eggs drop to the bottom as they flowed out of her.

The only difficulty with this method is that the animal doesn't like being upside down and immediately tries to right herself. I kept having to remove her from the beaker and replace her in the orientation we wanted. I designated this urchin as F1. She gave us a decent number of eggs. A second, smaller female (F2) spawned just a few eggs but we kept them all.

Sperm get a different treatment. I had only one male spawn this morning and he wasn't exactly a gusher. I pipetted off the concentrated sperm into a cold dish on ice, and didn't dilute the sperm until the eggs were ready for fertilization.

UP NEXT: Fertilization and subsequent events.

The juvenile sea urchins I've been raising this year are now nine months old. Back in June I put them on three different macroalgal diets and have been measuring their test diameters monthly. I do the measuring in the first week of every month, and today was the day for November. Over the past few weeks I lost a lot of my Ulva urchins, for no reason that I could discern. Judging from the poop production they were definitely eating, but on some days there would be a handful of corpses in the bowl when I changed the water. They all seemed healthy and happy today, including this beautiful creature:

Juvenile sea urchin (Strongylocentrotus purpuratus) that has been eating Ulva, age 9 months. 2 November 2015 © Allison J. Gong
Juvenile sea urchin (Strongylocentrotus purpuratus) that has been eating the green alga Ulva sp., age 9 months.
2 November 2015
© Allison J. Gong

Seriously, this has to be the most gorgeous photo of a sea urchin I've ever taken. This individual is the largest of my Ulva urchins, with a test diameter of 12.7 mm. I love the coloration of this animal: the younger spines are green, the older spines are pale purple, and the tube feet are beautifully transparent and tipped with purple suckers.

By contrast, the urchins eating Macrocystis continue to be a more uniformly golden color:

Juvenile sea urchin (Strongylocentrotus purpuratus) that has been eating the kelp Macrocystis pyrifera, age 9 months. 2 November 2015 © Allison J. Gong
Juvenile sea urchin (Strongylocentrotus purpuratus) that has been eating the kelp Macrocystis pyrifera, age 9 months.
2 November 2015
© Allison J. Gong

This Macrocystis urchin is actually a tad bigger than the Ulva urchin and has a test diameter of 13.0 mm. It looks smaller because its tube feet are fully extended, so I had to zoom out a bit to get the entire body in the frame. It was also crawling around very fast and I had to hold it down to get it centered, then remove the forceps and take the picture quickly before it walked out of the picture. Every photo of this individual that I managed to get is a little blurry because of the movement.

Last but not least, the urchins eating coralline algae are hanging in there. None of them died in the past month and they are growing. Their color patterns are qualitatively different from the those of urchins eating Ulva or Macrocystis. To my eye there is more contrast in the coralline urchins; they all seem to have prominent dark coloration in the lines that radiate outward from the apical region. The other urchins have it too, but in the coralline urchins this dark pigmentation is concentrated into more clearly defined streaks and contrasts more strongly with the paler background color.

Juvenile sea urchin (Strongylocentrotus purpuratus) that has been eating coralline algae, age 9 months. 2 November 2015 © Allison J. Gong
Juvenile sea urchin (Strongylocentrotus purpuratus) that has been eating coralline algae, age 9 months.
2 November 2015
© Allison J. Gong

This animal, with a test diameter of 6.08 mm, is about half the diameter of the largest of its full siblings in each of the other food treatments. Food quality definitely has an effect on size, as these data indicate:

Test diameters of juvenile sea urchins (Strongylocentrotus purpuratus) on three food treatments. 2 November 2015 © Allison J. Gong
Test diameters of juvenile sea urchins (Strongylocentrotus purpuratus) on three food treatments.
2 November 2015
© Allison J. Gong

It remains to be seen whether or not I'll be able to provide Ulva and Macrocystis to these animals throughout the winter. If we get the strong El Niño storms that are predicted, the nearshore algae could be wiped out for a while. I'll make sure that if I run out of one food then urchins in the other treatment will also fast until I can feed both of them again. In the meantime, because the coralline urchins are so far behind in their growth, I'll continue to give them access to food. I don't want any of them to die of starvation, and the coralline eaters are the most vulnerable, I think.

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