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I am astonished at how quickly my Pisaster larvae are growing and developing. This week saw their 3-week birthday, and today they are all of 24 days old. And look at how much they've changed since Monday!

Brachiolaria larva of Pisaster ochraceus, age 24 days. 26 June 2015. © Allison J. Gong
Brachiolaria larva of Pisaster ochraceus, age 24 days. 26 June 2015.
© Allison J. Gong

This individual measures 1500 µm long, not including the length of those two long brachiolar arms on the posterior end. The main part of the body barely fits in the field of view under the lowest magnification of my compound microscope. Those long arms are very flexible, and today I observed that the animal reacts to sudden bright light by flipping them up towards the anterior end. The body can be scrunched into a surprisingly small ball, too, as I saw when I sucked it up into a pipet. Lacking the skeletal arm rods of the sea urchin's pluteus larva, this brachiolaria's entire body can be squished and flexed along any axis.

This video shows a little of how the brachiolaria larva moves around. I've trapped it under a cover slip in a large drop of water on a depression slide so it can't swim away, but isn't being harmed. If you look closely you can see how tiny food cells are swept along by the current generated by the ciliated band. This is a ventral view, so you are looking down on the larva's front. The anterior end is to the left.

I've also been playing around with darkfield lighting, just because it's fun. Everybody should do things just because they're fun. Sometimes the fun stuff is also really cool:

Brachiolaria larva of Pisaster ochraceus, age 24 days. 26 June 2015. © Allison J. Gong
Brachiolaria larva of Pisaster ochraceus, age 24 days. 26 June 2015.
© Allison J. Gong

While I had the darkfield lighting working I shot another video, this time focusing through various focal planes to show the three-dimensional structure a bit more. You can still see the food particles zooming around.

The next major developmental hurdle for me to look for is the formation of the juvenile rudiment. I expected to have another couple of weeks before rudiments would start forming, but given how fast things are happening I might not have much more time at all.

Why are these larvae developing so quickly? We know that development and growth rates for many marine invertebrates are temperature-dependent: both occur more quickly at higher temperatures. Surface seawater temperatures at the marine lab have been elevated for the past few weeks, hovering at 14-16.5°C for as long as these larvae have been alive. The warmer water increases metabolic rate, thus faster growth and development.

Is this a good thing or a bad thing? Well, that's what I'm not sure of. My gut feeling is that it could be either, depending on food availability. One risk of higher metabolic rate is that the animal burns through its food supply more quickly. We can mitigate that risk by making sure that the larvae get fed every day and that their guts remain full at all times. Another risk of fast growth is the larvae could reach the developmental stage at which they could undergo metamorphosis, but not have had time to stockpile enough energy reserves to make it through the metamorphic process or survive long enough post-metamorphosis to grow the juvenile gut and begin feeding.

I can't do anything about the elevated water temperatures, so will just have to wait and see what happens with these larvae.

1

A few months ago, a former student invited me to participate in an activity with local Girl Scouts. The Scouts have a camp this weekend at Henry Cowell Redwoods State Park, and this year their theme is "Commotion in the Ocean." The former student, whose name is Thomas, works for the Squids for Kids program run jointly by the Hopkins Marine Station (the marine lab facility of Stanford University) in Pacific Grove and the NOAA Southwest Fisheries lab here in Santa Cruz. Squids for Kids provides Humboldt squids (Dosidicus gigas) to schools and other kid-focused programs around the country, along with instructions on how to dissect the squids and identify their parts. I think the way it worked is that the Girl Scouts applied for squid and Thomas was assigned the event. He invited me to join him because the Scouts thought it would be good for the girls to see a woman doing the dissection and getting all dirty.

Standard disclaimer: I feel very uncomfortable when people ask me to be a role model for girls, boys, women, men, whoever. It makes me feel self-conscious, as though I'm being scrutinized for a certain intangible quality of role-model-ishness and could somehow come up failing, and that I have to be better than I actually am. So I always go into these things with a little apprehension.

The thing about dissecting a Humboldt squid is that you can't go just part of the way into a squid; you have to dive in with both hands and resign yourself to the smell. Humboldts are large animals, compared to the ones I'm used to working with, and are easy to dissect:  All you do is make a cut open the mantle and all the internal organs are there to observe.

Thomas shows the girls what the Humboldt squid (Dosidicus gigas) is all about, 26 June 2015. © Allison J. Gong
Thomas shows the girls what the Humboldt squid (Dosidicus gigas) is all about, 26 June 2015.
© Allison J. Gong

Problem with just diving into a squid is that once you do, you can't take any more pictures because your hands get all gunked up. This is the only photo I snapped of the morning's activities before things got very smelly. I really didn't want to smell it on me for the next three days so I wore a lab coat and a glove on my left hand, leaving my right hand "clean" so I could drink my tea and keep an eye on the time. Even so, my right hand still has a bit of squid stink after several hours of near-continual dunking in either seawater or hot freshwater. Maybe I'm just imagining that I still smell it.

Experiences like today remind me that I'm not very good with young kids. I am simply not accustomed to dealing with their short attention spans and don't know how to distill an explanation into 25 words or fewer, which is what seems necessary for the youngest Girls Scouts at camp today.

That said, there was one girl I found very intriguing. I don't know her real name but her camp name was Rockcod. She was maybe 9 or 10 years old. She didn't want to touch any part of the squids even though her friends were getting in there and touching the gills, eyeballs, tentacles, and innards. Rockcod told me that her dad does a lot of fishing and she goes with him. They've never caught squids but catch lingcods and various rockfishes, which they take home and eat. Her uncle once caught a yellowfin tuna that was "as big as the table," probably about four feet long.

Despite adamantly not wanting to touch the squids, Rockcod was clearly fascinated by them. She left our station to participate in other activities but kept coming back and asking questions. She wanted to know what all the parts were and really wanted to be around when we opened up the next squid. She asked all the right questions:

  • How do you know if it's a boy or a girl?
  • Where is the ink? Can you write with it?
  • How come two of its arms are so much longer than the others?
  • Where is the mouth?
  • A squid has three hearts? No way!
  • Can you eat it?
  • What do they eat?

Several of the other girls (and most of their adult chaperones) were a bit squeamish and/or offended by the smell. I heard "Ew, that stinks!" more than a few times. Well, they do stink, there's no getting around it. Still, I'd rather smell an honestly dead squid than one that has been preserved in formaldehyde. And you do get used to the smell after a while. Except that I still catch a whiff of it emanating from somewhere on my body every once in a while. Hopefully it goes away with my next shower.

And I did get a thank-you gift!

IMG_4516

 

You know how the saying goes. I just wanted to share how beautiful this larva is.

Brachiolaria larva of Pisaster ochraceus, age 20 days. 22 June 2015. © Allison J. Gong
Brachiolaria larva of Pisaster ochraceus, age 20 days. 22 June 2015.
© Allison J. Gong

I have nothing to add. More on Friday, probably.

 

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.

Early brachiolaria larvae of Pisaster ochraceus, age 17 days. 19 June 2015. © Allison J. Gong
Early brachiolaria larvae of Pisaster ochraceus, age 17 days. 19 June 2015.
© Allison J. Gong

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?

Brachiolaria larvae of Pisaster ochraceus, aged 17 days. 19 June 2015. © Allison J. Gong
Brachiolaria larvae of Pisaster ochraceus, aged 17 days. 19 June 2015.
© Allison J. Gong

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:

Brachiolaria larva of Pisaster ochraceus, age 17 days. 19 June 2015. © Allison J. Gong
Brachiolaria larva of Pisaster ochraceus, age 17 days. 19 June 2015.
© Allison J. Gong

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.

1

THE ANSWER TO YESTERDAY'S PUZZLE IS . . .

. . . drum roll, please . . .

Microcladia coulteri!

I showed you this:

Mystery red alga, 18 June 2015. © Allison J. Gong
Mystery red alga, 18 June 2015.
© Allison J. Gong

but what you really needed to be certain of the ID was the rest of the photo:

A mystery no more! Microcladia coulteri growing epiphytically on another red alga, 18 June 2015. © Allison J. Gong
A mystery no more! Microcladia coulteri growing epiphytically on another red alga, 18 June 2015.
© Allison J. Gong

Huzzah again for natural history! I love it when natural history provides the answer to a taxonomic or identification question. Sometimes you need to see the organism where it lives in order to understand what it's all about. Quite a lot of modern biology has to do with grinding up organisms and examining their DNA; while I do appreciate the evolutionary and ecological insights these data provide, it's really not my cup of tea. I'd rather spend my time looking at intact organisms than their molecules, and getting outdoors to see them in nature instead of running gels and staring at computer algorithms. As in most other walks of life, it takes many kinds of work to get at the whole picture in ecology, and I am grateful to be able to contribute a little piece to the puzzle.

9

If you visit the California rocky intertidal in the spring or summer, one of the first things you notice will be the macroalgae, or seaweeds. They are incredibly abundant and diverse this time of year, covering just about every bit of rock. In fact, in a landscape sense the only visible organisms are macroalgae and surfgrass:

Algae-covered rocks at Pistachio Beach, 18 June 2015. © Allison J Gong
Algae-covered rocks at Pistachio Beach, 18 June 2015.
© Allison J Gong

Of the three major divisions of algae (the greens, browns, and reds), the red algae are the most diverse. We have several hundred species along the California coast, and while they don't get as big as the kelps (which are brown algae) they display an astonishing assortment of morphologies, colors, and life history complexities. Almost all of the algae in the photo above are reds. The olive-greenish stuff? Yep, those are reds; multiple genera of reds, in fact. The dark brown things? Those are also reds, again representing more than one genus.

Within the incredible diversity of red algae, today I want to focus on two species: Microcladia coulteri and Plocamium pacificum. Both of these algae have delicate branching forms that make beautiful pressings. But despite their apparently similar morphologies, they represent different taxonomic orders and have completely different lifestyles. Let's take a look at how similar they actually are:

Two unrelated but morphologically similar red algae, 18 June 2015. © Allison J. Gong
Two taxonomically unrelated but morphologically similar red algae, 18 June 2015.
© Allison J. Gong

Pretty tough to distinguish, aren't they? The specimen on the left is a bit more robust in comparison, but if you had only one of these in front of you and nothing to compare it to you'd probably be hard-pressed to determine which species it is.

This is where an understanding of natural history becomes invaluable. Since these species are morphologically so similar to each other, an extremely helpful piece of information is where (and how) each one lives. In terms of habitat, these species can be found pretty much right next to each other, so that doesn't help much. However, the surface on which each species grows tells you exactly what you need to know.

The specimen on the left in the photo above is Plocamium pacificum, a member of the taxonomic order Plocamiales. It lives from the mid-low intertidal to the shallow subtidal and is always attached to rocks, as you can see below:

Plocamium pacificum at Davenport Landing, 17 June 2015. © Allison J. Gong
Plocamium pacificum at Davenport Landing, 17 June 2015.
© Allison J. Gong

The specimen on the left was taken from a thallus that was growing on a rock. This means that it is Plocamium pacificum. Now we can label our photograph with one name.

Plocamium pacificum (left) and a mystery look-alike (right), 18 June 2015. © Allison J. Gong
Plocamium pacificum (left) and a mystery look-alike (right), 18 June 2015.
© Allison J. Gong

The specimen on the right was growing as an epiphyte ("on plant") on a large blade of another red alga. This epiphytic lifestyle tells me that it is not Plocamium, but a species in the genus Microcladia in the taxonomic order Ceramiales. When I brought it into the lab to key it out I was able to identify it as Microcladia coulteri. Three cheers for natural history!

Here's a picture of M. coulteri growing on blades of another red alga, Mazzaella sp. See how green the Mazzaella looks? Color isn't the only factor that determines which major group an alga belongs to, and can in fact be quite deceiving!

Microcladia coulteri growing epiphytically on Mazzaella sp. 18 June 2015. © Allison J. Gong
Microcladia coulteri growing epiphytically on Mazzaella sp. at Pistachio Beach, 18 June 2015.
© Allison J. Gong

Finally, we have both specimens identified:

Plocamium pacificum (left) and Microcladia coulteri (right), 18 June 2015. © Allison J. Gong
Plocamium pacificum (left) and Microcladia coulteri (right), 18 June 2015.
© Allison J. Gong

Which is all well and good when you have two specimens in hand that you can compare directly to each other. But what if all you have is this little bit?

Mystery red alga, 18 June 2015. © Allison J. Gong
Mystery red alga, 18 June 2015.
© Allison J. Gong

Would you say this is Plocamium, or Microcladia? What would you base your decision on? And how certain would you be?

Submit answers (with justifications!) in the Comments, and I'll give you the answer tomorrow.

Today I decided to look at some scuzz growing in one of the seawater tables at the marine lab. This table is populated mostly by coralline rocks, although I have some pet chitons running around in it.

Coralline rocks in seawater table at Long Marine Lab, 16 June 2015. © Allison J. Gong
Coralline rocks in seawater table at Long Marine Lab, 16 June 2015.
© Allison J. Gong

I picked out a promising rock and examined it under some decent light. Most of the rocks have at least some fuzzy red filamentous algae growing on them; this one also had a bit of a filamentous green, which made it a promising subject for photography. I already knew what the green was (Bryopsis corticulans) but didn't recognize the filamentous red. The Bryopsis is in the lower right corner of the rock in the photo below:

Coralline rock bearing red and green filamentous algae, 16 June 2015. © Allison J. Gong
Coralline rock bearing red and green filamentous algae, 16 June 2015.
© Allison J. Gong

What was noticeable about the Bryopsis and the mystery red is the difference in size. Bryopsis looks positively dainty until you compare it with the red. Wanting to take a closer look at the red, I plucked off a bit and mounted it on a microscope slide. This is really the only way to see what's going on with these filamentous algae, and it works like a charm. You don't have to make a cross-section or anything; you just put the piece in a drop of water, add a cover slip, and look at what you can get:

Apical tip of Antithamnion defectum, 16 June 2015. © Allison J. Gong
Apical tip of Antithamnion defectum, 16 June 2015.
© Allison J. Gong

What first caught my eye was the rather simple branching pattern. The central axis is made up of roughly rectangular cells, each of which has two side branches that are opposite each other. Each of the side branches has branchlets on only the upper surface. Branching like this is relatively easy to draw (things spiralling around in three dimensions are really difficult for me), although my drawing isn't nearly as pretty as the real thing.

This microscope view, along with my little sketches, provided me with enough information to key out this alga even though it didn't have any reproductive structures. According to the dichotomous keys in Marine Algae of California* (the book that marine biologists refer to as the MAC, our Bible for identifying the algae) it is Antithamnion defectum. The MAC says that this species is common on other algae and can be found both intertidally and subtidally from southern British Columbia to Baja California. It could very well be that I see this species in the field, but these filamentous reds look pretty much the same, at least to my inexpert eye. It really does take a microscope to figure out what I'm looking at.


*Abbott, Isabella A. and George J. Hollenberg. Marine Algae of California. Stanford: Stanford University Press, 1976. Print.

1

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:

10-day-old bipinnaria larva of Pisaster ochraceus, 12 June 2015. © Allison J. Gong
Ventral view of 10-day-old bipinnaria larva of Pisaster ochraceus, 12 June 2015.
© Allison J. Gong

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!

10-day-old bipinnaria larva of Pisaster ochraceus, drawn from life. 12 June 2015. © Allison J. Gong
10-day-old bipinnaria larva of Pisaster ochraceus, drawn from life. 12 June 2015.
© Allison J. Gong

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!

Part of what makes the marine algae so fascinating to me is their life cycles. I'm intrigued by organisms that do things differently from us. And to be honest, from the perspective of someone who studies invertebrates and their life cycles, we humans are rather boring: we're born into in one body, reproduce (maybe), and then die, all in the same body. Ulva, on the other hand, follows the typical plant example and has a life cycle that includes alternation of generations.

Without going into too much detail, let's just say that Ulva has two generations within a single life cycle, one called a sporophyte and the other called a gametophyte. The difference between the sporophyte and gametophyte is the number of chromosome sets found in the cells of the respective generations: sporophytes have two sets of chromosomes per cell, a condition which we describe as being diploid (2n), while gametophytes are haploid (1n) and have only one set of chromosomes per cell. The diagram below lays it out nicely. Note that the gametophyte in the diagram is white, while the sporophyte is green.

Alternation_of_generations_simpler.svgThe little white circles in the diagram above are the reproductive cells. These cells are produced by either the gametophyte (in the case of gametes) or the sporophyte (in the case of spores).

Now, determining if what you're looking at is a sporophyte or gametophyte can be easy or difficult, depending on whether your species is isomorphic ('same form') or heteromorphic ('other' or 'different form'). Unfortunately for us, Ulva happens to be isomorphic, which means that the sporophyte and gametophyte are for the most part morphologically indistinguishable. However, if you knew what kind of reproductive cells a particular generation produces, you could deduce whether that generation is a sporophyte or a gametophyte, right? So, is there any way to determine whether a 2.5 µm cell is a spore or a gamete?

Yes, there is! In the group of algae that includes Ulva the spores are quadriflagellate, which is just a fancy way of saying that each one bears four flagella. The gametes are biflagellate, having (you guessed it) two flagella. Now it's just a matter of counting flagella on these tiny reproductive cells released by the specimen of Ulva in my bowl.

And voilà!

Biflagellated gametes of Ulva sp., 11 June 2015. © Allison J. Gong
Biflagellated gametes of Ulva sp., 11 June 2015.
© Allison J. Gong

It's clear that these cells have only two flagella, right? This means that they are gametes, not spores, and the thallus that produced them was the gametophyte!

Pretty dang nifty, isn't it?

I was making my last run through the wet lab today, about to head off to forage for lunch before a meeting elsewhere, when I saw this in one of my bowls:

Specimen of Ulva sp. spawning, 11 June 2015. © Allison J. Gong
Specimen of Ulva sp. spawning, 11 June 2015.
© Allison J. Gong

This is one of my feeding treatments for the juvenile urchins. The sheet of green stuff is Ulva sp., a green alga several species of which grow locally in the intertidal. You also see it in harbors and estuaries. This particular bit was growing ferally in one of the large outdoor tanks in an area of the marine lab called the tank farm.

You can see that the algal body (called a thallus) has a fairly distinct edge, except for the parts that the urchins have munched through. Can you also see the cloudy pale green water that runs sort of horizontally across the middle third of the bowl? That's the stuff that caught my eye. After glancing at the clock I figured I had just enough time to take a quick peek under the scope, and if I really didn't care about eating lunch I could even snap a few pictures and still make it to my meeting on time. Anyone who knows me personally understands that I organize my life around food and the next time I get to eat. The fact that I was willing to forego lunch to look at this green spooge should tell you how exciting this was.

(It turns out that a few minutes later the person I was supposed to meet with e-mailed me and asked to postpone our meeting until next week. Yes! This means actual quality time with the microscope and the spooge.)

Here's what a spawning green alga looks like:

That undulating column on the left side is a stream of reproductive cells being released by the thallus. And yes, those are my little urchins chowing down. They like eating Ulva much better than the coralline rocks they'd been subsisting on until recently.

Under the compound scope at 400X magnification, the reproductive cells look like this:

The tiny little cells zooming around are about 2.5 µm long. The way they swim suggests that they have flagella. Do they look familiar?

They should. They look a lot your typical flagellated animal sperms! I don't think it's a coincidence that my first thought upon seeing the green stuff in the bowl was "Spooge!"

But here's where it gets tricky. For algae, looking and acting like sperm doesn't mean that something is sperm. More on that in the next post.

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