Notes from a California naturalist

My most recent batch of sea urchin larvae continues to do well, having gotten through the dreaded Day 24. I haven’t written about them lately because they’re not doing very differently from the group that I followed last winter/spring. However, I’ve been taking photos of the larvae twice a week and it seems a shame to let them go to waste, so I’ve put together a progression of larval development. As a reminder, the last time I wrote about these larvae they were six days old.

Age 9 days: The larvae had four arms and were growing their skeletal arm rods. Their stomachs, which we keep an eye on because their size can tell us whether or not we’re feeding them enough, were a bit small but not so much so that I worried.

Age 12 days: The larvae were growing their third pair of arms. Some had just begun growing the fourth pair of arms. Red pigment spots also start appearing all over the body. Some larvae develop lots of red spots, others have very few. Notice that the stomach is slightly pear-shaped; this is normal.

Age 17 days:  This larva doesn’t look appreciably different from the previous one. This photograph, though, is a bit clearer. The stomach has taken on a pink tinge, due to the red color of the food the animal is eating, and the mouth is the large rounded triangular in the in-focus plane. The pair of skeletal arm rods that are in focus are protruding from the ends of the arms, which raises is something to be concerned about. Sometimes the first sign of imminent doom is the shriveling of the arms, so seeing the rods sticking out makes me think “Uh-oh. . .”

Age 24 days: This is about the time in larval development when things often start to go wonky. I’ve looked back at my notes from previous spawnings of S. purpuratus, and seven of the 20 cultures that crashed did so in the week between days 20-28 of development. Some of these cultures were doing well right up to the point that they all died. They were literally there one day and gone the next.

Nonetheless, the current batch of larvae continued to do well. The fourth pair of arms were slow to grow but otherwise the larvae look fine. The top larva in the picture below is lying on its back, so you are looking onto the ventral surface. On the left side of the stomach there’s a little upward-facing invagination; this is part of the initial water vascular system forming. Note also that the overall shape of the larvae is changing a bit. They are becoming less pointy and a bit rounder.

Age 30 days:  At this stage the juvenile rudiment is clearly visible. You can see it as a rather nondescript blob of stuff to the left of the gut. The fourth pair of arms have also grown quite a bit but are still considerably shorter than the others. This individual has two bands of cilia, called epaulettes, that encircle the body. These epaulettes will become more conspicuous as the larva approaches competency.

Age 33 days: Today I got lucky! The larvae looked good when I changed their water this morning <knock on wood> and although I’m keeping my fingers crossed I have high hopes for these guys. They’re about as big as they’re going to get, measuring 760-800 µm in length. They will get heavier and more opaque as the juvenile rudiment continues to develop.

The really cool thing is that one of the larvae landed on the slide exactly as I wanted it to. It happened to fall onto its left side and stayed there, so I was able to focus up and down through the body to get the rudiment into focus.

Do you see five small roundish blobs that are evenly spaced around the larger golden circular blob? The large blob is the stomach, seen in side view. Those smaller blobs are tube feet! Don’t believe me? Then take a look at this close-up:

Now if those don’t look like tube feet, then I’ll eat my hat. What’s also noteworthy about this larva is that its epaulette bands are both visible, especially the posterior-most one.

So far, so good. I won’t know how successful larval development is for these guys until they either make it through metamorphosis, or not. In a very real sense, I won’t be able to draw any conclusions about the success of larval development until they either become established as juvenile urchins, or not. One of my graduate advisors inherited a couple of sayings that he passed on to me, as well as to a whole generation of aspiring invertebrate zoologists:

The animal is always right.

and

The life cycle is the organism.

The first is a given, right? The animal knows what it is and what it’s doing, even if we humans have no clue about what’s going on and can’t decide what its name should be.

The second saying might be a little less intuitive. What it means is that, for organisms with a multi-stage life cycle, you have to consider all of the stages if you want to understand them. This is a much more holistic view of biology, and it’s the one that appeals most strongly to me. When I’m thinking as a naturalist, I find my thought process constantly switching between “forest” and “trees” as I seek to understand even a teensy bit of the world around me. While it’s easy to get distracted by all the cool details of organisms, it’s important to step back and ask myself, “What does it all mean? What is the big picture here?” So yeah. Perhaps when (if!) these larvae turn into urchins and I’ve got them feeding on macroalgae in a few months, I’ll be able to say whether or not larval development was successful. If all goes well this larval phase, as all-consuming and fascinating as it is to me, will be only a small part of these animals’ lives.

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.

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:

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.