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Playing matchmaker

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.

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