Starting at the beginning, again

According to my notes at the lab, the last time I spawned urchins was December of 2016, making it four years ago. It has always been something I enjoyed doing, but I didn’t have a reason to until now.

When the coronavirus pandemic began almost a year ago now, access to all facilities at the marine lab was restricted to a group of people deemed essential. In my case, “essential” had to do with the fact that I keep animals alive. There were many hoops to jump through and inane questions to answer—for example, “What will happen if you don’t go in to check on water and food?” and “How many animals will die if you do not have access to the lab, and how much effort [i.e., $$$] would it take to replace them?”—but in the end someone higher up in the food chain exercised some common sense and decided to let me have continuous access to the lab. So I’ve been at the lab pretty much every day, to check on things and make sure that air and water are flowing.

So over the summer we were running sort of bare-bones operations at the lab. There were many fewer people looking after everyday things. The autoclave broke and wasn’t fixed until September. One of the casualties of this less-than-normal vigilance was one of the cultures in the phytoplankton lab. Our Rhodomonas flasks had been contaminated since late 2019, and we were struggling to rescue them. I tried so hard to keep them going ahead of the contamination, but ultimately failed. As of this writing all of the old Rhodomonas cultures have died.

In October, after the autoclave had been repaired, I decided to take action and replace our inevitably doomed Rhodomonas cultures. I found a company that sells small aliquots of many marine microalgae and ordered a strain of Rhodomonas that was isolated in Pacific Grove. May as well see if a local strain of algae works as a food for local larvae, right? The new Rhodomonas cultures seem to be growing well and it’s time to see of urchin larvae will eat and thrive on it.

About a month ago I collected 10 urchins to spawn. Yesterday was their lucky day! Purple sea urchins (Strongylocentrotus purpuratus) are broadcast spawners, and spawning is both inducible and synchronous. We can take advantage of the inducibility to make them spawn when we want, as long as they have ripe gonads. The difficulty is that we can’t tell by looking whether or not an urchin is gravid, so all we can do is try to induce them and then hope for the best.

As I’ve written before, we induce spawning in sea urchins by injecting them with a solution of potassium chloride (KCl). KCl is a salt solution that causes an urchin’s gonopores to open and release gametes if the gonads are ripe. I shot up 10 urchins yesterday, and eight of them spawned. An 80% spawning rate isn’t bad, but only two of the eight were female and neither of them had a lot of eggs to give.

Since the gonopores are located on the aboral (top) of the urchin, the easiest way to collect eggs is to invert the animal on a beaker of seawater, like so:

In nature the eggs, which are a pale orange color, would be whisked away by currents to be (hopefully) fertilized in the water column. In the lab we can collect the eggs in the beaker, as follows:

This is much less damaging to the animal than trying to pipet eggs off the top of the urchin.

We try to collect sperm and keep it dry, so there is no putting males upside-down on beakers of water. Instead we pipet up the sperm and keep it dry in dishes on ice. When it’s time to fertilize the eggs we dilute the sperm with filtered seawater and add a small amount to the eggs.

One of my favorite things ever is watching fertilization take place in real time, under the microscope. It truly is one of nature’s most amazing phenomena. It is a great thrill to watch the creation of new beings.

In the video you see eggs being bombarded with sperm, probably at much higher concentrations than they would encounter in the wild. It is common knowledge that it takes only one sperm to fertilize an egg, but what would happen if two sperm penetrated an egg at the same time? I’ve written about polyspermy and the fast and slow blocks thereto, in case you’d like to refresh your memory about what is happening in the video.

A successfully fertilized egg is easily recognized by its fertilization envelope, which is the slow block to polyspermy.

After fertilization, the next step to watch for is the first cleavage division, which occurs about two hours later.

Aren’t they pretty?

Over the next day or so the cleavage divisions continue, resulting in the stage that hatches out of the fertilization envelope. This stage is a blastula, which is a hollow ball of ciliated cells. The hollow space inside is called the blastocoel, and it is here that the larval gut will soon develop.

It’s easier to see the 3-dimensional structure of the blastula by watching it spin around.

As the blastula rotates under the coverslip, you can see the ciliary currents that would propel it through the water. You also see some objects that look like sperm and are, in fact, dead sperm, getting caught up in the currents.

The blastula is the same size as the egg. The embryo can’t begin to grow until it eats, which won’t happen until it has a gut. Over the next few days an invagination will begin at a certain location on the blastula which is called the blastopore; this invagination will eventually form the first larval gut. At that point I will have to start feeding them and calling them larvae.

And just to remind you of our humble beginnings, we begin life in much the same way as sea urchins. That blastopore, or initial opening to the larval gut, is the anus. The mouth doesn’t exist until the invagination breaks through to the opposite end of the embryo. So yes, like the sea urchin, you had an anus before you had a mouth!