From zero to cleavage in. . . nine hours

A recent college graduate and fellow marine lab denizen (Scott) and I are collaborating on a project to quantify growth rates in juvenile Pisaster orchraceus stars. This is one of the intertidal species whose populations in the field and in the lab were decimated by the most recent outbreak of sea star wasting syndrome (SSWS). We are interested in seeing how quickly the stars grow once they metamorphose and recruit to the benthos, and hope that the information will help researchers guesstimate the age of the little stars that are now being seen in the field. This would in turn tell us whether the little stars are survivors of SSWS or post-plague recruits. I keep seeing people refer to them as “babies,” but they could very well be several years old. We just don’t know, hence this study.

But before we get to measure juvenile growth we have to get through larval development, which is perfectly fine by me because I’m always up for observing marine invertebrate larvae. Two weeks ago Scott and I ventured into the field in search of prospective parents. We brought back eight individuals from two different sites, making sure to leave many more in place than we took away. It was actually rather gratifying to see how many hand-sized-or-larger P. ochraceus there were. This morning we met at 07:30 to shoot up the stars with magic juice and then wait for them to spawn.

It has been a while since I tried to induce spawning in Pisaster, and I had forgotten how much longer everything takes compared to the urchins. For one thing, the magic juice itself isn’t the same stuff that we use on the urchins, and works by an entirely different mechanism. The stars’ response to the magic juice takes 1.5-2 hours, whereas if the urchins aren’t doing anything 30 minutes after getting shot up they either need another injection or simply don’t have gametes to share.

However, despite my misgivings the animals spawned. Two large females gave us enormous quantities of eggs, and three more donated trivial amounts that we didn’t end up using.

This purple individual is the one we designated Female 1. See the huge piles of salmon-pink eggs?

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Although we had to wait for a male to spawn, we finally did get some sperm and fertilized the eggs at about 12:30. Another thing I had forgotten was that Pisaster eggs, when shed, are lumpy and strange. I was used to the urchin eggs, which are usually almost all beautifully spherical and small. The stars’ eggs are about twice as big, at ~160 µm in diameter. The lumpiness doesn’t seem to hamper the fertilization process, as you can see below.

In this photo you can see the fertilization envelope surrounding most of the eggs. In stars the perivitelline space (the space between the egg surface and the fertilization envelope) is very narrow, which makes it difficult to see the envelope; in urchins the space is much larger, and as a result the envelope quite conspicuous. The rising of the fertilization envelope off the surface of the egg is referred to as the slow block to polyspermy, a mechanical barrier that keeps multiple sperms from penetrating any individual egg. There’s also a fast block to polyspermy, but it happens on a molecular level milliseconds after a sperm makes contact with the egg surface; you can’t see it happen in real time.

Cleavage in stars proceeds much more slowly than it does in urchins, too. In embryological terms, “cleavage” refers to the first several divisions of the zygote, during which the cell number increases as the cell size decreases. This inverse relationship between cell size and number logically has to occur because the embryo can’t get any larger until it has a mouth and begins to feed, which won’t happen for at least a couple of days. It took our zygotes about four hours to undergo the first cleavage division.

I left the slide on the microscope to warm up and speed development a bit, and about 45 minutes later was rewarded with this mishmash of embryos at different stages. Nine hours after we started this whole process, there were 2-cell, 4-cell, and 8-cell embryos, as well as eggs that had not divided yet.

This asynchrony in early development is another way that stars differ from urchins, and it takes some getting used to. I expect that development will become more synchronized as the embryos continue to cleave, and that hatching will occur for all of them at about the same time, probably before Thursday. At least it won’t take another 9-hour day to see how far they’ve come.