Seastars, Lasers, and Blue Stains

Hello! Last time I posted I was just finishing my first few trials of blastomere separations on the seastar, Evasterias. While the seastars were extremely reproductive (one female, ‘Big Mama’ gave us over 50 million eggs!), we weren’t able to perform full blastomere separations as the eggs did not develop and we lost a trial due to a seatable flooding. Female seastars are only able to spawn once in a season, so with all of the failed trials, we ran out of female Evasterias to spawn. Luckily, there is another seastar here, Pisaster ochraceous, which we could use instead of the Evasterias. Pisaster egg sizes are comparable with Evasterias eggs, and the two species have a similar development, allowing us to use Pisaster for our comparative study.

The first spawning of the Pisaster yielded us more eggs than we would need (20 million eggs), so we attempted to remove the fertilization envelope of the eggs with a urea treatment and then separate the cells at the two cell stage to reduce the egg size. Unfortunately, the Pisaster eggs were more affected by the urea treatment and did not cleave into two cells, so we were unable to perform any blastomere separations. Luckily, a colleague in the lab we are working in, who is working with Dr. Allen on a different aspect of this project, Dr. Bruno Pernet, had a laser that we were able to use to see if we could kill one of the cells at the two cell stage instead of using a urea treatment. This method of reducing egg size was much more viable, so it is the method we ultimately decided to use.

In order to have enough larvae to rear to metamorphosis that had been zapped with the laser (which is all done by hand), I placed 40-50 eggs on a slide at a time, and Dr. Allen then zapped the eggs with the laser. These eggs were rinsed into 17 different petri dishes as our replicates, with 40-50 eggs in each dish. We allowed the embryos to develop and hatch into larvae. Following hatching, we counted the larvae in each petri dish, stained half of our control replicates and half of our zapped replicates with a blue vital stain. We are now rearing 10 replicates of 20 half larvae and 20 whole larvae to metamorphosis (which should take another 30 days or so) to see what the effect of halving the eggs will be on the larvae and juveniles. We’re also about to start a new project, which I’ll post about soon, where we are doing a very similar experiment on Pteraster tesselatus, a seastar with very large eggs compared to both Pisaster and Evasterias and a different mode of development.

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A Pisaster we found in the field during a low tide.

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A Pisaster spawning, all of the yellow are eggs

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A seastar larva after it has hatched

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All of my petri dishes filled with larvae. The red labels were embryos that were halved with the laser and the green labels are controls.

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The stain we are using to stain the larvae blue so we can tell them apart

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The view from our lab after a sunshower