Shaking

### Introduction

The intertidal is a rough environment. During winter storms, waves can crash 30 feet into the air. Each year several tourists in the Monterey area are drowned. The winter is also the time that the intertidal sea urchin S. Purpuratus spawns. How do the embryos survive in these conditions? One hypothesis is that the fertilization membrane offers some protection from mechanical damage. How can we test this hypothesis?

### Procedure

Unfertilized eggs do not have a fertilization membrane, but fertilized eggs do. Thus, unfertilized eggs are the control and fertilized eggs are the variable. Next we need to duplicate the wave action. This can be done by putting a quantity of eggs and seawater into a closed container and shaking it. Sand can be added for additional scouring effect. Your data chart might look something like this:

 SCORING FOR INTACT EGGS Condition Unfertilized Eggs 15 Minute Fertilized Standing water, no shaking Gentle shaking, 1 min. Moderate shaking, 1 min. High shaking, 1 min. High shaking with sand, 1 min.

Why 15 minute embryos? This is because the time just after fertilization is when everything is changing and the embryo is most vulnerable. Waiting 15 minutes or longer, allows the fertilization membrane to harden and the plasma membrane to recover. If you have time and materials (or help from classmates), you might add a 3-5 minute fertilized column.

After the shacking or no shaking do two things:

1) Score the number of intact eggs per sample per field of view in each environment.

2) Fertilize the unfertilized eggs and let the embryos settle in small petri dishes until the next day. Score the number of swimming blastula per sample per field of view in each environment and compare to the already fertilized samples. This will give you a second chart:

 SCORING FOR SWIMMING BLASTULA Condition Unfertilized Eggs 15 Minute Fertilized Standing water, no shaking Gentle shaking, 1 min. Moderate shaking, 1 min. High shaking, 1 min. High shaking with sand, 1 min.

### Questions

1) How did you control:

• The amount of shaking?
• For the size and amount of sand?
• The size of the "wave chamber" and amount of water?
• The number of eggs added to each condition?
• The amount of sperm added afterwards to the unfertilized eggs?

2) How did you judge "intactness" or "swimming blastula"? Did you have a standard?

3) Does the fertilization membrane help protect the egg?

• Summarize the evidence supporting your conclusion?

4) How would you do this experiment differently next time?