Post #13 - 3 x 3 - we did it!!

Today ended with a bit of a celebration.  Our goal for the COT fertilization study was to cross each of three female Acanthaster with each of three males, and today we ran the 9th experiment!    We still have tubes of embryos and sperm to analyze, but I should be able to do that in the next couple of days.  Then, being forever curious, we get to decide what additional crosses we should do while we have the opportunity.

I have told you a little bit about the fertilization experiment in bits and pieces,  let me try to present a more complete picture.  After removing some testes and ovaries from adult Acanthaster, I collect the eggs and sperm and make separate suspensions of known concentrations.  (I have to be really careful not to get any sperm in with the eggs for that would ruin everything!)  This is what the suspensions look like:

The dots in the right tube are eggs, each being about 160 um (that's 0.160 mm or 0.0063 inches).  I'll show pictures of sperm some other time.  The actual mixing of eggs and sperm occurs in the aquarium building:

On the left is a locker for dive gear, and inside the gray/white box on the right is a chiller that helps regulate the temperature of the water in the aquaria.  Around here, we call the aquarium room the "Aqualogic" room because of the company that makes the system.

For each aquarium, the system allows us to control water temperature by using both heaters and chillers,  and the pH of the water by bubbling carefully controlled gas mixtures.   In an adjacent room is the equipment for controlling the mixture of air and CO2 that gets pumped into each aquarium:

The six aquaria on the left have just air bubbling into them, while the tanks on the right receive air enriched with CO2.  As you know, since the CO2 dissolves in the water forming carbonic acid, the right tanks have a lower pH (are more acidic) than the left tanks.  In addition, for Emily's experiments half of each set of tanks also have a slightly higher temperature.

Water from the tanks is filtered to remove bacteria, then pipetted into a series of culture tubes.  This can be done with or without feline help.

Each run involves eight tubes, plus three more for pH measurements.  A serial dilution of sperm is made in the eight tubes, designed so that the first tube has way too many sperm (causing some eggs to be fertilized by more than one sperm and develop abnormally) while the last tube may not receive any sperm at all (resulting in no fertilization).  The reason for doing this is that ocean acidification may affect fertilization success in different ways, and these ways might be revealed by using such a huge range of sperm densities.  Once the tubes have their sperm, then approximately the same number of eggs are added to each:

The tubes are floated in one of the aquaria, as seen in a previous post, for six hours.  At this point, the embryos are preserved.  When examined under the microscope, the results are pretty cool!

All the embryos in the row at the bottom are normal blastulae (hollow balls of small cells), except for the second one from the left that is abnormal, presumably from polyspermy (too many sperm entering the egg).  The embryo at the top is also abnormal.   For every experiment, one set of tubes is filled with regular sea water, while another set of 8 is filled with high pCO2 water.  The results from both series will be compared to see if the more acidic sea water affected fertilization.

So even though there is work yet to be done, having actually finished running our 9th cross was a good reason to celebrate - at a nearby creperie!