Today we checked to see what development occurred since Wednesday’s strip spawn and KCl-treatment trials. There were so few eggs in the tripours to begin with, that there ended up being way too small of numbers of D-hinge larvae that I ended up dumping the trial and we’ll have to rethink this and re-do it. Details in post.

Counts using coulter counter

We started out using the coulter counter to see if it would work. We screened the tripours (used 31-33 because those are the extras) on 60micron screens. We realized quickly that there was a lot of larger junk that stayed in the sample, including pollen because the table wasn’t covered over the two days. We then swithed to screening with a 90micron screen into a 60 in order to catch the big stuff. I suspended them in 10ml seawater.

The first counts of the coulter counter were pretty high, but then the next two tripours were very low. Counts listed below:

Counts are #cells/ml in 10ml samples.

Tripour No. Trtmnt Count 1 Count 2 Count 3 Avg
1 0mM KCl 86 75 85 82
16 50mM KCl with hydration step 13 12 11 12
25 80mM KCl 29 43 34 36

The coulter counter was likely counting things that weren’t D-hinge.

Counts by hand

We then decided to do counts by hand. I screened them the same way and suspended them in 10ml seawter just like with the coulter counter.

I sampled out 1ml after mixing well by flipping contatiner upside-down a few times. I put the sample on a slide and put two drops of lugols in order to preserve the organisms so that they wouldn’t be moving around when I was trying to count them.

The counts were also very low. I didn’t count all tripours - I just started out by looking at a bunch of different treatments to see if it was worth me continuing and counting them all. Counts are below:

Counts are #D-hinge/1ml in 10ml samples

Tripour No. Trtmnt Count/ml Total No.
2 0mM KCl 0 0
4 10mM KCl 4 40
8 20mM KCl 6 60
17 50mM KCl + hydration step 7 70
18 50mM KCl + hydration step 10 100
19 60mM KCl 3 30
26 80mM KCl 0 0
27 80mM KCl 3 30
29 50mM KCl without hydration 3 30

The numbers are way too small to have too much meaning. Each tripour originally had ~10,000 eggs, so in terms of percentages, the number of eggs that made it to D-hinge range from 0%-0.01%.

Notes from this trial/things to change for next time

  • Didn’t have a true “negative control”. A true negative control would have been having a tripour of just eggs (no sperm added to fertilize)
  • 10,000 eggs/tripour is sticking with the 10eggs/ml rule that the hatchery has, but maybe it’s too small a scale for capturing any differences in this experiment. Maybe try putting more eggs in the tripours…. or using larger buckets?
  • Keep male and female geoduck separate after biopsy punching gonads to determine sex - may be cause of early polar body sightings (see this post).
  • Use less treatment groups, easier to manage and may be better to start on a much smaller scale, then get more complex as we learn what works