Jan 28, 2022
Staging colonial ascidians
- 1University of Fribourg
- Blanchoud lab, UNIFR
Protocol Citation: Simon Blanchoud, Marta Wawrzyniak 2022. Staging colonial ascidians. protocols.io https://dx.doi.org/10.17504/protocols.io.bsgznbx6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: February 17, 2021
Last Modified: January 28, 2022
Protocol Integer ID: 47353
Keywords: colonial ascidians, Staging, Botrylloides diegensis, tunicates
Abstract
This protocol has been successfully used to stage Botrylloides diegensis in our laboratory and was based on the following publication:
Materials
1 glass Petri dish with a slide holder
high magnification stereoscope
Artificial Seawater (ASW)
Staging
Staging
30s
30s
Select the slides to be staged.
Clean the colony. See Cleaning colonial ascidians V.2
Fill the Petri dish with ASW. See Artificial sea water V.2
Observe the organization of the zooids and the systems. Check for signs of takeover:
No signs of active filter-feeding.
Zooids within systems are not properly fused yet.
Remnants of resorbed zooids are still visible.
Turn the colony upside-down. Look for buds to stage.
Start at low magnification, look for well-visible buds (the one that are not too much underneath the zooid-parent, typically posterior to the adult).
Once the buds are identified, zoom in.
Monitor the activity of the heart. Check for signs of regular beating:
- Flow will be flowing through the bud at all stages, even without heart beats.
- At early stages, heart will be erratic and weak.
- Every minute or so, flow will be reversed and heart beat will stop transiently.
Look for secondary budlets to stage.
Budlets are often hidden or difficult to observe, focus on the most evident ones.
Variability can be observed within a colony. Monitor multiple buds and use the average stage as your reference.
Use the provided identification scheme to infer the stage from your observations. 
staging_scheme.png
staging_scheme.png The best method to ensure the current stage is to confirm it by staging on consecutive days. Stages should proceed approximately by one per day (at 20°C).
Take a couple of representative images for archiving.