I’m trying to find the standard on differentiating L1 from L2 from L3 from L4. I can only find rough estimates and between papers they don’t seem consistent. Is there a standard for when a worm is L2 v L3 or L3 v L4? I know the change occurs after lethargus, but I don’t have a way to monitor my worms in real time streaming. I’m looking for a morphological or temporal standard I can apply to make my baseline measurements less subjective. Thanks
Just before each lethargus the animal has begun to lay down a new layer of cuticle that will suit it better in the next larval stage.
During lethargus, the old cuticle is shed (the molt), and the animal is back to being dressed in just one cuticle.
But except by TEM, I am not aware of a method to quickly detect the extra cuticle before it is shed? Perhaps by polarized light?
Once upon a time, I spent many days watching worms undergo molts, as there was then no literature describing the larval stages of C. elegans.
I found that the exact timing of molts was somewhat variable from animal to animal, and the exact length of an animal at the time of molt varied too.
I was culturing each animal on a separate well in a 96 well plate, so the shed cuticles could be viewed directly, confirming each molting event.
I used a micrometer eyepiece to view the animal lengths, and missed a lot of sleep. Not sure that I’d recommend going back to this method,
but today it could become simpler with access to a digital camera and a motorized stage to follow each animal’s molts over time.
Given this variation, it is easy to understand why the literature offers a range of sizes for each larval stage, with the size range of successive stages showing some overlap.
The common tactic is to synchronize animals according to one timepoint (fertilization, egg laying, hatch, or recovery from starvation),
and to work on animals at set timepoints thereafter to yield animals at a consistent developmental stage.
The better your synchronization, the more tightly your population will sit within a uniform developmental status.
During this development, you need consistent temperature and access to food, or the animals will vary among themselves even further.
Hope this helps.
David Hall
Hi,
you could (depending upon your set up) use a stage-specific promoter::gfp strain to follow development in real time?
For example, unc-95::gfp to determine the time point for the L2/L3 molt in individual worms;
http://celladhesionlab.com/wp-content/uploads/2010/05/ZaidelBar2010.pdf
Armed with this info, and assuming that all the worms have access to the same amount of food and are kept at the same temperature, you could effectively
count back/count forward in your video stream footage to establish the time
points for the other developmental stage transistions for each worm you are filming, or even use other markers.
Just a thought.
Steve