postembryonic development. L1 larvae are very small and
fragile and are often best manipulated in liquid.- Every 48 h, deposit 10μL aliquots onto freshly seeded NGM
plates (with OP50). Make at least two duplicates per condition
and strain. - Allow time for the liquid to disappear, and then count the
number of L1 larvae per plate (initial) (seeNote 7). - The following day, count the number of moving animals as
viable (those that are moving or those that made a trail in the
bacterial lawn). - The survival rate can be calculated as the number of viable
animals/initial number seeded.
3.1.2 Post-diapause
Recovery Defects
and Determination of Adult
Survival
- To monitor for growth defects induced by L1 starvation, keep
the plates at 20C, and let the animals develop. - It is important to monitor the animals periodically throughout
postembryonic development for various growth or develop-
mental phenotypes (i.e., 24 h, 48 h after being placed on
food and then at the L4, L4þ1d, L4þ2d, and L4þ3d). - To calculate adult survival, transfer 50–100 L4 stage larvae
onto 2–3 OP50-seeded plates (seeNote 8). - Count the initial number of animals at L4 stage (day 0).
- Record the number of adult animals that are present on
the plate every 2 days from day 0 (L4þ2d to L4þ8d) (see
Note 9). - Compare with the initial number of animals transferred onto
the plate.
3.1.3 Fertility
Phenotyping: Sterility
and Brood Size
l Adult sterility: adult hermaphrodites that were subjected to
varying durations of starvation during the early L1 stage
(L1 diapause) demonstrate a substantially high frequency of
sterility. We use the following method to quantify the defects
in fertility that arise after acute starvation (Fig.1):- Transfer 100–150μL of L1 stage larvae that were subjected
to varying durations of L1 diapause onto OP50-seeded
plates. - Allow them to grow and develop for 2–3 days at 20C, until
they reach the L4 larval stage (seeNote 10). - Single 50–100 L4 stage larvae that were subjected to vary-
ing durations in the L1 diapause to individual bacteria-
seeded plates (seeNote 11). - Let them grow so they can eventually self-fertilize and gen-
erate an F 1 generation of progeny.
570 Emilie Demoinet and Richard Roy