AMPK Methods and Protocols

progeny, in this first selection, we assessed brood size in descen-

dants from AMPK mutant parents that had a reduced number of

progeny (<100 progeny), and although they were not the most

severely affected (dead or sterile), they were among the most

affected by the acute starvation during the L1 diapause that were

able to reproduce. In each subsequent generation, we selected

descendants for analysis that, like their parents, also had a

reduced brood size.

1. Follow the previously described Subheading3.1.1: bleach
   healthy gravid AMPK null mutants (aak-1; aak-2), and
   maintain them 3 days without food in the L1 diapause.


2. Place 3-day-starved L1 larvae on OP50-seeded plates, and
   allow them to grow at 20C.


3. Once most of the larvae reach the L4 stage, single out
   50–100 larvae onto fresh OP50-seeded plates, and let
   them develop in order to evaluate both fertility and brood
   size as described above. This will allow you to determine if
   the parents generated a normal brood size or whether the
   reproduction/brood size was compromised.


4. Select four plates from the affected parental plates, i.e., those
   that produce small broods (<100 F 1 progeny), and single
   out between 30 and 100 F 1 L4 larvae from each of these
   four plates to individual plates seeded with OP50.


5. Incubate these plates at 20C, and allow them to reproduce
   (120–400 individual animals from 4 initial F 1 isolates) (see
   Note 14).


6. 4–6 days after the isolation, count the number of F 2 progeny
   generated by each F 1 descendant to determine its brood size
   phenotype: affected or unaffected.


7. Continue the experiment into the next generation by
   repeating the steps described above: randomly select four
   affected plates (<100 F 2 progeny).


8. From each of these plates, single out 30–100 F 2 L4 larvae in
   individual plates and allow them to reproduce (120–400
   individual animals from 4 F 2 isolates) an F 3 brood.


9. Estimate the brood size when the F 3 progeny reaches the
   L4/young adult stage as described above. These steps were
   repeated successively such that the brood size of ~120–400
   individual animals can be assessed and used for downstream
   analyses.

l Method 2: alternatively, we could not rule out that animals may

be initially fertile, producing a brood of normal size, but the

effects of the acute starvation are recorded and worsen with each

successive generation. To test this possibility, we chose to select

Quantifying Starvation-inducible Transgenerational Defects 573