AMPK Methods and Protocols

5. Record the number of plates that still contain single animals
   96 h after being transferred as L4: these animals can be
   considered as sterile. Plates that generate progeny will have
   several smaller larvae on the bacterial lawn.
   l Quantification of brood size: by counting the number of
   animals on each plate, the brood size can be determined for
   each individual parent (P 0 ) that had been subjected to the initial
   diapause.


6. Fertile animals are allowed to reproduce and are then trans-
   ferred daily to a new seeded OP50 plate throughout their
   entire reproductive period (3–4 days) (seeNote 12).


7. Count the number of F 1 offspring that were present on
   each plate when they reach the L4/young adult stage (see
   Note 13).

Bleaching and o/n hatching in M9 at 20°C

Control (no starvation) L1 starvation (3 days)

L1 back on OP50, growth

Then, single 50 - 100 L4 (P 0 ) on OP50

Development

PHENOTYPING(at L2 + 4-6 days)

Affected Visibly unaffected:

Brood size <100, sterile Brood size ≥100

[P 0 ]

... P 0

...

Hypothesis 1
Affected parents have an increased
chance of transmitting brood size
defects in a transgenerational manner

Hypothesis 2 and 3

All animals (even unaffected parents)

can transmit transgenerational defects:

P 0 Parental Generation Phenotyping

and “plan” of the interesting experiment

Aff Aff Aff Aff Unaffected Unaffected

(<100) (<100) Ste (<100) (≥100) (≥100)

L1 survival

L1 immunostaining

Fertility phenotyping

Preparation for transgenerational

analysis

Fig. 1Determination of the P 0 parental phenotypes through inference from the subsequent filial generation.
Parents (P 0 ) are grown to adulthood and subjected to alkaline hypochlorite treatment to recover embryos. The
embryos are hatched overnight to yield emergent L1 stage larvae. These animals are then allowed to grow and
are counted to confirm whether the reproductive fitness of the parents is “affected (Aff)” or “Unaffected” by
the initial starvation based on brood size; Ste is sterile

Quantifying Starvation-inducible Transgenerational Defects 571