visibly unaffected parents (those that generated more than
100 F 1 progeny), and then using the same type of multigenera-
tional analysis described above, we then select the affected prog-
eny (born from an F 1 parent that produced a<100 progeny), as
described below:- Allow P 0 parents to self-fertilize (as described instep 3
Subheading3.2.1). - Randomly select four plates not affected (more than
100 F 1 ), and single out 50 F 1 L4 larvae to individual plates
seeded with OP50. - Incubate these plates at 20C, and allow them to reproduce
(200 individual animals from 4 initial F 1 isolates), and esti-
mate the brood size of each of the F 1 parents as affected or
unaffected plates. Some of these parents have a low brood
size, while others are almost normal. - Randomly select four affected plates (that produced a low
brood size), and we redo this selection at every subsequent
generation. - Single out 50 F 1 L4 larvae to individual plates seeded with
OP50 from each of these four affected plates, let them
develop, and estimate their brood size as affected or
unaffected. - For every subsequent generation, select progeny from four
affected plates, let them develop, and estimate their brood
size.
l Method 3: it is also possible that the animals that are born from
normal parents and themselves produce a normal brood might
eventually demonstrate the brood size defect only in later gen-
erations. This would be typical of a progressive loss of reproduc-
tive fitness over multiple generations. If we constantly select
animals from parents that produce normal brood sizes and eval-
uate their brood size, we could potentially determine if this
epigenetic phenomenon is recapitulated even in animals that
appear to be reproductively normal. - Derive the transgenerational analysis from the four ran-
domly selected unaffected P 0 plates, as we described above
in Method 2. - Single out 50 L4 F 1 larvae from each of these four plates that
harbored the parents that had normal brood sizes and
appeared unaffected based on the above criteria.
574 Emilie Demoinet and Richard Roy