Figure 10.—

Relaxed stringency of early selection allows some persistence of silencing in the F₄ generation. (A) Schematic of assay. We designed a selection process to evaluate the relationship between the strength of the silencing response measure by the silencing frequency in a particular pedigree, to the persistence of the silencing across generations. Degrees of silencing efficacy were determined by the silencing frequency and brood size of the selected animals. We used the frequency of silencing to classify pedigrees as transmitting at highest, intermediate, or low silencing efficacy. When then used brood size as a second criterion to guide the selection of individuals to analyze the silencing frequency of the next generation. In the “highest silencing efficacy” group, we selected from plates with the largest brood sizes (>90). In the intermediate silencing efficacy group, we selected individuals from plates with broods between 30 and 80. Animals where most siblings have no viable progeny represent low-silencing-efficacy groups and were not used. (B) Intermediate silencing efficacy populations overcome the F₄ bottleneck. We followed the less stringent selection scheme of intermediate silencing efficacy and found that 7/10 F₄ sibling groups had at least some viable F₅ progeny. This is in contrast to the F₄ bottleneck that we observed when we used the “highest silencing efficacy” selection (data in Figure 3 and data not shown). Error bars represent 1 SD. As the manner in which animals are chosen to carry forward the silencing trait is critical in determining the behavior of descendant populations, we describe the selection process for the intermediate silencing efficacy group in some detail as follows: The F₄ animals, classified as descendants of continuous intermediate silencing efficacy selection, were derived from one of five injected animals. Of the original five injected animals, we picked all viable progeny and arbitrarily assigned each a color (purple, red, green, orange, and blue). Three days later, all injected animals had viable progeny. We individually plated the F₁ animals and scored the frequency of viable F₂ progeny. Only the orange F₁ family had no viable progeny (n = 15). All F₁ plates from blue (n = 40), red (n = 54), purple (n = 91), and green (n = 20) had viable progeny. We selected F₂ animals from eight F₁ families: two blue, one green, two purple, and three red. Each F₁ family gave rise to an F₂ sibling group (designated by two letters). From the blue family, the BD group had 100% plates with viable progeny while the BE group had only 7.8%. From the green family, GF had 80%; from the purple families, both PH and PJ had 100% transmission; and from the red families, RA had 94.7%, RB had 80.7%, and RC had 100%. The RA and RB lineages fulfilled the criteria for selection of intermediate silencing efficacy. To extract the populations with smaller brood sizes, we removed the F₂ animals at day 2. On day 3, we scored the F₂ plates. This allowed us to better assign a generation to animals by increasing the age difference between F₃ adults and young F₄ larvae. Two days after removing the F₂ adults, we surveyed all plates of F₂ animals with F₃ broods. Plates with large brood sizes had depleted the bacterial lawns. Plates with “smaller” F₃ broods were not depleted of bacteria (fewer worms on plate, more food per worm) and their growth was uninterrupted. We used F₄ animals from small broods to represent the intermediate silencing efficacy groups.