Physiological Sexual Disorders

Worm lifespan assays were performed at 20°C as described previously (33 ) unless noted otherwise. For each lifespan assay, worms were transferred to new plates at least every other day and were scored dead or alive. Worms were scored dead if they did not respond to repeated prodding with a worm pick. Worms were scored as censored if they died because of bagging, vulval rupture, or if they crawled off the plate. Data from the censored worms were included up to the day of censorship. For each hermaphrodite lifespan assay, at least 90 hermaphrodite worms per condition were divided evenly among three plates (≥30 hermaphrodites/plate) unless otherwise noted.
In assays in which the lifespan of hermaphrodites was assessed in the presence of males, at least 45 hermaphrodite worms and an equal number of male worms were divided evenly among three plates (≥15 hermaphrodites/plate) as to control for total number of worms/plate. Each mixed sex condition begins with an equal number of hermaphrodite to male ratio (ex. 15 hermaphrodites: 15 males). The number of males on the plate stayed fixed throughout the assay. Wild-type male worms between adult day 1 and adult day 4 were used and they were freshly replaced every other day at the time the hermaphrodites were transferred to new plates. Male stocks were set up every other day for the entirety of the lifespan assay. Males were added to the hermaphrodites upon hatching for all experiments besides the RNAi screen, in which males were added to young adult hermaphrodites.
For the RNAi screen, adult worms were placed on NGM plates containing ampicillin (100 mg.ml⁻¹) and IPTG (0.4 mM) seeded with the respective bacteria and removed after 4–6 h to obtain synchronized populations of worms. HT115 bacteria transformed with vectors expressing RNAi to the genes of interest were all obtained from the Ahringer library (a gift from A. Fire). To avoid potential developmental defects, RNAi was initiated after larval development in young adult worms obtained from these synchronized populations. To each “hermaphrodite only” plate, 35 Punc-119::sid-1 young adult hermaphrodites were added. To each “hermaphrodite + male” plate, 15 wild-type males were added to 20 Punc-119::sid-1 young adult hermaphrodites. 15 wild-type male worms between adult day 1 and adult day 4 were replaced every other day at the time the living hermaphrodites were transferred to new plates. The lifespan assay for the RNAi screen was performed and scored as described above.
In lifespan assays in which the plates were conditioned with males prior to adding hermaphrodites (Fig 3A for schematic), each time a plate is conditioned, 30 males (day 1–4 of adulthood) were placed on an OP50 seeded plate and maintained at 20°C for 2 days. Males were removed from the plates (red tildes in Fig 3A) and at least 30 L1 hermaphrodites were added to the male-conditioned plates (black tildes in Fig 3A). These hermaphrodites were transferred to a male-conditioned plate every other day until the completion of the lifespan assay.