Eclipse e400 compound microscope

Mating assays were based on procedures described^{7 (link)}. Males were picked at the L4 stage and kept apart from hermaphrodites for 24 hours, either following 24 hours of starvation during L1. One male was transferred to a plate covered with a thin fresh OP50 lawn containing 10–15 adult unc-31(e928) hermaphrodites. These hermaphrodites move very little, allowing for an easy recording of male behavior. Hermaphrodites were also isolated from opposite sex at the L4 stage and used 24 hours later, and were always well-fed. Animals were monitored and sequence of events was recorded within a 10 min window or until the male ejaculated, whichever occurred first. Males were digitally recorded using the Exo Labs model 1 camera mounted on Nikon Eclipse E400 compound microscope with long-distance X20 lenses. % efficiency (per mating behavior step)=100×(the number of successful performances of mating behavior step/the number of times the male attempted the mating behavior step). % loss of hermaphrodite contact=100×(the number of times male lost contact with hermaphrodite and eventually re-initiated the mating sequence/the number of hermaphrodites the male contacted during assay). Two biological and technical replicates were performed for each experiment.

Mating assays were based on procedures described previously ^{12 (link),35} . Males were picked at an early L4 stage and kept apart from hermaphrodites for 24 hours. One male was transferred to a plate covered with a thin fresh OP50 lawn containing 10–15 adult unc-31(e928) hermaphrodites. These hermaphrodites move very little, allowing for an easy recording of male behavior. Hermaphrodites were also isolated from opposite sex at the L4 stage and used 24 hours later. Animals were monitored and sequence of events was recorded within a 15 min window or until the male ejaculated, whichever occurred first. Males were tested for their ability to locate vulva in a mating assay, calculated as location efficiency (L.E.)³⁶ . The number of passes or hesitations at the vulva until the male first stops at the vulva were counted. Location Efficiency = 1 / # encounters to stop. PHB silenced males were digitally recorded using the Exo Labs model 1 camera mounted on Nikon Eclipse E400 compound microscope with long-distance X20 lenses. These videos were analyzed for vulva location efficiency and % of successful contact response, which requires tail apposition and initiation of backward locomotion. % response to contact = 100x [the number of times a male exhibited contact response/the number of times the male makes contact with a hermaphrodite via the rays^{13 (link)}.