Sperm motility assays

Spermathecae were excised from females 24 hrs post-copulation with either Spi⁺ and Spi^- males and placed into 10 μl of HEPES-buffered saline solution [145 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1.3 mM CaCl2, 5 mM D-glucose, 10 mM 4-(2-hydroxyethyl)-1-piperazineethane- sulfonic acid (HEPES), pH 7.4] in the well of a concave glass microscope slide. Spermathecae were gently poked with a fine needle and sperm were allowed to exude from the organ for 5 minutes. Sperm beating was recorded using an inverted microscope (10x phase contrast, Zeiss Primovert) that housed a charge-coupled camera (Zeiss Axiocam ERc 5s). Two sperm tails per sample were analyzed. Recordings were acquired at a rate of 30 frames per second, and beat frequency was analyzed using FIJI and the ImageJ plugin SpermQ [74].