2.3. Dissection and staining of testes

Between 21 and 28 days post‐adult emergence, the testes were removed from males. At this age, males are sexually mature. In O. fasciatus, most testis development occurs in juveniles, with some sperm maturation during sexual maturation (Economopoulos & Gordon, 1971). Once sexual maturation is reached, sperm production is at a “steady state” until mating. Thus, changes with age (days past adult emergence) in virgin males were not predicted for virgin sexually mature males. So for convenience, we grouped males at this stage as a single developmental stage, sexual maturation, rather than by age (days past adult emergence). Individual tubules were separated from the outer membrane for fixation and staining. Testis tubules from males within a treatment were pooled for staining if they were dissected on the same day. The testis tubules were fixed in 4% formaldehyde in PBS plus 0.1% Triton X‐100 (PBT) for 30 min. The tubules were stained with α‐phosphohistone H3 Ser10 (pHH3) primary antibody (Millipore antibody 06‐570, Sigma‐Aldrich). α‐pHH3 stains for chromosome condensation in preparation for mitosis and meiosis (Hans & Dimitrov, 2001; Prigent & Dimitrov, 2003). The secondary antibody was an Alexa Fluor goat anti‐rabbit 647 (Thermo Fisher Scientific). Following antibody staining, the tubules were stained with DAPI (0.5 μg/mL PBT) to visualize nucleic acids. Stained tubules were mounted in Mowiol 4–88 mounting medium (Sigma‐Aldrich). Slides were kept in boxes to limit light exposure and stored at 4℃ until visualized. The testis tubules were visualized with a Zeiss LSM 710 Confocal Microscope (Zeiss) at the UGA Biomedical Microscopy Core Facility. All testis tubules were photographed and included in the analysis.