Melanosome aggregation assays evaluated melanophores present on scales isolated from the dorsal flank, just anterior to the dorsal fin. Single scales were isolated from the dorsal flank of hypermelanic (“Asty66”; n = 10 individuals) and surface fish (“Asty 155 and 152”; n = 12 individuals) by gentle removal with the #5 fine forceps (World Precision Instruments, Sarasota, FL). Scales were immediately placed in 1 mL of sterile 1× Danieau’s solution (Cold Springs Harbor Protocols) in a 48-well plate. Each scale was imaged using montage imaging under identical bright field conditions using a Leica stereomicroscope (M205FA) at 80× magnification (brightness = 60 %, saturation = 1.25, automatic exposure; Leica Application Suite v3.8). Each scale was then immersed in 1 μM of melanin concentrating hormone (“MCH”; Sigma-Aldrich) and re-imaged following 5 min of treatment. Control scales were treated with 1× Danieau’s solution. Melanophore areas were measured from pre- and post-treatment images using the polygon selection and measurement tools in ImageJ. Three melanophores were measured on each scale for pre- and post-treatment with MCH. Additionally, we measured relative darkness (white = 0, black = 255) of each melanophore using the “mean” function in ImageJ, as previously described [12 (link)]. Surface and hypermelanic scales were compared using a Student’s T-test (StatPlus v5.8).
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