Calcium accumulation

Exp 1: To investigate calcium accumulation, larvae between 4 and 40 hpf from N = 5 replicate families were collected on a 20 µm mesh and transferred to a 2 ml Eppendorf tube using a plastic Pasteur pipette. For each development stage, 90,000–300,000 individuals were collected. Samples were centrifuged and the supernatant was discarded. The samples were then vortexed in 500 µl distilled water and centrifuged for 10 min at 21,800 r.p.m. The supernatant was discarded and the larval pellet was dissolved in 500 µl 10 M HCl. Samples were analyzed using a flame photometer (EFOX 5053, Eppendorf) that was calibrated using urine standards (Biorapid). Calcium concentrations were normalized per larvae following a background subtraction for the HCl larvae were dissolved in. Larvae from each developmental stage were sampled and fixed in 4% PFA buffered to pH 8.1. Larval samples were subsequently analyzed to assess mineralization using an inverted microscope (Leica DMi8) equipped with crossed polarized light filters.