Thiocarbohydrazide

For egg sections, fixed eggs were washed with filtered (pore size = 0.2 µm) artificial seawater (Rohto Marine, Iwaki, Tokyo, Japan) (FASW) and post-fixed with 2.0% osmium tetroxide dissolved in FASW for 2 h at 4°C. After washing with 8.0% sucrose aqueous solution, the conductive staining was performed by incubating 0.5% thiocarbohydrazide (Thermo Fisher Scientific, Waltham, MA, USA) aqueous solution for 30 min and 1.0% osmium tetraoxide aqueous solution for 1 h at 4°C. The samples were washed, dehydrated in a graded series (30, 50, 70, 80, 90 and 100%) of N,N-dimethylformamide, cleared in n-butyl-glycidyl-ether (Nisshin EM, Tokyo, Japan) and embedded in Quetol 651 (Nisshin EM). Ultra-thin sections (60 nm thickness) were cut with a diamond knife on an Ultracut S ultra-microtome (Leica Microsystems, Wetzlar, Germany), stained with 2.0% uranyl acetate solution and 2.0% lead citrate solution, and observed using a Tecnai G² 20 electron microscope (FEI, Hillsboro, OR, USA) operated at 120 kV. Thin sections of the ovary and gill were prepared as described above with modification in the dehydration process by using a graded series (30, 50, 70, 80, 90 and 100%) of ethanol.