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Animals were exposed throughout the entire larval period, from hatching [Niewkwoop–Faber (NF) Stage 48 (16)] until complete tail reabsorption (NF Stage 66).In all experiments, all treatments and analyses were conducted blindly with color-coded tanks and treatments and number-coded specimens.
Initially, the sex of all individuals was determined based on gross gonadal morphology (Fig. Sex identification was confirmed by histology for 10 animals per tank.Histologically, the ovary is distinguished by the ovarian vesicle (hole in the center) along its entire length and the internal ring of connective tissue (in blue).Note the melanin granules (black) in the connective tissue in Serial transverse histological sectioning was conducted on the larynges of 10 males and 10 females from each replicate from all treatments in both experiments. To estimate the size of the larynx, the was measured.1) until a region approximately one-third through the larynx was repeatedly determined to be the largest section.For the final analysis this region was identified by shape.