Female bluehead wrasses were induced to change sex by removing dominant TP males from established social groups on patch reefs off the coast of Key Largo, FL in May 2012 and May to July 2014 (5). On each reef, IP males and females larger than the 45-mm standard length were captured and sexed by examination of the sexually dimorphic genital papilla and extrusion of gametes by gentle abdominal pressure. Females were tagged and returned to home reefs. Between 0 and 15 IP males were captured from experimental reefs and relocated to distant reefs to prevent competition with females for social dominance. Within 2 days following tagging and IP male removal, TP males were removed to allow females to compete for dominance and undergo sex change. Females exhibiting TP male–typical behaviors (18) were captured at increasing time points to produce a time series of samples across the sex-change process. Sequential removal of sex changers also served to initiate sex change in the next female (5). Tagged females showing no signs of behavioral or gonadal sex change (verified by histology) were captured as controls on a range of days following TP male/sex changer removal to control for varying degrees of social upheaval. Control TP males used in reported analyses were captured from unmanipulated reefs and served as a reference. All samples were collected around the daily spawning period.

Fish were euthanized with an overdose of MS-222 (Tricaine methanesulfonate) (Sigma-Aldrich) within 2 min of capture, and the brain and gonads were dissected immediately. The brain and one gonadal lobe were preserved in RNAlater (Life Technologies Inc.) on ice, followed by storage at −20°C overnight and then −80°C until RNA extraction. The second gonadal lobe was fixed for histological analysis in 4% paraformaldehyde/1× phosphate-buffered saline (PBS) overnight at 4°C, followed by storage in 1× PBS before fixation in paraffin for histological sectioning with hematoxylin and eosin staining [Histology Laboratory, College of Veterinary Medicine, North Carolina State University (NCSU)]. Experiments were approved by the Institutional Animal Care and Use Committee at NCSU.

Gonadal sections were examined under a light microscope to determine sex change status. In total, 41 samples were partitioned into successive stages (Fig. 1) based on gonadal histology (17) and behaviors observed at the time of capture (18). Females showing no signs of behavioral or gonadal sex change (healthy ovaries with mature follicles and intact zona pellucida) served as control females. Behavioral and histological characteristics of sex change stages are summarized in Fig. 1. Sex changers at stage 5 (ongoing spermatogenesis) were further divided into stages 5a and 5b to reflect their divergent global gene expression patterns (Fig. 2B). Sample sizes for each stage were as follows: six control females (CF), three stage 1 (S1), seven stage 2 (S2), three stage 3 (S3), three stage 4 (S4), three stage 5a (S5a), five stage 5b (S5b), three stage 6 (S6), and eight TP males.

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