**Background**
The Japanese flounder (Paralichthys olivaceus) undergoes dramatic metamorphosis from a pelagic, symmetrical larva to a benthic, asymmetrical juvenile, with one eye migrating to the other side of the body. This transition involves a shift from bright upper-water to dimmer deeper-water light environments. Ten opsin genes have been identified in this species, but the molecular mechanism of visual adaptation during metamorphosis remained unknown. Thyroid hormone (TH) is known to accelerate flounder metamorphosis, while thiourea (TU) inhibits it by suppressing TH synthesis, but how TH signaling regulates opsin expression during this process had not been characterized.
**Methods**
Flounder larvae were cultured at 19–21 °C in seawater with salinity 33. At 16 days post-hatching (dph), larvae were divided into three groups: normal control (NC), TH-treated (0.1 mg/L T3), and TU-treated (30.0 mg/L). Larval samples were collected at 17, 20, 24, 28, 32, 36, and 41 dph (n=3). For rescue experiments at 36 dph, TU-treated larvae were subdivided into TU, TU+NC (natural seawater), and TU+TH groups. Adult tissues (brain, eye, gill, heart, liver, stomach, kidney, intestine, gonads, muscle; n=3) were collected. qRT-PCR with β-actin as reference quantified opsin genes (rh1, lws, rh2b-1, sws2aβ, sws1) and TR isoforms (trαa, trαb, trβ). T3 levels were measured by ELISA. Dual luciferase reporter assays in 293T cells tested T3 (75 nM) and TR (Trαa, Trαb, Trβ) effects on opsin promoter activity. Phylogenetic analysis used MEGA 7.0 with neighbor-joining and 1000 bootstrap replicates. Statistical analysis used one-way ANOVA with Dunnett's post-hoc test; p<0.05 was considered significant.
**Key Results**
All five opsin genes were expressed at significantly highest levels in eye tissue compared with other adult tissues (p<0.05). During metamorphosis, rh2b-1 was highly expressed at 17–20 dph and significantly decreased afterward (p<0.05). rh1 and lws peaked at 28 dph then gradually declined until 41 dph. sws2aβ increased rapidly from 20 to 28 dph and remained high in late metamorphosis. sws1 peaked at 24 dph, then significantly decreased and remained low (p<0.05).
TH treatment significantly up-regulated rh1 at 20, 24, 32, and 36 dph; lws at 20 and 24 dph; and sws2aβ at 17, 20, and 24 dph (p<0.05). TU treatment significantly down-regulated rh1 at 24 and 28 dph; lws from 24 to 41 dph; and sws2aβ from 24 to 41 dph (p<0.05). rh2b-1 was significantly decreased by TH from 17–28 dph but up-regulated by TU from 28–41 dph (p<0.05). sws1 was significantly down-regulated by both TH and TU at 24 and 28 dph, and up-regulated by TU at 32, 36, and 41 dph (p<0.05). T3 levels in eyes peaked at 24 dph in TH-treated larvae and were continuously suppressed by TU.
In rescue experiments, sws2aβ and lws expression was significantly higher in rescue groups compared with continued TU treatment (p<0.05), comparable to NC and TH groups at 41 dph. sws1 and rh2b-1 significantly decreased in rescue groups versus TU group (p<0.05). rh1 showed no significant differences among rescue groups.
Dual luciferase assays showed significantly increased activity (p<0.05) for pro-rh1+TRs, pro-lws+Trαb/Trβ groups (with or without T3), and pro-sws2aβ+Trαa+T3, pro-sws2aβ+Trαb/Trβ, and pro-sws2aβ+Trαb/Trβ+T3 groups. rh2b-1 and sws1 promoter activities showed no significant differences across conditions. Phylogenetic analysis showed flounder opsins closely related to Hippoglossus hippoglossus (Rh2b-1 96%, Sws2aβ 98%, Sws1 100%, Lws 75%).
**Clinical Implications**
This is a basic fish biology study with no direct clinical human implications. However, it advances understanding of TH-regulated photoreceptor differentiation, which has broader relevance for comparative vertebrate visual development and may inform aquaculture practices for flounder cultivation by clarifying how TH manipulations affect visual system maturation during critical metamorphic windows.