DOI: 10.5061/dryad.fn2z34tw9
title: Data from: Thyroid hormone tinkering elicits integrated phenotypic changes potentially explaining rapid adaptation of color vision in cichlid fish

description: Vision is critical for most vertebrates, including fish. One challenge
 that aquatic habitats pose is the high variability in spectral properties
 depending on depth, turbidity and composition of the water body. By
 altering opsin gene expression and chromophore usage, cichlid fish
 modulate visual sensitivities to maximize sensory input from the available
 light in their respective habitat. Thyroid hormone (TH) has been proposed
 to play a role in governing adaptive diversification in visual sensitivity
 in Nicaraguan Midas cichlids, which evolved in less than ~3,100
 generations. As suggested by indirect measurements of TH levels (i.e.,
 expression of deiodinases), populations adapted to short wavelength light
 in clear lakes have lower TH levels than ones inhabiting turbid lakes
 enriched in long-wavelength light. We experimentally manipulated TH levels
 by exposing two-week-old Midas cichlids to exogenous TH or a TH-inhibitor
 and measured opsin gene expression and chromophore usage (via cyp27c1
 expression). Whereas exogenous TH induces long-wavelength sensitivity by
 changing opsin gene expression and chromophore usage in a concerted
 manner, TH-inhibited fish exhibit a visual phenotype with sensitivities
 shifted to shorter-wavelengths. Tinkering with TH levels in eyes results
 in concerted phenotypic changes that can provide a rapid mechanism of
 adaptation to novel light environments. --
description: <em>Thyroid hormone and
 thiourea
 treatments</em> Thyroid hormone (TH) levels were artificially manipulated in 149 individuals from eight separate broods (Table S1-2) of laboratory-reared <em>Amphilophus citrinellus </em>from great Lake Nicaragua (one tank per brood per treatment; 24 tanks in total), which is the source population to the colonization of clear-water crater lakes (Kautt et al. 2016; Kautt et al. 2020). To increase TH concentrations, we dissolved L-thyroxine in the tank water to a final concentration of 300 ng/ml (Prazdnikov and Shkil 2019; Volkov et al. 2020). To decrease endogenous TH concentrations, we dissolved thiourea to 0.01% (Prazdnikov and Shkil 2019). Concentrations were chosen to induce phenotypic differences without increased mortalities in either treatment group. Midas cichlid juveniles were raised together until 14 days post hatching. Afterwards, fish were equally distributed among three treatments: one untreated control, thiourea and thyroid hormone treatments (see Table S1 for details). Fish were kept in the treatments for 14 days. To circumvent degradation of exogenous L-thyroxine or thiourea, water with respective additives was replaced every three days. Fish were maintained under a 12:12 hour light:dark cycle under full spectrum white light and fed with <em>Artemia</em> spp. nauplii <em>ad libitum</em>. <em>Gene expression analysis</em> After 14 days in the treatments (i.e., 28 dph), we euthanized all fish simultaneously and at the same time of day for all broods, after six hours of light exposure (Yourick et al. 2019), with an overdose of MS-222 and stored samples in RNAlater (Sigma-Aldrich, St. Louis, Missouri) at -20°C until RNA-extraction. RNA was extracted from whole fish with the Tissue MiniPrep kit (Qiagen, Hilden, Germany), since extraocular opsin gene expression is negligible (Fig. S1; Karagic et al. 2018) and cDNA was synthesized with the GoScript Reverse Transcription system (Promega, Madison, Wisconsin). Expression levels were measured with quantitative real-time PCR for six cone opsin genes commonly expressed by Midas cichlids (<em>sws1, sws2b, sws2a, rh2b, rh2a, lws</em>), <em>cyp27c1</em>, as well as two housekeeping genes (<em>gapdh2</em>,<em>imp2</em>; housekeeping gene expression levels are given in Table S2 and efficiencies as well as primer sequences are given in Table S3). One of the two green-sensitive paralogs, <em>rh2a</em><em>a</em>, is not expressed in Midas cichlids (Härer et al. 2017; Torres-Dowdall et al. 2017), hence, it was not investigated in this study. Expression of cone opsin genes is given as proportional expression of each gene compared to the expression of all opsin genes and was calculated with the equation  Proportional expression = (1/(1+E<sub>ti</sub>)<sup>C<sub>ti</sub></sup>)/Σ(1/(1+E<sub>ti</sub>)<sup>C<sub>tn</sub></sup>) with <em>E<sub>i</sub> </em>and <em>C<sub>ti</sub> </em>as the efficiency and critical cycle number of the gene of interest, respectively. <em>E<sub>n</sub></em> and <em>C<sub>tn</sub></em> represent efficiencies and critical cycle numbers for all other opsin genes. Expression of <em>cyp27c1</em> was normalized to housekeeping gene expression using their geometric mean according to the following equation  Relative expression = Efficiency<sup>(C<sub>tgm</sub>-C<sub>ti</sub>)</sup> with <em>C<sub>tgm</sub></em> being the geometric mean of the critical cycle numbers of the housekeeping genes and <em>C<sub>ti</sub></em> the critical cycle number of the gene of interest. We computed the mean of each brood per gene and treatment to perform statistical analysis using a linear model with gene expression as the response and treatment as the explanatory variable. Using an ANOVA we tested for the effect of the treatment on gene expression and then performed pairwise comparisons to determine significant differences between treatments for each gene using the estimated marginal means and a post-hoc Tukey test (a = 0.05). <em>Estimations of predicted visual sensitivities</em> Predicted visual sensitivities of control, TH- and thiourea treated fish were estimated according to Rennison et al. (2016)using the mean expression levels of each opsin gene per treatment and absorbance spectra templates from Govardovskii et al. (2000). Additionally, we took chromophore usage into account based on <em>cyp27c1</em> expression levels using the threshold of 1 which we assumed to be mostly vitamin A2-derived chromophore usage based on Torres-Dowdall et al. (2017). Even though variation in chromophore usage is continuous, it is likely that below the threshold of relative expression of <em>cyp27c1</em> mainly vitamin A1-derived chromophores are used. Wavelengths of maximum absorbance for Midas cichlids were used from Torres-Dowdall et al. (2017), except for <em>sws1</em> which was taken from Spady et al. (2006) for tilapia. For the comparison between ancestral (<em>A. citrinellus) </em>and derived (<em>A. astorquii</em>) visual sensitivities, expression data were taken from Härer et al. (2017).