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. --

Thyroid hormone and thiourea treatments

Thyroid hormone (TH) levels were artificially manipulated in 149 individuals from eight separate broods (Table S1-2) of laboratory-reared Amphilophus citrinellus 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 Artemia spp. nauplii ad libitum.

Gene expression analysis

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 (sws1, sws2b, sws2a, rh2b, rh2a, lws), cyp27c1, as well as two housekeeping genes (gapdh2,imp2; 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, rh2aa, 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_ti)^C_ti)/Σ(1/(1+E_ti)^C_tn)

with E_i and C_ti as the efficiency and critical cycle number of the gene of interest, respectively. E_n and C_tn represent efficiencies and critical cycle numbers for all other opsin genes. Expression of cyp27c1 was normalized to housekeeping gene expression using their geometric mean according to the following equation 

Relative expression = Efficiency^(C_tgm-C_ti)

with C_tgm being the geometric mean of the critical cycle numbers of the housekeeping genes and C_ti 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).

Estimations of predicted visual sensitivities

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 cyp27c1 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 cyp27c1 mainly vitamin A1-derived chromophores are used. Wavelengths of maximum absorbance for Midas cichlids were used from Torres-Dowdall et al. (2017), except for sws1 which was taken from Spady et al. (2006) for tilapia. For the comparison between ancestral (A. citrinellus) and derived (A. astorquii) visual sensitivities, expression data were taken from Härer et al. (2017).