parison with the experimental groups. First experiment: a series of eight 76L/20gal tanks each contained one pair of adult CF (Florida) or SF, consisting of a male and female for each A. mexicanus morphotype. Four tanks were maintained under 8-hour daily treatments of high light conditions with 2500 cm2 LED grow lamps (Mars Hydro TS 3000) suspended ~15 cm above the water surface of the tanks (experimental group); four tanks were maintained under 8-hour daily exposure to ambient light conditions (control group). Light intensities at the water surface measured 696– 702 lux at the four treatment tanks, and 36–40 lux at the four ambient tanks. All tanks were connected to the RAS water (see above). Within each set of four tanks, pairs of CF and SF alternated, providing two tanks of each morphotype under experimental and control conditions. All fish were imaged live at the start of the experiment. The experiment was run for 72 days. All fish were imaged live at the end of the experiment. One additional 76L/20gal tank containing a group of 3 adult CF (Florida) was maintained under each of the two lighting conditions to provide biological materials for genetic and molecular data (see next section). Two 38L/10gal tanks were also maintained as additional no-light (dark) controls, with one tank containing a pair of adult CF (Florida), and a second tank containing a pair of adult SF. Second experiment: Three experimental groups of CF (Molino, Arizona, Florida) were each maintained separately in three 76L/20gal tanks, where they were exposed to daily cycles of combined illumination from wide-spectrum LED and full-spectrum, high-intensity halide light sources (see B; previous section). The experimental tanks were housed together in a separate room. Molino CF are juveniles that do not express black melanic pigmentation (eumelanin). Arizona CF are young adults that do express black melanin. Florida CF are adults from the first controlled experiment (72-day light treatments) that received additional exposure to combined illumination treatments. All three CF sources express other pigments (e.g. xanthophores, iridophores; see Figure 3). Additionally, the Molino CF group was exposed to three, 15-minute treatments per week under a 4-bulb tanning array (Sperti, FIJI SUN, KBD, Inc.) to stimulate pigment production. Control stocks for each CF group under combined illumination treatments were regularly maintained in ambient light for direct comparison with their respective treatment groups. D. Molecular biology Tissue and organ samples were collected on the 1st and 72nd day of the first controlled experiment (see previous section). On day 1, a dorsal subsection of the caudal fin was clipped from each CF and SF in the experimental groups, and from each CF and SF in the 38L/10gal dark tanks. Caudal fin samples were preserved in 95% EtOH at -20 ˚C. These samples will be processed for CF population identification through PCR and sequencing of barcoding genes (e.g. MT-CO1, MTCYB, 16S rDNA). On the 1st and 72nd day of the same experiment, one adult fish was sacrificed from each of the two groups of three CF (Florida) under each condition. The dorsum at the dorsal fin, caudal peduncle, caudal fin, gill and brain were removed by dissection and preserved in RNAlater™ (Invitrogen, Thermo Fisher Scientific) at -20 ˚C. These dissections provide molecular resources for the purification of DNA, purification of total RNA to synthesize cDNA templates for gene expression experiments, and as tissue samples for analyses with mass spectrometry. On the 1st day of the second controlled experiment with CF undergoing combined illumination treatments, dorsal subsections of the caudal fin were clipped and preserved from each of six CF (Arizona) for population identification; CF (Florida) were clipped previously. One adult CF (Arizona) was sacrificed at the end of illumination treatments to perform tissue and Figure 6. Comparative pigmentation of A. mexicanus surface fish under different environmental conditions. A. Adult surface fish in left-lateral view (see Fig. 1) maintained under ambient light. Notable areas of melanic pigmentation include the dorsal side (white arrows), posterior end of lateral line (dashed yellow arrow), posterior flank of body (yellow arrow), pigmented iris (red arrow) and a prominent arrow-shaped patch posterior to the gill chamber (dashed white arrow). B. Adult surface fish maintained under minimal light, lower than normal oxygen (0.9–4.0 mg/L) and moderately high CO2 (pH 5.8–6.0) for ~ 3 months. This ‘treated’ fish shows a visibly lower overall amount of melanic pigmentation (i.e. reduction of melanin) when directly compared with all body regions noted in ‘A’. Xanthophore pigments (yellow) are noticeably lower along the lateral line and tail in ‘B’. Macrophotographic images by Scott Arledge and Michael J. Boyle. full-spectrum light for 5 months, followed by daily cycles of LED lighting for 4 months. A group of their progeny (F1) were exposure to the same high intensity, full-spectrum light for ~40 days, followed by daily cycles of LED lighting for 4 months. The fish used for this experiment were from the original commercial (Florida) stock of cavefish. Morphological results of the first experiment are presented in Figure 5. Second experiment: five SF adults were maintained in a 340L/90gal tank with recirculating water and temperature regulation. Experimental conditions included minimal light, lower than normal levels of dissolved O2 (0.9–4.0 mg/L) and moderately high levels of CO2 (pH 5.8–6.0). Treatment was sustained for approximately 3 months. Morphological results of the second experiment are presented in Figure 6. Third experiment: four CF (Florida) were maintained in a 76L/20gal tank under ambient light, normal levels of dissolved O2 (6.5–8.0 mg/L) and high levels of CO2 (pH 5.3–5.5) for 6 weeks. See Results section for descriptive observations (not shown). CF and SF in all three preliminary experiments received a similar diet (see section on husbandry), regular water changes, and daily observations to assess health and changes in morphology. C. Controlled experiments Two controlled experiments were performed, each with a specific set(s) of untreated controls for direct visual and morphological comBOYLE, ARLEDGE, THOMAS, TOMKINS, AND GULIUZZA Testing the cavefish model 2023 ICC 127
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