Figure 5. Increase in melanic pigmentation of A. mexicanus cavefish from sustained exposure to treatments of high-intensity light. A–C. Adult male cavefish in left-lateral views. This fish was exposed to daily cycles of high intensity, full-spectrum light for 5 months, followed by daily cycles of LED lighting for 4 months. Pronounced increases in melanin pigment production (relative to adult cavefish in Fig. 1B) are detectable along the dorsal sides and dorsal midline (A, C, white arrows), the base of the dorsal fin (A, B, yellow arrows), dorsal flanks of head (A, dashed white arrow), within the adipose fin (A, C, red arrows), within and around olfactory pits (dashed red arrow), posterior end of the lateral line (A, dashed yellow arrow), in clusters of pigment along the dorsal body (C, dashed yellow arrows), and in fields along posterior body flanks (B, dashed yellow ellipse). Numerous iridescent cells also reveal linear accumulations of green and gold iridophores along the upper edge of the lateral line (A, B) and in clusters above the lateral line. D–F. First generation (F1) young adult offspring of the fish in ‘A’ in left-lateral views. This fish developed under exposure to high intensity, full-spectrum light for ~40 days, followed by daily cycles of LED lighting for 4 months. The F1 fish is approximately 20–30% smaller in overall size than the adult fish in ‘A’. There are general similarities in the amount and coverage of melanin pigment production between parent and offspring. However, In the F1, there is a more uniform distribution of pigment on the dorsal side and dorsal midline (D,F, white arrows), on the sides of the head (D, dashed white arrow), around the mouth (E, white arrow), chin (D, E, red arrows), underside (D, and E, white dashed arrow), olfactory pits (D, dashed red arrow; E, F), and posterior body flanks (D), and at the posterior end of the lateral line (D, dashed yellow arrow). The adipose fin also contains melanophores (D, F). In both fish (A, D) there is a common semi-circular pattern of melanophores following the contour of the apoptotic site of eye degeneration (yellow asterisks). Macrophotographic images by Scott Arledge and Michael J. Boyle. The primary CF stocks were purchased commercially from Florida. A secondary stock of Molino CF was generously provided by Dr. William R. Jeffery, University of Maryland, College Park. A third stock of CF was purchased commercially from Arizona. Original cave populations for the commercial CF stocks will be identified by PCR (DNA barcoding). SF and CF are maintained and cultured within individual aquarium tanks or a recirculating aquaculture system (RAS) of multiple aquaria. Prior to entering any tanks, source water is pumped through a multi-stage, 1.0 µm reverse osmosis and deionization filtration system (Bulk Reef Supply, RO Plus 200 GPD; LiquaGen 150 GPD) into a pre-RAS sump reservoir. Chemical treatments are added to pre-RAS water, which is then pumped into the RAS system following removal of equal amounts of system water (water changes). RAS water is further treated through continuous inline mechanical (200 µm ring-filter socks) and biological (AQUAMAXX) filtration. All water (individual tanks and RAS) is UV sterilized (Aqua Ultraviolet, Bulk Reef Supply). Conductivity, temperature and pH is monitored (bluelab® guardian) continuously. Water temperature is maintained at ~75 ˚F by automated regulation of temperatures measured below (EHEIM Thermocontrole150) and above (ARCTICA Titanium Chiller) the desired set point. Water chemistry (e.g. ammonia, nitrites, nitrates) is tested weekly and adjusted as required. Breeding and pre-experimental SF and CF stocks are maintained within individual 340L/90gal aquaria with dedicated water recirculation (FLUVAL 407), and temperature regulation systems. Individual tanks also receive periodic chemically-treated water exchanges as required. Surface fish and cavefish are fed daily with food pellets for adults (Xtreme NICE™ Aquatic Foods®) early juveniles and fry (Xtreme Nano™ Aquatic Foods®) or larvae (First Bites™ KYORIN Co., LTD). Fish are supplemented with brine shrimp cultured in the laboratory (www.brineshrimpdirect.com). Selected male and female SF and CF are maintained in pairs or small groups during, or outside of, experiments to monitor them for spawning events. When spawning occurs, embryos are collected from tank bottoms with serological pipettes and maintained in smaller tanks with regular water changes. Larval fish provide material for studies of development (Fig. 1) and for tracking morphological and genetic changes that may have been transferred to them through experimental treatments performed on their parental stages (Fig. 5). In vitro fertilization experiments are in progress to obtain reliable sources of lineage-specific materials for morphological, molecular, and epigenetic experiments during early development. All animals are treated humanely, as per the IACUC Handbook. B. Preliminary experiments Three preliminary experiments were performed to test for morphological responses of adult CF and SF to contrasting environmental conditions. First experiment: a pair of adult CF (male and female) were maintained in a 76L/20gal tank under continuous daily exposure to combined illumination from wide-spectrum LED (Hydra 32 HD Reef light, Bulk Reef Supply) and full-spectrum, high-intensity (VIVOSUN 10,000K metal halide bulb, Grower’s Choice) light sources. The pair of adult CF were exposed to daily cycles of high intensity, BOYLE, ARLEDGE, THOMAS, TOMKINS, AND GULIUZZA Testing the cavefish model 2023 ICC 126
RkJQdWJsaXNoZXIy MTM4ODY=