organ dissections (as above), and for comparison with corresponding samples from one adult CF from a stock tank; One month after arrival and acclimation of the Molino cavefish to the ICR, one juvenile fish was preserved in 95% EtOH at -20 ˚C, and one juvenile fish was preserved in RNAlater™ at -20 ˚C. These samples will be processed for gene-specific identification analyses, cDNA template synthesis, and mass spectrometry to assess presence or absence of chromatophores and proteins directly integral to the melanin synthesis pathway. E. Image acquisition and processing Macrophotographic images were captured with a Panasonic Lumix GH5 camera body, through a Cannon EF 24-70mm f/2.8L lens (camera settings are available upon request). Photographic lighting included two amaran P60c RGBWW LED Panels and a LS C300d II (Aputure Imaging Industries Co., Ltd.). Live SF and CF were individually placed within a 1.0 liter glass aquarium to record pre- and post-experimental morphology. The position of each fish within the aquarium was restricted toward one side, enabling photography of lateral, full-body profiles (Figs. 1, 4-6). Micrographs were captured with a Jenoptik Gryphax PROKYON digital microscope-dedicated camera, through a Zeiss SteREO Dicovery.V20 stereomicroscope under multiple objectives; these images were obtained as single captures, or as single image files extracted from a video series (see figure captions). Image editing and figure layouts were performed with Adobe Photoshop CC; all figures were formatted with Adobe Illustrator CC; video files were edited and extracted with Adobe Premiere Pro CC (Adobe.com; San Jose, CA). III. RESULTS A. General observations Spawning events were detectable in both stock and laboratory aquariums. Prior to spawning, male and female cavefish (CF) and surface fish (SF) begin to follow each other in close proximity, and at times in parallel profiles. Their coordinated movements indicate they are a mated pair. At the peak of their ‘dance-like’ behavior they simultaneously release a burst of gametes (sperm and eggs) and then either move off in different directions, or repeat the event before doing so. The egg cells are semi-transparent and tan (CF) or whitish-grey (SF) in color. Embryos have an approximate diameter of 960 µm, and reside within a larger egg envelope (chorion) with a diameter of ~1100–1200 µm (Fig. 2). Time of development from fertilization to the hatching of larval fish is ~25–28 hours (hrs) at 23 ˚C (see Materials and Methods for husbandry of larval and juvenile fish). Our commercial stock of Astyanax mexicanus CF from Florida come from a cave-dwelling population that exhibits melanic (black) pigmentation in early and late larval stages. The commercial CF from Arizona also produce black melanin, and observations of their larvae following spawning events confirm melanic pigmentation during early development. CF and SF under single or combined illumination treatments have not exhibited signs of stress, or either visible or behavioral symptoms of disease, during experimental periods. Similar observations have been made for both morphotypes when maintained in ambient light or in the dark. The multi-tank recirculating aquaculture system (RAS) is modifiable and accommodates a series of contrasting illumination treatments under shared, identical water conditions (chemistry, conductivity, temperature, pH). Regulation of narrow target ranges of CO2 (pH) and O2 are performed within individual, free-standing aquariums. We observed no obvious behavioral or physiological differences of CF or SF between these systems, or when moving fish from one system to another. B. Pigmentation Three chromatophores are visually identifiable on and within tissues of experimental surface fish (Guadalupe River, Texas) and cavefish (Molino, Arizona, Florida) models in this study. All surface fish specimens exhibit typical patterns of melanic pigmentation in A. mexicanus (see Fig. 1A). Under moderate stereoscopic magnification, patterns of dendritic melanocytes in SF (Fig. 3A–F) are observed within the head (encircling the eye, on upper and lower mouth parts, gill opercula), along the midbody (dorsum, behind gill chamber, along lateral stripe, scale margins) and within fins (e.g. adipose fin, caudal fin). The melanocytes are visible at both surface and subsurface positions along the body (Fig. 3B, C, F) and also deeper within regions of the brain cavity and heart (not shown). Xanthophores are most noticeable along the lateral stripe, in the adipose fin, and rays of the caudal fin (Fig. 1A; Fig. 3E). Iridescent iridophore pigments are primarily visible along the lateral stipe and peduncle (Fig. 1A, dashed white arrow). Along and within body regions of the cavefish models, black melanocytes are present in almost all body domains as observed in SF, with the exception of Molino CF. However, respective levels of expression in the commercial CF (Arizona, Florida) are visibly lower (Fig. 1B; Fig. 3G). In the Molino CF, regions of yellow-orange pigmentation are spatially similar to black melanic pigmentation patterns observed in commercial CF, and SF (Fig. 1A, B; Fig. 3H–L; Fig. 4B–C; Fig. 5; see Discussion). After high-light treatments, the expression and distribution of yellow-orange pigments in Molino CF are pronounced in head, body and fin cells (Fig. 3J–L). In the Molino CF, yellow orange pigments on the body are dendritic (not shown), and these same pigments are in dorsal cells of the optic tectum, and brain (Fig. 3H, I). As shown in SF, xanthophores are also expressed along the body in all three commercial CF, and in similar locations (Fig. 1B; Fig. 3G, H, J; Fig. 4A, B; Molino not shown), as are iridophores (Fig. 1B; Fig. 3J; Fig. 4A–C; Fig. 5A–C). Additionally, all three chromatophores are observable during early development of the Florida CF model (eye cup/pre-retinal tissues, head, olfactory pit, optic tectum, dorsum, viscera) in larval and pre-juvenile stages from 5–26 days (Fig. 7F–M) and beyond. We have not yet reared Arizona CF to juvenile stages, and have not induced or observed spawning in Molino CF. C. Preliminary experiments Surface fish (SF) from the Guadalupe River exhibit the common suite of external morphology, coloration and pigmentation patterns (Fig. 1A; Fig. 3) observed on SF from populations found in Mexico. These include dark melanic pigmentation along the dorsum, extending from their head to the tail. They express melanophores in a band between the dorsum and the lateral line that extends from their ‘shoulder’ to end of the peduncle (attachment site of caudal fin) at the tail. Melanophores are also expressed within the caudal fin, extending along central fin rays at the junction of dorsal and ventral sections of the forked tail, and along upper and lower fin rays at dorsal and ventral margins. Numerous melanophores are observed across posterior flanks, along the mid-body region, on the opercula (gill covers), in a dense vertical streak posterior to each operculum, on both upper and lower mouth sections, and encircling the iris (Fig.1A). All surface fish have a pair of functional eyes. In contrast, although melanophores are expressed to some extent within almost all analogous regions of the commercial cavefish (CF) model from Florida (Fig. 1B), the extent, density and overall pattern of melanic pigmentation is noticeably less. Their relatively size-conBOYLE, ARLEDGE, THOMAS, TOMKINS, AND GULIUZZA Testing the cavefish model 2023 ICC 128
RkJQdWJsaXNoZXIy MTM4ODY=