Figure 2. Selected stages of embryonic and larval development in an A. mexicanus surface fish. A. ~1.0 hpf (hours post fertilization); 1st cleavage stage embryo with two blastomeres (white arrow) extending from the nutritive yolk cell (dashed white arrow). Animal pole is to the top, vegetal pole is to the bottom. B. 1.25 hpf; 2nd cleavage stage with four blastomeres. A spherical, semi-transparent chorion (black arrow) surrounds the embryo. The original site of sperm penetration (micropyle) is visible on the animal pole (white arrowhead). C. 1.5 hpf; 3rd cleavage stage with eight blastomeres (white arrows). The embryo has begun to rotate toward the left side (relative to the micropyle). D. 2.0 hpf; embryo with more than ~ 30 undifferentiated blastomeres. E. 4.0 hpf; sphere stage undergoing epiboly, where the blastoderm (white arrow) is an enveloping layer (EVL) of motile cells that spread over the yolk syncytial layer (YSL, yellow arrow). Embryonic rotation is almost 90˚. F. 7.0 hpf; ~60% epiboly where the blastoderm is advancing (dashed yellow arrow) over the yolk cell (dashed white arrow) toward the vegetal pole. A bulge of blastoderm cells under the micropyle now provides a dorsal-ventral axis, with dorsal toward the outer edge (white arrow). G. 10 hpf; ~80% epiboly with notable elongation of the blastoderm and yolk cell. The future head end (white arrow) and tail end (dashed black arrow) are now defined. The micropyle (white arrowhead) has remained in the same original orientation during all stages of development thus far. H. 23 hpf; late pre-hatch larval stage. Relative to the image in ‘G’, this larva has rotated horizontally 180˚, with the anterior-posterior axis extending from the head in a counterclockwise orientation with the tail pointed back toward the head at the top left corner of the panel. Almost all larval organ systems are fully developed. I. 28 hpf; the swimming, non-feeding larva remains dependent on nutrition from the yolk cell. The developmental staging and process identification follow Hinaux et al. 2011. Stereomicrographs were extracted as individual files from a timelapse video series of images produced by Scott Arledge and Michael J. Boyle. embryos and larvae in a single spawning event. Therefore, access to early stages of development (Fig. 2) are readily available for experimental applications on comparative developmental morphology, genetic and transcriptomic expression patterns, environmental treatment conditions, and a diversity of micrographic imaging techniques – all of which facilitate in-depth research on the biochemistry and physiology underlying the process of adaptation. The cavefish forms of A. mexicanus are found within 29 known caves across the Sierra del Abra of Northeastern Mexico (Krishnan and Rohner 2017; Jeffery 2020). Often, cave-dwelling populations live in close proximity to conspecific surface-dwelling morphotypes. Genetic studies suggest there have been at least five cave invasions by surface-dwelling fish along with persistent gene-flow among cave-dwelling populations (Maldonado 2020; McGaugh 2020). Both surface fish and cavefish genomes have been sequenced and assembled. Although each morphotype exhibits a unique set of anatomical, physiological and behavioral traits, there is a limited amount of overall genetic variability between the surface and cave variants, and their genomes are nearly identical. As Jeffery (2019) states, “based on the molecular studies and the ability to produce fertile offspring, all cavefish populations and nearby surface fish are usually considered to be a single genetic species: A. mexicanus”. Tomkins discussed a widely-divergent range of evolutionary time intervals published by NDT advocates for the development of dark-adaptive traits in Astyanax (Tomkins 2022). NDT frames the adaptation of those traits as a gradual, haphazard or “hit and miss” process. Early estimates suggested that blind Astyanax morphotypes diverged from sighted surface populations between 3.1 – 8.1 million years ago (Strecker 2004; Ornelas-Garcia 2008). A decade later, Jeffery reported a much younger age, stating that “surface fish and cavefish split from a common ancestor very recently, within the past million years or less” (Jeffery 2019). Several additional studies suggested more recent deep-time estimates. And there is increased recognition for relatively fast rates of morphological change in which “recent studies have shown that the cave-dwelling form evolved rapidly within the last 200,000 years from an ancestor that lived at the surface” (Bilandzija 2020). Yet another contemporaneous study arrived at a divergence time between 115,000 – 190,000 years ago (Herman 2018). Although such estimates may reflect differences in methodology, they clearly indicate a decreasing trend in the time of production for cavefish adaptations. However, a new analytical approach considering the geographic distribution of mitochondrial and nuclear DNA polymorphisms are raising questions about all of the age estimates for development of troglomorphic traits, as well as the current assignment of cavefish into the so-called “old” and “new” lineages, and whether several caves have been independently populated at different times (Fumey 2018). Fumey “found that microsatellite polymorphism strongly supports a very recent origin of cave populations (< 20,000 years)” adding that “the only safe conclusion is that these cave populations are not millions of years old. The large uncertainty associated with these [prior] estimations is probably the reason why they are rarely cited by investigators working on these cavefish.” (Fumey 2018). In this manuscript we present a series of preliminary (single test) and controlled (treated and untreated specimens) experiments designed to test for phenotypic responses in A. mexicanus cavefish and surface fish morphotypes. Experimental treatment conditions consisted of exposure to high intensity light, elevated CO2 (low pH) and low dissolved O2 levels. In three different cavefish populations (strains), light exposure stimulated a noticeable increase in chromatophore BOYLE, ARLEDGE, THOMAS, TOMKINS, AND GULIUZZA Testing the cavefish model 2023 ICC 124
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