for external applications with transcriptomics, genomics, epigenetics and mass spectroscopy; and an (3) Imaging Center equipped with state-of-the-art stereo, compound and confocal microscopes, each with dedicated computer and digital camera systems. We will pursue multilevel research experiments with the Astyanax model to investigate genetic, cellular, organ-system and physiological evidence underlying organism-driven mechanisms of adaptation. In addition to ongoing experiments on pigmentation, and the associated components of pigment synthesis pathways, we will focus on the developmental processes of eye degeneration (Fig. 7), and restoration, in different cavefish strains. To do this, we will move beyond testing commercial cavefish – currently used to explore and establish technical protocols – to culturing distinct lines of cavefish (e.g. Pachón, Molino, Tinaja) utilized by prominent academic laboratories (see: Riddle et al. 2018; Baumann and Ingalls, 2019). There is a considerable amount of literature available on the process of eye degeneration in cavefish, and eye development in Astyanax mexicanus and Danio rerio (Zebrafish). Although, very few, if any, of the treatments we are exploring have been pursued in these fish. Therefore, we will begin to repeat several of the conventional studies, which provide invaluable protocols and methods to guide our work. It is also our intention to investigate multiple organ systems that exhibit adaptive traits, including nervous, respiratory, circulatory, muscular, digestive, epidermal, and others, as discussed above (see: Jeffery 2020). Notably, we will characterize the expression of gene transcripts in embryonic and larval stages when all cell types, tissues and organs are moving from specification to differentiation and functionality. We will also begin to ramp up reproductive output in different cavefish lines, providing not only material for developmental protocols, but also to assess trans-generational (genetic, epigenetic, phenotypic) signatures of adaptation. All of the above will guide necessary adjustments to ongoing experiments and future directions with the cavefish model, and potential alternative models going forward (e.g. reptiles, birds, freshwater and marine invertebrates). Most importantly, we approach this research from the most essential perspective of all: The intimate scriptural and working knowledge of the Creator of life, and through careful application of an original model of engineered adaptability that serves to honor His creative power and wisdom. V. CONCLUSIONS Based upon qualitative investigations thus far, we are able to conclude the following. The Astyanax model is tolerant of broad variations in light intensity, pH, oxygen, physical handling and tissue sampling without obvious signs of stress in behavior or physiology. These observations are applicable when maintaining cavefish under high-intensity light, or maintaining surface fish in total darkness. Upon treatment with high-intensity light, three different strains of cavefish show an increase in the amount, distribution and expression level of pigment cells (chromatophores) across multiple body regions. Two of our commercial cavefish strains express melanin, in addition to xanthophores and iridophores. In those fish, all three chromatophores show increased expression and distribution within weeks to months after exposure to light. In one commercial strain, the F1 progeny of parents under light treatments, showed increased pigmentation in less time than their parents, when reared under the same treatment. Juvenile Molino cavefish do not express melanin under sustained high-intensity light treatments; however, they exhibit conspicuous increases in both xanthophore and iridophore pigmentation patterns. When cavefish are transitioned to lower pH (~5.3–5.5), there is a noticeable reduction in melanic pigmentation, but no adverse physiological reactions; these cavefish are acclimated to low pH environments. When surface fish transition into simulated cave environments with low pH and low O2, they are stressed and do not show obvious signs of acclimation; they do exhibit decreases in melanic pigmentation. Commercial cavefish in controlled experiments under light reveal a range in the levels of increased melanin pigmentation across specimens, most likely due to genetic variation within unknown parent lineages from which they were reared. The above observations, and the conclusions drawn from them, indicate rapid responses to experimental treatments by A. mexicanus within short timeframes from days or weeks, to several months. These experiments imply that A. mexicanus may undergo relatively rapid transitions between surface and cave environments, suggesting the reversible character of adaptive traits in this model. While we are still in the early days of cavefish research, it should not be surprising to find that animals are adaptive to specific conditions in contrasting environments. Moreover, that their responses are rapid, reversible and appropriate. Human engineered systems are prepared with forethought to sense and collect information, interpret that information, and respond in accordance with the intention and purpose for which they were constructed. How much more should we then expect divinely engineered creatures to be prepared with unapproachable precision to thrive in the environments for which they were created. In line with our model of Continuous Environmental Tracking (CET), we should expect that if and when surface fish migrated into caves, they would actively track conditions within such unique environments and self-adjust by reintegrating latent sources of biological functionality. We predict that because all animals are adaptive, they would all have this capacity. They would not adjust and adapt through a popularized evolutionary scheme promoting a random mutation-selection process under the trackless agency of nature. As noted by Shapiro (2022), “To give Natural Selection deterministic power over the evolutionary process, it was necessary to assume that genetic changes were random, of small phenotypic effect, and generated significant adaptive differences by accumulation over long periods of time due to selective advantages they conferred”. The most notable outcome of our research thus far, is that adult cavefish and surface fish respond to experimental conditions within weeks of treatment, with responses not limited to multigenerational genetic inheritance. However, we envision that multigenerational epigenetic inheritance may confer certain capacities in subsequent generations that do not yet track with simple treatment conditions (i.e. light-induced restoration of eyes). As stated above, these are early days with the cavefish model, and we are already encouraged to present a new direction in experimental science for the ICR, and a vital new approach for Creation Science that envisions every organism as a divinely engineered creation with wondrous potential. Our research will confirm that life is thoughtfully and intentionally prepared by the infinite wisdom of our Creator, “in whose hand is the life of every living thing, and the breath of all mankind” (Job 12:10). ACKNOWLEDGMENTS We are grateful for all ICR administrators and technicians who assisted with construction and technical assistance during the establishment and operational maintenance of our live animal, microscopy and molecular research laboratories. In particular, we thank Chris Kinman, Michael Lane, Bill West, Daryl Robbins, Reed Arledge, and facilities support staff. We also acknowledge anonymous reviewers for their comments and suggestions, which improved this manuscript. Importantly, we are very appreciative of the numerous donors that continue to keep ICR equipped, operational and inspired BOYLE, ARLEDGE, THOMAS, TOMKINS, AND GULIUZZA Testing the cavefish model 2023 ICC 139
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