‘loss-of-function’ mutation of oca2 in melanic albino cavefish (see Fig. 3H–L), the melanin pathway is interrupted prior to tyrosinase function, which prevents melanin synthesis (Bilandžija et al. 2013). This increases the availability of L-tyrosine, dopamine and norepinephrine in pre-feeding larval cavefish, which in turn increases the level of catecholamines (CAT) in the brain and kidneys, relative to surface fish (Bilandžija et al. 2013). As with the shh pathway, the CAT pathway could promote adaptive physiological and behavioral traits in cave environments, and therefore provides another inferred example of pleiotropy in Astyanax. Furthermore, oca2 mutant surface fish present a pleiotropic function with duel effects on albinism and sleep loss (O’Gorman et al. 2021). This study implies that oca2 has yet another role as a regulator of “adaptive evolution” in cavefish (O’Gorman et al. 2021). The trend of uncovering coupled adaptive processes in the cavefish model is growing. This will likely challenge conventional science to reconcile random, mutational, evolutionary trade-offs with inferences for purposeful, organism-centered deployment of complex adaptive traits, of which most, or all, will be confirmed to be experimentally reversible. Are pleiotropic events truly selective gain and loss modalities? Tradeoffs also link other trait gains and losses. The relationship between the olfactory and lens placodes is impacted by a tradeoff controlled by Shh, Fgf8, and BMP4 signaling, antagonism between eyes and number of teeth may be controlled by Fgf8, BMP4, and pitx2, the enlargement of the hypothalamus is mediated by re-deployment of cells from the ventral retina, and VAB, and increased cranial neuromast density may be facilitated by the extra space created by eye loss. The precise mechanisms responsible for sensory trait linkages are still poorly understood. (Jeffery 2020). From an engineering perspective, “trait gains and losses”, “tradeoffs”, “antagonism”, “re-deployments” and “sensory trait linkages” would actually point to highly-integrated, innate adjustments that are pre-programmed responses by organisms to changing environments. In other words, coupled pleiotropic effects reflect built-in adaptive mechanisms, not fortuitous unguided evolutionary by-products of natural selection. To use Dr. Jeffery’s own words, “precise mechanisms” are certainly “responsible for sensory trait linkages” within cavefish and surface fish. However, they will remain “poorly understood” if their origins and functionality are pursued within the mutation-selection paradigm. With regards to adaptation, interpretations from continuous environmental tracking (CET) necessarily include the premise that organisms become acclimated to their environments. When traits are coupled, or multiplexed as described above, then those trait adjustments regardless of their direction (e.g. reduced, latent, enhanced) contribute to the process of acclimation. Furthermore, we should expect to find evidence that trait adjustments are integrated across molecular, genetic, physiological and anatomical scales. This would also apply to a broad number of traits and their genetic regulatory mechanisms that have yet to be investigated. In our second preliminary experiment, surface fish (SF) were maintained under low light conditions at lower than normal levels of dissolved O2 (0.9–4.0 mg/L) and moderately high levels of CO2 (pH 5.8–6.0) for 3 months. These fish were agitated, bumped into aquarium walls on multiple occasions, and at the lowest O2 levels they were ‘gasping’ for oxygen – gill opercula were flapping in coordination with rapid jaw movements. These fish also began to exhibit reductions in pigmentation around the iris, and along the dorsum and lateral flanks of the body (Fig. 6). In contrast, cavefish (CF) in our third preliminary experiment were maintained 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. These CF showed no outward signs of stress in feeding, breathing, swimming or navigation. Although O2 levels were not intentionally low, pH levels were set low to emulate aquatic pH levels within natural cave systems. The pH setting was derived from expected limestone mineral contributions (CaCO3) to the water, suggesting acidic levels in karst caves where CF are found. Accordingly, the CF in our experiment exhibited behavior indicative of acclimation to low pH, as predicted. However, the SF exhibited no evidence of acclimation to low oxygen or darkness. Several investigations provide fundamental examples of coupled trait adjustments that clearly point to the necessary process of acclimation in cave environments. In addition to the absence of light and limited food resources, karst caves commonly contain low oxygen environments – regions of hypoxia. Multiple studies report that Astyanax cavefish “likely consume less oxygen than surface fish” through “stable oxygen consumption” across a 24-hour day (Boggs and Gross 2021). Thus, cavefish are hypoxia-tolerant or hypoxia-acclimated. Compared with Astyanax surface fish, molecular and anatomical evidence supporting acclimation in karst caves include increased gill size for more efficient gas exchange, greater numbers of mature red blood cells (erythrocytes) during development, higher expression levels of hemoglobin subunit adult alpha 1 (hbaa1), genes involved in “oxygen transport” and “oxygen binding”, and resistance to hypoxia during “both development and adulthood” (Boggs and Gross 2021). We also learn from van der Weele and Jeffery (2022) that cavefish adjust to hypoxia with increased “erythrocyte development and constitutive overexpression” of multiple hypoxia-inducible factor one (hif1) gene subunits, and demonstrate the capacity to “carry and distribute essential oxygen to tissues and organs early in development”. Furthermore, cave populations not only “harbor significantly higher blood hemoglobin concentrations” than surface fish, but also possess “significantly larger erythrocytes” than surface fish (Boggs et al. 2022). When compared with surface fish, these hemoglobin-rich erythrocytes bind, transport and deliver more oxygen per blood cell, thus optimizing respiration at genetic, molecular, cellular and physiological levels within hypoxic environments. The anatomy, morphology and function of their hearts are optimized as well. Astyanax cave-dwelling morphs exhibit a slower heart rate than river-dwelling morphs, with “shape and size differences of the heart” arising during early development, “suggesting that such traits are genetically determined” (Tang et al. 2018). Also during early development, there are noticeable differences between CF and SF in heart size, morphology, beating frequency, melanophores, and adipocyte cells, and they all “become increasingly apparent over life” (Tang et al. 2018). Further, cavefish exhibit spongier heart morphologies that correlate with rounder ventricles and lower wall-to-trabecula area ratios. According to Tang et al. (2018), “a heart with more trabeculae has a larger surface area exposed to the blood.” And to emphasize the genetics behind many of the differences listed above, Tang et al. (2018) suggest there is an uncoupling of heart-related phenotypes “with atrial size and adipocyte number similar to surface fish, and ventricular size, shape and sponginess similar to Pachón [cavefish]”. From all of the essential systems described above, we are told that Astyanax cavefish have evolved hypoxic adaptations as a consequence of life in a low-oxygen environment. Clearly, such a complex series of highly-specified adjustments must be pre-planned and integrated on multiple scales in order to facilitate the stringent mechanisms of gas exchange and circulation in hypoxic environments. Astyanax cavefish function efficiently with this circulatory BOYLE, ARLEDGE, THOMAS, TOMKINS, AND GULIUZZA Testing the cavefish model 2023 ICC 134
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