molecular, genetic and physiological mechanisms that may account for those results, in cavefish, and other organisms, and present both ongoing experiments and future directions for research with the A. mexicanus model organism. A. Reproduction and development provide experimental access to early mechanisms of adaptation Development from fertilization through embryogenesis to the formation of larvae is considered one of the most critical periods in the life of fishes, as also observed in most major lineages across the Metazoa. During this period, numerous genomic elements are expressed, and distinct types of cells, tissues and organ-systems of the body plan become specified, differentiated and functional. It is also an influential interval of time when genetic and epi-genetic architectures are inherited, and/or initiated in a new generation. Thus, molecular and cellular signatures of adaption(s) would likely be detectable during development. Furthermore, the physical scale of cell types (neuron, blood), tissues (epidermis, muscle) and organs (brain, eye) are optimal for experimental manipulation and micrographic evaluation and imaging during early stages of development (Fig. 2). We have begun to take advantage of spawning events from both surface fish and cavefish morphotypes; however, such events have been unpredictable. Therefore progress is underway to establish breeding tanks and associated infrastructure for collecting, rearing and handling embryos and larvae based upon published protocols (Riddle et al. 2018; Baumann and Ingalls 2022). As mentioned above, the primary targets of experimental research include changes in pigmentation (next section) and restoration of sight, both of which are predicted by CET as repeatable and reversable adaptations. Through applied techniques in molecular biology, riboprobe synthesis, and both whole mount in situ hybridization (WMISH) and immunohistochemistry (IHC), we will initiate experiments to characterize gene expression during embryonic development of the visual system. This approach includes a microscopic comparison of eye development and eye degeneration in surface fish and cavefish, respectively. Primary molecular targets include genes that are shown to regulate optic cup and stalk development (pax6, shh, pax2), genes effecting loss and rescue of retinal tissue (Fgf8, Lhx2, rx3), and genes that are essential during lens formation (sox2, cryaa, crybb1c, cryba1l) that inhibit the apoptotic process following initial eye development in late embryonic and early larval stages (Yamamoto et al. 2004; Pottin et al. 2011; Krishnan and Rohner 2017; Sifuentes-Romero et al. 2020; Warren et al. 2021). There is a four-fold purpose in pursuing these and other molecular experiments. First, we intend to repeat multiple experiments that were published from the conventional perspective, and thus motivated by evolutionary thinking. This is not a redundant exercise, for we approach our questions from a completely different worldview with an original model that is formulated to test adaptations as pre-engineered systems in accord with that worldview. Second, the CET model proposes that adaptative mechanisms integrate multiple functions across different scales (molecular, cellular, physiological). For example, the loss of eyes in cavefish is not organ-specific, but involves the dual effect of shh expression on eye loss with a concomitant role in the enhancement of feeding anatomy (Yamamoto et al. 2009). Third, eye loss or eye restoration should be investigated to assess comparative gene expression patterns between generations. This will require WMISH of ‘eye genes’ within early developmental stages obtained from parental cavefish reared under high light treatments, the progeny of their subsequent F1 generation, the progeny of untreated cavefish from the same strain, and similar stages of development from different strains (e.g. commercial, Molino). And fourth, because conventional research with the Astyanax model is focused on “the gain and loss of traits” in cavefish (Jeffery 2001, 2020), random genetic mistakes are commonly invoked to explain those changes, whether they are regressive or constructive. Although, we also find conventional explanations that are illuminating: “To our knowledge, there is no case of a viable vertebrate embryo that would never develop eyes.” (Pottin et al. 2011). This is partly acknowledged in the fact that, “tight temporal regulation of signaling systems during early embryogenesis has a crucial impact on the size and shape of a structure . . . the neural plate-derived component of the CF eye defect.” (Pottin et al. 2011). Is there another explanation for eye loss, and the “gain and loss” of other traits in cavefish? Through molecular developmental experiments we aim to assess the conventional narrative that loss-of-function mutations (e.g. indels, frameshift, transposition) and natural selection have produced the natural cave mutants of Astyanax mexicanus. We think that CET would account for a series of highly-integrated molecular and developmental mechanisms regulating adaptive eye loss and restoration. B. Patterns of pigmentation demonstrate rapid and distinct responses to environmental change Experimental targets of chromatophore expression include light-induced changes in the amount, density and pattern distribution of melanophores – the melanin-producing chromatophores in fish. The size and shape of melanophores is known to be correlated with the extent of aggregation or dispersal of their melanosomes, which are the pigment-containing organelles within melanophores. In surface fish, melanosomes are abundant and widely dispersed within melanophores. In all cavefish that have been thoroughly examined, melanophores are detectable, even when they do not convey an obvious pigmentation pattern, or even lack melanin (see below). This leads many investigators to describe cavefish as unpigmented, having reduced pigmentation, or exhibiting a loss of melanin pigmentation (Klaassen et al. 2018; Jeffery 2020). The term ‘albinism’ is also utilized to indicate the absence of melanin or low production of melanin. But it does not imply the absence of pigmentation produced by other chromatophores. Before we initiated any experimental treatments, we observed a detectable distribution of black pigment in the form of small dendritic (stellate or star-shaped) melanophores in our commercial cavefish stocks (Fig 1, Fig. 3A–G, Fig. 4A, Fig. 5). The expression of melanin is notably low in these cavefish, and geographical locations for the cave systems of our commercial cavefish – purchased through suppliers in Florida and Arizona – have not yet been identified. However, they do possess both epidermal and subdermal melanocytes across their bodies that produce melanin pigment, as well as distinct expression patterns of pigments from other chromatophores (xanthophores, iridophores). It is possible that sources of ambient and/or incandescent room lighting on proprietor stocks, and on our own stock tanks after purchase, may have induced some pigmentation prior to experimentation. When we treat these cavefish with daily cycles of combined high intensity, full-spectrum and LED light sources, there is a pronounced increase in melanin pigment production. The pattern does vary among cavefish under treatment, which may indicate that source fish stocks are a genetic mosaic of cavefish populations, and/or there are undisclosed or untraceable patterns of hybridization events across surface fish and cavefish leading to the stocks we possess (Fumey et al. 2018; Jeffery 2020; Moran et al. 2022). Yet the response patterns are unmistakable. Those patterns include increased melanic pigmentation in the adult fish over short periods of weeks (Fig. BOYLE, ARLEDGE, THOMAS, TOMKINS, AND GULIUZZA Testing the cavefish model 2023 ICC 131
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