next steps in order to confirm the presence or absence of eumelanin and other pigments responding to treatments in cavefish. Melanin synthesis and correlated biosynthetic pathways are fundamental and extensive across animals. Functionally, pigmentation is clearly required in all vertebrate classes inhabiting sunlit environments, and is apparently either nonessential or necessarily downregulated in cave habitats, as observed among global cavefish varieties and a broad diversity of terrestrial representatives found within troglobitic communities (White et al. 2019). In the context of pigment-specific cave adaptations the most pertinent questions include (1) whether melanin synthesis is truly ‘lost’ and irreversible in cavefish, or (2) is the downregulation of melanin production one of several reversible traits within a system of adaptive responses by cavefish to changing environments? Molino and Pachón are the only two A. mexicanus populations with experimentally characterized “loss-function-mutations” that inactivate oca2 genes in cavefish (Protas et al. 2006). The proposed mutations have been traced to separate deletions of exons 21 and 24 in Molino and Pachón cavefish, respectively. In both cases, these ‘mutations’ cause albinism (no eumelanin production), with no evidence of genetic complementation through reproduction by fish that possess them. And although both of these cavefish populations have melanophores with functionally intact melanosomes, the first substrate in the melanin synthesis pathway – conversion of L-Tyrosine to L-DOPA – is blocked (Bilandžija et al. 2013). Melanophores can be induced to produce melanin in these cavefish with the addition of exogenous L-DOPA (Klaassen et al. 2018). The product of the oca2 gene “encodes a putative 12-pass membrane protein” (Bilandžija et al. 2013) that is considered “solely responsible for the evolution of albinism in multiple cavefish populations” (Klaassen et al. 2018). Of special interest, an oca2 deletion in exon 24 through the 3´ UTR (untranslated region) was also found in captive-bred Micos cavefish where it causes albinism (Gross and Wilkens 2013). It is the same deletion as found in albino Pachón cavefish. However, this particular loss-of-function oca2 allele does not cause albinism in the wild population of Micos cavefish (Gross and Wilkins 2013). Their explanation? “Perhaps a loss-of-function oca2 allele harbors a ‘cryptic’ selective value for cave-dwelling fish”. The same authors also imply that, “albinism can arise remarkably quickly in captive-bred fish drawn from cave populations that do not express albinism in nature.” (Gross and Wilkins 2013). And it has been suggested that many cave-related traits can appear within a single generation by phenotypic plasticity (Bilandžija et al. 2020). These interpretations suggests that oca2 may not act alone, but within larger networks of rapid and distinct responses to environmental change. Accordingly, although the “loss or modification of melanin” may indicate the action of single genes, reduction in the overall numbers of melanophores appears to require the action of “ten or more genes in each population” (Borowsky 2018). Thus, there is much more to the story on how this and other genes influence a diversity of traits in cavefish. C. Genomic and genetic regulatory architecture indicate multi-level controls underlying CET 1) Epigenetic mechanisms and transposable elements In a recent groundbreaking study on epigenetic mechanisms of eye loss in Astyanax cavefish (CF), the authors state that, “recent sequencing of the Pachón cavefish genome and other studies revealed no inactivating null mutations in essential eye development genes” (Gore et al. 2018). Null ‘mutations’ are defined as changes that interrupt gene transcription (nonsense or frameshift) or lead to the absence of gene products. Without such ‘mutations’, epigenetic 4), and continual increase in the sizes and distribution patterns of melanophores with continued treatment for several months (Fig. 5). If melanic pigmentation is an adaptive trait, then our observations support the possibility that it is not only a rapid response to an environmental stimulus, but also that pigmentation is a reversable and potentially repeatable trait in cavefish (Fig. 6). Note, we observed melanic pigmentation increases along the dorsum, at the bases of fins, around the mouth, olfactory pits and gill opercula. And there is a consistent increase in subdermal pigmentation surrounding the brain – multiple cell layers below epidermal cells – in both cavefish stocks. This deeper pattern suggests a requirement for protection of the optic tectum and its primary cavity, and that the other patterns likely provide a similar protective function. Furthermore, when we allowed the F1 generation to develop under the same light treatment, they produced a more pronounced pattern of increased pigmentation in the same areas as their parent cavefish within less than half the time (Fig. 5). These F1 fish also expressed melanic pigmentation in new areas, including undersides of the head and belly. Their visible increase in the density of melanophores may be due in part to their concentrations within and upon a smaller body, although specific areas of comparative increase suggest otherwise. Collectively, the F1 cavefish progeny exhibit a putative phenotypic transition between their original untreated commercial cavefish stock, and the A. mexicanus surface fish morphotype (Fig.1, Fig. 5). We also maintain a stock of Molino cavefish, and have reared them from post-larval stages to early juveniles. These fish were obtained from cultures research stocks (see Materials and Methods), and do not exhibit any evidence of melanic pigmentation (Fig. 3H–L). They were bred from one of only two original known cavefish populations that exhibit melanic albinism – a complete absence of melanin (Protas et al. 2006; Klaassen et al. 2018). Both Pachón and Molino cavefish are missing part or all, respectively, of an exon in the oculocutaneous albinism type 2 (oca2) gene and the inferred ‘deletions’ are not from the same exon (see below). Importantly, these cavefish do have melanophores (melanoblasts); however those cells are not producing melanin pigment (Klaassen et al. 2018). As stated above, our Molino cavefish juveniles do not produce black melanin, but they do produce both yellow-orange and iridescent pigments on and within their body (Fig. 3H–L). We intend to purify and analyze oca2 allele sequences from multiple specimens within our Molino cavefish stocks, and all other stocks (SF and CF), and also submit samples for mass spectrometry to identify the yellow-orange pigments and any other pigments they produce. Under a high-magnification stereomicroscope, the yellow-orange pigments appear to be small dendritic xanthophores; the identity of iridophores is not in question. We have also treated our Molino cavefish juveniles with high intensity, full-spectrum and LED light sources, along with bi-weekly pulses of 15-minute tanning treatments. This combination of light treatments has notably increased the amount and distribution of yellow-orange pigmentation in multiple regions, including oral, olfactory, brain-cavity, fin and in multiple epidermal cells and tissues (Fig. 3J–L). There has been no evidence yet of melanin production, although prominent yellow pigmentation patterns increased in similar locations where melanin increased in commercial cavefish under the same treatment protocols. Are they performing similar or related functions in cavefish as melanin? Does pheomelanin or another pigment (e.g. xanthophores) provide an alternative response to downregulation of black or brown melanic pigmentation? Thus far, pheomelanin has not been definitively confirmed in fish (Adachi et al. 2005; Kottler et al. 2015; Cal et al. 2017; Stocker et al. 2020). Comparative histology, mass spectrometry and developmental transcriptomics are appropriate BOYLE, ARLEDGE, THOMAS, TOMKINS, AND GULIUZZA Testing the cavefish model 2023 ICC 132
RkJQdWJsaXNoZXIy MTM4ODY=