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Insights from organisms, which have evolved natural strategies for promoting survivability under extreme environmental pressures, may help guide future research into novel approaches for enhancing human longevity. The cave-adapted Mexican tetra, Astyanax mexicanus , has attracted interest as a model system for metabolic resilience , a term we use to denote the property of maintaining health and longevity under conditions that would be highly deleterious in other organisms (Figure 1). Cave-dwelling populations of Mexican tetra exhibit elevated blood glucose, insulin resistance and hypertrophic visceral adipocytes compared to surface-dwelling counterparts. However, cavefish appear to avoid pathologies typically associated with these conditions, such as accumulation of advanced-glycation-end-products (AGEs) and chronic tissue inflammation. The metabolic strategies underlying the resilience properties of A. mexicanus cavefish, and how they relate to environmental challenges of the cave environment, are poorly understood. Here, we provide an untargeted metabolomics study of long- and short-term fasting in two A. mexicanus cave populations and one surface population. We find that, although the metabolome of cavefish bears many similarities with pathological conditions such as metabolic syndrome, cavefish also exhibit features not commonly associated with a pathological condition, and in some cases considered indicative of an overall robust metabolic condition. These include a reduction in cholesteryl esters and intermediates of protein glycation, and an increase in antioxidants and metabolites associated with hypoxia and longevity. This work suggests that certain metabolic features associated with human pathologies are either not intrinsically harmful, or can be counteracted by reciprocal adaptations. We provide a transparent pipeline for reproducing our analysis and a Shiny app for other researchers to explore and visualize our dataset. 

Abstract Cell lines have become an integral resource and tool for conducting biological experiments ever since the Hela cell line was first developed (Scherer et al. in J Exp Med 97:695–710, 1953). They not only allow detailed investigation of molecular pathways but are faster and more cost-effective than most in vivo approaches. The last decade saw many emerging model systems strengthening basic science research. However, lack of genetic and molecular tools in these newer systems pose many obstacles.Astyanax mexicanusis proving to be an interesting new model system for understanding metabolic adaptation. To further enhance the utility of this system, we developed liver-derived cell lines from both surface-dwelling and cave-dwelling morphotypes. In this study, we provide detailed methodology of the derivation process along with comprehensive biochemical and molecular characterization of the cell lines, which reflect key metabolic traits of cavefish adaptation. We anticipate these cell lines to become a useful resource for theAstyanaxcommunity as well as researchers investigating fish biology, comparative physiology, and metabolism. 

Physical inactivity is a scourge to human health, promoting metabolic disease and muscle wasting. Interestingly, multiple ecological niches have relaxed investment into physical activity, providing an evolutionary perspective into the effect of adaptive physical inactivity on tissue homeostasis. One such example, the Mexican cavefishAstyanax mexicanus,has lost moderate-to-vigorous activity following cave colonization, reaching basal swim speeds ~3.7-fold slower than their river-dwelling counterpart. This change in behavior is accompanied by a marked shift in body composition, decreasing total muscle mass and increasing fat mass. This shift persisted at the single muscle fiber level via increased lipid and sugar accumulation at the expense of myofibrillar volume. Transcriptomic analysis of laboratory-reared and wild-caught cavefish indicated that this shift is driven by increased expression ofpparγ—the master regulator of adipogenesis—with a simultaneous decrease in fast myosin heavy chain expression. Ex vivo and in vivo analysis confirmed that these investment strategies come with a functional trade-off, decreasing cavefish muscle fiber shortening velocity, time to maximal force, and ultimately maximal swimming speed. Despite this, cavefish displayed a striking degree of muscular endurance, reaching maximal swim speeds ~3.5-fold faster than their basal swim speeds. Multi-omic analysis suggested metabolic reprogramming, specifically phosphorylation of Pgm1-Threonine 19, as a key component enhancing cavefish glycogen metabolism and sustained muscle contraction. Collectively, we reveal broad skeletal muscle changes following cave colonization, displaying an adaptive skeletal muscle phenotype reminiscent to mammalian disuse and high-fat models while simultaneously maintaining a unique capacity for sustained muscle contraction via enhanced glycogen metabolism. 

Image-based cell classification has become a common tool to identify phenotypic changes in cell populations. However, this methodology is limited to organisms possessing well characterized species-specific reagents (e.g., antibodies) that allow cell identification, clustering and convolutional neural network (CNN) training. In the absence of such reagents, the power of image-based classification has remained mostly off-limits to many research organisms. We have developed an image-based classification methodology we named Image3C (Image-Cytometry Cell Classification) that does not require species-specific reagents nor pre-existing knowledge about the sample. Image3C combines image-based flow cytometry with an unbiased, high-throughput cell cluster pipeline and CNN integration. Image3C exploits intrinsic cellular features and non-species-specific dyes to perform de novo cell composition analysis and to detect changes in cellular composition between different conditions. Therefore, Image3C expands the use of imaged-based analyses of cell population composition to research organisms in which detailed cellular phenotypes are unknown or for which species-specific reagents are not available. 

Reduced parasitic infection rates in the developed world are suspected to underlie the rising prevalence of autoimmune disorders. However, the long-term evolutionary consequences of decreased parasite exposure on an immune system are not well understood. We used the Mexican tetra Astyanax mexicanus to understand how loss of parasite diversity influences the evolutionary trajectory of the vertebrate immune system, by comparing river with cave morphotypes. Here, we present field data affirming a strong reduction in parasite diversity in the cave ecosystem, and show that cavefish immune cells display a more sensitive pro-inflammatory response towards bacterial endotoxins. Surprisingly, other innate cellular immune responses, such as phagocytosis, are drastically decreased in cavefish. Using two independent single-cell approaches, we identified a shift in the overall immune cell composition in cavefish as the underlying cellular mechanism, indicating strong differences in the immune investment strategy. While surface fish invest evenly into the innate and adaptive immune systems, cavefish shifted immune investment to the adaptive immune system, and here, mainly towards specific T-cell populations that promote homeostasis. Additionally, inflammatory responses and immunopathological phenotypes in visceral adipose tissue are drastically reduced in cavefish. Our data indicate that long-term adaptation to low parasite diversity coincides with a more sensitive immune system in cavefish, which is accompanied by a reduction in the immune cells that play a role in mediating the pro-inflammatory response. 

Abstract Identifying the genetic factors that underlie complex traits is central to understanding the mechanistic underpinnings of evolution. Cave-dwelling Astyanax mexicanus populations are well adapted to subterranean life and many populations appear to have evolved troglomorphic traits independently, while the surface-dwelling populations can be used as a proxy for the ancestral form. Here we present a high-resolution, chromosome-level surface fish genome, enabling the first genome-wide comparison between surface fish and cavefish populations. Using this resource, we performed quantitative trait locus (QTL) mapping analyses and found new candidate genes for eye loss such as dusp26 . We used CRISPR gene editing in A. mexicanus to confirm the essential role of a gene within an eye size QTL, rx3 , in eye formation. We also generated the first genome-wide evaluation of deletion variability across cavefish populations to gain insight into this potential source of cave adaptation. The surface fish genome reference now provides a more complete resource for comparative, functional and genetic studies of drastic trait differences within a species. 

Abstract Studying how different genotypes respond to environmental variation is essential to understand the genetic basis of adaptation. The Mexican tetra,Astyanax mexicanus, has cave and surface‐dwelling morphotypes that have adapted to entirely different environments in the wild, and are now successfully maintained in lab conditions. While this has enabled the identification of genetic adaptations underlying a variety of physiological processes, few studies have directly compared morphotypes between lab‐reared and natural populations. Such comparative approaches could help dissect the varying effects of environment and morphotype, and determine the extent to which phenomena observed in the lab are generalizable to conditions in the field. To this end, we take a transcriptomic approach to compare the Pachón cavefish and their surface fish counterparts in their natural habitats and the lab environment. We identify key changes in expression of genes implicated in metabolism and physiology between groups of fish, suggesting that morphotype (surface or cave) and environment (natural or lab) both alter gene expression. We find gene expression differences between cave and surface fish in their natural habitats are much larger than differences in expression between morphotypes in the lab environment. However, lab‐raised cave and surface fish still exhibit numerous gene expression changes, supporting genetically encoded changes in livers of this species. From this, we conclude that a controlled laboratory environment may serve as an ideal setting to study the genetic underpinnings of metabolic and physiological differences between the cavefish and surface fish. 

Abstract BackgroundAstyanax mexicanusis a well‐established fish model system for evolutionary and developmental biology research. These fish exist as surface forms that inhabit rivers and 30 different populations of cavefish. Despite important progress in the deployment of new technologies, deep mechanistic insights into the genetic basis of evolution, development, and behavior have been limited by a lack of transgenic lines commonly used in genetic model systems. ResultsHere, we expand the toolkit of transgenesis by characterizing two novel stable transgenic lines that were generated using the highly efficientTol2system, commonly used to generate transgenic zebrafish. A stable transgenic line consisting of the zebrafish ubiquitin promoter expresses enhanced green fluorescent protein ubiquitously throughout development in a surface population ofAstyanax. To define specific cell‐types, a Cntnap2‐mCherry construct labels lateral line mechanosensory neurons in zebrafish. Strikingly, both constructs appear to label the predicted cell types, suggesting many genetic tools and defined promoter regions in zebrafish are directly transferrable to cavefish. ConclusionThe lines provide proof‐of‐principle for the application ofTol2transgenic technology inA. mexicanus. Expansion on these initial transgenic lines will provide a platform to address broadly important problems in the quest to bridge the genotype‐phenotype gap.