Astyanax Mexicanus Research Articles

Reduced Oxygen as an Environmental Pressure in the Evolution of the Blind Mexican Cavefish

Extreme environmental features can drive the evolution of extreme phenotypes. Over the course of evolution, certain environmental changes may be so drastic that they lead to extinction. Conversely, if an organism adapts to harsh environmental changes, the adaptations may permit expansion of a novel niche. The interaction between environmental stressors and adaptive changes is well-illustrated by the blind Mexican cavefish, Astyanaxmexicanus, which has recurrently adapted to the stark subterranean environment. The transition from terrestrial rivers and streams (occupied by extant surface morphs of the same species) to the cave has been accompanied by the resorption of eyes, diminished pigmentation and reduced metabolism in cave-dwelling morphs. The principal features of caves most often associated with evolution of these common cave features are the absence of light and limited nutrition. However, a putatively essential cave feature that has received less attention is the frequently low concentration of oxygen within natural karst environments. Here, we review the potential role of limited oxygen as a critical environmental feature of caves in the Sierra de El Abra. Additionally, we review evidence that Astyanax cavefish may have evolved adaptive features enabling them to thrive in lower oxygen compared to their surface-dwelling counterparts.

A Supernumerary “B-Sex” Chromosome Drives Male Sex Determination in the Pachón Cavefish, <i>Astyanax mexicanus</i>

Sex chromosomes are generally derived from a pair of classical type-A chromosomes, and relatively few alternative models have been proposed up to now. B chromosomes (Bs) are supernumerary and dispensable chromosomes with non-Mendelian inheritance found in many plant and animal species, that have often been considered as selfish genetic elements that behave as genome parasites. The observation that in some species Bs can be either restricted or predominant in one sex raised the interesting hypothesis that Bs could play a role in sex determination. The characterization of putative B master sex-determining (MSD) genes, however, has not yet been provided to support this hypothesis. Here, in Astyanax mexicanus cavefish originating from Pachon cave, we show that Bs are strongly male-predominant. Based on a high-quality genome assembly of a B-carrying male, we characterized the Pachon cavefish B sequence and found that it contains two duplicated loci of the putative MSD gene growth differentiation factor 6b gdf6b). Supporting its role as an MSD gene, we found that the Pachon cavefish gdf6b gene is expressed specifically in differentiating male gonads, and that its knockout induces male-to-female sex reversal in B-carrying males. This demonstrates that gdf6b is necessary for triggering male sex determination in Pachon cavefish. Altogether these results bring multiple and independent lines of evidence supporting the conclusion that the Pachon cavefish B is a “B-sex” chromosome containing duplicated copies of the gdf6b gene acting as an MSD gene promoting male sex determination in this species.

The subterranean catfish Phreatobius cisternarum provides insights into visual adaptations to the phreatic environment

Vertebrate eyes share the same general organization, though species have evolved morphological and functional adaptations to diverse environments. Cave-adapted animals are characterized by a variety of features including eye reduction, loss of body pigmentation, and enhanced non-visual sensory systems. Species that live in perpetual darkness have also evolved sensory mechanisms that are independent of light stimuli. The subterranean catfish Phreatobius cisternarum lives in the Amazonian phreatic zone and displays a diversity of morphological features that are similar to those observed in cavefish and appear to be adaptations to life in the dark. Here we combine histological and transcriptome analyses to characterize sensory adaptations of P. cisternarum to the subterranean environment. Histological analysis showed that the vestigial eyes of P. cisternarum contain a rudimentary lens. Transcriptome analysis revealed a repertoire of eleven visual and non-visual opsins and the expression of 36 genes involved in lens development and maintenance. In contrast to other cavefish species, such as Astyanax mexicanus, Phreatichthys andruzzii, Sinocyclocheilus anophthalmus and Sinocyclocheilus microphthalmus, DASPEI neuromast staining patterns did not show an increase in the number of sensory hair cells. Our work reveals unique adaptations in the visual system of P. cisternarum to underground habitats and helps to shed light into troglomorphic attributes of subterranean animals.

An Asymmetric Genetic Signal Associated with Mechanosensory Expansion in Cave-Adapted Fish.

A key challenge in contemporary biology is connecting genotypic variation to phenotypic diversity. Quantitative genetics provides a powerful technique for identifying regions of the genome that covary with phenotypic variation. Here, we present a quantitative trait loci (QTL) analysis of a natural freshwater fish system, Astyanax mexicanus, that harbors two morphs corresponding to a cave and surface fish. Following their divergence ~500 Kya, cavefish have adapted to the extreme pressures of the subterranean biome. As a consequence, cavefish have lost numerous features, but evolved gains for a variety of constructive features including behavior. Prior work found that sensory tissues (neuromasts) present in the “eye orbit” region of the skull associate with sensitivity to vibrations in water. This augmented sensation is believed to facilitate foraging behavior in the complete darkness of a cave, and may impact on evolved lateral swimming preference. To this point, however, it has remained unclear how morphological variation integrates with behavioral variation through heritable factors. Using a QTL approach, we discovered the genetic architecture of neuromasts present in the eye orbit region, demonstrating that this feature is under genetic control. Interestingly, linked loci were asymmetric-signals were detected using only data collected from the right, but not left, side of the face. This finding may explain enhanced sensitivity and/or feedback of water movements mediating a lateral swimming preference. The locus we discovered based on neuromast position maps near established QTL for eye size and a facial bone morphology, raising the intriguing possibility that eye loss, sensory expansion, and the cranial skeleton may be integrated for evolving adaptive behaviors. Thus, this work will further our understanding of the functional consequence of key loci that influence the evolutionary origin of changes impacting morphology, behavior, and adaptation.

Vibration attraction response is a plastic trait in blind Mexican tetra (Astyanax mexicanus), variable within subpopulations inhabiting the same cave.

Animals evolve their sensory systems and foraging behaviours to adapt and colonize new and challenging habitats such as the dark cave environment. Vibration attraction behaviour (VAB) gives fish the ability to locate the source of a water disturbance in the darkness. VAB evolved in the blind Mexican cave tetra, Astyanax mexicanus. VAB is triggered in cavefish by vibration stimuli peaking at 35 Hz, which is within the main spectrum of water fluctuations produced by many prey crustaceans and insects. VAB has a genetic component and is correlated to an increased number of head mechanosensory neuromasts in the eye orbital region when compared to surface fish. Previous competitive prey capture assays have supported the advantage of VAB for foraging in the dark. Despite its putative adaptive function, VAB has been described as absent in some Astyanax cave populations (Tinaja and Molino) but present in others (Pachón, Piedras, Toro and Sabinos). Here we have tested the occurrence of VAB in the field and in multiple cave populations using a vibrating device in natural pools. Our results confirmed the presence of VAB in caves such as Pachón, Toro and Sabinos but showed that VAB is also present in the Tinaja and Molino cave populations, previously reported as VAB-negative in laboratory experiments. Thus, VAB is available throughout the range of hypogean A. mexicanus. However, and most notably, within a given cave the levels of VAB were highly variable among different pools. Fish at one pool may express no VAB, while fish at another nearby pool of the same cave may actively show VAB. While a variety of environmental conditions may foster this diversity, we found that individuals inhabiting pools with a high abundance of organic matter have reduced expression of VAB. In contrast, in pools with little organic debris where fish probably depend more on hunting than on scavenging, VAB is enhanced. Our results suggest that expression of VAB is a plastic trait whose variability can depend on local conditions. Such plasticity may be required within and among caves where high environmental variability between pools results in a diverse availability of food.

Evolutionary and homeostatic changes in morphology of visual dendrites of Mauthner cells in Astyanax blind cavefish.

Mauthner cells are the largest neurons in the hindbrain of teleost fish and most amphibians. Each cell has two major dendrites thought to receive segregated streams of sensory input: the lateral dendrite receives mechanosensory input while the ventral dendrite receives visual input. These inputs, which mediate escape responses to sudden stimuli, may be modulated by the availability of sensory information to the animal. To understand the impact of the absence of visual information on the morphologies of Mauthner cells during developmental and evolutionary time scales, we examined the teleost Astyanax mexicanus. This species of tetra is found in two morphs: a seeing surface fish and a blind cavefish. We compared the structure of Mauthner cells in surface fish raised under daily light conditions, in surface fish raised in constant darkness, and in two independent lineages of cave populations. The length of ventral dendrites of Mauthner cells in dark-raised surface fish larvae were longer and more branched, while in both cave morphs the ventral dendrites were smaller or absent. The absence of visual input in surface fish with normal eye development leads to a homeostatic increase in dendrite size, whereas over evolution, the absence of light led to the loss of eyes and a reduction in dendrite size.

Spooky Interaction at a Distance in Cave and Surface Dwelling Electric Fishes.

Glass knifefish (Eigenmannia) are a group of weakly electric fishes found throughout the Amazon basin. Their electric organ discharges (EODs) are energetically costly adaptations used in social communication and for localizing conspecifics and other objects including prey at night and in turbid water. Interestingly, a troglobitic population of blind cavefish Eigenmannia vicentespelea survives in complete darkness in a cave system in central Brazil. We examined the effects of troglobitic conditions, which includes a complete loss of visual cues and potentially reduced food sources, by comparing the behavior and movement of freely behaving cavefish to a nearby epigean (surface) population (Eigenmannia trilineata). We found that the strengths of electric discharges in cavefish were greater than in surface fish, which may result from increased reliance on electrosensory perception, larger size, and sufficient food resources. Surface fish were recorded while feeding at night and did not show evidence of territoriality, whereas cavefish appeared to maintain territories. Surprisingly, we routinely found both surface and cavefish with sustained differences in EOD frequencies that were below 10 Hz despite being within close proximity of about 50 cm. A half century of analysis of electrosocial interactions in laboratory tanks suggest that these small differences in EOD frequencies should have triggered the “jamming avoidance response,” a behavior in which fish change their EOD frequencies to increase the difference between individuals. Pairs of fish also showed significant interactions between EOD frequencies and relative movements at large distances, over 1.5 m, and at high differences in frequencies, often >50 Hz. These interactions are likely “envelope” responses in which fish alter their EOD frequency in relation to higher order features, specifically changes in the depth of modulation, of electrosocial signals.

Diversity of Olfactory Responses and Skills in Astyanax Mexicanus Cavefish Populations Inhabiting different Caves

Animals in many phyla are adapted to and thrive in the constant darkness of subterranean environments. To do so, cave animals have presumably evolved mechano- and chemosensory compensations to the loss of vision, as is the case for the blind characiform cavefish, Astyanax mexicanus. Here, we systematically assessed the olfactory capacities of cavefish and surface fish of this species in the lab as well as in the wild, in five different caves in northeastern Mexico, using an olfactory setup specially developed to test and record olfactory responses during fieldwork. Overall cavefish showed lower (i.e., better) olfactory detection thresholds than surface fish. However, wild adult cavefish from the Pachón, Sabinos, Tinaja, Chica and Subterráneo caves showed highly variable responses to the three different odorant molecules they were exposed to. Pachón and Subterráneo cavefish showed the highest olfactory capacities, and Chica cavefish showed no response to the odors presented. We discuss these data with regard to the environmental conditions in which these different cavefish populations live. Our experiments in natural settings document the diversity of cave environments inhabited by a single species of cavefish, A. mexicanus, and highlight the complexity of the plastic and genetic mechanisms that underlie cave adaptation.

From the darkness, light

From the darkness, light

Cloning and characterization of nicotinic acetylcholine receptor γ-like gene in adult transparent Pristella maxillaris.

Nicotinic acetylcholine receptors (nAChRs) play an important role in regulating the development and function of nervous system. The muscle AChR is composed of four homologous glycoprotein subunits with a stoichiometry α2βγδ in fetal or α2βεδ in adult. But the mechanism controlling the transition of fetal AChR γ-subunit to adult AChR ε is still unknown. Here a gene annoted AChR γ-like in Pristella maxillaris was first cloned by rapid amplification of cDNA ends (RACE) based on a transcriptome of dorsal fins. The full length of AChR γ-like was 1984 bp and it encoded 518 amino acids from 100 bp to 1653 bp. The multiple alignment analysis showed that AChR γ-like had 98% protein identity to AChR γ-like in Astyanax mexicanus. Then an 11647 bp DNA from 5'-UTR to 3'-UTR was cloned based on gene structure of AChR γ-like in A.mexicanus. Additionally a 2768 bp DNA upstream 5'-UTR was cloned by chromosome walking method. Furthermore, the results from semi-quantitative PCR showed that AChR γ-like was highly expressed in embryo and adult tissues, such as the muscle, eye, heart and intestine. While it showed low expression in the brain and gill. Significantly, the results of in situ hybridization showed strong diffused expression of AChR γ-like in the muscle of 1 dpf (day post-fertilization) embryo. And weak signal was observed in the muscle of 2-4 dpf embryos. All these data indicated that AChR γ-like could be one subunit of AChRs in the muscle and it could be used to study the development of the neuromuscular junction in adult transparent Pristella maxillaris. Thus our work will lay the foundation for using Pristella maxillaris to analyze the in vivo function of the nAChRs in adult vertebrate.

Identification of novel fish sialidase genes responsible for KDN-cleaving activity.

2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a minor component of sialic acids detected in vertebrates, such as human cancer cells, rat liver, and fish tissues. Although the enzyme activity of KDN-cleaving sialidase (KDN-sialidase) has been detected in rainbow trout, the gene responsible for its expression has not been identified in vertebrates. We evaluated sialidases in human and various fish for their KDN-cleaving activity using an artificial substrate, methylumbelliferyl-KDN (MU-KDN). Four of the human sialidases tested (NEU1, NEU2, NEU3, and NEU4) did not hydrolyze MU-KDN. Although most fish Neu1s showed negligible KDN-sialidase activity, two Neu1b sialidases from Oreochromis niloticus and Astyanax mexicanus, a paralog of Neu1, exhibited a potent KDN-sialidase activity. Further, O. niloticus andOryzias latipesNeu3a exhibited a drastically high KDN-sialidase activity, while Danio rerio Neu3.1 showed moderate activities and other Neu3 proteins exhibited little activity. All the Neu4 sialidases tested in fish cleaved KDN and Neu5Ac from MU-KDN and MU-Neu5Ac, respectively, with equivalent potential. To our knowledge, this is the first report to identify KDN-sialidase genes in vertebrates and we believe that KDN-sialidase activity could be conserved among fish Neu4s.

B chromosomes of multiple species have intense evolutionary dynamics and accumulated genes related to important biological processes

BackgroundOne of the biggest challenges in chromosome biology is to understand the occurrence and complex genetics of the extra, non-essential karyotype elements, commonly known as supernumerary or B chromosomes (Bs). The non-Mendelian inheritance and non-pairing abilities of B chromosomes make them an interesting model for genomics studies, thus bringing to bear different questions about their genetic composition, evolutionary survival, maintenance and functional role inside the cell. This study uncovers these phenomena in multiple species that we considered as representative organisms of both vertebrate and invertebrate models for B chromosome analysis.ResultsWe sequenced the genomes of three animal species including two fishes Astyanax mexicanus and Astyanax correntinus, and a grasshopper Abracris flavolineata, each with and without Bs, and identified their B-localized genes and repeat contents. We detected unique sequences occurring exclusively on Bs and discovered various evolutionary patterns of genomic rearrangements associated to Bs. In situ hybridization and quantitative polymerase chain reactions further validated our genomic approach confirming detection of sequences on Bs. The functional annotation of B sequences showed that the B chromosome comprises regions of gene fragments, novel genes, and intact genes, which encode a diverse set of functions related to important biological processes such as metabolism, morphogenesis, reproduction, transposition, recombination, cell cycle and chromosomes functions which might be important for their evolutionary success.ConclusionsThis study reveals the genomic structure, composition and function of Bs, which provide new insights for theories of B chromosome evolution. The selfish behavior of Bs seems to be favored by gained genes/sequences.

Incipient genetic divergence or cryptic speciation? Procamallanus (Nematoda) in freshwater fishes (Astyanax)

AbstractHosts provide the main environmental traits parasites have to deal with, resulting in covariation between both associates at both micro‐ and macro‐evolutionary scales; phylogenetic analyses of highly host‐specific parasites have shown that parasite and host phylogeny might be highly congruent, and adaptation of a host species to new environments may lead to concordant changes of their parasites. Procamallanus (Spirocamallanus) neocaballeroi is a highly host‐specific parasitic nematode of the Neotropical freshwater fish genus Astyanax in Mexico. One of the host species of the nematode is the emblematic Mexican tetra, A. mexicanus, which exhibits two contrasting phenotypes, a cave‐dwelling morph (with troglomorphic features), and the surface‐dwelling morph; other congeneric species inhabit rivers and lakes, and some of them occur in sympatry, displaying trophic specializations. Here, we explored the hypothesis that contrasting environments (surface rivers vs cave rivers), and host morphological divergence (sympatric ecomorphs in a lacustrine environment) might result in the divergence of their parasites, even though the hosts maintain a cohesive genetic structure as the same species. To test the hypothesis, several populations of Astyanax spp. were sampled to search for P. (S.) neocaballeroi. The nematode was found in 10 of the 52 sampled sites; two localities corresponded to cave populations. The phylogenetic analysis based on COI sequences yielded three major lineages for P. (S.) neocaballeroi. We found no concordance between the three lineages and the habitat where they occur in Astyanax mexicanus, even considering those living in drastic environmental conditions (caves), or between these lineages and lacustrine ecomorphs of Astyanax aeneus and A. caballeroi occurring in sympatry. Instead, genetic lineages of the nematode exhibit a clearer pattern of host species association and geographical distribution; our results showed that P. (S.) neocaballeroi is experiencing an incipient divergence although the morphological study of lineages shows no conspicuous differences.

Hydrodynamic Perception Using an Artificial Lateral Line Device with an Optimized Constriction Canal

To perform flow-related behaviors in darkness, blind cavefish have evolved Lateral Line Systems (LLSs) with constriction canals to enhance hydrodynamic sensing capabilities. Mimicking the design principles, we developed a Canal-type Artificial Lateral Line (CALL) device featuring a biomimetic constriction canal. The hydrodynamic characterization results revealed that the sensitivity of the canal LLS increases with the decrease in the width (from 1 mm to 0.6 mm) and length (from 3 mm to 1 mm) of the constriction canal, which is in accordance with the modeling results of canal mechanics. The CALL device was characterized in Karman vortex streets generated by a cylinder in a laminar flow. The CALL device was able to identify the diameter of the cylinder, with a mean identification error of approximately 2.5%. It also demonstrated the identification ability of wake width using the CALL device, indicating the potential for application in hydrodynamic perception.

Evolution of the acoustic startle response of Mexican cavefish.

The ability to detect threatening stimuli and initiate an escape response is essential for survival and under stringent evolutionary pressure. In diverse fish species, acoustic stimuli activate Mauthner neurons, which initiate a C-start escape response. This reflexive behavior is highly conserved across aquatic species and provides a model for investigating the neural mechanism underlying the evolution of escape behavior. Here, we characterize evolved differences in the C-start response between populations of the Mexican cavefish, Astyanax mexicanus. Cave populations of A. mexicanus inhabit an environment devoid of light and macroscopic predators, resulting in evolved differences in various morphological and behavioral traits. We find that the C-start is present in river-dwelling surface fish and multiple populations of cavefish, but that response kinematics and probability differ between populations. The Pachón population of cavefish exhibits an increased response probability, a slower response latency and speed, and reduction of the maximum bend angle, revealing evolved differences between surface and cave populations. Analysis of the responses of two other independently evolved populations of cavefish, revealed the repeated evolution of reduced angular speed. Investigation of surface-cave hybrids reveals a correlation between angular speed and peak angle, suggesting these two kinematic characteristics are related at the genetic or functional levels. Together, these findings provide support for the use of A. mexicanus as a model to investigate the evolution of escape behavior.

Adaptation to low parasite abundance affects immune investment and immunopathological responses of cavefish.

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.

Genomic Analysis of the Only Blind Cichlid Reveals Extensive Inactivation in Eye and Pigment Formation Genes.

Trait loss represents an intriguing evolutionary problem, particularly when it occurs across independent lineages. Fishes in light-poor environments often evolve “troglomorphic” traits, including reduction or loss of both pigment and eyes. Here, we investigate the genomic basis of trait loss in a blind and depigmented African cichlid, Lamprologus lethops, and explore evolutionary forces (selection and drift) that may have contributed to these losses. This species, the only known blind cichlid, is endemic to the lower Congo River. Available evidence suggests that it inhabits deep, low-light habitats. Using genome sequencing, we show that genes related to eye formation and pigmentation, as well as other traits associated with troglomorphism, accumulated inactivating mutations rapidly after speciation. A number of the genes affected in L. lethops are also implicated in troglomorphic phenotypes in Mexican cavefish (Astyanax mexicanus) and other species. Analysis of heterozygosity patterns across the genome indicates that L. lethops underwent a significant population bottleneck roughly 1 Ma, after which effective population sizes remained low. Branch-length tests on a subset of genes with inactivating mutations show little evidence of directional selection; however, low overall heterozygosity may reduce statistical power to detect such signals. Overall, genome-wide patterns suggest that accelerated genetic drift from a severe bottleneck, perhaps aided by directional selection for the loss of physiologically expensive traits, caused inactivating mutations to fix rapidly in this species.

Dark world rises: The emergence of cavefish as a model for the study of evolution, development, behavior, and disease.

A central question in biology is how naturally occurring genetic variation accounts for morphological and behavioral diversity within a species. The Mexican tetra, Astyanax mexicanus, has been studied for nearly a century as a model for investigating trait evolution. In March of 2019, researchers representing laboratories from around the world met at the Sixth Astyanax International Meeting in Santiago de Querétaro, Mexico. The meeting highlighted the expanding applications of cavefish to investigations of diverse aspects of basic biology, including development, evolution, and disease-based applications. A broad range of integrative approaches are being applied in this system, including the application of state-of-the-art functional genetic assays, brain imaging, and genome sequencing. These advances position cavefish as a model organism for addressing fundamental questions about the genetics and evolution underlying the impressive trait diversity among individual populations within this species.

Repeated evolution of eye loss in Mexican cavefish: Evidence of similar developmental mechanisms in independently evolved populations.

Evolution in similar environments often leads to convergence of behavioral and anatomical traits. A classic example of convergent trait evolution is the reduced traits that characterize many cave animals: reduction or loss of pigmentation and eyes. While these traits have evolved many times, relatively little is known about whether these traits repeatedly evolve through the same or different molecular and developmental mechanisms. The small freshwater fish, Astyanax mexicanus, provides an opportunity to investigate the repeated evolution of cave traits. A. mexicanus exists as two forms, a sighted, surface-dwelling form and at least 29 populations of a blind, cave-dwelling form that initially develops eyes that subsequently degenerate. We compared eye morphology and the expression of eye regulatory genes in developing surface fish and two independently evolved cavefish populations, Pachón and Molino. We found that many of the previously described molecular and morphological alterations that occur during eye development in Pachón cavefish are also found in Molino cavefish. However, for many of these traits, the Molino cavefish have a less severe phenotype than Pachón cavefish. Further, cave-cave hybrid fish have larger eyes and lenses during early development compared with fish from either parental population, suggesting that some different changes underlie eye loss in these two populations. Together, these data support the hypothesis that these two cavefish populations evolved eye loss independently, yet through some of the same developmental and molecular mechanisms.

Genetic architecture underlying changes in carotenoid accumulation during the evolution of the blind Mexican cavefish, Astyanax mexicanus.

Carotenoids are lipid-soluble yellow to orange pigments produced by plants, bacteria, and fungi. They are consumed by animals and metabolized to produce molecules essential for gene regulation, vision, and pigmentation. Cave animals represent an interesting opportunity to understand how carotenoid utilization evolves. Caves are devoid of light, eliminating primary production of energy through photosynthesis and, therefore, limiting carotenoid availability. Moreover, the selective pressures that favor carotenoid-based traits, like pigmentation and vision, are relaxed. Astyanax mexicanus is a species of fish with multiple river-adapted (surface) and cave-adapted populations (i.e., Tinaja, Pachón, Molino). Cavefish exhibit regressive features, such as loss of eyes and melanin pigment, and constructive traits, like increased sensory neuromasts and starvation resistance. Here, we show that, unlike surface fish, Tinaja and Pachón cavefish accumulate carotenoids in the visceral adipose tissue. Carotenoid accumulation is not observed in Molino cavefish, indicating that it is not an obligatory consequence of eye loss. We used quantitative trait loci mapping and RNA sequencing to investigate genetic changes associated with carotenoid accumulation. Our findings suggest that multiple stages of carotenoid processing may be altered in cavefish, including absorption and transport of lipids, cleavage of carotenoids into unpigmented molecules, and differential development of intestinal cell types involved in carotenoid assimilation. Our study establishes A. mexicanus as a model to study the genetic basis of natural variation in carotenoid accumulation and how it impacts physiology.