Astyanax Mexicanus Research Articles

The nature and distribution of putative non-functional alleles suggest only two independent events at the origins of Astyanax mexicanus cavefish populations

BackgroundSeveral studies suggested that cavefish populations of Astyanax mexicanus settled during the Late Pleistocene. This implies that the cavefish’s most conspicuous phenotypic changes, blindness and depigmentation, and more cryptic characters important for cave life, evolved rapidly.ResultsUsing the published genomes of 47 Astyanax cavefish from la Cueva de El Pachón, El Sótano de la Tinaja, La Cueva Chica and El Sótano de Molino, we searched for putative loss-of-function mutations in previously defined sets of genes, i.e., vision, circadian clock and pigmentation genes. Putative non-functional alleles for four vision genes were identified. Then, we searched genome-wide for putative non-functional alleles in these four cave populations. Among 512 genes with segregating putative non-functional alleles in cavefish that are absent in surface fish, we found an enrichment in visual perception genes. Among cavefish populations, different levels of shared putative non-functional alleles were found. Using a subset of 12 genes for which putative loss-of-function mutations were found, we extend the analysis of shared pseudogenes to 11 cave populations. Using a subset of six genes for which putative loss-of-function mutations were found in the El Sótano del Toro population, where extensive hybridization with surface fish occurs, we found a correlation between the level of eye regression and the amount of putative non-functional alleles.ConclusionsWe confirm that very few putative non-functional alleles are present in a large set of vision genes, in accordance with the recent origin of Astyanax mexicanus cavefish. Furthermore, the genome-wide analysis indicates an enrichment of putative loss-of-function alleles in genes with vision-related GO-terms, suggesting that visual perception may be the function chiefly impacted by gene losses related to the shift from a surface to a cave environment.The geographic distribution of putative loss-of-function alleles newly suggests that cave populations from Sierra de Guatemala and Sierra de El Abra share a common origin, albeit followed by independent evolution for a long period. It also supports that populations from the Micos area have an independent origin. In El Sótano del Toro, the troglomorphic phenotype is maintained despite massive introgression of the surface genome.

Starvation-resistant cavefish reveal conserved mechanisms of starvation-induced hepatic lipotoxicity.

Starvation causes the accumulation of lipid droplets in the liver, a somewhat counterintuitive phenomenon that is nevertheless conserved from flies to humans. Much like fatty liver resulting from overfeeding, hepatic lipid accumulation (steatosis) during undernourishment can lead to lipotoxicity and atrophy of the liver. Here, we found that although surface populations of Astyanax mexicanus undergo this evolutionarily conserved response to starvation, the starvation-resistant cavefish larvae of the same species do not display an accumulation of lipid droplets upon starvation. Moreover, cavefish are resistant to liver atrophy during starvation, providing a unique system to explore strategies for liver protection. Using comparative transcriptomics between zebrafish, surface fish, and cavefish, we identified the fatty acid transporter slc27a2a/fatp2 to be correlated with the development of fatty liver. Pharmacological inhibition of slc27a2a in zebrafish rescues steatosis and atrophy of the liver upon starvation. Furthermore, down-regulation of FATP2 in Drosophila larvae inhibits the development of starvation-induced steatosis, suggesting the evolutionarily conserved importance of the gene in regulating fatty liver upon nutrition deprivation. Overall, our study identifies a conserved, druggable target to protect the liver from atrophy during starvation.

In preprints: Shh signaling activity predicts cardiac laterality in Astyanax mexicanus populations.

In preprints: Shh signaling activity predicts cardiac laterality in Astyanax mexicanus populations.

Telencephalic eversion in embryos and early larvae of four teleost species.

The telencephalon of ray-finned fishes undergoes eversion, which is very different to the evagination that occurs in most other vertebrates. Ventricle morphogenesis is key to build an everted telencephalon. Thus, here we use the apical marker zona occludens 1 to understand ventricle morphology, extension of the tela choroidea and the eversion process during early telencephalon development of four teleost species: giant danio (Devario aequipinnatus), blind cavefish (Astyanax mexicanus), medaka (Oryzias latipes), and paradise fish (Macroposus opercularis). In addition, by using immunohistochemistry against tubulin and calcium-binding proteins, we analyze the general morphology of the telencephalon, showing changes in the location and extension of the olfactory bulb and other telencephalic regions from 2 to 5 days of development. We also analyze the impact of abnormal eye and telencephalon morphogenesis on eversion, showing that cyclops mutants do undergo eversion despite very dramatic abnormal eye morphology. We discuss how the formation of the telencephalic ventricle in teleost fish, with its characteristic shape, is a crucial event during eversion.

Telomere length and dynamics in Astyanax mexicanus cave and surface morphs.

Telomeres are non-coding DNA repeats at the chromosome ends and their shortening is considered one of the major causes of aging. However, they also serve as a biomarker of environmental exposures and their length and attrition is affected by various stressors. In this study, we examined the average telomere length in Astyanax mexicanus, a species that has both surface-dwelling and cave-adapted populations. The cave morph descended from surface ancestors and adapted to a markedly different environment characterized by specific biotic and abiotic stressors, many of which are known to affect telomere length. Our objective was to explore whether telomere length differs between the two morphs and whether it serves as a biological marker of aging or correlates with the diverse environments the morphs are exposed to. We compared telomere length and shortening between laboratory-reared Pachón cavefish and Rio Choy surface fish of A. mexicanus across different tissues and ages. Astyanax mexicanus surface fish exhibited longer average telomere length compared to cavefish. In addition, we did not observe telomere attrition in either cave or surface form as a result of aging in adults up to 9 years old, suggesting that efficient mechanisms prevent telomere-mediated senescence in laboratory stocks of this species, at least within this time frame. Our results suggest that telomere length in Astyanax may be considered a biomarker of environmental exposures. Cavefish may have evolved shorter and energetically less costly telomeres due to the absence of potential stressors known to affect surface species, such as predator pressure and ultra-violet radiation. This study provides the first insights into telomere dynamics in Astyanax morphs and suggests that shorter telomeres may have evolved as an adaptation to caves.

Defining the Unseen: Population-Specific Markers for Astyanax mexicanus Blind Cavefish.

Astyanax mexicanus is an emerging model system used to study development, evolution, and behavior of multiple cavefish populations that have repeatedly evolved from conspecific surface fish. Although surface and cavefish live and breed in the laboratory, there are no rapid methods for distinguishing between different cavefish populations. We present 2 methods for genotyping fish for a total of 16 population-specific markers using methods that are easy and inexpensive to implement in a basic molecular biology laboratory. This resource will help researchers maintain independent stocks within the laboratory and distinguish between fish from different populations.

Unraveling stress resilience: Insights from adaptations to extreme environments by Astyanax mexicanus cavefish.

Extreme environmental conditions have profound impacts on shaping the evolutionary trajectory of organisms. Exposure to these conditions elicits stress responses, that can trigger phenotypic changes in novel directions. The Mexican Tetra, Astyanax mexicanus, is an excellent model for understanding evolutionary mechanisms in response to extreme or new environments. This fish species consists of two morphs; the classical surface-dwelling fish and the blind cave-dwellers that inhabit dark and biodiversity-reduced ecosystems. In this review, we explore the specific stressors present in cave environments and examine the diverse adaptive strategies employed by cave populations to not only survive but thrive as successful colonizers. By analyzing the evolutionary responses of A. mexicanus, we gain valuable insights into the genetic, physiological, and behavioral adaptations that enable organisms to flourish under challenging environmental conditions.

The long-read assembly of Apareiodon sp., a neotropical fish with a ZZ/ZW sex chromosome system.

Neotropical fishes emerge as an extremely diverse group of vertebrates where genomic strategies to evaluate structural and functional features are still beginning. Here, we present a second draft genome of Apareiodon sp. (2n=54, ZZ/ZW), adding PacBio technology whole genome sequencing, and assembling by combining two technologies (long and short reads). Using a detailed strategy for genome assembly with fish genomes of Pygocentrus nattereri, Carassius auratus, and Astyanax mexicanus as references, the final assembly of the Apareiodon sp. genome generated 93 scaffolds, an N50 of 37,200,078 bases, and a size estimate considering 28 scaffolds (26 autosomes+ZW) of ~945 Mb. In Apareiodon sp., this second genome draft confirmed that ~36% of the genome is composed of repetitive DNA. Furthermore, the new draft genome has improved genomic quality assessments, allowing the annotation of 36,290 genes and 15,683 proteins, which presented similarities to reference genomes. The second draft genome of Apareiodon sp. will be useful for research on integrative cytogenetic and genomic data. It will open perspectives for analyzing sex-determining genes in Neotropical fish with a ZZ/ZW sex chromosome system.

An Astyanax mexicanus mao knockout line uncovers the developmental roles of monoamine homeostasis in fish brain.

Monoaminergic systems are conserved in vertebrates, yet they present variations in neuroanatomy, genetic components and functions across species. MonoAmine Oxidase, or MAO, is the enzyme responsible for monoamine degradation. While mammals possess two genes, MAO-A and MAO-B, fish possess one single mao gene. To study the function of MAO and monoamine homeostasis on fish brain development and physiology, here we have generated a mao knockout line in Astyanax mexicanus (surface fish), by CRISPR/Cas9 technology. Homozygote mao knockout larvae died at 13 days post-fertilization. Through a time-course analysis, we report that hypothalamic serotonergic neurons undergo fine and dynamic regulation of serotonin level upon loss of mao function, in contrast to those in the raphe, which showed continuously increased serotonin levels - as expected. Dopaminergic neurons were not affected by mao loss-of-function. At behavioral level, knockout fry showed a transient decrease in locomotion that followed the variations in the hypothalamus serotonin neuronal levels. Finally, we discovered a drastic effect of mao knockout on brain progenitors proliferation in the telencephalon and hypothalamus, including a reduction in the number of proliferative cells and an increase of the cell cycle length. Altogether, our results show that MAO has multiple and varied effects on Astyanax mexicanus brain development. Mostly, they bring novel support to the idea that serotonergic neurons in the hypothalamus and raphe of the fish brain are different in nature and identity, and they unravel a link between monoaminergic homeostasis and brain growth.

The biomimetic potential of novel adaptations in subterranean animals

Subterranean habitats can be considered harsh conditions with lack of light, low nutrients levels and constant high humidity. To thrive under such conditions, cave-adapted species have evolved a range of novel morphological, physiological and behavioural adaptations. These adaptations might have significant biomimetic potential. Biomimetics or biologically inspired design is a relatively new interdisciplinary field that aims to harness the processes and mechanisms in nature that have been optimised over millions of years’ evolution to improve our own technology. There are two main approaches to biologically inspired design—the problem-driven approach starting with an engineering problem and searching through biological equivalents and the solution-driven approach, which starts with a biological example or solution followed by the identification of a suitable engineering application. While the former approach is the most popular and is favoured by engineers, the latter remains the most successful and is typically driven by fundamental biological research. However, few biomimetic solutions or concepts have so far been described from subterranean habitats despite the rich potential. In this review paper, I first outline the theory behind biologically inspired design before I review the few biomimetic related studies of cave adapted organisms mainly based on the exceptional lateral line systems in blind cave fish. However, the main body of the review focuses on identifying and discussing subterranean adaptations with a particular strong potential including biomimetics sensors, adhesion in high humidity and wet conditions and biomaterials

Biogeographical affinities of the aquatic community of Refugio Cave, a newly discovered Astyanax cave

Pachón cave in the Sierra de El Abra, in Northeast Mexico, stands out as hosting the world’s most widely studied cavefish population – with over 500 scholarly articles published about the population. Refugio Cave was recently discovered in the El Abra region. This cave hosts the mysid cave shrimp Spelaeomysis quinterensis and the blind cave tetra fish, Astyanax mexicanus. This study aims to understand how the aquatic community of Refugio Cave es related to other cave populations in the area. For this purpose, the Histone H3 gene of mysid shrimps and the OCA2 gene that confers albinism in Astyanax fish was sequenced. Results support that the Refugio and Pachón aquatic communities, which are only 4.5 km away apart, are closely related. Thus, the Refugio Cave population may contribute to better understand the evolutionary history of such an important population and, perhaps, help with Pachon’s cavefish conservation.

Phylogeographic relationships and morphological evolution between cave and surface Astyanax mexicanus populations (De Filippi 1853) (Actinopterygii, Characidae).

The Astyanax mexicanus complex includes two different morphs, a surface- and a cave-adapted ecotype, found at three mountain ranges in Northeastern Mexico: Sierra de El Abra, Sierra de Guatemala and Sierra de la Colmena (Micos). Since their discovery, multiple studies have attempted to characterize the timing and the number of events that gave rise to the evolution of these cave-adapted ecotypes. Here, using RADseq and genome-wide sequencing, we assessed the phylogenetic relationships, genetic structure and gene flow events between the cave and surface Astyanax mexicanus populations, to estimate the tempo and mode of evolution of the cave-adapted ecotypes. We also evaluated the body shape evolution across different cave lineages using geometric morphometrics to examine the role of phylogenetic signal versus environmental pressures. We found strong evidence of parallel evolution of cave-adapted ecotypes derived from two separate lineages of surface fish and hypothesize that there may be up to four independent invasions of caves from surface fish. Moreover, a strong congruence between the genetic structure and geographic distribution was observed across the cave populations, with the Sierra de Guatemala the region exhibiting most genetic drift among the cave populations analysed. Interestingly, we found no evidence of phylogenetic signal in body shape evolution, but we found support for parallel evolution in body shape across independent cave lineages, with cavefish from the Sierra de El Abra reflecting the most divergent morphology relative to surface and other cavefish populations.

Eye convergence is evoked during larval prey capture (LPC) without visual stimulus and in blind cavefish

In zebrafish larvae, the first response when detecting prey is an oculomotor behavior; eye convergence. Eye convergence increases the overlap between the visual fields of the left and right eyes to prepare for tracking prey. A high vergence angle is maintained throughout the prey-tracking and capture swim phases, enhancing binocular depth. Since the discovery of eye convergence, hundreds of articles reporting on this behavior in zebrafish have been published. In this study, we found that the larvae of blind tetra cavefish, Astyanax mexicanus, despite being adapted to the absence of visual stimuli due to the lack of light in the cave, have retained the oculomotor behavior of eye convergence in their vestigial eyes. In Astyanax, eye convergence responses can be triggered singlehandedly by vibrations elicited with a glass rod at frequencies similar to those generated by its prey (10–35 Hz). The blind cave tetra offers an intriguing combination of regression of the eye structure, while retaining several of the physiological functions and actions performed in the eye, including light-entrained retinomotor rhythms and eye convergence.

Genetic mapping of craniofacial traits in the Mexican tetra reveals loci associated with bite differences between cave and surface fish

BackgroundThe Mexican tetra, Astyanax mexicanus, includes interfertile surface-dwelling and cave-dwelling morphs, enabling powerful studies aimed at uncovering genes involved in the evolution of cave-associated traits. Compared to surface fish, cavefish harbor several extreme traits within their skull, such as a protruding lower jaw, a wider gape, and an increase in tooth number. These features are highly variable between individual cavefish and even across different cavefish populations.ResultsTo investigate these traits, we created a novel feeding behavior assay wherein bite impressions could be obtained. We determined that fish with an underbite leave larger bite impressions with an increase in the number of tooth marks. Capitalizing on the ability to produce hybrids from surface and cavefish crosses, we investigated genes underlying these segregating orofacial traits by performing Quantitative Trait Loci (QTL) analysis with F2 hybrids. We discovered significant QTL for bite (underbite vs. overbite) that mapped to a single region of the Astyanax genome. Within this genomic region, multiple genes exhibit coding region mutations, some with known roles in bone development. Further, we determined that there is evidence that this genomic region is under natural selection.ConclusionsThis work highlights cavefish as a valuable genetic model for orofacial patterning and will provide insight into the genetic regulators of jaw and tooth development.

Chronic Thermal Acclimation Effects on Critical Thermal Maxima (CTmax) and Oxidative Stress Differences in White Epaxial Muscle between Surface and Cave Morphotypes of the Mexican Cavefish (Astyanax mexicanus).

AbstractIn the face of increasing environmental temperatures, operative differences between mitochondrial function and whole-animal phenotypic response to the environment are underrepresented in research, especially in subtemperate ectothermic vertebrates. A novel approach to exploring this connection is to examine model species that are genetically similar but that have different whole-animal phenotypes, each of which inhabits different environments. The blind Mexican cavefish (Astyanax mexicanus) has the following two morphotypes: a surface form found in aboveground rivers and an obligate cave-dwelling form. Each morphotype inhabits vastly different thermal and oxygen environments. Whole-animal and mitochondrial responses to thermal acclimation and oxidative stress, with respect to increasing temperatures, have not been previously determined in either morphotype of this species. Here, we chronically acclimated both morphotypes to three temperatures (14°C, 25°C, and 31°C) to establish potential for acclimation and critical thermal maxima (CTmax) for each morphotype of this species. After measuring CTmax in six cohorts, we additionally measured enzymatic antioxidant capacity (catalase, superoxide dismutase, and glutathione peroxidase activities), peroxyl scavenging capacity, and lipid peroxidation damage in white epaxial muscle for each individual. We found a significant effect of acclimation temperature on CTmax (, ) but no effect of morphotype on CTmax (, ). Additionally, we found that morphotype had a significant effect on glutathione peroxidase activity, with the surface morphotype having increased glutathione peroxidase activity compared with the cave morphotype (, ). No other oxidative stress variable demonstrated significant differences. Increases in CTmax with chronic thermal acclimation to higher temperatures suggests that there is some degree of phenotypic plasticity in this species that nominally occupies thermally stable environments. The decreased glutathione peroxidase activity in the cave morphotype may be related to decreased environmental oxygen concentration and decreased metabolic rate in this environmentally constrained morphotype compared to in its surface-living counterparts.

Acoustic signatures in Mexican cavefish populations inhabiting different caves.

Complex patterns of acoustic communication exist throughout the animal kingdom, including underwater. The river-dwelling and the Pachón cave-adapted morphotypes of the fish Astyanax mexicanus are soniferous and share a repertoire of sounds. Their function and significance is mostly unknown. Here, we explored whether and how sounds produced by blind cavefishes inhabiting different Mexican caves may vary. We compared "Clicks" and "Serial Clicks" produced by cavefish in six different caves distributed in three mountain ranges in Mexico. We also sampled laboratory-bred cavefish lines originating from four of these caves. Sounds were extracted and analyzed using both a manual method and a machine learning-based automation tool developed in-house. Multi-parametric analyses suggest wild cave-specific acoustic signatures, or "accents". An acoustic code also existed in laboratory cavefish lines, suggesting a genetic basis for the evolution of this trait. The variations in acoustic parameters between caves of origin did not seem related to fish phenotypes, phylogeography or ecological conditions. We propose that the evolution of such acoustic signatures would progressively lead to the differentiation of local accents that may prevent interbreeding and thus contribute to speciation.

A brain-wide analysis maps structural evolution to distinct anatomical module.

The vertebrate brain is highly conserved topologically, but less is known about neuroanatomical variation between individual brain regions. Neuroanatomical variation at the regional level is hypothesized to provide functional expansion, building upon ancestral anatomy needed for basic functions. Classically, animal models used to study evolution have lacked tools for detailed anatomical analysis that are widely used in zebrafish and mice, presenting a barrier to studying brain evolution at fine scales. In this study, we sought to investigate the evolution of brain anatomy using a single species of fish consisting of divergent surface and cave morphs, that permits functional genetic testing of regional volume and shape across the entire brain. We generated a high-resolution brain atlas for the blind Mexican cavefish Astyanax mexicanus and coupled the atlas with automated computational tools to directly assess variability in brain region shape and volume across all populations. We measured the volume and shape of every grossly defined neuroanatomical region of the brain and assessed correlations between anatomical regions in surface fish, cavefish, and surface × cave F2 hybrids, whose phenotypes span the range of surface to cave. We find that dorsal regions of the brain are contracted, while ventral regions have expanded, with F2 hybrid data providing support for developmental constraint along the dorsal-ventral axis. Furthermore, these dorsal-ventral relationships in anatomical variation show similar patterns for both volume and shape, suggesting that the anatomical evolution captured by these two parameters could be driven by similar developmental mechanisms. Together, these data demonstrate that A. mexicanus is a powerful system for functionally determining basic principles of brain evolution and will permit testing how genes influence early patterning events to drive brain-wide anatomical evolution.

White epaxial muscle aerobic and anaerobic potential and muscle fiber structure in surface and cave morphotypes of the Mexican cavefish (Astyanax mexicanus).

Proper muscle function and muscle fiber structures that match the environmental demands of organisms are imperative to their success in any ecosystem. The Mexican cavefish, Astyanax mexicanus, has two morphotypes: an obligate cave-dwelling form that lives in thermally insulated caves and an O2 poor environment, and a surface form that lives in a more thermally variable, but O2 rich river environment. As environment can determine physiological adaptations, it is of interest to compare the aerobic and anaerobic metabolic profiles of white muscle metabolism in both morphotypes of this species, as well as their muscle structures. Here, we used white muscle of both morphotypes of the Mexican cavefish to determine citrate synthase (CS) activity as a measure of aerobic potential, and lactate concentration as a measure of anaerobic potential at three different chronic acclimation temperatures (14°C, 25°C, and 31°C). By examining aerobic and anaerobic potential in both morphs, we sought to link environmental thermal flexibility to muscle metabolism. We found that the surface morphotype had higher CS activity and lower lactate concentration, suggesting an overall more efficient usage of aerobic metabolism; whereas the cave morphotype showed lower CS activity and higher lactate concentration, suggesting a stronger reliance on anaerobic pathways. We also measured white muscle histological variables that have been previously linked to whole-animal metabolism: fiber diameter, number of nuclei per mm of fiber and myonuclear domain (MND) of both morphotypes at 25°C to examine cell-level differences in muscle morphology. However, we found no differences in fiber diameter, number of nuclei per mm of fiber or MND between the two morphotypes. Thus, although the cellular morphology is similar in these species, the environmental differences in the evolution of the two morphs has led to differences in their metabolic profiles.

Discovery of putative long non-coding RNAs expressed in the eyes of Astyanax mexicanus (Actinopterygii: Characidae)

Astyanax mexicanus is a well-known model species, that has two morphotypes, cavefish, from subterranean rivers and surface fish, from surface rivers. They are morphologically distinct due to many troglomorphic traits in the cavefish, such as the absence of eyes. Most studies on A. mexicanus are focused on eye development and protein-coding genes involved in the process. However, lncRNAs did not get the same attention and very little is known about them. This study aimed to fill this knowledge gap, identifying, describing, classifying, and annotating lncRNAs expressed in the embryo’s eye tissue of cavefish and surface fish. To do so, we constructed a concise workflow to assemble and evaluate transcriptomes, annotate protein-coding genes, ncRNAs families, predict the coding potential, identify putative lncRNAs, map them and predict interactions. This approach resulted in the identification of 33,069 and 19,493 putative lncRNAs respectively mapped in cavefish and surface fish. Thousands of these lncRNAs were annotated and identified as conserved in human and several species of fish. Hundreds of them were validated in silico, through ESTs. We identified lncRNAs associated with genes related to eye development. This is the case of a few lncRNAs associated with sox2, which we suggest being isomorphs of the SOX2-OT, a lncRNA that can regulate the expression of sox2. This work is one of the first studies to focus on the description of lncRNAs in A. mexicanus, highlighting several lncRNA targets and opening an important precedent for future studies focusing on lncRNAs expressed in A. mexicanus.

Changes in local interaction rules during ontogeny underlie the evolution of collective behavior

Collective motion emerges from individual interactions which produce group-wide patterns in behavior. While adaptive changes to collective motion are observed across animal species, how local interactions change when these collective behaviors evolve is poorly understood. Here, we use the Mexican tetra, Astyanax mexicanus, which exists as a schooling surface form and a non-schooling cave form, to study differences in how fish alter their swimming in response to neighbors across ontogeny and between evolutionarily diverged populations. We find that surface fish undergo a transition to schooling mediated by changes in the way fish modulate speed and turning relative to neighbors. This transition begins with the tendency to align to neighbors emerging by 28days post-fertilization and ends with the emergence of robust attraction by 70days post-fertilization. Cavefish exhibit neither alignment nor attraction at any stage of development. These results reveal how evolution alters local interactions to produce striking differences in collective behavior.