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The teleost Astyanax mexicanus consists of surface dwelling (surface fish) and cave dwelling (cavefish) forms. Cavefish have evolved in subterranean habitats characterized by reduced oxygen levels (hypoxia) and exhibit a subset of phenotypic traits controlled by increased Sonic hedgehog (Shh) signaling along the embryonic midline. The enhancement of primitive hematopoietic domains, which are formed bilaterally in the anterior and posterior lateral plate mesoderm, are responsible for the development of more larval erythrocytes in cavefish relative to surface fish. In this study, we determine the role of hypoxia and Shh signaling in the development and evolution of primitive hematopoiesis in cavefish. We show that hypoxia treatment during embryogenesis increases primitive hematopoiesis and erythrocyte development in surface fish. We also demonstrate that upregulation of Shh midline signaling by the Smoothened agonist SAG increases primitive hematopoiesis and erythrocyte development in surface fish, whereas Shh downregulation via treatment with the Smoothened inhibitor cyclopamine decreases these traits in cavefish. Together these results suggest that hematopoietic enhancement is regulated by hypoxia and Shh signaling. Lastly, we demonstrate that hypoxia enhances expression of Shh signaling along the midline of surface fish embryos. We conclude that hypoxia-mediated Shh plasticity may be a driving force for the adaptive evolution of primitive hematopoiesis and erythrocyte development in cavefish., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The ability of organisms to adapt to sudden extreme environmental changes produces some of the most drastic examples of rapid phenotypic evolution. The Mexican Tetra, Astyanax mexicanus, is abundant in the surface waters of northeastern Mexico, but repeated colonizations of cave environments have resulted in the independent evolution of troglomorphic phenotypes in several populations. Here, we present three chromosome-scale assemblies of this species, for one surface and two cave populations, enabling the first whole-genome comparisons between independently evolved cave populations to evaluate the genetic basis for the evolution of adaptation to the cave environment. Our assemblies represent the highest quality of sequence completeness with predicted protein-coding and noncoding gene metrics far surpassing prior resources and, to our knowledge, all long-read assembled teleost genomes, including zebrafish. Whole-genome synteny alignments show highly conserved gene order among cave forms in contrast to a higher number of chromosomal rearrangements when compared with other phylogenetically close or distant teleost species. By phylogenetically assessing gene orthology across distant branches of amniotes, we discover gene orthogroups unique to A. mexicanus. When compared with a representative surface fish genome, we find a rich amount of structural sequence diversity, defined here as the number and size of insertions and deletions as well as expanding and contracting repeats across cave forms. These new more complete genomic resources ensure higher trait resolution for comparative, functional, developmental, and genetic studies of drastic trait differences within a species., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Animals are adapted to their natural habitats and lifestyles. Their brains perceive the external world via their sensory systems, compute information together with that of internal states and autonomous activity, and generate appropriate behavioral outputs. However, how do these processes evolve across evolution? Here, focusing on the sense of olfaction, we have studied the evolution in olfactory sensitivity, preferences, and behavioral responses to six different food-related amino acid odors in the two eco-morphs of the fish Astyanax mexicanus . To this end, we have developed a high-throughput behavioral setup and pipeline of quantitative and qualitative behavior analysis, and we have tested 489 six-week-old Astyanax larvae. The blind, dark-adapted morphs of the species showed markedly distinct basal swimming patterns and behavioral responses to odors, higher olfactory sensitivity, and a strong preference for alanine, as compared to their river-dwelling eyed conspecifics. In addition, we discovered that fish have an individual 'swimming personality', and that this personality influences their capability to respond efficiently to odors and find the source. Importantly, the personality traits that favored significant responses to odors were different in surface fish and cavefish. Moreover, the responses displayed by second-generation cave × surface F2 hybrids suggested that olfactory-driven behavior and olfactory sensitivity is a quantitative genetic trait. Our findings show that olfactory processing has rapidly evolved in cavefish at several levels: detection threshold, odor preference, and foraging behavior strategy. Cavefish is therefore an outstanding model to understand the genetic, molecular, and neurophysiological basis of sensory specialization in response to environmental change., Competing Interests: MB, LV, MK, MP, SR No competing interests declared, (© 2023, Blin et al.)

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.

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., (© 2024 The Authors. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution published by Wiley Periodicals LLC.)

In vitro assays are crucial tools for gaining detailed insights into various biological processes, including metabolism. Cave morphs of the river-dwelling fish species, Astyanax mexicanus, have adapted their metabolism allowing them to thrive in the biodiversity-deprived and nutrient-limited environment of caves. Liver-derived cells from the cave and river morphs of A. mexicanus have proven to be excellent in vitro resources to better understand the unique metabolism of these fish. However, the current 2D cultures have not fully captured the complex metabolic profile of the Astyanax liver. It is known that 3D culturing can modulate the transcriptomic state of cells when compared to its 2D monolayer culture. Therefore, to broaden the possibilities of the in vitro system by modeling a wider gamut of metabolic pathways, we cultured the liver-derived Astyanax cells of both surface and cavefish into 3D spheroids. We successfully established 3D cultures at various cell seeding densities for several weeks and characterized the resultant transcriptomic and metabolic variations. We found that the 3D cultured Astyanax cells exhibit an altered transcriptomic profile and consequently represent a wider range of metabolic pathways, including cell cycle changes and antioxidant activities, associated with liver functioning as compared to its monolayer culture. Enzymatic assay measuring antioxidants in 2D culture and 3D spheroids also revealed enhanced antioxidative capacity of 3D cultured spheroids, in line with the differential gene expression data. Additionally, the spheroids also exhibited surface and cave-specific metabolic signatures, making it a suitable system for evolutionary studies associated with cave adaptation. Notably, cavefish derived spheroids enriched for genes responding to xenobiotic stimulus, while the ones from surface enriched for immune response, both of which resonated with known physiologically adaptations associated with each morph. Taken together, the liver-derived spheroids prove to be a promising in vitro model for widening our understanding of metabolism in A. mexicanus and of vertebrates in general., (© 2024 The Authors. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution published by Wiley Periodicals LLC.)

The ecological and genetic changes that underlie the evolution of host-microbe interactions remain elusive, primarily due to challenges in disentangling the variables that alter microbiome composition. To understand the impact of host habitat, host genetics, and evolutionary history on microbial community structure, we examined gut microbiomes of river- and three cave-adapted morphotypes of the Mexican tetra, Astyanax mexicanus , in their natural environments and under controlled laboratory conditions. Field-collected samples were dominated by very few taxa and showed considerable interindividual variation. We found that lab-reared fish exhibited increased microbiome richness and distinct composition compared to their wild counterparts, underscoring the significant influence of habitat. Most notably, however, we found that morphotypes reared on the same diet throughout life developed distinct microbiomes suggesting that genetic loci resulting from cavefish evolution shape microbiome composition. We observed stable differences in Fusobacteriota abundance between morphotypes and demonstrated that this could be used as a trait for quantitative trait loci mapping to uncover the genetic basis of microbial community structure., Competing Interests: The authors declare no competing interests., (© 2024 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

How genotype determines phenotype is a well-explored question, but genotype-environment interactions and their heritable impact on phenotype over the course of evolution are not as thoroughly investigated. The fish Astyanax mexicanus , consisting of surface and cave ecotypes, is an ideal emerging model to study the genetic basis of adaptation to new environments. This model has permitted quantitative trait locus mapping and whole-genome comparisons to identify the genetic bases of traits such as albinism and insulin resistance and has helped to better understand fundamental evolutionary mechanisms. In this review, we summarize recent advances in A. mexicanus genetics and discuss their broader impact on the fields of adaptation and evolutionary genetics., Competing Interests: Declaration of Interests The authors declare no competing interests.

How genotype determines phenotype is a well-explored question, but genotype-environment interactions and their heritable impact on phenotype over the course of evolution are not as thoroughly investigated. The fish Astyanax mexicanus, comprising surface and cave ecotypes, is an ideal emerging model to study the genetic basis of adaptation to new environments. This model has permitted quantitative trait locus (QTL) mapping and whole-genome comparisons to identify the genetic bases of traits such as albinism and insulin resistance and has helped in the better understanding of fundamental evolutionary mechanisms. In this review, we summarize recent advances in A. mexicanus genetics and discuss its broader impact on the fields of adaptation and evolutionary genetics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Most animals on earth have evolved under daily light-dark cycles and consequently possess a circadian clock which regulates much of their biology, from cellular processes to behaviour. There are however some animals that have invaded dark ecosystems and have adapted to an apparently arrhythmic environment. One such example is the Mexican blind cavefish Astyanax mexicanus , a species complex with over 30 different isolated cave types, including the founding surface river fish. These cavefish have evolved numerous fascinating adaptations to the dark, such as loss of eyes, reduced sleep phenotype and alterations in their clock and light biology. While cavefish are an excellent model for studying circadian adaptations to the dark, their rarity and long generational time makes many studies challenging. To overcome these limitations, we established embryonic cell cultures from cavefish strains and assessed their potential as tools for circadian and light experiments. Here, we show that despite originating from animals with no eyes, cavefish cells in culture are directly light responsive and show an endogenous circadian rhythm, albeit that light sensitivity is relatively reduced in cave strain cells. Expression patterns are similar to adult fish, making these cavefish cell lines a useful tool for further circadian and molecular studies.

Monoamine oxidases (MAO; MAO-A and MAO-B in mammals) are enzymes catalyzing the degradation of biogenic amines, including monoamine neurotransmitters. In humans, coding mutations in MAOs are extremely rare and deleterious. Here, we assessed the structural and biochemical consequences of a point mutation (P106L) in the single mao gene of the blind cavefish, Astyanax mexicanus. This mutation decreased mao enzymatic activity by ∼3-fold and affected the enzyme kinetics parameters, in line with potential structure-function alterations. HPLC measurements in brains of four A. mexicanus genetic lines (mutant and non-mutant cavefish, and mutant and non-mutant surface fish) showed major disturbances in serotonin, dopamine, noradrenaline and metabolite levels in mutants and demonstrated that the P106L mao mutation is responsible for monoaminergic disequilibrium in the P106L mao mutant cavefish brain. The outcomes of the mutation were different in the posterior brain (containing the raphe nucleus) and the anterior brain (containing fish-specific hypothalamic serotonergic clusters), revealing contrasting properties in neurotransmitter homeostasis in these different neuronal groups. We also discovered that the effects of the mutation were partially compensated by a decrease in activity of TPH, the serotonin biosynthesis rate-limiting enzyme. Finally, the neurochemical outcomes of the mao P106L mutation differed in many respects from a treatment with deprenyl, an irreversible MAO inhibitor, showing that genetic and pharmacological interference with MAO function are not the same. Our results shed light on our understanding of cavefish evolution, on the specificities of fish monoaminergic systems, and on MAO-dependent homeostasis of brain neurochemistry in general., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

AbstractGenetic divergence in the presence of gene flow has been well documented, but there is little information on the specific factors maintaining divergence. The present study investigates this in the Mexican tetra ( Astyanax mexicanus ), an excellent model for studying this question because surface and cave populations differ markedly in phenotype and genotype but are interfertile. Previous population studies documented significant gene flow among cave and surface populations, but they focused on analyses of neutral markers whose evolutionary dynamics likely differ from those of genes involved in cave adaptation. The present study advances our understanding of this question by focusing specifically on the genetics responsible for eye and pigmentation reduction, signature traits of cave populations. Direct observations of two cave populations over the course of 63 years verify that surface fish frequently move into the caves and even hybridize with the cave fish. Importantly, however, historical records show that surface alleles for pigmentation and eye size do not persist but are rapidly eliminated from the cave gene pool. It has been argued that regression of eyes and pigmentation was driven by drift, but the results of this study suggest that strong selection actively eliminates surface alleles from the cave populations.

Animal model systems are dependent on the standardization of husbandry protocols that maximize growth and reduce generation time. The Mexican tetra, Astyanax mexicanus , exists as eyed surface and blind cave dwelling populations. The opportunity for comparative approaches between independently evolved populations has led to the rapid growth of A. mexicanus as a model for evolution and biomedical research. However, a slow and inconsistent growth rate remains a major limitation to the expanded application of A. mexicanus . Fortunately, this temporal limitation can be addressed through husbandry changes that accelerate growth rates while maintaining optimal health outcomes. Here, we describe a husbandry protocol that produces rapid growth rates through changes in diet, feeding frequency, growth sorting and progressive changes in tank size. This protocol produced robust growth rates and decreased the age of sexual maturity in comparison to our previous protocol. To determine whether changes in feeding impacted behavior, we tested fish in exploration and schooling assays. We found no difference in behavior between the two groups, suggesting that increased feeding and rapid growth will not impact the natural variation in behavioral traits. Taken together, this standardized husbandry protocol will accelerate the development of A. mexicanus as a genetic model.

Chronically high blood glucose (hyperglycemia) leads to diabetes and fatty liver disease. Obesity is a major risk factor for hyperglycemia, but the underlying mechanism is unknown. Here, we show that a high-fat diet (HFD) in mice causes early loss of expression of the glycolytic enzyme Hexokinase 2 (HK2) specifically in adipose tissue. Adipose-specific knockout of Hk2 reduced glucose disposal and lipogenesis and enhanced fatty acid release in adipose tissue. In a non-cell-autonomous manner, Hk2 knockout also promoted glucose production in liver. Furthermore, we observed reduced hexokinase activity in adipose tissue of obese and diabetic patients, and identified a loss-of-function mutation in the hk2 gene of naturally hyperglycemic Mexican cavefish. Mechanistically, HFD in mice led to loss of HK2 by inhibiting translation of Hk2 mRNA. Our findings identify adipose HK2 as a critical mediator of local and systemic glucose homeostasis, and suggest that obesity-induced loss of adipose HK2 is an evolutionarily conserved mechanism for the development of selective insulin resistance and thereby hyperglycemia., Competing Interests: MS, AT, SS, IF, DW, AV, MP, ND, DR, TM, NH, RP, SF, NR, MH No competing interests declared, BW, AM is affiliated with St. Clara Research Ltd. The author has no other competing interests to declare, (© 2023, Shimobayashi et al.)

Cavefish are vertebrates living in extreme subterranean environments with no light, temperature changes, and limited food. Circadian rhythms in these fish are suppressed in natural habitats. However, they can be found in artificial light-dark cycles and other zeitgebers. The molecular circadian clock has its peculiarities in cavefish. In Astyanax mexicanus , the core clock mechanism is tonically repressed in the caves due to the overactivation of the light input pathway. A lack of functional light input pathway but rather the entrainment of circadian genes' expression by scheduled feeding were revealed in more ancient Phreatichthys andruzzii . Different evolutionarily determined irregularities in the functioning of molecular circadian oscillators can be expected in other cavefish. The unique property of some species is the existence of surface and cave forms. Along with the ease of maintenance and breeding, it made cavefish a promising model for chronobiological studies. At the same time, a divergence of the circadian system between cavefish populations requires the strain of origin to be indicated in further research.

Background: Aggression is observed across the animal kingdom, and benefits animals in a number of ways to increase fitness and promote survival. While aggressive behaviors vary widely across populations and can evolve as an adaptation to a particular environment, the complexity of aggressive behaviors presents a challenge to studying the evolution of aggression. The Mexican tetra, Astyanax mexicanus exists as an aggressive river-dwelling surface form and multiple populations of a blind cave form, some of which exhibit reduced aggression, providing the opportunity to investigate how evolution shapes aggressive behaviors., Results: To define how aggressive behaviors evolve, we performed a high-resolution analysis of multiple social behaviors that occur during aggressive interactions in A. mexicanus. We found that many of the aggression-associated behaviors observed in surface-surface aggressive encounters were reduced or lost in Pachón cavefish. Interestingly, one behavior, circling, was observed more often in cavefish, suggesting evolution of a shift in the types of social behaviors exhibited by cavefish. Further, detailed analysis revealed substantive differences in aggression-related sub-behaviors in independently evolved cavefish populations, suggesting independent evolution of reduced aggression between cave populations. We found that many aggressive behaviors are still present when surface fish fight in the dark, suggesting that these reductions in aggression-associated and escape-associated behaviors in cavefish are likely independent of loss of vision in this species. Further, levels of aggression within populations were largely independent of type of opponent (cave vs. surface) or individual stress levels, measured through quantifying stress-like behaviors, suggesting these behaviors are hardwired and not reflective of population-specific changes in other cave-evolved traits., Conclusion: These results reveal that loss of aggression in cavefish evolved through the loss of multiple aggression-associated behaviors and raise the possibility that independent genetic mechanisms underlie changes in each behavior within populations and across populations. Taken together, these findings reveal the complexity of evolution of social behaviors and establish A. mexicanus as a model for investigating the evolutionary and genetic basis of aggressive behavior., (© 2022. The Author(s).)

The growing use of genomics in diverse organisms provides the basis for identifying genomic and transcriptional differences across species and experimental conditions. Databases containing genomic and functional data have played critical roles in the development of numerous genetic models but most emerging models lack such databases. The Mexican tetra, Astyanax mexicanus exists as 2 morphs: surface-dwelling and cave-dwelling. There exist at least 30 cave populations, providing a system to study convergent evolution. We have generated a web-based analysis suite that integrates datasets from different studies to identify how gene transcription and genetic markers of selection differ between populations and across experimental contexts. Results of diverse studies can be analyzed in conjunction with other genetic data (e.g. Gene Ontology information), to enable biological inference from cross-study patterns and identify future avenues of research. Furthermore, the framework that we have built for A. mexicanus can be adapted for other emerging model systems., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

The chemical senses of olfaction and taste are well developed in fish and play a vital role in its various activities such as navigation, mate recognition, and food detection. The small teleost fish Astyanax mexicanus consists of interfertile river-dwelling and cave-dwelling populations, referred to as "surface fish" and "cavefish" respectively. An important anatomical feature of cavefish is the lack of eyes leading them to be referred to as blind fish and suggesting an enhanced functional role for other senses such as taste. In this study, we characterize the expression of bitter taste receptors (T2Rs or Tas2Rs ) in A. mexicanus and investigate their functionality in a heterologous expression system. The genome database of A. mexicanus (ensemble and NCBI) showed 7 Tas2Rs , among these Tas2R1, Tas2R3, Tas2R4 , and Tas2R114 are well characterized in humans and mice but not in A. mexicanus . Therefore, the 4 Tas2Rs were selected for further analysis and their expression in A. mexicanus was confirmed by in situ hybridization and RT-PCR in early developmental stages. These Tas2Rs are expressed in various oral and extraoral organs (liver, fins, jaws, and gills) in A. mexicanus , and Tas2R1 has maximum expression and is localized throughout the fish body. Using the heterologous expression of A. mexicanus T2Rs in HEK293T cells coupled with cell-based calcium mobilization assays, we show that A. mexicanus T2Rs are activated by commonly used fish food and known bitter agonists, including quinine. This study provides novel insights into the extraoral expression of T2Rs in A. mexicanus and suggests their importance in extraoral food detection., Competing Interests: The authors declare no competing financial interests., (© 2022 The Authors. FASEB BioAdvances published by Wiley Periodicals LLC on behalf of The Federation of American Societies for Experimental Biology.)

Animals can evolve dramatic sensory functions in response to environmental constraints, but little is known about the neural mechanisms underlying these changes. The Mexican tetra, Astyanax mexicanus , is a leading model to study genetic, behavioral, and physiological evolution by comparing eyed surface populations and blind cave populations. We compared neurophysiological responses of posterior lateral line afferent neurons and motor neurons across A. mexicanus populations to reveal how shifts in sensory function may shape behavioral diversity. These studies indicate differences in intrinsic afferent signaling and gain control across populations. Elevated endogenous afferent activity identified a lower response threshold in the lateral line of blind cavefish relative to surface fish leading to increased evoked potentials during hair cell deflection in cavefish. We next measured the effect of inhibitory corollary discharges from hindbrain efferent neurons onto afferents during locomotion. We discovered that three independently derived cavefish populations have evolved persistent afferent activity during locomotion, suggesting for the first time that partial loss of function in the efferent system can be an evolutionary mechanism for neural adaptation of a vertebrate sensory system., Competing Interests: EL, AP, AK, JL No competing interests declared, (© 2022, Lunsford et al.)

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., Competing Interests: JM, JP, TB, LO, RP, JK, SX, NR No competing interests declared, (© 2022, Medley, Persons et al.)

Nutrient availability varies seasonally and spatially in the wild. While many animals, such as hibernating animals or migrating birds, evolved strategies to overcome periods of nutrient scarcity, 1 , Here, we used Astyanax mexicanus, which includes river-dwelling surface fish and cave-adapted cavefish populations, to study the genetic adaptation to nutrient limitations. 2 the cellular mechanisms of these strategies are poorly understood. Cave environments represent an example of nutrient-deprived environments, since the lack of sunlight and therefore primary energy production drastically diminishes the nutrient availability. 3 Here, we used Astyanax mexicanus, which includes river-dwelling surface fish and cave-adapted cavefish populations, to study the genetic adaptation to nutrient limitations. 4-9 We show that cavefish populations store large amounts of fat in different body regions when fed ad libitum in the lab. We found higher expression of lipogenesis genes in cavefish livers when fed the same amount of food as surface fish, suggesting an improved ability of cavefish to use lipogenesis to convert available energy into triglycerides for storage into adipose tissue. 10-12 Moreover, the lipid metabolism regulator, peroxisome proliferator-activated receptor γ (Pparγ), is upregulated at both transcript and protein levels in cavefish livers. Chromatin immunoprecipitation sequencing (ChIP-seq) showed that Pparγ binds cavefish promoter regions of genes to a higher extent than surface fish and inhibiting Pparγ in vivo decreases fat accumulation in A. mexicanus. Finally, we identified nonsense mutations in per2, a known repressor of Pparγ, providing a possible regulatory mechanism of Pparγ in cavefish. Taken together, our study reveals that upregulated Pparγ promotes higher levels of lipogenesis in the liver and contributes to higher body fat accumulation in cavefish populations, an important adaptation to nutrient-limited environments., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The vertebrate retinas originate from a specific anlage in the anterior neural plate called the eye field. Its identity is conferred by a set of 'eye transcription factors', whose combinatorial expression has been overlooked. Here, we use the dimorphic teleost Astyanax mexicanus, which develops proper eyes in the wild type and smaller colobomatous eyes in the blind cavefish embryos, to unravel the molecular anatomy of the eye field and its variations within a species. Using a series of markers (rx3, pax6a, cxcr4b, zic1, lhx2, emx3 and nkx2.1a), we draw a comparative 3D expression map at the end of gastrulation/onset of neurulation, which highlights hyper-regionalization of the eye field into sub-territories of distinct sizes, shapes, cell identities and combinatorial gene expression levels along the three body axes. All these features show significant variations in the cavefish natural mutant. We also discover sub-domains within the prospective telencephalon and characterize cell identities at the frontiers of the eye field. We propose putative fates for some of the characterized eye-field subdivisions, and suggest the existence of a trade-off between some subdivisions in the two Astyanax morphs on a micro-evolutionary scale., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Advances in sequencing and assembly technology have led to the creation of genome assemblies for a wide variety of non-model organisms. The rapid production and proliferation of updated, novel assembly versions can create vexing problems for researchers when multiple-genome assembly versions are available at once, requiring researchers to work with more than one reference genome. Multiple-genome assemblies are especially problematic for researchers studying the genetic makeup of individual cells, as single-cell RNA sequencing (scRNAseq) requires sequenced reads to be mapped and aligned to a single reference genome. Using the Astyanax mexicanus , this study highlights how the interpretation of a single-cell dataset from the same sample changes when aligned to its two different available genome assemblies. We found that the number of cells and expressed genes detected were drastically different when aligning to the different assemblies. When the genome assemblies were used in isolation with their respective annotations, cell-type identification was confounded, as some classic cell-type markers were assembly-specific, whilst other genes showed differential patterns of expression between the two assemblies. To overcome the problems posed by multiple-genome assemblies, we propose that researchers align to each available assembly and then integrate the resultant datasets to produce a final dataset in which all genome alignments can be used simultaneously. We found that this approach increased the accuracy of cell-type identification and maximised the amount of data that could be extracted from our single-cell sample by capturing all possible cells and transcripts. As scRNAseq becomes more widely available, it is imperative that the single-cell community is aware of how genome assembly alignment can alter single-cell data and their interpretation, especially when reviewing studies on non-model organisms.

Adaptation to novel environments often involves the evolution of multiple morphological, physiological, and behavioral traits. One striking example of multi-trait evolution is the suite of traits that has evolved repeatedly in cave animals, including regression of eyes, loss of pigmentation, and enhancement of non-visual sensory systems. 1 The Mexican tetra, Astyanax mexicanus, consists of fish that inhabit at least 30 caves in Mexico and ancestral-like surface fish that inhabit the rivers of Mexico and southern Texas. , 2 The Mexican tetra, Astyanax mexicanus, consists of fish that inhabit at least 30 caves in Mexico and ancestral-like surface fish that inhabit the rivers of Mexico and southern Texas. 3 Cave A. mexicanus are interfertile with surface fish and have evolved a number of traits, including reduced pigmentation, eye loss, and alterations to behavior. 4-6 To define relationships between different cave-evolved traits, we phenotyped 208 surface-cave F2 hybrid fish for numerous morphological and behavioral traits. We found differences in sleep between pigmented and albino hybrid fish, raising the possibility that these traits share a genetic basis. In cavefish and other species, mutations in oculocutaneous albinism 2 (oca2) cause albinism. 7-12 Surface fish with mutations in oca2 displayed both albinism and reduced sleep. Further, this mutation in oca2 fails to complement sleep loss when surface fish harboring this engineered mutation are crossed to independently evolved populations of albino cavefish with naturally occurring mutations in oca2. Analysis of the oca2 locus in wild-caught cave and surface fish suggests that oca2 is under positive selection in 3 cave populations. Taken together, these findings identify oca2 as a novel regulator of sleep and suggest that a pleiotropic function of oca2 underlies the adaptive evolution of albinism and sleep loss., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Despite a longstanding interest in understanding how animals adapt to environments with limited nutrients, we have incomplete knowledge of the genetic basis of metabolic evolution. The Mexican tetra, Astyanax mexicanus, is a species of fish that consists of two morphotypes; eyeless cavefish that have adapted to a low-nutrient cave environment, and ancestral river-dwelling surface fish with abundant access to nutrients. Cavefish have evolved altered blood sugar regulation, starvation tolerance, increased fat accumulation, and superior body condition. To investigate the genetic basis of cavefish metabolic evolution we carried out a quantitative trait loci (QTL) analysis in surface/cave F2 hybrids. We genetically mapped seven metabolism-associated traits in hybrids that were challenged with a nutrient restricted diet., Results: We found that female F2 hybrids are bigger than males and have a longer hindgut, bigger liver, and heavier gonad, even after correcting for fish size. Although there is no difference between male and female blood sugar level, we found that high blood sugar is associated with weight gain in females and lower body weight and fat level in males. We identified a significant QTL associated with 24-h-fasting blood glucose level with the same effect in males and females. Differently, we identified sex-independent and sex-dependent QTL associated with fish length, body condition, liver size, hindgut length, and gonad weight. We found that some of the genes within the metabolism QTL display evidence of non-neutral evolution and are likely to be under selection. Furthermore, we report predicted nonsynonymous changes to the cavefish coding sequence of these genes., Conclusions: Our study reveals previously unappreciated genomic regions associated with blood glucose regulation, body condition, gonad size, and internal organ morphology. In addition, we find an interaction between sex and metabolism-related traits in A. mexicanus. We reveal coding changes in genes that are likely under selection in the low-nutrient cave environment, leading to a better understanding of the genetic basis of metabolic evolution.

The fish species Astyanax mexicanus with its sighted and blind eco-morphotypes has become an original model to challenge vertebrate developmental evolution. Recently, we demonstrated that phenotypic evolution can be impacted by early developmental events starting from the production of oocytes in the fish ovaries. A. mexicanus offers an amenable model to test the influence of maternal determinants on cell fate decisions during early development, yet the mechanisms by which the information contained in the eggs is translated into specific developmental programs remain obscure due to the lack of specific tools in this emergent model. Here we describe methods for the generation of pescoids from yolkless-blastoderm explants to test the influence of embryonic and extraembryonic tissues on cell fate decisions, as well as the production of chimeric embryos obtained by intermorph cell transplantations to probe cell autonomous or non-autonomous processes. We show that Astyanax pescoids have the potential to recapitulate the main ontogenetic events observed in intact embryos, including the internalization of mesodermal progenitors and eye development, as followed with zic:GFP reporter lines. In addition, intermorph cell grafts resulted in proper integration of exogenous cells into the embryonic tissues, with lineages becoming more restricted from mid-blastula to gastrula. The implementation of these approaches in A. mexicanus will bring new light on the cascades of events, from the maternal pre-patterning of the early embryo to the evolution of brain regionalization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Torres-Paz and Rétaux.)

Background: Astyanax mexicanus from the river basins of the Gulf of Mexico slope are small freshwater fish that usually live in large groups in different freshwater environments. The group is considered successful due to its high capacity for dispersal and adaptation to different habitats, and the species present high morphological variability throughout their distribution in Mexico. This has produced the most extreme morphotype of the group; the hypogeous or troglobite, which has no eyes or coloration, and is probably the cause of taxonomic uncertainty in the recognition of species across the entire range. Most studies of A. mexicanus have mainly focused on cave individuals, as well as their adjacent surface locations, providing an incomplete evolutionary history, particularly in terms of factors related to dispersal and the potential corridors used, barriers to gene flow, and distribution of genetic variability. The aim of the present study is to determine the population structure and the degree and direction of genetic flow in this complex taxonomic group, incorporating geographic locations not previously included in analyses using microsatellite loci. Our aim is to contribute to the knowledge of the intricate evolutionary history of A. mexicanus throughout most of its range., Methods: The present study included a set of several cave and surface locations of A. mexicanus , which have been widely sampled along the Gulf of Mexico slope, in a genetic population analysis using 10 microsatellite loci., Results: Ten genetic populations or lineages were identified. In these populations, gene flow was recorded at two time periods. Historical gene flow, both inter and intra-basin, was observed among surface populations, from surface to cave populations, and among cave populations, whereas recording of contemporary gene flow was limited to intra-basin exchanges and observed among surface populations, surface to cave populations, and cave populations., Competing Interests: The authors declare there are no competing interests., (©2021 Pérez-Rodríguez et al.)

Morphological diversification during adaptive radiation may depend on factors external or internal to the lineage. We provide evidence for the latter in characiform fishes (tetras and piranhas), which exhibit extensive dental diversity. Phylogenetic character mapping supported regain of lost teeth as contributing to this diversity. To test for latent potential for dentition that would facilitate its evolutionary expansion, we overexpressed a tooth initiation signal, the tumour necrosis factor pathway ligand ectodysplasin , in a model characiform, the Mexican tetra ( Astyanax mexicanus ). This manipulation resulted in extensive ectopic dentition, in contrast with its previously reported limited effect in the zebrafish ( Danio rerio ). Tooth location in the order Cypriniformes, to which the zebrafish belongs, is much more restricted than in characiforms, a pattern that may be explained by differences in the retention of ancestral developmental potential. Our results suggest that differences in evolvability between lineages may lead to contrasting patterns of diversification.

The adult human heart cannot repair itself after injury and, instead, forms a permanent fibrotic scar that impairs cardiac function and can lead to incurable heart failure. The zebrafish, amongst other organisms, has been extensively studied for its innate capacity to repair its heart after injury. Understanding the signals that govern successful regeneration in models such as the zebrafish will lead to the development of effective therapies that can stimulate endogenous repair in humans. To date, many studies have investigated cardiac regeneration using a reverse genetics candidate gene approach. However, this approach is limited in its ability to unbiasedly identify novel genes and signalling pathways that are essential to successful regeneration. In contrast, drawing comparisons between different models of regeneration enables unbiased screens to be performed, identifying signals that have not previously been linked to regeneration. Here, we will review in detail what has been learnt from the comparative approach, highlighting the techniques used and how these studies have influenced the field. We will also discuss what further comparisons would enhance our knowledge of successful regeneration and scarring. Finally, we focus on the Astyanax mexicanus , an intraspecies comparative fish model that holds great promise for revealing the secrets of the regenerating heart.

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., (© 2020 Wiley Periodicals LLC.)

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., (© 2020 Wiley Periodicals LLC.)

The ability of fishes to adapt to any aquatic environment seems limitless. It is enthralling how new species keep appearing at the deep sea or in subterranean environments. There are close to 230 known species of cavefishes, still today the best-known cavefish is Astyanax mexicanus, a Characid that has become a model organism, and has been studied and scrutinized since 1936. There are two morphotypes for A. mexicanus, a surface fish and a cavefish. The surface fish lives in central and northeastern Mexico and south of the United States, while the cavefish is endemic to the "Sierra del Abra-Tanchipa region" in northeast Mexico. The extensive genetic and genomic analysis depicts a complex origin for Astyanax cavefish, with multiple cave invasions and persistent gene flow among cave populations. The surface founder population prevails in the same region where the caves are. In this review, we focus on both morphotype's main morphological and physiological differences, but mainly in recent discoveries about behavioral and metabolic adaptations for subterranean life. These traits may not be as obvious as the troglomorphic characteristics, but are key to understand how Astyanax cavefish thrives in this environment of perpetual darkness., (© 2020 Wiley Periodicals, Inc.)

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., (© 2020 Wiley Periodicals, Inc.)

Animals respond to sleep loss with compensatory rebound sleep, and this is thought to be critical for the maintenance of physiological homeostasis. Sleep duration varies dramatically across animal species, but it is not known whether evolutionary differences in sleep duration are associated with differences in sleep homeostasis. The Mexican cavefish, Astyanax mexicanus, has emerged as a powerful model for studying the evolution of sleep. While eyed surface populations of A. mexicanus sleep approximately 8 hr each day, multiple blind cavefish populations have converged on sleep patterns that total as little as 2 hr each day, providing the opportunity to examine whether the evolution of sleep loss is accompanied by changes in sleep homeostasis. Here, we examine the behavioral and molecular response to sleep deprivation across four independent populations of A. mexicanus. Our behavioral analysis indicates that surface fish and all three cavefish populations display robust recovery sleep during the day following nighttime sleep deprivation, suggesting sleep homeostasis remains intact in cavefish. We profiled transcriptome-wide changes associated with sleep deprivation in surface fish and cavefish. While the total number of differentially expressed genes was not greater for the surface population, the surface population exhibited the highest number of uniquely differentially expressed genes than any other population. Strikingly, a majority of the differentially expressed genes are unique to individual cave populations, suggesting unique expression responses are exhibited across independently evolved cavefish populations. Together, these findings suggest sleep homeostasis is intact in cavefish despite a dramatic reduction in overall sleep duration., (© 2020 Wiley Periodicals, Inc.)

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., (© 2020 Wiley Periodicals LLC.)

The small teleost fish Astyanax mexicanus has emerged as an outstanding model for studying many biological topics in the context of evolution. A major attribute is conspecific surface dwelling (surface fish) and blind cave dwelling (cavefish) morphs that can be raised in the laboratory and spawn large numbers of transparent and synchronously developing embryos. More than 30 cavefish populations have been discovered, mostly in northeastern Mexico, and some are thought to have evolved independently from surface fish ancestors, providing excellent models of parallel and convergent evolution. Cavefish have evolved eye and pigmentation regression, as well as modifications in brain morphology, behaviors, heart regenerative capacity, metabolic processes, and craniofacial organization. Thus, the Astyanax model provides researchers with natural "mutants" to study life in the challenging cave environment. The application of powerful genetic approaches based on hybridization between the two morphs and between the different cavefish populations are key advantages for deciphering the developmental and genetic mechanisms regulating trait evolution. QTL analysis has revealed the genetic architectures of gained and lost traits. In addition, some cavefish traits resemble human diseases, offering novel models for biomedical research. Astyanax research is supported by genome assemblies, transcriptomes, tissue and organ transplantation, gene manipulation and editing, and stable transgenesis, and benefits from a welcoming and interactive research community that conducts integrated community projects and sponsors the International Astyanax Meeting (AIM)., Competing Interests: Competing interestsThe author declares no competing interests., (© The Author(s) 2020.)

A widely accepted model for the evolution of cave animals posits colonization by surface ancestors followed by the acquisition of adaptations over many generations. However, the speed of cave adaptation in some species suggests mechanisms operating over shorter timescales. To address these mechanisms, we used Astyanax mexicanus , a teleost with ancestral surface morphs (surface fish, SF) and derived cave morphs (cavefish, CF). We exposed SF to completely dark conditions and identified numerous altered traits at both the gene expression and phenotypic levels. Remarkably, most of these alterations mimicked CF phenotypes. Our results indicate that many cave-related traits can appear within a single generation by phenotypic plasticity. In the next generation, plasticity can be further refined. The initial plastic responses are random in adaptive outcome but may determine the subsequent course of evolution. Our study suggests that phenotypic plasticity contributes to the rapid evolution of cave-related traits in A. mexicanus ., Competing Interests: HB, BH, MS, GM, MN, AK, RG, HĆ, MP, KR, WJ No competing interests declared, (© 2020, Bilandžija et al.)

Natural model systems evolving under extreme environmental pressures provide the opportunity to advance our knowledge of how the craniofacial complex evolves in nature. Unlike traditional models, natural systems are less inbred, and, therefore, better model the complex variation of the human population. Owing to the nature of certain craniofacial aberrations in blind Mexican cavefish, we suggest that this organism can provide new insights to a variety of craniofacial changes. Diverse cranial features have evolved in natural cave-dwelling Astyanax fish, which have thrived in the unforgiving darkness and nutrient-poor environment of the cave for countless generations. While the genetic and environmental underpinnings of various cranial anomalies have been investigated for decades, a comprehensive characterization of their molecular and developmental origins remains incomplete. Cavefish provide numerous advantages given the availability of genomic resources, developmental and molecular tools, and the presence of a normative surface-dwelling "ancestral" surrogate for comparative studies. By leveraging the frequency of abnormal and asymmetric cranial features in cavefish, we anticipate advances in our knowledge of the etiologies of irregular cranial features. Extreme adaptations in cavefish are expected to offer new insights into the complex and multifactorial nature of craniofacial disorders and facial asymmetry. Anat Rec, 2018. © 2018 American Association for Anatomy., (© 2018 American Association for Anatomy.)

Sequential developmental events, starting from the moment of fertilization, are crucial for the acquisition of animal body plan. Subtle modifications in such early events are likely to have major impacts in later morphogenesis, bringing along morphological diversification. Here, comparing the blind cave and the surface morphotypes of Astyanax mexicanus fish, we found heterochronies during gastrulation that produce organizer and axial mesoderm tissues with different properties (including differences in the expression of dkk1b ) that may have contributed to cavefish brain evolution. These variations observed during gastrulation depend fully on maternal factors. The developmental evolution of retinal morphogenesis and hypothalamic patterning are among those traits that retained significant maternal influence at larval stages. Transcriptomic analysis of fertilized eggs from both morphotypes and reciprocal F 1 hybrids showed a strong and specific maternal signature. Our work strongly suggests that maternal effect genes and developmental heterochronies that occur during gastrulation have impacted morphological brain change during cavefish evolution., Competing Interests: JT, JL, SR No competing interests declared, (© 2019, Torres-Paz et al.)

Most animals and plants live on the planet exposed to periods of rhythmic light and dark. As such, they have evolved endogenous circadian clocks to regulate their physiology rhythmically, and non-visual light detection mechanisms to set the clock to the environmental light-dark cycle. In the case of fish, circadian pacemakers are not only present in the majority of tissues and cells, but these tissues are themselves directly light-sensitive, expressing a wide range of opsin photopigments. This broad non-visual light sensitivity exists to set the clock, but also impacts a wide range of fundamental cell biological processes, such as DNA repair regulation. In this context, Astyanax mexicanus is a very intriguing model system with which to explore non-visual light detection and circadian clock function. Previous work has shown that surface fish possess the same directly light entrainable circadian clocks, described above. The same is true for cave strains of Astyanax in the laboratory, though no daily rhythms have been observed under natural dark conditions in Mexico. There are, however, clear alterations in the cave strain light response and changes to the circadian clock, with a difference in phase of peak gene expression and a reduction in amplitude. In this study, we expand these early observations by exploring the development of non-visual light sensitivity and clock function between surface and cave populations. When does the circadian pacemaker begin to oscillate during development, and are there differences between the various strains? Is the difference in acute light sensitivity, seen in adults, apparent from the earliest stages of development? Our results show that both cave and surface populations must experience daily light exposure to establish a larval gene expression rhythm. These oscillations begin early, around the third day of development in all strains, but gene expression rhythms show a significantly higher amplitude in surface fish larvae. In addition, the light induction of clock genes is developmentally delayed in cave populations. Zebrafish embryonic light sensitivity has been shown to be critical not only for clock entrainment, but also for transcriptional activation of DNA repair processes. Similar downstream transcriptional responses to light also occur in Astyanax. Interestingly, the establishment of the adult timing profile of clock gene expression takes several days to become apparent. This fact may provide mechanistic insight into the key differences between the cave and surface fish clock mechanisms., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Astyanax mexicanus consists of two different populations: a sighted surface-dwelling form (surface fish) and a blind cave-dwelling form (cavefish). In the cavefish, embryonic expression of sonic hedgehog a (shha) in the prechordal plate is expanded towards the anterior midline, which has been shown to contribute to cavefish specific traits such as eye degeneration, enhanced feeding apparatus, and specialized brain anatomy. However, it is not clear how this expanded expression is achieved and which signaling pathways are involved. Nodal signaling has a crucial role for expression of shh and formation of the prechordal plate. In this study, we report increased expression of prechordal plate marker genes, nodal-related 2 (ndr2) and goosecoid (gsc) in cavefish embryos at the tailbud stage. To investigate whether Nodal signaling is responsible for the anterior expansion of the prechordal plate, we used an inhibitor of Nodal signaling and showed a decreased anterior expansion of the prechordal plate and increased pax6 expression in the anterior midline in treated cavefish embryos. Later in development, the lens and optic cup of treated embryos were significantly larger than untreated embryos. Conversely, increasing Nodal signaling in the surface fish embryo resulted in the expansion of anterior prechordal plate and reduction of pax6 expression in the anterior neural plate together with the formation of small lenses and optic cups later in development. These results confirmed that Nodal signaling has a crucial role for the anterior expansion of the prechordal plate and plays a significant role in cavefish eye development. We showed that the anterior expansion of the prechordal plate was not due to increased total cell number, suggesting the expansion is achieved by changes in cellular distribution in the prechordal plate. In addition, the distribution of presumptive prechordal plate cells in Spemann's organiser was also altered in the cavefish. These results suggested that changes in the cellular arrangement of Spemann's organiser in early gastrulae could have an essential role in the anterior expansion of the prechordal plate contributing to eye degeneration in the cavefish., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cavefish populations of Astyanax mexicanus have increased body fat compared to surface fish populations of the same species when fed ad libitum in the laboratory. We have previously shown that some cavefish populations display hyperphagia (elevated appetite) to increase food consumption, fat deposition and starvation resistance. However, not all cavefish populations display hyperphagia, yet all previously tested cavefish display elevated body fat levels. Here we have extended this analysis by focusing on visceral fat acquisition in three independently derived cavefish populations. We show that cavefish from two independently derived cavefish populations (Pachón and Tinaja) display increased amounts of visceral adipose tissue (VAT) due to hypertrophy of visceral adipocytes while Molino cavefish display hypertrophy but only slightly elevated VAT levels compared to surface fish. Furthermore, we show that Pachón and Tinaja cavefish develop increased VAT even when food intake is matched to surface fish, suggesting appetite independent mechanisms. We show that in the Pachón population, the differences in the visceral fat in adults correlates with changes in the timing of visceral development, making a developmental contribution likely. Visceral fat development in surface fish starts between 10 and 11 dpf, while in Pachón cavefish, visceral fat cells become visible as early as 8 dpf and develop significantly higher amounts of lipid droplets before surface fish start visceral fat accumulation. We further show that this developmental difference is unique to the Pachón cavefish population, while the Tinaja cavefish population - which displays hyperphagia - starts to develop visceral fat similar to surface fish. We suggest the differences in early adipogenesis in the Pachón population as an additional strategy of increased fat gain in cavefish to adapt to food scarcity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The role of maternal factors in the evolution of development is poorly understood. Here we describe the use of reciprocal hybridization between the surface dwelling (surface fish, SF) and cave dwelling (cavefish, CF) morphs of the teleost Astyanax mexicanus to investigate the roles of maternal genetic effects in cavefish development. Reciprocal hybridization, a procedure in which F1 hybrids are generated by fertilizing SF eggs with CF sperm (SF × CF hybrids) and CF eggs with SF sperm (CF × SF hybrids), revealed that the CF degenerative eye phenotype showed maternal genetic effects. The eyes of CF × SF hybrids resembled the degenerate eyes of CF in showing ventral reduction of the retina and corresponding displacement of the lens within the optic cup, a smaller lens and eyeball, more lens apoptosis, a smaller cartilaginous sclera, and lens-specific gene expression characteristics compared to SF × CF hybrids, which showed eye and lens gene expression phenotypes resembling SF. In contrast, reciprocal hybridization failed to support roles for maternal genetic effects in the CF regressive pigmentation phenotype or in CF constructive changes related to enhanced jaw development. Maternal transcripts encoded by the pou2f1b, runx2b, and axin1 genes, which are involved in determining ventral embryonic fates, were increased in unfertilized CF eggs. In contrast, maternal mRNAs encoded by the ß-catenin and syntabulin genes, which control dorsal embryonic fates, showed similar expression levels in unfertilized SF and CF eggs. Furthermore, maternal transcripts of a sonic hedgehog gene were detected in SF and CF eggs and early cleaving embryos. This study reveals that CF eye degeneration is controlled by changes in maternal factors produced during oogenesis and introduces A. mexicanus as a model system for studying the role of maternal changes in the evolution of development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Understanding the genetic basis of trait evolution is critical to identifying the mechanisms that generated the immense amount of diversity observable in the living world. However, genetically manipulating organisms from natural populations with evolutionary adaptations remains a significant challenge. Astyanax mexicanus exists in two interfertile forms, a surface-dwelling form and multiple independently evolved cave-dwelling forms. Cavefish have evolved a number of morphological and behavioral traits and multiple quantitative trait loci (QTL) analyses have been performed to identify loci underlying these traits. These studies provide a unique opportunity to identify and test candidate genes for these cave-specific traits. We have leveraged the CRISPR/Cas9 genome editing techniques to characterize the effects of mutations in oculocutaneous albinism II (oca2), a candidate gene hypothesized to be responsible for the evolution of albinism in A. mexicanus cave populations. We generated oca2 mutant surface A. mexicanus. Surface fish with oca2 mutations are albino due to a disruption in the first step of the melanin synthesis pathway, the same step that is disrupted in albino cavefish. Hybrid offspring from crosses between oca2 mutant surface and cavefish are albino, definitively demonstrating the role of this gene in the evolution of albinism in this species. This research elucidates the role oca2 plays in pigmentation in fish, and establishes that this gene is solely responsible for the evolution of albinism in multiple cavefish populations. Finally, it demonstrates the utility of using genome editing to investigate the genetic basis of trait evolution., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Little is known about the genetic basis of behavioral choice, such as temperature preference, especially in natural populations. Thermal preference can play a key role in habitat selection, for example in aquatic species. Examining this behavior on a genetic level requires access to individuals or populations of the same species that display distinct temperature preferences. Caves provide a uniquely advantageous setting to tackle this problem, as animals colonizing caves encounter an environment that generally has a different, and far more stable, annual temperature than what is encountered on the outside. Here, we focus on cave and surface populations of Astyanax mexicanus, the Mexican tetra, and examine temperature preference and strength of temperature preference (reflected in the percent of time spent at the optimal temperature). We used a tank with a stable temperature gradient and automated tracking software to follow individual fish from each population. We found that distinct populations of A. mexicanus display differences in both temperature preference and strength of preference. Hybrid crosses established that these are multigenic traits that segregate independently from one another. Temperature preference in many aquatic animals is known to shift towards warmer temperatures following infection with parasites (akin to a fever response in humans). While surface fish infected by the ectoparasite Gyrodactylus turnbulli (a gill fluke) displayed a strong fever response, cavefish showed a significantly attenuated fever response. This work establishes A. mexicanus as a genetically tractable system in which differences in temperature preference can be studied in naturally evolved populations., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Through the course of evolution, the gastrointestinal (GI) tract has been modified to maximize nutrient absorption, forming specialized segments that are morphologically and functionally distinct. Here we show that the GI tract of the Mexican tetra, Astyanax mexicanus, has distinct regions, exhibiting differences in morphology, motility, and absorption. We found that A. mexicanus populations adapted for life in subterranean caves exhibit differences in the GI segments compared to those adapted to surface rivers. Cave-adapted fish exhibit bi-directional churning motility in the stomach region that is largely absent in river-adapted fish. We investigated how this motility pattern influences intestinal transit of powdered food and live prey. We found that powdered food is more readily emptied from the cavefish GI tract. In contrast, the transit of live rotifers from the stomach region to the midgut occurs more slowly in cavefish compared to surface fish, consistent with the presence of churning motility. Differences in intestinal motility and transit likely reflect adaptation to unique food sources available to post-larval A. mexicanus in the cave and river environments. We found that cavefish grow more quickly than surface fish when fed ad libitum, suggesting that altered GI function may aid in nutrient consumption or absorption. We did not observe differences in enteric neuron density or smooth muscle organization between cavefish and surface fish. Altered intestinal motility in cavefish could instead be due to changes in the activity or patterning of the enteric nervous system. Exploring this avenue will lead to a better understanding of how the GI tract evolves to maximize energy assimilation from novel food sources., (Copyright © 2018. Published by Elsevier Inc.)

Regulation of heart size and shape is one of the least understood processes in developmental biology. We have for the first time analysed the hearts of Astyanax mexicanus and identified several differences in heart morphology between the surface (epigean morph) and cave-dwelling (troglomorph) morphs. Examination of the adult revealed that the troglomorph possesses a smaller heart with a rounder ventricle in comparison to the epigean morph. The size differences identified appear to arise early in development, as early as 24 h post-fertilisation (hpf), while shape differences begin to appear at 2 days post-fertilisation. The heart of the first-generation cross between the cave-dwelling and river-dwelling morph shows uncoupling of different phenotypes observed in the parental populations and indicates that the cardiac differences have become embedded in the genome during evolution. The differences in heart morphology are accompanied by functional changes between the two morphs, with the cave-dwelling morph exhibiting a slower heart rate than the river-dwelling morph. The identification of morphological and functional differences in the A. mexicanus heart could allow us to gain more insight into how such parameters are regulated during cardiac development, with potential relevance to cardiac pathologies in humans., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The Mexican tetra, Astyanax mexicanus , comes in two forms: a classical river-dwelling fish and a blind and depigmented cave-dwelling fish. The two morphotypes are used as models for evolutionary biology, to decipher mechanisms of morphological and behavioural evolution in response to environmental change. Over the past 40 years, insights have been obtained from genetics, developmental biology, physiology and metabolism, neuroscience, genomics, population biology and ecology. Here, we promote the idea that A. mexicanus , as a model, has reached a stage where an integrated approach or a multi-disciplinary method of analysis, whereby a phenomenon is examined from several angles, is a powerful tool that can be applied to understand general evolutionary processes. Mexican cavefish have undergone considerable selective pressure and extreme morphological evolution, an obvious advantage to contribute to our understanding of evolution through comparative analyses and to pinpoint the specific traits that may have helped their ancestors to colonize caves., (© 2018 The Author(s).)

Background: The phenotypes of sperm are generally believed to be under the control of the diploid genotype of the male producing them rather than their own haploid genotypes, because developing spermatids share cytoplasm through intercellular bridges. This sharing is believed to homogenize their content of gene products. However, not all developing spermatids have identical gene products and estimates are that alleles at numerous gene loci are unequally expressed in sperm. This provides scope for the hypothesis that sperm phenotypes might be influenced by their unique haplotypes. Here we test a key prediction of this hypothesis., Results: The haploid hypothesis predicts that phenotypically different sperm subpopulations should be genetically distinct. We tested this by genotyping different sperm subpopulations that were generated by exposing sperm to a chemical dye challenge (Hoechst 33342). Dye treatment caused the cells to swell and tend to clump together. The three subpopulations of sperm we distinguished in flow cytometry corresponded to single cells, and clumps of two or three. Cell clumping in the presence of the dye may reflect variation in cell adhesivity. We found that allelic contents differed among the three populations. Importantly, the subpopulations with clumped sperm cells were significantly enriched in allelic combinations that had previously been observed to have significantly lower transmission success., Conclusions: We show that at least one sperm phenotype is correlated with its haploid genotype. This supports a broader hypothesis that the haploid genotypes of sperm cells may influence their fitness, with potentially significant implications for the transmission of deleterious alleles or combinations of alleles to their offspring.