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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.

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

Cave‐dwelling animals evolve distinct troglomorphic traits, such as loss of eyes, skin pigmentation, and augmentation of senses following long‐term adaptation to perpetual darkness. However, the molecular genetic mechanisms underlying these phenotypic variations remain unclear. In this study, we conducted comparative histology and comparative transcriptomics study of the skin of eight Sinocyclocheilus species (Cypriniformes: Cyprinidae) that included surface‐ and cave‐dwelling species. We analyzed four surface and four cavefish species by using next‐generation sequencing, and a total of 802,798,907 clean reads were generated and assembled into 505,495,009 transcripts, which contributed to 1,037,334 unigenes. Bioinformatic comparisons revealed 10,629 and 6,442 significantly differentially expressed unigenes between four different surface‐cave fish groups. Further, tens of differentially expressed genes (DEGs) potentially related to skin pigmentation were identified. Most of these DEGs (including GNAQ, PKA, NRAS, and p38) are downregulated in cavefish species. They are involved in key signaling pathways of pigment synthesis, such as the melanogenesis, Wnt, and MAPK pathways. This trend of downregulation was confirmed through qPCR experiments. This study will deepen our understanding of the formation of troglomorphic traits in cavefishes., We conducted a comparative histology and comparative transcriptomics study on the skin of eight Sinocyclocheilus species, including four surface and four cavefish species. The result suggests that these differentially expressed genes (DEGs) may be responsible for pigmentation loss in the Sinocyclocheilus cavefish species.

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

The adaptive evolution of visual systems has been observed in many cavefish. However, little is known about the molecular mechanisms underlying these adaptations, which include regressive changes such as eye degeneration. Here, we analyzed phylogenetic and expression patterns of 6 eye-related genes (crx, foxg1b, opn1sw2, otx2, rho and sox2) in 12 Sinocyclocheilus species from China, including 8 stygobionts and 4 stygophiles, and examined photoreceptor cell morphology of these species. Those eye-degenerated species of Sinocyclocheilus were polyphyletic and showed different degrees of photoreceptor defects in responses to cave environments. The eye loss and degeneration are the result of convergent evolution. Although S. anophthalmus grouped with the eye-normal species, it displayed not only a high degree of eye degeneration but also significant expression differences in eye-related genes compared with the eye-normal species. The gene foxg1b, which was determined to be under positive selection, might play an important role in the process of eye degeneration in S. anophthalmus based on differential expression. Eye-related gene expression and selection may have contributed to the polyphyly of the cave species. We examined gene expression and duplication in 6 eye-related genes and revealed that these genes displayed considerable diversity in relative expression in Sinocyclocheilus fishes. Otx2 and sox2 were significantly up-regulated in individual cave species, while the other 4 genes (crx, foxg1b, opn1sw2 and rho) were significantly down-regulated. These findings provide a valuable resource for elucidating molecular mechanisms associated with visual system evolution in cavefish.

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.

Cavefishes represent one of the most bizarre and intriguing life forms inhabiting groundwater environments. One‐third of the known cavefishes worldwide is endemic to China, and almost half of those belongs to a single genus, Sinocyclocheilus (Cypriniformes: Cyprinidae). Analyzing the morphometrics of three Sinocyclocheilus species, we aimed to assess whether variability among conspecific populations exists. We predict that populations inhabiting different subterranean habitats (shallow vs. deep) show divergences in specific morphological traits to better cope with the local ecological conditions. Our results showed that the populations showing bigger eyes and reduced humpback were those occurring close to the cave entrance (habitats with light and high food availability), while specimens with smaller eyes and increased humpback were collected from deeper groundwater areas (habitats laying in darkness with food scarcity). This explorative study paves the way for further researches aiming to collect novel data on Chinese cavefishes and highlights the usefulness of these species in evolutionary studies., Chinese cavefish are among the less studied vertebrates species. Conspecific populations show variability for some morphological traits, which are related to the ecological features of the inhabited environment.

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 river-adapted (surface) and multiple 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 (VAT). Carotenoid accumulation is not observed in Molino cavefish, indicating that it is not an obligatory consequence of eye loss. We used quantitative trait loci (QTL) 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 un-pigmented 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.

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 ofA. mexicanussleep approximately eight hours each day, multiple blind cavefish populations have converged on sleep patterns that total as little as two hours 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 ofA. 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.

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.

The genetic regulatory network governing vertebrate gonadal differentiation appears less conserved than previously thought. Here, we investigated the gonadal development of Astyanax mexicanus Pachón cavefish by looking at primordial germ cells (PGCs) migration and proliferation, gonad histology, and gene expression patterns. We showed that PGCs are first detected at the 80% epiboly stage and then reach the gonadal primordium at 1 day post-fertilization (dpf). However, in contrast to the generally described absence of PGCs proliferation during their migration phase, PGCs number in cavefish doubles between early neurula and 8–9 somites stages. Combining both gonadal histology and vasa (germ cell marker) expression patterns, we observed that ovarian and testicular differentiation occurs around 65 dpf in females and 90 dpf in males, respectively, with an important inter-individual variability. The expression patterns of dmrt1, gsdf, and amh revealed a conserved predominant male expression during cavefish gonadal development, but none of the ovarian differentiation genes, i. e., foxl2a, cyp19a1a, and wnt4b displayed an early sexually dimorphic expression, and surprisingly all these genes exhibited predominant expression in adult testes. Altogether, our results lay the foundation for further research on sex determination and differentiation in A. mexicanus and contribute to the emerging picture that the vertebrate sex differentiation downstream regulatory network is less conserved than previously thought, at least in teleost fishes.

Deciphering the genetic code of organisms with unusual phenotypes can help answer fundamental biological questions and provide insight into mechanisms relevant to human biomedical research. The cave salamander Proteus anguinus (Urodela: Proteidae), also known as the olm, is an example of a species with unique morphological and physiological adaptations to its subterranean environment, including regenerative abilities, resistance to prolonged starvation, and a life span of more than 100 years. However, the structure and sequence of the olm genome is still largely unknown owing to its enormous size, estimated at nearly 50 gigabases. An international Proteus Genome Research Consortium has been formed to decipher the olm genome. This perspective provides the scientific and biomedical rationale for exploring the olm genome and outlines potential outcomes, challenges, and methodological approaches required to analyze and annotate the genome of this unique amphibian.

The morphogenesis of the vertebrate eye consists of a complex choreography of cell movements, tightly coupled to axial regionalization and cell type specification processes. Disturbances in these events can lead to developmental defects and blindness. Here, we have deciphered the sequence of defective events leading to coloboma in the embryonic eye of the blind cavefish of the species Astyanax mexicanus. Using comparative live imaging on targeted enhancer-trap Zic1:hsp70:GFP reporter lines of both the normal, river-dwelling morph and the cave morph of the species, we identified defects in migratory cell behaviours during evagination that participate in the reduced optic vesicle size in cavefish, without proliferation defect. Further, impaired optic cup invagination shifts the relative position of the lens and contributes to coloboma in cavefish. Based on these results, we propose a developmental scenario to explain the cavefish phenotype and discuss developmental constraints to morphological evolution. The cavefish eye appears as an outstanding natural mutant model to study molecular and cellular processes involved in optic region morphogenesis., Summary: We show that the developing embryonic eye of the blind Mexican cavefish suffers multiple tissue morphogenesis and cell behaviour defects that lead to coloboma.

Sex chromosomes are generally derived from a pair of classical type-A chromosomes, and relatively few alternative models have been proposed up to now.(1,2) B chromosomes (Bs) are supernumerary and dispensable chromosomes with non-Mendelian inheritance found in many plant and animal species(3,4) that have often been considered as selfish genetic elements that behave as genome parasites.(5,6) The observation that in some species Bs can be either restricted or predominant in one sex(7–14) raised the interesting hypothesis that Bs could play a role in sex determination.(15) The characterization of putative B master sex-determining (MSD) genes, however, has not yet been provided to support this hypothesis. Here, in Astyanax mexicanus cavefish originating from Pachón cave, we show that Bs are strongly male predominant. Based on a high-quality genome assembly of a B-carrying male, we characterized the Pachón cavefish B sequence and found that it contains two duplicated loci of the putative MSD gene growth differentiation factor 6b (gdf6b). Supporting its role as an MSD gene, we found that the Pachón cavefish gdf6b gene is expressed specifically in differentiating male gonads, and that its knockout induces male-to-female sex reversal in B-carrying males. This demonstrates that gdf6b is necessary for triggering male sex determination in Pachón cavefish. Altogether these results bring multiple and independent lines of evidence supporting the conclusion that the Pachón cavefish B is a “B-sex” chromosome that contains duplicated copies of the gdf6b gene, which can promote male sex determination in this species.

Circadian rhythms are nearly ubiquitous throughout nature, suggesting they are critical for survival in diverse environments. Organisms inhabiting largely arrhythmic environments, such as caves, offer a unique opportunity to study the evolution of circadian rhythms in response to changing ecological pressures. Populations of the Mexican tetra, Astyanax mexicanus, have repeatedly invaded caves from surface rivers, where individuals must contend with perpetual darkness, reduced food availability, and limited fluctuations in daily environmental cues. To investigate the molecular basis for evolved changes in circadian rhythms, we investigated rhythmic transcription across multiple independently-evolved cavefish populations. Our findings reveal that evolution in a cave environment has led to the repeated disruption of the endogenous biological clock, and its entrainment by light. The circadian transcriptome shows widespread reductions and losses of rhythmic transcription and changes to the timing of the activation/repression of core-transcriptional clock. In addition to dysregulation of the core clock, we find that rhythmic transcription of the melatonin regulator aanat2 and melatonin rhythms are disrupted in cavefish under darkness. Mutants of aanat2 and core clock gene rorca disrupt diurnal regulation of sleep in A. mexicanus, phenocopying circadian modulation of sleep and activity phenotypes of cave populations. Together, these findings reveal multiple independent mechanisms for loss of circadian rhythms in cavefish populations and provide a platform for studying how evolved changes in the biological clock can contribute to variation in sleep and circadian behavior., Author summary Biological rhythms are molecular, physiological, and behavioral changes that follow a daily cycle and allow for animals to coordinate critical biological processes with their external environment. While these clocks are ubiquitous from unicellular life through humans, little is known about how they evolve in the absence of daily cycling within an environment. In this study, we sought to understand the evolutionary response of the biological clock when organisms become established in an environment that lacks daily fluctuations in light, temperature, and other environmental factors. Astyanax mexicanus have repeatedly moved from surface rivers into caves where they live in complete darkness. We find that multiple populations of cavefish have disrupted biological clocks compared to their surface relatives, but that these clocks are disrupted via different molecular mechanisms in different populations. Our results suggest that changes to the biological clock in these populations may also affect aspects of cavefish behavior, like the sleep-wake cycle. This study demonstrates that moving into an environment without daily cycles has led to predictable disruptions to the biological clock among cavefish populations, but that the clock itself can be broken multiple ways.

The direction of visceral organ asymmetry is highly conserved during vertebrate evolution with heart development biased to the left and pancreas and liver development restricted to opposing sides of the midline. Here we show that reversals in visceral organ asymmetry have evolved in Astyanax mexicanus, a teleost species with interfertile surface-dwelling (surface fish) and cave-dwelling (cavefish) forms. Visceral organ asymmetry is conventional in surface fish but some cavefish have evolved reversals in heart, liver, and pancreas development. Corresponding changes in the normally left-sided expression of the Nodal-Pitx2/Lefty signaling system are also present in the cavefish lateral plate mesoderm (LPM). The Nodal antagonists lefty1 (lft1) and lefty2 (lft2), which confine Nodal signaling to the left LPM, are expressed in most surface fish, however, lft2, but not lft1, expression is absent during somitogenesis of most cavefish. Despite this difference, multiple lines of evidence suggested that evolutionary changes in L-R patterning are controlled upstream of Nodal-Pitx2/Lefty signaling. Accordingly, reciprocal hybridization of cavefish and surface fish showed that modifications of heart asymmetry are present in hybrids derived from cavefish mothers but not from surface fish mothers. The results indicate that changes in visceral asymmetry during cavefish evolution are influenced by maternal genetic effects.

Organisms living in the subterranean biome evolve extreme characteristics including vision loss and sensory expansion. Despite prior work linking certain genes to Mendelian traits, the genetic basis for complex cave-associated traits remains unknown. Moreover, it is unclear if certain forms of genetic variation (e.g., indels, copy number variants) are more common in regressive evolution. Progress in this area has been limited by a lack of suitable natural model systems and genomic resources. In recent years, the Mexican tetra, Astyanax mexicanus, has advanced as a model for cave biology and regressive evolution. Here, we present the results of a genome-wide screen for in-frame indels using alignments of RNA-sequencing reads to the draft cavefish genome. Mutations were discovered in three genes associated with blood physiology (mlf1, plg, and wdr1), two genes associated with growth factor signaling (ghrb, rnf126), one gene linked to collagen defects (mia3), and one gene which may have a global epigenetic impact on gene expression (mki67). With one exception, polymorphisms were shared between Pachón and Tinaja cavefish lineages, and different from the surface-dwelling lineage. We confirmed the presence of mutations using direct Sanger sequencing and discovered remarkably similar developmental expression in both morphs despite substantial coding sequence alterations. Further, three mutated genes mapped near previously established quantitative trait loci associated with jaw size, condition factor, lens size, and neuromast variation. This work reveals previously unappreciated traits evolving in this species under environmental pressures (e.g., blood physiology) and provides insight to genetic changes underlying convergence of organisms evolving in complete darkness.

Astyanax mexicanus is a well-established and widely used fish model system for evolutionary and developmental biology research. These fish exist as surface forms that inhabit rivers and 30 different populations of cavefish. The establishment of A. mexicanus as an emergent model organism for understanding the evolutionary basis of development and behavior has been accelerated by an increasing availability of genomic approaches to identify genotype-phenotype associations. Despite important progress in the deployment of new technologies, deep mechanistic insights into A. mexicanus evolution and development have been limited by a lack of transgenic lines commonly used in genetic model systems. Here, we expand the toolkit of transgenesis by characterizing two novel stable transgenic lines that were generated using the highly efficient Tol2 system, commonly used to generate transgenic zebrafish. A stable transgenic line consisting of the zebrafish ubiquitin promoter fused to eGFP expressed eGFP ubiquitously throughout development in a surface population of Astyanax. To define specific cell-types, we injected fish with a Cntnap2-mCherry construct that labels lateral line mechanosensory neurons in zebrafish. Strikingly, both constructs appear to label the predicted cell types, suggesting many genetic tools and defined promoter regions in zebrafish are directly transferrable to cavefish. The lines provide proof-of-principle for the application of Tol2 transgenic technology in A. mexicanus. Expansion on these initial transgenic lines will provide a platform to address broadly important problems in the quest to bridge the genotype to phenotype gap.

Scleral ossicles are bony elements found along the eyes of many fishes, amphibians, and reptiles. These bones provide a superficial layer of support to the eye and may facilitate visual acuity. Previous research has shown that scleral ossicle diversity is generally limited among teleosts, but that scleral ossicles have been lost numerous times among teleosts inhabiting benthopelagic habitats (Franz-Odendaal. Anat Rec 291 (2008) 161-168). In this study, we further investigate these patterns of intraspecific and interspecific variation by examining eyes from multiple individuals of 10 riverine teleosts native to Kentucky as well as one population of the Mexican blind cavefish, Astyanax mexicanus, and by re-analyzing a quantitative database of scleral ossicle number and depth preference from over 100 teleosts using newly resolved teleost phylogenies. Consistent with the limited diversity of most teleost families, we find that intraspecific variation in scleral ossicle number and size is virtually nonexistent among the species sampled, although we do find evidence of additional interspecific variation among the Cyprinodontiformes, as well as dramatic intrapopulation variation among cavefish from Chica Cave. Although our data replicates the negative relationship between scleral ossicle number and the depth preference previously found among teleosts (Franz-Odendaal. Anat Rec 291 (2008) 161-168), even when accounting for phylogenetic relationships, our results further reveal that this relationship is relatively weak. We conclude that further sampling may reveal additional interspecific and even intraspecific variation among some groups of teleosts, and that depth could serve as a proxy for other life history traits that more directly influence teleost scleral ossicle diversity such as prey-capture strategies. Anat Rec, 302:1238-1249, 2019. © 2019 Wiley Periodicals, Inc.

A blind fish of Sinocyclocheilus (Cypriniformes: Cyprinidae)was caught in open water in the Three Gorges (Sanxia)reservoir, at a depth of 20 m in the mainstream of YangtzeRiver in Zigui County, Hubei Province, China. This fish can beeasily distinguished from all other congeners by externalmorphological characteristics, and is estimated to havediverged from its sister group about 0.55 million years ago(Ma). The geologically well separated locality of this specieshas expanded the distribution of Sinocyclocheilus cavefishfrom around N25° (latitude) to above N30° . Herein, wedescribe this new species as Sinocyclocheilus sanxiaensis sp.nov ., and discuss the possible reasons why the speciesappears, surprisingly, in the Three Gorges reservoir.

Fish are good for your health in more ways than you may expect. For one, eating fish is a common dietary recommendation for a healthy diet. However, fish have much more to provide than omega-3 fatty acids to your circulatory system. Some fish species now serve as important and innovative model systems for diabetes research, providing novel and unique advantages compared with classical research models. Not surprisingly, the largest share of diabetes research in fish occurs in the laboratory workhorse among fish, the zebrafish (Danio rerio). Established as a genetic model system to study development, these small cyprinid fish have eventually conquered almost every scientific discipline and, over the past decade, have emerged as an important model system for metabolic diseases, including diabetes mellitus. In this review we highlight the practicability of using zebrafish to study diabetes and hyperglycemia, and summarize some of the recent research and breakthroughs made using this model. Equally exciting is the appearance of another emerging discipline, one that is taking advantage of evolution by studying cases of naturally occurring insulin resistance in fish species. We briefly discuss two such models in this review, namely the rainbow trout (Oncorhynchus mykiss) and the cavefish (Astyanax mexicanus).

We describe a new species of cave-dwelling loach, Triplophysa wulongensis sp. nov., based on specimens collected in a subterranean pool in a cave in Wulong County, Chongqing, Southwest China. The pool is connected to the Wujiang River drainage. Triplophysa wulongensis differs from its congeners by the following combination of characters: eyes present, caudal fin with 18 branched rays; posterior chamber of the air bladder degenerate; stomach U-shaped; intestine without bends or loops immediately posterior to stomach; body smooth and scaleless, and lateral line complete. The mitochondrial cytochrome b sequence differs from those of other published sequences of species of Triplophysa by 14.9–24.9% in K2P distance. Phylogenetic analysis based on cytochrome b gene sequences recovered T. wulongensis as sister taxon to all other cave-dwelling species of Triplophysa .

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

Made available in DSpace on 2018-11-26T17:33:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-01-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Complete mitochondrial genome of the characiform fish Astyanax paranae was characterized in the present study. The whole mitogenome was 16,707 bp long and consisted of 13 protein-coding genes, 22 tRNAs, 2 rRNAs genes, a control region and origin of light-strand replication. The gene order is similar to those of the congeneric blind cavefish A. mexicanus. The nucleotide content of A. paranae mitogenome was 29.5% for A, 27.6% for T, 15.8% for G, 27.1% for C. Nucleotide identity between A. paranae and A. mexicanus across all the 37 genic regions ranged from 74.9% (ND2) to 90.3% (COX3). Univ Estadual Paulista, Inst Biociencias, Dept Morfol, Botucatu, SP, Brazil Univ Granada, Dept Genet, Granada, Spain Univ Estadual Paulista, Inst Biociencias, Dept Morfol, Botucatu, SP, Brazil FAPESP: 2013/24367-0

The study of swimming behavior is an important part of fish biology research and the swim tunnel is used to study swimming performance as well as metabolism of fish. In this investigation, we have developed a user-friendly, automated, modular, and low-cost swim tunnel that permits to study the performance of one or more fish separately, as well as a small group of individuals. To validate our swim tunnel, we assessed swimming activity of four different species (zebrafish, medaka, guppy, and cavefish) recording reliable data of swimming behavior and performance. Because swimming behavior has been recently used in different fields from physiology to ecotoxicology, our setup could help researchers with a low-cost solution.

The fish speciesAstyanax mexicanuswith 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. mexicanusoffers 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 thatAstyanaxpescoids have the potential to recapitulate the main ontogenetic events observed in intact embryos, including the internalization of mesodermal progenitors and eye development, as followed withzic:GFPreporter 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 inA. mexicanuswill bring new light on the cascades of events, from the maternal pre-patterning of the early embryo to the evolution of brain regionalization.

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

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

Cavefish evolved a series of distinct survival mechanisms for adaptation to cave habitat. Such mechanisms include loss of eyesight and pigmentation, sensitive sensory organs, unique dietary preferences, and predation behavior. Thus, it is of great interest to understand the mechanisms underlying these adaptability traits of troglobites. The teleost genus Sinocyclocheilus (Cypriniformes: Cyprinidae) is endemic to China and has more than 70 species reported (including over 30 cavefish species). High species diversity and diverse phenotypes make the Sinocyclocheilus as an outstanding model for studying speciation and adaptive evolution. In this study, we conducted a comparative transcriptomics study on the brain tissues of two Sinocyclocheilus species (surface-dwelling species – Sinocyclocheilus malacopterus and semi-cave-dwelling species – Sinocyclocheilus rhinocerous living in the same water body. A total of 425,188,768 clean reads were generated, which contributed to 102,839 Unigenes. Bioinformatic analysis revealed a total of 3,289 differentially expressed genes (DEGs) between two species Comparing to S. malacopterus, 2,598 and 691 DEGs were found to be respectively, down-regulated and up-regulated in S. rhinocerous. Furthermore, it is also found tens of DEGs related to cave adaptability such as insulin secretion regulation (MafA, MafB, MafK, BRSK, and CDK16) and troglomorphic traits formation (CEP290, nmnat1, coasy, and pqbp1) in the cave-dwelling S. rhinocerous. Interestingly, most of the DEGs were found to be down-regulated in cavefish species and this trend of DEGs expression was confirmed through qPCR experiments. This study would provide an appropriate genetic basis for future studies on the formation of troglomorphic traits and adaptability characters of troglobites, and improve our understanding of mechanisms of cave adaptation.

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, 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, 5, 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, 8, 9, 10, 11, 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.

The Astyanax species complex has two morphs: a blind, depigmented morph which inhabits caves in M&eacute, xico and an eyed, pigmented surface-dwelling morph. The eyed morph can also be found in a few caves, sometimes hybridizing with the cave morph. This species complex has arguably become the most prominent model system among cave organisms for the study of evolutionary development and genomics. Before this study, 32 caves were known to be inhabited by the cave morph, 30 of them within the El Abra region. The purpose of this study was to conduct new surveys of the area and to assess some unconfirmed reports of caves presumably inhabited by troglomorphic fish. We describe two new localities, S&oacute, tano del Toro #2 and S&oacute, tano de La Calera. These two caves comprise a single hydrologic system together with the previously described cave of S&oacute, tano del Toro. The system is inhabited by a mixed population of troglomorphic, epigeomorphic, and presumably hybrid fish. Furthermore, Astyanax cavefish and the mysid shrimp Spelaeomysis quinterensis show a phylogeographic convergence that supports the notion that the central Sierra de El Abra is a biogeographical region that has influenced the evolutionary history of its aquatic community across species. The presumptive location of the boundaries of this biogeographical region are identified.

Brain atlases reveal neuroanatomical and activity signatures associated with behavioral evolution in cavefish., Environmental perturbation can drive behavioral evolution and associated changes in brain structure and function. The Mexican fish species, Astyanax mexicanus, includes eyed river-dwelling surface populations and multiple independently evolved populations of blind cavefish. We used whole-brain imaging and neuronal mapping of 684 larval fish to generate neuroanatomical atlases of surface fish and three different cave populations. Analyses of brain region volume and neural circuits associated with cavefish behavior identified evolutionary convergence in hindbrain and hypothalamic expansion, and changes in neurotransmitter systems, including increased numbers of catecholamine and hypocretin/orexin neurons. To define evolutionary changes in brain function, we performed whole-brain activity mapping associated with behavior. Hunting behavior evoked activity in sensory processing centers, while sleep-associated activity differed in the rostral zone of the hypothalamus and tegmentum. These atlases represent a comparative brain-wide study of intraspecies variation in vertebrates and provide a resource for studying the neural basis of behavioral evolution.

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

Vestigial structures are key indicators of evolutionary descent, but the mechanisms underlying their development are poorly understood. This study examines vestigial eye formation in the teleost Astyanax mexicanus, which consists of a sighted surface-dwelling morph and multiple populations of blind cave morphs. Cavefish embryos initially develop eyes, but they subsequently degenerate and become vestigial structures embedded in the head. The mutated genes involved in cavefish vestigial eye formation have not been characterized. Here we identify cystathionine ß-synthase a (cbsa), which encodes the key enzyme of the transsulfuration pathway, as one of the mutated genes responsible for eye degeneration in multiple cavefish populations. The inactivation of cbsa affects eye development by increasing the transsulfuration intermediate homocysteine and inducing defects in optic vasculature, which result in aneurysms and eye hemorrhages. Our findings suggest that localized modifications in the circulatory system may have contributed to the evolution of vestigial eyes in cavefish., The teleost Astyanax mexicanus has sighted morphs living in surface rivers and various blind morphs living in caves. Here, the authors suggest that loss of eyes in cave morphs is linked to mutations in the cystathionine ß-synthase a (cbsa) gene, which cause eye degeneration by disrupting function of the optic circulatory system.

Extreme environments often result in the evolution of dramatic adaptive features. The Mexican tetra, Astyanax mexicanus, includes 30 different populations of cave-dwelling forms that live in perpetual darkness. As a consequence, many populations have evolved eye loss, reduced pigmentation, and amplification of nonvisual sensory systems. Closely-related surface-dwelling morphs demonstrate typical vision, pigmentation, and sensation. Transcriptomic assessments in this system have revealed important developmental changes associated with the cave morph, however, they have not accounted for photic rearing conditions. Prior studies reared individuals under a 12:12 hr light/dark (LD) cycle. Here, we reared cavefish under constant darkness (DD) for 5+ years. From these experimental individuals, we performed mRNA sequencing and compared gene expression of surface fish reared under LD conditions to cavefish reared under DD conditions to identify photic-dependent gene expression differences. Gene Ontology enrichment analyses revealed a number of previously underappreciated cave-associated changes impacting blood physiology and olfaction. We further evaluated the position of differentially expressed genes relative to QTL positions from prior studies and found several candidate genes associated with these ecologically relevant lighting conditions. In sum, this work highlights photic conditions as a key environmental factor impacting gene expression patterns in blind cave-dwelling fish.

Background Natural model systems are indispensable for exploring adaptations in response to environmental pressures. Sinocyclocheilus of China, the most diverse cavefish clade in the world (75 species), provide unique opportunities to understand recurrent evolution of stereotypic traits (such as eye loss and sensory expansion) in the context of a deep and diverse phylogenetic group. However, they remain poorly understood in terms of their morphological evolution. Therefore, we explore key patterns of morphological evolution, habitat utilization and geographic distribution in these fishes. Results We constructed phylogenies and categorized 49 species based on eye-related condition (Blind, Micro-eyed, and Normal-eyed), habitat types (Troglobitic—cave-restricted; Troglophilic—cave-associated; Surface—outside caves) and existence of horns. Geometric-morphometric analyses show Normal-eyed morphs with fusiform shapes segregating from Blind/Micro-eyed deeper bodied morphs along the first principal-component axis; second axis accounts for shape complexity related to horns. The body shapes showed a significant association with eye-related condition and horn, but not habitat types. Ancestral reconstructions suggest at least three independent origins of Blind morphs, each with different levels of modification in relation to their ancestral Normal-eyed morphs; Sinocyclocheilus are also pre-adapted for cave dwelling. Our geophylogeny shows an east-to-west diversification spanning Pliocene and Pleistocene, with early-diversifying Troglobitic species dominating subterranean habitats of karstic plains whereas predominantly Surface forms inhabit hills to the west. Evolutionary rates analyses suggest that lineages leading to Blind morphs were characterized by significant rate shifts, such as a slowdown in body size evolution and a 5–20 fold increase in rate of eye regression, possibly explained by limited resource availability. Body size and eye size have undergone reversals, but not horns, a trait entailing considerable time to form. Conclusions Sinocyclocheilus occupied cave habitats in response to drying associated with aridification of China during late Miocene and the Pliocene. The prominent cave-adaptations (eye-regression, horn-evolution) occur in clades associated with the extensive subterranean cave system in Guangxi and Guizhou provinces. Integration of morphology, phylogeny, rate analyses, molecular-dating and distribution show not only several remarkable patterns of evolution, but also interesting exceptions to these patterns signifying the diversification of Sinocyclocheilus as an invaluable model system to explore evolutionary novelty.

The neurotransmitter serotonin controls a great variety of physiological and behavioral processes. In humans, mutations affecting the monoamine oxidase or MAO, the serotonin-degrading enzyme, are highly deleterious. Yet, blind cavefish of the speciesA. mexicanuscarry a partial loss-of-function mutation in MAO (P106L) and seem to thrive in their subterranean environment. Here, we established 4 fish lines, corresponding to the blind cave-dwelling and the sighted river-dwelling morphs of this species, with or without the mutation, in order to decipher the exact contribution ofmaoP106L in the evolution of cavefish neuro-behavioral traits. Unexpectedly, althoughmaoP106L appeared as an excellent candidate for the genetic determinism of the loss of aggressive and schooling behaviors in cavefish, we demonstrated that it was not the case. Similarly, the anatomical variations in monoaminergic systems observed between cavefish and surface fish brains were independent frommaoP106L, and rather due other, morph-dependent developmental processes. On the other hand, we found thatmaoP106L strongly affected anxiety-like behaviors. Cortisol measurements showed lower basal levels and an increased amplitude of stress response after a change of environment in fish carrying the mutation. Finally, we studied the distribution of the P106Lmaoallele in wild populations of cave and riverA. mexicanus, and discovered that the mutant allele was present - and sometimes fixed - in all populations inhabiting caves of the Sierra de El Abra. The possibility that this partial loss-of-functionmaoallele evolves under a selective or a genetic drift regime in the particular cave environment is discussed.

Stress responses are conserved physiological and behavioral outcomes as a result of facing potentially harmful stimuli, yet in pathological states, stress becomes debilitating. Stress responses vary considerably throughout the animal kingdom, but how these responses are shaped evolutionarily is unknown. The Mexican cavefish has emerged as a powerful system for examining genetic principles underlying behavioral evolution. Here, we demonstrate that cave Astyanax have reduced behavioral and physiological measures of stress when examined at larval stages. We also find increased expression of the glucocorticoid receptor, a repressible element of the neuroendocrine stress pathway. Additionally, we examine stress in three different cave populations, and find that some, but not all, show reduced stress measures. Together, these results reveal a mechanistic system by which cave-dwelling fish reduced stress, presumably to compensate for a predator poor environment.Research HighlightCavefish populations of A. mexicanus have reduced stress relative to surface conspecific at larval stagesWe show that a glucocorticoid receptor, a negative regulator of the neuroendocrine stress axis, is upregulated in stress-resistant cavefishThere exists much ontological heterogeneity between different cavefish populations.

Articles Discussed: Stahl, B. A. et al. Manipulation of Gene Function in Mexican Cavefish. Journal of Visualized Experiments. (146) (2019). Peus, R. et al. Gamete Collection and In Vitro Fertilization of Astyanax mexicanus. Journal of Visualized Experiments. (147) (2019). Worsham, M. et al. Behavioral Tracking and Neuromast Imaging of Mexican Cavefish.Journal of Visualized Experiments. (147) (2019). Jaggard, J.B., Lloyd, E., Lopatto, A., Duboue, E.R., Keene, A.C. Automated Measurements of Sleep and Locomotor Activity in Mexican Cavefish. Journal of Visualized Experiments. (145) (2019). Luc, H., Sears, C., Raczka, A., Gross, J.B. Wholemount In Situ Hybridization for Astyanax Embryos. Journal of Visualized Experiments. (145) (2019). Riddle, M., Martineau, B., Peavey, M., Tabin, C. Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry. Journal of Visualized Experiments. (142) (2018).

Life in complete darkness has driven the evolution of a suite of troglobitic features in the blind Mexican cavefish Astyanax mexicanus, such as eye and pigmentation loss. While regressive evolution is a hallmark of obligate cave-dwelling organisms, constructive (or augmented) traits commonly arise as well. The cavefish cranium has undergone extensive changes compared with closely-related surface fish. These alterations are rooted in both cranial bones and surrounding sensory tissues such as enhancements in the gustatory and lateral line systems. Cavefish also harbor numerous cranial bone asymmetries: fluctuating asymmetry of individual bones and directional asymmetry in a dorsal bend of the skull. This asymmetry is mirrored by the asymmetrical patterning of mechanosensory neuromasts. We explored the relationship between facial bones and neuromasts using in vivo fluorescent colabeling and microcomputed tomography. We found an increase in neuromast density within dermal bone boundaries across three distinct populations of cavefish compared to surface-dwelling fish. We also show that eye loss disrupts early neuromast patterning, which in turn impacts the development of dermal bones. While cavefish exhibit alterations in cranial bone and neuromast patterning, each population varied in the severity. This variation may reflect observed differences in behavior across populations. For instance, a bend in the dorsal region of the skull may expose neuromasts to water flow on the opposite side of the face, enhancing sensory input and spatial mapping in the dark.

Colonization of novel habitats often results in the evolution of diverse behaviors. Comparisons between individuals from closely related populations that have evolved divergent behaviors in different environments can be used to investigate behavioral evolution. However, until recently, functionally connecting genotypes to behavioral phenotypes in these evolutionarily relevant organisms has been difficult. The development of gene editing tools will facilitate functional genetic analysis of genotype–phenotype connections in virtually any organism, and has the potential to significantly transform the field of behavioral genetics when applied to ecologically and evolutionarily relevant organisms. The blind cavefish Astyanax mexicanus provides a remarkable example of evolution associated with colonization of a novel habitat. These fish consist of a single species that includes sighted surface fish that inhabit the rivers of Mexico and southern Texas and at least 29 populations of blind cavefish from the Sierra Del Abra and Sierra de Guatemala regions of Northeast Mexico. Although eye loss and albinism have been studied extensively in A. mexicanus, derived behavioral traits including sleep loss, alterations in foraging and reduction in social behaviors are now also being investigated in this species to understand the genetic and neural basis of behavioral evolution. Astyanax mexicanus has emerged as a powerful model system for genotype–phenotype mapping because surface and cavefish are interfertile. Further, the molecular basis of repeated trait evolution can be examined in this species, as multiple cave populations have independently evolved the same traits. A sequenced genome and the implementation of gene editing in A. mexicanus provides a platform for gene discovery and identification of the contributions of naturally occurring variation to behaviors. This review describes the current knowledge of behavioral evolution in A. mexicanus with an emphasis on the molecular and genetic underpinnings of evolved behaviors. Multiple avenues of new research that can be pursued using gene editing tools are identified, and how these will enhance our understanding of behavioral evolution is discussed.

Eye degeneration is one of the most obvious characteristics of organisms restricted to subterranean habitats. In cavefish, eye degeneration has evolved independently numerous times and each process is associated with different genetic mechanisms. To gain a better understanding of these mechanisms, we compared the eyes of adult individuals of the cave loach Triplophysa rosa and surface loach Triplophysa bleekeri. Compared with the normal eyes of the surface loach, those of the cave loach were found to possess a small abnormal lens and a defective retina containing photoreceptor cells that lack outer segments. Sequencing of the transcriptomes of both species to identify differentially expressed genes (DEGs) and genes under positive selection revealed 4,802 DEGs and 50 genes under positive selection (dN/dS > 1, FDR < 0.1). For cave loaches, we identified one Gene Ontology category related to vision that was significantly enriched in downregulated genes. Specifically, we found that many of the downregulated genes, including pitx3, lim2, crx, gnat2, rx1, rho, prph2, and β|γ-crystallin are associated with lens/retinal development and maintenance. However, compared with those in the surface loach, the lower dS rates but higher dN rates of the protein-coding sequences in T. rosa indicate that changes in amino acid sequences might be involved in the adaptation and visual degeneration of cave loaches. We also found that genes associated with light perception and light-stimulated vision have evolved at higher rates (some genes dN/dS > 1 but FDR > 0.1). Collectively, the findings of this study indicate that the degradation of cavefish vision is probably associated with both gene expression and amino acid changes and provide new insights into the mechanisms underlying the degeneration of cavefish eyes.

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.\ud \ud 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.

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 X CF hybrids) and CF eggs with SF sperm (CF X SF hybrids), revealed that the CF degenerative eye phenotype showed maternal genetic effects. The eyes of CF X 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 X 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.

Feeding strategies are dependent on multi-modal sensory processing, that integrates visual, chemosensory, and mechanoreceptive cues. In many fish species, local environments and food availability dramatically influence the evolution of sensory and morphological traits that underlie feeding. The Mexican cavefish, Astyanax mexicanus, have developed robust changes in sensory-dependent behaviors, but the impact on prey detection and feeding behavior is not known. In the absence of eyes, cavefish have evolved enhanced sensitivity of the lateral line, comprised of mechanosensory organs that sense water flow and detect prey. Here, we identify evolved differences in prey capture behavior of larval cavefish that are dependent on lateral line sensitivity. Under lighted conditions, cavefish strike Artemia prey at a wider angle than surface fish; however, this difference is diminished under dark conditions. In addition, the strike distance is greater in cavefish than surface fish, revealing an ability to capture, and likely detect, prey at greater distances. Experimental ablation of the lateral line disrupts prey capture in cavefish under both light and dark conditions, while it only impacts surface fish under dark conditions. Together, these findings identify an evolutionary shift towards a dependence on the lateral line for prey capture in cavefish, providing a model for investigating how loss of visual cues impacts multi-modal sensory behaviors.

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.

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.

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 Queretaro, 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.

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

The teleost Astyanax mexicanus is a single species with surface dwelling (surface fish) and cave dwelling (cavefish) morphs. Constructive and regressive traits have evolved in cavefish as adaptations for survival in perpetual darkness. In addition to darkness, cavefish must cope with de-oxygenated aquatic environments. Blood cell quantification and expression of hematopoietic marker genes indicated that cavefish have more erythrocytes than surface fish, and that this increase has a developmental basis. In contrast to zebrafish and other teleost embryos, in which erythrocyte formation is restricted to the posterior lateral mesoderm, the anterior as well as the posterior lateral mesoderm is involved in red blood cell formation in Astyanax embryos, and both of these hematopoietic domains are expanded in cavefish embryos. Erythroid development in the anterior mesoderm may be a pre-adaptation for surface fish to successfully colonize hypoxic cave environments. We also show that cavefish are less sensitive to phenylhydrazine-induced erythrocyte ablation than surface fish, suggesting a functional advantage of increased red blood cells. By mimicking a hypoxic cave environment in the laboratory, we further demonstrate that cavefish respond to hypoxia differently than surface fish. Surface fish with fewer red blood cells use overall metabolic depression to counteract hypoxia, whereas cavefish with larger numbers of erythrocytes respond to hypoxia by switching to anaerobic metabolism. These results suggest that cavefish may have adapted to hypoxic environments by enhancing the capacity to form erythrocytes and reprogramming metabolism. Summary Astyanax mexicanus cavefish adapt to life in hypoxic cave environments by evolving the capacity to increase red blood cell numbers during early development and reprogramming metabolism to favor anaerobic processes.