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Developmental evolution and diversification of morphology can arise through changes in the regulation of gene expression or protein-coding sequence. To unravel mechanisms underlying early developmental evolution in cavefish of the species Astyanax mexicanus, we compared transcriptomes of surface-dwelling and blind cave-adapted morphs at the end of gastrulation. Twenty percent of the transcriptome was differentially expressed. Allelic expression ratios in cave X surface hybrids showed that cis-regulatory changes are the quasi-exclusive contributors to inter-morph variations in gene expression. Among a list of 108 genes with change at the cis-regulatory level, we explored the control of expression of rx3, which is a master eye gene. We discovered that cellular rx3 levels are cis-regulated in a cell-autonomous manner, whereas rx3 domain size depends on non-autonomous Wnt and Bmp signalling. These results highlight how uncoupled mechanisms and regulatory modules control developmental gene expression and shape morphological changes. Finally, a transcriptome-wide search for fixed coding mutations and differential exon use suggested that variations in coding sequence have a minor contribution. Thus, during early embryogenesis, changes in gene expression regulation are the main drivers of cavefish developmental evolution., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The circadian clock represents a key timing system entrained by various periodic signals that ensure synchronization with the environment. Many investigations have pointed to the existence of two distinct circadian oscillators: one regulated by the light-dark cycle and the other set by feeding time. Blind cavefish have evolved under extreme conditions where they completely lack light exposure and experience food deprivation. Here, we have investigated feeding regulated clocks in two cavefish species, the Somalian cavefish Phreatichthys andruzzii and the Mexican cavefish Astyanax mexicanus , in comparison with the surface-dwelling zebrafish Danio rerio . Our results reveal that feeding represents an extremely strong synchronizer for circadian locomotor rhythmicity in subterranean cavefish. Indeed, we showed that consuming just one meal every 4 days is sufficient to entrain circadian rhythmicity in both cavefish species, but not in zebrafish. These profound adaptations to an extreme environment provide insight into the connections between feeding and circadian clocks., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

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.)

Background: Several studies suggested that cavefish populations of Astyanax mexicanus settled during the Late Pleistocene. This implies that the cavefish's most conspicuous phenotypic changes, blindness and depigmentation, and more cryptic characters important for cave life, evolved rapidly., Results: Using the published genomes of 47 Astyanax cavefish from la Cueva de El Pachón, El Sótano de la Tinaja, La Cueva Chica and El Sótano de Molino, we searched for putative loss-of-function mutations in previously defined sets of genes, i.e., vision, circadian clock and pigmentation genes. Putative non-functional alleles for four vision genes were identified. Then, we searched genome-wide for putative non-functional alleles in these four cave populations. Among 512 genes with segregating putative non-functional alleles in cavefish that are absent in surface fish, we found an enrichment in visual perception genes. Among cavefish populations, different levels of shared putative non-functional alleles were found. Using a subset of 12 genes for which putative loss-of-function mutations were found, we extend the analysis of shared pseudogenes to 11 cave populations. Using a subset of six genes for which putative loss-of-function mutations were found in the El Sótano del Toro population, where extensive hybridization with surface fish occurs, we found a correlation between the level of eye regression and the amount of putative non-functional alleles., Conclusions: We confirm that very few putative non-functional alleles are present in a large set of vision genes, in accordance with the recent origin of Astyanax mexicanus cavefish. Furthermore, the genome-wide analysis indicates an enrichment of putative loss-of-function alleles in genes with vision-related GO-terms, suggesting that visual perception may be the function chiefly impacted by gene losses related to the shift from a surface to a cave environment. The geographic distribution of putative loss-of-function alleles newly suggests that cave populations from Sierra de Guatemala and Sierra de El Abra share a common origin, albeit followed by independent evolution for a long period. It also supports that populations from the Micos area have an independent origin. In El Sótano del Toro, the troglomorphic phenotype is maintained despite massive introgression of the surface genome., (© 2024. The Author(s).)

Monoaminergic systems are conserved in vertebrates, yet they present variations in neuroanatomy, genetic components and functions across species. MonoAmine Oxidase, or MAO, is the enzyme responsible for monoamine degradation. While mammals possess two genes, MAO-A and MAO-B, fish possess one single mao gene. To study the function of MAO and monoamine homeostasis on fish brain development and physiology, here we have generated a mao knockout line in Astyanax mexicanus (surface fish), by CRISPR/Cas9 technology. Homozygote mao knockout larvae died at 13 days post-fertilization. Through a time-course analysis, we report that hypothalamic serotonergic neurons undergo fine and dynamic regulation of serotonin level upon loss of mao function, in contrast to those in the raphe, which showed continuously increased serotonin levels - as expected. Dopaminergic neurons were not affected by mao loss-of-function. At behavioral level, knockout fry showed a transient decrease in locomotion that followed the variations in the hypothalamus serotonin neuronal levels. Finally, we discovered a drastic effect of mao knockout on brain progenitors proliferation in the telencephalon and hypothalamus, including a reduction in the number of proliferative cells and an increase of the cell cycle length. Altogether, our results show that MAO has multiple and varied effects on Astyanax mexicanus brain development. Mostly, they bring novel support to the idea that serotonergic neurons in the hypothalamus and raphe of the fish brain are different in nature and identity, and they unravel a link between monoaminergic homeostasis and brain growth., (© 2023 Japanese Society of Developmental Biologists.)

Complex patterns of acoustic communication exist throughout the animal kingdom, including underwater. The river-dwelling and the Pachón cave-adapted morphotypes of the fish Astyanax mexicanus are soniferous and share a repertoire of sounds. Their function and significance is mostly unknown. Here, we explored whether and how sounds produced by blind cavefishes inhabiting different Mexican caves may vary. We compared "Clicks" and "Serial Clicks" produced by cavefish in six different caves distributed in three mountain ranges in Mexico. We also sampled laboratory-bred cavefish lines originating from four of these caves. Sounds were extracted and analyzed using both a manual method and a machine learning-based automation tool developed in-house. Multi-parametric analyses suggest wild cave-specific acoustic signatures, or "accents". An acoustic code also existed in laboratory cavefish lines, suggesting a genetic basis for the evolution of this trait. The variations in acoustic parameters between caves of origin did not seem related to fish phenotypes, phylogeography or ecological conditions. We propose that the evolution of such acoustic signatures would progressively lead to the differentiation of local accents that may prevent interbreeding and thus contribute to speciation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Hyacinthe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The sizes of Astyanax mexicanus blind cavefish populations of North-East Mexico are demographic parameters of great importance for investigating a variety of ecological, evolutionary, and conservation issues. However, few estimates have been obtained. For these mobile animals living in an environment difficult to explore as a whole, methods based on capture-mark-recapture are appropriate, but their feasibility and interpretation of results depend on several assumptions that must be carefully examined. Here, we provide evidence that minimally invasive genetic identification from captures at different time intervals (three days and three years) can give insights into cavefish population size dynamics as well as other important demographic parameters of interest. We also provide tools to calibrate sampling and genotyping efforts necessary to reach a given level of precision. Our results suggest that the El Pachón cave population is currently very small, of an order of magnitude of a few hundreds of individuals, and is distributed in a relatively isolated area. The probable decline in population size in the El Pachón cave since the last census in 1971 raises serious conservation issues.

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.)

Background: Ray-finned fishes (Actinopterygii) perceive their environment through a range of sensory modalities, including olfaction. Anatomical diversity of the olfactory organ suggests that olfaction is differentially important among species. To explore this topic, we studied the evolutionary dynamics of the four main gene families (OR, TAAR, ORA/VR1 and OlfC/VR2) coding for olfactory receptors in 185 species of ray-finned fishes., Results: The large variation in the number of functional genes, between 28 in the ocean sunfish Mola mola and 1317 in the reedfish Erpetoichthys calabaricus, is the result of parallel expansions and contractions of the four main gene families. Several ancient and independent simplifications of the olfactory organ are associated with massive gene losses. In contrast, Polypteriformes, which have a unique and complex olfactory organ, have almost twice as many olfactory receptor genes as any other ray-finned fish., Conclusions: We document a functional link between morphology of the olfactory organ and richness of the olfactory receptor repertoire. Further, our results demonstrate that the genomic underpinning of olfaction in ray-finned fishes is heterogeneous and presents a dynamic pattern of evolutionary expansions, simplifications, and reacquisitions., (© 2022. The Author(s).)

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.)

The fish Astyanax mexicanus comes in two very different forms: a "normal" river morph, and a blind, depigmented cave morph, living in the total and permanent darkness of Mexican caves. This species is on the way to becoming a model of choice in evolutionary and comparative biology, both for the study of the evolution of behavior, physiology or morphology, and for molecular genetics or population genetics. Here, I present the advancement of knowledge in the field of the developmental evolution of the eye of the cave morph. By rewinding back in time its development from the eye of the larva to the retinal field at the end of gastrulation, the cave-dwelling Astyanax embryo reveals mechanisms and processes likely to contribute to evolutionary variations between species, but also to pathological variations in the morphogenesis of the optic region., (© Société de Biologie, 2022.)

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., Competing Interests: Declaration of interests The authors declare no competing interests, (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Teleost fishes perceive their environment through a range of sensory modalities, among which olfaction often plays an important role. Richness of the olfactory repertoire depends on the diversity of receptors coded by homologous genes classified into four families: OR, TAAR, VR1, and VR2. Herein, we focus on the OR gene repertoire. While independent large contractions of the OR gene repertoire associated with ecological transitions have been found in mammals, little is known about the diversity of the OR gene repertoire and its evolution in teleost fishes, a group that includes more than 34,000 living species. We analyzed genomes of 163 species representing diversity in this large group. We found a large range of variation in the number of functional OR genes, from 15 in the Broad-nose Pipefish Syngnathus typhle and the Ocean Sunfish Mola mola, to 429 in the Zig-zag Eel Mastacembelus armatus. The number of OR genes was higher in species when a multilamellar olfactory rosette was present. Moreover, the number of lamellae was correlated with the richness of the OR gene repertoire. While a slow and balanced birth-and-death process generally drives the evolution of the OR gene repertoire, we inferred several episodes of high rates of gene loss, sometimes followed by large gains in the number of OR genes. These gains coincide with morphological changes of the olfactory organ and suggest a strong functional association between changes in the morphology and the evolution of the OR gene repertoire., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and 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.)

Evolution sometimes proceeds by loss, especially when structures and genes become dispensable after an environmental shift relaxes functional constraints. Subterranean vertebrates are outstanding models to analyze this process, and gene decay can serve as a readout. We sought to understand some general principles on the extent and tempo of the decay of genes involved in vision, circadian clock, and pigmentation in cavefishes. The analysis of the genomes of two Cuban species belonging to the genus Lucifuga provided evidence for the largest loss of eye-specific genes and nonvisual opsin genes reported so far in cavefishes. Comparisons with a recently evolved cave population of Astyanax mexicanus and three species belonging to the Chinese tetraploid genus Sinocyclocheilus revealed the combined effects of the level of eye regression, time, and genome ploidy on eye-specific gene pseudogenization. The limited extent of gene decay in all these cavefishes and the very small number of loss-of-function mutations per pseudogene suggest that their eye degeneration may not be very ancient, ranging from early to late Pleistocene. This is in sharp contrast with the identification of several vision genes carrying many loss-of-function mutations in ancient fossorial mammals, further suggesting that blind fishes cannot thrive more than a few million years in cave ecosystems., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Animals evolve their sensory systems and foraging behaviours to adapt and colonize new and challenging habitats such as the dark cave environment. Vibration attraction behaviour (VAB) gives fish the ability to locate the source of a water disturbance in the darkness. VAB evolved in the blind Mexican cave tetra, Astyanax mexicanus. VAB is triggered in cavefish by vibration stimuli peaking at 35 Hz, which is within the main spectrum of water fluctuations produced by many prey crustaceans and insects. VAB has a genetic component and is correlated to an increased number of head mechanosensory neuromasts in the eye orbital region when compared to surface fish. Previous competitive prey capture assays have supported the advantage of VAB for foraging in the dark. Despite its putative adaptive function, VAB has been described as absent in some Astyanax cave populations (Tinaja and Molino) but present in others (Pachón, Piedras, Toro and Sabinos). Here we have tested the occurrence of VAB in the field and in multiple cave populations using a vibrating device in natural pools. Our results confirmed the presence of VAB in caves such as Pachón, Toro and Sabinos but showed that VAB is also present in the Tinaja and Molino cave populations, previously reported as VAB-negative in laboratory experiments. Thus, VAB is available throughout the range of hypogean A. mexicanus. However, and most notably, within a given cave the levels of VAB were highly variable among different pools. Fish at one pool may express no VAB, while fish at another nearby pool of the same cave may actively show VAB. While a variety of environmental conditions may foster this diversity, we found that individuals inhabiting pools with a high abundance of organic matter have reduced expression of VAB. In contrast, in pools with little organic debris where fish probably depend more on hunting than on scavenging, VAB is enhanced. Our results suggest that expression of VAB is a plastic trait whose variability can depend on local conditions. Such plasticity may be required within and among caves where high environmental variability between pools results in a diverse availability of food., (© 2020 Fisheries Society of the British Isles.)

The neurotransmitter serotonin controls a variety of physiological and behavioral processes. In humans, mutations affecting monoamine oxidase (MAO), the serotonin-degrading enzyme, are highly deleterious. Yet, blind cavefish of the species Astyanax mexicanus carry a partial loss-of-function mutation in MAO (P106L) and thrive in their subterranean environment. Here, we established four 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 of mao P106L in the evolution of cavefish neurobehavioral traits. Unexpectedly, although mao P106L appeared to be 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 from mao P106L, and rather due to other, morph-dependent developmental processes. However, we found that mao P106L 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 P106L mao allele in wild populations of cave and river A. 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-function mao allele evolves under a selective or a neutral regime in the particular cave environment is discussed., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Astyanax mexicanus has gained importance as a laboratory model organism for evolutionary biology. However, little is known about its intermediary metabolism, and feeding regimes remain variable between laboratories holding this species. We thus aimed to evaluate the intermediary metabolism response to nutritional status and to low (NC) or high (HC) carbohydrate diets in various organs of the surface-dwelling form of the species. As expected, glycaemia increased after feeding. Fish fed the HC diet had higher glycaemia than fish fed the NC diet, but without displaying hyperglycaemia, suggesting that carbohydrates are efficiently used as an energy source. At molecular level, only fasn ( Fatty Acid Synthase ) transcripts increased in tissues after refeeding, suggesting an activation of lipogenesis. On the other hand, we monitored only moderate changes in glucose-related transcripts. Most changes observed were related to the nutritional status, but not to the NC versus HC diet. Such a metabolic pattern is suggestive of an omnivorous-related metabolism, and this species, at least at adult stage, may adapt to a fish meal-substituted diet with high carbohydrate content and low protein supply. Investigation to identify molecular actors explaining the efficient use of such a diet should be pursued to deepen our knowledge on this species., Competing Interests: The authors declare that there are no conflicts/competing of interests., (© 2020 The Authors.)

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., (© 2021 S. Karger AG, Basel.)

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.)

Acoustic communication allows the exchange of information within specific contexts and during specific behaviors. The blind, cave-adapted and the sighted, river-dwelling morphs of the species Astyanax mexicanus have evolved in markedly different environments. During their evolution in darkness, cavefish underwent a series of morphological, physiological and behavioral changes, allowing the study of adaptation to drastic environmental change. Here we discover that Astyanax is a sonic species, in the laboratory and in the wild, with sound production depending on the social contexts and the type of morph. We characterize one sound, the "Sharp Click", as a visually-triggered sound produced by dominant surface fish during agonistic behaviors and as a chemosensory-, food odor-triggered sound produced by cavefish during foraging. Sharp Clicks also elicit different reactions in the two morphs in play-back experiments. Our results demonstrate that acoustic communication does exist and has evolved in cavefish, accompanying the evolution of its behaviors.

Fish reproductive patterns are very diverse in terms of breeding frequency, mating system, sexual dimorphisms and selection, mate choice, spawning site choice, courtship patterns, spawning behaviors and parental care. Here we have compared the breeding behavior of the surface-dwelling and cave-dwelling morphs of the characiform A. mexicanus, with the goals of documenting the spawning behavior in this emerging model organism, its possible evolution after cave colonization, and the sensory modalities involved. Using infrared video recordings, we showed that cave and surface Astyanax spawning behavior is identical, occurs in the dark, and can be divided into 5 rapid phases repeated many times, about once per minute, during spawning sessions which last about one hour and involve one female and several males. Such features may constitute "pre-adaptive traits" which have facilitated fish survival after cave colonization, and may also explain how the two morphs can hybridize in the wild and in the laboratory. Accordingly, cross-breeding experiments involving females of one morphotype and males of the other morphotype showed the same behavior including the same five phases. However, breeding between cavefish females and surface fish males was more frequent than the reverse. Finally, cavefish female pheromonal solution was able to trigger strong behavioral responses in cavefish males-but not on surface fish males. Lastly, egg production seemed higher in surface fish females than in cavefish females. These results are discussed with regards to the sensory modalities involved in triggering reproductive behavior in the two morphs, as well as its possible ongoing evolution., Competing Interests: The authors have declared that no competing interests exist.

Symbiotic relationships between host and microbiome can play a major role in local adaptation. Previous studies with freshwater organisms have shown that microbiome performs numerous important biochemical functions for the host, playing a key role in metabolism, physiology or health. Experimental studies in fish groups have found an effect of enzymatic activity of gut microbiota on a variety of metabolic processes. The goal of this study was to compare stomach microbiome from cave and surface Astyanax mexicanus, in order to evaluate the potential response of microbiota to contrasting environmental conditions and physiological adaptations of the host. Stomach microbiota was obtained from three different populations: Pachón cave, and two surface rivers (Rascón and Micos rivers). The stomach microbiome was analyzed using the Ion 16S Metagenomic kit considering seven variable regions: V2, V3, V4, V6-7, V8 and V9. A high diversity was observed across samples, including 16 phyla, 120 families and 178 genera. Gammaproteobacteria, Firmicutes, Bacteroidetes and Betaproteobacteria were the most abundant phyla across the samples. Although the relative abundance of the core OTUs at genus level were highly contrasting among populations, we did not recover differences in stomach microbiome between contrasting habitats (cave vs. surface rivers). Rather, we observed a consistent association between β-diversity and dissolved oxygen concentration in water. Therefore, and unexpectedly, the microbiota of A. mexicanus is not linked with the contrasting conditions of the habitat considered here but is related to water parameters., Competing Interests: The authors declare there are no competing interests.

The fish Astyanax mexicanus comes in two forms: the normal surface-dwelling (SF) and the blind depigmented cave-adapted (CF) morphs. Among many phenotypic differences, cavefish show enhanced olfactory sensitivity to detect amino-acid odors and they possess large olfactory sensory organs. Here, we questioned the relationship between the size of the olfactory organ and olfactory capacities. Comparing olfactory detection abilities of CF, SF and F1 hybrids with various olfactory epithelium (OE) sizes in behavioral tests, we concluded that OE size is not the only factor involved. Other possibilities were envisaged. First, olfactory behavior was tested in SF raised in the dark or after embryonic lens ablation, which leads to eye degeneration and mimics the CF condition. Both absence of visual function and absence of visual organs improved the SF olfactory detection capacities, without affecting the size of their OE. This suggested that developmental plasticity occurs between the visual and the olfactory modalities, and can be recruited in SF after visual deprivation. Second, the development of the olfactory epithelium was compared in SF and CF in their first month of life. Proliferation, cell death, neuronal lifespan, and olfactory progenitor cell cycling properties were identical in the two morphs. By contrast, the proportions of the three main olfactory sensory neurons subtypes (ciliated, microvillous and crypt) in their OE differed. OMP-positive ciliated neurons were more represented in SF, TRPC2-positive microvillous neurons were proportionately more abundant in CF, and S100-positive crypt cells were found in equal densities in the two morphs. Thus, general proliferative properties of olfactory progenitors are identical but neurogenic properties differ and lead to variations in the neuronal composition of the OE in SF and CF. Together, these experiments suggest that there are at least two components in the evolution of cavefish olfactory skills: (1) one part of eye-dependent developmental phenotypic plasticity, which does not depend on the size of the olfactory organ, and (2) one part of developmental evolution of the OE, which may stem from embryonic specification of olfactory neurons progenitor pools., (Copyright © 2018 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: Cavefish populations belonging to the Mexican tetra species Astyanax mexicanus are outstanding models to study the tempo and mode of adaptation to a radical environmental change. They are currently assigned to two main groups, the so-called "old" and "new" lineages, which would have populated several caves independently and at different times. However, we do not have yet accurate estimations of the time frames of evolution of these populations., Results: We reanalyzed the geographic distribution of mitochondrial and nuclear DNA polymorphisms and we found that these data do not support the existence of two cavefish lineages. Using IMa2, a program that allows dating population divergence in addition to demographic parameters, we found that microsatellite polymorphism strongly supports a very recent origin of cave populations (< 20,000 years). We identified a large number of single-nucleotide polymorphisms (SNPs) in transcript sequences of pools of embryos (Pool-seq) belonging to Pachón cave population and a surface population from Texas. Based on summary statistics that can be computed with this SNP data set together with simulations of evolution of SNP polymorphisms in two recently isolated populations, we looked for sets of demographic parameters that allow the computation of summary statistics with simulated populations that are similar to the ones with the sampled populations. In most simulations for which we could find a good fit between the summary statistics of observed and simulated data, the best fit occurred when the divergence between simulated populations was less than 30,000 years., Conclusions: Although it is often assumed that some cave populations have a very ancient origin, a recent origin of these populations is strongly supported by our analyses of independent sets of nuclear DNA polymorphism. Moreover, the observation of two divergent haplogroups of mitochondrial and nuclear genes with different geographic distributions support a recent admixture of two divergent surface populations, before the isolation of cave populations. If cave populations are indeed only several thousand years old, many phenotypic changes observed in cavefish would thus have mainly involved the fixation of genetic variants present in surface fish populations and within a very short period of time.

Background: Life in the darkness of caves is accompanied, throughout phyla, by striking phenotypic changes including the loss or severe reduction in eyes and pigmentation. On the other hand, cave animals have undergone constructive changes, thought to be adaptive, to survive in this extreme environment. The present study addresses the question of the evolution of growth in caves, taking advantage of the comparison between the river-dwelling and the cave-dwelling morphs of the Mexican tetra, Astyanax mexicanus ., Results: A sclerochronology approach was undertaken to document the growth of the species in these two very distinct habitats. Scales from 158 wild Astyanax mexicanus specimens were analyzed from three caves (Pachón, Tinaja and Subterráneo) and two rivers (Rio Gallinas and Arroyo Lagarto) in San Luis Potosi and Tamaulipas, Mexico. A 10-13% reduction in scales size was observed in the cave morphs compared to the surface morphs. Age could be reliably inferred from annual growth increments on the scales from the two morphs of the species. Further comparisons with growth curves in laboratory conditions, obtained using the von Bertalanffy growth model, were also performed. In the wild and in the laboratory, cavefish originating from the Pachón cave reached smaller sizes than surface fish from three different locations: Rio Gallinas and Arroyo Lagarto (wild sampling) and Texas (laboratory population), respectively. Wild Pachón cavefish also seemed to grow to smaller sizes than the two other wild cavefish populations studied, Tinaja and Subterráneo. Finally, growth in the laboratory was faster than in the wild, particularly in the two first years of life., Conclusions: These data suggest that cavefish originating from the Pachón cave are subjected to an intrinsic limitation of their final size, which is at least in part independent from energy/food availability. This growth limitation may be an advantageous way of limiting energy expenditure and food needs in the cave environment. Moreover, growth regulation evolved differently in independently evolved cave populations. These results are discussed with regard to the sources of energy or general ecological conditions present in caves, and to the differences in behavior or feeding skills known in cavefish.

Blindness is a convergent trait in many cave animals of various phyla. Astyanax mexicanus cavefish is one of the best studied cave animals; however the mechanisms underlying eye degeneration in this species are not yet completely understood. The lens seems to play a central role, but only relatively late differentiation defects have been implicated in the cavefish lens apoptosis phenotype so far. Here, we used genetic crosses between Astyanax cavefish and surface fish to confirm that during development, lens size is independent of retina size. We then investigated whether the small size of the cavefish lens could directly cause cell death. Laser ablation experiments of lens placode cells in surface fish embryos showed that a small lens size is not sufficient to trigger lens apoptosis. We further examined potential lens morphogenesis defects through classical histology and live-imaging microscopy. From lens placode to lens ball, we found that lens invagination and formation of the lens epithelium and fiber cells occur normally in cavefish. We conclude that the main and deleterious defect in the Astyanax cavefish lens must concern the molecular control of lens cell function.

Natural variations in sensory systems constitute adaptive responses to the environment. Here, we compared sensory placode development in the blind cave-adapted morph and the eyed river-dwelling morph of Astyanax mexicanus Focusing on the lens and olfactory placodes, we found a trade-off between these two sensory components in the two morphs: from neural plate stage onwards, cavefish have larger olfactory placodes and smaller lens placodes. In a search for developmental mechanisms underlying cavefish sensory evolution, we analyzed the roles of Shh, Fgf8 and Bmp4 signaling, which are known to be fundamental in patterning the vertebrate head and are subtly modulated in space and time during cavefish embryogenesis. Modulating these signaling systems at the end of gastrulation shifted the balance toward a larger olfactory derivative. Olfactory tests to assess potential behavioral outcomes of such developmental evolution revealed that Astyanax cavefish are able to respond to a 10 5 -fold lower concentration of amino acids than their surface-dwelling counterparts. We suggest that similar evolutionary developmental mechanisms may be used throughout vertebrates to drive adaptive sensory specializations according to lifestyle and habitat., (© 2016. Published by The Company of Biologists Ltd.)

Considerable progress has been made in the understanding of molecular and cellular mechanisms controlling the development of the mammalian cortex. The proliferative and neurogenic properties of cortical progenitors located in the ventricular germinal zone start being understood. Little is known however on the cis-regulatory control that finely tunes gene expression in these progenitors. Here, we undertook an in silico-based approach to address this question, followed by some functional validation. Using the Eurexpress database, we established a list of 30 genes specifically expressed in the cortical germinal zone, we selected mouse/human conserved non-coding elements (CNEs) around these genes and we performed motif-enrichment search in these CNEs. We found an over-representation of motifs corresponding to binding sites for Pax, Sox, and Lhx transcription factors, often found as pairs and located within 100bp windows. A small subset of CNEs (n=7) was tested for enhancer activity, by ex-vivo and in utero electroporation assays. Two showed strong enhancer activity in the germinal zone progenitors. Mutagenesis experiments on a selected CNE showed the functional importance of the Pax, Sox, and Lhx TFBS for conferring enhancer activity to the CNE. Overall, from a cis-regulatory viewpoint, our data suggest an input from Pax, Sox and Lhx transcription factors to orchestrate corticogenesis. These results are discussed with regards to the known functional roles of Pax6, Sox2 and Lhx2 in cortical development., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The fish Astyanax mexicanus presents, within the same species, populations of river-dwelling surface fish (SF) and blind cave-living fish. In cavefish (CF), the eyes develop almost normally during embryogenesis. But 40 h after fertilization, the lens enters apoptosis, triggering the progressive degeneration of the entire eye. Before apoptosis, the CF lens expresses early differentiation factors correctly. Here, we searched for possible late differentiation defects that would be causal in CF lens degeneration. We reasoned that crystallins, the major lens structural proteins, could be defective or misregulated. We surveyed the CF and SF transcriptomes and uncovered 14 Astyanax crystallins from the beta, gamma, lambda, mu, and zeta families. These proteins are less polymorphic and accumulate more fixed mutations, some at highly conserved positions, in CF than in SF, suggesting relaxed selection at these loci in CF. In situ hybridizations and qPCR show that crybb1c, crybgx, crygm5 are expressed at much lower levels or are not expressed in the CF lens. For the best crystallin candidates, we tested a potential causal role in CF lens apoptosis. Crybgx, crybb1c (not expressed in CF from very early on), and cryaa (previously shown to be faintly expressed in CF) failed to induce any defect when knocked-down in zebrafish embryos. However, the anti-apoptotic cryaa protected lens cells from apoptosis when reexpressed by transgenesis in CF, suggesting a cell-autonomous effect of cryaa on lens cell survival. Altogether, these data suggest that crystallin sequence evolution and expression defects may contribute to the loss of eyes in CF., (© 2014 Wiley Periodicals, Inc.)

We aimed to identify cis-regulatory elements that control gene expression in progenitors of the cerebral cortex. A list of 975 putative enhancers were retrieved from a ChIP-Seq experiment performed in NS5 mouse stem cells with antibodies to Sox2, Brn2/Pou3f2, or Brn1/Pou3f3. Through a selection pipeline including gene ontology and expression pattern, we reduced the number of candidate enhancer sequences to 20. Ex vivo electroporation of green fluorescent pProtein (GFP) reporter constructs in the telencephalon of mouse embryos showed that 35% of the 20 selected candidate sequences displayed enhancer activity in the developing cortex at E13.5. In silico transcription factor binding site (TFBS) searches and mutagenesis experiments showed that enhancer activity is related to the presence of Sox/Pou TFBS pairs in the sequence. Comparative genomic analyses showed that enhancer activity is not related to the evolutionary conservation of the sequence. Finally, the combination of in utero electroporation of GFP reporter constructs with immunostaining for Tbr2 (basal progenitor marker) and phospho-histoneH3 (mitotic activity marker) demonstrated that each enhancer is specifically active in precise subpopulations of progenitors in the cortical germinal zone, highlighting the heterogeneity of these progenitors in terms of cis-regulation., (© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Natural populations subjected to strong environmental selection pressures offer a window into the genetic underpinnings of evolutionary change. Cavefish populations, Astyanax mexicanus (Teleostei: Characiphysi), exhibit repeated, independent evolution for a variety of traits including eye degeneration, pigment loss, increased size and number of taste buds and mechanosensory organs, and shifts in many behavioural traits. Surface and cave forms are interfertile making this system amenable to genetic interrogation; however, lack of a reference genome has hampered efforts to identify genes responsible for changes in cave forms of A. mexicanus. Here we present the first de novo genome assembly for Astyanax mexicanus cavefish, contrast repeat elements to other teleost genomes, identify candidate genes underlying quantitative trait loci (QTL), and assay these candidate genes for potential functional and expression differences. We expect the cavefish genome to advance understanding of the evolutionary process, as well as, analogous human disease including retinal dysfunction.

Background: Enhanced food-finding efficiency is an obvious adaptive response to cave environments. Here, we have compared the food-finding abilities of Astyanax surface fish and blind cavefish young larvae in their first month of life, in the dark., Results: Our results show that enhanced prey capture skills of cavefish are already in effect in fry soon after the yolk is depleted and the young larvae must find food for themselves. Moreover, using prey capture competition assays on surface fish fry with lensectomies, we showed that eye-dependent developmental processes are not the main determinant for enhanced prey capture skills. Finally, using F2 hybrid larvae resulting from crosses between surface fish and cavefish, we found that reduced eyes do not confer a selective advantage for prey capture by fry in the dark., Conclusion: We discuss these data with regards to our current developmental and genetic understanding of cavefish morphological and behavioral evolution.

Astyanax mexicanus, a teleost fish comprising both sighted river-dwelling and blind cave-dwelling morphs, is becoming increasingly used in the field of developmental and evolutionary biology. Thus, new experimental and technological tools are needed on this emerging fish model by the expanding scientific community. Here, we describe Astyanax husbandry and egg spawning habits, a prerequisite to the successful establishment of Astyanax transgenic lines. We then compare two different transgenesis methods on both surface and cave Astyanax. Both meganuclease (I-SceI)- and transposase (Tol2)-mediated transgenesis are equivalently efficient, resulting in ∼40% mosaic transgenic fish in F0. Furthermore, the transmission rate was analyzed in F1 in the case of the I-SceI method and was found to be 16%. Finally, the transgene was found stable up the F3 generation, demonstrating the feasibility of generating stable transgenic lines in Astyanax and opening a wide range of possibilities for this fish model.