The Mexican Tetra, Astyanax mexicanus, as a Model System in Cell and Developmental Biology.

Our understanding of cell and developmental biology has been greatly aided by a focus on a small number of model organisms. However, we are now in an era where techniques to investigate gene function can be applied across phyla, allowing scientists to explore the diversity and flexibility of developmental mechanisms and gain a deeper understanding of life. Researchers comparing the eyeless cave-adapted Mexican tetra, Astyanax mexicanus, with its river-dwelling counterpart are revealing how the development of the eyes, pigment, brain, cranium, blood, and digestive system evolves as animals adapt to new environments. Breakthroughs in our understanding of the genetic and developmental basis of regressive and constructive trait evolution have come from A. mexicanus research. They include understanding the types of mutations that alter traits, which cellular and developmental processes they affect, and how they lead to pleiotropy. We review recent progress in the field and highlight areas for future investigations that include evolution of sex differentiation, neural crest development, and metabolic regulation of embryogenesis.

Natural reversal of cavefish heart asymmetry is controlled by Sonic Hedgehog effects on the left-right organizer.

The direction of left-right visceral asymmetry is conserved in vertebrates. Deviations of the standard asymmetric pattern are rare, and the underlying mechanisms are not understood. Here, we use the teleost Astyanax mexicanus, consisting of surface fish with normal left-oriented heart asymmetry and cavefish with high levels of reversed right-oriented heart asymmetry, to explore natural changes in asymmetry determination. We show that Sonic Hedgehog (Shh) signaling is increased at the posterior midline, Kupffer's vesicle (the teleost left-right organizer) is enlarged and contains longer cilia, and the number of dorsal forerunner cells is increased in cavefish. Furthermore, Shh increase in surface fish embryos induces asymmetric changes resembling the cavefish phenotype. Asymmetric expression of the Nodal antagonist Dand5 is equalized or reversed in cavefish, and Shh increase in surface fish mimics changes in cavefish dand5 asymmetry. Shh decrease reduces the level of right-oriented heart asymmetry in cavefish. Thus, naturally occurring modifications in cavefish heart asymmetry are controlled by the effects of Shh signaling on left-right organizer function.

Next-generation plasmids for transgenesis in zebrafish and beyond.

Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible modular system. Here, we establish several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse beta-globin minimal promoter coupled to several fluorophores, CreERT2 and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein mCerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3' vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Finally, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker that is active before hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish and other models.

Optic cup morphogenesis across species and related inborn human eye defects.

The vertebrate eye is shaped as a cup, a conformation that optimizes vision and is acquired early in development through a process known as optic cup morphogenesis. Imaging living, transparent teleost embryos and mammalian stem cell-derived organoids has provided insights into the rearrangements that eye progenitors undergo to adopt such a shape. Molecular and pharmacological interference with these rearrangements has further identified the underlying molecular machineries and the physical forces involved in this morphogenetic process. In this Review, we summarize the resulting scenarios and proposed models that include common and species-specific events. We further discuss how these studies and those in environmentally adapted blind species may shed light on human inborn eye malformations that result from failures in optic cup morphogenesis, including microphthalmia, anophthalmia and coloboma.

Hypoxia-sonic hedgehog axis as a driver of primitive hematopoiesis development and evolution in cavefish.

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

Advancing human disease research with fish evolutionary mutant models.

Model organism research is essential to understand disease mechanisms. However, laboratory-induced genetic models can lack genetic variation and often fail to mimic the spectrum of disease severity. Evolutionary mutant models (EMMs) are species with evolved phenotypes that mimic human disease. EMMs complement traditional laboratory models by providing unique avenues to study gene-by-environment interactions, modular mutations in noncoding regions, and their evolved compensations. EMMs have improved our understanding of complex diseases, including cancer, diabetes, and aging, and illuminated mechanisms in many organs. Rapid advancements of sequencing and genome-editing technologies have catapulted the utility of EMMs, particularly in fish. Fish are the most diverse group of vertebrates, exhibiting a kaleidoscope of specialized phenotypes, many that would be pathogenic in humans but are adaptive in the species' specialized habitat. Importantly, evolved compensations can suggest avenues for novel disease therapies. This review summarizes current research using fish EMMs to advance our understanding of human disease.

3D spheroid culturing of Astyanax mexicanus liver-derived cell lines recapitulates distinct transcriptomic and metabolic states of in vivo tissue environment.

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

A New Species of the Blind Cave Loach Genus Protocobitis (Cypriniformes: Cobitidae), Protocobitis longicostatus sp. nov., from Guangxi, China.

Protocobitis species are typical cave-dwelling fish, exhibiting distinctive morphological adaptations such as colorless body, lack of eyes, and reduced scales and ribs in response to their extreme cave habitats. Distinct from the recorded species, P. anteroventris, P. polylepis, and P. typhlops, a new species, Protocobitis longicostatus sp. nov., is described from Guangxi Zhuang Autonomous Region, China. Protocobitis longicostatus sp. nov. can easily be distinguished from all known congeners by the following characteristics: whole body covered by scales except head, 12 branched caudal fin rays, and long ribs. These species face threats from habitat degradation, hydrological changes, and environmental pollution. Thus, the conservation of cavefish in China has become an urgent issue.

Metabolic shift toward ketosis in asocial cavefish increases social-like affinity.

BACKGROUND: Social affinity and collective behavior are nearly ubiquitous in the animal kingdom, but many lineages feature evolutionarily asocial species. These solitary species may have evolved to conserve energy in food-sparse environments. However, the mechanism by which metabolic shifts regulate social affinity is not well investigated. RESULTS: In this study, we used the Mexican tetra (Astyanax mexicanus), which features riverine sighted surface (surface fish) and cave-dwelling populations (cavefish), to address the impact of metabolic shifts on asociality and other cave-associated behaviors in cavefish, including repetitive turning, sleeplessness, swimming longer distances, and enhanced foraging behavior. After 1 month of ketosis-inducing ketogenic diet feeding, asocial cavefish exhibited significantly higher social affinity, whereas social affinity regressed in cavefish fed the standard diet. The ketogenic diet also reduced repetitive turning and swimming in cavefish. No major behavioral shifts were found regarding sleeplessness and foraging behavior, suggesting that other evolved behaviors are not largely regulated by ketosis. We further examined the effects of the ketogenic diet via supplementation with exogenous ketone bodies, revealing that ketone bodies are pivotal molecules positively associated with social affinity. CONCLUSIONS: Our study indicated that fish that evolved to be asocial remain capable of exhibiting social affinity under ketosis, possibly linking the seasonal food availability and sociality.

Quantitative Lipidomics and Spatial MS-Imaging Uncovered Neurological and Systemic Lipid Metabolic Pathways Underlying Troglomorphic Adaptations in Cave-Dwelling Fish.

Sinocyclocheilus represents a rare, freshwater teleost genus endemic to China that comprises the river-dwelling surface fish and the cave-dwelling cavefish. Using a combinatorial approach of quantitative lipidomics and mass-spectrometry imaging (MSI), we demonstrated that neural compartmentalization of lipid distribution and lipid metabolism is associated with the evolution of troglomorphic traits in Sinocyclocheilus. Attenuated docosahexaenoic acid (DHA) biosynthesis via the Δ4 desaturase pathway led to reductions in DHA-phospholipids in cavefish cerebellum. Instead, cavefish accumulates arachidonic acid-phospholipids that may disfavor retinotectal arbor growth. Importantly, MSI of sulfatides coupled with immunostaining of myelin basic protein and transmission electron microscopy images of hindbrain axons revealed demyelination in cavefish raphe serotonergic neurons. Demyelination in cavefish parallels the loss of neuroplasticity governing social behavior such as aggressive dominance. Outside the brain, quantitative lipidomics and qRT-PCR revealed systemic reductions in membrane esterified DHAs in the liver, attributed to suppression of genes along the Sprecher pathway (elovl2, elovl5, and acox1). Development of fatty livers was observed in cavefish; likely mediated by an impeded mobilization of storage lipids, as evident in the diminished expressions of pnpla2, lipea, lipeb, dagla, and mgll; and suppressed ß-oxidation of fatty acyls via both mitochondria and peroxisomes as reflected in the reduced expressions of cpt1ab, hadhaa, cpt2, decr1, and acox1. These neurological and systemic metabolic adaptations serve to reduce energy expenditure, forming the basis of recessive evolution that eliminates nonessential morphological and behavioral traits and giving cavefish a selective advantage to thrive in caves where proper resource allocation becomes a major determinant of survival.

Blind fish have cells that see light.

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

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

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

Evolution of left-right asymmetry in the sensory system and foraging behavior during adaptation to food-sparse cave environments.

BACKGROUND: Laterality in relation to behavior and sensory systems is found commonly in a variety of animal taxa. Despite the advantages conferred by laterality (e.g., the startle response and complex motor activities), little is known about the evolution of laterality and its plasticity in response to ecological demands. In the present study, a comparative study model, the Mexican tetra (Astyanax mexicanus), composed of two morphotypes, i.e., riverine surface fish and cave-dwelling cavefish, was used to address the relationship between environment and laterality. RESULTS: The use of a machine learning-based fish posture detection system and sensory ablation revealed that the left cranial lateral line significantly supports one type of foraging behavior, i.e., vibration attraction behavior, in one cave population. Additionally, left-right asymmetric approaches toward a vibrating rod became symmetrical after fasting in one cave population but not in the other populations. CONCLUSION: Based on these findings, we propose a model explaining how the observed sensory laterality and behavioral shift could help adaptation in terms of the tradeoff in energy gain and loss during foraging according to differences in food availability among caves.

Characterizing the genetic basis of trait evolution in the Mexican cavefish.

Evolution in response to a change in ecology often coincides with various morphological, physiological, and behavioral traits. For most organisms little is known about the genetic and functional relationship between evolutionarily derived traits, representing a critical gap in our understanding of adaptation. The Mexican tetra, Astyanax mexicanus, consists of largely independent populations of fish that inhabit at least 30 caves in Northeast Mexico, and a surface fish population, that inhabit the rivers of Mexico and Southern Texas. The recent application of molecular genetic approaches combined with behavioral phenotyping have established A. mexicanus as a model for studying the evolution of complex traits. Cave populations of A. mexicanus are interfertile with surface populations and have evolved numerous traits including eye degeneration, insomnia, albinism, and enhanced mechanosensory function. The interfertility of different populations from the same species provides a unique opportunity to define the genetic relationship between evolved traits and assess the co-evolution of behavioral and morphological traits with one another. To define the relationships between morphological and behavioral traits, we developed a pipeline to test individual fish for multiple traits. This pipeline confirmed differences in locomotor activity, prey capture, and startle reflex between surface and cavefish populations. To measure the relationship between traits, individual F2 hybrid fish were characterized for locomotor behavior, prey-capture behavior, startle reflex, and morphological attributes. Analysis revealed an association between body length and slower escape reflex, suggesting a trade-off between increased size and predator avoidance in cavefish. Overall, there were few associations between individual behavioral traits, or behavioral and morphological traits, suggesting independent genetic changes underlie the evolution of the measured behavioral and morphological traits. Taken together, this approach provides a novel system to identify genetic underpinnings of naturally occurring variation in morphological and behavioral traits.

Novel genome sequence of Chinese cavefish (Triplophysa rosa) reveals pervasive relaxation of natural selection in cavefish genomes.

All cavefishes, living exclusively in caves across the globe, exhibit similar phenotypic traits, including the characteristic loss of eyes. To understand whether such phenotypic convergence shares similar genomic bases, here we investigated genome-wide evolutionary signatures of cavefish phenotypes by comparing whole-genome sequences of three pairs of cavefishes and their surface fish relatives. Notably, we newly sequenced and generated a whole-genome assembly of the Chinese cavefish Triplophysa rosa. Our comparative analyses revealed several shared features of cavefish genome evolution. Cavefishes had lower mutation rates than their surface fish relatives. In contrast, the ratio of nonsynonymous to synonymous substitutions (ω) was significantly elevated in cavefishes compared to in surface fishes, consistent with the relaxation of purifying selection. In addition, cavefish genomes had an increased mutational load, including mutations that alter protein hydrophobicity profiles, which were considered harmful. Interestingly, however, we found no overlap in positively selected genes among different cavefish lineages, indicating that the phenotypic convergence in cavefishes was not caused by positive selection of the same sets of genes. Analyses of previously identified candidate genes associated with cave phenotypes supported this conclusion. Genes belonging to the lipid metabolism functional ontology were under relaxed purifying selection in all cavefish genomes, which may be associated with the nutrient-poor habitat of cavefishes. Our work reveals previously uncharacterized patterns of cavefish genome evolution and provides comparative insights into the evolution of cave-associated phenotypic traits.

Evolving in the darkness: Phylogenomics of Sinocyclocheilus cavefishes highlights recent diversification and cryptic diversity.

Troglomorphism-any morphological adaptation enabling life to the constant darkness of caves, such as loss of pigment, reduced eyesight or blindness, over-developed tactile and olfactory organs-has long intrigued biologists. However, inferring the proximate and ultimate mechanisms driving the evolution of troglomorphism (stygomorphism) in freshwater fish requires a sound understanding of the evolutionary relationships between surface and stygomorphic lineages. We use Restriction Site Associated DNA Sequencing (RADseq) to better understand the evolution of the Sinocyclocheilus fishes of China. With a remarkable array of derived stygomorphic traits, they comprise the largest cavefish diversification in the world, emerging as a multi-species model system to study evolutionary novelty. We sequenced a total of 120 individuals throughout the Sinocyclocheilus distribution. The data comprised a total of 646,497 bp per individual, including 4378 loci and 67,983 SNPs shared across a minimum of 114 individuals at a given locus. Phylogenetic analyses using either the concatenated RAD loci (RAxML) or the SNPs under a coalescent model (SVDquartets, SNAPP) showed a high degree of congruence with similar topologies and high node support (>95 for most nodes in the phylogeny). The major clades recovered conform to a pattern previously established using Sanger-based mt-DNA sequences, with a few notable exceptions. We now recognize six major clades in this group, elevating the blind cavefish S. tianlinensis and the micro-eyed S. microphthalmus as two new distinct clades due to their deep divergence from other clades. PCA plots of the SNP data also support the recognition of six major clusters of species congruent with the identified clades in ordination space. A Bayes factor delimitation (BFD) analysis showed support for 21 species, recognizing 19 previously described species and two putative new cryptic ones. Two species whose identities were previously disputed, S. furcodorsalis and S. tianeensis, are supported here as distinct species. In addition, our multi-species calibrated tree in SNAPP suggests that the genus Sinocyclocheilus originated around 10.16 Mya, with most speciation events occurring in the last 2 Mya, likely favored by the uplift of the Qinghai-Tibetan Plateau and cave occupation induced by climate-driven aridification during this period. These results provide a firm basis for future comparative studies on the evolution of Sinocyclocheilus and its adaptations to cave life.

Quantification and comparison of teleost scleral cartilage development and growth.

The ocular skeleton is composed of the scleral cartilage and the scleral ossicles. Teleost scleral cartilage is composed of a single layer of chondrocytes embedded in the sclera of the eye. The teleost scleral cartilage ring can vary in depth across teleost families and species, from a narrow ring a few cells wide to a deeper ring that resembles a cup and surrounds the entire sclera. However, very little research has been conducted on the development and morphology of teleost scleral cartilage. Thus, this study aims to characterize the development of the scleral cartilage in the zebrafish and Mexican tetra, with respect to the timing of emergence, depth throughout development, and positioning within the eye. We hypothesized that the scleral cartilage would first emerge in the scleral tissue closely abutting the ora serrata, and that growth would proceed in an anterior-to-posterior direction, resulting in differences in scleral cartilage depth between different fish species. We found that the scleral cartilage ring does not develop uniformly along its circumference, and that its relationship to the ora serrata varies between the rostral and caudal regions. Furthermore, distinct differences in the growth trajectory of the scleral cartilage indicate that the deep scleral cartilage of the Pachón cavefish is the result of both decreased eye size and prolonged cartilage growth. A significant difference in the size of the scleral chondrocytes was also noted. Overall, this study provides the first characterization of early scleral cartilage development in teleost fish and indicates that some aspects of scleral cartilage development and morphology are highly conserved while others are not.

Morphological description of gametes in cave and surface populations of Astyanax mexicanus (De Filippi, 1853).

The Mexican tetra Astyanax mexicanus presents two contrasting morphs, a widely distributed surface morph and a cave-adapted morph. These cave-adapted morphs have evolved independently from two different lineages (i.e. 'old' and 'new' lineages); therefore, this model system gives a unique opportunity to explore parallel adaptive evolution in biological traits. The present study corresponds to the first morphological description of the Astyanax mexicanus maturation process of the spermatozoa and oocytes, using thermal and hormonal stimuli to promote spermatogenesis and oogenesis, considering surface and cave morphs from both lineages. We corroborate the relevance of thermal and hormonal stimuli to promote gamete maturation. The hormone Ovaprim (GnRHa + Domperidone) is an effective promoter of ovarian development, maturation end in oocytes and spawning in Astyanax mexicanus. The sperm morphology of Astyanax mexicanus includes the sperm head, the midpiece, and tail or flagellum. We found differences in the spermatozoan total length between environments (F = 9.929, P = 0.05) and linages (F = 49.86, P = 0.005). The oocytes showed a spherical conformation with a mean diameter of 822.4 ± 194.1 µm for the surface populations, and 604.6 ± 38.3 µm for the cave populations. The oocyte chorion presents ridges and grooves that are arranged radially towards the micropyle. A plug in the micropyle zone was observed after fertilization, confirmed by the outer membrane of the chorion, which provides some weak adhesiveness to the substrate. We observed differences in chorion thickness between the contrasting environmental conditions. This is the first morphological characterization of the Sótanos Vázquez, Escondido and Tigre, which previous to this study were only known from speleological expeditions, with no previous biological information available.

Testicular miRNAs and tsRNAs provide insight into gene regulation during overwintering and reproduction of Onychostoma macrolepis.

The late overwintering period and breeding period are two important developmental stages of testis in Onychostoma macrolepis. Small non-coding RNAs (sncRNAs) are well-known regulators of biological processes associated with numerous biological processes. This study aimed to elucidate the roles of four sncRNA classes (microRNAs [miRNAs], Piwi-interacting RNAs [piRNAs], tRNA-derived small RNAs [tsRNAs], and rRNA-derived small RNAs [rsRNAs]) across testes in the late overwintering period (in March) and breeding period (in June) by high-throughput sequencing. The testis of O. macrolepis displayed the highest levels of piRNAs and lowest levels of rsRNAs. Compared with miRNAs and tsRNAs in June, tsRNAs in March had a higher abundance, while miRNAs in March had a much lower abundance. Bioinformatics analysis identified 1,362 and 1,340 differentially expressed miRNAs and tsRNAs, respectively. Further analysis showed that miR-200-1, miR-143-1, tRFi-Lys-CTT-1, and tRFi-Glu-CTC-1 could play critical roles during the overwintering and breeding periods. Our findings provided an unprecedented insight to reveal the epigenetic mechanism underlying the overwintering and reproduction process of male O. macrolepis.

Subterranean life: Behavior, metabolic, and some other adaptations of Astyanax cavefish.

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