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.

From darkness to discovery: evolutionary, adaptive, and translational genetic insights from cavefish.

How genotype determines phenotype is a well-explored question, but genotype-environment interactions and their heritable impact on phenotype over the course of evolution are not as thoroughly investigated. The fish Astyanax mexicanus, consisting of surface and cave ecotypes, is an ideal emerging model to study the genetic basis of adaptation to new environments. This model has permitted quantitative trait locus mapping and whole-genome comparisons to identify the genetic bases of traits such as albinism and insulin resistance and has helped to better understand fundamental evolutionary mechanisms. In this review, we summarize recent advances in A. mexicanus genetics and discuss their broader impact on the fields of adaptation and evolutionary genetics.

Evolution of the regulation of developmental gene expression in blind Mexican cavefish.

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.

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.

Metabolic reprogramming underlies cavefish muscular endurance despite loss of muscle mass and contractility.

Physical inactivity is a scourge to human health, promoting metabolic disease and muscle wasting. Interestingly, multiple ecological niches have relaxed investment into physical activity, providing an evolutionary perspective into the effect of adaptive physical inactivity on tissue homeostasis. One such example, the Mexican cavefish Astyanax mexicanus, has lost moderate-to-vigorous activity following cave colonization, reaching basal swim speeds ~3.7-fold slower than their river-dwelling counterpart. This change in behavior is accompanied by a marked shift in body composition, decreasing total muscle mass and increasing fat mass. This shift persisted at the single muscle fiber level via increased lipid and sugar accumulation at the expense of myofibrillar volume. Transcriptomic analysis of laboratory-reared and wild-caught cavefish indicated that this shift is driven by increased expression of pparγ-the master regulator of adipogenesis-with a simultaneous decrease in fast myosin heavy chain expression. Ex vivo and in vivo analysis confirmed that these investment strategies come with a functional trade-off, decreasing cavefish muscle fiber shortening velocity, time to maximal force, and ultimately maximal swimming speed. Despite this, cavefish displayed a striking degree of muscular endurance, reaching maximal swim speeds ~3.5-fold faster than their basal swim speeds. Multi-omic analysis suggested metabolic reprogramming, specifically phosphorylation of Pgm1-Threonine 19, as a key component enhancing cavefish glycogen metabolism and sustained muscle contraction. Collectively, we reveal broad skeletal muscle changes following cave colonization, displaying an adaptive skeletal muscle phenotype reminiscent to mammalian disuse and high-fat models while simultaneously maintaining a unique capacity for sustained muscle contraction via enhanced glycogen metabolism.

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.

The prevalence of copy number increase at multiallelic copy number variants associated with cave colonization.

Copy number variation is a common contributor to phenotypic diversity, yet its involvement in ecological adaptation is not easily discerned. Instances of parallelly evolving populations of the same species in a similar environment marked by strong selective pressures present opportunities to study the role of copy number variants (CNVs) in adaptation. By identifying CNVs that repeatedly occur in multiple populations of the derived ecotype and are not (or are rarely) present in the populations of the ancestral ecotype, the association of such CNVs with adaptation to the novel environment can be inferred. We used this paradigm to identify CNVs associated with recurrent adaptation of the Mexican tetra (Astyanax mexicanus) to cave environment. Using a read-depth approach, we detected CNVs from previously re-sequenced genomes of 44 individuals belonging to two ancestral surfaces and three derived cave populations. We identified 102 genes and 292 genomic regions that repeatedly diverge in copy number between the two ecotypes and occupy 0.8% of the reference genome. Functional analysis revealed their association with processes previously recognized to be relevant for adaptation, such as vision, immunity, oxygen consumption, metabolism, and neural function and we propose that these variants have been selected for in the cave or surface waters. The majority of the ecotype-divergent CNVs are multiallelic and display copy number increases in cavefish compared to surface fish. Our findings suggest that multiallelic CNVs - including gene duplications - and divergence in copy number provide a fast route to produce novel phenotypes associated with adaptation to subterranean life.

Selecting reference genes for RT-qPCR studies involving the presence of B chromosomes in Psalidodon (Characiformes, Characidae).

BACKGROUND: B chromosomes are extra non-essential elements present in several eukaryotes. Unlike A chromosomes which are essential and present in all individuals of a species, B chromosomes are not necessary for normal functioning of an organism. Formerly regarded as genetically inactive, B chromosomes have been discovered to not only express their own genes, but also to exert influence on gene expression in A chromosomes. Recent studies have shown that, in some Psalidodon (Characiformes, Characidae) species, B chromosomes might be associated with phenotypic effects, such as changes in the reproductive cycle and gene expression. METHODS AND RESULTS: In this study, we aimed to establish stable reference genes for RT-qPCR experiments conducted on gonads of three fish species within Psalidodon genus, both in the presence and absence of B chromosomes. The stability of five selected reference genes was assessed using NormFinder, geNorm, BestKeeper, and RefFinder algorithms. We determined ppiaa and pgk1 as the most stable genes in P. fasciatus, whereas ppiaa and hmbsa showed the highest stability in P. bockmanni. For P. paranae, tbp and hprt1 were the most stable genes in females, and ppiaa and hprt1 were the most stable in males. CONCLUSIONS: We determined the most stable reference genes in gonads of three Psalidodon species considering the presence of B chromosomes. This is the first report of reference gene stability in the genus and provides valuable tools to better understand the effects of B chromosomes at gene expression level.

Repeated evolution of circadian clock dysregulation in cavefish populations.

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.

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.

All-triploid offspring in the yellowtail tetra Astyanax altiparanae Garutti & Britski 2000 (Teleostei, Characidae) derived from female tetraploid × male diploid crosses.

This study aimed to evaluate the ploidy and survival of larvae resulting from crosses between tetraploid females and diploid males of yellowtail tetra Astyanax altiparanae, both females (three diploids and three tetraploids) and males (n = 3 diploids). Breeders were subjected to hormonal induction with pituitary gland extract from common carp fish (Cyprinus carpio). Females received two doses at concentrations of 0.3 and 3.0 mg/kg -1 body weight and at intervals of 6 h. Males were induced with a single dose of 3.0 mg/kg -1 applied simultaneously with the second dose in females. Oocytes from each diploid and tetraploid female were fertilized with semen from the same male, resulting in two crosses: cross 1 (diploid male and diploid female) and cross 2 (diploid male and tetraploid female). The procedures were performed with separate females (diploid and tetraploid) and diploid males for each repetition (n = 3). For ploidy determination, 60 larvae from each treatment were analyzed using flow cytometry and cytogenetic analyses. As expected, flow cytometry analysis showed that progenies from crosses 1 and 2 presented diploid and triploid individuals, respectively, with a 100% success rate. The same results were confirmed in the cytogenetic analysis, in which the larvae resulting from cross 1 had 50 metaphase chromosomes and those from cross 2 had 75 chromosomes. The oocytes have a slightly ovoid shape at the time of extrusion. Diploid oocytes had a size of 559 ± 20.62 µm and tetraploid of 1025.33 ± 30.91 µm. Statistical differences were observed between eggs from crosses 1 and 2 (P = 0.0130). No significant differences between treatments were observed for survival at the 2-cell stage (P = 0.6174), blastula (P = 0.9717), gastrula (P = 0.5301), somite (P = 0.3811), and hatching (P = 0.0984) stages. In conclusion, our results showed that tetraploid females of the yellowtail tetra A. altiparanae are fertile, present viable gametes after stripping and fertilization using the 'dry method', and may be used for mass production of triploids. This is the first report of these procedures within neotropical characins, and which can be applied in other related species of economic importance.

Fish models for investigating nutritional regulation of embryonic development.

In recent decades, biologist have focused on the spatiotemporal regulation and function of genes to understand embryogenesis. It is clear that maternal diet impacts fetal development but how nutrients, like lipids and vitamins, modify developmental programs is not completely understood. Fish are useful research organisms for such investigations. Most species of fish produce eggs that develop outside the mother, dependent on a finite amount of yolk to form and grow. The developing embryo is a closed system that can be readily biochemically analyzed, easily visualized, and manipulated to understand the role of nutrients in tissue specification, organogenesis, and growth. Natural variation in yolk composition observed across fish species may be related to unique developmental strategies. In this review, we discuss the reasons that teleost fishes are powerful models to understand nutritional control of development and highlight three species that are particularly valuable for future investigations: the zebrafish, Danio rerio, the African Killifish, Nothobranchius furzeri, and the Mexican tetra, Astyanax mexicanus. This review is a part of a special issue on nutritional, hormonal, and metabolic drivers of development.

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.

Mitogenome of Knodus borki (Cypriniformes: Characidae): genomic characterization and phylogenetic analysis.

BACKGROUND: The taxonomic status of Knodu in the family Characidae is not yet clear. This study aimed to address this by sequencing and annotating Knodu borki Zarske, 2008. MATERIALS AND RESULTS: K. borki Zarske, 2008 was sequenced using a Hiseq platform and the complete mitogenome was assembled in SPAdes v3.15.2 and SOAPdenovo2 v.2.01. The mitogenome of K. borki from Guangzhou, the first sequenced species of the genus Knodu, is 16,837 bp in length and contains 13 protein-coding genes (PCGs), two ribosomal (r) RNAs, 22 transfer (t) RNAs, and one D-loop. Among these 37 genes, 28 are encoded by the heavy strand, while nine are encoded by the light strand. Twenty-one of the tRNAs can form typical cloverleaf secondary structures, except tRNA-Ser1, which lacks dihydrouridine arms. All PCGs have the same start codon (ATG), with the exception of COI (GTG). Four PCGs (ND1, ATP8, ND4L, and ND5) have TAA as the stop codon, ND6 has TAG as the stop codon, COI has AGG as the stop codon, and the remaining seven genes have incomplete stop codons of TA-/T-(ND2, COII, COIII, ND3, ND4, and Cyt b as T-, ATP6 as TA-). Phylogenetic analysis showed that K. borki belongs to the family Characidae. CONCLUSIONS: Our findings demonstrate that K. borki belongs to the family Characidae, due to consistency with the morphological identification. This study provides molecular information for further research on the phylogeny of the genus Knodus and for analyses of the taxonomic status of Characidae.

Long-term persistence of supernumerary B chromosomes in multiple species of Astyanax fish.

BACKGROUND: Eukaryote genomes frequently harbor supernumerary B chromosomes in addition to the "standard" A chromosome set. B chromosomes are thought to arise as byproducts of genome rearrangements and have mostly been considered intraspecific oddities. However, their evolutionary transcendence beyond species level has remained untested. RESULTS: Here we reveal that the large metacentric B chromosomes reported in several fish species of the genus Astyanax arose in a common ancestor at least 4 million years ago. We generated transcriptomes of A. scabripinnis and A. paranae 0B and 1B individuals and used these assemblies as a reference for mapping all gDNA and RNA libraries to quantify coverage differences between B-lacking and B-carrying genomes. We show that the B chromosomes of A. scabripinnis and A. paranae share 19 protein-coding genes, of which 14 and 11 were also present in the B chromosomes of A. bockmanni and A. fasciatus, respectively. Our search for B-specific single-nucleotide polymorphisms (SNPs) identified the presence of B-derived transcripts in B-carrying ovaries, 80% of which belonged to nobox, a gene involved in oogenesis regulation. Importantly, the B chromosome nobox paralog is expressed > 30× more than the A chromosome paralog. This indicates that the normal regulation of this gene is altered in B-carrying females, which could potentially facilitate B inheritance at higher rates than Mendelian law prediction. CONCLUSIONS: Taken together, our results demonstrate the long-term survival of B chromosomes despite their lack of regular pairing and segregation during meiosis and that they can endure episodes of population divergence leading to species formation.

Chromosomal Instability and Origin of B Chromosomes in the Amazonian Glass Tetra Moenkhausia oligolepis (Günther, 1864) (Characiformes, Characidae).

B chromosomes occur in different species of the small characid fishes of the genus Moenkhausia. These supernumerary elements, that do not recombine with chromosomes of the standard A complement and follow their own evolutionary mechanism vary in number, morphology, and distribution. Here, we show karyotypic data of individuals of 2 populations of Moenkhausia oligolepis of the Brazilian Amazon (Pedro Correia and Taboquinha streams, Tocantins river basin), both with a diploid number of 50 chromosomes and karyotypic formula of 10m + 32sm + 8a. In addition to the normal complement, we also observed the occurrence of B chromosomes in the 2 populations with intra- and interindividual variation ranging from 0 to 10 Bs, independent of sex. The C-banding pattern evidenced heterochromatic blocks located mainly in the pericentromeric region of the chromosomes, while the B chromosomes appeared euchromatic. Silver-stained nucleolus organizer regions were identified in multiples sites, and some of these blocks were positive when stained with chromomycin A3. The karyotype analysis and the application of whole-chromosome painting in populations of M. oligolepis reinforce the conservation of the basal diploid number for the genus, as well as the evolutionary tendency in these fishes to carry B chromosomes. Both populations turned out to be in different stages of stability and expansion of their B chromosomes. We further suggest that the origin of these chromosomes is due to the formation of isochromosomes. Here, we identified a pair of complement A chromosomes involved in this process.

Effects of triploid induction on innate immunity and hematology in Astyanax altiparanae.

Triploid induction is a promising biotechnique that could be used to enhance aquaculture yields in the near future. However, studies conducted with several fish species have demonstrated that the presence of an extra set of chromosomes may result in deleterious health effects. Furthermore, studies of fish immune responses still need to be conducted before these specimens can be readily commercialized. In the study presented herein, we evaluated the effects of triploid induction on hematology, erythrocyte morphometry and morphology, phagocytosis, and the expression levels of IL-1ß and TGF-ß using specimens of the Neotropical species, Astyanax altiparanae. In general, the cell counts of erythrocytes, leukocytes, and neutrophils in triploid fish were lower than those in diploid fish. The erythrocytes of triploid fish were larger than those found in diploid fish, but also demonstrated considerably higher frequencies of cellular and nuclear abnormalities. Although not statistically significant, triploid induction resulted in a phagocytic capacity (PC) 20% lower than that found with diploid fish. No notable differences were observed in phagocytic index (PI). Gene expression levels for the cytokine IL-1 were lower in tissues from the head kidney, liver, and spleen of triploid fish with respect to diploid fish. Gene expression levels of TGF-ß were lower only in the spleen of triploids compared to diploids. In conclusion, triploid induction resulted in A. altiparanae specimens with immune impairments and potentially lower resistances to disease and low-quality environments.

Evolutionary emergence of the rac3b/rfng/sgca regulatory cluster refined mechanisms for hindbrain boundaries formation.

Developmental programs often rely on parallel morphogenetic mechanisms that guarantee precise tissue architecture. While redundancy constitutes an obvious selective advantage, little is known on how novel morphogenetic mechanisms emerge during evolution. In zebrafish, rhombomeric boundaries behave as an elastic barrier, preventing cell intermingling between adjacent compartments. Here, we identify the fundamental role of the small-GTPase Rac3b in actomyosin cable assembly at hindbrain boundaries. We show that the novel rac3b/rfng/sgca regulatory cluster, which is specifically expressed at the boundaries, emerged in the Ostariophysi superorder by chromosomal rearrangement that generated new cis-regulatory interactions. By combining 4C-seq, ATAC-seq, transgenesis, and CRISPR-induced deletions, we characterized this regulatory domain, identifying hindbrain boundary-specific cis-regulatory elements. Our results suggest that the capacity of boundaries to act as an elastic mesh for segregating rhombomeric cells evolved by cooption of critical genes to a novel regulatory block, refining the mechanisms for hindbrain segmentation.

Moenkhausia andrica (Characiformes: Characidae): a new species from the rio Tapajós basin, Brazil, with minute fin hooklets in females.

In this study, a new species of Moenkhausia is described from the upper rio Juruena, rio Tapajós basin, Brazil. It is distinguished from all congeners by the presence of minute bony hooks in all fins of both mature females and males and combination of a prepelvic region flattened, dorsal portion of the humeral blotch extending two scales horizontally and vertically, lateral line with 28-32 scales, five scale series above and below lateral line; circumpeduncular scales 13-14, anal-fin rays 16-19 and dorsal portion of eyes blue in live specimens. The new species is also supported by high divergence in the mitochondrial gene cytochrome c oxidase subunit I (COI). The presence of minute fin bony hooks in both females and males, population variations and late development of the lateral line in Moenkhausia andrica are discussed.

Genotoxic effects of urban pollution in the Iguaçu River on two fish populations.

Environmental impacts on water resources and discharges of pollutants are some of the main problems of aquatic ecosystems. Currently, the Iguaçu River is considered the second most polluted river in Brazil, therefore, given the importance of understanding the effects of water pollution on living organisms, this study aimed to analyze the possible genotoxic effects in Astyanax bifasciatus (Characiformes; Characidae) and Geophagus brasiliensis (Perciformes: Cichlidae) in response to the water quality of the Iguaçu River. Four sampling points were determined on the Iguaçu River, with different levels of environmental impact. The micronuclei and nuclear abnormalities were quantified by counting 2000 erythrocytes from each specimen. There was a statistical difference in the frequency of nuclear abnormalities both for A. bifasciatus (Kruskal-Wallis 3;30 = 10.9; p = 0.01) and for G. brasiliensis (Kruskal-Wallis 3;26 = 12.27; p = 0.006), with point 1 (critically degraded) showing a higher frequency of nuclear abnormalities and point 4 (moderately degraded) showing a lower frequency of nuclear abnormalities for both species, showing genotoxic potential in fish erythrocytes. This result demonstrates the need for further discussion on the conservation of the Iguaçu River as well as broadening the discussion on its potability.