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Purpose: This work described a new species of Ceratomyxa, based on morphological and phylogenetic analyzes of myxospores collected from the gallbladder of the fish Astyanax mexicanus. Methods: Sixty-two specimens were captured, between December 2022 and February 2024, in the Flexal River, in the community of Tessalônica, state of Amapá. The specimens were transported alive to the Laboratory of Morphophysiology and Animal Health, at the State University of Amapá, where the studies were carried out. Results: Eleven specimes of A. mexicanus was parasitized with Ceratomyxa tessaloniensis n. sp. Morphological and morphometric analyses revealed strongly arched spores that measured 3.13 ± 0.5 μm in length and 12.18 ± 1.9 μm in thickness with a posterior angle of 74º ± 10.6º and two polar capsules measuring 1.74 ± 0.1 μm long and 1.5 ± 0.16 μm wide. The partial sequence of the 18 S gene from spores of Ceratomyxa tessaloniensis n. sp., comprised 932 base pairs. Conclusion: Phylogenetic analysis grouped Ceratomyxa tessaloniensis n. sp., with strong nodal support for the exclusive clade of freshwater species, being a new species described for the State of Amapá, the first described in A. mexicanus and the first species with the presence of non-wormlike plasmodia in South America. [ABSTRACT FROM AUTHOR]

Batesian mimicry occurs when palatable mimics gain protection from predators by evolving a phenotypic resemblance to an aposematic model species. While common in nature, the mechanisms maintaining mimicry are not fully understood. Patterns of temporal synchrony (i.e. temporal co-occurrence) and model first occurrence have been observed in several mimicry systems, but the hypothesis that predator foraging decisions can drive the evolution of prey phenology has not been experimentally tested. Here, using phenotypically accurate butterfly replicas, we measured predation rates on the chemically defended model species Battus philenor and its imperfect Batesian mimic Limenitis arthemis astyanax under four different phenological conditions to understand the importance of temporal synchrony and model first occurrence in mimicry complexes. We predicted that protection for mimics increases when predators learn to avoid the models' aposematic signal right before encountering the mimic, and that learned avoidance breaks down over time in the model's absence. Surprisingly, we found that asynchronous model first occurrence, even on short time scales, did not provide increased protection for mimics. Mimics were only protected under conditions of temporal synchrony, suggesting that predators rely on current information, not previously learned information, when making foraging decisions. [ABSTRACT FROM AUTHOR]

[Display omitted] • Neotropical Astyanax fishes migrated north in the Great American Biotic Exchange. • Gyrodactylus evolved in Gondwana and co-migrated with their characid fish hosts. • Astyanax mexicanus and its gyrodactylid parasites are distributed north of the Trans-Mexican Volcanic Belt (TMVB) • Parasites found past the TMVB are the most derived clades. • Gyrodactylus spp. infecting Astyanax in Mexico are morphologically cryptic species. The neotropical fish genus Astyanax (Characidae) and its associated helminths migrated northward from South America following the Great American Biotic Interchange (GABI): ca. 150 Astyanax spp. are found throughout South and Central America, up to the Mexico-USA border. Most characids are distributed south of the Trans-Mexican Volcanic Belt (TMVB), which bisects the country and represents a major transition zone between the neotropical and nearctic realms. Here, we characterize parasites of the monogenean genus Gyrodactylus infecting Astyanax spp. in Mexico: Astyanax aeneus south of the TMBV, Astyanax mexicanus north of it. Based on morphological, phylogenetic (internal transcribed spacer (ITS) and cytochrome oxidase subunit II (cox 2)) and statistical analyses of morphometric data, we confirmed the validity of Gyrodactylus pakan and Gyrodactylus teken , and erected two new species, Gyrodactylus aphaa n. sp. and Gyrodactylus ricardoi n. sp. These four gyrodactylids are part of a complex of morphologically cryptic species, which are phylogenetically closely related to each other, and sister species to Gyrodactylus carolinae and Gyrodactylus heteracanthus , parasites of characins in Brazil. Four gyrodactylid lineages (G. pakan , G. ricardoi n. sp., G. teken , Gyrodactylus sp. A) are distributed north of the TMVB; G. pakan is also widely distributed south of the TMVB, together with G. aphaa n. sp. Based on the ITS phylogeny, Brazilian parasites form a sister clade to all Mexican gyrodactylids, whose derived clades are distributed in progressively more northerly latitudes in Mexico – the three most-derived species north of the TMVB. This would suggest that gyrodactylid species diverged gradually, presumably as their characid fish hosts colonized and adapted to new environments north of the TMVB. [ABSTRACT FROM AUTHOR]

Reactive dyes used in the textile industry can be harmful to aquatic organisms. This study aimed to evaluate the potential cyto/genotoxic effects of the Novacron® Bold Deep Navy reactive dye on fish Astyanax lacustris before and after treatment through a vertical flow constructed wetland system (VFCW). Micronucleus test (MN), cellular morphological changes (CMC), and the comet assay were used to assess the effects of the dye in erythrocytes. During the experiment, A. lacustris specimens were exposed to sublethal concentrations (10, 20, 30, and 40 mg/L) of Novacron® Bold Deep Navy dye for 96 h. Results showed that the dye caused MN formation (27), DNA damage, and several types of CMC (3513) in all tested concentrations on A. lacustris. Notably, analyses were significant (p < 0.05) for all concentrations, except in the frequency mean of DNA damage at 10 mg/L. The bioremediation of the dye carried out by the VFCW system proved to be efficient, as confirmed by UV–Vis spectrophotometry, liquid chromatography-mass spectrometry (LC–MS), and physicochemical analysis. Thus, the Novacron® Bold Deep Navy dye poses a potential risk of genotoxicity and cytotoxicity for aquatic organisms, and the VFCW system proved to be efficient in treating the dye. [ABSTRACT FROM AUTHOR]

The ability to follow the evolutionary trajectories of specific neuronal cell types has led to major insights into the evolution of the vertebrate brain. Here, we study how cave life in the Mexican tetra (Astyanax mexicanus) has affected an identified giant multisensory neuron, the Mauthner neuron (MN). Because this neuron is crucial in driving rapid escapes, the absence of predation risk in the cave forms predicts a massive reduction in this neuron. Moreover, the absence of functional eyes in the A. mexicanus Pachón form predicts an even stronger reduction in the cell's large ventral dendrite that receives visual inputs in sighted fish species. We succeeded in recording in vivo from this neuron in the blind cavefish and two surface tetra (A. mexicanus and Astyanax aeneus), which offers unique chances to simultaneously study evolutionary changes in morphology and function in this giant neuron. In contrast to the predictions, we find that cave life, while sufficient to remove vision, has neither affected the cell's morphology nor its functional properties. This specifically includes the cell's ventral dendrite. Furthermore, cave life did not increase the variance in morphological or functional features. Rather, variability in surface and cave forms was the same, which suggests a complex stabilizing selection in this neuron and a continued role of its ventral dendrite. We found that adult cavefish are potent predators that readily attack smaller fish. So, one of the largely unknown stabilizing factors could be using the MN in such attacks and, in the young fish, escaping them. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Hypoxia increases hematopoiesis and erythrocytes in surface fish. • Shh upregulation increases hematopoiesis and erythrocytes in surface fish. • Shh inhibition decreases hematopoiesis and erythrocytes in cavefish. • Hypoxia upregulates Shh along the embryonic midline in surface fish. [ABSTRACT FROM AUTHOR]

Aluminum (Al) can cause endocrine disruption in aquatic animals, but assessments of animal social behavior in neotropical teleost fish species with importance for Brazilian aquaculture have still not been addressed so far, which can further complete this ecotoxicological knowledge. In order to evaluate the social behavior and plasma cortisol concentration of fish exposed to Al, we performed a 1 h acute exposure with Astyanax lacustris couples in three different experimental groups: control in neutral pH (CTL/n group), acid pH (pH/ac group), and aluminum in acid pH (Al/ac group; 2.0 mg L−1). An ethogram of social interactions between males and females and swimming activities were performed. Furthermore, the cortisol plasma concentration was measured by enzyme-linked immunosorbent, and the gonadal maturation stage of the animals was evaluated by histology. Adult and mature females in the CTL/n and pH/ac groups were more aggressive and active than mature males, including several attacks on the male. Moreover, males did not present attack behavior in these groups at any time, but did show submission behavior and constant avoidance of female attacks. In the Al/ac, females did not attack males, couple decreased swimming activity, a repetitive movement toward the aquarium surface, and high mucus production were observed, making the water cloudy. Regarding cortisol plasma concentration, males had higher cortisol plasma concentrations than females in the CTL/n and pH/ac groups, which was not observed in the Al/ac group. Therefore, Al in addition to being described in the literature as an endocrine disruptor, it can be considered as behavioral disrupter in A. lacustris in this important freshwater species cultivated in South America. [ABSTRACT FROM AUTHOR]

Background: Allocreadiids are relatively small digeneans that appear to be restricted to freshwater systems distributed across the world. Allocreadiids are highly diverse in the Americas, particularly in the Neotropical biogeographical region. Their taxonomic history has been rather controversial, with several taxonomic reassessments and the description of new genera and species. Methods: We sampled Creptotrematina specimens from a characid collected in the Pardo River, Paranapanema River basin in Brazil, and specimens of C. aguirrepequenoi, from Astyanax spp. in several localities between northern Mexico and Costa Rica. The specimens were studied through integrative approaches using morphological and molecular analyses of the 28S rDNA gene and two different regions of the COI mtDNA gene. Results: We describe a new species of Creptotrematina which is differentiated from other congeners by the overall body size, but in particular by the size and position of the cirrus-sac, distribution of the vitelline follicles, and extension of uterine loops in the posterior end of body. Phylogenetic analyses of the 28S rDNA and COI mtDNA genes placed the new species in a monophyletic clade together with all other sequenced species of Creptotrematina, and as a sister species of C. batalhensis. Genetic divergences between the new species and other Creptotrematina spp. varied from 1.1 to 1.2% for the 28S rDNA and 12.4–14.3% for the COI mtDNA. Phylogenetic analysis based on COI mtDNA showed the isolates of C. aguirrepequenoi grouped in four monophyletic clades representing populations geographically separated along a wide geographical range spanning between northern Mexico and Costa Rica, with an estimated genetic divergence between 3.9% and 8.9%. Conclusions: Our findings based on integrative analyses recognize Creptotrematina saltograndensis n. sp. from a characid collected in the Pardo River, Paranapanema River basin in Brazil and provide validation of the wide geographical distribution of C. aguirrepequenoi across Middle-America parasitizing Astyanax spp.; the genetic divergence of the species through the analysis of two regions of COI mtDNA points towards considering it represent a species complex, although we refrain at the moment on describing a new species, awaiting for further verification using other molecular markers, and obtaining fresh material for a more detailed taxonomic analyses. This study increases the known diversity of allocreadiids and contributes to the understanding of evolutionary relationships, host-parasite relationships, and biogeographic history of the group. [ABSTRACT FROM AUTHOR]

Organochlorine pesticides (OCPs) are persistent pollutants previously used in agriculture, known for their ability to bioaccumulate and pose health risks. This study analyzed samples of roe, viscera, and muscle from Astyanax altiparanae fish collected from the Alagados reservoir in Paraná, Brazil. Samples were prepared through extraction and purification, then analyzed using gas chromatography with an electron capture detector (GC/ECD), chosen for its sensitivity in detecting OCPs. The method was validated for precision, accuracy, and detection limits. Detected OCPs included Aldrin (17.1 to 50.6 ng/g in roe), α-endosulfan (3.4 to 23.5 ng/g), p, p'-DDE (4.2 to 134.7 ng/g), Dieldrin (84.7 to 183.1 ng/g), β-endosulfan (6.0 to 51.6 ng/g), and p, p'-DDT (56.6 to 286.8 ng/g). In viscera, concentrations ranged from Aldrin (19.8 to 93.3 ng/g) to p, p'-DDT (52.3 to 89.2 ng/g). Muscle samples showed similar trends. Principal component analysis indicated a link between higher OCP concentrations and increased abdominal width of the fish. While OCP levels were below FAO and WHO limits, risk quotient calculations suggest potential health risks from consuming these fish. [ABSTRACT FROM AUTHOR]

The São Francisco River (SFR) is a watershed of extreme socioeconomic importance and rich biodiversity, notable for its unique ichthyofauna and high levels of endemism. However, human interference threatens several fish species, putting them at risk of extinction. Genetic knowledge about the ichthyofauna of the SFR is still lacking. To address this gap, we used DNA barcoding of 94 taxa from six orders, 32 families, and 73 genera, sourced from the BOLD Systems platform and samples in the middle and lower SFR regions. Analyses revealed distinct patterns of genetic divergence. The BOLD platform identified 111 bins, 75 as MATCH, 32 as SPLIT, two as MERGE and two MIXTURE. We detected taxonomic confusions in the genera Astyanax and Psalidodon. Fourteen taxa exhibited high cryptic diversity, corroborated by lineage delimitation methods (BINs, ASAP, and GMYC). A significant portion of this cryptic diversity (eight lineages) is concentrated in the upper SFR, a region with tectonic faults, geomorphological reactivations, and the transposition of another basin, which likely contributed to genetically divergent lineages. Our data fill shortfalls in Linnaean and Darwinian knowledge about the SFR ichthyofauna, considering its hydrogeological formation. We hope this information will serve as a foundation for future management and conservation plans for this important ichthyofauna. [ABSTRACT FROM AUTHOR]

Sensing and acquiring dissolved oxygen is crucial for nearly all aquatic life. This may become even more vital as dissolved oxygen concentrations continue to decline in many aquatic environments. While certain phenotypes that enable fish to live in low oxygen have been characterized, adaptations that arise following sudden, drastic reductions in dissolved oxygen are relatively unknown. Here, we assessed the blind Mexican cavefish, Astyanax mexicanus, for alterations to gill morphology that may be adaptive for life in hypoxic caves. The Astyanax system provides the unique opportunity to compare gill morphology between stereotypical "surface" adapted morphotypes and obligate cave‐dwelling conspecifics. While the surface environment is well‐oxygenated, cavefish must cope with significantly reduced oxygen. We began by quantifying traditional morphological gill traits including filament number and length as well as lamellar density and height in surface fish and two distinct cave populations, Pachón and Tinaja. This enabled us to estimate total lamellar height, a proxy for gill surface area. We then used immunohistochemical staining to label 5‐HT‐positive neuroepithelial cells (NECs), which serve as key oxygen sensors in fish. We discovered an increase in gill surface area for both cavefish populations compared to surface, which may enable a higher capacity of oxygen acquisition. Additionally, we found more NECs in Pachón cavefish compared to both surface fish and Tinaja cavefish, suggesting certain selective pressures may be cave‐specific. Collectively, this work provides evidence that cavefish have adapted to low oxygen conditions via alterations to gill morphology and oxygen sensing, and informs evolutionary mechanisms of rapid adaptation to dramatic, chronic hypoxia. Research Highlights: Astyanax cavefish have significantly greater surface area for oxygen exchange based on increased lamellar height. Certain cavefish have substantially elevated numbers of oxygen sensing cells in gill filaments. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The most studied cavefish in the world is Astyanax mexicanus, and the most frequently used specimens in research come from the Pachón cave in Northeast Mexico. A recent study using the capture–mark–recapture technique revealed that the Pachón population size is only in the hundreds of individuals, not in the thousands. This result raises serious concerns about their long-term survival and highlights the need to accurately estimate the population size of other Astyanax caves. In our study, we used a well-established, non-invasive method for estimating population size: the transect sampling method, which counts the abundance of organisms along a line at regular intervals. We used the 310 m long Sabinos Cave's Pool #1 for measurements and estimated a population of 1142 individuals. Most importantly, we found that cavefish are not distributed uniformly: 68% of the individuals were clustered under a bat roost. To corroborate this observation, we used drones to explore submerged galleries inaccessible to scientists. Results from the Toro/Calera Cave system confirmed that most galleries were essentially void of cavefish, except for in those few areas with high amounts of nutrients. Scientists visiting the most studied Astyanax localities (Pachón, Sabinos, Tinaja, and Chica) experience what appear to be high-density populations of about 10 fish/m2, possibly because the pool they visit happens to be under bat roosts. Our study shows that one cannot extrapolate the density at these localities to the rest of the habitat, where densities might be two orders of magnitude lower. Future studies should consider that the apparent high density at visited sites is overrepresenting the total population size when calculating the number of fish specimens to be captured. [ABSTRACT FROM AUTHOR]

Major tailings dam failures have occurred recently around the world and resulted in severe environmental impacts, such as metal contamination. Manganese is a metal highly associated with mining activities, largely detected in mining dam collapses. This metal is considered necessary for different organisms, but it can be toxic and cause oxidative stress and genetic damage in fishes. In this study, we investigated the toxic effects of manganese on Astyanax lacustris, by exposing the fish individually to different concentrations of this metal (2.11, 5.00, and 10.43 mg/L) for 96 h. To assess the effects of manganese, we used biochemical biomarkers (glutathione S-transferase, catalase, and acetylcholinesterase enzyme activity) and the manganese bioaccumulation in different tissues (liver and gills). The obtained data showed that only at concentrations of 5.00 mg/L and 10.43 mg/L the activity of glutathione S-transferase differed significantly. Additionally, the acetylcholinesterase activity in the brain tissue was inhibited. The highest level of manganese bioaccumulation was observed in the liver and branchial tissue. Overall, we concluded that high concentrations of manganese may cause physiological changes in Astyanax lacustris. [ABSTRACT FROM AUTHOR]

The ability of organisms to adapt to sudden extreme environmental changes produces some of the most drastic examples of rapid phenotypic evolution. The Mexican Tetra, Astyanax mexicanus , is abundant in the surface waters of northeastern Mexico, but repeated colonizations of cave environments have resulted in the independent evolution of troglomorphic phenotypes in several populations. Here, we present three chromosome-scale assemblies of this species, for one surface and two cave populations, enabling the first whole-genome comparisons between independently evolved cave populations to evaluate the genetic basis for the evolution of adaptation to the cave environment. Our assemblies represent the highest quality of sequence completeness with predicted protein-coding and noncoding gene metrics far surpassing prior resources and, to our knowledge, all long-read assembled teleost genomes, including zebrafish. Whole-genome synteny alignments show highly conserved gene order among cave forms in contrast to a higher number of chromosomal rearrangements when compared with other phylogenetically close or distant teleost species. By phylogenetically assessing gene orthology across distant branches of amniotes, we discover gene orthogroups unique to A. mexicanus. When compared with a representative surface fish genome, we find a rich amount of structural sequence diversity, defined here as the number and size of insertions and deletions as well as expanding and contracting repeats across cave forms. These new more complete genomic resources ensure higher trait resolution for comparative, functional, developmental, and genetic studies of drastic trait differences within a species. [ABSTRACT FROM AUTHOR]

This study tested the use of the essential oil of Zingiber officinale (EOZO) as an anesthetic for Astyanax lacustris and its possible histopathological effects on the gills. Two experiments were carried out. Experiment 1 determined anesthesia induction time and recovery time and the ventilatory frequency of animals of two size classes exposed to different concentrations of EOZO (0, 75, 150, 300, 450, and 600 μL L−1): Adults I (18.86 ± 4.81 g; 10.76 ± 0.87 cm) and Adults II (7.39 ± 2.57 g; 8.33 ± 0.86 cm). Ten fish were used for each size class and anesthetic concentration, distributed in a completely randomized design considering each animal as a replicate. Experiment II evaluated the gill histopathology of Adults II exposed to the same EOZO concentrations as used in Experiment I. Experiment I found concentrations of 300–600 μL L−1 EOZO and 150–600 μL L−1 EOZO to cause deep anesthesia for Adults I and Adults II, respectively. These recommended anesthetic concentrations provided induction and recovery times within acceptable limits for fish. Ventilatory frequency was reduced in the minimum concentrations indicated for each size class. No mortality was observed within 24 h post-test. Experiment II found irreversible damage to gill tissue for fish exposed above 300 μL L−1 EOZO. Therefore, EOZO concentrations of 300 μL L−1 and 150 μL L−1 are recommended for Adults I and Adults II, respectively. [ABSTRACT FROM AUTHOR]

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 fishmimics 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. [ABSTRACT FROM AUTHOR]

Intending to compare in vitro cell growth in different conditions, we established cell cultures using fin biopsies of two freshwater fishes, Astyanax bimaculatus and Geophagus proximus. Three different culture media (Leibovitz–L‐15, Dulbecco's Modified Eagle Medium [DMEM], and 199) were employed, with or without the addition of AmnioMax, maintaining a standard temperature of 29°C. Based on the results obtained, we standardized a cell growth protocol in which medium 199 was less efficient for both species. Notably, G. proximus cells exhibited superior proliferation in DMEM and L‐15 media, whereas A. bimaculatus cells demonstrated better parameters exclusively in the DMEM medium. Successful subculturing of cells with good proliferation index was observed, accompanied by preserved morphological characteristics. Therefore, the methodology outlined in this study represents an advancement in establishing fish cell cultures. [ABSTRACT FROM AUTHOR]

With 75 known species, the freshwater fish genus Sinocyclocheilus is the largest cavefish radiation in the world and shows multiple adaptations for cave‐dwelling (stygomorphic adaptations), which include a range of traits such as eye degeneration (normal‐eyed, micro‐eyed and eyeless), depigmentation of skin, and in some species, the presence of "horns". Their behavioural adaptations to subterranean environments, however, are poorly understood. Wall‐following (WF) behaviour, where an organism remains in close contact with the boundary demarcating its habitat when in the dark, is a peculiar behaviour observed in a wide range of animals and is enhanced in cave dwellers. Hence, we hypothesise that wall‐following is also present in Sinocyclocheilus, possibly enhanced in eyeless species compared to eye bearing (normal‐/micro‐eyed species). Using 13 species representative of Sinocyclocheilus radiation and eye morphs, we designed a series of assays, based on pre‐existing methods for Astyanax mexicanus behavioural experiments, to examine wall‐following behaviour under three conditions. Our results indicate that eyeless species exhibit significantly enhanced intensities of WF compared to normal‐eyed species, with micro‐eyed forms demonstrating intermediate intensities in the WF distance. Using a mtDNA based dated phylogeny (chronogram with four clades A–D), we traced the degree of WF of these forms to outline common patterns. We show that the intensity of WF behaviour is higher in the subterranean clades compared to clades dominated by normal‐eyed free‐living species. We also found that eyeless species are highly sensitive to vibrations, whereas normal‐eyed species are the least sensitive. Since WF behaviour is presented to some degree in all Sinocyclocheilus species, and given that these fishes evolved in the late Miocene, we identify this behaviour as being ancestral with WF enhancement related to cave occupation. Results from this diversification‐scale study of cavefish behaviour suggest that enhanced wall‐following behaviour may be a convergent trait across all stygomorphic lineages. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In nature, animals must navigate to forage according to their sensory inputs. Different species use different sensory modalities to locate food efficiently. For teleosts, food emits visual, mechanical, chemical, and/or possibly weak-electrical signals, which can be detected by optic, auditory/lateral line, and olfactory/taste buds sensory systems. However, how fish respond to and use different sensory inputs when locating food, as well as the evolution of these sensory modalities, remain unclear. We examined the Mexican tetra, Astyanax mexicanus, which is composed of two different morphs: a sighted riverine (surface fish) and a blind cave morph (cavefish). Compared with surface fish, cavefish have enhanced non-visual sensory systems, including the mechanosensory lateral line system, chemical sensors comprising the olfactory system and taste buds, and the auditory system to help navigate toward food sources. We tested how visual, chemical, and mechanical stimuli evoke food-seeking behavior. In contrast to our expectations, both surface fish and cavefish did not follow a gradient of chemical stimulus (food extract) but used it as a cue for the ambient existence of food. Surface fish followed visual cues (red plastic beads and food pellets), but, in the dark, were likely to rely on mechanosensors—the lateral line and/or tactile sensor—as cavefish did. Our results indicate cavefish used a similar sensory modality to surface fish in the dark, while affinity levels to stimuli were higher in cavefish. In addition, cavefish evolved an extended circling strategy to forage, which may yield a higher chance to capture food by swimming-by the food multiple times instead of once through zigzag motion. In summary, we propose that ancestors of cavefish, similar to the modern surface fish, evolved extended food-seeking behaviors, including circling motion, to adapt to the dark. [ABSTRACT FROM AUTHOR]

Astyanax caucanus is an endemic fish species to the Magdalena-Cauca basin in Colombia. It is considered a Least Concern species by the International Union for Conservation of Nature, and currently, it is not a fishery resource. Its fertilized eggs may drift up to 4–5 days before hatching and can be carried up to 340 km given the water velocity of the river. Although A. caucanus is listed as short -migratory species (< 50 km), this study hypothesized that it exhibits gene flow along the middle and lower section of the Cauca River due to the great potential for larval dispersal. To test this hypothesis, we developed a set of species-specific microsatellite primers suitable for population genetic studies. Genetic structure analyses with 193 samples evidenced two genetic stocks that coexist, comigrate, and exhibit gene flow along the study area. Both stocks show high genetic diversity indices (Na and HE) and effective population sizes (Ne > 1000), but also show evidence of bottlenecked populations and high values of the inbreeding coefficient (FIS). Finally, these results are useful to understand the effects of other anthropic activities, besides fishing pressure on population bottlenecks found for other fish species cohabiting the area. [ABSTRACT FROM AUTHOR]

Extreme environmental conditions have profound impacts on shaping the evolutionary trajectory of organisms. Exposure to these conditions elicits stress responses, that can trigger phenotypic changes in novel directions. The Mexican Tetra, Astyanax mexicanus, is an excellent model for understanding evolutionary mechanisms in response to extreme or new environments. This fish species consists of two morphs; the classical surface‐dwelling fish and the blind cave‐dwellers that inhabit dark and biodiversity‐reduced ecosystems. In this review, we explore the specific stressors present in cave environments and examine the diverse adaptive strategies employed by cave populations to not only survive but thrive as successful colonizers. By analyzing the evolutionary responses of A. mexicanus, we gain valuable insights into the genetic, physiological, and behavioral adaptations that enable organisms to flourish under challenging environmental conditions. Research Highlights: Unique adaptations in cave populations of Astyanax mexicanus makes them a remarkable model to study the mechanisms underlying stress adaptation to environmental challenges. This review explores the remarkable adaptive strategies that allows the cavefish to successfully survive the challenging environmental shift at the time of cave colonization. [ABSTRACT FROM AUTHOR]

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. Highlights: 3D spheroids derived in vitro from Astyanax liver cells exhibit metabolic state resembling multiple aspects of liver tissue. [ABSTRACT FROM AUTHOR]

We aimed to examine the responses of pollution biomarkers in feral fish from Astyanax genus collected at three hydrographic regions in southern Brazil and the capacity of these tools to differentiate between various levels of contamination. To achieve this, levels of organochlorine pesticides (liver), as well as the biomarkers AChE (muscle and brain), TBARS (liver), and EROD (liver) were assessed. Collections were conducted in four municipalities (Alegrete, Caraá, Lavras, and Santa Vitória) during 1 year, encompassing winter and summer. Fish from Alegrete were the most contaminated overall, but animals sampled in Caraá, and Lavras also displayed elevated levels of current-use pesticides. Elevated levels of endosulfans, DDTs, HCHs, and current-use pesticides were accompanied by elevated levels of TBARS in the liver. Conversely, fish from Santa Vitória exhibited the highest levels of PAHs, accompanied by elevated levels of EROD in the liver and reduced levels of AChE in muscle and brain. TBARS proved to be a reliable biomarker for assessing impacts arising from pesticide accumulation, while EROD and AChE served as valuable indicators of impacts resulting from PAHs accumulation. Ultimately, the results obtained in this study demonstrate the reliable use of the proposed biomarkers for tracking biological impacts stemming from aquatic pollution using feral Astyanax as biomonitoring species. [ABSTRACT FROM AUTHOR]

Cave‐adapted animals evolve a suite of regressive and constructive traits that allow survival in the dark. Most studies aiming at understanding cave animal evolution have focused on the genetics and environmental underpinnings of regressive traits, with special emphasis on vision loss. Possibly as a result of vision loss, other non‐visual sensory systems have expanded and compensated in cave species. For instance, in many cave‐dwelling fish species, including the blind cavefish of the Mexican tetra, Astyanax mexicanus, a major non‐visual mechanosensory system called the lateral line, compensated for vision loss through morphological expansions. While substantial work has shed light on constructive adaptation of this system, there are still many open questions regarding its developmental origin, synaptic plasticity, and overall adaptive value. This review provides a snapshot of the current state of knowledge of lateral line adaption in A. mexicanus, with an emphasis on anatomy, synaptic plasticity, and behavior. Multiple open avenues for future research in this system, and how these can be leveraged as tools for both evolutionary biology and evolutionary medicine, are discussed. [ABSTRACT FROM AUTHOR]

The ecological and genetic changes that underlie the evolution of host–microbe interactions remain elusive, primarily due to challenges in disentangling the variables that alter microbiome composition. To understand the impact of host habitat, host genetics, and evolutionary history on microbial community structure, we examined gut microbiomes of river‐ and three cave‐adapted morphotypes of the Mexican tetra, Astyanax mexicanus, in their natural environments and under controlled laboratory conditions. Field‐collected samples were dominated by very few taxa and showed considerable interindividual variation. We found that lab‐reared fish exhibited increased microbiome richness and distinct composition compared to their wild counterparts, underscoring the significant influence of habitat. Most notably, however, we found that morphotypes reared on the same diet throughout life developed distinct microbiomes suggesting that genetic loci resulting from cavefish evolution shape microbiome composition. We observed stable differences in Fusobacteriota abundance between morphotypes and demonstrated that this could be used as a trait for quantitative trait loci mapping to uncover the genetic basis of microbial community structure. [ABSTRACT FROM AUTHOR]

We studied the effects of water-soluble (vitamin C, VC) and fat-soluble antioxidants (oregano essential oil, OEO) on the stress responses and redox status of Astyanax aff. bimaculatus exposed to air. Ten isoproteic and isoenergetic diets were formulated, with different levels of VC (0, 40, 80, 120, and 160 mg kg−1), with or without OEO (1,000 mg kg−1). Fish were fed these diets for 55 days, and then 18 fish from each group were exposed to air. VC at concentrations of 120 and 160 mg kg−1 reduced glycemia. In the gills, VC at 120 and 160 mg kg−1, only in combination with OEO, reduced oxidative damage (malondialdehyde, MDA, and carbonylated proteins, PC) and superoxide dismutase (SOD) activity. In the absence of VC, OEO increased PC and SOD. In the liver, all VC concentrations, without OEO, reduced MDA while in the presence of OEO, only VC at 40 and 160 mg kg−1 reduced MDA. Regardless of OEO, all VC concentrations reduced PC levels and SOD and catalase (CAT) activity. However, OEO increased CAT and glutathione S-transferase (GST) activity regardless of VC level. While VC at a concentration of 40 with 1,000 mg kg−1 OEO reduced oxidative damage to lipids in the gills and liver, a reduction in protein and lipid damage in the gills was only observed with a least VC 120 + OEO. Therefore, we recommend simultaneous supplementation of VC 120 + 1,000 OEO, as their combined use is more effective in reducing oxidative damage in A. aff. bimaculatus exposed to air. [ABSTRACT FROM AUTHOR]

The Astyanax fish exhibits two morphs: an eyed, pigmented surface morph and an eyeless, depigmented cave morph. Previous studies have shown that blind morphs swim nearly parallel to the wall and can sense detailed information about objects by gliding alongside them and sensing changes in the flow field around their body using their lateral line sensory system. Hence, cavefish can build hydrodynamic images of their surroundings. Field observations showed that one of their presumptive prey, mysid shrimp, is predominately found not on the floor, but crawling on the walls. In our study, the angle of the body axis with respect to a vertical wall was measured while fish swam in a tank. Results show that when swimming by a wall, cavefish incline the vertical axis of their body away from the wall. But most significantly, this angle is different when the right side or the left side of their body is oriented towards the wall. Intriguingly, cavefish have a leftward-biased dorso-cranial bend, where the convex side of the head is towards their right side. Other studies have shown behavioral "handedness". When exhibiting Vibration Attraction Behavior (VAB), cavefish in the field show laterality on the preponderant side they circle to explore a vibrating stimulus. Likewise in larval prey capture (LPC) behavior, larvae strike towards prey preferentially located on one side. Our results support that cavefish also express behavioral lateralization during passive swimming by walls and/or when searching for food that is perched on the walls, such as mysid shrimp. [ABSTRACT FROM AUTHOR]

Objective: Ectothermic fish are directly affected by temperature changes in the environment. The aim of this study was to evaluate the metabolic responses in the gills of Yellowtail Lambari Astyanax lacustris under thermal stress. Methods: To this end, we used spectrophotometry to evaluate the biomarkers of carbohydrate and protein metabolism, antioxidant defense, and oxidative damage in fish subjected to low (15°C) and high (31°C) temperatures, with control groups held at 23°C, for 2, 6, 12, 24, 48, and 96 h. Result: The results showed that cold thermal stress did not change the energy demand, and the antioxidant defense was reduced; therefore, the gills were vulnerable to the action of reactive oxygen species (ROS), presenting increased protein carbonylation at 12 h. With heat thermal stress, a higher energy demand was observed, which was verified by an increase in aerobic metabolism by glycolysis and the citric acid cycle. High‐temperature stress also increased the antioxidant defenses, as verified by the increased activities of glutathione peroxidase, glutathione reductase, and glutathione S‐transferase. However, the antioxidant defense system could not protect tissues from the action of ROS, as protein carbonylation increased at 6 and 24 h, indicating oxidative stress. Conclusion: The results showed that (1) temperature variations caused metabolic adjustments in the gills of Yellowtail Lambari, (2) the adaptive responses were different for winter and summer temperatures, and (3) Yellowtail Lambari recovered homeostasis when subjected to thermal stress, even with the occurrence of oxidative stress. Impact statementThis study explores cellular stress responses in terms of metabolism, providing a comparison between low and high temperatures within an evolutionary and environmental context. These findings are crucial for fish farming and conservation efforts, revealing possible impacts of global climate change. [ABSTRACT FROM AUTHOR]

Background. Telomeres are non-coding DNA repeats at the chromosome ends and their shortening is considered one of the major causes of aging. However, they also serve as a biomarker of environmental exposures and their length and attrition is affected by various stressors. In this study, we examined the average telomere length in Astyanax mexicanus, a species that has both surface-dwelling and cave-adapted populations. The cave morph descended from surface ancestors and adapted to a markedly different environment characterized by specific biotic and abiotic stressors, many of which are known to affect telomere length. Our objective was to explore whether telomere length differs between the two morphs and whether it serves as a biological marker of aging or correlates with the diverse environments the morphs are exposed to. Methods. We compared telomere length and shortening between laboratory-reared Pachón cavefish and Rio Choy surface fish of A. mexicanus across different tissues and ages. Results. Astyanax mexicanus surface fish exhibited longer average telomere length compared to cavefish. In addition, we did not observe telomere attrition in either cave or surface form as a result of aging in adults up to 9 years old, suggesting that efficient mechanisms prevent telomere-mediated senescence in laboratory stocks of this species, at least within this time frame. Our results suggest that telomere length in Astyanax may be considered a biomarker of environmental exposures. Cavefish may have evolved shorter and energetically less costly telomeres due to the absence of potential stressors known to affect surface species, such as predator pressure and ultra-violet radiation. This study provides the first insights into telomere dynamics in Astyanax morphs and suggests that shorter telomeres may have evolved as an adaptation to caves. [ABSTRACT FROM AUTHOR]

We analyzed two morpho-species, Astyanax aeneus and Astyanax caballeroi, coexisting in Lake Catemaco, with ecological differences previously associated with different trophic niches. While A. aeneus is a widely distributed species, A. caballeroi is endemic to Lake Catemaco. Due to the contrasting morphology between these two sympatric species, they were originally assigned to different genera, but little is known about their genetic differentiation. We hypothesized that these two species, which present differentiation in morphology and in trophic niches, co-occur in concomitance with assortative mating and form distinct genetic clusters. We tested this prediction by typing a set of 12 microsatellites in 348 individuals of the two morpho-species, comparing their genetic structure patterns with that of an allopatric population of A. aeneus (i.e., the Maquinas population). Genetic structure analysis, assignment analyses, and estimation of gene flow between the sympatric morpho-species were carried out. Contrary to expectations, there is a lack of genetic differentiation between the A. aeneus and A. caballeroi morpho-species. However, there was a genetic differentiation between Lake Catemaco and Río Maquinas populations of A. aeneus. In addition, we found an asymmetric gene flow pattern, with a larger migration rate from A. aeneus to A. caballeroi than in the other direction. Finally, the allopatric population of A. aeneus showed lower levels of genetic diversity than those reported for both morpho-species in the lacustrine system. Consequently, our results support the notion that these morpho-species are either in an early phase of speciation or represent a single polymorphic species. This model provides relevant information to understanding the presence of polymorphisms under gene flow and their potential impact on the speciation process. [ABSTRACT FROM AUTHOR]

Here we describe a new species of the genus Saccocoelioides found parasitizing Astyanax dissimilis Garavello & Sampaio, Psalidodon bifasciatus (Garavello and Sampaio) and Bryconamericus ikaa Casciotta, Almirón & Azpelicueta from the Iguazu National Park, Misiones province, Argentina. Saccocoelioides miguelmontesi n. sp. was studied based on morphological and molecular (28S rDNA and COI mtDNA sequences) data. The COI mtDNA tree indicated that the specimens collected from the three fish hosts are conspecific, with an intragroup p-distance of 0%. The new species shows an intermediate morphological configuration between the diminutive and robust forms described for Saccocoelioides by Curran (2018). Although, in the 28S rDNA tree, it is placed in a well-supported clade with the two robust species analysed (S. elongatus and S. magnus ; p-distance of 1 and 2%, respectively), it differs from the robust group by the range of body size, mature egg size, oral and ventral sucker size, sucker ratio, oral sucker to pharynx ratio, and post-cecal or post-testis/body length percentage. Our results led us to redefine the robust group as having eggs shorter or equal in length to the pharynx. Saccocoelioides miguelmontesi n. sp. the 10 th species reported from Argentina and the 7 th species within the robust group. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Amongst aquatic animals, fish are potentially vulnerable to the ingestion—accidental or intentional—of microplastics (MPs) discarded in the environment, due to aspects such as their similarity to food and the buoyancy of these materials, as well as their attractive coloration. By analyzing toxicological parameters (mortality, malformations, and changes in weight gain) and monitoring the ingestion and excretion of MPs from two different polymers, it was noted that exposure to a diet containing these particles was responsible for causing mortality, as well as accumulation of the ingested MPs, which were not fully excreted and remained retained in the gastrointestinal tract of the lambari rosa fish. Therefore, the results of the current study should be combined with further research, covering different species, to improve understanding of the bioaccumulation of MPs and to help devise attempts at environmental mitigation, since the harmful potential of these particles is clear, especially for the aquatic organisms that ingest them. Microplastics are a class of contaminants that pose a threat to aquatic biota, as they are easily found in aquatic ecosystems and can be ingested by a wide variety of organisms, such as fish. The lambari rosa (Astyanax altiparanae) is a microphage fish, which feeds on microscopic beings and particles, making it potentially susceptible to ingesting MPs discarded in the environment. In addition, this fish is of great economic and food importance, as it is used for human consumption. This study aimed to evaluate the accumulation and possible toxicological effects caused to lambari rosa (n = 450) by the ingestion of polyethylene (PE) and polyethylene terephthalate (PET) MPs, since the MPs of these polymers in the form of granules, fragments, and fibers are the most commonly reported in the aquatic environment. The parameters investigated here were the quantitative analysis of ingested MPs using microscopic and staining techniques, as well as the mortality rate, malformations/injuries, and impaired weight gain. At the end of the experiment, it was concluded that MPs from both polymers accumulated in the gastrointestinal tract of the lambari rosa, and that dietary exposure, especially to the PET polymer, was responsible for increasing the mortality rate in this species. [ABSTRACT FROM AUTHOR]

Proper muscle function and muscle fiber structures that match the environmental demands of organisms are imperative to their success in any ecosystem. The Mexican cavefish, Astyanax mexicanus, has two morphotypes: an obligate cave‐dwelling form that lives in thermally insulated caves and an O2 poor environment, and a surface form that lives in a more thermally variable, but O2 rich river environment. As environment can determine physiological adaptations, it is of interest to compare the aerobic and anaerobic metabolic profiles of white muscle metabolism in both morphotypes of this species, as well as their muscle structures. Here, we used white muscle of both morphotypes of the Mexican cavefish to determine citrate synthase (CS) activity as a measure of aerobic potential, and lactate concentration as a measure of anaerobic potential at three different chronic acclimation temperatures (14°C, 25°C, and 31°C). By examining aerobic and anaerobic potential in both morphs, we sought to link environmental thermal flexibility to muscle metabolism. We found that the surface morphotype had higher CS activity and lower lactate concentration, suggesting an overall more efficient usage of aerobic metabolism; whereas the cave morphotype showed lower CS activity and higher lactate concentration, suggesting a stronger reliance on anaerobic pathways. We also measured white muscle histological variables that have been previously linked to whole‐animal metabolism: fiber diameter, number of nuclei per mm of fiber and myonuclear domain (MND) of both morphotypes at 25°C to examine cell‐level differences in muscle morphology. However, we found no differences in fiber diameter, number of nuclei per mm of fiber or MND between the two morphotypes. Thus, although the cellular morphology is similar in these species, the environmental differences in the evolution of the two morphs has led to differences in their metabolic profiles. Research highlights: Using white muscle of both morphotypes of the Mexican cavefish to determine citrate synthase (CS) activity as a measure of aerobic potential, and lactate concentration as a measure of anaerobic potential at three different chronic acclimation temperatures (14°C, 25°C, and 31°C), we found that the surface morphotype had higher CS activity and lower lactate concentration, suggesting an overall more efficient usage of aerobic metabolism; whereas the cave morphotype showed lower CS activity and higher lactate concentration, suggesting a stronger reliance on anaerobic pathways. We also measured white muscle fiber diameter, number of nuclei per mm of fiber and myonuclear domain (MND) of both morphotypes at 25°C to examine cell‐level differences in muscle morphology. But, we found no differences in fiber diameter, number of nuclei per mm of fiber or MND between the two morphotypes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Psalidodon jequitinhonhae was originally proposed as a variety of Tetragonopterus rutilus, based on the analysis of 14 specimens from the Jequitinhonha River, Brazil. In 1910 it was relocated in Astyanax, as A. fasciatus jequitinhonhae in Tetragonopterinae and in 2020 in Psalidodon, as Stethaprioninae member. However, in none of these revisions, P. jequitinhonhae was morphologically redescribed. A short sequence of the Cytochrome c oxidase subunit I (COI) gene obtained from one of the syntypes is compared to sequences obtained from new samples, allowing the recognition of the species and its morphological redescription based on new specimens. Both morphological and molecular data converged and corroborated P. jequitinhonhae as a valid species, occurring in the Jequitinhonha and Pardo river basins in Brazil. The syntype that provided the analyzed COI sequence is referred to as the lectotype by present designation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Rudimentary structures are variable, which is most impressively demonstrated by the eyes and dark body pigmentation of species evolving in caves. As these can often still be crossed with their ancestral surface sister forms, variability is usually attributed to hybridization. For two invertebrate and two vertebrate cave species, I show here that in spite of genetic separation between surface and cave forms, variability of the rudimentary structures is nonetheless present. This probably results from the loss of stabilizing selection, because neutral deleterious mutations are no longer eliminated. In contrast, compensatory traits in caves do not differ from their surface forms with respect to variability, because both are subject to selection. In the phylogenetically young Astyanax cave populations, traits compensatory to cave life have achieved different stages of adaptation compared to the phylogenetically old populations. They may show the same degree of differentiation, be intermediate or do not differ from the surface ancestor. This is attributed to different intensities of selection. [ABSTRACT FROM AUTHOR]

In the face of increasing environmental temperatures, operative differences between mitochondrial function and whole-animal phenotypic response to the environment are underrepresented in research, especially in subtemperate ectothermic vertebrates. A novel approach to exploring this connection is to examine model species that are genetically similar but that have different whole-animal phenotypes, each of which inhabits different environments. The blind Mexican cavefish (Astyanax mexicanus) has the following two morphotypes: a surface form found in aboveground rivers and an obligate cave-dwelling form. Each morphotype inhabits vastly different thermal and oxygen environments. Whole-animal and mitochondrial responses to thermal acclimation and oxidative stress, with respect to increasing temperatures, have not been previously determined in either morphotype of this species. Here, we chronically acclimated both morphotypes to three temperatures (14°C, 25°C, and 31°C) to establish potential for acclimation and critical thermal maxima (CTmax) for each morphotype of this species. After measuring CTmax in six cohorts, we additionally measured enzymatic antioxidant capacity (catalase, superoxide dismutase, and glutathione peroxidase activities), peroxyl scavenging capacity, and lipid peroxidation damage in white epaxial muscle for each individual. We found a significant effect of acclimation temperature on CTmax (F = 29.57 , P < 0.001) but no effect of morphotype on CTmax (F = 2.092 , P = 0.162). Additionally, we found that morphotype had a significant effect on glutathione peroxidase activity, with the surface morphotype having increased glutathione peroxidase activity compared with the cave morphotype (F = 6.270 , P = 0.020). No other oxidative stress variable demonstrated significant differences. Increases in CTmax with chronic thermal acclimation to higher temperatures suggests that there is some degree of phenotypic plasticity in this species that nominally occupies thermally stable environments. The decreased glutathione peroxidase activity in the cave morphotype may be related to decreased environmental oxygen concentration and decreased metabolic rate in this environmentally constrained morphotype compared to in its surface-living counterparts. [ABSTRACT FROM AUTHOR]

The vertebrate brain is highly conserved topologically, but less is known about neuroanatomical variation between individual brain regions. Neuroanatomical variation at the regional level is hypothesized to provide functional expansion, building upon ancestral anatomy needed for basic functions. Classically, animal models used to study evolution have lacked tools for detailed anatomical analysis that are widely used in zebrafish and mice, presenting a barrier to studying brain evolution at fine scales. In this study, we sought to investigate the evolution of brain anatomy using a single species of fish consisting of divergent surface and cave morphs, that permits functional genetic testing of regional volume and shape across the entire brain. We generated a high-resolution brain atlas for the blind Mexican cavefish Astyanax mexicanus and coupled the atlas with automated computational tools to directly assess variability in brain region shape and volume across all populations. We measured the volume and shape of every grossly defined neuroanatomical region of the brain and assessed correlations between anatomical regions in surface fish, cavefish, and surface × cave F2 hybrids, whose phenotypes span the range of surface to cave. We find that dorsal regions of the brain are contracted, while ventral regions have expanded, with F2 hybrid data providing support for developmental constraint along the dorsal-ventral axis. Furthermore, these dorsal-ventral relationships in anatomical variation show similar patterns for both volume and shape, suggesting that the anatomical evolution captured by these two parameters could be driven by similar developmental mechanisms. Together, these data demonstrate that A. mexicanus is a powerful system for functionally determining basic principles of brain evolution and will permit testing how genes influence early patterning events to drive brain-wide anatomical evolution. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Antibiotics are present in the environment and can be bioaccumulated by organisms and transferred through the food chain, which poses a problem when considering fish meat consumption. The study focused on lambari, a fish with potential in aquaculture because of its ease of rearing in small-scale operations. The objective of the study was to measure the bioaccumulation and clearance of the antibiotic sulfadiazine (SDZ) in lambaris. The tests were divided into two phases: exposure and depletion. During exposure, the fish were fed medicated feed for seven days, and during depletion they were transferred to clean tanks and fed uncontaminated feed for another seven days. SDZ concentrations increased in the fish over the days, with the greatest accumulation occurring on day seven. After the depletion phase, SDZ concentrations decreased. The results showed that there was little bioaccumulation of SDZ in the fish, but that the compound was more present in the water. The results also indicated that the concentrations of SDZ were below the established maximum limit. This study contributes to the understanding of the dynamics of SDZ in an aquatic species native to Brazil. Antibiotics are present in the environment, primarily due to their release through wastewater treatment plants, agricultural practices, and improper disposal of unused medications. In the environment, these drugs can be bioaccumulated by organisms and transferred along the food chain. This is a problem when considering the consumption of fish meat. In the United States, legislation stipulates that the maximum residue limit for sulfadiazine (SDZ) should not exceed 100 μg kg−1. Lambari fishes have potential economic importance in aquaculture, as they are relatively easy to breed and can be raised in small-scale operations. Finally, studying the biology and ecology of lambari could provide valuable information about freshwater ecosystems and their inhabitants. The current work aimed to measure the bioaccumulation and depletion of the antibiotic SDZ 14C in lambari (Astyanax bimaculatus). For this purpose, the tests were divided into two stages; seven days of exposure and seven days of depletion, where one fish was randomly selected and sampled every day. In the exposure phase, the fish were fed the medicated feed three times a day at a concentration of 2.5 mg·g−1. The control fish were fed uncontaminated feed. For the depletion phase, the remaining lambari were transferred to clean tanks and fed uncontaminated feed three times a day. The fish samples were burned in the Oxidizer and the reading of radioactivity was performed in a liquid scintillation spectrometer. It is worth noting that on day 7 and day 14, the water in the aquariums was filtered through filter paper to collect the metabolic excrement. SDZ concentrations increased over the days and accumulation occurred in the fish, with day seven presenting the maximum accumulation value of 91.7 ng·g−1 due to feeding uptake. After the depletion phase on day 13, the value found was 0.83 ng·g−1. The bioconcentration factor calculated was 20 L·kg−1. After the bioaccumulation period, the concentrations of SDZ in the water and excreta were 4.5 µg·L−1 and 363.5 ng·g−1, respectively. In the depletion period, the concentrations in the water and excreta were 0.01 µg·L−1 and 5.96 ng·g−1, respectively. These results imply that there was little SDZ bioaccumulation in the fish, but that it was distributed in larger amounts in the water. This is due to the physicochemical properties of the molecule with the low Log P value. Regarding the maximum residue limit, the value was below the established value. This study contributes to understanding SDZ dynamics in an aquatic species native to Brazil. [ABSTRACT FROM AUTHOR]

Reduction or complete loss of traits is a common occurrence throughout evolutionary history. In spite of this, numerous questions remain about why and how trait loss has occurred. Cave animals are an excellent system in which these questions can be answered, as multiple traits, including eyes and pigmentation, have been repeatedly reduced or lost across populations of cave species. This review focuses on how the blind Mexican cavefish, Astyanax mexicanus , has been used as a model system for examining the developmental, genetic, and evolutionary mechanisms that underlie eye regression in cave animals. We focus on multiple aspects of how eye regression evolved in A. mexicanus , including the developmental and genetic pathways that contribute to eye regression, the effects of the evolution of eye regression on other traits that have also evolved in A. mexicanus , and the evolutionary forces contributing to eye regression. We also discuss what is known about the repeated evolution of eye regression, both across populations of A. mexicanus cavefish and across cave animals more generally. Finally, we offer perspectives on how cavefish can be used in the future to further elucidate mechanisms underlying trait loss using tools and resources that have recently become available. [ABSTRACT FROM AUTHOR]

Astyanax mexicanus is a well-known model species, that has two morphotypes, cavefish, from subterranean rivers and surface fish, from surface rivers. They are morphologically distinct due to many troglomorphic traits in the cavefish, such as the absence of eyes. Most studies on A. mexicanus are focused on eye development and protein-coding genes involved in the process. However, lncRNAs did not get the same attention and very little is known about them. This study aimed to fill this knowledge gap, identifying, describing, classifying, and annotating lncRNAs expressed in the embryo's eye tissue of cavefish and surface fish. To do so, we constructed a concise workflow to assemble and evaluate transcriptomes, annotate protein-coding genes, ncRNAs families, predict the coding potential, identify putative lncRNAs, map them and predict interactions. This approach resulted in the identification of 33,069 and 19,493 putative lncRNAs respectively mapped in cavefish and surface fish. Thousands of these lncRNAs were annotated and identified as conserved in human and several species of fish. Hundreds of them were validated in silico, through ESTs. We identified lncRNAs associated with genes related to eye development. This is the case of a few lncRNAs associated with sox2, which we suggest being isomorphs of the SOX2-OT, a lncRNA that can regulate the expression of sox2. This work is one of the first studies to focus on the description of lncRNAs in A. mexicanus, highlighting several lncRNA targets and opening an important precedent for future studies focusing on lncRNAs expressed in A. mexicanus. [ABSTRACT FROM AUTHOR]

Herbicides are used in agriculture to control harmful crop weeds, prevent algae proliferation, and enhance macrophyte growth. Herbicide contamination of water bodies might exert toxic effects on fish in different development stages. Sperm, embryos, and adults of Astyanax altiparanae were used as a model to examine the detrimental effects of the following herbicide formulations: Roundup Transorb® (glyphosate), Arsenal® NA (imazapyr), and Reglone® (diquat). The lethal concentration 50 (LC50) values for adults using glyphosate and imazapyr were 3.14 mg/L and 4.59 mg/L, respectively, while the LC50 was higher than 28 mg/L for diquat. For the initial stages of embryo development, LC50 values were 16.52 mg/L glyphosate, 9.33 mg/L imazapyr, and 1084 mg/L diquat. Inhibition of sperm motility was noted at 252 mg/L glyphosate, 137 mg/L imazapyr, and 11,300 mg/L diquat, with an average sperm viability of 12.5%, 73.2%, and 89.3%, respectively, compared to 87.5% detected to control. A.altiparanae exhibited different sensitivities to the herbicide formulations investigated in the developmental stages evaluated. Roundup Transorb® exposure was more toxic for adults, while Arsenal® NA was most harmful for early embryonic development and inhibited sperm motility. Reglone® demonstrated low toxicity for A.altiparanae compared to Roundup Transorb® and Arsenal® NA. A.altiparanae may be considered an emerging fish model for toxicological studies for the neotropical region due to its wide distribution and biological characteristics. [ABSTRACT FROM AUTHOR]

This study aimed to verify whether the inclusion of omega-3 polyunsaturated fatty acids (PUFAs-ô3) in the diet of Astyanax lacustris males improves seminal quality. Four hundred fish were arranged in 20 boxes of 180 L in the recirculation system. These were fed with isoprotein diets (32% CP), until apparent satiation, with four levels of marine fish oil rich in PUFAs-ô3 inclusion (In% = 0, 3, 6, and 9), for 105 days. After, males were hormonally induced to spermiation, and semen was collected after 226 ATUs. Parameters evaluated were osmolality, seminal volume and color, spermatic concentration, sperm morphology, membrane integrity and, sperm motility (total and progressive motility, velocity curvilinear, straight line and average path, linearity and rectilinearity coefficients, trajectory oscillation, head lateral displacement amplitude, and cross-beat frequency). Seminal volume was greater in the In-0 and In-3 groups. Inclusion of PUFAs-ô3 positively influenced the kinetic parameters, as treatment with 6% and 9% of inclusion fish oil resulted in higher values for most of these parameters and did not differ statistically from each other. Thus, it is concluded that the addition of PUFAs-ô3 to the feed of breeders significantly improved the seminal quality of A. lacustris males. [ABSTRACT FROM AUTHOR]

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