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.

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

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

A 3D molecular map of the cavefish neural plate illuminates eye-field organization and its borders in vertebrates.

The vertebrate retinas originate from a specific anlage in the anterior neural plate called the eye field. Its identity is conferred by a set of 'eye transcription factors', whose combinatorial expression has been overlooked. Here, we use the dimorphic teleost Astyanax mexicanus, which develops proper eyes in the wild type and smaller colobomatous eyes in the blind cavefish embryos, to unravel the molecular anatomy of the eye field and its variations within a species. Using a series of markers (rx3, pax6a, cxcr4b, zic1, lhx2, emx3 and nkx2.1a), we draw a comparative 3D expression map at the end of gastrulation/onset of neurulation, which highlights hyper-regionalization of the eye field into sub-territories of distinct sizes, shapes, cell identities and combinatorial gene expression levels along the three body axes. All these features show significant variations in the cavefish natural mutant. We also discover sub-domains within the prospective telencephalon and characterize cell identities at the frontiers of the eye field. We propose putative fates for some of the characterized eye-field subdivisions, and suggest the existence of a trade-off between some subdivisions in the two Astyanax morphs on a micro-evolutionary scale.

Reinterpreting the work of Charles Breder: Sensory neuromasts and orbital skeleton variation in eyeless Astyanax cavefish.

Charles Breder, a pioneering researcher of blind Mexican cavefish was the first to note extreme variation in the facial skeleton of this intriguing subterranean-dwelling organism. Using a system of polar coordinate plots, he identified substantial dysmorphic changes affecting bones of the orbital skeleton. A complication of his landmark publication from 1944 was an error in the number of orbital bones depicted for this species. Intriguingly, however, he proposed an unknown "organizing force" likely influences final bone position and associated dysmorphia. At the time this was merely hypothetical. Roughly eight decades since its publication, however, insights into sensory influences on facial bone development may explain dysmorphia and variation in bone numbers for Astyanax cavefish. A morphological association between mechano-sensory neuromasts of the lateral line and dermal bones of the facial skeleton had been appreciated in the classical literature, but the polarity of this interaction has long remained unclear. Here, we propose that sensory-skeletal integration between sensory neuromasts and bones explain the incomplete numbers of bones, and dysmorphic features such as fusion between neighboring elements. We propose that in closely-related surface fish (and most teleost fish) this developmental coupling enables the sensory and skeletal systems to become integrated into a functional unit over the course of life history. In this opinion article, we discuss the relevance of this (poorly understood) phenomenon as a potential evolutionary source of variation in the facial bone structures of taxa across deep geologic time. We provide three potential explanations for the error in Breder's drawings, that may be explained by natural developmental variation documented in other related species. Moreover, we argue that the natural variation in this "evolutionary" model system is useful for explaining diverse cranial features by uniting aberrations occurring during embryogenesis with long-term adult dysmorphia.

Unraveling stress resilience: Insights from adaptations to extreme environments by Astyanax mexicanus cavefish.

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.

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.

Primordial germ cells of Astyanax altiparanae, isolated and recovered intact after vitrification: A preliminary study for potential cryopreservation of Neotropical fish germplasm.

Primordial germ cells (PGCs) constitute an important cell lineage that directly impacts genetic dissemination and species conservation through the creation of cryobanks. In order to advance the field of animal genetic cryopreservation, this work aimed to recover intact PGCs cryopreserved in embryonic tissues during the segmentation phase for subsequent in vitro maintenance, using the yellow-tailed tetra (Astyanax altiparanae) as a model organism. For this, a total of 202 embryos were distributed in two experiments. In the first experiment, embryos in the segmentation phase were dissociated, and isolated PGCs were maintained in vitro. They were visualized using gfp-Pm-ddx4 3'UTR labeling. The second experiment aimed to vitrify PGCs using 3 cryoprotective agents or CPAs (dimethyl sulfoxide, ethylene glycol, and 1,2 propanediol) at 3 molarities (2, 3, and 4 M). The toxicity, somatic cell viability, and recovery of intact PGCs were evaluated. After cryopreservation and thawing, 2 M ethylene glycol produced intact PGCs and somatic cells (44 ± 11.52 % and 42.35 ± 0.33 %, respectively) post-thaw. The recovery of PGCs from frozen embryonic tissues was not possible without the use of CPAs. Thus, the vitrification of PGCs from an important developmental model and Neotropical species such as A. altiparanae was achieved, and the process of isolating and maintaining PGCs in a culture medium was successful. Therefore, to ensure the maintenance of genetic diversity, PGCs obtained during embryonic development in the segmentation phase between 25 and 28 somites were stored through vitrification for future applications in the reconstitution of species through germinal chimerism.

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.

Immunological effects of DNA vaccination and interleukin utilization as an adjuvant in Astyanax lacustris immunized against Ichthyophthirius multifiliis.

The increasing significance of the aquaculture sector and commercially valuable species underscores the need to develop alternatives for controlling diseases such as Ichthyophthirius multifiliis-induced ichthyophthiriasis. This ciliated protozoan parasite threatens nearly all freshwater fish species, causing substantial losses in the fishery industry. Despite this, effective large-scale treatments are lacking, emphasizing the necessity of adopting preventive strategies. While the pathogenesis of ichthyophthiriasis and its immune stimulation allows for vaccination strategies, precise adjustments are crucial to ensure the production of an effective vaccine compound. Therefore, this study aimed to evaluate the impact of immunizing Astyanax lacustris with a genetic vaccine containing IAG52A from I. multifiliis and the molecular adjuvant IL-8 from A. lacustris. Transcript analysis in immunized A. lacustris indicated mRNA production in fish muscles, demonstrating an expression of this mRNA. Fish were divided into five groups, receiving different vaccine formulations, and all groups received a booster dose 14 days after the initial immunization. Samples from vaccinated fish showed increased IL-1ß mRNA expression in the spleen within 6 h post the second dose and after 14 days. In the head kidney, IL-1ß mRNA expression showed no significant difference at 6 and 24 h but an increase was noted in fish injected with IAG and IAG + IL-8 after 14 days. IL-8 mRNA expression in the spleen and kidney did not significantly differ from the control group. Histological analysis revealed no variation in leukocyte concentration at 6 and 24 h post-vaccination; however, after 14 days, the groups injected with IAG and IAG + IL-8 exhibited a higher leukocyte density at the application sites than the control. The obtained data suggest that the used vaccine is transcribed, indicating its potential to stimulate innate immune response parameters through mRNA cytokine expression and leukocyte migration.

Effect of water calcium, copper, and silver on branchial Na+ permeability in a characid and cichlid fish.

We tested the hypothesis that water Ca2+ is involved in control of branchial Na+ permeability in low pH tolerant convict cichlids and black neon tetras. We measured Na+ efflux in water with different Ca2+ concentrations during exposure to low pH, silver, and copper, at levels which are known to stimulate Na+ efflux. For convict cichlids at pH 7.5 exposure to 0 µmol L-1 Ca2+caused Na+ efflux to rise 2.5 times above controls at 100 µmol L-1 Ca2+. However, raising [Ca2+] to 500 µmol L-1 had no effect. Upon exposure to pH 3.5 (control [Ca2+]) Na+ efflux rose almost 5× and increasing the [Ca2+] 5-fold did not reduce the magnitude of stimulation. Exposure to 1 µmol L-1 silver and 25 µmol L-1 copper stimulated Na+ efflux 7×, and 2×, respectively. Raising [Ca2+] concentration during metal exposure halved the stimulation of Na+ efflux caused by silver, and eliminated the stimulation elicited by copper. For black neon tetras raising or lowering water [Ca2+] had no effect on Na+ efflux at pH 7.5. Exposure to pH 3.5 caused Na+ efflux to rise 2.5× but changing [Ca2+] had no effect. Exposure to 1 µmol L-1 silver, or 25 µmol L-1 copper caused Na+ efflux of tetras to rise 4-fold and 3-fold, respectively. Raising [Ca2+] during silver exposure reduced the stimulation of Na+ efflux by about 50%, but during copper exposure increased [Ca2+] had no effect on stimulation of Na+ efflux. These results suggest water Ca2+ plays a role in control of branchial Na+ permeability in cichlids, but perhaps not tetras. In addition, the silver and copper concentrations required to inhibit Na+ uptake and stimulate Na+ efflux were higher than the concentrations used on non-characids and non-cichlids, which indicates that our fish are much more tolerant of these metals.

Biochemical changes and bioaccumulation of manganese in Astyanax lacustris (Teleostei: Characidae).

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.

Trophic plasticity of tetra-fish, Psalidodon aff. fasciatus (Cuvier, 1819) (Characidae), in an Atlantic Forest stream of Northeast Brazil.

Trophic plasticity is a distinctive feature of freshwater fishes, representing an essential strategy for fish living in resource-variable environments. We analyzed the stomach contents of individuals sampled in two Atlantic Forest streams to identify the primary food sources consumed by Psalidodon aff. fasciatus and verify the existence of spatial, seasonal, and ontogenetic variations. The diet was determined by analyzing the stomach contents using the Volume Method to quantify the importance of food items. In general, Psalidodon aff. fasciatus was classified as an omnivorous species, consuming mainly insects, plant material, and filamentous algae. The results also showed significant effects for all factors considered (spatial, seasonal, and ontogenetic). Finally, Psalidodon aff. fasciatus demonstrated considerable trophic plasticity, which can result in better use of available resources in the environment and improved resource partitioning, reducing intraspecific and interspecific competition.

Growth and carbon turnover of Piaractus mesopotamicus Holmberg, 1887 (Osteicthyes: Characidae): contribution of extruded feed and natural food.

Piaractus mesopotamicus, is a fish usually farmed in semi-intensive systems with access to natural food and supplementary feed. This study evaluates effects of feed allowance on the productive performance, carbon turnover and proportions of nutrient (carbon) contribution of feed and natural food for the growth of pacu. Juvenile fish were stocked in fiberglass tanks and fed to 100, 75, 50, 25, 0% apparent satiety (ApS), with a practical, extruded (C4 photosynthetic pathway) feed in a randomized design trial (n=3); plankton production for simulated semi-intensive farming system condition was induced by chemical fertilization. A control treatment was set up in tanks devoid of natural food. Data on muscle stable carbon isotope ratios were used to study carbon turnover using a relative growth-based model. Low variation of the δ13C impaired fitting a turnover model curve for the 0 and 25 % ApS treatments. Fish of the 100% and 75% ApS treatments reached circa 95% and 82.85% of the carbon turnover, respectively. Extruded feed was the main nutrient source for the growth of pacu in the semi-intensive, simulated farming condition. The current study contributes to the knowledge of the relationship between feeding rates and carbon turnover rates in the pacu muscle.

Effect of dietary caffeine supplementation on the carcass composition of pacu Piaractus mesopotamicus.

Pacu (Piaractus mesopotamicus) is a fish with a high production potential in Brazil. However, one limitation is the excessive amount of ether extract in its carcass, an undesirable characteristic for the consumer. One approach to overcome this limitation is to improve carcass quality through zootechnical additives such as caffeine. The aim of this study was to evaluate the effect of supplementing the diet of pacu with caffeine on cut yield, biological indices, and carcass composition. Two hundred pacu with an initial weight of 1,687 g were used. The animals were allocated to 20 aquaculture cages of 1 m³, with 10 animals per cage. A completely randomized design with four treatments and five replicates was used. The treatments evaluated consisted of four inclusion levels of caffeine: T1 = 0.00 g; T2 = 0.16 g; T3 = 0.32 g, and T4 = 0.48 g caffeine.kg-1 of feed. The findings show that caffeine can be recommended as a diet supplement for carcass improvement of pacu, reducing the fat content and increasing the protein content of the carcass. Caffeine up to 0.32 g.kg-1 of feed can be added to the diet of pacu without affecting its performance or cut yield.