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.

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

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

Loss of behavioral stress response in blind cavefish reduces energy expenditure.

The stress response is essential for animal self-defense and survival. However, species may exhibit stress response variation depending on their specific environmental and selection pressures. Blind cavefish dwell in cave environments, which differ markedly in stressors and resource availability compared to surface aquatic environments. However, whether blind cavefish exhibit differences in stress response as an adaptation to their cave environments remains unclear. Here, we investigated differences in stress response in six closely related Triplophysa species, including three blind cavefish (T. longibarbata, T. jiarongensis, and T. rosa) and three normal-sighted river fish (T. nasobarbatula, T. dongsaiensis, and T. bleekeri). Results showed that blind cavefish exhibited a range of distinct behavioral responses compared to sighted river fish, including greater levels of activity, shorter duration of freezing, absence of erratic movements or thrashing behavior, and opposite behavioral trends over time. Furthermore, the cavefish species demonstrated attenuated increases in metabolic rate in response to stressors related to novel environments. Cave-dwelling T. rosa also exhibited lower basal hypothalamic-pituitary-inter-renal (HPI) axis-related gene expression levels and stress hormone concentrations compared to river-dwelling T. bleekeri. These results suggest that blind cavefish may have lost their behavioral stress response, potentially mediated by a reduction in basal activity of the HPI axis, thus enabling the conservation of energy by reducing unnecessary expenditure in energy-limited caves.

Effects of temperature on food intake and the expression of appetite regulators in three Characidae fish: The black-skirted tetra (Gymnocorymbus ternetzi), neon tetra (Paracheirodon innesi) and Mexican cavefish (Astyanax mexicanus).

The Characidae family of fish is composed of commercially important species for which little is known about the regulation of feeding. Fish are ectotherms so that their body temperature fluctuates with the temperature of the surrounding water. Changes in water temperature can thus have major effects on the physiology of fish, in particular their feeding. The mechanisms by which appetite is influenced by changes in temperatures in fish remain unclear. In this study, we examined the effects of temperature on feeding behavior, food intake and the expression of appetite regulators in three characid fish (black tetra, neon tetra and cavefish) by submitting them to four different temperatures for 2 weeks (20°C, 24°C, 28°C, 32°C). In all species, food intake increased with increasing temperature. In neon and black tetras, increasing temperatures decreased expressions of orexin and leptin and increased that of cocaine and amphetamine regulated transcript (CART). In cavefish, temperature had no effect on brain orexin, leptin or CART. In all three species, higher temperatures induced increases in intestine expression of cholecystokinin (CCK), but no effects were seen for intestine ghrelin and peptide YY expressions. Our results show that temperature affects feeding in Characidae fish and induces species-specific changes in the expression of appetite regulators.

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

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

Blind cavefish retain functional connectivity in the tectum despite loss of retinal input.

Sensory systems display remarkable plasticity and are under strong evolutionary selection. The Mexican cavefish, Astyanax mexicanus, consists of eyed river-dwelling surface populations and multiple independent cave populations that have converged on eye loss, providing the opportunity to examine the evolution of sensory circuits in response to environmental perturbation. Functional analysis across multiple transgenic populations expressing GCaMP6s showed that functional connectivity of the optic tectum largely did not differ between populations, except for the selective loss of negatively correlated activity within the cavefish tectum, suggesting positively correlated neural activity is resistant to an evolved loss of input from the retina. Furthermore, analysis of surface-cave hybrid fish reveals that changes in the tectum are genetically distinct from those encoding eye loss. Together, these findings uncover the independent evolution of multiple components of the visual system and establish the use of functional imaging in A. mexicanus to study neural circuit evolution.

Evolutionary convergence of a neural mechanism in the cavefish lateral line system.

Animals can evolve dramatic sensory functions in response to environmental constraints, but little is known about the neural mechanisms underlying these changes. The Mexican tetra, Astyanax mexicanus, is a leading model to study genetic, behavioral, and physiological evolution by comparing eyed surface populations and blind cave populations. We compared neurophysiological responses of posterior lateral line afferent neurons and motor neurons across A. mexicanus populations to reveal how shifts in sensory function may shape behavioral diversity. These studies indicate differences in intrinsic afferent signaling and gain control across populations. Elevated endogenous afferent activity identified a lower response threshold in the lateral line of blind cavefish relative to surface fish leading to increased evoked potentials during hair cell deflection in cavefish. We next measured the effect of inhibitory corollary discharges from hindbrain efferent neurons onto afferents during locomotion. We discovered that three independently derived cavefish populations have evolved persistent afferent activity during locomotion, suggesting for the first time that partial loss of function in the efferent system can be an evolutionary mechanism for neural adaptation of a vertebrate sensory system.

Liver-derived cell lines from cavefish Astyanax mexicanus as an in vitro model for studying metabolic adaptation.

Cell lines have become an integral resource and tool for conducting biological experiments ever since the Hela cell line was first developed (Scherer et al. in J Exp Med 97:695-710, 1953). They not only allow detailed investigation of molecular pathways but are faster and more cost-effective than most in vivo approaches. The last decade saw many emerging model systems strengthening basic science research. However, lack of genetic and molecular tools in these newer systems pose many obstacles. Astyanax mexicanus is proving to be an interesting new model system for understanding metabolic adaptation. To further enhance the utility of this system, we developed liver-derived cell lines from both surface-dwelling and cave-dwelling morphotypes. In this study, we provide detailed methodology of the derivation process along with comprehensive biochemical and molecular characterization of the cell lines, which reflect key metabolic traits of cavefish adaptation. We anticipate these cell lines to become a useful resource for the Astyanax community as well as researchers investigating fish biology, comparative physiology, and metabolism.

Kinematic analysis of social interactions deconstructs the evolved loss of schooling behavior in cavefish.

Fish display a remarkable diversity of social behaviors, both within and between species. While social behaviors are likely critical for survival, surprisingly little is known about how they evolve in response to changing environmental pressures. With its highly social surface form and multiple populations of a largely asocial, blind, cave-dwelling form, the Mexican tetra, Astyanax mexicanus, provides a powerful model to study the evolution of social behavior. Here we use motion tracking and analysis of swimming kinematics to quantify social swimming in four Astyanax mexicanus populations. In the light, surface fish school, maintaining both close proximity and alignment with each other. In the dark, surface fish no longer form coherent schools, however, they still show evidence of an attempt to align and maintain proximity when they find themselves near another fish. In contrast, cavefish from three independently-evolved populations (Pachón, Molino, Tinaja) show little preference for proximity or alignment, instead exhibiting behaviors that suggest active avoidance of each other. Two of the three cave populations we studied also slow down when more fish are present in the tank, a behavior which is not observed in surface fish in light or the dark, suggesting divergent responses to conspecifics. Using data-driven computer simulations, we show that the observed reduction in swimming speed is sufficient to alter the way fish explore their environment: it can increase time spent exploring away from the walls. Thus, the absence of schooling in cavefish is not merely a consequence of their inability to see, but may rather be a genuine behavioral adaptation that impacts the way they explore their environment.

Alterations to cavefish red blood cells provide evidence of adaptation to reduced subterranean oxygen.

Animals inhabiting extreme environments allow the powerful opportunity to examine adaptive evolution in response to diverse pressures. One such pressure is reduced oxygen, commonly present at high-altitude and subterranean environments. Cave-dwelling animals must also deal with darkness and starvation, both of which have been rigorously studied as key forces driving the evolution of cave-associated traits. Interestingly, hypoxia as an environmental pressure has received less attention. Here we examined putatively adaptive phenotypes evolving in a freshwater teleost fish, Astyanax mexicanus, which includes both surface- and cave-dwelling forms. This model system also provides the opportunity to identify convergent responses to hypoxia, owing to the presence of numerous natural and independently-colonised cave populations, alongside closely-related surface conspecifics. The focus of this study is hemoglobin, an essential molecule for oxygen transport and delivery. We found that multiple cave populations harbor a higher concentration of hemoglobin in their blood, which is coincident with an increase in cave morph erythrocyte size compared to surface fish. Interestingly, both cave and surface morphs have comparable numbers of erythrocytes per unit of blood, suggesting elevated hemoglobin is not due to overproduction of red blood cells. Alternatively, owing to an increased cell area of erythrocytes in cavefish, we reason that they contain more hemoglobin per erythrocyte. These findings support the notion that cavefish have adapted to hypoxia in caves through modulation of both hemoglobin production and erythrocyte size. This work reveals an additional adaptive feature of Astyanax cavefish, and demonstrates that coordinated changes between cellular architecture and molecular changes are necessary for organisms evolving under intense environmental pressure.

Fish can use hydrostatic pressure to determine their absolute depth.

Hydrostatic pressure is a global cue that varies linearly with depth which could provide crucial spatial information for fish navigating vertically; however, whether fish can determine their depth using hydrostatic pressure has remained unknown. Here we show that Mexican tetras (Astyanax mexicanus) can learn the depth of a food site and consistently return to it with high fidelity using only hydrostatic pressure as a cue. Further, fish shifted their search location vertically as predicted if using pressure alone to signal depth. This study uncovers new sensory information available to fish which allows them to resolve their absolute depth on a fine scale.

Social-like responses are inducible in asocial Mexican cavefish despite the exhibition of strong repetitive behavior.

Social behavior is a hallmark of complex animal systems; however, some species appear to have secondarily lost this social ability. In these non-social species, whether social abilities are permanently lost or suppressed is unclear. The blind cavefish Astyanax mexicanus is known to be asocial. Here, we reveal that cavefish exhibited social-like interactions in familiar environments but suppressed these interactions in stress-associated unfamiliar environments. Furthermore, the level of suppression in sociality was positively correlated with that of stereotypic repetitive behavior, as seen in mammals. Treatment with a human antipsychotic drug targeting the dopaminergic system induced social-like interactions in cavefish, even in unfamiliar environments, while reducing repetitive behavior. Overall, these results suggest that the antagonistic association between repetitive and social-like behaviors is deeply shared from teleosts through mammals.

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.

Modulation of immunity and hepatic antioxidant defense by corticosteroids in pacu (Piaractus mesopotamicus).

We investigated the impact of both the oral administration of hydrocortisone (HC) and an acute stressor on stress, innate immune responses and antioxidant system/oxidative stress responses of juvenile Piaractus mesopotamicus. Fish were either 1) given a commercial feed (C), 2) given a feed supplemented with 400 mg/kg HC, or 3) fed a commercial feed, chased for 2 min and exposed to air for 4 min (S). After initial sampling, fish C and HC were fed and sampled 1, 3, 6, 24 and 72 h post-feeding. Fish S were fed at the same time as the other groups, exposed to a stressor, and sampled 1, 3, 6, 24 and 72 h after. Exposure to the stressor increased circulating glucose and cortisol levels (at 1 and 3 h, respectively), while oral HC increased circulating cortisol at 1 h and glucose at 3 h. The stressor activated respiratory activity of leukocytes (RAL) at 3 h and reduced it at 6 h. HC did not activate RAL, but it did impair it at 6 h. The serum hemolytic activity of the complement system (HAC50) was impaired by the stressor at 1 and 3 h and by HC at 1 h. Regarding the antioxidant system, exposure to the stressor reduced glutathione peroxidase (GPx) and catalase (CAT) activity and decreased concentrations of reduced glutathione (GSH) in the liver up to 6 h. HC only impaired GPx. Additionally, stress induced the accumulation of melano-macrophage (MM) and melano-macrophage centers (MMC), which are biomarkers of oxidative stress, in the spleen. Differences in biomarkers in fish given cortisol and exposed to stress indicate that exogenous hormone was unable to precisely reproduce stress responses.

Endocrine disruption caused by the aquatic exposure to aluminum and manganese in Astyanax altiparanae (Teleostei: Characidae) females during the final ovarian maturation.

Aluminum (Al) and manganese (Mn) can be toxic to aquatic biota and cause endocrine disruption in fish, affecting reproduction. This study evaluates the physiological responses of the ray-finned teleost fish Astyanax altiparanae vitellogenic females after acute exposure (96 h) to Al and Mn (alone and combined) in acid pH followed by the same period of exposure to metal-free water in neutral pH. The aim of this second period of exposure was to assess the recovery capacity from the toxic effects these metals. Five experimental groups were established: a control in neutral pH (Ctrl), and acidic pH (Ac), aluminum (Al), manganese (Mn), and Al + Mn groups, maintaining the acidic pH in the groups to which metals were added. The following biological parameters were evaluated: metal tissue concentration, relative fecundity (RF: absolute fecundity/body mass). Plasma levels of cortisol (proxy for stress) and 17α hydroxyprogesterone (17α-OHP), and gene expression of pituitary lhß mRNA (proxies for final maturation) were measured to evaluate endocrine disruption. In the synchronic exposure, the presence of Mn potentiated the accumulation of Al in gills. The females from acidic pH and Al groups showed a reduced RF. Exposure to Al and Mn triggered an endocrine disruption response, evidenced by a decrease in the plasma concentration of 17α-OHP and cortisol. Despite this anti-steroidogenic effect, no changes occurred in the pituitary gene expression of lhß. The endocrine changes and the metal accumulation were temporary, while the impacts on RF under the experimental conditions suggest permanent impairment in the reproduction of this species.

Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus.

The Mexican tetra, Astyanax mexicanus, is an emerging model system for studies in development and evolution. The existence of eyed surface (surface fish) and blind cave (cave fish) morphs in this species presents an opportunity to interrogate the mechanisms underlying morphological and behavioral evolution. Cave fish have evolved novel constructive and regressive traits. The constructive changes include increases in taste buds and jaws, lateral line sensory organs, and body fat. The regressive changes include loss or reduction of eyes. melanin pigmentation, schooling behavior, aggression, and sleep. To experimentally interrogate these changes, it is crucial to obtain large numbers of spawned embryos. Since the original A. mexicanus surface fish and cave fish were collected in Texas and Mexico in the 1990s, their descendants have been routinely stimulated to breed and spawn large numbers of embryos bimonthly in the Jeffery laboratory. Although breeding is controlled by food abundance and quality, light-dark cycles, and temperature, we have found that incremental temperature changes play a key role in stimulating maximal spawning. The gradual increase of temperature from 72 °F to 78 °F in the first three days of a breeding week provides two-three consecutive spawning days with maximal numbers of high-quality embryos, which is then followed by a gradual decrease of temperature from 78 °F to 72 °F during the last three days of the spawning week. The procedures shown in this video outline the workflow before and during a laboratory breeding week for incremental temperature stimulated spawning.

Vibration attraction response is a plastic trait in blind Mexican tetra (Astyanax mexicanus), variable within subpopulations inhabiting the same cave.

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

The influence of increased water temperature on the duration of spermatogenesis in a neotropical fish, Astyanax altiparanae (Characiformes, Characidae).

In view of the established climate change scenario and the consequent changes in global temperature, it is essential to study its effects on animal spermatogenesis. Therefore, the aim of this study was to verify the duration of spermatogenesis at different temperatures. For this purpose, 96 male and adult specimens of Astyanax altiparanae were kept in a closed circulation system with water temperature stabilized at 27 °C and 32 °C. Subsequently, the specimens received pulses of BrdU (bromodeoxyuridine) at a concentration of 100 mg/kg/day for 2 consecutive days, and the samples were collected daily for a period of 15 days. Their testes were removed, fixed, processed in historesin, and sectioned in 3 µm, submitted to hematoxylin/eosin staining and to bromodeoxyuridine immunodetection. Partial results of the optimum temperature experiments allowed the classification of A. altiparanae spermatogenic cells in Aund, Adiff, and type B spermatogonia, spermatocytes, spermatids, and spermatozoa. The duration of spermatogenesis was determined as approximately 6 days for animals at a temperature of 27 °C and 1 day for animals at 32 °C. The elevated temperature was also responsible for increasing cell proliferation, resulting in an increase in the number of spermatocytes, spermatids, spermatozoa, and cell death (cell pyknotic). The duration of spermatogenesis in A. altiparanae was directly affected by the elevated water temperature, causing a reduction in the estimated time of spermatogenesis.

New insights into patterns and rates of tooth replacement in serrasalmid and characid fishes, with implications for the subsistence fishery of Peru's remote ribereños villages.

We explored patterns, rates and unexpected socio-ecological consequences of tooth replacement in serrasalmids and characids of the Peruvian Amazon using microcomputed tomography. Of 24 specimens collected in February 2019, representing a mix of red-bellied piranha Pygocentrus nattereri, redeye piranha Serrasalmus rhombeus, silver dollar fish Ctenobrycon hauxwellianus and mojara Astyanax abramis, six individuals possessed edentulous jaw quadrants. On average, 22.9% of fish collected per day from these species featured incomplete dentition, a value three to five times higher than anticipated based on replacement rates estimated from captive fish, differences that may be driven by ontogeny, seasonality or environmental quality.

Daily variation feeding of Astyanax lacustris (Lütken 1875) in a subtropical river

This study aimed to know the daily variation of Astyanax lacustris (Lütken 1875) feeding in a lotic environment. Fish were caught with a net for three days every three hours in the summer to capture ten individuals per hour in a stretch of the Ijuí River, Middle Uruguay River, Brazil. The captured specimens had their stomachs removed and the content analyzed and separated into seven food categories with the aid of stereomicroscope. For the analysis of food items were used the frequency of occurrence methods, volumetric method and applied the Alimentary Index (IAi). Two hundred stomachs were analyzed, of which 95% had food content. The most abundant items were algae and autochthonous insects. Astyanax lacustris feeds throughout the day especially early in the morning (9 hours) and reduces its feeding at night (24 hours and 3 hours). It was also verified variation of AI of different food items throughout the day. It is concluded that A. lacustris feeds throughout the day, especially in the daytime and that throughout the 24 hours it varies its diet due to photoperiod and food availability.