Effects of environmental hypoxia on the goldfish skeletal muscle: Focus on oxidative status and mitochondrial dynamics.

The skeletal muscle is a highly plastic tissue. Its ability to respond to external stimuli and challenges allows it to face the functional needs of the organism. In the goldfish Carassius auratus, a model of hypoxia resistance, exposure to reduced oxygen is accompanied by an improvement of the swimming performance, relying on a sustained contractile behavior of the skeletal muscle. At the moment, limited information is available on the mechanisms underlying these responses. We here evaluated the effects of short- (4 days) and long- (20 days) term exposure to moderate water hypoxia on the goldfish white skeletal muscle, focusing on oxidative status and mitochondrial dynamics. No differences in lipid peroxidation, measured as 2-thiobarbituric acid-reacting substances (TBARS), and oxidatively modified proteins (OMP) were detected in animals exposed to hypoxia with respect to their normoxic counterparts. Exposure to short-term hypoxia was characterized by an enhanced SOD activity and expression, paralleled by increased levels of Nrf2, a regulator of the antioxidant cell response, and HSP70, a chaperone also acting as a redox sensor. The expression of markers of mitochondrial biogenesis (TFAM) and abundance (VDAC) and of the mtDNA/nDNA ratio was similar under normoxia and under both short- and long-term hypoxia, thus excluding a rearrangement of the mitochondrial apparatus. Only an increase of PGC1α (a transcription factor involved in mitochondrial dynamics) was detected after 20 days of hypoxia. Our results revealed novel aspects of the molecular mechanisms that in the goldfish skeletal muscle may sustain the response to hypoxia, thus contributing to adequate tissue function to organism requirements.

Functional, structural, and molecular remodelling of the goldfish (Carassius auratus) heart under moderate hypoxia.

The goldfish (Carassius auratus) is known for its physiologic ability to survive even long periods of oxygen limitation (hypoxia), adapting the cardiac performance to the requirements of peripheral tissue perfusion. We here investigated the effects of short-term moderate hypoxia on the heart, focusing on ventricular adaptation, in terms of hemodynamics and structural traits. Functional evaluations revealed that animals exposed to 4 days of environmental hypoxia increased the hemodynamic performance evaluated on ex vivo cardiac preparations. This was associated with a thicker and more vascularized ventricular compact layer and a reduced luminal lacunary space. Compared to normoxic animals, ventricular cardiomyocytes of goldfish exposed to hypoxia showed an extended mitochondrial compartment and a modulation of proteins involved in mitochondria dynamics. The enhanced expression of the pro-fission markers DRP1 and OMA1, and the modulation of the short and long forms of OPA1, suggested a hypoxia-related mitochondria fission. Our data propose that under hypoxia, the goldfish heart undergoes a structural remodelling associated with a potentiated cardiac activity. The energy demand for the highly performant myocardium is supported by an increased number of mitochondria, likely occurring through fission events.

Benzodiazepine Delorazepam Induces Locomotory Hyperactivity and Alterations in Pedal Mucus Texture in the Freshwater Gastropod Planorbarius corneus.

Benzodiazepines, psychotropic drugs, are ubiquitous in the aquatic environment due to over-consumption and inefficient removal by sewage treatment plants. Bioaccumulation with consequent behavioral and physiological effects has been reported in many aquatic species. However, the responses are species-specific and still poorly understood. To improve the knowledge, we exposed the freshwater snail Planorbarius corneus to 1, 5, or 10 µg/L of delorazepam, the most widely consumed benzodiazepine in Italy. Conventional behavioral tests were used to assess the effects on locomotor and feeding behavior. Histological and biochemical analyses were also performed to detect possible changes in the structure and composition of the foot mucus and glands. The results show a paradoxical response with reduced feeding activity and locomotor hyperactivity. Pedal mucus was altered in texture but not in composition, becoming particularly rich in fibrous collagen-like material, and a significant change in the protein composition was highlighted in the foot. In conclusion, exposure to delorazepam induces disinhibited behavior in Planorbarius corneus, potentially increasing the risk of predation, and an increase in mucus protein production, which, together with reduced feeding activity, would severely compromise energy resources.

Nitric Oxide Function and Nitric Oxide Synthase Evolution in Aquatic Chordates.

Nitric oxide (NO) is a key signaling molecule in almost all organisms and is active in a variety of physiological and pathological processes. Our understanding of the peculiarities and functions of this simple gas has increased considerably by extending studies to non-mammal vertebrates and invertebrates. In this review, we report the nitric oxide synthase (Nos) genes so far characterized in chordates and provide an extensive, detailed, and comparative analysis of the function of NO in the aquatic chordates tunicates, cephalochordates, teleost fishes, and amphibians. This comprehensive set of data adds new elements to our understanding of Nos evolution, from the single gene commonly found in invertebrates to the three genes present in vertebrates.

1,3-Butanediol Administration Increases ß-Hydroxybutyrate Plasma Levels and Affects Redox Homeostasis, Endoplasmic Reticulum Stress, and Adipokine Production in Rat Gonadal Adipose Tissue.

Ketone bodies (KBs) are an alternative energy source under starvation and play multiple roles as signaling molecules regulating energy and metabolic homeostasis. The mechanism by which KBs influence visceral white adipose tissue physiology is only partially known, and our study aimed to shed light on the effects they exert on such tissue. To this aim, we administered 1,3-butanediol (BD) to rats since it rapidly enhances ß-hydroxybutyrate serum levels, and we evaluated the effect it induces within 3 h or after 14 days of treatment. After 14 days of treatment, rats showed a decrease in body weight gain, energy intake, gonadal-WAT (gWAT) weight, and adipocyte size compared to the control. BD exerted a pronounced antioxidant effect and directed redox homeostasis toward reductive stress, already evident within 3 h after its administration. BD lowered tissue ROS levels and oxidative damage to lipids and proteins and enhanced tissue soluble and enzymatic antioxidant capacity as well as nuclear erythroid factor-2 protein levels. BD also reduced specific mitochondrial maximal oxidative capacity and induced endoplasmic reticulum stress as well as interrelated processes, leading to changes in the level of adipokines/cytokines involved in inflammation, macrophage infiltration into gWAT, adipocyte differentiation, and lipolysis.

Structural and functional damage to the retina and skeletal muscle in Xenopus laevis embryos exposed to the commonly used psychotropic benzodiazepine delorazepam.

Benzodiazepines, psychotropic drugs, are among the most frequently found pharmaceuticals in aquatic matrices. An increasing number of studies are reporting their harmful effects on adults' behaviour and physiology, while little information is available regarding developing organisms exposed since early stages. Improper activation of GABA receptors during embryonic development is likely to induce relevant consequences on the morphogenesis and, at later stages, on behaviour. This study investigated the negative effects of three increasing concentrations of delorazepam on Xenopus laevis retinal and skeletal muscle morphogenesis. Morphological and ultrastructural investigations were correlated with gene expression, while Raman spectroscopy highlighted the main biochemical components affected. Conventional phototactic response and orientation in the magnetic field were assessed as indicators of proper interaction between sensory organs and the nervous system. Results confirm the profound impact of delorazepam on development and return an alarming picture of the amphibians' survival potentialities in a benzodiazepine-contaminated environment.

Aluminum induces a stress response in zebrafish gills by influencing metabolic parameters, morphology, and redox homeostasis.

Environmental air pollution and resulting acid rain have the effect of increasing aluminum levels in water bodies. We studied the effects of aluminum on fish gills, the tissue most exposed to aluminum, using zebrafish as an experimental model. Adult zebrafish were exposed to an aluminum concentration found in polluted environments (11 mg/L) for 10, 15 and 20 days and the effects on gill morphology, redox homeostasis (ROS content, NADPH oxidase, NOX, activity, oxidative damage, antioxidant enzymes, total antioxidant capacity, in vitro susceptibility to oxidants) and on behavioural and metabolic parameters (routine respiratory oxygen consumption rMO2, tail-beating frequency, cytochrome oxidase activity and muscle lactate content) were evaluated. Exposure to aluminum affects branchial histology, inducing alterations in primary and secondary lamellae and redox homeostasis, modifying ROS levels, NOX activity, lipid and protein oxidative damage, antioxidant enzymes, and total antioxidant capacities, and increases rMO2. The effects exhibited a time-dependent behaviour, suggesting the activation of an adaptive response. These changes are associated with a transition of muscle metabolism from aerobic to anaerobic, as suggested by the increase in muscle lactate content, which is probably functional to preserve locomotor performance. Overall, the results here reported provide new insights into the toxicity mechanisms of Al exposure on gill tissue and the subsequent adaptive response of aquatic species.

Behavioral alterations and gills damage in Mytilus galloprovincialis exposed to an environmental concentration of delorazepam.

Psychoactive compounds, and benzodiazepines (BZPs) in particular, represent an important class of emerging pollutants due to their large (ab)use and high resistance to degradation. Nowadays it is known that sewage treatment does not completely eliminate these substances and, therefore, BZPs and their metabolites reach concern levels in most aquatic environments all over Europe, ranging from µg/L to ng/L. In this study, we investigated the effects of delorazepam on Mytilus galloprovincialis, a model organism in toxicity testing and a key species in coastal marine ecosystems. Given its psychoactive activity, the study primarily addressed discovering the effects on behavior, by conventional valve opening and closure tests. Possible cytotoxic activity was also investigated by analyzing valve abductor muscles, gills histology, and correlated oxygen consumption. Results demonstrate negative effects on mussel behavior, interference with metabolism, and alteration of gill morphology and protein content. In conclusion, delorazepam confirms its toxicity to aquatic environments, highlighting the possibility that BZDs can ultimately affect the structure of the food web and the functions of the coastal ecosystems.

How sea urchins face microplastics: Uptake, tissue distribution and immune system response.

Plastic pollution represents one of the major threats to the marine environment. A wide range of marine organisms has been shown to ingest microplastics due to their small dimensions (less than 1 mm). This negatively affects some biological processes, such as feeding, energy reserves and reproduction. Very few studies have been performed on the effect of microplastics on sea urchin development and virtually none on adults. The aim of this work was to evaluate the uptake and distribution of fluorescent labelled polystyrene microbeads (micro-PS) in the Mediterranean sea urchin Paracentrotus lividus and the potential impact on circulating immune cells. Differential uptake was observed in the digestive and water vascular systems as well as in the gonads based on microbeads size (10 and 45 µm in diameter). Treatment of sea urchins with particles of both sizes induced an increase of the total number of immune cells already after 24 h. No significant differences were observed among immune cell types. However, the ratio between red and white amoebocytes, indicative of sea urchin healthy status, increased with both particles. This effect was detectable already at 24 h upon exposure to smaller micro-PS (10 µm). An increase of intracellular levels of reactive oxygen and nitrogen species was observed at 24 h upon both micro-PS exposure, whereas at later time these levels became comparable to those of controls. A significant increase of total antioxidant capacity was observed after treatment with 10 µm micro-PS. Overall data provide the first evidence on polystyrene microbeads uptake and tissue distribution in sea urchins, indicating a stress-related impact on circulating immune cells.

Effects of four food dyes on development of three model species, Cucumis sativus, Artemia salina and Danio rerio: Assessment of potential risk for the environment.

Food dyes, or color additives, are chemicals added to industrial food products and in domestic cooking to improve the perceived flavor and attractiveness. Of natural and synthetic origin, their safety has been long discussed, and concern for human safety is now clearly manifested by warnings added on products labels. Limited attention, however, has been dedicated to the effects of these compounds on aquatic flora and fauna. For this reason, the toxicity of four different commercially available food dyes (cochineal red E120, Ponceau red E124, tartrazine yellow E102 and blue Patent E131) was assessed on three different model organisms, namely Cucumis sativus, Artemia salina and Danio rerio that occupy diverse positions in the trophic pyramid. The evidence collected indicates that food dyes may target several organs and functions, depending on the species. C. sativus rate of germination was increased by E102, while root/shoot ratio was ∼20% reduced by E102, E120 and E124, seed total chlorophylls and carotenoids were 15-20% increased by E120 and 131, and total antioxidant activity was ∼25% reduced by all dyes. Mortality and low mobility of A. salina nauplii were increased by up to 50% in presence of E124, E102 and E131, while the nauplii phototactic response was significantly altered by E102, E120 and E124. Two to four-fold increases in the hatching percentages at 48 h were induced by E124, E102 and E131 on D. rerio, associated with the occurrence of 20% of embryos showing developmental defects. These results demonstrated that the food dyes examined are far from being safe for the aquatic organisms as well as land organisms exposed during watering with contaminated water. The overall information obtained gives a realistic snapshot of the potential pollution risk exerted by food dyes and of the different organism' ability to overcome the stress induced by contamination.

Assessing the Influence of Personality on Sensitivity to Magnetic Fields in Zebrafish.

To orient themselves in their environment, animals integrate a wide array of external cues, which interact with several internal factors, such as personality. Here, we describe a behavioral protocol designed for the study of the influence of zebrafish personality on their orientation response to multiple external environmental cues, specifically water currents and magnetic fields. This protocol aims to understand whether proactive or reactive zebrafish display different rheotactic thresholds (i.e., the flow speed at which the fish start swimming upstream) when the surrounding magnetic field changes its direction. To identify zebrafish with the same personality, fish are introduced in the dark half of a tank connected with a narrow opening to a bright half. Only proactive fish explore the novel, bright environment. Reactive fish do not exit the dark half of the tank. A swimming tunnel with low flow rates is used to determine the rheotactic threshold. We describe two setups to control the magnetic field in the tunnel, in the range of the earth's magnetic field intensity: one that controls the magnetic field along the flow direction (one dimension) and one that allows a three-axial control of the magnetic field. Fish are filmed while experiencing a stepwise increase of the flow speed in the tunnel under different magnetic fields. Data on the orientation behavior are collected through a video-tracking procedure and applied to a logistic model to allow the determination of the rheotactic threshold. We report representative results collected from shoaling zebrafish. Specifically, these demonstrate that only reactive, prudent fish show variations of the rheotactic threshold when the magnetic field varies in its direction, while proactive fish do not respond to magnetic field changes. This methodology can be applied to the study of magnetic sensitivity and rheotactic behavior of many aquatic species, both displaying solitary or shoaling swimming strategies.

Living in future ocean acidification, physiological adaptive responses of the immune system of sea urchins resident at a CO2 vent system.

The effects of ocean acidification, a major anthropogenic impact on marine life, have been mainly investigated in laboratory/mesocosm experiments. We used the CO2 vents at Ischia as a natural laboratory to study the long-term effects of ocean acidification on the sea urchin Paracentrotus lividus population resident in low-pH (7.8 ±â€¯0.2) compared to that at two control sites (pH 8.02 ±â€¯0.00; 8.02 ±â€¯0.01). The novelty of the present study is the analysis of the sea urchin immune cells, the sentinels of environmental stress responses, by a wide-ranging approach, including cell morphology, biochemistry and proteomics. Immune cell proteomics showed that 311 proteins were differentially expressed in urchins across sites with a general shift towards antioxidant processes in the vent urchins. The vent urchin immune cells showed higher levels of total antioxidant capacity, up-regulation of phagosome and microsomal proteins, enzymes of ammonium metabolism, amino-acid degradation, and modulation of carbon metabolism proteins. Lipid-hydroperoxides and nitric oxide levels were not different in urchins from the different sites. No differences in the coelomic fluid pH, immune cell composition, animal respiration, nitrogen excretion and skeletal mineralogy were observed. Our results reveal the phenotypic plasticity of the immune system of sea urchins adapted to life at vent site, under conditions commensurate with near-future ocean acidification projections.

Earth-strength magnetic field affects the rheotactic threshold of zebrafish swimming in shoals.

Rheotaxis, the unconditioned orienting response to water currents, is a main component of fish behavior. Rheotaxis is achieved using multiple sensory systems, including visual and tactile cues. Rheotactic orientation in open or low-visibility waters might also benefit from the stable frame of reference provided by the geomagnetic field, but this possibility has not been explored before. Zebrafish (Danio rerio) form shoals living in freshwater systems with low visibility, show a robust positive rheotaxis, and respond to geomagnetic fields. Here, we investigated whether a static magnetic field in the Earth-strength range influenced the rheotactic threshold of zebrafish in a swimming tunnel. The direction of the horizontal component of the magnetic field relative to water flow influenced the rheotactic threshold of fish as part of a shoal, but not of fish tested alone. Results obtained after disabling the lateral line of shoaling individuals with Co2+ suggest that this organ system is involved in the observed magneto-rheotactic response. These findings constitute preliminary evidence that magnetic fields influence rheotaxis and suggest new avenues for further research.

Probiotic modulation of the microbiota-gut-brain axis and behaviour in zebrafish.

The gut microbiota plays a crucial role in the bi-directional gut-brain axis, a communication that integrates the gut and central nervous system (CNS) activities. Animal studies reveal that gut bacteria influence behaviour, Brain-Derived Neurotrophic Factor (BDNF) levels and serotonin metabolism. In the present study, we report for the first time an analysis of the microbiota-gut-brain axis in zebrafish (Danio rerio). After 28 days of dietary administration with the probiotic Lactobacillus rhamnosus IMC 501, we found differences in shoaling behaviour, brain expression levels of bdnf and of genes involved in serotonin signalling/metabolism between control and treated zebrafish group. In addition, in microbiota we found a significant increase of Firmicutes and a trending reduction of Proteobacteria. This study demonstrates that selected microbes can be used to modulate endogenous neuroactive molecules in zebrafish.

On the genome base composition of teleosts: the effect of environment and lifestyle.

BACKGROUND: The DNA base composition is well known to be highly variable among organisms. Bio-physic studies on the effect of the GC increments on the DNA structure have shown that GC-richer DNA sequences are more bendable. The result was the keystone of the hypothesis proposing the metabolic rate as the major force driving the GC content variability, since an increased resistance to the torsion stress is mainly required during the transcription process to avoid DNA breakage. Hence, the aim of the present work is to test if both salinity and migration, suggested to affect the metabolic rate of teleostean fishes, affect the average genomic GC content as well. Moreover, since the gill surface has been reported to be a major morphological expression of metabolic rate, this parameter was also analyzed in the light of the above hypothesis. RESULTS: Teleosts living in different environments (freshwater and seawater) and with different lifestyles (migratory and non-migratory) were analyzed studying three variables: routine metabolic rate, gill area and genomic GC-content, none of them showing a phylogenetic signal among fish species. Routine metabolic rate, specific gill area and average genomic GC were higher in seawater than freshwater species. The same trend was observed comparing migratory versus non-migratory species. Crossing salinity and lifestyle, the active migratory species living in seawater show coincidentally the highest routine metabolic rate, the highest specific gill area and the highest average genomic GC content. CONCLUSIONS: The results clearly highlight that environmental factors (salinity) and lifestyle (migration) affect not only the physiology (i.e. the routine metabolic rate), and the morphology (i.e. gill area) of teleosts, but also basic genome feature (i.e. the GC content), thus opening to an interesting liaison among the three variables in the light of the metabolic rate hypothesis.

Structural and functional changes in the zebrafish (Danio rerio) skeletal muscle after cadmium exposure.

This report describes the alterations induced by an environmentally realistic concentration of cadmium in skeletal muscle fibre organization, composition, and function in the teleost zebrafish. Results demonstrate that the ion induces a significant quantitative and qualitative deterioration, disrupting sarcomeric pattern and altering glycoprotein composition. These events, together with a mitochondrial damage, result in a significant reduction in swimming performance. In conclusion, the evidence here collected indicate that in presence of an environmental cadmium contamination, important economic (yields in fisheries/aquaculture), consumer health (fish is an important source of proteins), and ecological (reduced fitness due to reduced swimming performance) consequences can be expected.

Length and GC content variability of introns among teleostean genomes in the light of the metabolic rate hypothesis.

A comparative analysis of five teleostean genomes, namely zebrafish, medaka, three-spine stickleback, fugu and pufferfish was performed with the aim to highlight the nature of the forces driving both length and base composition of introns (i.e., bpi and GCi). An inter-genome approach using orthologous intronic sequences was carried out, analyzing independently both variables in pairwise comparisons. An average length shortening of introns was observed at increasing average GCi values. The result was not affected by masking transposable and repetitive elements harbored in the intronic sequences. The routine metabolic rate (mass specific temperature-corrected using the Boltzmann's factor) was measured for each species. A significant correlation held between average differences of metabolic rate, length and GC content, while environmental temperature of fish habitat was not correlated with bpi and GCi. Analyzing the concomitant effect of both variables, i.e., bpi and GCi, at increasing genomic GC content, a decrease of bpi and an increase of GCi was observed for the significant majority of the intronic sequences (from ∼ 40% to ∼ 90%, in each pairwise comparison). The opposite event, concomitant increase of bpi and decrease of GCi, was counter selected (from <1% to ∼ 10%, in each pairwise comparison). The results further support the hypothesis that the metabolic rate plays a key role in shaping genome architecture and evolution of vertebrate genomes.

Metabolic rate and genomic GC: what we can learn from teleost fish.

Teleosts are a highly diverse group of animals occupying all kind of aquatic environment. Data on routine mass specific metabolic rate were re-examined correcting them for the Boltzmann's factor. Teleostean fish were grouped in five broad groups, corresponding to major environmental classifications: polar, temperate, sub-tropical, tropical and deep-water. The specific routine metabolic rate, temperature-corrected using the Boltzmann's factor (MR), and the average base composition of genomes (GC%) were calculated in each group. Fish of the polar habitat showed the highest MR. Temperate fish displayed a significantly higher MR than tropical fish, which had the lowest average value. These results were apparently in agreement with the cold adaptation hypothesis. In contrast with this hypothesis, however, the MR of fish living in deep-water environment turned out to be not significantly different from that of fish living in tropical habitats. Most probably, the amount of oxygen dissolved in the water directly affects MR adaptation. Regarding the different habitats, the genomic GC levels showed a decreasing trend similar to that of MR. Indeed, both polar and temperate fish showed a GC level significantly higher than that of both sub-tropical and tropical fish. Plotting the genomic GC levels versus the MR a significant positive correlation was found, supporting the hypothesis that metabolic rate can explain not only the compositional transition mode (e.g. amphibian/mammals), but also the compositional shifting mode (e.g. fish/fish) of evolution observed for vertebrate genomes.

Nonsomatotopic organization of the higher motor centers in octopus.

Hyperredundant limbs with a virtually unlimited number of degrees of freedom (DOFs) pose a challenge for both biological and computational systems of motor control. In the flexible arms of the octopus, simplification strategies have evolved to reduce the number of controlled DOFs. Motor control in the octopus nervous system is hierarchically organized. A relatively small central brain integrates a huge amount of visual and tactile information from the large optic lobes and the peripheral nervous system of the arms and issues commands to lower motor centers controlling the elaborated neuromuscular system of the arms. This unique organization raises new questions on the organization of the octopus brain and whether and how it represents the rich movement repertoire. We developed a method of brain microstimulation in freely behaving animals and stimulated the higher motor centers-the basal lobes-thus inducing discrete and complex sets of movements. As stimulation strength increased, complex movements were recruited from basic components shared by different types of movement. We found no stimulation site where movements of a single arm or body part could be elicited. Discrete and complex components have no central topographical organization but are distributed over wide regions.

ATP release and extracellular nucleotidase activity in erythrocytes and coronary circulation of rainbow trout.

The present study tested the hypothesis that rainbow trout erythrocytes release ATP upon deoxygenation, a mechanism that enables mammalian erythrocytes to produce local vasodilation. We also investigated ATP release and ectonucleotidase activity in the coronary circulation of the isolated trout heart. Erythrocytes suspended in an albumin-containing saline and equilibrated at physiological Pco2 showed negligible hemolysis (<0.1%), and notably they released small amounts of ATP. The elevation of extracellular [ATP] was higher in the presence of the ectonucleotidase inhibitor ARL 67156 than in its absence, revealing the presence of ectonucleotidase activity. The induction of either a slow (minutes) or a fast (seconds) decrease in hemoglobin O2 saturation did not lead to additional ATP release. An elevation of Pco(2) was also without influence on erythrocyte ATP release. In the saline-perfused coronary circulation, [ATP] increased as the perfusate moved through the vessels in the presence of ARL 67156. When ATP was added to the inflowing saline, most ATP disappeared during passage of the coronary bed when ARL 67156 was absent but not when it was present. We conclude that rainbow trout erythrocytes and vasculature possess the key elements for ATP signaling, i.e. cellular ATP release and balanced ATP degradation by ectonucleotidases, but that erythrocyte ATP release is not influenced by oxygenation degree. The latter is suggested to be related to the lack of a deoxygenation-dependent interaction of trout hemoglobin with the cytoplasmic domain of band 3.