Urea Excretion and Arginase Activity as New Biomarkers for Nitrite Stress in Freshwater Aquatic Animals

Background: In recent years, the concern has been growing on increasing aquatic nitrite levels due to anthropogenic activities. Crustaceans and fish easily uptake nitrite via the chloride uptake system of gills. High nitrite body levels may interfere with nitric oxide (NO) production by nitric oxide synthase (NOS). The arginase, which catalyzes arginine conversion to ornithine and urea, is central to NO homeostasis. In vivo, changes in the arginase activity alter urea body levels and urea excretion and modulate NOS by altering arginine availability for NO synthesis. Excess arginase activity may uncouple NOS and induce oxidative stress. Methods: We tested muscle arginase activity and urea excretion in two fish species, zebrafish and convict cichlid, and the crustacean Yamato shrimp, under sub-lethal nitrite stress. Results: Exposure to nitrite (2 mM in the fish, 1 mM in the shrimp) significantly increased blood nitrite concentration in all species. Concomitantly, nitrite stress significantly increased arginase activity, urea excretion, and urea levels in the blood. In Yamato shrimp, urea levels also increased in muscle. Conclusion: Our results agree with the hypothesis that nitrite stress affects NO homeostasis by arginase stimulation and urea excretion. These parameters might function as markers of sub-lethal nitrite stress in freshwater fish and crustaceans

Presence of Gambusia affinis (Baird & Girard, 1853) in a freshwater ecosystem of Campania region (Italy)

The western mosquitofish, Gambusia affinis, is a small fish native to the southeastern United States. In the past century this species, and its congener, the eastern mosquitofish, Gambusia holbrooki, have been stocked in permanent and temporary waters throughout the world for mosquito control. These two species, very similar in appearance and biology, quickly became invasive with a strong ecological impact on ecosystems. They are considered responsible for the decline of several native amphibians and small fish in the Mediterranean region. Previous studies on European population conducted from Portugal to Greece reported the presence of only G. holbrooki in Italy, with report on Sicily (Catania) and Tuscany (Coltano) (Vidal, 2010). During an experimental trawl survey in 2010, samples of mosquito fishes were collected with nets from a pond near Cancello Arnone (Campania, Caserta, Italy). In order to define the Gambusia species, identification through dichotomous keys and DNA based methods were conducted. In particular, gonopodia morphology of preserved male individuals along with dorsal and anal fin rays were used to species differentiation (Walters and Feeman, 2000; Veenvliet, 2007), in our case giving uncertain results. For the molecular characterization, DNA from muscle tissue was isolated and two primer sets were used based on the conserved regions of the 12S and 16S rRNA loci as described by Kitano et al. (2007). PCR amplification and sequencing showed a 100% of maximum identity with G. affinis sequences in Genbank. These results, while contributing to unriddle the ambiguities in Gambusia taxonomy (see Vidal, 2010), call for further studies in order to define Gambusia affinis distribution in the Campania region and its impact on freshwater population

Angitola lake sediments: preliminary data and biotic indices

The Angitola lake is an artificial basin located in Calabria Region, in South Italy, part of the “Natural Regional Park of Serre”. Its surface area is 1.96 km2 and it is approximately 3 km away from Sant’Eufemia gulf. The basin was created in 1966 by damming the homonymous river. Four major rivers enter the lake: three in the far southeast, the fourth, smaller and active only during the winter period, in the southwest area. The international Ramsar Convention includes the basin and surroundings; in light of the Rio Convention, its directives have been transposed in the “Birds Directive” (BD) and “Habitats Directive” (HD). The Angitola lake, entrusted to the WWF Calabria, is one of the 2299 Italian SIC (code IT9340086): this area significantly contributes in maintaining and restoring the endangered freshwater habitat listed in HD, in protecting biodiversity of the region and it is part of the Natura 2000 network. The present study is part of the Angitola FISH2O project (European Fisheries Fund/FEP code 02/BA/12) and it aimed to examine the benthic macro-invertebrates community of the southeast part of Angitola lake. The first 15 cm in depths of sediments have a very variable composition, from fine sand to mud (rich in organic matter). This variability can be explained by the different characteristic of the chosen sampling transepts. Fauna sampling, carried out by core drills and plankton nets, shows presence of Diptera and Tricoptera larvae and some Nematoda, Polichaeta and Mollusca. Preliminary conclusion is that the Angitola lake is a diversified environment in which areas with different anthropic interference are present. The benthic macro-invertebrates community might be involved by possible disturbances induced by chemicals (e.g. water pollution) and/or physical variations (e.g. high sedimentation). Medium and long-term investigations are imperative to protect and promote the lake biodiversity, to verify the effects of seasonal fluctuations and how these are related to human activities such as tourism, demographic increase and industrial activities

Towards sustainable aquaculture systems: Biological and environmental impact of replacing fishmeal with Arthrospira platensis (Nordstedt) (spirulina)

Sustainable fish food production is crucial for aquaculture. Microalgae, such as spirulina (Arthrospira platensis), can supplement diet antioxidants or replace expensive fishmeal with high-quality proteins. In this study, we tested fish growth and wellbeing by feeding fish on a diet in which 5% of fishmeal was replaced by spirulina (SP5 diet). The low level of spirulina in the diet was intended as supplementation and was effective in ameliorating the redox state of a model fish species (juvenile Koi Carp, Cyprinus carpio L.) in a preliminary lab protocol in a six-week trial. When compared with both the control diet (no Spirulina) and a diet containing 30% spirulina replacing fishmeal (SP30 diet), SP5 was able to reduce the muscle levels of reactive oxygen species (ROS), oxidative damage, and susceptibility to oxidative stress, while increasing glutathione reductase and peroxidase activity. However, high production costs and impacts still limit the use of spirulina in fish diet. Recent studies focused on growing spirulina on urban or agro-industrial wastewater, with appropriate profiles for the alga growth. Therefore, in a circular economy context, a possibility still to be tested and exploited is feeding farmed fish with spirulina produced on output wastewater recirculated back from the same farming plant. Life Cycle Assessment (LCA) was applied to estimate the sustainability of such “circular” fish farming. The LCA ReCiPe Midpoint (H) impact assessment method was used. Firstly, the LCA environmental impacts associated with the production of spirulina grown on aquaculture wastewater as well as on the standard culture medium (Zarrouk medium) were assessed and compared by means of a “gate to gate” analysis. Then, the LCA impacts of an SP5 diet for fish, in which spirulina grown on aquaculture wastewater was used to replace 5% fishmeal (SP5ww), were compared to the diet containing spirulina grown on a standard medium (SP5st) and that one without spirulina (control diet). Results indicated that SP5ww was significantly less impacting, by avoiding the treatment and disposal of wastewater and the need for the highly impacting standard culture medium. In conclusion, the proposed approach for using spirulina in aquaculture represents a valid solution for aquaculture circular economy scenario while at the same time improving fish welfare

1,3-Butanediol Administration Increases β-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 β-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

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\u27s 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

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.Open access funding provided by Università della Calabria within the CRUI-CARE Agreement. This research was funded by the MIUR (Ministero dell’Istruzione dell’Università e della Ricerca) of Italy (ex 60%). M. F. is supported by PON “Ricerca e Innovazione” 2014–2020, Azione IV.6, “Contratti di ricerca su tematiche Green” (D. M. 1062 del 10.08.2021), C. I.: 1062_R6_GREEN, CUP: H25F21001230004

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

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

Commercial Red Food Dyes Preparations Modulate the Oxidative State in Three Model Organisms (Cucumis sativus, Artemia salina, and Danio rerio)

The growing environmental spreading of food synthetic dyes and bio-colors have the potential for altering organisms’ redox states. Here, three model species for aquatic pollution trials, Cucumis sativus seeds, Artemia salina cysts, and Danio rerio embryos, were short-term exposed to a fixed concentration of the artificial red E124, and two red bio-colors, cochineal E120, and vegan red (VEGR). In the animal models, we evaluated the total reactive oxygen species (ROS) and the susceptibility to in vitro oxidative stress, and in C. sativus, H2O2 production and antioxidant capacity. We also measured organismal performance indices (routine oxygen consumption in the animal models, dark oxygen consumption, and photosynthetic efficiency in C. sativus). In C. sativus, only E124 increased ROS and affected dark oxygen consumption and photosynthetic efficiency, while all dyes enhanced the antioxidant defenses. In the A. salina nauplii, all dyes increased ROS, while E120 and E124 reduced the susceptibility to oxidative stress. In D. rerio, treatments did not affect ROS content, and reduced oxidative stress susceptibility. Our data show that red food dyes affect the redox state of the developing organisms, in which ROS plays a significant role. We suggest a potentially toxic role for red food dyes with environmentally relevant consequences