T-cells and CD45-cells discovery in the central nervous system of healthy and nodavirus-infected teleost fish Dicentrarchuslabrax.

To achieve insights in antiviral immune defense of the central nervous system (CNS), we investigated T cells and CD45 cells in the marine fish model Dicentrarchus labrax infected with the CNS-tropic virus betanodavirus. By employing markers for pan-T cells (mAb DLT15) and CD45-cells (mAb DLT22) in immunofluorescence (IIF) of leukocytes from brain, we obtained 3,7 ± 2.3 % of T cells and 7.3 ± 3.2 % of CD45+ cells. Both IIF and immunoelectron microscopy confirmed a leukocyte/glial morphology for the immunoreactive cells. Quantitative immunohistochemistry (qIHC) of brain/eye sections showed 1.9 ± 0.8 % of T+ cells and 2 ± 0.9 % of CD45+ cells in the brain, and 3.6 ± 1.9 % and 4.1 ± 2.2 % in the eye, respectively. After in vivo RGNNV infection the number of T cells/CD45+ leukocytes in the brain increased to 8.3 ± 2.1 % and 11.6 ± 4.4 % (by IIF), and 26.1 ± 3.4 % and 45.6 ± 5.9 % (by qIHC), respectively. In the eye we counted after infection 8.5 ± 4.4 % of T cells and 10.2 ± 5.8 % of CD45 cells. Gene transcription analysis of brain mRNA revealed a strong increase of gene transcripts coding for: antiviral proteins Mx and ISG-12; T-cell related CD3ε/δ, TcRß, CD4, CD8α, CD45; and for immuno-modulatory cytokines TNFα, IL-2, IL-10. A RAG-1 gene product was also present and upregulated, suggesting somatic recombination in the fish brain. Similar transcription data were obtained in the eye, albeit with differences. Our findings provide first evidence for a recruitment and involvement of T cells and CD45+ leukocytes in the fish eye-brain axis during antiviral responses and suggest similarities in the CNS immune defense across evolutionary distant vertebrates.

Exposure to the Natural Compound Climacostol Induces Cell Damage and Oxidative Stress in the Fruit Fly Drosophila melanogaster.

The ciliate Climacostomum virens produces the metabolite climacostol that displays antimicrobial activity and cytotoxicity on human and rodent tumor cells. Given its potential as a backbone in pharmacological studies, we used the fruit fly Drosophila melanogaster to evaluate how the xenobiotic climacostol affects biological systems in vivo at the organismal level. Food administration with climacostol demonstrated its harmful role during larvae developmental stages but not pupation. The midgut of eclosed larvae showed apoptosis and increased generation of reactive oxygen species (ROS), thus demonstrating gastrointestinal toxicity. Climacostol did not affect enteroendocrine cell proliferation, suggesting moderate damage that does not initiate the repairing program. The fact that climacostol increased brain ROS and inhibited the proliferation of neural cells revealed a systemic (neurotoxic) role of this harmful substance. In this line, we found lower expression of relevant antioxidant enzymes in the larvae and impaired mitochondrial activity. Adult offsprings presented no major alterations in survival and mobility, as well the absence of abnormal phenotypes. However, mitochondrial activity and oviposition behavior was somewhat affected, indicating the chronic toxicity of climacostol, which continues moderately until adult stages. These results revealed for the first time the detrimental role of ingested climacostol in a non-target multicellular organism.

Transcriptome Analysis Reveals Early Hemocyte Responses upon In Vivo Stimulation with LPS in the Stick Insect Bacillus rossius (Rossi, 1788).

Despite a growing number of non-model insect species is being investigated in recent years, a greater understanding of their physiology is prevented by the lack of genomic resources. This is the case of the common European stick insect Bacillus rossius (Rossi, 1788): in this species, some knowledge is available on hemocyte-related defenses, but little is known about the physiological changes occurring in response to natural or experimental challenges. Here, the transcriptional signatures of adult B. rossius hemocytes were investigated after a short-term (2 h) LPS stimulation in vivo: a total of 2191 differentially expressed genes, mostly involved in proteolysis and carbohydrate and lipid metabolic processes, were identified in the de novo assembled transcriptome and in-depth discussed. Overall, the significant modulation of immune signals-such as C-type lectins, ML domain-containing proteins, serpins, as well as Toll signaling-related molecules-provide novel information on the early progression of LPS-induced responses in B. rossius.

Comparative Analysis of the pIgR Gene from the Antarctic Teleost Trematomus bernacchii Reveals Distinctive Features of Cold-Adapted Notothenioidei.

The IgM and IgT classes were previously identified and characterized in the Antarctic teleost Trematomus bernacchii, a species belonging to the Perciform suborder Notothenoidei. Herein, we characterized the gene encoding the polymeric immunoglobulin receptor (pIgR) in the same species and compared it to the pIgR of multiple teleost species belonging to five perciform suborders, including 11 Antarctic and 1 non-Antarctic (Cottoperca gobio) notothenioid species, the latter living in the less-cold peri-Antarctic sea. Antarctic pIgR genes displayed particularly long introns marked by sites of transposable elements and transcription factors. Furthermore, analysis of T. bernacchii pIgR cDNA unveiled multiple amino acid substitutions unique to the Antarctic species, all introducing adaptive features, including N-glycosylation sequons. Interestingly, C. gobio shared most features with the other perciforms rather than with the cold-adapted relatives. T. bernacchii pIgR transcripts were predominantly expressed in mucosal tissues, as indicated by q-PCR and in situ hybridization analysis. These results suggest that in cold-adapted species, pIgR preserved its fundamental role in mucosal immune defense, although remarkable gene structure modifications occurred.

TiO2-NPs and cadmium co-exposure: in vitro assessment of genetic and genomic DNA damage on Dicentrarchus labrax embryonic cells.

The increased titanium dioxide nanoparticles (TiO2-NPs) spread and their interaction with organic and inorganic pollutants arouses concern for the potential hazards for organisms and environment. This study tested in vitro the genotoxic effects of TiO2-NPs (1 µg/mL) and cadmium (Cd) (0.1 µg/mL) co-exposure using Dicentrarchus labrax embryonic cells (DLEC) as experimental model. The genotoxicity tests (Comet assay, Diffusion Assay and Random Amplification of Polymorphic DNA (RAPD-PCR) were conducted after 3, 24 and 48 hours of exposure to TiO2-NPs and Cd alone and in combination. The results showed that the percentage of DNA damage and apoptotic cells increases following 48 hours TiO2-NPs exposure, while DNA instability was detected for all the times tested. Cd induced genotoxic effects starting from 3 hour-exposure and for all the treatment times. Cd + TiO2-NPs co-exposure did not cause any genomic damage or apoptosis for all the exposure times. The possibility that Cd and TiO2-NPs form aggregates no longer able of penetrating the nucleus and damaging the genetic material is discussed.

Molecular, Cellular and Functional Analysis of TRγ Chain along the European Sea Bass Dicentrarchus labrax Development.

In jawed vertebrates, adaptive immune responses are enabled by T cells. Two lineages were characterized based on their T cell receptor (TcR) heterodimers, namely αß or γδ peptide chains, which display an Ig domain-type sequence that is somatically rearranged. γδ T cells have been less extensively characterized than αß and teleost fish, in particular, suffer from a severe scarcity of data. In this paper, we worked on the well-known model, the European sea bass Dicentrarchus labrax, to broaden the understanding of teleost γδ-T cells. The T cell receptor chain (TR) γ transcript was expressed at a later developmental stage than TRß, suggesting a layered appearance of fish immune cells, and the thymus displayed statistically-significant higher mRNA levels than any other organ or lymphoid tissue investigated. The polyclonal antibody developed against the TRγ allowed the localization of TRγ-expressing cells in lymphoid organs along the ontogeny. Cell positivity was investigated through flow cytometry and the highest percentage was found in peripheral blood leukocytes, followed by thymus, gut, gills, spleen and head kidney. Numerous TRγ-expressing cells were localized in the gut mucosa, and the immunogold labelling revealed ultrastructural features that are typical of T cells. At last, microalgae-based diet formulations significantly modulated the abundance of TRγ+ cells in the posterior intestine, hinting at a putative involvement in nutritional immunity. From a comparative immunological perspective, our results contribute to the comprehension of the diversity and functionalities of γδ T cells during the development of a commercially relevant marine teleost model.

Attacins: A Promising Class of Insect Antimicrobial Peptides.

Insects produce a large repertoire of antimicrobial peptides (AMPs) as the first line of defense against bacteria, viruses, fungi or parasites. These peptides are produced from a large precursor that contains a signal domain, which is cleaved in vivo to produce the mature protein with antimicrobial activity. At present, AMPs from insects include several families which can be classified as cecropins, ponericins, defensins, lebocins, drosocin, Metchnikowin, gloverins, diptericins and attacins according to their structure and/or function. This short review is focused on attacins, a class of glycine-rich peptides/proteins that have been first discovered in the cecropia moth (Hyalophora cecropia). They are a rather heterogeneous group of immunity-related proteins that exhibit an antimicrobial effect mainly against Gram-negative bacteria. Here, we discuss different attacin and attacin-like AMPs that have been discovered so far and analyze their structure and phylogeny. Special focus is given to the physiological importance and mechanism of action of attacins against microbial pathogens together with their potential pharmacological applications, emphasizing their roles as antimicrobials.

State-of-the-Art Vaccine Research for Aquaculture Use: The Case of Three Economically Relevant Fish Species.

In the last three decades, the aquaculture sector has experienced a 527% growth, producing 82 million tons for a first sale value estimated at 250 billion USD. Infectious diseases caused by bacteria, viruses, or parasites are the major causes of mortality and economic losses in commercial aquaculture. Some pathologies, especially those of bacterial origin, can be treated with commercially available drugs, while others are poorly managed. In fact, despite having been recognized as a useful preventive measure, no effective vaccination against many economically relevant diseases exist yet, such as for viral and parasitic infections. The objective of the present review is to provide the reader with an updated perspective on the most significant and innovative vaccine research on three key aquaculture commodities. European sea bass (Dicentrarchus labrax), Nile tilapia (Oreochromis niloticus), and Atlantic salmon (Salmo salar) were chosen because of their economic relevance, geographical distinctiveness, and representativeness of different culture systems. Scientific papers about vaccines against bacterial, viral, and parasitic diseases will be objectively presented; their results critically discussed and compared; and suggestions for future directions given.

Molecular and cellular characterization of European sea bass CD3ε+ T lymphocytes and their modulation by microalgal feed supplementation.

The CD3 coreceptor is a master T cell surface marker, and genes encoding CD3ζ, γδ, and ε chains have been reported in several teleost fish. Here, a complete cDNA sequence of CD3ɛ chain was identified from a sea bass (Dicentrarchus labrax L.) gill transcriptome. Its basal expression was quantified in both lymphoid and non-lymphoid organs of sea bass juveniles with real-time qPCR analysis. After either in vitro stimulation of head kidney leukocytes with the T-cell mitogen phytohaemagglutinin or in vivo stimulation with an orally administered Vibrio anguillarum vaccine, CD3ε expression levels increased in head kidney leukocytes, confirming that CD3ε T cells may play important roles in fish systemic protection against pathogens. Further, three peptides were designed on the CD3ɛ cytoplasmic tail region and employed as immunogens for antibody production in rabbit. One antiserum so obtained, named RACD3/1, immunostained a band of the expected size in a western blot of a sea bass thymocyte lysate. The distribution of CD3ε+ lymphocyte population in the lymphoid organs and mucosal tissues was addressed in healthy fish by IHC. In decreasing percentage order, CD3ε+ lymphocytes were detected by flow cytometry in thymus, peripheral blood leukocytes, gills, head kidney, gut, and spleen. Finally, a significant in vivo enhancement of CD3ε+ T intestinal lymphocytes was found in fish fed on diets in which 100% fish meal was replaced by the microalgae Nannochloropsis sp. biomass. These results indicate that CD3ε+ T cells are involved in nutritional immune responses.

Differential Effects of Dietary Supplementation of Krill Meal, Soybean Meal, Butyrate, and Bactocell® on the Gene Expression of Atlantic Salmon Head Kidney.

The head kidney is a key organ that plays a fundamental role in the regulation of the fish immune response and in the maintenance of endocrine homeostasis. Previous studies indicate that the supplementation of exogenous dietary components, such as krill meal (KM), soybean meal (SM), Bactocell® (BA), and butyrate (BU), can have a significant effect on the immune function of the head kidney. The aim of this study was to investigate the differential effect of these four dietary ingredients on the transcriptional profiles of the head kidney of the Atlantic salmon. This study revealed that just a small number of genes were responsive to the feeding regime after a long-term (12 weeks) treatment, and evidenced that the most significant alterations, both in terms of the number of affected genes and magnitude of changes in gene expression, were detectable in the BU- and KM-fed groups compared with controls, while the SM diet had a nearly negligible effect, and BA had no significant effects at all. Most of the differentially expressed genes were involved in the immune response and, in line with data previously obtained from pyloric caeca, major components of the complement system were significantly affected. These alterations were accompanied by an increase in the density of melanomacrophage centers in the KM- and SM-fed group and their reduction in the BU-fed group. While three types of dietary supplements (BU, KM, and SM) were able to produce a significant modulation of some molecular players of the immune system, the butyrate-rich diet was revealed as the one with the most relevant immune-stimulating properties in the head kidney. These preliminary results suggest that further investigations should be aimed towards the elucidation of the potential beneficial effects of butyrate and krill meal supplementation on farmed salmon health and growth performance.

The Natural Compound Climacostol as a Prodrug Strategy Based on pH Activation for Efficient Delivery of Cytotoxic Small Agents.

We synthesized and characterized MOMO as a new small molecule analog of the cytotoxic natural product climacostol efficiently activated in mild extracellular acidosis. The synthesis of MOMO had a key step in the Wittig olefination for the construction of the carbon-carbon double bond in the alkenyl moiety of climacostol. The possibility of obtaining the target (Z)-alkenyl MOMO derivative in very good yield and without presence of the less active (E)-diastereomer was favored from the methoxymethyl ether (MOM)-protecting group of hydroxyl functions in aromatic ring of climacostol aldehyde intermediate. Of interest, the easy removal of MOM-protecting group in a weakly acidic environment allowed us to obtain a great quantity of climacostol in biologically active (Z)-configuration. Results obtained in free-living ciliates that share the same micro-environment of the climacostol natural producer Climacostomum virens demonstrated that MOMO is well-tolerated in a physiological environment, while its cytotoxicity is rapidly and efficiently triggered at pH 6.3. In addition, the cytostatic vs. cytotoxic effects of acidified-MOMO can be modulated in a dose-dependent manner. In mouse melanoma cells, MOMO displayed a marked pH-sensitivity since its cytotoxic and apoptotic effects become evident only in mild extracellular acidosis. Data also suggested MOMO being preferentially activated in the unique extra-acidic microenvironment that characterizes tumoural cells. Finally, the use of the model organism Drosophila melanogaster fed with an acidic diet supported the efficient activity and oral delivery of MOMO molecule in vivo. MOMO affected oviposition of mating adults and larvae eclosion. Reduced survival of flies was due to lethality during the larval stages while emerging larvae retained their ability to develop into adults. Interestingly, the gut of eclosed larvae exhibited an extended damage (cell death by apoptosis) and the brain tissue was also affected (reduced mitosis), demonstrating that orally activated MOMO efficiently targets different tissues of the developing fly. These results provided a proof-of-concept study on the pH-dependence of MOMO effects. In this respect, MOM-protection emerges as a potential prodrug strategy which deserves to be further investigated for the generation of efficient pH-sensitive small organic molecules as pharmacologically active cytotoxic compounds.

Fish-derived antimicrobial peptides: Activity of a chionodracine mutant against bacterial models and human bacterial pathogens.

The increasing resistance to conventional antibiotics is an urgent problem that can be addressed by the discovery of new antimicrobial drugs such as antimicrobial peptides (AMPs). AMPs are components of innate immune system of eukaryotes and are not prone to the conventional mechanisms that are responsible of drug resistance. Fish are an important source of AMPs and, recently, we have isolated and characterized a new 22 amino acid residues peptide, the chionodracine (Cnd), from the Antarctic icefish Chionodraco hamatus. In this paper we focused on a new Cnd-derived mutant peptide, namely Cnd-m3a, designed to improve the selectivity against prokaryotic cells and the antimicrobial activity against human pathogens of the initial Cnd template. Cnd-m3a was used for immunization of rabbits, which gave rise to a polyclonal antibody able to detect the peptide. The interaction kinetic of Cnd-m3a with the Antarctic bacterium Psychrobacter sp. (TAD1) was imaged using a transmission electron microscopy (TEM) immunogold method. Initially the peptide was associated with the plasma membrane, but after 180 min of incubation, it was found in the cytoplasm interacting with a DNA target inside the bacterial cells. Using fluorescent probes we showed that the newly designed mutant can create pores in the outer membrane of the bacteria E. coli and Psychrobacter sp. (TAD1), confirming the results of TEM analysis. Moreover, in vitro assays demonstrated that Cnd-m3a is able to bind lipid vesicles of different compositions with a preference toward negatively charged ones, which mimics the prokaryotic cell. The Cnd-m3a peptide showed quite low hemolytic activity and weak cytotoxic effect against human primary and tumor cell lines, but high antimicrobial activity against selected Gram - human pathogens. These results highlighted the high potential of the Cnd-m3a peptide as a starting point for developing a new human therapeutic agent.

Bioactivity and Structural Properties of Novel Synthetic Analogues of the Protozoan Toxin Climacostol.

Climacostol (5-[(2Z)-non-2-en-1-yl]benzene-1,3-diol) is a resorcinol produced by the protozoan Climacostomum virens for defence against predators. It exerts a potent antimicrobial activity against bacterial and fungal pathogens, inhibits the growth of several human and rodent tumour cells, and is now available by chemical synthesis. In this study, we chemically synthesized two novel analogues of climacostol, namely, 2-methyl-5 [(2Z)-non-2-en-1-yl]benzene-1,3-diol (AN1) and 5-[(2Z)-non-2-en-1-yl]benzene-1,2,3-triol (AN2), with the aim to increase the activity of the native toxin, evaluating their effects on prokaryotic and free-living protists and on mammalian tumour cells. The results demonstrated that the analogue bearing a methyl group (AN1) in the aromatic ring exhibited appreciably higher toxicity against pathogen microbes and protists than climacostol. On the other hand, the analogue bearing an additional hydroxyl group (AN2) in the aromatic ring revealed its ability to induce programmed cell death in protistan cells. Overall, the data collected demonstrate that the introduction of a methyl or a hydroxyl moiety to the aromatic ring of climacostol can effectively modulate its potency and its mechanism of action.

Vaccination and immune responses of European sea bass (Dicentrarchus labrax L.) against betanodavirus.

This review summarizes the available knowledge on the immune defences of European sea bass against antigenic preparations derived from the viral encephalopathy and retinopathy virus (betanodavirus), which represents a major threat to the health of this fish species. The nodavirus is widely present and differentiates into several strains that infect invertebrates (in insects, alphanodavirus) and teleost fish, and thus may represent a great problem for farmed fish species. Many efforts have been directed to discovering new immunizations to induce protection in sea bass, especially at young stages, and these efforts have included employing diverse betanodavirus strains, antigen preparation, vaccination routes, and the addition of adjuvants and/or immunostimulants. The obtained results showed that inactivated preparations of betanodavirus that were administered intraperitoneally may induce both immune recognition and protection. Attempts at performing mucosal immunization by immersion and/or oral administration, which is a vaccination route that is highly preferred for sea bass, have shown intriguing results, and more studies are necessary for its improvement. Overall, the objective of identifying a reliable vaccine that also cross-protects against different genotypes or reassortant viruses for use in European sea bass against betanodavirus appears to be an attainable goal in the near future.

Dysfunctional autophagy induced by the pro-apoptotic natural compound climacostol in tumour cells.

Autophagy occurs at a basal level in all eukaryotic cells and may support cell survival or activate death pathways. Due to its pathophysiologic significance, the autophagic machinery is a promising target for the development of multiple approaches for anti-neoplastic agents. We have recently described the cytotoxic and pro-apoptotic mechanisms, targeting the tumour suppressor p53, of climacostol, a natural product of the ciliated protozoan Climacostomum virens. We report here on how climacostol regulates autophagy and the involvement of p53-dependent mechanisms. Using both in vitro and in vivo techniques, we show that climacostol potently and selectively impairs autophagy in multiple tumour cells that are committed to die by apoptosis. In particular, in B16-F10 mouse melanomas climacostol exerts a marked and sustained accumulation of autophagosomes as the result of dysfunctional autophagic degradation. We also provide mechanistic insights showing that climacostol affects autophagosome turnover via p53-AMPK axis, although the mTOR pathway unrelated to p53 levels plays a role. In particular, climacostol activated p53 inducing the upregulation of p53 protein levels in the nuclei through effects on p53 stability at translational level, as for instance the phosphorylation at Ser15 site. Noteworthy, AMPKα activation was the major responsible of climacostol-induced autophagy disruption in the absence of a key role regulating cell death, thus indicating that climacostol effects on autophagy and apoptosis are two separate events, which may act independently on life/death decisions of the cell. Since the activation of p53 system is at the molecular crossroad regulating both the anti-autophagic action of climacostol and its role in the apoptosis induction, it might be important to explore the dual targeting of autophagy and apoptosis with agents acting on p53 for the selective killing of tumours. These findings also suggest the efficacy of ciliate bioactive molecules to identify novel lead compounds in drug discovery and development.

Fish Lymphocytes: An Evolutionary Equivalent of Mammalian Innate-Like Lymphocytes?

Lymphocytes are the responsible of adaptive responses, as they are classically described, but evidence shows that subpopulations of mammalian lymphocytes may behave as innate-like cells, engaging non-self rapidly and without antigen presentation. The innate-like lymphocytes of mammals have been mainly identified as γδT cells and B1-B cells, exert their activities principally in mucosal tissues, may be involved in human pathologies and their functions and tissue(s) of origin are not fully understood. Due to similarities in the morphology and immunobiology of immune system between fish and mammals, and to the uniqueness of having free-living larval stages where the development can be precisely monitored and engineered, teleost fish are proposed as an experimental model to investigate human immunity. However, the homology between fish lymphocytes and mammalian innate-like lymphocytes is an issue poorly considered in comparative immunology. Increasing experimental evidence suggests that fish lymphocytes could have developmental, morphological, and functional features in common with innate-like lymphocytes of mammals. Despite such similarities, information on possible links between conventional fish lymphocytes and mammalian innate-like lymphocytes is missing. The aim of this review is to summarize and describe available findings about the similarities between fish lymphocytes and mammalian innate-like lymphocytes, supporting the hypothesis that mammalian γδT cells and B1-B cells could be evolutionarily related to fish lymphocytes.

Influence of Probiotics Administration on Gut Microbiota Core: A Review on the Effects on Appetite Control, Glucose, and Lipid Metabolism.

An increasing number of studies has shown that dietary probiotics exert beneficial health effects in both humans and animals. It is well established that gut microbiota play a pivotal role in regulating host metabolism, and a growing number of studies has elucidated that probiotics positively interfere with gut microbiota. Accumulating evidence shows that probiotics, through their metabolic activity, produce metabolites that in turn contribute to positively affect host physiology. For these reasons, probiotics have shown significant potential as a therapeutic tool for a diversity of diseases, but the mechanisms through which probiotics act has not been fully elucidated yet. The goal of this review was to provide evidence on the effects of probiotics on gut microbiota changes associated with host metabolic variations, specifically focusing on feed intake and lipid and glucose metabolism. In addition, we review probiotic interaction with the gut microbiota. The information collected here will give further insight into the effects of probiotics on the gut microbiota and their action on metabolite release, energy metabolism, and appetite. This information will help to improve knowledge to find better probiotic therapeutic strategies for obesity and eating disorders.

Oestrogen receptor distribution related to functional thymus anatomy of the European sea bass, Dicentrarchus labrax.

In jawed vertebrates, the crosstalk between immune and endocrine system as well as many fundamental mechanisms of T cell development are evolutionary conserved. Oestrogens affect mammalian thymic function and plasticity, but the mechanisms of action and the oestrogen receptors involved remain unclear. To corroborate the oestrogenic regulation of thymic function in teleosts and to identify the implicated oestrogen receptor subtypes, we examined the distribution of nuclear and membrane oestrogen receptors within the thymus of the European Sea bass, Dicentrarchus labrax, in relation to its morpho-functional organisation. Immunohistological analysis specified thymus histology and organisation in teleosts and described, for the first time, Hassall's corpuscle like structures in the medulla of sea bass. All oestrogen receptors were expressed at the transcript and protein level, both in T cells and in stromal cells belonging to specific functional areas. These observations suggest complex regulatory actions of oestrogen on thymic function, notably through the stromal microenvironment, comprising both, genomic and non-genomic pathways that are likely to affect T cell maturation and trafficking processes. Comparison with birds, rodents and humans supports the thymic localization of oestrogen receptors and suggests that oestrogens modulate T cell maturation in all gnathostomes.

Dietary lipid content reorganizes gut microbiota and probiotic L. rhamnosus attenuates obesity and enhances catabolic hormonal milieu in zebrafish.

In the present study, we explored whether dietary lipid content influences the gut microbiome in adult zebrafish. Diets containing three different lipid levels (high [HFD], medium [MFD], and low [LFD]) were administered with or without the supplementation of Lactobacillus rhamnosus (P) to zebrafish in order to explore how the dietary lipid content may influence the gut microbiome. Dietary lipid content shifted the gut microbiome structure. The addition of L. rhamnosus in the diets, induced transcriptional reduction of orexigenic genes, upregulation of anorexigenic genes, and transcriptional decrease of genes involved in cholesterol and triglyceride (TAG) metabolism, concomitantly with lower content of cholesterol and TAG. Probiotic feeding also decreased nesfatin-1 peptide in HFD-P and attenuated weight gain in HFD-P and MFD-P fed zebrafish, but not in LFD-P group. Intestinal ultrastructure was not affected by dietary fat level or probiotic inclusion. In conclusion, these findings underline the role of fat content in the diet in altering gut microbiota community by shifting phylotype composition and highlight the potential of probiotics to attenuate high-fat diet-related metabolic disorder.

Immunoglobulin T from sea bass (Dicentrarchus labrax L.): molecular characterization, tissue localization and expression after nodavirus infection.

BACKGROUND: Immunoglobulins (Igs) are fundamental components of the adaptive immune system of vertebrates, with the IgT/IgZ isotype specific of Teleosts. In this paper we describe the identification of an IgT heavy chain from the European sea bass (Dicentrarchus labrax L.), its molecular characterization and tissue mRNA localization by in situ hybridization. RESULTS: Sea bass IgT consists of 552 aa (Accession Number KM410929) and it contains a putative 19 amino acids long signal peptide and one potential N-glycosylation site. The C-region consists of four CH domains; each contains the cysteine and tryptophan residues required for their correct folding. Based on the recent sequencing of sea bass genome, we have identified five different genomic contigs bearing exons unequivocally pertaining to IgT (CH2, CH3 and CH4), but none corresponded to a complete IgH locus as IgT sequences were found in the highly fragmented assembled genomic regions which could not be assigned to any major scaffold. The 3D structure of sea bass IgT has been modelled using the crystal structure of a mouse Ig gamma as a template, thus showing that the amino acid sequence is suitable for the expected topology referred to an immunoglobulin-like architecture. The basal expression of sea bass IgT and IgM in different organs has been analysed: gut and gills, important mucosal organs, showed high IgT transcripts levels and this was the first indication of the possible involvement of sea bass IgT in mucosal immune responses. Moreover, sea bass IgT expression increased in gills and spleen after infection with nodavirus, highlighting the importance of IgT in sea bass immune responses. In situ hybridization confirmed the presence of IgT transcripts in the gut and it revealed a differential expression along the intestinal tract, with a major expression in the posterior intestine, suggesting the hindgut as a site for the recruitment of IgT+ cells in this species. IgT transcripts were also found in gill filaments and parallel lamellae and, for the first time, we identified scattered IgT positive cells in the liver, with a strong signal in the hepatic parenchyma. CONCLUSIONS: In conclusion, we performed a full molecular characterization of IgT in sea bass that points out its possible involvement in mucosal immune responses of this species.