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.

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.

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.

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.

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.

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.

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.

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.

Structure and membrane interactions of chionodracine, a piscidin-like antimicrobial peptide from the icefish Chionodraco hamatus.

Chionodracine (Cnd) is a 22-residue peptide of the piscidin family expressed in the gills of the Chionodraco hamatus as protection from bacterial infections. Here, we report the effects of synthetic Cnd on both Psychrobacter sp. TAD1 and Escherichia coli bacteria, as well as membrane models. We found that Cnd perforates the inner and outer membranes of Psychrobacter sp. TAD1, making discrete pores that cause the cellular content to leak out. Membrane disruption studies using intrinsic and extrinsic fluorescence spectroscopy revealed that Cnd behaves similarly to other piscidins, with comparable membrane partition coefficients. Membrane accessibility assays and structural studies using NMR in detergent micelles show that Cnd adopts a canonical topology of antimicrobial helical peptides, with the hydrophobic face toward the lipid environment and the hydrophilic face toward the bulk solvent. The analysis of Cnd free energy of binding to vesicles with different lipid contents indicates a preference for charged phospholipids and a more marked binding to native E. coli extracts. Taken with previous studies on piscidin-like peptides, we conclude that Cnd first adsorbs to the membrane, and then forms pores together with membrane fragmentation. Since Cnd has only marginal hemolytic activity, it constitutes a good template for developing new antimicrobial agents.

Natural products from aquatic eukaryotic microorganisms for cancer therapy: Perspectives on anti-tumour properties of ciliate bioactive molecules.

Several modern drugs, including those for cancer therapy, have been isolated from natural sources, are based on natural products and its derivatives, or mime natural products. Some of them are in clinical use, others in clinical trials. The success of natural products in drug discovery is related to their biochemical characteristics and to the technologic methods used to study their feature. Natural compounds may acts as chemo-preventive agents and as factors that increase therapeutic efficacy of existing drugs, thus overcoming cancer cell drug resistance that is the main factor determining the failure in conventional chemotherapy. Water environment, because of its physical and chemical conditions, shows an extraordinary collection of natural biological substances with an extensive structural and functional diversity. The isolation of bioactive molecules has been reported from a great variety of aquatic organisms; however, the therapeutic application of molecules from eukaryotic microorganisms remains inadequately investigated and underexploited on a systematic basis. Herein we describe the biological activities in mammalian cells of selected substances isolated from ciliates, free-living protozoa common almost everywhere there is water, focusing on their anti-tumour actions and their possible therapeutic activity. In particular, we unveil the cellular and molecular machine mediating the effects of cell type-specific signalling protein pheromone Er-1 and secondary metabolites, i.e. euplotin C and climacostol, in cancer cells. To support the feasibility of climacostol-based approaches, we also present novel findings and report additional mechanisms of action using both in vitro and in vivo models of mouse melanomas, with the scope of highlighting new frontiers that can be explored also in a therapeutic perspective. The high skeletal chemical difference of ciliate compounds, their sustainability and availability, also through the use of new organic synthesis/modifications processes, and the results obtained so far in biological studies provide a rationale to consider some of them a potential resource for the design of new anti-cancer drugs.

A formalin-inactivated immunogen against viral encephalopathy and retinopathy (VER) disease in European sea bass (Dicentrarchus labrax): immunological and protection effects.

The European sea bass (Dicentrarchus labrax) is an important farmed fish species in the Mediterranean area, very sensitive to the infection by encephalopathy and retinopathy virus (VERv), or Betanodavirus, which causes massive mortalities. Effective vaccines to fight the pathology are not yet available and in this work we describe a promising intraperitoneal immunization route against VERv of sea bass juveniles. We performed intraperitoneal and immersion immunization trials with a VERv (isolate 283.2009 RGNNV) inactivated by formalin, ß-propiolactone and heat treatment. Interestingly, the intraperitoneal immunization with formalin-inactivated VERv induced a significant antigen-specific IgM production, differently from other inactivation protocols. However, the same formalin-inactivated antigen resulted in very low IgM antibodies when administered by immersion. Following the intraperitoneal injection with formalin-inactivated virus, the quantitative expression of the antiviral MxA gene showed a modulation of transcripts in the gut after 48 h and on head kidney after 24 h, whereas ISG12 gene was significantly up-regulated after 48 h on both tissues. In immersion immunization with formalin-inactivated VERv, a modulation of MxA and ISG12 genes after 24 h post-treatment was detected in the gills. An effective uptake of VERv particles in the gills was confirmed by immunohistochemistry using anti-VERv antibodies. Lastly, in challenge experiments using live VERv after intraperitoneal immunization with formalin-inactivated VERv, we observed a significant increase (81.9%) in relative survival percentage with respect to non-immunized fish, whereas immersion immunization resulted in no protection. Our results suggest that intraperitoneal immunization with formalin-inactivated VERv could be a safe and effective strategy to fight Betanodavirus infection in European sea bass.

The protein pheromone Er-1 of the ciliate Euplotes raikovi stimulates human T-cell activity: involvement of interleukin-2 system.

Water-soluble protein signals (pheromones) of the ciliate Euplotes have been supposed to be functional precursors of growth factors and cytokines that regulate cell-cell interaction in multi-cellular eukaryotes. This work provides evidence that native preparations of the Euplotes raikovi pheromone Er-1 (a helical protein of 40 amino acids) specifically increases viability, DNA synthesis, proliferation, and the production of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1ß, IL-2, and IL-13 in human Jurkat T-cells. Also, Er-1 significantly decreases the mRNA levels of the ß and γ subunits of IL-2 receptor (IL-2R), while the mRNA levels of the α subunit appeared to be not affected. Jurkat T-cell treatments with Er-1 induced the down-regulation of the IL-2Rα subunit by a reversible and time-dependent endocytosis, and increased the levels of phosphorylation of the extracellular signal-regulated kinases (ERK). The cell-type specificity of these effects was supported by the finding that Er-1, although unable to directly influence the growth of human glioma U-373 cells, induced Jurkat cells to synthesize and release factors that, in turn, inhibited the U-373 cell proliferation. Overall, these findings imply that Er-1 coupling to IL-2R and ERK immuno-enhances T-cell activity, and that this effect likely translates to an inhibition of glioma cell growth.

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.

A piscidin-like antimicrobial peptide from the icefish Chionodraco hamatus (Perciformes: Channichthyidae): molecular characterization, localization and bactericidal activity.

Antimicrobial peptides (AMPs) are considered one of the most ancient components of the innate immune system. They are able to exert their protection activity against a variety of microorganisms, and are widely distributed in both vertebrates and invertebrates. In this paper we focused on an AMP identified in the Antarctic teleost Chionodraco hamatus, an icefish species. The cDNA sequence of the AMP, named chionodracine, is comprised of 515 bp and translates for a putative protein precursor of 80 amino acids, with a signal peptide of 22 amino acids. The structural features evidenced in the primary sequence of chionodracine lead to the inclusion of the peptide in the antimicrobial family of piscidins. The analysis by real-time PCR of the basal gene transcripts of chionodracine in different icefish tissues showed that the highest expression was found in gills, followed by head kidney. The chionodracine expression levels in head kidney leukocytes were up-regulated in vitro both by LPS and poly I:C, and in vivo by LPS. A putative chionodracine mature peptide was synthesized and employed to obtain a polyclonal antiserum, which was used in immunohistochemistry of gills sections and revealed a significant positivity associated with mast cells. The bactericidal activity of the peptide was investigated and found significant against Antarctic psychrophilic bacteria strains (Psychrobacter sp. TAD1 and TA144), the Gram-positive Bacillus cereus, and at a lesser extent against the Gram-negative Escherichia coli. Interestingly, the haemolytic activity of chionodracine was tested in vitro on human erythrocytes and no significant lysis occurred until peptide concentration of 50 µM.

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.

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.

Effect of dietary alginic acid on juvenile tilapia (Oreochromis niloticus) intestinal microbial balance, intestinal histology and growth performance.

The aim of the present study was to assess the effect of a commercial alginic acid source (Ergosan) on tilapia Oreochromis niloticus intestinal microbial balance, intestinal morphology, and growth parameters. Fish were fed a basal control diet or the basal diet plus a source of alginic acid (5 g kg(-1) Ergosan; Schering-Plough Aquaculture, UK) for 9 weeks. At the end of the trial, light and electron microscopy demonstrated that the morphology of the intestinal tract at the gross and ultra-structural level was not affected by dietary alginic acid inclusion. Both groups of fish displayed healthy, normal morphology with no signs of disease, cell or tissue damage. Intestinal epithelial leucocyte infiltration was not affected by dietary alginic acid. Molecular bacterial profiles derived from PCR-DGGE illustrated highly similar microbial communities (both within the lumen and associated with the intestinal mucosa) in the respective treatment groups. Microbial ecological parameters (e.g. species diversity and richness) also remained unaffected. Although not significant, trends towards elevated survival and body protein content were observed in the alginic acid-fed fish. These results are suggestive that alginic acid does not adversely impact the indigenous gastrointestinal microbial balance and subsequently does not impact upon the epithelial brush border integrity. Validation of non-detrimental impacts of immunostimulatory products on gastric microbiota and epithelial integrity should be pursued in future studies as maintaining microbial balance and epithelial integrity is essential for proper gut functionality.

The insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) alters the membrane raft location of the TSH receptor stably expressed in Chinese hamster ovary cells.

DDT is a highly lipophilic molecule known to deplete membrane rafts of their phosphoglycolipid and cholesterol contents. However, we have recently shown that DDT can also alter the thyroid homeostasis by inhibiting TSH receptor (TSHr) internalization. The present study was undertaken to verify whether DDT goitrogenic effects are due to the insecticide acting directly on TSHr or via alteration of the membrane rafts hosting the receptor itself. Our results demonstrate that, in CHO-TSHr transfected cells, TSHr is activated in the presence of TSH, while it is inhibited following DDT exposure. DDT can also reduce the endocytic vesicular traffic, alter the extension of multi-branched microvilli along their plasma membranes and induce TSHr shedding in vesicular forms. To verify whether TSHr displacement might depend on DDT altering the raft constitution of CHO-TSHr cell membranes the extent of TSHr and lipid raft co-localization was examined by confocal microscopy. Evidence shows that receptor/raft co-localization increased significantly upon exposure to TSH, while receptors and lipid rafts become dislodged on opposite cell poles in DDT-exposed CHO-TSHr cells. As a control, under similar culturing conditions, diphenylethylene, which is known to be a lipophilic substance that is structurally related to DDT, did not affect the extent of TSHr and lipid raft co-localization in CHO-TSHr cells treated with TSH. These findings corroborate and extend our view that, in CHO cells, the DDT disrupting action on TSHr is primarily due to the insecticide acting on membranes to deplete their raft cholesterol content, and that the resulting inhibition on TSHr internalization is due to receptor dislodgement from altered raft microdomains of the plasma membrane.

Teleost intestinal immunology.

Teleosts clearly have a more diffuse gut associated lymphoid system, which is morphological and functional clearly different from the mammalian GALT. All immune cells necessary for a local immune response are abundantly present in the gut mucosa of the species studied and local immune responses can be monitored after intestinal immunization. Fish do not produce IgA, but a special mucosal IgM isotype seems to be secreted and may (partly) be the recently described IgZ/IgT. Fish produce a pIgR in their mucosal tissues but it is smaller (2 ILD) than the 4-5 ILD pIgR of higher vertebrates. Whether teleost pIgR is transcytosed and cleaved off in the same way needs further investigation, especially because a secretory component (SC) is only reported in one species. Teleosts also have high numbers of IEL, most of them are CD3-ɛ+/CD8-α+ and have cytotoxic and/or regulatory function. Possibly many of these cells are TCRγδ cells and they may be involved in the oral tolerance induction observed in fish. Innate immune cells can be observed in the teleost gut from first feeding onwards, but B cells appear much later in mucosal compartments compared to systemic sites. Conspicuous is the very early presence of putative T cells or their precursors in the fish gut, which together with the rag-1 expression of intestinal lymphoid cells may be an indication for an extra-thymic development of certain T cells. Teleosts can develop enteritis in their antigen transporting second gut segment and epithelial cells, IEL and eosinophils/basophils seem to play a crucial role in this intestinal inflammation model. Teleost intestine can be exploited for oral vaccination strategies and probiotic immune stimulation. A variety of encapsulation methods, to protect vaccines against degradation in the foregut, are reported with promising results but in most cases they appear not to be cost effective yet. Microbiota in fish are clearly different from terrestrial animals. In the past decade a fast increasing number of papers is dedicated to the oral administration of a variety of probiotics that can have a strong health beneficial effect, but much more attention has to be paid to the immune mechanisms behind these effects. The recent development of gnotobiotic fish models may be very helpful to study the immune effects of microbiota and probiotics in teleosts.

Microbial manipulations to improve fish health and production--a Mediterranean perspective.

The interactions between the endogenous gut microbiota and the fish host are integral in mediating the development, maintenance and effective functionality of the intestinal mucosa and gut associated lymphoid tissues (GALTs). These microbial populations also provide a level of protection against pathogenic visitors to the gastrointestinal (GI) tract and aid host digestive function via the production of exogenous digestive enzymes and vitamins. Manipulation of these endogenous populations may provide an alternative method to antibiotics to control disease and promote health management. Applications of probiotics for Mediterranean teleosts can stimulate immune responses, enhance growth performance, feed utilisation, digestive enzyme activities, antioxidant enzyme activities, gene expression, disease resistance, larval survival, gut morphology, modulate GI microbiota and mediate stress responses. Although considerably less information is available regarding prebiotic applications for Mediterranean teleosts, prebiotics also offer benefits with regards to improving immune status and fish production. Despite the promising potential benefits demonstrated in current literature, obtaining consistent and reliable results is often difficult due to our incomplete understanding of indigenous fish GI microbiota and their subsequent host interactions which mediate and drive both localised and systemic host immunological responses. Additionally, the probiotic and prebiotic (biotics) mechanisms which mediate host benefits at the mucosal interface are poorly understood. Future studies focused on these interactions utilising gnotobiotic techniques should provide a better understanding of how to extract the full potential of biotic applications to promote immune function of Mediterranean teleosts.