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.

Increased water temperature contributes to a chondrogenesis response in the eyes of spotted wolffish.

Adult vertebrate cartilage is usually quiescent. Some vertebrates possess ocular scleral skeletons composed of cartilage or bone. The morphological characteristics of the spotted wolffish (Anarhichas minor) scleral skeleton have not been described. Here we assessed the scleral skeletons of cultured spotted wolffish, a globally threatened marine species. The healthy spotted wolffish we assessed had scleral skeletons with a low percentage of cells staining for the chondrogenesis marker sex-determining region Y-box (Sox) 9, but harboured a population of intraocular cells that co-express immunoglobulin M (IgM) and Sox9. Scleral skeletons of spotted wolffish with grossly observable eye abnormalities displayed a high degree of perochondrial activation as evidenced by cellular morphology and expression of proliferating cell nuclear antigen (PCNA) and phosphotyrosine. Cells staining for cluster of differentiation (CD) 45 and IgM accumulated around sites of active chondrogenesis, which contained cells that strongly expressed Sox9. The level of scleral chondrogenesis and the numbers of scleral cartilage PCNA positive cells increased with the temperature of the water in which spotted wolffish were cultured. Our results provide new knowledge of differing Sox9 spatial tissue expression patterns during chondrogenesis in normal control and ocular insult paradigms. Our work also provides evidence that spotted wolffish possess an inherent scleral chondrogenesis response that may be sensitive to temperature. This work also advances the fundamental knowledge of teleost ocular skeletal systems.

CD45 in ocular tissues during larval and juvenile stages and early stages of V. anguillarum infection in young lumpfish (Cyclopterus lumpus).

Immune responses to infectious diseases impacting lumpfish (Cyclopterus lumpus) eye tissue are only starting to be studied at a molecular and histopathological level. In this study, we extend our understanding of lumpfish sensory organ anatomy, of components of the lumpfish nasal and ocular immune system and the nature of the intraocular response to Vibrio anguillarum infection. We have evaluated the expression of cluster of differentiation (CD) 45 protein, a tyrosine phosphatase, in larval and juvenile lumpfish tissues in order to spatially survey ocular and related head structures that may participate in early stages of intraocular immune responses. We provide here a histological mapping of the larval lumpfish nasal chamber system since its connectively with the eye though mucosal epithelia have not been explored. These results build upon our growing understanding of the lumpfish intraocular immune response to pathogens, exemplified herein by experimental nasally delivered V. anguillarum infection. CD45 is developmentally regulated in lumpfish eyes and periocular anatomy with early expression appearing in larvae in corneal epithelium and in nasal structures adjacent to the eye. Normal juvenile and adult lumpfish eyes express CD45 in the corneal epithelium, in leukocyte cells within blood vessel lumens of the rete mirabile, choroid body and choriocapillaris vasculatures. Experimental nasally delivered V. anguillarum infection led to qualitative and quantitative changes in CD45 expression in head kidney renal tubule tissues by 7 days post infection (dpi). The same animals showed redistribution and upregulation of corneal epithelial CD45 expression, corneal epithelial dysplasia and an increased frequency of CD45+ cells in ocular vasculature. Interestingly, while CD45 upregulation and/or CD45+ cell infiltration into inner ocular and retinal tissues was not observed under this experimental scenario, subtle neural retinal changes were observed in infected fish. This work provides new fundamental knowledge on North Atlantic teleost visual systems and vision biology in general.

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.

The Anti-SARS-CoV-2 Antibody Response in a Centenarian Woman: A Case of Long-Term Memory?

SARS-CoV-2 is the virus responsible for the COVID-19 pandemic, causing respiratory syndrome and other manifestations. The clinical consequences of the SARS-CoV-2 infection are highly heterogeneous, ranging from asymptomatic and mild to severe and fatal conditions, with the highest mortality rate reached among elderly people. Such heterogeneity appears strongly influenced by the host immune response, which in turn is profoundly affected by aging. In fact, the occurrence of a low-grade inflammation and a decline in specific immune defense is generally reported in older people. Although the low ability of B cells to provide primary and secondary specific responses with a consequent increase in susceptibility to and severity of virus infections is generally described in elderly people, we would like to present here the particular case of a 100-year-old woman, who recovered well from COVID-19 and developed a long-term memory against SARS-CoV-2. Following the infection, the patient's blood was tested with both a classical ELISA and a specific Cell-ELISA addressed to measure the anti-spike S1 specific IgG released in plasma or produced in vitro by memory B cells, respectively. While showing negative on classical serological testing, the patient's blood was positive in Cell-ELISA up to 1 year after the infection. Our observation highlights a potential mechanism of B cell-dependent, long-term protection in response to SARS-CoV-2 infection, suggesting that in a case of successful aging, the absence of specific antibodies in serum does not necessarily mean the absence of immune memory.

Prepubertal gonad investment modulates thymus function: evidence in a teleost fish.

Thymus plasticity following gonadectomy or sex hormone replacement has long since exemplified sex hormone effects on the immune system in mammals and, to a lesser extent, in 'lower vertebrates', including amphibians and fish. Nevertheless, the underlying physiological significances as well as the ontogenetic establishment of this crosstalk remain largely unknown. Here, we used a teleost fish, the European sea bass, Dicentrarchus labrax, to investigate: (1) whether the regulation of thymus plasticity relies on resource trade-off with somatic growth and reproductive investment and (2) if the gonad-thymus interaction takes place during gonadal differentiation and development. Because gonadal development and, supposedly, thymus function in sea bass depend on environmental changes associated with the winter season, we evaluated thymus changes (foxn1 expression, and thymocyte and T cell content) in juvenile D. labrax raised for 1 year under either constant or fluctuating photoperiod and temperature. Importantly, in both conditions, intensive gonadal development following sex differentiation coincided with a halt of thymus growth, while somatic growth continued. To the best of our knowledge, this is the first study showing that gonadal development during prepuberty regulates thymus plasticity. This finding may provide an explanation for the initiation of the thymus involution related to ageing in mammals. Comparing fixed and variable environmental conditions, our work also demonstrates that the extent of the effects on the thymus, which are related to reproduction, depend on ecophysiological conditions, rather than being directly related to sexual maturity and sex hormone levels.

Prepubertal gonad investment modulates thymus function: evidence in a teleost fish.

Thymus plasticity following gonadectomy or sex hormone replacement has long since exemplified sex hormone effects on the immune system in mammals and, to a lesser extent, in 'lower vertebrates', including amphibians and fish. Nevertheless, the underlying physiological significances as well as the ontogenetic establishment of this crosstalk remain largely unknown. Here, we used a teleost fish, the European sea bass, Dicentrarchus labrax, to investigate: (1) whether the regulation of thymus plasticity relies on resource trade-off with somatic growth and reproductive investment and (2) if the gonad-thymus interaction takes place during gonadal differentiation and development. Because gonadal development and, supposedly, thymus function in sea bass depend on environmental changes associated with the winter season, we evaluated thymus changes (foxn1 expression, and thymocyte and T cell content) in juvenile D. labrax raised for 1 year under either constant or fluctuating photoperiod and temperature. Importantly, in both conditions, intensive gonadal development following sex differentiation coincided with a halt of thymus growth, while somatic growth continued. To the best of our knowledge, this is the first study showing that gonadal development during prepuberty regulates thymus plasticity. This finding may provide an explanation for the initiation of the thymus involution related to ageing in mammals. Comparing fixed and variable environmental conditions, our work also demonstrates that the extent of the effects on the thymus, which are related to reproduction, depend on ecophysiological conditions, rather than being directly related to sexual maturity and sex hormone levels.

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.

Cold Adaptation in Antarctic Notothenioids: Comparative Transcriptomics Reveals Novel Insights in the Peculiar Role of Gills and Highlights Signatures of Cobalamin Deficiency.

Far from being devoid of life, Antarctic waters are home to Cryonotothenioidea, which represent one of the fascinating cases of evolutionary adaptation to extreme environmental conditions in vertebrates. Thanks to a series of unique morphological and physiological peculiarities, which include the paradigmatic case of loss of hemoglobin in the family Channichthyidae, these fish survive and thrive at sub-zero temperatures. While some of the distinctive features of such adaptations have been known for decades, our knowledge of their genetic and molecular bases is still limited. We generated a reference de novo assembly of the icefish Chionodraco hamatus transcriptome and used this resource for a large-scale comparative analysis among five red-blooded Cryonotothenioidea, the sub-Antarctic notothenioid Eleginops maclovinus and seven temperate teleost species. Our investigation targeted the gills, a tissue of primary importance for gaseous exchange, osmoregulation, ammonia excretion, and its role in fish immunity. One hundred and twenty genes were identified as significantly up-regulated in Antarctic species and surprisingly shared by red- and white-blooded notothenioids, unveiling several previously unreported molecular players that might have contributed to the evolutionary success of Cryonotothenioidea in Antarctica. In particular, we detected cobalamin deficiency signatures and discussed the possible biological implications of this condition concerning hematological alterations and the heavy parasitic loads typically observed in all Cryonotothenioidea.

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.

A Cell-Based ELISA to Improve the Serological Analysis of Anti-SARS-CoV-2 IgG.

Knowledge of the antibody-mediated immune response to SARS-CoV-2 is crucial to understand virus immunogenicity, establish seroprevalence, and determine whether subjects or recovered patients are at risk for infection/reinfection and would therefore benefit from vaccination. Here, we describe a novel and simple cell-ELISA specifically designed to measure viral spike S1-specific IgG produced in vitro by B cells in peripheral blood mononuclear cell (PBMC) cultures from a cohort of 45 asymptomatic (n = 24) and symptomatic (n = 21) individuals, with age ranging from 8 to 99 years. All subjects underwent ELISA serological screening twice, at the same time as the cell-ELISA (T2) as well as 35-60 days earlier (T1). Cryopreserved PBMCs of healthy donors obtained years before the COVID-19 pandemic were also included in the analysis. The preliminary results presented here show that out of 45 tested subjects, 16 individuals (35.5%) were positive to the cell-ELISA, 11 (24.5%) were concomitantly positive in the serological screening (T1 and/or T2), and only one person was exclusively positive in ELISA (T1) and negative in cell-ELISA, though values were close to the cutoff. Of note, five individuals (11.2%) tested negative in ELISA but positive in cell-ELISA and thus, they appear to have circulating B cells that produce antibodies against SARS-CoV-2, likely at levels that are undetectable in the serum, which challenges the negative results of the serological screening. The relative level of in vitro secreted IgG was measurable in positive subjects, ranging from 7 to 50 ng/well. Accordingly, all anti-SARS-CoV-2 antibody-positive subjects previously reported moderate to severe symptoms attributable to COVID-19, even though the RT-PCR data were rarely available to confirm viral infection. Overall, the described cell-ELISA might be an effective method for detecting subjects who encountered the virus in the past, and thus helpful to improve serological ELISA tests in the case of undetectable/equivocal circulating IgG levels, and a suitable and improved tool to better evaluate SARS-CoV-2-specific humoral immunity in the COVID-19 pandemic.

Identification of an IgD/IgT chimera in the European sea bass (Dicentrarchus labrax L.).

Three classes of immunoglobulins have been identified in Teleosts: IgM, IgT/Z and IgD. They are fundamental for fish immune responses and, therefore, their functional activities are heavily investigated. In this paper, we describe the identification of a new IgD/IgT chimera in sea bass (Dicentrarchus labrax) from a gills transcriptome. This transcript joined the first six constant domains of the IgD chain with the two terminal constant domains of IgT, generating a long in-frame coding sequence with a junction between the canonical δ6 exon splicing donor site and the τ3 exon splicing acceptor site. Studies performed on genomic DNA confirmed the presence of the sequence and identifies and intronic region of 656 bp within this joining region. The basal expression of the IgD/IgT chimera was investigated both in silico and in vivo: high level of expression was found in gills, gut and head kidney. Moreover, IgD/IgT transcripts were up-regulated after in vitro stimulation of sea bass HK leukocytes with LPS. The IgD/IgT chimera was found also in two congener species, Morone saxatilis and Morone chrysops. It is not possible to have a precise idea on the evolutionary scenario that lead to the appearance of this sequence due to the lack of genomic information, but we could speculate that an ancestral duplication of the entire IgH locus was followed by the chimerization of Cδ/Cτ in one of the two loci. Finally, the IgD/IgT high basal expression in tissues and organs fundamental for sea bass immune response and its modulation after LPS stimulation provide a very preliminary indication that this unusual Ig variant could have a functional activity.

Trematocine, a Novel Antimicrobial Peptide from the Antarctic Fish Trematomus bernacchii: Identification and Biological Activity.

Antimicrobial peptides (AMPs) are short peptides active against a wide range of pathogens and, therefore, they are considered a useful alternative to conventional antibiotics. We have identified a new AMP in a transcriptome derived from the Antarctic fish Trematomus bernacchii. This peptide, named Trematocine, has been investigated for its expression both at the basal level and after in vivo immunization with an endemic Antarctic bacterium (Psychrobacter sp. TAD1). Results agree with the expected behavior of a fish innate immune component, therefore we decided to synthesize the putative mature sequence of Trematocine to determine the structure, the interaction with biological membranes, and the biological activity. We showed that Trematocine folds into a α-helical structure in the presence of both zwitterionic and anionic charged vesicles. We demonstrated that Trematocine has a highly specific interaction with anionic charged vesicles and that it can kill Gram-negative bacteria, possibly via a carpet like mechanism. Moreover, Trematocine showed minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against selected Gram-positive and Gram-negative bacteria similar to other AMPs isolated from Antarctic fishes. The peptide is a possible candidate for a new drug as it does not show any haemolytic or cytotoxic activity against mammalian cells at the concentration needed to kill the tested bacteria.

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.

Identification, molecular characterization and functional analysis of interleukin (IL)-2 and IL-2like (IL-2L) cytokines in sea bass (Dicentrarchus labrax L.).

In mammals, interleukin (IL)-2, initially known as a T-cell grow factor, is an immunomodulatory cytokine involved in the proliferation of T cells upon antigen activation. In bony fish, some IL-2 orthologs have been identified, but, recently, an additional IL-2like (IL-2L) gene has been found. In this paper, we report the presence of these two divergent IL-2 isoforms in sea bass (Dicentrarchus labrax L.). Genomic analyses revealed that they originated from a gene duplication event, as happened in most percomorphs. These two IL-2 paralogs show differences in the amino acid sequence and in the exon 4 size, and these features could be an indication that they bind preferentially to different specific IL-2 receptors. Sea bass IL-2 paralogs are highly expressed in gut and spleen, which are tissues and organs involved in fish T cell immune functions, and the two cytokines could be up-regulated by both PHA stimulation and vaccination with a bacterial vaccine, with IL-2L being more inducible. To investigate the functional activities of sea bass IL-2 and IL-2L we produced the corresponding recombinant molecules in E. coli and used them to in vitro stimulate HK and spleen leukocytes. IL-2L is able to up-regulate the expression of markers related to different T cell subsets (Th1, Th2 and Th17) and to Treg cells in HK, whereas it has little effect in spleen. IL-2 is not active on these markers in HK, but shows an effect on Th1 markers in spleen. Finally, the stimulation with recombinant IL-2 and IL-2L is also able to induce in vitro proliferation of HK- and spleen-derived leukocytes. In conclusion, we have demonstrated that sea bass possess two IL-2 paralogs that likely have an important role in regulating T cell development in this species and that show distinct bioactivities.

Molecular and Structural Characterization of MHC Class II ß Genes Reveals High Diversity in the Cold-Adapted Icefish Chionodraco hamatus.

This study reports the presence of two distinct MHC class II ß genes in the Antarctic icefish Chionodraco hamatus, belonging to the classical (ChhaDAB) and nonclassical (ChhaDBB) evolutionary lineages. By the application of targeted sequencing approach, a remarkable molecular diversity in the exon 2 sequence of the highly expressed gene ChhaDAB has been observed, resulting in an estimate of 92 different variants translated in 87 different peptides from 54 analysed icefish individuals. A highly conservative estimate, based on a 95% sequence identity threshold clustering, translate this variability in 41 different peptide clusters belonging to four different clades and showing the signature of different kinds of selection. In stark contrast, the poorly expressed ChhaDBB gene displayed a very low level of molecular diversity within exon 2, in agreement with expectations for a nonclassical MHC class II ß gene.

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.

Dietary fatty acid source has little effect on the development of the immune system in the pyloric caeca of Atlantic salmon fry.

The quality and relative amounts of dietary lipids may affect the health and growth of cultured Atlantic salmon. So far, little is known about their effects on the performance of the fish immune system during early life stages and, in particular their importance in the transition from endogenous nutrition (yolk) in the alevin stage to exogenous nutrition in the later fry stage. We investigated the immunomodulatory effects of fish oil, vegetable oil and phospholipid-rich oil in feeds for farmed Atlantic salmon using a transcriptomic approach. The experiment allowed a fine-scale monitoring of gene expression profiles in two tissues, the pyloric caeca of the intestine and the liver, in a 94 days-long first feeding experiment. The analysis of transcriptional profiles revealed that first feeding induced a strong immunomodulation in the pyloric caeca after 48 days of feeding, lasting up to day 94 and possibly beyond. On the other hand, the differential effect of the three dietary regimes was negligible. We interpret this upregulation, undetectable in liver, as a potentiation of the immune system upon the first contact of the digestive system with exogenous feed. This process involved a complex network of gene products involved in both cellular and humoral immunity. We identified the classical pathway of the complement system, acting at the crossroads between innate and adaptive immunity, as a key process modulated in response to the switch from endogenous to exogenous nutrition.

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.