An ex vivo Approach in European Seabass Leucocytes Supports the in vitro Regulation by Postbiotics of Aip56 Gene Expression of Photobacterium damselae subsp. piscicida.

Shewanella putrefaciens Pdp11 (SpPdp11) is a probiotic strain assayed in aquaculture; however, its postbiotic potential is unknown. Postbiotics are bacterial metabolites, including extracellular products (ECPs) that improve host physiology and immunity. Their production and composition can be affected by different factors such as the growing conditions of the probiotics. Photobacterium damselae subsp. piscicida strain Lg 41/01 (Phdp) is one of the most important pathogens in marine aquaculture. The major virulent factor of this bacterium is the exotoxin aip56, responsible for inducing apoptosis of fish leucocytes. Viable SpPdp11 cells have been reported to increase resistance to challenges with Phdp. This work aimed to evaluate the effect of two ECPs, T2348-ECP and FM1548-ECP, obtained from SpPdp11 grown under different culture conditions that previously demonstrated to exert different degradative and non-cytotoxic activities, as well as the effect on pathogens biofilm formation. These SpPdp11-ECPs were then analyzed by their effect on the viability, phagocytosis, respiratory burst and apoptogenic activity against European sea bass leucocytes infected or not with Phdp supernatant. Both ECPs, T2348-ECP and FM1548-ECP, were not cytotoxic against leucocytes and significantly reduced their apoptosis. Phagocytosis and respiratory burst of leucocytes were significantly reduced by incubation with Phdp supernatant, and not influenced by incubation with T2348-ECP or FM1548-ECP. However, both activities were significantly increased after leucocyte incubation with combined T2348-ECP and FM1548-ECP with Phdp supernatant, compared to those incubated only with Phdp supernatant. Finally, both T2348-ECP and FM1548-ECP significantly reduced the relative in vitro expression of the Phdp aip56 encoding gene.

In vitro immune-depression and anti-inflammatory activities of cantharidin on gilthead seabream (Sparus aurata) leucocytes activated by λ-carrageenan.

Cantharidin is a natural compound with known therapeutic applications in humans. The aim of this study was to investigate the in vitro effects of cantharidin on gilthead seabream (Sparus aurata) head kidney leucocytes (HKL) stimulated with λ-carrageenan. HKLs were incubated for 24 h with cantharidin (0, 2.5 and 5 µg mL-1) and λ-carrageenan (0 and 1000 µg mL-1). The results showed that HKL viability only decreased by 15.2% after incubated with 5 µg mL-1 of cantharidin and λ-carrageenan. Cantharidin increased the peroxidase activity of HKLs only when incubated in combination with λ-carrageenan. Besides this, cantharidin inhibited the respiratory burst and phagocytic activities. Furthermore, cantharidin induced morphological changes in HKLs (apoptotic and vacuolization signs) that were enhanced when incubated with λ-carrageenan. Considering the analysis of the selected gene expression studied in HKLs [NF-κB subunits (rela, relb, crel, nfkb1, nfkb2), proinflammatory cytokines (il1b, tnfa), anti-inflammatory cytokines (il10, tgfb) and caspases (casp1, casp3, casp8, casp9)], although λ-carrageenan up-regulated the expression of the proinflammatory gene il1b, λ-carrageenan and cantharidin down-regulated its expression in HKLs. In addition, cantharidin up-regulated casp3 and casp9 expression. The casp3 and casp9 gene expression was down-regulated while casp1 gene expression was up-regulated in HKLs incubated with both cantharidin and λ-carrageenan. All the effects of cantharidin are related to its inhibitory effect on protein phosphatases, which induce apoptosis at long exposure times, and minimize the effects of λ-carrageenan. The present results provide detailed insight into the immune-depressive and anti-inflammatory properties of cantharidin on immune cells, which could be of interest to the aquaculture sector.

Biology and functions of fish thrombocytes: A review.

This comprehensive review examines the role of fish thrombocytes, cells considered functionally analogous to platelets in terms of coagulation, but which differ in their origin and morphology. Despite the evolutionary distance between teleosts and mammals, genomic studies reveal conserved patterns in blood coagulation, although there are exceptions such as the absence of factors belonging to the contact system. Beyond coagulation, fish thrombocytes have important immunological functions. These cells express both proinflammatory genes and genes involved in antigen presentation, suggesting a role in both innate and adaptive immune responses. Moreover, having demonstrated their phagocytic abilities, crucial in the fight against pathogenic microorganisms, underscores their multifaceted involvement in immunity. Finally, the need for further research on the functions of these cells is highlighted, in order to better understand their involvement in maintaining the health of aquaculture fish. The use of standardized and automated methods for the analysis of these activities is advocated, emphaiszing their potential to facilitate the early detection of stress or infection, thus minimizing the economic losses that these adverse situations can generate in the field of aquaculture.

Participation of Hepcidins in the Inflammatory Response Triggered by λ-Carrageenin in Gilthead Seabream (Sparus aurata).

The role of hepcidins, antimicrobial peptides involved in iron metabolism, immunity, and inflammation, is studied. First, gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs) were incubated with λ-carrageenin to study the expression of hepcidin and iron metabolism-related genes. While the expression of most of the genes studied was upregulated, the expression of ferroportin gene (slc40a) was downregulated. In the second part of the study, seabream specimens were injected intramuscularly with λ-carrageenin or buffer (control). The expression of the same genes was evaluated in the head kidney, liver, and skin at different time points after injection. The expression of Hamp1m, ferritin b, and ferroportin genes (hamp1, fthb, and slc40a) was upregulated in the head kidney of fish from the λ-carrageenin-injected group, while the expression of Hamp2C and Hamp2E genes (hamp2.3 and hamp2.7) was downregulated. In the liver, the expression of hamp1, ferritin a (ftha), slc40a, Hamp2J, and Hamp2D (hamp2.5/6) genes was downregulated in the λ-carrageenin-injected group. In the skin, the expression of hamp1 and (Hamp2A Hamp2C) hamp2.1/3/4 genes was upregulated in the λ-carrageenin-injected group. A bioinformatic analysis was performed to predict the presence of transcription factor binding sites in the promoter region of hepcidins. The primary sequence of hepcidin was conserved among the different mature peptides, although changes in specific amino acid residues were identified. These changes affected the charge, hydrophobicity, and probability of hepcidins being antimicrobial peptides. This study sheds light on the poorly understood roles of hepcidins in fish. The results provide insight into the regulatory mechanisms of inflammation in fish and could contribute to the development of new strategies for treat inflammation in farm animals.

Evaluation of the Differential Postbiotic Potential of Shewanella putrefaciens Pdp11 Cultured in Several Growing Conditions.

The increased knowledge of functional foods has led to the development of a new generation of health products, including those containing probiotics and products derived from them. Shewanella putrefaciens Pdp11 (SpPdp11) is a strain described as a probiotic that exerts important beneficial effects on several farmed fish. However, the use of live probiotic cells in aquaculture has limitations such as uncertain survival and shelf life, which can limit their efficacy. In addition, its efficacy can vary across species and hosts. When probiotics are administered orally, their activity can be affected by the environment present in the host and by interactions with the intestinal microbiota. Furthermore, live cells can also produce undesired substances that may negatively impact the host as well as the risk of potential virulence reversion acquired such as antibiotic resistance. Therefore, new alternatives emerged such as postbiotics. Currently, there is no knowledge about the postbiotic potential of SpPdp11 in the aquaculture industry. Postbiotic refers to the use of bacterial metabolites, including extracellular products (ECPs), to improve host physiology. However, the production of postbiotic metabolites can be affected by various factors such as cultivation conditions, which can affect bacterial metabolism. Thus, the objective of this study was to evaluate the postbiotic potential of ECPs from SpPdp11 under different cultivation conditions, including culture media, temperature, growth phase, and salinity. We analyzed their hydrolytic, antibacterial, antiviral, and cytotoxic capacity on several fish cell lines. The results obtained have demonstrated how each ECP condition can exert a different hydrolytic profile, reduce the biofilm formation by bacterial pathogens relevant to fish, lower the titer of nervous necrosis virus (NNV), and exert a cytotoxic effect on different fish cell lines. In conclusion, the ECPs obtained from SpPdp11 have different capacities depending on the cultivation conditions used. These conditions must be considered in order to recover the maximum number of beneficial capacities or to choose the appropriate conditions for specific activities.

A review of soluble factors and receptors involved in fish skin immunity: The tip of the iceberg.

The immune system of fish possesses soluble factors, receptors, pathways and cells very similar to those of the other vertebrates' immune system. Throughout evolutionary history, the exocrine secretions of organisms have accumulated a large reservoir of soluble factors that serve to protect organisms from microbial pathogens that could disrupt mucosal barrier homeostasis. In parallel, a diverse set of recognition molecules have been discovered that alert the organism to the presence of pathogens. The known functions of both the soluble factors and receptors mentioned above encompass critical aspects of host defense, such as pathogen binding and neutralization, opsonization, or modulation of inflammation if present. The molecules and receptors cooperate and are able to initiate the most appropriate immune response in an attempt to eliminate pathogens before host infection can begin. Furthermore, these recognition molecules, working in coordination with soluble defence factors, collaboratively erect a robust and perfectly coordinated defence system with complementary specificity, activity and tissue distribution. This intricate network constitutes an immensely effective defence mechanism for fish. In this context, the present review focuses on some of the main soluble factors and recognition molecules studied in the last decade in the skin mucosa of teleost fish. However, knowledge of these molecules is still very limited in all teleosts. Therefore, further studies are suggested throughout the review that would help to better understand the functions in which the proteins studied are involved.

Dietary Inclusion of Halobacterium salinarum Modulates Growth Performances and Immune Responses in Farmed Gilthead Seabream (Sparus aurata L.).

The use of natural immunostimulants is considered the most promising alternative to promote fish health, productive performance and quality, increasing the aquaculture profitability, sustainability and social acceptance. The purpose of this study was to evaluate the effect of the integration of a potential probiotic strain, Halobacterium salinarum, belonging to the Archaea domain, in the formulated diets of farmed gilthead seabream (Sparus aurata L.) in terms of growth performances and immunity responses. The experiment was set up to test two different levels of inclusion of the bacteria in the diet: 0.05% (D1) and 0.1% (D2). The effects on fish growth performances; humoral (peroxidase, protease, antiprotease and IgM levels) and cellular immunity parameters (phagocytosis, respiratory burst and myeloperoxidase), along with bactericidal activity, were evaluated after 15 and 30 days of experimental feeding. The obtained results showed that the inclusion of H. salinarum at the highest concentration (D2 0.1%) improved growth performances, bactericidal activity against Vibrio anguillarum and some parameters related both to the humoral and cellular immune response, suggesting exploring other aspects of welfare in view of future supplementations of this probiotic strain in the diet of S. aurata.

Non-nosocomial Healthcare-Associated Infective Endocarditis: A Distinct Entity? Data From the GAMES Series (2008-2021).

Background: Patients who acquire infective endocarditis (IE) following contact with the healthcare system, but outside the hospital, are classified as having non-nosocomial healthcare-associated IE (HCIE). Our aim was to characterize HCIE and establish whether its etiology, diagnosis, and therapeutic approach suggest it should be considered a distinct entity. Methods: This study retrospectively analyzes data from a nationwide, multicenter, prospective cohort including consecutive cases of IE at 45 hospitals across Spain from 2008 to 2021. HCIE was defined as IE detected in patients in close contact with the healthcare system (eg, patients receiving intravenous treatment, hemodialysis, or institutionalized). The prevalence and main characteristics of HCIE were examined and compared with those of community-acquired IE (CIE) and nosocomial IE (NIE) and with literature data. Results: IE was diagnosed in 4520 cases, of which 2854 (63%) were classified as CIE, 1209 (27%) as NIE, and 457 (10%) as HCIE. Patients with HCIE showed a high burden of comorbidities, a high presence of intravascular catheters, and a predominant staphylococcal etiology, Staphylococcus aureus being identified as the most frequent causative agent (35%). They also experienced more persistent bacteremia, underwent fewer surgeries, and showed a higher mortality rate than those with CIE (32.4% vs 22.6%). However, mortality in this group was similar to that recorded for NIE (32.4% vs 34.9%, respectively, P = .40). Conclusions: Our data do not support considering HCIE as a distinct entity. HCIE affects a substantial number of patients, is associated with a high mortality, and shares many characteristics with NIE.

Combined effects of rearing temperature regime (thermocycle vs. constant temperature) during early development and thermal treatment on Nile tilapia (Oreochromis niloticus) sex differentiation.

In nature, water temperature experiences daily variations known as thermocycles. Temperature is the main environmental factor that influences sex determination in most teleost fish. The purpose of this study was to examine the effects of rearing temperature (thermocycle (TC) vs. constant (CTE)) on development and a posterior thermal shock throughout the period of sex differentiation of Nile tilapia (Oreochromis niloticus). Embryos and larvae were kept under two temperature regimes: TC of 31 °C:25 °C day:night vs. CTE of 28 °C from 0 to 11 dpf. After this period, the larvae from each group were subjected to either heat treatment (HT, 36 °C for 12 days) or kept under the same rearing temperatures until 23 dpf (Control, C). Then all the groups remained at constant temperature until 270 dpf, when blood and gonads were collected. Larval samples were used to examine the expression of genes related to male (amh, ara, sox9a, dmrt1a) and female (cyp19a1a, foxl2, era) sexual differentiation. In juveniles, sex was characterized by histology, the gonadal expression of the genes involved in the sex steroid synthesis was analyzed by qPCR, and plasma testosterone (T) and estradiol (E2) levels were analyzed by ELISA. In larvae, daily TCs increased the survival rate against HT and up-regulated the expression of ovarian differentiation genes. In juveniles, TC + C induced a higher proportion of females and higher cyp19a1a expression compared to CTE + C. HT induced changes in the CTE group by up-regulating testicular differentiation genes and down-regulating female promoting genes, which did not occur in the TC group. Juveniles from TC + C group presented a higher proportion of females with higher E2 and cyp19a1a than CTE + HT. Fish from the CTE + HT group showed a higher percentage of males with highest T and amh. These findings indicate that daily TCs during larval development promote ovarian differentiation and diminish the masculinizing effects of HT.

Contaminant-induced oxidative stress underlies biochemical, molecular and fatty acid profile changes, in gilthead seabream (Sparus aurata L.).

Chemical contaminants such as heavy metals, polybrominated diphenyl ethers (PBDEs) and drugs, are constantly found in the marine environment determining the interest of the scientific community for their side effects on animal welfare, food safety and security. Few studies have analyzed the effects of mix of contaminants in fish, in terms of molecular and nutritional composition response, beside it is indispensable to think more and more on effect of contaminants along the food web system. In this study, Sparus aurata specimens were exposed for 15 days, by diet, to a mixture of carbamazepine (Cbz), polybrominated diphenyl ether-47 (PBDE-47) and cadmium chloride (CdCl2), at two doses (0.375 µg g-1 D1; 37.5 µg g-1 D2) (T15). After, fish were fed with a control diet, without contaminants mix, for other 15 days (T30). The study explored the effect on oxidative stress in the liver, analyzing specific molecular markers and effects on quality, by fatty acid profile and lipid peroxidation. Molecular markers involved in ROS scavenging, such as superoxide dismutase (sod), catalase (cat) and glutathione peroxidase (gpx) were evaluated by gene expression; as markers of quality and lipid peroxidation, the fatty acids (FAs) profile and the level of malondyaldeide (MDA) were assessed. Sod and cat genes underwent to up-regulation after 15 days of diet containing contaminants and showed down-regulation after the next 2 weeks of detoxification (T30). At T15, the FAs profile showed an increase of the saturated fatty acids (SFA), and a decrease of the polyunsatured fatty acids (PUFA). The MDA levels increased over time, indicating an ongoing radical damage. These results suggest that the effects of the contaminants can be perceived not only at molecular but also at nutritional level and that the molecular and biochemical markers adopted could be differently used to monitor the health of aquatic organisms in the marine environment.

Design of a new mortality indicator in acute coronary syndrome on admission to the Intensive Care Unit.

OBJECTIVE: To design a mortality indicator in acute coronary syndrome (ACS) in the intensive care unit (ICU). DESIGN: A multicenter, observational descriptive study was carried out. PARTICIPANTS: Patients with ACS admitted to the ICUs included in the ARIAM-SEMICYUC registry between January 2013 and April 2019. INTERVENTIONS: None. MAIN VARIABLES OF INTEREST: Demographic parameters, time of access to the healthcare system, and clinical condition. Revascularization therapy, drugs and mortality were analyzed. Cox regression analysis was performed, followed by the design of a neural network. A receiver operating characteristic curve (ROC) was plotted to calculate the power of the new score. Lastly, the clinical utility or relevance of the ARIAM indicator (ARIAM's) was assessed using a Fagan test. RESULTS: A total of 17,258 patients were included in the study, with a mortality rate of 3.5% (n = 605) at discharge from the ICU. The variables showing statistical significance (P < .001) were entered into the supervised predictive model, an artificial neural network. The new ARIAM's yielded a mean of 0.0257 (95%CI: 0.0245-0.0267) in patients discharged from the ICU versus 0.27085 (95%CI: 0.2533-0.2886) in those who died (P < .001). The area under the ROC curve of the model was 0.918 (95%CI: 0.907-0.930). Based on the Fagan test, the ARIAM's showed the mortality risk to be 19% (95%CI: 18%-20%) when positive and 0.9% (95%CI: 0.8%-1.01%) when negative. CONCLUSIONS: A new mortality indicator for ACS in the ICU can be established that is more accurate and reproducible, and periodically updated.

Skin mucus metabolomics provides insights into the interplay between diet and wound in gilthead seabream (Sparus aurata).

The molecular processes underlying skin wound healing in several fish species have been elucidated in the last years, however, metabolomic insights are scarce. Here we report the skin mucus metabolome of wounded and non-wounded gilthead seabream (Sparus aurata) fed with silk fibroin microparticles, a functional additive considered to accelerate the wound healing process. The three experimental diets (commercial diet enriched with 0 mg (control), 50 mg or 100 mg of silk fibroin microparticles Kg-1) were administered for 30 days and thereafter, a skin wound was inflicted. Skin mucus was collected on day 30 of feeding and 7 days post-wounding and subjected to metabolomic analysis by Ultra Performance Liquid Chromatography coupled with a high-resolution quadrupole-orbitrap mass spectrometry. The most enriched metabolite class was amino acids and derivatives, followed by nucleotides, nucleosides and analogues and carbohydrates and their derivatives. Metabolomic profiles revealed that the diet had a more profound effect than wounding in skin mucus. Metabolic pathway analysis of significantly affected metabolites revealed perturbations in the aminoacyl t-RNA biosynthesis in the skin. In particular, skin wound resulted in a decreased methionine level in mucus. Further, silk fibroin supplementation increased methionine level in skin mucus, which correlated with several wound morphometric parameters that characterized the epithelial healing capacity in seabream. The results provided new insight into the physiological consequences of skin wounds and how these processes could be influenced by dietary manipulation.

Molecular characterization of the cytotoxic and regulatory T cell coreceptor (CRTAM), and its ligand CADM1, in the European seabass and gilthead seabream.

T cell activation is a multifaceted process that depends on the activation of the T cell receptor (TCR). However, other coreceptors are also strictly necessary to provide co-signals and modulate the immune response. However, to date, most of these coreceptors are unknown in fish or their information is very limited. Therefore, in this work, we have identified the cytotoxic and regulatory T cell molecule, CRTAM, and its ligand, the cell adhesion molecule 1, CADM1, in European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata); and evaluated their transcriptional levels. Both putative proteins showed the canonical architecture observed in mammals, where CRTAM exhibited two immunoglobulin domains and CADM1, both the a and b forms, exhibited three of these domains. In addition, phylogeny and synteny analyses showed their conservation throughout vertebrate evolution. We found constitutive expression of all three genes, with crtam and cadm1a being predominant in immune tissues such as spleen, thymus and head-kidney (HK), while cadm1b expression was more limited to the brain. In vitro, only the T cell mitogen phytohemagglutinin (PHA) up-regulated the transcription of crtam and cadm1a in HK leucocytes. Nodavirus (NNV) infection elicited an up-regulation of crtam and cadm1a in brain and HK, appearing earlier in seabream than in seabass, which could explain the resistance of seabream to the development of nodavirus disease. In addition, they are up-regulated during the innate cell-mediated cytotoxic response in seabream but not in seabass. Altogether, our data seem to indicate that CRTAM is more related to the innate cytotoxicity in seabream and more in the specific and T cell-mediated cytotoxicity in seabass. Our results highlight the importance of CRTAM and CADM1 as important molecules in the activation of T lymphocytes in seabass and seabream, but further studies are needed.

Comparative study of immunological biomarkers in the carpet shell clams (Ruditapes decussatus) from metal-contaminated sites in the South Lagoon of Tunis (Tunisia).

The South Lagoon of Tunis (Tunisia) is a Mediterranean lagoon adversely affected by industrial contaminants, harbour activity and untreated urban sewage. In this lagoon, the clam Ruditapes decussatus has been widely used as a biomonitor of seawater pollution through measurements of parameters related to oxidative stress and neurotoxicity. However, few studies have considered parameters of the immune system of this species in the South Lagoon of Tunis. Therefore, the aim of the present work was to evaluate several immune-related parameters in the cell-free haemolymph of carpet shell clams sampled during August and February from three polluted sites in the South Lagoon of Tunis (S1, S2 and S3) and one less polluted site as a reference site (RS) in order to identify suitable biomarkers for environmental quality assessments of this ecosystem. Concerning the immune-related parameters, seasonal factors modulated phenoloxidase, lysozyme, protease and esterase activity, with lower values measured for samples collected in August than for samples collected in February. In fact, bactericidal activity against two of the pathogenic bacteria tested and the activity of most immune-related enzymes were reduced in the cell-free haemolymph of clams collected from the most sampling sites in August compared to February one. In addition, values of abiotic parameters (temperature, salinity and pH) and metal (cadmium, copper, iron, lead and zinc) concentrations in the clams' soft tissues, previously obtained and published by the authors, as well as the values of immune-related parameters were integrated using principal component analyses. Results indicated that the values of all measured immune-related parameters were negatively correlated with the temperature values and the variations most of these parameters highlighted that the chemical industrial area (S3) was the most impacted location within the South Lagoon of Tunis. The present study illustrates that the immune-related parameters measured in carpet shell clam cell-free haemolymph represent suitable biomarkers for environmental quality assessments because they provide effective seasonal and spatial discrimination.

Phytochemical compounds, antiradical capacity, and in vitro inhibitory effect against fish pathogenic bacteria of okra fruits (Abelmoschus esculentus L.) at different maturity stages.

Background: Abelmoschus esculentus L., okra, has been known as a healthy plant and classically employed in food and folk medicine for several human and animal diseases. Aim: The in vitro antioxidant and antibacterial capacities, in addition to the phytochemical compounds of the okra fruit extracts gathered at three maturity stages, were the objectives of this study. Methods: This study examined the modifications in total phenolic content (TPC), total flavonoid content (TFC), and antioxidant and antibacterial capacities of three okra fruit hydroalcoholic extracts during three comestible maturity stages. The different maturity stages of okra pods were demonstrated as early-stage, mid-stage, and late-stage maturation. Results: The mid-stage of okra fruit maturity had the highest TPC (43.27 ± 2.029 mg GAE g-1), TFC (29.96 ± 0.19 mg RE g-1), and antioxidant capacity (75.64% ± 0.79%). Moreover, at mid-stage maturity, okra fruit extracts exhibited a major antibacterial effect against Vibrio anguillarum. The phenolic content was significantly increased at the mid-stage maturity, while the flavonoid level and the antioxidant activity were greatly decreased at the end of fruit maturity. Conclusion: The results confirmed that A. esculentus L. fruits at mid-stage maturity are an excellent source of biomolecules with high antiradical and bactericidal activities, which could be used as functional foods and as an option for chemical compounds for fish farming to prevent and treat numerous marine animal diseases.

Implication of adipocytes from subcutaneous adipose tissue and fatty acids in skin inflammation caused by λ-carrageenin in gilthead seabream (Sparusaurata).

The role of subcutaneous adipose tissue adipocytes and the effects of fatty acids on carrageenan-induced skin inflammation in gilthead seabream (Sparus aurata) were studied. Fish were injected intramuscularly with phosphate-buffered saline (control) or λ-carrageenin (1%), and skin samples collected at the injection site at 3 and 6 h post-injection (p.i.) were processed for histological study. In addition, the presence and levels of lipid classes, fatty acid methyl esters (FAME) and eicosanoids were evaluated in the skin samples obtained from the injected areas. Histological results indicated an increase in adipocyte area in fish sampled at 3 h p.i. with λ-carrageenin compared to fish in the control group. Furthermore, the frequency of adipocytes between 4500 and 5000 µm2 was increased at 6 h in the λ-carrageenin group compared to the control group. Analysis of lipid classes found that fish injected with λ-carrageenan showed increased free fatty acid (FFA) and sphingomyelin content at 3 and 6 h, respectively, compared to the control group. An increase in saturated fatty acids (SFA), n-6 polyunsaturated fatty acids (PUFA), and a decrease in the values of monounsaturated fatty acids (MUFA), n-3 PUFA and minor fatty acids were observed in fish skin at 6 h after λ-carrageenin injection, with respect to the values obtained in the control group. Regarding the analysis of eicosanoids, an increase in hydroxyeicosatetraenoic acid (5-HETE) was detected in the skin of fish at 6 h post-carrageenin injection compared to the control group. The presented results indicate the contribution of adipocytes and fatty acids in the development and regulation of the inflammatory response triggered by λ-carrageenin in gilthead seabream skin.

Vitamin D3 deficiency induced intestinal inflammatory response of turbot through nuclear factor-κB/inflammasome pathway, accompanied by the mutually exclusive apoptosis and autophagy.

Vitamin D3 (VD3) participated widely in the nuclear factor-κB (NF-κB)-mediated inflammation, apoptosis, and autophagy through the vitamin D receptor (VDR). However, the molecular mechanisms remain not understood in teleost. The present study investigated the functions of VD3/VDR on intestinal inflammation, autophagy, and apoptosis of turbot in vivo and in vitro. Triple replicates of 30 fish were fed with each of three diets with graded levels of 32.0 (D0), 1012.6 (D1), and 3978.2 (D2) IU/kg VD3. Obvious intestinal enteritis was observed in the D0 group and followed with dysfunction of intestinal mucosal barriers. The intestinal inflammatory response induced by VD3 deficiency was regulated by the NF-κB/inflammasome signalling. The promotion of intestinal apoptosis and suppression of intestinal autophagy were also observed in the D0 group. Similarly, VD3 deficiency in vitro induced more intense inflammation regulated by NF-κB/inflammasome signalling. The mutually exclusive apoptosis and autophagy were also observed in the group without 1,25(OH)2D3 in vitro, accompanied by similar changes in apoptosis and autophagy increased apoptosis. The gene expression of VDRs was significantly increased with the increasing VD3 supplementation both in vivo and in vitro. Moreover, VDR knockdown in turbot resulted in intestinal inflammation, and this process relied on the activation of inflammasome mediated by NF-κB signalling. Simultaneously, intestinal apoptosis was promoted, whereas intestinal autophagy was inhibited. In conclusion, VD3 deficiency could induce intestinal inflammation via activation of the NF-κB/inflammasome pathway, intestinal apoptosis, and autophagy formed a mutually exclusive relation in teleost. And VDR is the critical molecule in those processes.

Chitosan-nanoparticles effects on mucosal immunity: A systematic review.

Nanoparticles-based treatments is of utmost importance for aquaculture. In this scenario, chitosan-based nanoparticles have been proposed due to the properties of chitosan, which include mucoadhesiveness. Nevertheless, pivotal parameters of chitosan, such as degree of acetylation and molecular weight, are commonly underestimated in the available literature despite the influence they seem to have on the properties of chitosan-based nanoparticles. In this systematic review, the immunomodulator capacity of chitosan nanoparticles used as mucosal vaccines on teleost fish has been evaluated paying special attention to the chitosan properties. Four databases were used for literature search, yielding 486 documents, from which 14 meet the inclusion criteria. Only 21% of the available studies reported properly chitosan properties, which should be improved in future works to generate reproducible data as well as valuable information. To the best of our knowledge, this work objectively compares for the first time, by quantifying the mg of chitosan/g of fish applied in each study, the chitosan nanoparticle preparation and doses applied to fish, as well as the effects of the treatments applied on fish immune status.

In vitro effects of λ-carrageenin in the head-kidney leucocytes of gilthead seabream (Sparus aurata).

The λ-carrageenin is a sulphated mucopolysaccharide that has been used for decades to induce experimental inflammation in mammals. However, it has been little considered in fish. We studied the in vitro effects of λ-carrageenin on gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs). For this purpose, HKLs were incubated with serial concentrations (from 0 to 1,000 µg mL-1) of λ-carrageenin for 3, 6, 12 and 24 h to assess its influence on cell viability and morphology, cell activity and modulation of several selected inflammation-related genes. The viability results demonstrated that λ-carrageenin has no negative effects on HKLs. The respiratory burst activity and phagocytic ability of HKLs after being incubated with λ-carrageenin (100 and 1,000 µg mL-1) for 24 h were increased, whereas the phagocytic capacity was inhibited by the higher dose at the same experimental time compared with control samples. However, the peroxidase activity of HKLs was not changed by incubation with λ-carrageenin. According to transmission electron microscopy results, incubation of HKLs with the higher dose of λ-carrageenin appeared to activate the cells being evident different morphological changes without sign of cell death. Furthermore, up-regulation of three proinflammatory cytokines (il1b, tnfa, and il6) and down-regulation of anti-inflammatory genes (tgfb) were denoted in HKLs incubated with carrageenin. The present results provide a detailed approach to the effects of λ-carrageenin on fish leucocytes, which could have some impact on how we understand the response of these cells when inducing an inflammatory process in fish.