The myxozoans Myxobolus cerebralis and Tetracapsuloides bryosalmonae modulate rainbow trout immune responses: quantitative shotgun proteomics at the portals of entry after single and co-infections.

Introduction: Little is known about the proteomic changes at the portals of entry in rainbow trout after infection with the myxozoan parasites, Myxobolus cerebralis, and Tetracapsuloides bryosalmonae. Whirling disease (WD) is a severe disease of salmonids, caused by the myxosporean M. cerebralis, while, proliferative kidney disease (PKD) is caused by T. bryosalmonae, which instead belongs to the class Malacosporea. Climate change is providing more suitable conditions for myxozoan parasites lifecycle, posing a high risk to salmonid aquaculture and contributing to the decline of wild trout populations in North America and Europe. Therefore, the aim of this study was to provide the first proteomic profiles of the host in the search for evasion strategies during single and coinfection with M. cerebralis and T. bryosalmonae. Methods: One group of fish was initially infected with M. cerebralis and another group with T. bryosalmonae. After 30 days, half of the fish in each group were co-infected with the other parasite. Using a quantitative proteomic approach, we investigated proteomic changes in the caudal fins and gills of rainbow trout before and after co-infection. Results: In the caudal fins, 16 proteins were differentially regulated post exposure to M. cerebralis, whereas 27 proteins were differentially modulated in the gills of the infected rainbow trout post exposure to T. bryosalmonae. After co-infection, 4 proteins involved in parasite recognition and the regulation of host immune responses were differentially modulated between the groups in the caudal fin. In the gills, 11 proteins involved in parasite recognition and host immunity, including 4 myxozoan proteins predicted to be virulence factors, were differentially modulated. Discussion: The results of this study increase our knowledge on rainbow trout co-infections by myxozoan parasites and rainbow trout immune responses against myxozoans at the portals of entry, supporting a better understanding of these host-parasite interactions.

Ontogeny of myeloperoxidase (MPO) positive cells in flounder (Paralichthys olivaceus).

Neutrophils represent an important asset of innate immunity. Neutrophils express myeloperoxidase (MPO) which is a heme-containing peroxidase involved in microbial killing. In this study, by using real-time quantitative PCR and Western blot analysis, the flounder MPO (PoMPO) was observed to be highly expressed in the head kidney, followed by spleen, gill, and intestine during ontogeny - during developmental stages from larvae to adults. Furthermore, PoMPO positive cells were present in major immune organs of flounder at all developmental stages, and the number of neutrophils was generally higher as the fish grew to a juvenile stage. In addition, flow cytometry analysis revealed that the proportion of PoMPO positive cells relative to leukocytes, in the peritoneal cavity, head kidney, and peripheral blood of flounder juvenile stage was 18.3 %, 34.8 %, and 6.0 %, respectively, which is similar to the adult stage in flounder as previously reported. The presence and tissue distribution of PoMPO during ontogeny suggests that PoMPO positive cells are indeed a player of the innate immunity at all developmental stages of flounder.

Mucosal barrier status in Atlantic salmon fed rapeseed oil and Schizochytrium oil partly or fully replacing fish oil through winter depression.

The study was designed to investigate the effects of replacing fish oil by algal oil and rapeseed oil on histomorphology indices of the intestine, skin and gill, mucosal barrier status and immune-related genes of mucin and antimicrobial peptide (AMP) genes in Atlantic salmon (Salmo salar). For these purposes, Atlantic salmon smolts were fed three different diets. The first was a control diet containing fish oil but no Schizochytrium oil. In the second diet, almost 50 % of the fish oil was replaced with algal oil, and in the third diet, fish oil was replaced entirely with algal oil. The algal oil contained mostly docosahexaenoic acid (DHA) and some eicosapentaenoic acid (EPA). The study lasted for 49 days in freshwater (FW), after which some fish from each diet group were transferred to seawater (SW) for a 48-h challenge test at 33 ppt to test their ability to tolerate high salinity. Samples of skin, gills, and mid intestine [both distal (DI) and anterior (AI) portions of the mid intestine] were collected after the feeding trial in FW and after the SW-challenge test to assess the effects of the diets on the structure and immune functions of the mucosal surfaces. The results showed that the 50 % VMO (Veramaris® algal oil) dietary group had improved intestinal, skin, and gill structures. Principal component analysis (PCA) of the histomorphological parameters demonstrated a significant effect of the algal oil on the intestine, skin, and gills. In particular, the mucosal barrier function of the intestine, skin, and gills was enhanced in the VMO 50 % dietary group after the SW challenge, as evidenced by increased mucous cell density. Immunolabelling of heat shock protein 70 (HSP70) in the intestine (both DI and AI) revealed downregulation of the protein expression in the 50 % VMO group and a corresponding upregulation in the 100 % VMO group compared to 0 % VMO. The reactivity of HSP70 in the epithelial cells was higher after the SW challenge compared to the FW phase. Immune-related genes related to mucosal defense, such as mucin genes [muc2, muc5ac1 (DI), muc5ac1 (AI), muc5ac2, muc5b (skin), and muc5ac1 (gills)], and antimicrobial peptide genes [def3 (DI), def3 (AI), and cath1 (skin)] were significantly upregulated in the 50 % VMO group. PCA of gene expression demonstrated the positive influences on gene regulation in the 50 % VMO dietary group. In conclusion, this study demonstrated the positive effect of substituting 50 % of fish oil with algal oil in the diets of Atlantic salmon. The findings of histomorphometry, mucosal mapping, immunohistochemistry, and immune-related genes connected to mucosal responses all support this conclusion.

Severe Natural Outbreak of Cryptocaryon irritans in Gilthead Seabream Produces Leukocyte Mobilization and Innate Immunity at the Gill Tissue.

The protozoan parasite Cryptocaryon irritans causes marine white spot disease in a wide range of fish hosts, including gilthead seabream, a very sensitive species with great economic importance in the Mediterranean area. Thus, we aimed to evaluate the immunity of gilthead seabream after a severe natural outbreak of C. irritans. Morphological alterations and immune cell appearance in the gills were studied by light microscopy and immunohistochemical staining. The expression of several immune-related genes in the gills and head kidney were studied by qPCR, including inflammatory and immune cell markers, antimicrobial peptides (AMP), and cell-mediated cytotoxicity (CMC) molecules. Serum humoral innate immune activities were also assayed. Fish mortality reached 100% 8 days after the appearance of the C. irritans episode. Gill filaments were engrossed and packed without any space between filaments and included parasites and large numbers of undifferentiated and immune cells, namely acidophilic granulocytes. Our data suggest leukocyte mobilization from the head kidney, while the gills show the up-regulated transcription of inflammatory, AMPs, and CMC-related molecules. Meanwhile, only serum bactericidal activity was increased upon infection. A potent local innate immune response in the gills, probably orchestrated by AMPs and CMC, is triggered by a severe natural outbreak of C. irritans.

Molecular characterisation and biological activity of an antiparasitic peptide from Sciaenops ocellatus and its immune response to Cryptocaryon irritans.

Cryptocaryon irritans, a holotrichous ciliate parasitic protozoan, can trigger marine white spot disease and cause substantial economic losses in mariculture. However, methods of preventing and curing the disease have negatively affect fish, human, other organisms, and the natural environment. The antiparasitic activity of some antimicrobial peptides (AMPs) has garnered extensive attention of scholars. In this study, we identified and characterised a novel antiparasitic peptide, named So-pis, from Sciaenops ocellatus. The sequence analysis, structural features, and tissue distribution suggested that So-pis is genetically related to the piscidins family. However, So-pis showed a relatively low overall conservation compared with other known piscidins. So-pis is abound in glycine residues (22.7 %) and it has a neutral isoelectric point, weak amphipathicity, relatively long α-helix, and high hydrophobicity. These key elements are responsible for its biological activity. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicated that So-pis is a typically gill-expressed peptide. The expression of So-pis in the gill, skin, spleen, and head kidney could be regulated during C. irritans infection, thereby implicating a role of So-pis in immune defence against C. irritans. The synthetic So-pis had limited or no antimicrobial activity against bacterial and yeasts but exhibited potent antiparasitic activity against C. irritans in vitro. The activity of synthetic So-pis against erythrocytes was less potent than its antiparasitic activity against C. irritans. These results indicated that So-pis might be one of the crucial defence cytokines against C. irritans in the red drum. Cumulatively, our data suggested that So-pis might be a potential candidate for developing a novel, effective, and safe therapeutic agent against marine white spot disease.

The Expression of CD28 and Its Synergism on the Immune Response of Flounder (Paralichthys olivaceus) to Thymus-Dependent Antigen.

CD28 is well known as a critical T-cell costimulatory receptor involved in T cell activation by binding to its ligands. In this study, CD28 was cloned, and its expression profiles were characterized in flounder (Paralichthys olivaceus); variations of CD28+ cells after being stimulated with different types of antigens and the function of the CD28 costimulatory pathway on T-cell activation were investigated in vitro. fCD28 consists of four exons and three introns, and the full-length cDNA of fCD28 was 675-bp encoded 224 amino acids. The conserved motif (121TFPPPF126) binding to the CD80/86 ligand exists in the Ig-superfamily homology domain. The high expression of fCD28 is in gills, PBLs, head kidney, and spleen. CD28+ cells were co-localized with CD4+ T lymphocytes but not on IgM+ B lymphocyte cells. Moreover, the expression of CD28 was significantly varied in flounder after being stimulated by keyhole limpet hemocyanin (KLH) at both the transcriptional and cellular levels, while no significant differences were observed between lipopolysaccharide (LPS) stimulation and the control group. Notably, treatment of PBLs cultured in vitro with CD28 molecule-specific antibody (anti-CD28 Abs) and PHA produced more cell colonies and stimulated the proliferation of cultured leukocytes compared to PHA stimulation alone and the control group, and a higher level of IL-2 was detected in the culture medium. Meanwhile, anti-CD28 Abs increased the percent of CD28+ cells (10.41 ± 1.35%), CD4+ T lymphocytes (18.32 ± 2.15%), and CD28+/CD4+ double-positive cells (6.24 ± 1.52%). This effect also resulted in significant variations in the genes of cell membrane-bound molecules, cytokines, and related signaling pathways in cultured leukocytes, with significant changes in the genes of interleukin-2 (IL-2) and nuclear factor of activated T cells (NFAT) in the early stages of culture, and the expression of other molecules increased over time. These results proved the localization of the CD28 molecule on T lymphocytes in flounder, and anti-CD28 may act as the B7 ligand involved in T cell activation after antigen stimulation. These data provide a basis for a more in-depth study of the mechanism of the CD28 costimulatory pathway in T cell activation.

Gill Transcriptomic Responses to Toxin-producing Alga Prymnesium parvum in Rainbow Trout.

The gill of teleost fish is a multifunctional organ involved in many physiological processes, including protection of the mucosal gill surface against pathogens and other environmental antigens by the gill-associated lymphoid tissue (GIALT). Climate change associated phenomena, such as increasing frequency and magnitude of harmful algal blooms (HABs) put extra strain on gill function, contributing to enhanced fish mortality and fish kills. However, the molecular basis of the HAB-induced gill injury remains largely unknown due to the lack of high-throughput transcriptomic studies performed on teleost fish in laboratory conditions. We used juvenile rainbow trout (Oncorhynchus mykiss) to investigate the transcriptomic responses of the gill tissue to two (high and low) sublethal densities of the toxin-producing alga Prymnesium parvum, in relation to non-exposed control fish. The exposure time to P. parvum (4-5 h) was sufficient to identify three different phenotypic responses among the exposed fish, enabling us to focus on the common gill transcriptomic responses to P. parvum that were independent of dose and phenotype. The inspection of common differentially expressed genes (DEGs), canonical pathways, upstream regulators and downstream effects pointed towards P. parvum-induced inflammatory response and gill inflammation driven by alterations of Acute Phase Response Signalling, IL-6 Signalling, IL-10 Signalling, Role of PKR in Interferon Induction and Antiviral Response, IL-8 Signalling and IL-17 Signalling pathways. While we could not determine if the inferred gill inflammation was progressing or resolving, our study clearly suggests that P. parvum blooms may contribute to the serious gill disorders in fish. By providing insights into the gill transcriptomic responses to toxin-producing P. parvum in teleost fish, our research opens new avenues for investigating how to monitor and mitigate toxicity of HABs before they become lethal.

High-Resolution, 3D Imaging of the Zebrafish Gill-Associated Lymphoid Tissue (GIALT) Reveals a Novel Lymphoid Structure, the Amphibranchial Lymphoid Tissue.

The zebrafish is extensively used as an animal model for human and fish diseases. However, our understanding of the structural organization of its immune system remains incomplete, especially the mucosa-associated lymphoid tissues (MALTs). Teleost MALTs are commonly perceived as diffuse and scattered populations of immune cells throughout the mucosa. Yet, structured MALTs have been recently discovered in Atlantic salmon (Salmo salar L.), including the interbranchial lymphoid tissue (ILT) in the gills. The existence of the ILT was only recently identified in zebrafish and other fish species, highlighting the need for in-depth characterizations of the gill-associated lymphoid tissue (GIALT) in teleosts. Here, using 3-D high-resolution microscopy, we analyze the GIALT of adult zebrafish with an immuno-histology approach that reveals the organization of lymphoid tissues via the labeling of T/NK cells with an antibody directed to a highly conserved epitope on the kinase ZAP70. We show that the GIALT in zebrafish is distributed over at least five distinct sub-regions, an organization found in all pairs of gill arches. The GIALT is diffuse in the pharyngeal part of the gill arch, the interbranchial septum and the filaments/lamellae, and structured in two sub-regions: the ILT, and a newly discovered lymphoid structure located along each side of the gill arch, which we named the Amphibranchial Lymphoid Tissue (ALT). Based on RAG2 expression, neither the ILT nor the ALT constitute additional thymi. The ALT shares several features with the ILT such as presence of abundant lymphoid cells and myeloid cells embedded in a network of reticulated epithelial cells. Further, the ILT and the ALT are also a site for T/NK cell proliferation. Both ILT and ALT show structural changes after infection with Spring Viraemia of Carp Virus (SVCV). Together, these data suggest that ALT and ILT play an active role in immune responses. Comparative studies show that whereas the ILT seems absent in most neoteleosts ("Percomorphs"), the ALT is widely present in cyprinids, salmonids and neoteleosts, suggesting that it constitutes a conserved tissue involved in the protection of teleosts via the gills.

The immune response of pIgR and Ig to Flavobacterium columnare in grass carp (Ctenopharyngodon idellus).

The polymeric immunoglobulin receptor (pIgR) plays an important role in mediating the transcytosis of polymeric immunoglobulins (pIgs) to protect organisms against pathogen invasion. Here, a polyclonal antibody against grass carp (Ctenopharyngodon idellus) recombinant pIgR was developed by immunizing New Zealand white rabbit, and the responses of pIgR, IgM and IgZ were analyzed after bath immunization and intraperitoneal administration with Flavobacterium columnare. The results showed that pIgR transcription level was similar to IgM and IgZ, but pIgR rose much faster and peaked earlier than IgM and IgZ; the pIgR mRNA levels were higher in the skin and spleen for both immunized groups, while IgM and IgZ mRNA expression were higher in skin, gills, and intestines in bath immersion group, or spleen and head kidney in intraperitoneal immunization group. ELISA revealed that the IgM, IgZ and pIgR protein levels were up-regulated in skin mucus, gill mucus, gut mucus and bile, reaching a higher peak level earlier in skin mucus and gill mucus in bath immersion group, but a higher peak level in bile in injection group. Moreover, secretory component molecules were detected in grass carp's skin, gill and intestine mucus and bile, but not in serum, which molecular mass was near the theoretical mass obtained from the sequence of grass carp pIgR. These results demonstrated that bath and intraperitoneal immunization up-regulated pIgR and secretory Ig expression in secretions, which provided more insights into the role of pIgR in immunity and offer insight into ways of protecting teleost against pathogen invasion.

Dual-Organ Transcriptomic Analysis of Rainbow Trout Infected With Ichthyophthirius multifiliis Through Co-Expression and Machine Learning.

Ichthyophthirius multifiliis is a major pathogen that causes a high mortality rate in trout farms. However, systemic responses to the pathogen and its interactions with multiple organs during the course of infection have not been well described. In this study, dual-organ transcriptomic responses in the liver and head kidney and hemato-serological indexes were profiled under I. multifiliis infection and recovery to investigate systemic immuno-physiological characteristics. Several strategies for massive transcriptomic interpretation, such as differentially expressed genes (DEGs), Poisson linear discriminant (PLDA), and weighted gene co-expression network analysis (WGCNA) models were used to investigate the featured genes/pathways while minimizing the disadvantages of individual methods. During the course of infection, 6,097 and 2,931 DEGs were identified in the head kidney and liver, respectively. Markers of protein processing in the endoplasmic reticulum, oxidative phosphorylation, and the proteasome were highly expressed. Likewise, simultaneous ferroptosis and cellular reconstruction was observed, which is strongly linked to multiple organ dysfunction. In contrast, pathways relevant to cellular replication were up-regulated in only the head kidney, while endocytosis- and phagosome-related pathways were notably expressed in the liver. Moreover, interestingly, most immune-relevant pathways (e.g., leukocyte trans-endothelial migration, Fc gamma R-mediated phagocytosis) were highly activated in the liver, but the same pathways in the head kidney were down-regulated. These conflicting results from different organs suggest that interpretation of co-expression among organs is crucial for profiling of systemic responses during infection. The dual-organ transcriptomics approaches presented in this study will greatly contribute to our understanding of multi-organ interactions under I. multifiliis infection from a broader perspective.

Ozone nanobubble treatments improve survivability of Nile tilapia (Oreochromis niloticus) challenged with a pathogenic multi-drug-resistant Aeromonas hydrophila.

A rapid increase in multi-drug-resistant (MDR) bacteria in aquaculture highlights the risk of production losses due to diseases and potential public health concerns. Previously, we reported that ozone nanobubbles (NB-O3 ) were effective at reducing concentrations of pathogenic bacteria in water and modulating fish immunity against pathogens; however, multiple treatments with direct NB-O3 exposures caused alterations to the gills of exposed fish. Here, we set up a modified recirculation system (MRS) assembled with an NB-O3 device (MRS-NB-O3 ) to investigate whether MRS-NB-O3 (a) were safe for tilapia (Oreochromis niloticus), (b) were effective at reducing bacterial load in rearing water and (c) improved survivability of Nile tilapia following an immersion challenge with a lethal dose of MDR Aeromonas hydrophila. The results showed no behavioural abnormalities or mortality of Nile tilapia during the 14-day study using the MRS-NB-O3 system. In the immersion challenge, although high bacterial concentration (~2 × 107  CFU/ml) was used, multiple NB-O3 treatments in the first two days reduced the bacteria between 15.9% and 35.6% of bacterial load in water, while bacterial concentration increased from 13.1% to 27.9% in the untreated control. There was slight up-regulation of non-specific immune-related genes in the gills of the fish receiving NB-O3 treatments. Most importantly, this treatment significantly improved survivability of Nile tilapia with relative percentage survival (RPS) of 64.7% - 66.7% in treated fish and surviving fish developed specific antibody against MDR A. hydrophila. In summary, the result suggests that NB-O3 is a promising non-antibiotic approach to control bacterial diseases, including MDR bacteria, and has high potential for application in recirculation aquaculture system (RAS).

Mucosal and Systemic Immune Responses to Salmon Gill Poxvirus Infection in Atlantic Salmon Are Modulated Upon Hydrocortisone Injection.

Salmon Gill Poxvirus Disease (SGPVD) has emerged as a cause of acute mortality in Atlantic salmon (Salmo salar L.) presmolts in Norwegian aquaculture. The clinical phase of the disease is associated with apoptotic cell death in the gill epithelium causing acute respiratory distress, followed by proliferative changes in the regenerating gill in the period after the disease outbreak. In an experimental SGPV challenge trial published in 2020, acute disease was only seen in fish injected with hydrocortisone 24 h prior to infection. SGPV-mediated mortality in the hydrocortisone-injected group was associated with more extensive gill pathology and higher SGPV levels compared to the group infected with SGPV only. In this study based on the same trial, SGPV gene expression and the innate and adaptive antiviral immune response was monitored in gills and spleen in the presence and absence of hydrocortisone. Whereas most SGPV genes were induced from day 3 along with the interferon-regulated innate immune response in gills, the putative SGPV virulence genes of the B22R family were expressed already one day after SGPV exposure, indicating a potential role as early markers of SGPV infection. In gills of the hydrocortisone-injected fish infected with SGPV, MX expression was delayed until day 10, and then expression skyrocketed along with the viral peak, gill pathology and mortality occurring from day 14. A similar expression pattern was observed for Interferon gamma (IFNγ) and granzyme A (GzmA) in the gills, indicating a role of acute cytotoxic cell activity in SGPVD. Duplex in situ hybridization demonstrated effects of hydrocortisone on the number and localization of GzmA-containing cells, and colocalization with SGPV infected cells in the gill. SGPV was generally not detected in spleen, and gill infection did not induce any corresponding systemic immune activity in the absence of stress hormone injection. However, in fish injected with hydrocortisone, IFNγ and GzmA gene expression was induced in spleen in the days prior to acute mortality. These data indicate that suppressed mucosal immune response in the gills and the late triggered systemic immune response in the spleen following hormonal stress induction may be the key to the onset of clinical SGPVD.

CCL19 variants mediate chemotactic response via CCR7 in grass carp Ctenopharyngodon idella.

CC chemokine ligand 19 (CCL19) plays a key role in the regulation of immune responses including homeostasis, inflammation, and immune tolerance. In this study, two variants of CCL19 homologues (CCL19a2 and CCL19b) and CCR7 were investigated in grass carp Ctenopharyngodon idella. The three genes were widely expressed in immune tissues and could be modulated by stimulation with LPS, PHA and poly(I:C), and infection with Flavobacterium columnare and grass carp reovirus. In an in vitro chemotaxis assay, the recombinant CCL19a2 and CCL19b were active to promote the migration of HEK293 T cells expressing CCR7 and leucocytes isolated from the gills, head kidney and spleen. Moreover, their chemotactive effects were validated in vivo. We found that the cells recruited by CCL19a2 and CCl19b are mainly monocytes/macrophages expressing high levels of IL-1ß, IFN-γ, colony stimulating factor 1 receptor (CSF1R) and MHC II. Our work suggests that CCL19a2 and CCl19b are involved in recruitment of antigen presenting cells in fish.

Immunologic Profiling of the Atlantic Salmon Gill by Single Nuclei Transcriptomics.

Anadromous salmonids begin life adapted to the freshwater environments of their natal streams before a developmental transition, known as smoltification, transforms them into marine-adapted fish. In the wild, smoltification is a photoperiod-regulated process, involving radical remodeling of gill function to cope with the profound osmotic and immunological challenges of seawater (SW) migration. While prior work has highlighted the role of specialized "mitochondrion-rich" cells (MRCs) and accessory cells (ACs) in delivering this phenotype, recent RNA profiling experiments suggest that remodeling is far more extensive than previously appreciated. Here, we use single-nuclei RNAseq to characterize the extent of cytological changes in the gill of Atlantic salmon during smoltification and SW transfer. We identify 20 distinct cell clusters, including known, but also novel gill cell types. These data allow us to isolate cluster-specific, smoltification-associated changes in gene expression and to describe how the cellular make-up of the gill changes through smoltification. As expected, we noted an increase in the proportion of seawater mitochondrion-rich cells, however, we also identify previously unknown reduction of several immune-related cell types. Overall, our results provide fresh detail of the cellular complexity in the gill and suggest that smoltification triggers unexpected immune reprogramming.

Upregulation of polymeric immunoglobulin receptor expression in flounder (Paralichthys olivaceus) gill cells by cytokine tumor necrosis factor-α via activating PI3K and NF-κB signaling pathways.

The polymeric immunoglobulin receptor (pIgR) transports secretory immunoglobulins across mucosal epithelial cells into external secretions, playing critical roles in mucosal surface defenses, but the regulation mechanism of pIgR expression is not clarified in teleost fish. In this study, the dynamic changes of flounder (Paralichthys olivaceus) pIgR (fpIgR) and pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) mRNA expression in mucosal tissues were first analyzed post inactivated Vibrio anguillarum immunization, and increased production of TNF-α was found to correlate with increased expression of fpIgR. To determine that cytokine TNF-α influenced fpIgR expression, following confirming that natural fpIgR expressed on flounder gill (FG) cells, FG cells were incubated with various concentrations of recombinant TNF-α for different time, the results showed that the expressions of fpIgR were significantly upregulated at gene and protein levels in a dose-dependent and time-dependent manner, and similar change trend was observed for free secretory component (SC) secreted by fpIgR into the culture supernatant. After FG cells were treated with TNF-α, specific phosphoinositide 3-kinase (PI3K) inhibitor wortmannin, nuclear factor kappa-B (NF-κB) inhibitor Bay11-7082, and the mixtures of TNF-α and wortmannin / Bay11-7082 respectively, the fpIgR protein and mRNA levels, together with SC secretion, obviously decreased in wortmannin- and Bay11-7082-treated cells compared with the untreated control, and cotreatment with wortmannin / Bay11-7082 plus TNF-α resulted in lower expression compared with that upon treatment with TNF-α alone, indicating that the inhibition of PI3K and NF-κB both blocked the ability of TNF-α to increase cellular fpIgR and SC levels. Furthermore, the gene expressions of PI3K and NF-κB were upregulated and present a tendency to increase first and then decrease after TNF-α treatment of FG cells; However, the expression of PI3K mRNA was inhibited significantly by wortmannin but not by Bay11-7082, and the expression of NF-κB mRNA was suppressed obviously by Bay11-7082 but not by wortmannin, suggesting that inhibition of PI3K or NF-κB had no influence on each other. All these results collectively revealed that TNF-α could transcriptionally upregulate fpIgR expression and SC production, and this TNF-α-induced pIgR expression was regulated by complex mechanisms that involved PI3K and NF-κB signaling pathways, which provided evidences for pro-inflammatory cytokine TNF-α acting as a regulator in pIgR expression and better understanding of regulation mechanism of pIgR expression in teleost fish.

Two fibrinogen-related proteins (FREPs) in the razor clam (Sinonovacula constricta) with a broad recognition spectrum and bacteria agglutination activity.

Fibrinogen-related proteins (FREPs) that contain only the fibrinogen-related domain are likely involved in pathogen recognition. In this study, we identified two FREPs from the razor clam (Sinonovacula constricta), called ScFREP-1 and ScFREP-2, and investigated their roles in the immune response. Both ScFREP-1 and ScFREP-2 contained a fibrinogen-related domain at the C-terminal. ScFREP-1 and ScFREP-2 mRNAs were detected in all adult clam tissues tested, with the highest expression levels in the gill and mantle, respectively. Their expression levels were significantly upregulated after microbe infection. Recombinant ScFREPs could bind Gram-positive and Gram-negative bacteria as well as some pathogen-associated molecular patterns (PAMPs), and they could agglutinate those bacteria. These results showed that ScFREPs functioned as potential pattern recognition receptors to mediate immune response by recognizing PAMPs and agglutinating invasive microbes.

Cadmium-induced Oxidative Stress and Immunosuppression Mediated Mitochondrial Apoptosis via JNK-FoxO3a-PUMA pathway in Common Carp (Cyprinus carpio L.) Gills.

Cadmium (Cd)-caused water environment pollution has become a matter of concern. Gill is an organ with respiratory and mucosal immune functions, and is also one of the organs directly attacked by pollutants. It was found that excess Cd could cause Cd accumulation and gill injury in carp. However, the mechanism of Cd-caused damage in common carp gills is still unclear. Oxidative stress, immunosuppression, and apoptosis took part in the mechanism of poisoning caused by some harmful substances. The aim of the study was to investigate complex molecular mechanism of apoptotic injury caused by Cd in common carp gills. Hence, in this study, we established a Cd poisoning model to explore whether excess Cd can induce apoptosis through observing histomorphology and apoptotic cells; and determining mineral elements, oxidative stress-related factors, immune-related, and apoptosis-related genes in common carp gills. Fifty-four fish were randomly separated into the control group and the Cd group and were cultured for 45 days. The water of the control group was drinking water and the water of the Cd group was CdCl2-added drinking water (0.26 mg/L Cd). In our results, we found that excess Cd increased Cd level, decreased the levels of essential mineral elements (Cu, Fe, Zn, and Mn), damaged mitochondria, and increased apoptotic cells in common carp gills, meaning that excess Cd caused Cd accumulation and apoptotic injury via mitochondrion in common carp gills. Furthermore, we found that Cd inhibited anti-apoptosis-related gene Bcl-2 and stimulated pro-apoptosis-related genes (JNK, FoxO3a, PUMA, Bax, Apaf-1, Caspase-9, and Caspase-3) on 15th, 30th, and 45th days. Above data meant that Cd exposure caused apoptosis via mitochondrion and JNK-FoxO3a-PUMA pathway in common carp gills. In addition, in our experiment, Cd treatment increased oxidants (H2O2 and MDA) and decreased antioxidants (CAT, GPx, GST, SOD, T-AOC, and GSH), indicating that Cd caused oxidative stress via oxidation/antioxidation imbalance. Meanwhile, compared to the control group, T-help 17 (Th17) cell-related factors (IL-17, TNF-α, and RORγ) were up-regulated, regulatory T (Treg) cell-related factors (IL-10 and Foxp3) were down-regulated, and IL-17/IL-10, TNF-α/IL-10, and RORγ/Foxp3 were increased in Cd-exposed group; meaning that excess Cd induced immunosuppression via the imbalance of Th17/Treg cells. Taken together, our findings indicated that JNK-FoxO3a-PUMA pathway and mitochondrion participated in oxidative stress and immunosuppression-mediated apoptosis caused by Cd in common carp (Cyprinus carpio L.) gills. Our data provided new perspectives on the negative effects of heavy metal pollutants on fish.

Anatomy of teleost fish immune structures and organs.

The function of a tissue is determined by its construction and cellular composition. The action of different genes can thus only be understood properly when seen in the context of the environment in which they are expressed and function. We now experience a renaissance in morphological research in fish, not only because, surprisingly enough, large structures have remained un-described until recently, but also because improved methods for studying morphological characteristics in combination with expression analysis are at hand. In this review, we address anatomical features of teleost immune tissues. There are approximately 30,000 known teleost fish species and only a minor portion of them have been studied. We aim our review at the Atlantic salmon (Salmo salar) and other salmonids, but when applicable, we also present information from other species. Our focus is the anatomy of the kidney, thymus, spleen, the interbranchial lymphoid tissue (ILT), the newly discovered salmonid cloacal bursa and the naso-pharynx associated lymphoid tissue (NALT).

An overview of the structural and functional aspects of immune cells in teleosts.

The immune system of fish consists of two main components, innate and adaptive immunities. Innate immunity is non-specific and acts as the primary line of protection against pathogen invasion, while adaptive immunity is more specific to a certain pathogen/following adaptation. The adaptive immune system consists of the humoral and cellular components. Cytotoxic T-lymphocyte cells are the major component of the cellular immunity that frequently kills viral-, bacterial- or parasitic-infected cells. According to the anatomical location, the mucosal-associated lymphoid tissue (MALT) in teleost fish subdivides into gut-associated lymphoid tissue (GALT), gill-associated lymphoid tissue (GIALT), and skin-associated lymphoid tissue (SALT). The MALTs contain various leukocytes; including, but not limited to, lymphocytes (T and B cells), plasma cells, macrophages, and granulocytes. Macrophages are multifunctional cells that are mainly involved in the immune response, including; phagocytosis and degradation of foreign antigens, tissue remodeling, and production of cytokines, chemokines and growth factors. An interesting feature of teleost macrophages is their ability to form melanomacrophage centers (MMC) in the hemopoietic tissues. Dendritic cells, rodlet cells, mast cells, eosinophilic granular cells (ECGs), telocytes, osteoclasts, club cells, as well as, barrier cells have been recorded in many fish species and have many immunological roles. This paper aims to summarize the current knowledge of the immune cells present in fish tissues serving as anatomical and physiological barriers against external hazards. Increased knowledge of fish immune systems will facilitate the development of novel vaccination strategies in fish.

Exogenous inositol ameliorates the effects of acute ammonia toxicity on intestinal oxidative status, immune response, apoptosis, and tight junction barriers of great blue-spotted mudskippers (Boleophthalmus pectinirostris).

Ammonia toxicity can disrupt the intestinal health of aquatic animals. It is important to find substances that alleviate these adverse effects. The present study explored the possible protective role of myo-inositol (MI) in ammonia-induced toxicity in the fish intestine. Great blue-spotted mudskippers (Boleophthalmus pectinirostris) accumulated in artificial seawater (15‰ salinity, n = 600) were randomly selected and intraperitoneally injected with NaCl (0.68%) or MI (2.5 mg/g fish in 0.68% NaCl) then exposed to artificial seawater alone (NaCl and MI group) or seawater containing 57.025 mmol/L ammonium chloride (NH3 and NH3 + MI group). After a 24-h experiment, it showed that ammonia exposure down-regulated the mRNA expression levels of intestinal barrier function proteins (Zo-1, Ocln, Cldn-5, Cldn-12, and Cldn-15) and anti-inflammatory cytokines (Tgf-ß and Il-10) while the acute ammonia stress up-regulated the apoptosis genes (p53, Bax, Caspase-3, and Caspase-9) and pro-inflammatory cytokines (Tnf-α and Il-1ß). Furthermore, ammonia challenge also induced oxidative stress, as the malondialdehyde and the protein carbonyl contents were increased. In addition, ammonia stress down-regulated the antioxidant enzymes (Cu/Zn-Sod, Cat, Gpx, and Gst) activities as well as their gene transcription levels. The administration of the exogenous myo-inositol greatly ameliorated the ammonia-induced changes in redox capacity, immune response, apoptosis, inflammation, and tight junction barrier function to levels similar to those of the NaCl group. Furthermore, fish injected with MI alone showed no significant changes compared with the NaCl group. Taken together, pretreatment with myo-inositol had no obvious side-effects and effectively protected the mudskippers' intestine from the toxicity caused by acute ammonia stress.