Discovery of an unconventional lamprey lymphocyte lineage highlights divergent features in vertebrate adaptive immune system evolution.

Lymphocyte receptors independently evolved in both jawed and jawless vertebrates with similar adaptive immune responses. However, the diversity of functional subtypes and molecular architecture in jawless vertebrate lymphocytes, comparable to jawed species, is not well defined. Here, we profile the gills, intestines, and blood of the lamprey, Lampetra morii, with single-cell RNA sequencing, using a full-length transcriptome as a reference. Our findings reveal higher tissue-specific heterogeneity among T-like cells in contrast to B-like cells. Notably, we identify a unique T-like cell subtype expressing a homolog of the nonlymphoid hematopoietic growth factor receptor, MPL-like (MPL-L). These MPL-L+ T-like cells exhibit features distinct from T cells of jawed vertebrates, particularly in their elevated expression of hematopoietic genes. We further discovered that MPL-L+ VLRA+ T-like cells are widely present in the typhlosole, gill, liver, kidney, and skin of lamprey and they proliferate in response to both a T cell mitogen and recombinant human thrombopoietin. These findings provide new insights into the adaptive immune response in jawless vertebrates, shedding new light on the evolution of adaptive immunity.

Aeromonas salmonicida AI-1 and AI-2 quorum sensing pathways are differentially regulated by rainbow trout mucins and during in vivo colonization.

Aeromonas salmonicida is an opportunistic pathogen with relevance for aquaculture. Fish epithelia are covered by a mucus layer, composed mainly by highly glycosylated mucins, which are the first point of contact between fish and pathogens. Quorum sensing (QS), a bacterial communication mechanism through secreted autoinducer signals that governs gene expression, influences bacterial growth and virulence. The main A. salmonicida autoinducers are mediated by the luxS and asaI genes, corresponding to inter- and intraspecies communication, respectively. The aim of this study was to determine the effect of the mucins that pathogens encounter during colonization of the gill and skin on A. salmonicida QS. We found that expression of A. salmonicida asaI, but not luxS, was increased after culture at 20 °C compared to 10 °C. Rainbow trout gill and skin mucins up-regulated asaI expression 2-fold but down-regulated luxS 10-fold. The downregulation of luxS was reflected by a reduction in autoinducer-2 secretion. Mucins isolated from skin had a stronger inhibitory effect than mucins isolated from gills on both luxS expression and A1-2 secretion, consistent with a higher relative abundance of N-Acetylneuraminic acid on skin mucins than on gill mucins. Reduction of AI-2 production by mucins or luxS-deletion lead to a reduced A. salmonicida auto-aggregation. Furthermore, after colonization of the gill, luxS was down regulated whereas asaI expression was upregulated. Both in vivo and in vitro, the expression of luxS and asaI were thus differentially regulated, frequently in an inverse manner. The strong AI-2 inhibiting effect of the skin mucins is likely part of the mucin-based defense against pathogens.

Plantago asiatica seed as a protective agent for mitigating metals toxicity on Penaeusvannamei.

Plantago asiatica seeds (PS) are commonly used as a medicinal plant. This study investigates the efficacy of PS against heavy metal toxicity in white shrimp (Penaeus vannamei). After feeding PS diet (5 g/kg) or basal diet (control group) for 7 days, shrimps were exposed to sublethal concentrations of heavy metals in seawater (As: 12 mg/L, Pb: 250 mg/L, Hg: 0.4 mg/L). The 7-day survival observation showed that the survival in groups fed with PS were significantly higher than that in the control group, revealing that dietary PS had the efficacy to mitigate heavy metal toxicity in white shrimp. Under the same feeding condition, white shrimps were exposed to safety dose of heavy metals (1/10 of sublethal concentrations) to understand the mechanism of mitigation. The metal accumulations in haemolymph, gills, hepatopancreas, and muscle tissues as well as the immune, anti-oxidative, stress related gene expressions in haemocytes, gills and hepatopancreas were measured for 14 days. The As accumulation in gills and hepatopancreas of groups fed with PS were significantly lower than those of control group on day 7 and 14, respectively; The Pb concentration in haemolymph of group fed with PS was significantly lower than that of control group on day 7 and 14; The Hg concentration in hepatopancreas of the group fed with PS was significantly lower than that of control group on day 7. Dietary PS could mitigate heavy metal-induced immune suppression, oxidative stress, and stress response by positively regulating immune (proPO I, Toll, IMD), antioxidant (SOD, GST, Trx), and negatively regulating stress response genes (HSP70, MT). The present study demonstrated that dietary PS could protect white shrimp against metal toxicity by reducing metal accumulations and regulating the immune, antioxidant, and stress response gene expressions in specific tissue. Therefore, PS may serve as a beneficial feed additive in the aquaculture.

Gilthead seabream mucus glycosylation is complex, differs between epithelial sites and carries unusual poly N-acetylhexosamine motifs.

Gilthead seabream (Sparus aurata) is a marine finfish of economic importance in aquaculture. Despite its adaptability to varying culture conditions, gilthead seabream culture can be affected by viral, bacterial or parasitic diseases. The main route of entry of pathogens is through mucosal surfaces. Teleost external and internal surfaces are covered by mucus, mainly comprised of highly glycosylated proteins called mucins. The mucin glycans regulate pathogen growth, adhesion, virulence and inter and intra species communication. Here, we characterized the gilthead seabream mucus glycosylation, compared it to previously described species and investigated associations with microbiota. 214 glycans were identified. The majority of the glycans were found at more than one epithelial surface, but 27, 22 and 89 O-glycan structures were unique to skin, gill and intestinal sample groups, respectively. Six O-glycan core types were observed. The majority of the seabream skin and gill O-glycans were neutral with unusual poly HexNAc motifs. In contrast, seabream intestinal O-glycans were highly acidic and not of the 'poly HexNAc' type observed in skin and gill. Furthermore, gilthead seabream gill mucosa had less oligomannose and more complex N-glycans compared to skin and intestine. The concentration and diversity of bacteria was similar in skin, gill and intestine, but the bacterial species differed between epithelia and co-varied with glycan epitopes. The presence of a complex mucus glycosylation with plenty of glycan epitopes for bacterial foraging, suggest that the skin mucosal defense in seabream includes an abundant resident microbiota. This large library of structures provides a platform for further studies, for example aiming to identifying glycans to use for diagnostic purposes, to study host-microbe interactions or disease intervention therapies.

Teleost Muc2 and Muc5ac: Key guardians of mucosal immunity in flounder (Paralichthys olivaceus).

Mucins secreted by mucous cells constitute a core part of the defense line against the invasion of pathogens. However, mucins' structure and immunological functions remain largely unknown in teleost fish. In this study, two typical mucins, Muc2 and Muc5ac of flounder (Paralichthys olivaceus), were cloned and their physicochemical properties, structure and conservation were analyzed. Notably, specific antibodies against flounder Muc2 and Muc5ac were developed. It was verified at the gene and protein level that Muc2 was expressed in the hindgut and gills but not in the skin, while Muc5ac was expressed in the skin and gills but not in the hindgut. After flounders were immunized by immersion with inactivated Edwardsiella tarda, Muc2 and Muc5ac were significantly upregulated at both the gene expression and protein levels, and Muc2+/Muc5ac+ mucous cells proliferated and increased secretion of Muc2 and Muc5ac. Moreover, Muc2 and Muc5ac exerted retention and clearance effects on E. tarda in a short period (within 1 dpi). These results revealed the characterization of fish mucins Muc2 and Muc5ac at the protein level and clarified the role of mucins as key guardians to maintain the mucus barrier, which advanced our understanding of teleost mucosal barrier.

IL-20 is produced by CD3γδ T cells and induced in the mucosal tissues of grass carp during infection with Aeromonas hydrophila.

Interleukin (IL) 20 is a multifunctional cytokine and plays a vital role in regulating autoimmune diseases, inflammation, and immune responses. IL-20 homologs have been described in fish. However, due to the lack of antibodies, cellular sources and immunological functions of fish IL-20 in response to infections have not been fully characterized. In this study, a monoclonal antibody (mAb) was generated against the recombinant grass carp (Ctenopharyngodon idella) IL-20 protein and characterized by immunoblotting, immunofluorescent microscopy and flow cytometry. It was shown that the IL-20 mAb specifically recognized recombinant IL-20 proteins expressed in the E. coli cells and HEK293 cells. Using confocal microscopy, the IL-20+ cells were identified in the head kidney, gills and intestine of grass carp, and induced after infection with Aeromonas hydrophila. Moreover, the IL-20 protein was found to be secreted mainly by CD3γδ T cells which were located predominantly in the gill filaments and intestinal mucosa. Taken together, our results suggest that IL-20 producing T cells are required for the mucosal immunity against bacterial infection in fish.

Host response to Aplysia Abyssovirus 1 in nervous system and gill.

The California sea hare (Aplysia californica) is a model for age associated cognitive decline. Recent researched identified a novel nidovirus, Aplysia Abyssovirus 1, with broad tropism enriched in the Aplysia nervous system. This virus is ubiquitous in wild and maricultured, young and old animals without obvious pathology. Here we re-evaluated gene expression data from several previous studies to investigate differential expression in the nervous system and gill in response to virus and aging as well as the mutational spectrum observed in the viral sequences obtained from these datasets. Viral load and age were highly correlated, indicating persistent infection. Upregulated genes in response to virus were enriched for immune genes and signatures of ER and proteostatic stress, while downregulated genes were enriched for mitochondrial metabolism. Differential expression with respect to age suggested increased iron accumulation and decreased glycolysis, fatty acid metabolism, and proteasome function. Interaction of gene expression trends associated with viral infection and aging suggest that viral infection likely plays a role in aging in the Aplysia nervous system. Mutation analysis of viral RNA identified signatures suggesting ADAR and AID/APOBEC like deaminase act as part of Aplysia anti-viral defense.

Novel insights into immune stress markers associated with myxosporeans gill infection in Nile tilapia (molecular and immunohistochemical studies).

Nile tilapia (Oreochromis niloticus) is valued in aquaculture because of its quick development and ability to thrive in various environments. Myxosporeans are among the fish parasites that affect fish productivity, as they impact fish growth and reproduction, resulting in large fish deaths in farms and hatcheries. This study has been focused on morpho-molecular identification for the myxosporean parasites infecting Nile tilapia from three governorates in Egypt and assessment of gene expression of different cytokines (Interleukin-1ßeta (IL-1ß), major histocompatibility complex class II (MHC-II), and clusters of differentiation 4 (CD-4) and 8 (CD-8)) in tissues. Additionally, this work aimed to correlate the developed histopathological alterations and inflammatory reactions in gills with immunohistochemical expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α). Finally, the infected fish's cortisol levels and blood glucose were assessed. Results of BLAST sequence analysis of the 18S rRNA for the collected protozoans confirmed Myxobolus agolus, M. brachysporus, M. tilapiae, and Henneguya species. The molecular characterization of the immunological status of gills revealed marked upregulation of different inflammatory cytokines in the gills of infected fish. There was a significantly increased serum cortisol and glucose level in infected fish compared with control, non-infected ones. Severe histopathological alterations were observed in the infected fish gills, associated with increased expression of iNOS and TNF-α and related to myxosporean infection. The present study provides new insights into oxidative stress biomarkers in Nile tilapia infected with Myxosporeans and elucidates the gill's immune status changes as a portal of entry for protozoa that contribute to tissue damage.

Histopathology and transcriptome profiling reveal features of immune responses in gills and intestine induced by Spring viremia of carp virus.

The immune system of bony fish closely resembles that of mammals, comprising both specific (adaptive) and non-specific (innate) components. Notably, the mucosa-associated lymphoid tissue (MALT) serves as the first line of defense within the non-specific immune system, playing a critical role in protecting these aquatic organisms against invading pathogens. MALT encompasses a network of immune cells strategically distributed throughout the gills and intestines, forming an integral part of the mucosal barrier that interfaces directly with the surrounding aquatic environment. Spring Viremia of Carp Virus（SVCV）, a highly pathogenic agent causing substantial harm to common carp populations, has been designated as a Class 2 animal disease by the Ministry of Agriculture and Rural Affairs of China. Utilizing a comprehensive array of research techniques, including Hematoxylin and Eosin (HE)、Alcian Blue Periodic Acid-Schiff (AB-PAS)、transcriptome analysis for global gene expression profiling and Reverse Transcription-Polymerase Chain Reaction (RT-qPCR), this study uncovered several key findings: SVCV is capable of compromising the mucosal architecture in the gill and intestinal tissues of carp, and stimulate the proliferation of mucous cells both in gill and intestinal tissues. Critically, the study revealed that SVCV's invasion elicits a robust response from the carp's mucosal immune system, demonstrating the organism's capacity to resist SVCV invasion despite the challenges posed by the pathogen.

Impact of freshwater rearing history on Atlantic salmon gill response to viral stimulation post seawater transfer.

Land-based recirculating aquaculture systems (RAS) have risen in prevalence in recent years for Atlantic salmon production, enabling intensive production which allows increased growth and environmental control, but also having the potential for reducing water use and eutrophication. The Atlantic salmon has an anadromous life history with juvenile stages in freshwater (FW) and on-growing in seawater (SW), enabled by a transformational process known as smoltification. The timing of smoltification and transfer of smolts from FW to SW is critical under commercial production with high mortalities during this period. The impact of FW rearing system on immune function following seawater transfer (SWT) is not well understood. In this study parr were raised in either RAS or a traditional open-LOCH system until smolting and then transferred to a common marine environment. Two-weeks post-SWT fish were immune stimulated with a viral mimic (poly I:C) for 24 h to assess the ability to mount an antiviral immune response, assessed by whole transcriptome analysis of gill tissue, an important immune organ in fish. We show that unstimulated smolts reared in the LOCH had higher immune gene expression than those reared in RAS as determined by functional analysis. However, following stimulation, smolts reared in the RAS mounted a greater magnitude of response with a suite of immune genes displaying higher fold induction of transcription compared to LOCH reared smolts. We suggest RAS smolts have a lower steady state immune-associated transcriptome likely due to an unvarying environment, in terms of environmental factors and lack of exposure to pathogens, which shows a compensatory mechanism following stimulation allowing immune 'catch-up' with those reared in the LOCH. Alternatively, the RAS fish are experiencing an excessive response to the immune stimulation.

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.

Hypoxia stress alters gene expression in the gills and spleen of greater amberjack (Serioladumerili).

Greater amberjack (Seriola dumerili) is a fish species that has significant economic and cultural value. It has a large size and grows rapidly. However, the intolerance to hypoxia poses a major obstacle to the growth of its aquaculture industry. This study focuses on the gills and spleen, two organs closely associated with the response to acute hypoxic stress. By simulating the acute hypoxic environment and using Illumina RNA-Seq technology, we explored the gills and spleen transcriptome changes in the acute hypoxia intolerant and tolerant groups of greater amberjack. It was discovered that gill tissues in the tolerant group may maintain a stable intracellular energy supply by promoting glycolysis and ß-oxidation compared to the intolerant group. Additionally, it promotes angiogenesis, enhances the ability to absorb dissolved oxygen, and accelerates oxygen transport to the mitochondria, adapting to the hypoxic environment. Anti-apoptotic genes were up-regulated in gill tissues in the tolerant group compared to the intolerant group, thereby minimizing the damage of acute hypoxia. On the other hand, the spleen inhibited the TCA and energy-consuming lipid synthesis pathways to supply energy under acute hypoxic stress. Pro-angiogenic genes were down-regulated in the spleen of individuals in the tolerant group compared to the intolerant group, which may be related to organ function. The suppressed reactive oxygen species (ROS) production and the impaired immune response function of the spleen were also found. The study explored the acute hypoxic stress response in greater amberjack and the molecular mechanisms underlying its tolerance to acute hypoxia.

Niclosamide subacute exposure alters the immune response and microbiota of the gill and gut in black carp larvae, Mylopharyngodon piceus.

Niclosamide (NIC) is a commonly used insecticide and molluscicide in the prevention and treatment of parasitic diseases in fish. The utilization of NIC has the potential to disrupt the microbial community present on the mucosal tissue of fish, leading to localized inflammatory responses. The objective of this study was to evaluate the impact of NIC on the immune system and bacterial populations within the gill and gut of Mylopharyngodon piceus. Fish were subjected to varying concentrations of NIC, including a control group (0 µg/L), a low NIC group (15% 96 h LC50, LNG, 9.8 µg/L), and a high NIC group (80% 96 h LC50, HNG, 52.5 µg/L). Gill and gut samples were collected 28 days post-exposure for analysis. The findings revealed that the 96-h LC50 for NIC was determined to be 65.7 µg/L, and histopathological examination demonstrated that exposure to NIC resulted in gill filament subepithelial edema, exfoliation, degeneration, and a decrease in gill filament length. Furthermore, the gut exhibited apical enterocyte degeneration and leucocyte infiltration following NIC exposure. Additionally, NIC exposure led to a significant elevation in the levels of immunoglobulin M (IgM), complement component 3 (C3), and complement component 4 (C4) in both gill and gut tissues. Moreover, the activity of lysozyme (LYZ) was enhanced in the gill, while the activities of peroxidase (POD) and immunoglobulin T (IgT) were increased in gut tissue. The exposure to NIC resulted in enhanced mRNA expression of c3, c9, tnfα, il6, il8, and il11 in the gill tissue, while decreasing c3 and il8 expression in the gut tissue. Furthermore, the natural resistance-associated macrophage protein (nramp) mRNA increased, and liver-expressed antimicrobial peptide 2 (leap2) mRNA decreased in gill and gut tissues. And hepcidin (hepc) mRNA levels rose in gill but fell in gut tissue. NIC exposure also led to a decrease in gill bacterial richness and diversity, which significantly differed from the control group, although this separation was not significant in the gut tissue. In conclusion, the administration of NIC resulted in alterations in both the immune response and mucosal microbiota of fish. Furthermore, it was noted that gills displayed a heightened vulnerability to sublethal effects of NIC in comparison to gut tissues.

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.

Temperature and immune challenges modulate the transcription of genes of the ubiquitin and apoptosis pathways in two high-latitude Notothenioid fish across the Antarctic Polar Front.

Thermal variations due to global climate change are expected to modify the distributions of marine ectotherms, with potential pathogen translocations. This is of particular concern at high latitudes where cold-adapted stenothermal fish such as the Notothenioids occur. However, little is known about the combined effects of thermal fluctuations and immune challenges on the balance between cell damage and repair processes in these fish. The aim of this study was to determine the effect of thermal variation on specific genes involved in the ubiquitination and apoptosis pathways in two congeneric Notothenioid species, subjected to simulated bacterial and viral infections. Adult fish of Harpagifer bispinis and Harpagifer antarcticus were collected from Punta Arenas (Chile) and King George Island (Antarctica), respectively, and distributed as follows: injected with PBS (control), LPS (2.5 mg/kg) or Poly I:C (2 mg/kg) and then submitted to 2, 5 and 8 °C. After 1 week, samples of gills, liver and spleen were taken to evaluate the expression by real-time PCR of specific genes involved in ubiquitination (E3-ligase enzyme) and apoptosis (BAX and SMAC/DIABLO). Gene expression was tissue-dependent and increased with increasing temperature in the gills and liver while showing an opposite pattern in the spleen. Studying a pair of sister species that occur across the Antarctic Polar Front can help us understand the particular pressures of intertidal lifestyles and the effect of temperature in combination with biological stressors on cell damage and repair capacity in a changing environment.

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.

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.