Molecular characterization, expression and immune functional analysis of cystatin 10 in turbot.

BACKGROUND: Cystatin is a protease inhibitor that also regulates genes expression linked to inflammation and plays a role in defense and regulation. METHODS AND RESULTS: Cystatin 10 (Smcys10) was cloned from Scophthalmus maximus and encodes a 145 amino acid polypeptide. The results of qRT-PCR showed that Smcys10 exhibited tissue-specific expression patterns, and its expression was significantly higher in the skin than in other tissues. The expression level of Smcys10 was significantly different in the skin, gill, head kidney, spleen and macrophages after Vibrio anguillarum infection, indicating that Smcys10 may play an important role in resistance to V. anguillarum infection. The recombinant Smcys10 protein showed binding and agglutinating activity in a Ca2+-dependent manner against bacteria. rSmcys10 treatment upregulated the expression of IL-10, TNF-α and TGF-ß in macrophages of turbot and hindered the release of lactate dehydrogenase (LDH) from macrophages after V. anguillarum infection, which confirmed that rSmcys10 reduced the damage to macrophages by V. anguillarum. The NF-κB pathway was suppressed by Smcys10, as demonstrated by dual-luciferase analysis. CONCLUSIONS: These results indicated that Smcys10 is involved in the host antibacterial immune response.

Zoonotic human liver flukes, a type 1 biocarcinogen, in freshwater fishes: genetic analysis and confirmation of molluscan vectors and reservoir hosts in Bangladesh.

BACKGROUND: Opisthorchiid flukes, particularly Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, and Metorchis spp. are the most common fish-borne zoonotic human liver flukes (hLFs). Liver fluke infections are more prevalent in resource-deprived and underprivileged areas. We herein estimated the prevalence of the metacercariae (MC) of major hLFs in common large freshwater fishes (lFWF) marketed for human consumption from some selected areas of Bangladesh along with detection of their molluscan vectors and reservoirs. METHODS: The current status of fish-borne zoonotic hLF infections in lFWF was investigated along with their molluscan vectors and mammalian reservoir hosts in Mymensingh and Kishoreganj in Bangladesh from July 2018-June 2022 using conventional and multiple molecular techniques, such as PCR, PCR-restriction fragment length polymorphism (RFLP), sequencing, and bioinformatic analyses. The infection rate of fishes was analyzed using the Z-test and the loads of MC were compared using the chi-squared (χ2) test. RESULTS: The MC of C. sinensis, Opisthorchis spp., and Metorchis spp. were detected in 11 species of common and popular lFWF. In lFWF, the estimated prevalence was 18.7% and the mean load was 137.4 ± 149.8 MC per 100 g of fish. The prevalence was the highest (P < 0.05) in spotted snakehead fishes (Channa punctata, 63.6%). The highest rate of infection (P < 0.05) was observed with the MC of C. sinensis (11.8%). Metacercariae were almost equally (P > 0.05) distributed between the head and body of fishes. The infection rate was slightly higher in cultured (19.6%) fishes. The MC of C. sinensis, O. felineus, O. viverrini, and Metorchis orientalis in fishes were confirmed using PCR, PCR-RFLP and bioinformatics. The cercariae of opisthorchiid (Pleurolophocercus cercariae) flukes were only recovered from Bithynia spp. (3.9%, 42 out of 1089). The ova of hLFs from dogs (4.3%, 5 out of 116) and cats (6.0%, 6 out of 100), and adult flukes (M. orientalis) from ducks (41.1% 113 out of 275) were detected. CONCLUSIONS: The MC of hLFs are highly prevalent in fresh water fishes in Bangladesh. Reservoir hosts, such as street dogs, cats, and ducks carried the patent infection, and residents of Bangladesh are at risk.

Database construction and comparative genomics analysis of genes involved in nutritional metabolic diseases in fish.

Nutritional metabolic diseases in fish frequently arise in the setting of intensive aquaculture. The etiology and pathogenesis of these conditions involve energy metabolic disorders influenced by both internal genetic factors and external environmental conditions. The exploration of genes associated with nutritional and metabolic disorder has sparked considerable interest within both the aquaculture scientific community and the industry. High-throughput sequencing technology offers researchers extensive genetic information. Effectively mining, analyzing, and securely storing this data is crucial, especially for advancing disease prevention and treatment strategies. Presently, the exploration and application of gene databases concerning nutritional and metabolic disorders in fish are at a nascent stag. Therefore, this study focused on the model organism zebrafish and five primary economic fish species as the subjects of investigation. Using information from KEGG, OMIM, and existing literature, a novel gene database associated with nutritional metabolic diseases in fish was meticulously constructed. This database encompassed 4583 genes for Danio rerio, 6287 for Cyprinus carpio, 3289 for Takifugu rubripes, 3548 for Larimichthys crocea, 3816 for Oreochromis niloticus, and 5708 for Oncorhynchus mykiss. Through a comparative systems biology approach, we discerned a relatively high conservation of genes linked to nutritional metabolic diseases across these fish species, with over 54.9 % of genes being conserved throughout all six species. Additionally, the analysis pinpointed the existence of 13 species-specific genes within the genomes of large yellow croaker, tilapia, and rainbow trout. These genes exhibit the potential to serve as novel candidate targets for addressing nutritional metabolic diseases.

Novel C-type lectin mediated non-specific cytotoxic cells killing activity through NCCRP-1 in nile tilapia (Oreochromis niloticus).

Non-specific cytotoxic cells (NCCs) are vital immune cells involved in teleost's non-specific immunity. As a receptor molecule on the NCCs' surface, the non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is known to play a crucial role in mediating their activity. Nevertheless, there have been limited studies on the signal molecule that transmits signals via NCCRP-1. In this study, a yeast two-hybrid (Y2H) library of tilapia liver and head kidney was constructed and subsequently screened with the bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1) to obtain a C-type lectin (On-CTL) with an interacting protein sequence. Consequently, the full-length sequence of On-CTL was cloned and analyzed. The expression analysis revealed that On-CTL is highly expressed in the liver and is widely distributed in other tissues. Furthermore, On-CTL expression was significantly up-regulated in the brain, intestine, and head kidney following a challenge with Streptococcus agalactiae. A point-to-point Y2H method was also used to confirm the binding between On-NCCRP-1 and On-CTL. The recombinant On-CTL (rOn-CTL) protein was purified. In vitro experiments demonstrated that rOn-CTL can up-regulate the expression of killer effector molecules in NCCs via its interaction with On-NCCRP-1. Moreover, activation of NCCs by rOn-CTL resulted in a remarkable enhancement in their ability to eliminate fathead minnow cells, indicating that rOn-CTL effectively modulates the killing activity of NCCs through the NCC receptor molecule On-NCCRP-1. These findings significantly contribute to our comprehension of the regulatory mechanisms governing NCC activity, paving the way for future research in this field.

Molecular cloning and functional analyses of C-C motif chemokine ligand 3 (CCL3) in mandarin fish Siniperca chuatsi.

Chemokines are critical molecules involved in immune reaction and immune system homeostasis, and some chemokines play a role in antiviral immunity. It is not known if the C-C motif chemokine ligand 3 (CCL3), a member of the CC chemokine family, possesses antiviral properties in fish. In this study, a ccl3 was cloned from the mandarin fish (Siniperca chuatsi), and it has an open reading frame (ORF) of 276 base pairs, which are predicted to encode a 91-amino acid peptide. Mandarin fish CCL3 revealed conserved sequence features with four cysteine residues and closely relationships with the CCL3s from other vertebrates based on the sequence alignment and phylogenetic analysis. The transcripts of ccl3 were notably enriched in immune-related organs, such as spleen and gills in healthy mandarin fish, and the ccl3 was induced in the isolated mandarin fish brain (MFB) cells following infection with infectious spleen and kidney necrosis virus (ISKNV). Moreover, in MFB cells, overexpression of CCL3 induced immune factors, such as IL1ß, TNFα, MX, IRF1 and IFNh, and exhibited antiviral activity against ISKNV. This study sheds light on the immune role of CCL3 in immune response of mandarin fish, and its antiviral defense mechanism is of interest for further investigation.

MOLECULAR PHYLOGENY OF THE LEECH GENUS PONTOBDELLA (HIRUDINIDA: PISCICOLIDAE) WITH NOTES ON PONTOBDELLA CALIFORNIANA AND PONTOBDELLA MACROTHELA.

Leech specimens of the genus Pontobdella (Hirudinida: Piscicolidae) were found off the coast of the state of Oaxaca (Pacific) as well as in Veracruz and Tabasco (Gulf of Mexico), Mexico. Based on the specimens collected in Oaxaca, a redescription of Pontobdella californiana is provided, with emphasis on the differences in the reproductive organs with the original description of the species. In addition, leech cocoons assigned to P. californiana were found attached to items hauled by gillnets and studied using scanning electron microscopy and molecular approaches. Samples of Pontobdella macrothela were found in both Pacific and Atlantic oceans, representing new geographic records. The phylogenetic position of P. californiana is investigated for the first time, and with the addition of Mexican samples of both species, the phylogenetic relationships within Pontobdella are reinvestigated. Parsimony and maximum-likelihood phylogenetic analysis were based on mitochondrial (cytochrome oxidase subunit I [COI] and 12S rRNA) and nuclear (18S rRNA and 28S rRNA) DNA sequences. Based on our results, we confirm the monophyly of Pontobdella and the pantropical distribution of P. macrothela with a new record in the Tropical Eastern Pacific.

PREVALENCE OF OPECOELID TREMATODES (TREMATODA: DIGENEA: OPECOELIDAE) IN THE WOOLLY SCULPIN CLINOCOTTUS ANALIS FROM THE PACIFIC COAST OF THE BAJA CALIFORNIA PENINSULA.

Prevalence values for 3 digenean species of the family Opecoelidae were calculated during a half-year period from 20 individuals per month of the fish species Clinocottus analis, collected from an intertidal environment of Baja California, Mexico. Trematode species recovered were Opecoelus adsphaericus, Opecoelus cameroni, and Opecoelus pacificus. Of these, only O. adsphaericus was present throughout the study, whereas O. pacificus and O. cameroni were recorded for 1 or 2 mo, respectively, exhibiting relatively low prevalence values. The decrease in prevalence of O. adsphaericus coincides with the appearance of O. pacificus and O. cameroni; these last 2 species were found only in the largest hosts, and their presence represents new records for Mexican marine fauna.

Chlorella vulgaris algae ameliorates chlorpyrifos toxicity in Nile tilapia with special reference to antioxidant enzymes and Streptococcus agalactiae infection.

BACKGROUND: Chlorpyrifos (CPF) is a widely used pesticide in the production of plant crops. Despite rapid CPF biodegradation, fish were exposed to wastewater containing detectable residues. Recently, medicinal plants and algae were intensively used in aquaculture to replace antibiotics and ameliorate stress impacts. METHODS AND RESULTS: An indoor experiment was conducted to evaluate the deleterious impacts of CPF pollution on Nile tilapia health and the potential mitigation role of Chlorella vulgaris algae. Firstly, the median lethal concentration LC50 - 72 h of CPF was determined to be 85.8 µg /L in Nile tilapia (35.6 ± 0.5 g body weight) at a water temperature of 27.5 °C. Secondly, fish were exposed to 10% of LC50 - 72 h for six weeks, and tissue samples were collected and examined every two weeks. Also, Nile tilapia were experimentally infected with Streptococcus agalactiae. Exposed fish were immunosuppressed expressed with a decrease in gene expressions of interleukin (IL) 1ß, IL-10, and tumor necrosis factor (TNF)-α. Also, a decline was recorded in glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) gene expression in the head kidney tissue. A high mortality rate (MR) of 100% was recorded in fish exposed to CPF for six weeks and challenged with S. agalactiae. Fish that received dietary C. vulgaris could restore gene expression cytokines and antioxidants compared to the control. After six weeks of CPF exposure, fish suffered from anemia as red blood cell count (RBCs), hemoglobin (Hb), and packed cell volume (PCV) significantly declined along with downregulation of serum total protein (TP), globulin (GLO), and albumin (ALB). Liver enzymes were significantly upregulated in fish exposed to CPF pollution, alanine aminotransferase (ALT) (42.5, 53.3, and 61.7 IU/L) and aspartate aminotransferase (AST) (30.1, 31.2, and 22.8) after 2, 4, and 6 weeks, respectively. On S. agalactiae challenge, high MR was recorded in Nile tilapia exposed to CPF (G3) 60%, 60%, and 100% in week 2, week 4, and week 6, and C. vulgaris provided a relative protection level (RPL) of 0, 14.29, and 20%, respectively. CONCLUSIONS: It was concluded that CPF pollution induces immunosuppressed status, oxidative stress, and anemic signs in Nile tilapia. In contrast, C. vulgaris at a 50 g/kg fish feed dose could partially ameliorate such withdrawals, restoring normal physiological parameters.

DC-SIGN of Largemouth Bass (Micropterus salmoides) Mediates Immune Functions against Aeromonas hydrophila through Collaboration with the TLR Signaling Pathway.

C-type lectins in organisms play an important role in the process of innate immunity. In this study, a C-type lectin belonging to the DC-SIGN class of Micropterus salmoides was identified. MsDC-SIGN is classified as a type II transmembrane protein. The extracellular segment of MsDC-SIGN possesses a coiled-coil region and a carbohydrate recognition domain (CRD). The key amino acid motifs of the extracellular CRD of MsDC-SIGN in Ca2+-binding site 2 were EPN (Glu-Pro-Asn) and WYD (Trp-Tyr-Asp). MsDC-SIGN-CRD can bind to four pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), glucan, peptidoglycan (PGN), and mannan. Moreover, it can also bind to Gram-positive, Gram-negative bacteria, and fungi. Its CRD can agglutinate microbes and displays D-mannose and D-galactose binding specificity. MsDC-SIGN was distributed in seven tissues of the largemouth bass, among which the highest expression was observed in the liver, followed by the spleen and intestine. Additionally, MsDC-SIGN was present on the membrane of M. salmoides leukocytes, thereby augmenting the phagocytic activity against bacteria. In a subsequent investigation, the expression patterns of the MsDC-SIGN gene and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) exhibited an up-regulated expression response to the stimulation of Aeromonas hydrophila. Furthermore, through RNA interference of MsDC-SIGN, the expression level of the DC-SIGN signaling pathway-related gene (RAF1) and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) was decreased. Therefore, MsDC-SIGN plays a pivotal role in the immune defense against A. hydrophila by modulating the TLR signaling pathway.

Mycolicibacterium cyprinidarum sp. nov., a rapidly growing species isolated from diseased koi carp, Cyprinus carpio.

A rapidly growing nontuberculous mycobacterium was isolated from diseased koi carp in Niigata, Japan, which was identified as representing a novel Mycolicibacterium species through whole genome sequence analysis. The bacterial isolates (NGTWS0302, NGTWS1803T and NGTWSNA01) were found to belong to the genus Mycolicibacterium through phylogenetic analysis using whole genome sequences of mycobacteria species. The bacterial colony was smooth, moist and non-chromogenic on 1% Ogawa medium at 30 °C. In biochemical characteristic tests, the bacterial isolates showed positive reactions for catalase activity, Tween 80 hydrolysis and tellurite reduction. The isolates were sensitive to 2-4 µg ml-1 ampicillin, kanamycin and rifampicin. Based on these results, we propose a novel Mycolicibacterium species, Mycolicibacterium cyprinidarum sp. nov. The type strain is NGTWS1803T (=JCM 35117T=ATCC TSD-289T).

Determination and Characterization of Novel Papillomavirus and Parvovirus Associated with Mass Mortality of Chinese Tongue Sole (Cynoglossus semilaevis) in China.

A massive mortality event concerning farmed Chinese tongue soles occurred in Tianjin, China, and the causative agent remains unknown. Here, a novel Cynoglossus semilaevis papillomavirus (CsPaV) and parvovirus (CsPV) were simultaneously isolated and identified from diseased fish via electron microscopy, virus isolation, genome sequencing, experimental challenges, and fluorescence in situ hybridization (FISH). Electron microscopy showed large numbers of virus particles present in the tissues of diseased fish. Viruses that were isolated and propagated in flounder gill cells (FG) induced typical cytopathic effects (CPE). The cumulative mortality of fish given intraperitoneal injections reached 100% at 7 dpi. The complete genomes of CsPaV and CsPV comprised 5939 bp and 3663 bp, respectively, and the genomes shared no nucleotide sequence similarities with other viruses. Phylogenetic analysis based on the L1 and NS1 protein sequences revealed that CsPaV and CsPV were novel members of the Papillomaviridae and Parvoviridae families. The FISH results showed positive signals in the spleen tissues of infected fish, and both viruses could co-infect single cells. This study represents the first report where novel papillomavirus and parvovirus are identified in farmed marine cultured fish, and it provides a basis for further studies on the prevention and treatment of emerging viral diseases.

Phylogenomic Characterization of Ranavirus Isolated from Wild Smallmouth Bass (Micropterus dolomieu).

In September 2021, 14 smallmouth bass (SMB; Micropterus dolomieu) with skin lesions were collected from Green Bay waters of Lake Michigan and submitted for diagnostic evaluation. All the skin samples tested positive for largemouth bass virus (LMBV) by conventional PCR. The complete genome of the LMBV (99,328 bp) isolated from a homogenized skin sample was determined using an Illumina MiSeq sequencer. A maximum likelihood (ML) phylogenetic analysis based on the 21 core iridovirus genes supported the LMBV isolated from SMB (LMBV-WVL21117) as a member of the species Santee-Cooper ranavirus. Pairwise nucleotide comparison of the major capsid protein (MCP) gene showed that LMBV-WVL21117 is identical to other LMBV reported from the United States and nearly identical to doctor fish virus and guppy virus 6 (99.2%) from Southeast Asia, as well as LMBV isolates from China and Thailand (99.1%). In addition, ML phylogenetic analysis based on the MCP gene suggests three genotypes of LMBV separated by region: genotype one from the United States, genotype two from Southeast Asia, and genotype three from China and Thailand. Additional research is needed to understand the prevalence and genetic diversity of LMBV strains circulating in wild and managed fish populations from different regions.

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.

Immunomodulatory, antioxidant, and growth-promoting activities of dietary fermented Moringa oleifera in Nile tilapia (Oreochromus niloticus) with in-vivo protection against Aeromonas hydrophila.

BACKGROUND: Moringa oleifera, a well-known medicinal plant, has been used in aquafeed as a dietary supplement. Based on previous studies, insufficient research is available on the dietary supplementation of Nile tilapia with M. oleifera leaf and seed mixtures, specifically the fermented form. Therefore, this study aimed to investigate the efficacy of fermented (FMO) versus non-fermented M. oleifera (MO) leaf and seed mixtures on immunological parameters, antioxidant activity, growth performance, and resistance to A. hydrophila infection after a 30-day feeding trial on Nile tilapia. METHODS: A total of 180 fingerlings were randomly divided into four groups in addition to the control group (36 fish each, in triplicate). Fish in the tested groups were fed on basal diet supplemented with MO5%, MO10%, FMO5%, and FMO10%, while those in control were fed on basal diet only. After the feeding trial, fish were challenged with A. hydrophila. The immunomodulatory activity of M. oleifera was evaluated in terms of phagocytic and lysozyme activities, immune-related cytokines and IgM gene expression. Antioxidants, and growth-promoting activities were also assessed. RESULTS: The results revealed that fish supplemented FMO markedly in FMO10% group followed by FMO5%, exhibited significant (P < 0.05) improvement in the tested immunological, hepatic antioxidants, and growth performance parameters. Furthermore, the highest survival rate post-challenge with mild clinical symptoms, and the lowest A. hydrophila bacterial count were reported in these groups. Meanwhile, MO10%-supplementation exhibited the opposite trend. CONCLUSIONS: The study' conclusion suggests that fermented M. oleifera leaf and seed mixture is a promising growth-promoting and immunostimulatory feed-additive candidate for Nile tilapia and could reduce the losses caused by A. hydrophila infection.

Analysis of tissue tropism of GCRV-II infection in grass carp using a VP35 monoclonal antibody.

Grass carp, one of the major freshwater aquaculture species in China, is susceptible to grass carp reovirus (GCRV). GCRV is a non-enveloped RNA virus and has a double-layered capsid, causing hemorrhagic disease and high mortalities in infected fish. However, the tropism of GCRV infection has not been investigated. In this study, monoclonal antibodies against recombinant VP35 protein were generated in mice and characterized. The antibodies exhibited specific binding to the N terminal region (1-155 aa) of the recombinant VP35 protein expressed in the HEK293 cells, and native VP35 protein in the GCRV-II infected CIK cells. Immunofluorescent staining revealed that viruses aggregated in the cytoplasm of infected cells. In vivo challenge experiments showed that high levels of GCRV-II viruses were present in the gills, intestine, spleen and liver, indicating that they are the major sites for virus infection. Our study showed that the VP35 antibodies generated in this study exhibited high specificity, and are valuable for the development of diagnostic tools for GCRV-II infection.

Diversification of Toll-like receptor 1 in swamp eel (Monopterus albus).

Toll-like receptor 1 (TLR1) is a pattern recognition receptor that plays critical roles in triggering immune activation via detecting bacterial lipoproteins and lipopeptides. In this study, the genetic characteristic of TLR1 was studied for an important aquaculture fish, swamp eel Monopterus albus. The eel has been seriously threatened by infectious diseases. However, a low level of genetic heterogeneity in the fish that has resulted from a demographic bottleneck presents further challenges in breeding for disease resistance. A comparison with the homologue of closely related species M. javanensis revealed that amino acid replacement (nonsynonymous) but not silent (synonymous) differences have accumulated nonrandomly over the coding sequences of the receptors at the early stage of their phylogenetic split. The combined results from comparative analyses of nonsynonymous-to-synonymous polymorphisms showed that the receptor has undergone significant diversification in M. albus driven by adaptive selection likely after the genetic bottleneck. Some of the changes reported here have taken place in the structures mediating heterodimerization with co-receptor TLR2, ligand recognition, and/or formation of active signaling complex with adaptor, which highlighted key structural elements and strategies of TLR1 in arms race against exogenous challenges. The findings of this study will add to the knowledge base of genetic engineering and breeding for disease resistance in the eel.

Ficus hirta Vahl. alleviate LPS induced apoptosis via down-regulating of miR-411 in orange-spotted grouper (Epinephelus coioides) spleen cell.

Ficus hirta Vahl. (FhV) has been shown to have antimicrobial and antiviral efficacy. To further ascertain the pharmacological properties of FhV., and to search for alternatives to antibiotics. An in vitro experiment was carried out to evaluate what influence FhV. would have on LPS-induced apoptosis. In this study, Fas, an apoptosis receptor, was cloned, which included a 5'-UTR of 39 bp, an ORF of 951 bp, a protein of 316 amino acids, and a 3'-UTR of 845 bp. EcFas was most strongly expressed in the spleen tissue of orange-spotted groupers. In addition, the apoptosis of fish spleen cells induced by LPS was concentration-dependent. Interestingly, appropriate concentrations of FhV. alleviated LPS-induced apoptosis. Inhibition of miR-411 further decreased the inhibitory effect of Fas on apoptosis, which reduced Bcl-2 expression and mitochondrial membrane potential, enhanced the protein expression of Bax and Fas. More importantly, the FhV. could activate miR-411 to improve this effect. In addition, luciferase reporter assays showed that miR-411 binds to Fas 3'-UTR to inhibit Fas expression. These findings provide evidence that FhV. alleviates LPS-induced apoptosis by activating miR-411 to inhibit Fas expression and, therefore, provided possible strategies for bacterial infections in fish.

Virus-specific antibody secreting cells reside in the peritoneal cavity and systemic immune sites of Atlantic salmon (Salmo salar) challenged intraperitoneally with salmonid alphavirus.

The development and persistence of antibody secreting cells (ASC) after antigenic challenge remain inadequately understood in teleosts. In this study, intraperitoneal (ip) injection of Atlantic salmon (Salmo salar) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3-6 weeks post injection (wpi) locally in the peritoneal cavity (PerC) and in systemic lymphoid tissues, while at 13 wpi the response was only elevated in PerC. At the same time point a specific ASC response was induced by WtSAV3 in PerC and systemic tissues, with the highest frequency in PerC, suggesting a local role. Inactivated SAV (InSAV1) induced comparatively lower ASC responses in all sites, and specific serum antibodies were only induced by WtSAV3 and not by InSAV1. An InSAV1 boost did not increase these responses. Expression of immune marker genes implies a role for PerC adipose tissue in the PerC immune response. Overall, the study suggests the Atlantic salmon PerC as a secondary immune site and an ASC survival niche.

RING finger protein 122-like (RNF122L) negatively regulates antiviral immune response by targeting STING in common carp (Cyprinus carpio L.).

Stimulator of interferon genes (STING), as an imperative adaptor protein in innate immune, responds to nucleic acid from invading pathogens to build antiviral responses in host cells. Aberrant activation of STING may trigger tissue damage and autoimmune diseases. Given the decisive role in initiating innate immune response, the activity of STING is intricately governed by several posttranslational modifications, including phosphorylation and ubiquitination. Here, we cloned and characterized a novel RNF122 homolog from common carp (named CcRNF122L). Expression analysis disclosed that the expression of CcRNF122L is up-regulated under spring viremia of carp virus (SVCV) stimulation in vivo and in vitro. Overexpression of CcRNF122L hampers SVCV- or poly(I:C)-mediated the expression of IFN-1 and ISGs in a dose-dependent way. Mechanistically, CcRNF122L interacts with STING and promotes the polyubiquitylation of STING. This polyubiquitylation event inhibits the aggregation of STING and the subsequent recruitment of TBK1 and IRF3 to the signaling complex. Additionally, the deletion of the TM domain abolishes the negative regulatory function of CcRNF122L. Collectively, our discoveries unveil a mechanism that governs the STING function and the precise adjustment of the innate immune response in teleost.

Functional role of TrIL-1ß in Takifugu rubripes defense against Cryptocaryon irritans infection.

The Japanese puffer, Takifugu rubripes, is a commercially important fish species in China that is under serious threat from white spot disease (cyptocaryoniasis), which leads to heavy economic losses. We previously found that interleukin-1ß (IL-1ß), an important cytokine with a potential role in resistance against pathogens, was one of the most significantly differentially up-regulated proteins in the gills and spleen of T. rubripes infected by the protozoan parasite Cryptocaryon irritans. In this study, we assessed the potential function of T. rubripes IL-1ß (TrIL-1ß) in fish infected with C. irritans. Phylogenetic analysis indicated that the TrIL-1ß protein sequence was most closely related to that of Atlantic salmon (Salmo salar) (67.2 %). The incubation experiments revealed that TrIL-1ß may reduce trophont activity by destroying membranes. Immunofluorescence experiments showed that recombinant TrIL-1ß promoted the expression of endogenous IL-1ß, which penetrated and disrupted the cell membranes of trophonts. Transmission electron microscopy showed that the IL-1ß group had less tissue damage compared with control groups of fish. IL-1ß-small interfering RNA and IL-1ß overexpression experiments were performed in head kidney primary cells, and challenge experiments were performed in vitro. Quantitative RT-PCR results showed that TrIL-1ß regulated and activated MyD88/NF-κB and MyD88/MAPK/p38 signaling pathways during C. irritans infection. TrIL-1ß also promoted the differential expression of IgM, showing that it was involved in humoral immunity of T. rubripes. The cumulative mortality experiment show that TrIL-1ß could protect fish against C. irritans infection. These results enrich current knowledge about the molecular structure of TrIL-1ß. They also suggested that recombinant TrIL-1ß could be used as an adjuvant in a subunit vaccine against C. irritans infection, which is of profound importance for the prevention and control of parasitic diseases in T. rubripes.