Cloning and expression analysis of Janus activated kinase family genes from spotted seabass (Lateolabrax maculatus).

Janus kinases (JAKs) are important components of the JAK-STAT signaling pathway and play vital roles in innate immunity, autoimmune diseases, and inflammation. However, information about JAKs remains largely unknown in the spotted seabass, a fish species of Perciformes with great commercial value in the aquaculture industry. The aims of this study are to obtain the complete cDNA sequences of JAKs (JAK1, JAK2A, JAK2B, JAK3 and TYK2) from spotted seabass and to investigate their roles upon stimulation with lipopolysaccharides (LPS) and Edwardsiella tarda, using RT-PCR, PCR and qRT-PCR methods. All five JAK genes from the spotted seabass, each encode more than 1100 amino acids residues. JAK1 and JAK3 consist of 24 exons and 23 introns, whereas JAK2A, JAK2B and TYK2 consist of 23 exons and 22 introns. Furthermore, these five spotted seabass JAKs share high sequence identities with those of other fish species in protein domain analysis, synteny analysis, and phylogenetic analysis. Moreover, these five JAK genes were ubiquitously expressed in all tissues examined from healthy fish, and inducible expressions of JAKs were observed in the intestine, gill, head kidney, and spleen following LPS treatment or E. tarda infection. These findings indicate that all these JAK genes are involved in the antibacterial immunity of the spotted seabass and provide a basis for further understanding the mechanism of JAKs antibacterial response in the spotted sea bass.

Disruption of a kasB homolog gene (kasB) causes attenuation of cell invasion and virulence of Nocardia seriolae.

Nocardia seriolae is the primary aetiological agent of nocardiosis in fish, which causes mass mortality in freshwater and marine fish. ß-ketoacyl-ACP synthase (KAS) is one of the essential enzymes in the synthesis of mycolic acids (MASs) in Mycobacterium spp. and has been chosen as the target for therapeutic intervention in mycobacterial diseases. In the present study, a kasB homologue gene (kasB) was identified in the genome of N. seriolae, and the gene-deficient mutant (ΔkasB) was generated based on a clinical isolate, XSYC-Ns. Compared to the wild-type (WT) strain, the ΔkasB showed a measurably growth defect in vitro but retained the acid-fastness in acid-fast staining. Observation of the cell ultrastructure showed some alterations in the cell wall of the ΔkasB strain. Compared to its original strain, the cell wall lipid layer seemed sparser, and a wider electron-transparent zone was observed in the cell wall of ΔkasB strain. Moreover, the ΔkasB strain showed impaired ability of cell invasion as well as intracellular survival in the cell line originating from the head-kidney of the large yellow croaker (LYC-hK), compared to its original strain. In addition, the deficiency of ΔkasB significantly attenuated the virulence of N. seriolae in largemouth bass. The present study suggested that the ΔkasB gene might be involved in the synthesis of extracellular cell-wall lipids in N. seriolae and play a crucial role in its pathogenicity.

The goldfish primary kidney macrophage system.

Miodrag (Mike) Belosevic and collaborators profoundly influenced the development of primary kidney macrophage culturing system (PKM) to study fish immunology in various aspects of comparative immunology. Their application of using PKM model, opened a new path for studying the development of macrophages, regulation of hematopoiesis, and cell specific response against various pathogens. By measuring histopathological and immunological outcomes, the biological implications of a variety of cytokines and signal transduction molecules could be elucidated with the established PKM system. A variety of growth factors mediating hematopoiesis and cytokines regulating the immune responses were functionally characterized, which served as a fundamental basis for making goldfish an excellent model to study fish immunology. Specifically, using in vivo and PKM based in vitro assays, the Belosevic lab advanced the goldfish-M. marinum model to study the anti-mycobacteria responses in teleosts, thus paving a way for the development of novel therapeutic approaches which could be applied in aquaculture settings or utilized as a model for human disease. In this review, we will look at the contribution of Dr. Mike Belosevic to teleost macrophage development, multiple cytokine functional characterization, and host-pathogen interactions.

Identification and functional characterization of Caspase-9 in goldfish (Carassius auratus L.) in response to Aeromonas hydrophila infection.

Apoptosis plays a pivotal role in the immune response to combat pathogen infections. In mammals, caspase-9, abbreviated as Casp9, plays an irreplaceable role in the initiation phase of the apoptotic cascade. To investigate the role of Casp9 in teleosts, we conducted a functional characterization of Casp9 in goldfish (Carassius auratus L.). The open reading frame of GfCasp9 spans 1296 base pairs (bp), encoding a protein composed of 431 amino acids. GfCasp9 was ubiquitously expressed in various tissues, with the spleen and brain showing the highest levels of expression. Subcellular localization analysis revealed that GfCasp9 is distributed in both the cytoplasm and nucleus. Overexpressing of GfCasp9 in HEK293 cells elicits a robust apoptotic response. Additionally, infection with Aeromonas hydrophila significantly increases the mRNA and protein expression of GfCasp9. These findings underscore the critical importance of GfCasp9 in immune responses and apoptosis against bacterial infections.

Molecular characterization and functional analysis of galectin-1 from silver pomfret (Pampus argenteus).

Galectins, as members of lectin families, exhibit a high affinity for ß-galactosides and play diverse roles in biological processes. They function as pattern recognition receptors (PRRs) with important roles in immune defense. In this study, galectin-1, designated as SpGal-1, was identified and characterized from silver pomfret (Pampus argenteus). The SpGal-1 comprises an open reading frame (ORF) spanning 396 base pairs (bp) and encodes a deduced amino acid (aa) sequence containing a single carbohydrate recognition domain (CRD). Sublocalization analysis revealed that SpGal-1 was mainly expressed in the cytoplasm. The mRNA transcripts of SpGal-1 were ubiquitously detected in various tissues, with a higher expression level in the intestine. In addition, when exposed to Photobacterium damselae subsp. damselae (PDD) infection, both the liver and head kidney exhibited significantly increased SpGal-1 mRNA expression. The recombinant protein of SpGal-1 (named as rSpGal-1) demonstrated hemagglutination against red blood cells (RBCs) from Larimichthys crocea and P. argenteus in a Ca2+ or ß-Mercaptoethanol (ß-ME)-independent manner. Notably, rSpGal-1 could bind with various pathogen-associated molecular patterns (PAMPs) including D-galactose, D-mannose, lipopolysaccharide (LPS), and peptidoglycan (PGN), with highest affinity to PGN. Moreover, rSpGal-1 effectively interacted with an array of bacterial types encompassing Gram-positive bacteria (Staphylococcus aureus and Nocardia seriolae) and Gram-negative bacteria (PDD and Escherichia coli, among others), with the most robust binding affinity towards PDD. Collectively, these findings highlight that SpGal-1 is a crucial PRR with involvement in the host immune defense of silver pomfret.

Identification and functional characterization of Interleukin-11 in goldfish (Carassius auratus L.).

Interleukin-11 (IL-11) is a versatile cytokine that modulates cellular differentiation and proliferation in various cell types and tissues. In this study, IL-11 gene from goldfish (Carassius auratus L.) has been identified and characterized. Goldfish IL-11 (gfIL-11) has an open reading frame (ORF) that spans 591 base pairs (bp). The ORF encodes a precursor protein consisting of 196 amino acids (aa), which includes a 26 aa signal peptide and a conserved domain belonging to the IL-11 superfamily. Based on phylogenetic analysis, gfIL-11 was found to be closely related to other IL-11 homologues identified in various fish species. The gfIL-11 transcript exhibited varied expression levels across all the analyzed tissues, with the highest expression observed in the gill and spleen. Treatment of goldfish head kidney leukocytes (HKLs) with LPS and live Aeromonas hydrophila, increased gfIL-11 mRNA expression level. Recombinant gfIL-11 protein (rgIL-11) induced a dose-dependent production of TNF-α and IFNγ from goldfish HKLs. Furthermore, the administration of rgIL-11 to goldfish HKLs triggered an increase in the expression of various transcription factors such as MafB, cJun, GATA2, and Egr1, which play a vital role in the differentiation of myeloid precursors into macrophages and monocytes. Our findings provide evidence that IL-11 is a crucial cytokine that promotes cell proliferation, immune response, and differentiation across various hematopoietic lineages and stages of goldfish.

Molecular characterization, gene expression and functional analysis of goldfish (Carassius auratus L.) macrophage colony stimulating factor 2.

Background: Macrophage colony-stimulating factor 2 (MCSF-2) is an important cytokine that controls how cells of the monocyte/macrophage lineage proliferate, differentiate, and survive in vertebrates. Two isoforms of MCSF have been identified in fish, each exhibiting distinct gene organization and expression patterns. In this study, we investigated a goldfish MCSF-2 gene in terms of its immunomodulatory and functional properties. Methods: In this study, goldfish were acclimated for 3 weeks and sedated with TMS prior to handling. Two groups of fish were used for infection experiments, and tissues from healthy goldfish were collected for RNA isolation. cDNA synthesis was performed, and primers were designed based on transcriptome database sequences. Analysis of gfMCSF-2 sequences, including nucleotide and amino acid analysis, molecular mass prediction, and signal peptide prediction, was conducted. Real-time quantitative PCR (qPCR) was used to analyze gene expression levels, while goldfish head kidney leukocytes (HKLs) were isolated using standard protocols. The expression of gfMCSF-2 in activated HKLs was investigated, and recombinant goldfish MCSF-2 was expressed and purified. Western blot analysis, cell proliferation assays, and flow cytometric analysis of HKLs were performed. Gene expression analysis of transcription factors and pro-inflammatory cytokines in goldfish head kidney leukocytes exposed to rgMCSF-2 was conducted. Statistical analysis using one-way ANOVA and Dunnett's post hoc test was applied. Results: We performed a comparative analysis of MCSF-1 and MCSF-2 at the protein and nucleotide levels using the Needleman-Wunsch algorithm. The results revealed significant differences between the two sequences, supporting the notion that they represent distinct genes rather than isoforms of the same gene. Sequence alignment demonstrated high sequence identity with MCSF-2 homologs from fish species, particularly C. carpio, which was supported by phylogenetic analysis. Expression analysis in various goldfish tissues demonstrated differential expression levels, with the spleen exhibiting the highest expression. In goldfish head kidney leukocytes, gfMCSF-2 expression was modulated by chemical stimuli and bacterial infection, with upregulation observed in response to lipopolysaccharide (LPS) and live Aeromonas hydrophila. Recombinant gfMCSF-2 (rgMCSF-2) was successfully expressed and purified, showing the ability to stimulate cell proliferation in HKLs. Flow cytometric analysis revealed that rgMCSF-2 induced differentiation of sorted leukocytes at a specific concentration. Moreover, rgMCSF-2 treatment upregulated TNFα and IL-1ß mRNA levels and influenced the expression of transcription factors, such as MafB, GATA2, and cMyb, in a time-dependent manner. Conclusion: Collectively, by elucidating the effects of rgMCSF-2 on cell proliferation, differentiation, and the modulation of pro-inflammatory cytokines and transcription factors, our findings provided a comprehensive understanding of the potential mechanisms underlying gfMCSF-2-mediated immune regulation. These results contribute to the fundamental knowledge of MCSF-2 in teleosts and establish a foundation for further investigations on the role of gfMCSF-2 in fish immune responses.

Transcriptome analysis of Cryptocaryon irritans tomont responding to Bacillus licheniformis treatment.

Cryptocaryon irritans is a ciliated obligate parasite that causes cryptocaryonosis (white spot disease) and poses great threat to marine fish farming. In recent years, the use of probiotics protects fish from pathogens, which has been identified as the sustainable and environmentally friendly tool to maintain the health and well-being of the host. Accordingly, Cryptocaryon irritans tomont and probiotic Bacillus strain (B.licheniformis, previously isolated from aquaculture water) were co-cultured to detect whether B. licheniformis has anti-C. irritants effect. The result showed that during 4-day incubation, B. licheniformi with 1 × 107 CFU/mL and 1 × 108 CFU/mL concentration effectively inhibited the incubation of C. irritans tomont, indicating that B. licheniformi could inhibit the transformation from reproductive tomont to infective theront of C. irritans. Later, C. irritans samples in the control (without B. licheniformi supplementation) and 1 × 107 CFU/mL B. licheniformi treatment group were sent for transcriptome analysis. Compare with the control group, a total of 3237 differentially expressed genes were identified, among which 626 genes were up-regulated and 2611 genes were down-regulated in 1 × 107 CFU/mL B. licheniformi group. Further Kyoto Encyclopedia of Genes and Genomes pathways analysis showed that anti-C. irritans mechanism of B. licheniformi was mainly involved in the energy metabolism (carbon metabolism, oxidative phosphorylation, biosynthesis of amino acids), transcription and translation (Ribosomes, spliceosomes, RNA transport, etc), lysosome-based degradation (lysosome, phagosome, protein processing in endoplasmic reticulum) and PI3K-Akt pathways. Our study findings raised the possibility of using marine microorganism B. licheniformi in handling aquaculture associated pathogen C. irritans, and preliminarily clarified the molecular mechanism.

Molecular and functional characterization of two granulocyte colony stimulating factors in goldfish (Carassius auratus L.).

Granulocyte colony-stimulating factor (GCSF) is a member of the hematopoietic growth factor family that acts primarily on neutrophils and neutrophilic precursors to promote cell proliferation and differentiation. Although multiple GCSF genes have been found in teleosts, knowledge of their functions during fish hematopoietic development is still limited. Here, we report for the first time the molecular and functional characterization of two goldfish GCSFs (gfGCSF-a and gfGCSF-b). The open reading frame (ORF) of the gfGCSF-a and gfGCSF-b cDNA transcript consisted respectively of 624 bp and 678 bp with its ORF encoding 207 and 225 amino acids (aa), with a 17 aa signal peptide for each gene and a conserved domain of the IL-6 superfamily. Treatment of goldfish head kidney leukocytes (HKLs) with LPS increased gfGCSF-a and gfGCSF-b mRNA expression levels, also exposure of HKLs to either heat-killed or live A. hydrophila, induced transcriptional upregulation of gfGCSF-a and gfGCSF-b levels. Recombinant gfGCSF-a and gfGCSF-b protein (rgGCSF-a and rgGCSF-b) induced a dose-dependent production of TNFα and IL-1ß from goldfish neutrophils. In vitro experiments showed rgGCSF-a and rgGCSF-b differentially promoted the proliferation and differentiation of leukocytes in goldfish. Furthermore, treatment of HKLs with rgGCSF-a showed significant upregulation of mRNA levels of the hematopoietic transcription factor GATA2, Runx1, MafB, and cMyb, while gfGCSF-b induces not only all four transcriptional factors mentioned above but also CEBPα. Our results indicate that goldfish GCSF-a and GCSF-b are important regulators of neutrophil proliferation and differentiation, which could stimulate different stages and lineages of hematopoiesis.

Transcriptome profiling and differential expression analysis of altered immune-related genes in goldfish (Carassius auratus) infected with Aeromonas hydrophila.

Goldfish (Carassius auratus) have been employed as a model organism to investigate the innate immune system and host-pathogen interactions. A Gram-negative bacterium called Aeromonas hydrophila has been found to cause mass mortality due to infection in a wide variety of fish species in the aquatic system. In this study, damages in Bowman's capsule, inflammatory tubular (proximal and distilled convoluted) structure, and glomerular necrosis were observed in A. hydrophila-infected head kidney of goldfish. To increase the better understanding of immune mechanisms of host defense against A. hydrophila, we performed a transcriptome analysis in head kidney of goldfish at 3 and 7 days of post-infection (dpi). Comparing to the control group, 4638 and 2580 differentially expressed genes (DEGs) were observed at 3 and 7 dpi, respectively. The DEGs were subsequently enriched in multiple immune-related pathways including Protein processing in endoplasmic reticulum, Insulin signaling pathway, and NOD-like receptor signaling pathway. The expression profile of immune-related genes such as TRAIL, CCL19, VDJ recombination-activating protein 1-like, Rag-1, and STING was validated by qRT-PCR. Furthermore, the levels of immune-related enzyme (LZM, AKP, SOD, and CAT) activities were examined at 3 and 7 dpi. The knowledge gained from the current study will be helpful for better understanding of early immune response in goldfish after A. hydrophila challenge, which will aid in future research on prevention strategies in teleost.

Characterization of the pro-inflammatory roles of the goldfish (Carassius auratus L.) M17 protein.

The interleukin-6 family of cytokines possesses a diversity of roles with significant redundancy. The roles of these molecules have been relatively well characterized in mammals, with limited attention in other species. Progress has been made in the discovery of homologous molecules in fish. Here we report the characterization of pro-inflammatory properties of recombinant goldfish M17. Recombinant goldfish M17 enhanced phagocytosis, primed production of reactive oxygen intermediates, and was chemotactic to macrophages. Treatment of goldfish macrophages with LPS, heat-killed and live Aeromonas hydrophila resulted in higher M17 mRNA levels. Recombinant M17 (RgM17) induced dose-dependent production of IFNγ and IL-1ß1 in goldfish macrophages. Furthermore, treatment of macrophages with rgM17 resulted in upregulation of transcription factors that were important in the differentiation of myeloid progenitors into monocytes/macrophages (Runx1 and GATA2). Our results indicate that goldfish M17 is an essential inflammatory cytokine for proliferation and differentiation of goldfish progenitor cells.

Nigericin treatment activates endoplasmic reticulum apoptosis pathway in goldfish kidney leukocytes.

Nigericin has been reported to induce apoptosis and pyroptosis in mammalian models. However, the effects and mechanism underlying the immune responses of teleost HKLs induced by nigericin remain enigmatic. To decipher the mechanism after nigericin treatment, the transcriptomic profile of goldfish HKLs was analyzed. The results demonstrated that a total of 465 differently expressed genes (DEGs) with 275 up-regulated and 190 down-regulated genes were identified between the control and nigericin treated groups. Among them, the top 20 DEG KEGG enrichment pathways were observed including apoptosis pathways. In addition, the expression level of selected genes (ADP4, ADP5, IRE1, MARCC, ALR1, DDX58) by quantitative real-time PCR showed a significant change after treatment with nigericin, which was generally identical to the expression patterns of the transcriptomic data. Furthermore, the treatment could induce cell death of HKLs, which was confirmed by LDH release and annexin V-FITC/PI assays. Taken together, our results support the idea that nigericin treatment might activate the IRE1-JNK apoptosis pathway in goldfish HKLs, which will provide insights into the mechanisms underlying HKLs immunity towards apoptosis or pyroptosis regulation in teleosts.

Effect of copper sulphate on Cryptocaryon irritans based on metabolome analysis.

Cryptocaryon irritans is one of the most harmful marine parasites in mariculture. Copper sulphate is often used to kill parasites and the influence of copper sulphate on the tomont stage of C. irritans was explored in this study. The results showed that excystment rate was not significantly affected when tomonts were exposed to 5 mg/L (76.7%) and 10 mg/L (78.9%) of copper sulphate for 3 h. However, excystment rate was significantly inhibited when exposed to 15 mg/L (33.3%) for 3 h and 5 mg/L (28.9%), 10 mg/L (33.3%) and 15 mg/L (33.3%) for 6 h. After treatment with high concentrations of copper sulphate, the interior of the tomonts was fuzzy under the microscope, and the division process could not be observed. Metabolomic results combined with preliminary transcriptome analysis results showed that the tomonts were induced to produce linoleate, riboflavin, inositol and other substances under the stress of Cu2+ , which affected the antioxidant mechanism of the body. Using MDA content determination and antioxidant enzyme activity analysis, copper sulphate was found to cause oxidative damage to tomonts by affecting the generation of metabolites, leading to the death of tomonts.

The galU gene is required for in vivo survival of pseudomonas plecoglossicida in large yellow croaker (Larimichthys crocea).

Pseudomonas plecoglossicida is an important pathogenic bacterium in aquaculture that causes visceral granulomas in large yellow croaker (Larimichthys crocea). Uridine diphosphate glucose phosphorylase encoded by galU plays a key role in biosynthesis of the bacterial envelope, particularly lipopolysaccharide and the capsule. In this study, we inactivated the galU gene in the P. plecoglossicida isolate XSDHY-P. The galU mutant strain showed impaired growth in the early exponential stage and lacked the O polysaccharide side chain in lipopolysaccharide, but almost no defect in biofilm formation was detected. The galU mutant strain also exhibited significantly more sensitivity to the bactericidal action of normal fish serum mediated by the complement system compared to the wild-type strain. In a cell model originating from the head kidney of large yellow croaker, the galU mutant strain showed lower capacities of adhesion, invasion, and intracellular survival compared to the wild-type strain. In addition, the deficiency of the galU mutant drastically decreased bacterial loads in tissues and attenuated P. plecoglossicida virulence in fish. These results suggest that the galU gene of P. plecoglossicida is required for in vivo survival in large yellow croaker.

Transcriptome profiling and differential expression analysis of the immune-related genes during the acute phase of infection with Photobacterium damselae subsp. damselae in silver pomfret (Pampus argenteus).

Silver pomfret has been widely cultured in China due to its high economic value. Photobacterium damselae subsp. damselae (PDD) is a Gram-negative bacterium that has been shown to infect many fish species. To increase knowledge of the molecular mechanisms of the host defense against PDD, we conducted transcriptome analysis of head kidney in silver pomfret at 24 h and 72 h post-infection (hpi) via Illumina sequencing. The de novo assembly resulted in the identification of 79,063 unigenes, with 59,386 (75.11%) successfully annotated in public databases (NR, NT, KO, Swiss-Prot, Pfam, GO, and KOG databases). Comparison of gene expression profiles between PBS-injected fish (sham control) and PDD-challenged fish revealed 329 and 570 differentially expressed genes (DEGs) were screened at 24 hpi and 72 hpi, respectively. The DEGs were enriched in multiple immune-related pathways such as Hepatitis C, Gastric acid secretion, CAMs and Leukocyte transendothelial migration pathways, Primary immunodeficieny, ECM-receptor interaction, PI3K-Akt signaling pathway. The data obtained in the present study offers valuable information for acute immune response of silver pomfret challenged with PDD, which will facilitate further investigations on strategies against Photobacterium spp. infection in teleosts.

Outbreak of Cryptocaryon irritans infection in silver pomfret Pampus argenteus cultured in China.

Silver pomfret Pampus argenteus is a major cultivated marine fish species with a high market value. In summer 2021, Cryptocaryon irritans, a ciliate parasite, infected the cultured silver pomfret in aquaculture ponds in Ningbo, Zhejiang Province, China. The symptoms of infected fish include white spots on the skin and fins, increased body surface mucus, loss of appetite, irritability, and shedding of scales. After collecting white spots from moribund fish, the 18S ribosomal RNA sequence of the pathogen on the fish skin was amplified by PCR; phylogenetic analysis showed that it was closely related to C. irritans strains from Ningde, Fujian, China. Four groups of silver pomfret were tested in an artificial infection experiment over the course of 72 h, consisting of 3 infected groups (1600, 4000, and 8000 theronts fish-1) and 1 healthy group. White spots were observed on the skin and fins of the infected fish, but not on their gills. Samples were taken from the gills, liver, kidney, and spleen of both infected and healthy fish and were compared to evaluate any significant histopathological differences. As the dose of infection increased, symptoms became more pronounced. At 72 h, mortality rates were 8.3, 50, and 66.7% for the 3 different concentrations, respectively. The median lethal concentration was calculated to be 366 theronts g-1 at 72 h, 298 theronts g-1 at 84 h, and 219 theronts g-1 at 96 h. This study emphasizes the importance of developing early diagnosis methods and appropriate prevention strategies to decrease the impact of C. irritans infection in the silver pomfret aquaculture industry.

Outbreak of vibriosis associated with Vibrio parahaemolyticus in the mud crab Scylla paramamosain cultured in China.

In this study, a Gram-negative bacterium was isolated from diseased Scylla paramamosain and tentatively named strain QX17. The bacterial isolate was identified as Vibrio parahaemolyticus based on morphological and biochemical characteristics and molecular identification with the 16S rRNA and HSP60 genes. In the challenge experiment, S. paramamosain injected intramuscularly with the V. parahaemolyticus isolate developed pathological signs similar to the naturally diseased mud crabs. The infection experiment also showed that the median lethal dosage (LD50) for QX17 was 4.79 × 102 CFU g-1 (crab weight). Histopathological analysis of the diseased mud crabs infected with V. parahaemolyticus showed deformation and basement membrane rupture of hepatopancreatic tubules in the hepatopancreas, and disordered and broken muscle fiber in the muscle. Antimicrobial susceptibility tests revealed that QX17 was highly sensitive to most of the tested aminoglycosides, tetracyclines, and quinolones. To the best of our knowledge, this is the first study reporting isolation and antibiotic sensitivities of V. parahaemolyticus from cultured mud crabs. The discovery of V. parahaemolyticus in cultured mud crabs not only adds to the growing list of emerging pathogens in crab aquaculture in China, but also highlights the necessity of developing early detection strategies and appropriate interventions to reduce the damage caused by V. parahaemolyticus in mud crab aquaculture.

Teleost antimicrobial peptide hepcidin contributes to host defense of goldfish (Carassius auratus L.) against Trypanosoma carassii.

Hepcidin is an antimicrobial peptide and an iron regulatory protein that prevents the release of excess iron in the blood. There is evidence suggesting that teleost hepcidin is a major player in antimicrobial defense against various bacteria species, but little is known regarding the effects of teleost hepcidin in protozoan parasitic infections. We examined the role of hepcidin during the course of infection of goldfish with Trypanosoma carassii. Quantitative real-time PCR was used to determine the expression of hepcidin in goldfish immune organs during the course of T. carassii infection. During the acute phase of the T. carassii infection, the mRNA levels of hepcidin were up-regulated in liver and kidney. In contrast, an up-regulation of hepcidin mRNA expression in spleen was observed during the chronic phase of the infection. Furthermore, a synthetic goldfish hepcidin peptide induced trypanosome lysis in vitro, and parasite surface disruption was confirmed by scanning electron microscopy (SEM) analysis. These results suggest that, in addition to well-characterized direct antibacterial activities, teleost hepcidin also exhibits trypanocidal activity.

Mechanisms of Fish Macrophage Antimicrobial Immunity.

Overcrowding conditions and temperatures shifts regularly manifest in large-scale infections of farmed fish, resulting in economic losses for the global aquaculture industries. Increased understanding of the functional mechanisms of fish antimicrobial host defenses is an important step forward in prevention of pathogen-induced morbidity and mortality in aquaculture setting. Like other vertebrates, macrophage-lineage cells are integral to fish immune responses and for this reason, much of the recent fish immunology research has focused on fish macrophage biology. These studies have revealed notable similarities as well as striking differences in the molecular strategies by which fish and higher vertebrates control their respective macrophage polarization and functionality. In this review, we address the current understanding of the biological mechanisms of teleost macrophage functional heterogeneity and immunity, focusing on the key cytokine regulators that control fish macrophage development and their antimicrobial armamentarium.

Characterization and functional assessment of the NLRC3-like molecule of the goldfish (Carassius auratus L.).

The NLRC3-like (NLRC3L) molecule from the goldfish transcriptome database was identified and characterized. Quantitative gene expression analysis revealed the highest mRNA levels of NLRC3L were in the spleen and intestine, with lower mRNA levels observed in muscle and liver. Goldfish NLRC3L was differentially expressed in goldfish immune cell populations with highest mRNA levels measured in PBLs and macrophages. We generated a recombinant form of the molecule (rgfNLRC3L) and an anti-CT-NLRC3L IgG. Treatment of goldfish primary kidney macrophages in vitro with ATP, LPS and heat-killed Aeromonas salmonicida up-regulated the NLRC3L mRNA and protein. Confocal microscopy and co-immunoprecipitation assays indicated that goldfish rgfNLRC3L interacted with apoptosis-associated spec-like protein (ASC) in eukaryotic cells, indicating that NLRC3L may participate in the regulation of the inflammasome responses. The dual-luciferase reporter assay showed that NLRC3L over-expression did not cause the activation of NF-κB, but that it cooperated with RIP2 to down-regulate NF-κB activation. Our results indicate that the NLRC3L may function as a regulator of NLR pathways in teleosts.