Molecular and ultrastructural characterization of a novel cryptic species of the Mesomycetozoea clade isolated from Manila clam, Ruditapes philippinarum, on the west coast of Korea.

In the present study, a cryptic species (IchX) was isolated from the hemolymph of the Manila clam, Ruditapes philippinarum, collected from the west coast region of South Korea. Following comprehensive molecular analysis, a partial sequence resembling the small subunit of the ribosomal RNA (SSU rRNA) gene was obtained, indicating that this species belonged to the class Mesomycetozoea, also known as Ichthyosporea. Detailed phylogenetic analyses based on SSU rRNA sequences placed IchX in a distinct clade within the order Dermocystida, class Mesomycetozoea, and showed that IchX is closely related to Ichthyosporea sp. Microscopic examination of in vitro cultured IchX cells revealed life-cycle stages of different sizes, from the endospore to sporangium through vegetative stages. An ameboid-like structure was observed in the early endospore stages as the characteristic feature of zoospores. Ultrastructural analyses using scanning electron microscopy revealed that all endospores and vegetative cell stages are spherical. Transmission electron microscopy revealed characteristic features, including a spindle pole body and membrane-decorated hyaline vesicles, consistent with those previously described in Mesomycetozoea. In addition, a prominent fibrillar structure was observed. Notably, the cell wall of mature IchX sporangia was digested with 2 M NaOH, while that of the endospores was resistant. This is the first report of a novel Mesomycetozoean from the Manila clams. Further taxonomic study of this organism and elucidation of its pathological characteristics are necessary.

First report of Perkinsus marinus occurrence associated with wild Pacific oysters Crassostrea gigas from the west coast of Korea.

This study reports the occurrence of Perkinsus marinus associated with wild Pacific oyster (Crassostrea gigas) specimens collected along the west coast of Korea. Confirmation of P. marinus presence was achieved by conventional PCR using World Organization of Animal Health (WOAH)-recommended primers that specifically targeted regions of the rDNA locus (ITS1, 5.8S, and ITS2). Sequencing of 10 samples revealed two distinct sequences differing by a single base pair, indicating potential haplotype variability. One sequence closely resembled the P. marinus strain found in Maryland, USA, whereas the other exhibited divergence, indicative of species diversity in the Korean strain, as was evident from the haplotype network analysis. Further validation involved the Ray's Fluid Thioglycollate Medium (RFTM) assay, which initially yielded inconclusive results, possibly due to low infection intensity. Subsequently, RFTM and 2 M NaOH assays conducted on the isolates in the present study, cultured P. marinus cells in standard DMEM/F12 medium, and a positive P. marinus strain (ATCC 50509), revealed characteristic hypnospores of P. marinus upon Lugol's iodine staining. These comprehensive investigations underscore the conclusive confirmation of P. marinus in Korean waters and mark a significant milestone in our understanding of the distribution and characteristics of this parasite in previously unreported regions.

Dataset of De novo leaf, salicylic induced leaf and flower transcriptome profiling of Datura metel plant.

Datura metel L (thorn's apple) is a popular plant belonging to the family Solanaceae, growing all around the year in humid and warm climates. The importance of D. metel as a medicinal marvel is due to secondary metabolites within various parts of the plant, which serve different therapeutic functions. The whole plant is considered a narcotic, anodyne, and antispasmodic, while the leaves, bark, and seeds are also separately used in extractions. The biological potency of the plant has been used in traditional medicine for over a century. Currently, plant parts are used as a rich source in pharmaceutical manufacturing of secondary metabolites such as flavonoids, saponins, alkaloids, steroids, tannins, and withanaloids. D. metel has proven advanced functions of antiviral effects, antibacterial and antifungal effects, anti-inflammatory, analgesic, antipyretic, hepatoprotective, nephroprotective effect, anticancer, and to treat chronic cardiovascular diseases, diabetes, and neurological ailments. This is the first report on transcriptome assembly for this plant. The raw RNA sequencing data for leaf, salicylic-induced leaf, and flower are available at the NCBI Sequence Read Archive (SRA) under the Bioproject access PRJNA838784. The raw RNA sequencing data that is currently accessible can be utilized to conduct differential gene expression investigations pertaining to various secondary metabolite pathways and diverse tissues, as well as for the research of gene expression related to stress induced by salicylic acid in leaf tissues of the plant. Gene functions can be evaluated and mostly utilized for gene clustering data analysis, gene characterizations, and the identification of genes involved in linked biological pathways in plant studies.

Molecular expression analysis and characterization of rockfish (Sebastes schlegelii) B cell activating factor.

B cell activating factor (BAFF) is recognized as a member of the TNF superfamily proteins that mediate the immune responses. In this study, BAFF from rockfish (Sebastes schlegelii) (SsBAFF) was characterized based on its functional aspects. The open reading frame of SsBAFF is 804 bp in length and encodes a 267 long amino acid residue protein with predicted molecular weight of 29.48 kDa. The deduced protein sequence comprises with transmembrane domain, furin cleavage site and TNF domain carrying Flap binding site that unique to TNF family. Recombinant SsBAFF (rSsBAFF) significantly enhanced rockfish lymphocytes proliferation and viability in a concentration dependent-manner according to the results from water soluble tetrazolium salt (WST-1) assay and flow cytometric assay. rSsBAFF also modulated the expression of genes involved in anti-inflammatory (IL-10 and NFκB-2) and anti-apoptotic (Bcl-2 and Bax) signal pathways. SsBAFF mRNA expression was detected ubiquitously in all analyzed rockfish tissues, with the highest levels in the spleen and head kidney. Further, the expression of SsBAFF in spleen were significantly induced following LPS, poly (I:C) and Streptococcus iniae challenges. These findings strongly suggest that SsBAFF might play an important role in rockfish immune system through regulating the inflammatory response and proliferation of immune cells.

Structural and functional analysis of three Iκb kinases (IKK) in disk abalone (Haliotis discus discus): Investigating their role in the innate immune responses.

The IκB kinases (IKK) are large multiprotein complexes that regulate the activation of the transcription factor NF-κB and are involved in a diverse range of biological processes, including innate immunity, inflammation, and development. To explore the potential roles of invertebrate IKKs on immunity, three IKK encoding genes have been identified from molluscan species disk abalone and designed as AbIKK1, AbIKK2 and AbIKK3 at the transcriptional level. Coding sequences of AbIKK1, AbIKK2 and AbIKK3 encode the peptides of 746, 751 and 713 amino acids with the predicted molecular mass of 86.16, 86.12 and 81.88 kDa respectively. All three AbIKKs were found to share conserved IKK family features including the kinase superfamily domain (KD), ubiquitin-like domain (ULD), and α-helical scaffold/dimerization domain (SDD), similar to their mammalian counterparts. Under normal physiological conditions, AbIKKs were ubiquitously detected in six different tissues, with the highest abundance in the digestive tract and gills. Temporal transcriptional profiles in abalone hemocytes revealed the induction of AbIKK1, AbIKK2, and AbIKK3 expression following exposure to Gram-negative (Vibrio parahemolyticus) and Gram-positive (Listeria monocytogenes) bacteria, viruses (viral hemorrhagic septicemia virus, VHSV), LPS, or poly I:C. The overexpression of AbIKKs in HEK293T or RAW264.7 murine macrophage cells induced NF-κB promoter activation independent of stimulation by TNF-α or LPS. Moreover, iNOS and COX2 expression was induced in AbIKK transfected RAW264.7 murine macrophage cells and the induced state was maintained post-LPS treatment. Furthermore, mRNA levels of three selected cytokine-encoding genes (IL-1ß, IL-6, and TNF-α) were found to be elevated in abalone IKK overexpressed RAW264.7 murine macrophage cells, both with and without LPS exposure. Overall, our findings demonstrated that AbIKKs identified in this study were positively involved in eliciting innate immune responses in abalone. In addition, the data revealed the presence of an evolutionarily conserved signaling mechanism for IKK mediated NF-κB activation in mollusks.

Transcriptome-wide identification, functional characterization, and expression analysis of two novel invertebrate-type Toll-like receptors from disk abalone (Haliotis discus discus).

Toll-like receptors (TLRs) are well-known pattern recognition receptors that play key immunological roles in a diverse range of organisms. In this study, two novel invertebrate TLRs from disk abalone (designated as AbTLR-A and AbTLR-B) were identified and functionally characterized for the first time. AbTLR-A and AbTLR-B comprised the typical TLR domain architecture containing an extracellular leucine-rich repeat domain, transmembrane domain, and Toll/interleukin-1 receptor domain. Expressional analysis revealed that both TLRs were constitutively expressed at all the early embryonic stages of disk abalone analyzed, with the highest level of AbTLR-A found at the 16-cell stage and AbTLR-B at the trochophore stage. According to tissue distribution analysis, prominent mRNA expression of AbTLR-A and AbTLR-B was detected in the hemocytes and gills, respectively. AbTLR-A and AbTLR-B mRNAs were significantly up-regulated in response to Gram-negative Vibrio parahemolyticus, Gram-positive Listeria monocytogenes, and viral hemorrhagic septicemia virus injections in abalone hemocytes and gills. Overexpression of AbTLR-A and AbTLR-B in HEK293T cells directly activated nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) responsive reporters. Neither TLRs showed a high response to pathogen-associated molecular patterns in vitro. Co-expression of AbTLR-A and AbTLR-B with AbMyD88-2 and AbMyD88-X activated NF-κB-responsive reporters in a synergetic manner. These findings demonstrate the involvement of AbTLR-A and AbTLR-B in abalone innate immunity.

Molecular characterization and expression analysis of big-belly seahorse (Hippocampus abdominalis) interleukin-10 and analysis of its potent anti-inflammatory properties in LPS-induced murine macrophage RAW 264.7 cells.

Interleukin-10 (IL-10) is a pleiotropic cytokine involved in the regulation of innate and adaptive immunity. In this study, IL-10 from big-belly seahorse (Hippocampus abdominalis) (HaIL-10) was characterized based on its molecular and functional aspects. The coding sequence of HaIL-10 is 570 bp in length and encodes a 189-amino acid residue protein (calculated molecular weight, 21.89 kDa). The deduced amino acid sequence comprises a typical signal peptide and a mature peptide domain sequence carrying four conserved Cys residues and two additional Cys residues specific to fish. Phylogenetic analysis indicated an evolutionary relationship between HaIL-10 and its counterparts in other vertebrates, with close clustering to the fish-specific homologs. Recombinant HaIL-10 (rHaIL-10) significantly reduced nitric oxide (NO) production by lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells in a concentration-dependent manner but had no effect on cell viability, suggestive of its involvement in immune response. The protein expressions of iNOS and COX-2 were significantly reduced by rHaIL-10 in LPS-induced murine macrophages RAW 264.7 cells. HaIL-10 mRNA expression was observed in all analyzed tissues, with the maximum expression being noted in the kidney and ovary. However, transcriptional levels of HaIL-10 were significantly higher in the blood, gill, and intestine upon in vivo induction with LPS, polyinosinic:polycytidylic acid [poly (I:C)], and Streptococcus iniae. To summarize, our findings help in the improved understanding of the biological functions of HaIL-10 and modulation of HaIL-10 mRNA expression in response to immune stress.

Transcriptional profiling, molecular cloning, and functional analysis of C1 inhibitor, the main regulator of the complement system in black rockfish, Sebastes schlegelii.

C1-inhibitor (C1inh) plays a crucial role in assuring homeostasis and is the central regulator of the complement activation involved in immunity and inflammation. A C1-inhibitor gene from Sebastes schlegelii was identified and designated as SsC1inh. The identified genomic DNA and cDNA sequences were 6837 bp and 2161 bp, respectively. The genomic DNA possessed 11 exons, interrupted by 10 introns. The amino acid sequence possessed two immunoglobulin-like domains and a serpin domain. Multiple sequence alignment revealed that the serpin domain of SsC1inh was highly conserved among analyzed species where the two immunoglobulin-like domains showed divergence. The distinctiveness of teleost C1inh from other homologs was indicated by the phylogenetic analysis, genomic DNA organization, and their extended N-terminal amino acid sequences. Under normal physiological conditions, SsC1inh mRNA was most expressed in the liver, followed by the gills. The involvement of SsC1inh in homeostasis was demonstrated by modulated transcription profiles in the liver and spleen upon pathogenic stress by different immune stimulants. The protease inhibitory potential of recombinant SsC1inh (rSsC1inh) and the potentiation effect of heparin on rSsC1inh was demonstrated against C1esterase and thrombin. For the first time, the anti-protease activity of the teleost C1inh against its natural substrates C1r and C1s was proved in this study. The protease assay conducted with recombinant black rockfish C1r and C1s proteins in the presence or absence of rSsC1inh showed that the activities of both proteases were significantly diminished by rSsC1inh. Taken together, results from the present study indicate that SsC1inh actively plays a significant role in maintaining homeostasis in the immune system of black rock fish.

A comparative study of three akirin genes from big belly seahorse Hippocampus abdominalis: Molecular, transcriptional and functional characterization.

Akirins, members of the NF-κB signaling pathway, are highly conserved nuclear proteins, which regulate gene expression in many physiological processes, including immunity, myogenesis, carcinogenesis, and embryogenesis. The akirin family in teleost fish consists of two to three genes. In the present study, three akirin genes from Hippocampus abdominalis were identified from a transcriptome database and designated as HaAkirin1, HaAkirin2(1), and HaAkirin2(2). The nuclear localization of HaAkirin1 and HaAkirin2(1) was confirmed by subcellular localization analysis. In contrast, diffused localization of HaAkirin2(2) was identified in the nucleus and cytoplasm that confirmed the aberrant nature of the nuclear localization signal. Phylogenetic analysis revealed a closer relationship of HaAkirins with other known teleost akirins. All three HaAkirin transcripts were ubiquitously expressed in all examined tissues with higher expression in ovary tissue. Immune challenge with LPS, poly I:C, and Streptococcus iniae exhibited a significant increase in the expression of all three HaAkirins in kidney and liver tissues. NF-κB luciferase assays revealed that relative luciferase activity was significantly higher for all three HaAkirin genes than mock controls. These results suggest that HaAkirin genes might play a role in regulating NF-κB dependent immune gene expression and their expression could be induced by bacterial and viral pathogen recognition molecular patterns.

Characterization of a Kazal-type serine protease inhibitor from black rockfish Sebastes schlegelii and its possible role in hepatic immune response.

Kazal-type serine protease inhibitors (KSPIs) play important roles in the regulation of endogenous proteases, cell development, blood coagulation, and immune response. In this study, we identified and characterized a KSPI homologue (SsKSPI) in black rockfish, Sebastes schlegelii. The full-length cDNA sequence of SsKSPI was 532 base pairs (bp), including an open reading frame (ORF) of 330 bp, which encodes a polypeptide of 110 amino acids with a signal peptide of 21 amino acids. The greatest value for identity (42.9%) and similarity (50.9%) was observed with Channa striata KSPI. We purified the recombinant protein of SsKSPI and performed protease inhibitory assays using three common serine proteases. The recombinant SsKSPI exhibited specific inhibitory activity against subtilisin A in a dose-dependent manner. Tissue distribution of SsKSPI mRNA has been examined amongst 10 important tissues in healthy rockfish and the liver was found to be the predominant expression organ of SsKSPI. The modulation of SsKSPI expression under immune challenges was also investigated in the liver. The SsKSPI mRNA expression was significantly up-regulated in response to both bacterial (Streptococcus iniae and lipopolysaccharide) and viral (polyinosinic:polycytidylic acid) challenges. Overall, we propose that SsKSPI is potentially involved in the hepatic immune response against bacterial and viral infections in black rockfish.

Molecular features and the transcriptional and functional delineation of complement system activators C1r and C1s from Sebastes schlegelii.

C1r and C1s are serine proteases responsible for activating the classical complement pathway to initiate the complement cascade, which plays a crucial role in eliminating invading pathogenic microbes. In this study, cDNA sequences of C1r and C1s were identified from black rockfish and designated as SsC1r and SsC1s, respectively. In both sequences, two CUB domains, an EGF-like domain, two CCP domains, and a trypsin-like serine protease domain were identified. Multiple sequence alignments with known vertebrate homologs demonstrated that both sequences were highly conserved and, especially, the catalytic and substrate binding residues were completely conserved. In the constructed phylogenetic tree, C1r and C1s formed two separate clusters, which further branched into groups of related organisms. SsC1r and SsC1s joined with their respective teleostean clusters. Transcriptional analysis showed that the highest mRNA expression level was in the liver under normal physiological conditions. Significantly upregulated expression of both mRNAs in spleen and liver after pathologic stress, by intraperitoneal injection with different stimuli, suggested their vital role in immunity. The serine protease domains of SsC1r and SsC1s were cloned and the recombinant proteins were expressed and purified. A protease assay, conducted to confirm their functionality, indicated that both recombinant proteins had proteolytic activity. Taken together, these results indicate that SsC1r and SsC1s have significant properties to aid in the immunity of black rockfish by activating the complement system by proteolytic cleavage.

Molecular identification and functional analysis of two variants of myeloid differentiation factor 88 (MyD88) from disk abalone (Haliotis discus discus).

Myeloid differentiation factor 88 (MyD88) is a crucial adaptor protein of the Toll-like receptor (TLR)- and interleukin 1 receptor-mediated signaling pathways and is involved in a diverse array of inflammatory responses via NF-κB activation. In the present study, two MyD88 variants were identified from disk abalone (Haliotis discus discus) and designated AbMyD88-2 and AbMyD88-X. The deduced AbMyD88-2 and AbMyD88-X comprised 433 and 354 amino acids with predicted molecular masses of 48.85 kDa and 40.17 kDa, respectively. AbMyD88-2 and AbMyD88-X possessed typical MyD88 domain structural features including an N-terminal death domain (DD) and C-terminal toll interleukin 1 receptor (TIR) domain similar to those in mammals. Expression analysis of AbMyD88-2 and AbMyD88-X mRNA at different early embryonic developmental stages of abalone by qPCR revealed that their constitutive expression at all developmental stages analyzed with the considerably higher values at the 16-cell (AbMyD88-2) and morula stages (AbMyD88-X). In unchallenged disk abalones, AbMyD88-2 was highly expressed in muscles, while AbMyD88-X mRNA was predominantly transcribed in hemocytes. Moreover, AbMyD88-2 and AbMyD88-X mRNA were differentially modulated in abalone hemocytes after a challenge with live bacteria (Vibrio parahaemolyticus, Listeria monocytogenes), virus (viral hemorrhagic septicemia virus), and pathogen-associated molecular patterns (lipopolysaccharides and Poly I:C). Overexpression of AbMyD88-2 and AbMyD88-X in HEK293T cells induced the activation of the NF-κB promoter. AbMyD88-2 and AbMyD88-X involvement in inflammatory responses was characterized by their overexpression in RAW264.7 murine macrophage cells. These results revealed comparatively higher NO (Nitric oxide) production, induction of inflammatory mediator genes (iNOS and COX2), and proinflammatory genes (IL1ß, IL6 and TNFα) expression in abalone MyD88s-overexpressing cells than in mock control in the presence or absence of LPS stimulation. Altogether, these results suggest that existence of a MyD88-dependent like signaling pathway in disk abalone and that both AbMyD88-2 and AbMyD88-X might be involved in innate immune and inflammatory responses.

Identification and characterization of molluscan caveolin-1 ortholog from Haliotis discus discus: Possible involvement in embryogenesis and host defense mechanism against pathogenic stress.

Caveolins are principal membrane proteins of caveolae that play a central role in signal transduction, substrate transport, and membrane trafficking in various cell types. Numerous studies have reported the crucial role of caveolin-1 (CAV1) in response to invading microbes; yet, very little is known about molluscan CAV1. In this study, we identified and characterized CAV1 ortholog from the disk abalone, Haliotis discus discus (HdCAV1). The cDNA sequence of HdCAV1 is 826 bp long and encodes a 127-amino acid polypeptide. Characteristic caveolin superfamily domain (Glu3 - Lys126) and two possible transmembrane domains (Cys48 - Tyr67 and Ile103 - Phe120) were identified in the HdCAV1 protein. Homology analysis revealed that HdCAV1 shared higher identity (>47%) with molluscans, but lower identity with other species. Phylogenetic tree constructed by the neighbor-joining (NJ) method revealed a distinct evolutionary pathway for molluscans. Transcriptional analysis by SYBR Green qPCR showed the highest expression of HdCAV1 mRNA in late veliger stage, as compared to that in other embryonic developmental stages of disk abalone. In adult animals, gill tissue showed highest HdCAV1 transcript levels under normal physiological condition. Stimulations with two bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), viral hemorrhagic septicemia virus, and two pathogen-associated molecular patterns (LPS and poly I:C) significantly modulated the expression of HdCAV1 transcripts. Collectively, these data suggest that CAV1 plays an important role in embryogenesis and host immune defense in disk abalone.

Molecular cloning, biochemical characterization, and expression analysis of two glutathione S-transferase paralogs from the big-belly seahorse (Hippocampus abdominalis).

Glutathione S-transferases (GSTs, EC 2.5.1.18) are important Phase II detoxifying enzymes that catalyze hydrophobic, electrophilic xenobiotic substance with the conjugation of reduced glutathione (GSH). In this study, GSTµ and GSTρ paralogs of GST in the big belly seahorse (Hippocampus abdominalis; HaGSTρ, HaGSTµ) were biochemically, molecularly, functionally characterized to determine their detoxification range and protective capacities upon different pathogenic stresses. HaGSTρ and HaGSTµ are composed of coding sequences of 681bp and 654bp, which encode proteins 225 and 217 amino acids, with predicted molecular masses of 26.06kDa and 25.74kDa respectively. Sequence analysis revealed that both HaGSTs comprise the characteristic GSH-binding site in the thioredoxin-like N-terminal domain and substrate binding site in the C-terminal domain. The recombinant HaGSTρ and HaGSTµ proteins catalyzed the model GST substrate 1-chloro-2, 4-dinitrobenzene (CDNB). Enzyme kinetic analysis revealed different Km and Vmax values for each rHaGST, suggesting that they have different conjugation rates. The optimum conditions (pH, temperature) and inhibitory assays of each protein demonstrated different optimal ranges. However, HaGSTµ was highly expressed in the ovary and gill, whereas HaGSTρ was highly expressed in the gill and pouch. mRNA expression of HaGSTρ and HaGSTµ was significantly elevated upon lipopolysaccharide, Poly (I:C), and Edwardsiella tarda challenges in liver and in blood cells as well as with Streptococcus iniae challenge in blood cells. From these collective experimental results, we propose that HaGSTρ and HaGSTµ are effective in detoxifying xenobiotic toxic agents, and importantly, their mRNA expression could be stimulated by immunological stress signals in the aquatic environment.

Molecular identification of disk abalone (Haliotis discus discus) tetraspanin 33 and CD63: Insights into potent players in the disk abalone host defense system.

Tetraspanins are a superfamily of transmembrane proteins involved in a diverse range of physiological processes including differentiation, adhesion, signal transduction, cell motility, and immune responses. In the present study, two tetraspanins, CD63 and tetraspanin 33 (TSPAN33) from disk abalone (AbCD63 and AbTSPAN33), were identified and characterized at the molecular level. The coding sequences for AbCD63 and AbTSPAN33 encoded polypeptides of 234 and 290 amino acids (aa) with predicted molecular mass of 25.3 and 32.5 kDa, respectively. The deduced AbCD63 and AbTSPAN33 protein sequences were also predicted to have a typical tetraspanin domain architecture, including four transmembrane domains (TM), short N- and C- terminal regions, a short intracellular loop, as well as a large and small extracellular loop. A characteristic CCG motif and cysteine residues, which are highly conserved across CD63 and TSPAN33 proteins of different species, were present in the large extracellular loop of both abalone tetraspanins. Phylogenetic analysis revealed that the AbCD63 and AbTSPAN33 clustered in the invertebrate subclade of tetraspanins, thus exhibiting a close relationship with tetraspanins of other mollusks. The AbCD63 and AbTSPAN33 mRNA transcripts were detected at early embryonic development stages of disk abalone with significantly higher amounts at the trochophore stage, suggesting the involvement of these proteins in embryonic development. Both AbCD63 and AbTSPAN33 were ubiquitously expressed in all the tissues of unchallenged abalones analyzed, with the highest expression levels found in hemocytes. Moreover, significant induction of AbCD63 and AbTSPAN33 mRNA expression was observed in immunologically important tissues, such as hemocytes and gills, upon stimulation with live bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), virus (viral hemorrhagic septicemia virus), and two potent immune stimulators [polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS)]. Collectively, these findings suggest that AbCD63 and AbTSPAN33 are involved in innate immune responses in disk abalone during pathogenic stress.

Molecular insights of two STAT1 variants from rock bream (Oplegnathus fasciatus) and their transcriptional regulation in response to pathogenic stress, interleukin-10, and tissue injury.

Signal transducers and activators of transcription 1 (STAT1) is critically involved in mediating cytokine-driven signaling, and triggers the transcription of target genes to activate cellular functions. Although the structural and functional aspects of STAT members have been well described in mammals, only limited information is available for the STAT genes in teleost fishes. In the present study, two variants of STAT1 genes (RbSTAT1 and RbSTAT1L) were identified from rock bream and characterized at the cDNA and genomic sequence levels. RbSTAT1 and RbSTAT1L were found to share a common domain architecture with mammalian STAT1. Phylogenetic analysis revealed that RbSTAT1 shows a common evolutionary trajectory with other STAT1 counterparts, whereas RbSTAT1L showed a separate path, implying that it could be a novel member of the STAT family. The genomic organizations of RbSTAT1 and RbSTAT1L illustrated a similar exon-intron pattern with 23 exons in the coding sequence. Transcription factor-binding sites, which are mostly involved in the regulation of immune responses, were predicted at the putative promoter regions of the RbSTAT1 and RbSTAT1L genes. SYBR Green qPCR analysis revealed the ubiquitous expression of RbSTAT1 and RbSTAT1L transcripts in different fish tissues with the highest level observed in peripheral blood cells. Significantly modulated transcripts were noted upon viral (rock bream iridovirus [RBIV]), bacterial (Edwardsiella tarda and Streptococcus iniae), and pathogen-associated molecular pattern (lipopolysaccharide and poly I:C) stimulations. The WST-1 cell viability assay affirmed the potential antiviral capacity of RbSTAT1 and RbSTAT1L against RBIV. A possible role of RbSTAT1 and RbSTAT1L in the wound healing process was revealed according to their modulated expression in injured fish. In addition, the transcriptional regulation of RbSTAT1 and RbSTAT1L was analyzed by qPCR following stimulation with rock bream interleukin-10. Taken together, these findings suggest that the STAT1-mediated Janus kinase/STAT pathway might at least in part be involved in the regulatory mechanisms underlying the immune defensive roles against microbial pathogens and the wound healing process.

Molecular cloning, transcriptional profiling, and subcellular localization of signal transducer and activator of transcription 2 (STAT2) ortholog from rock bream, Oplegnathus fasciatus.

Signal transducer and activator of transcription 2 (STAT2) is a key element that transduces signals from the cell membrane to the nucleus via the type I interferon-signaling pathway. Although the structural and functional aspects of STAT proteins are well studied in mammals, information on teleostean STATs is very limited. In this study, a STAT paralog, which is highly homologous to the STAT2 members, was identified from a commercially important fish species called rock bream and designated as RbSTAT2. The RbSTAT2 gene was characterized at complementary DNA (cDNA) and genomic sequence levels, and was found to possess structural features common with its mammalian counterparts. The complete cDNA sequence was distributed into 24 exons in the genomic sequence. The promoter proximal region was analyzed and found to contain potential transcription factor binding sites to regulate the transcription of RbSTAT2. Phylogenetic studies and comparative genomic structure organization revealed the distinguishable evolution for fish and other vertebrate STAT2 orthologs. Transcriptional quantification was performed by SYBR Green quantitative real-time PCR (qPCR) and the ubiquitous expression of RbSTAT2 transcripts was observed in all tissues analyzed from healthy fish, with a remarkably high expression in blood cells. Significantly (P<0.05) altered transcription of RbSTAT2 was detected after immune challenge experiments with viral (rock bream irido virus; RBIV), bacterial (Edwardsiella tarda and Streptococcus iniae), and immune stimulants (poly I:C and LPS). Antiviral potential was further confirmed by WST-1 assay, by measuring the viability of rock bream heart cells treated with RBIV. In addition, results of an in vitro challenge experiment signified the influence of rock bream interleukin-10 (RbIL-10) on transcription of RbSTAT2. Subcellular localization studies by transfection of pEGFP-N1/RbSTAT2 into rock bream heart cells revealed that the RbSTAT2 was usually located in the cytoplasm and translocated near to the nucleus upon poly I:C administration. Altogether, these findings suggest that RbSTAT2 is involved in various biologically crucial mechanisms, and provides immune protection to the rock bream.

A CXCL ortholog from Hippocampus abdominalis: Molecular features and functional delineation as a pro-inflammatory chemokine.

Chemokines are a family of chemotactic cytokines that regulate leukocyte migration. They are classified into four groups namely, CXC, CC, C and CX3C, based on the formation of a disulfide bridge. Among these, CXC chemokines have been identified as the largest group of chemokines in humans. In this study, we identified and functionally characterized a homolog of CXC chemokine from the big-belly seahorse, Hippocampus abdominalis, and designated it as ShCXCL. The cDNA of ShCXCL composed of a 342-bp open reading frame encoding 113 amino acids (aa). The CXC family-specific small cytokine domain (SCY) was identified from the mature peptide region, which comprised of a conserved CXC motif. As ShCXCL lacks an ELR (Glutamic acid-Leucine-Arginine) motif, it belongs to ELR- subfamily. The recombinant ShCXCL protein strongly induced the nitric oxide (NO) production in macrophage cells (RAW 264.7 cell line) and showed the chemotactic effect on flounder peripheral blood leukocytes. Tissue profiling showed a ubiquitous expression pattern in all examined tissues, with a high abundance in spleen. The up-regulated mRNA expression pattern of ShCXCL was observed in blood and kidney tissues after immune stimulation by live bacteria, such as Streptococcus iniae and Edwardsiella tarda, and mitogens, such as lipopolysaccharides (LPS) and polyinosinic:polycytidylic acid (poly I:C), suggesting its important role in host immune defense against microbial infection.

Molecular, transcriptional and functional insights into duplicated goose-type lysozymes from Sebastes schlegelii and their potential immunological role.

Black rockfish (Sebastes schlegelii), an important aquaculture species in Korea, has been affected by bacterial diseases leading to a drastic decline in production. Goose-type lysozyme (LysG) is a key enzyme of the innate immune system to eradicate bacterial infections. In this study, two isoforms of LysG from black rockfish, designated as RfLysG1 and RfLysG2, have been identified and characterized at the molecular, transcriptional, and functional levels. The deduced amino acid sequences had the LysG family characteristics and exhibited conserved properties, including active residues and domains. The cDNA sequences of RfLysG1 and RfLysG2 were 1514 bp and 900 bp in length, respectively. The 567-bp open reading frame (ORF) of RfLysG1 encoded a protein of 188 amino acids with molecular mass 20.11 kDa, and the 600-bp ORF of RfLysG2 encoded a polypeptide with 199 amino acids and molecular mass of 22.19 kDa. Homology studies indicated that RfLysG1 showed the highest identity (84.6%) with LysG-B of Oplegnathus fasciatus, while RfLysG2 showed the highest identity (74.4%) with LysG of Siniperca chuatsi. Both sequences possessed a soluble lytic trans-glycosylase domain. Both lacked signal peptide and they were not identified as proteins secreted by non-classical pathway by the SecretomeP server. Transcriptional analysis of the two genes showed constitutive expression, where both genes were highly expressed in blood under normal physiological conditions. In response to the immune challenges lipopolysaccharide (LPS), Streptococcus iniae, and poly I:C injection, the expression of RfLysG1 and RfLysG2 was significantly upregulated in blood and spleen tissues in a time-dependent manner. Turbidimetric assays indicated that both recombinant proteins tagged with maltose-binding protein (MBP) were reactive against several Gram-positive and Gram-negative bacteria, but MBP was inactive. Optimum temperatures for the recombinant RfLysG1 and RfLysG2 were 40 °C and 50 °C, respectively, and both were highly active at pH 3.0. The results provide evidence for the vital immunological role and bacteriolytic potential of RfLysG1 and RfLysG2.

A homolog of teleostean signal transducer and activator of transcription 3 (STAT3) from rock bream, Oplegnathus fasciatus: Structural insights, transcriptional modulation, and subcellular localization.

Signal transducer and activator of transcription 3 (STAT3) is one of the crucial transcription factors in the Janus kinase (JAK)/STAT signaling pathway, and it was previously considered as acute phase response factor. A number of interleukins (ILs) such as IL-5, IL-6, IL-9, IL-10, IL-12, and IL-22 are known to be involved in activation of STAT3. In addition, various growth factors and pathogenic or oxidative stresses mediate the activation of a wide range of functions via STAT3. In this study, a STAT3 homolog was identified and functionally characterized from rock bream (RbSTAT3), Oplegnathus fasciatus. In silico characterization revealed that the RbSTAT3 amino acid sequence shares highly conserved common domain architectural features including N-terminal domain, coiled coil domain, DNA binding domain, linker domain, and Src homology 2 (SH2) domains. In addition, a fairly conserved transcriptional activation domain (TAD) was located at the C-terminus. Comparison of RbSTAT3 with other counterparts revealed higher identities (>90%) with fish orthologs. The genomic sequence of RbSTAT3 was obtained from a bacterial artificial chromosome (BAC) library, and was identified as a multi-exonic gene (24 exons), as found in other vertebrates. Genomic structural comparison and phylogenetic studies have showed that the evolutionary routes of teleostean and non-teleostean vertebrates were distinct. Quantitative real time PCR (qPCR) analysis revealed that the spatial distribution of RbSTAT3 mRNA expression was ubiquitous and highly detectable in blood, heart, and liver tissues. Transcriptional modulation of RbSTAT3 was examined in blood and liver tissues after challenges with bacteria (Edwardsiella tarda and Streptococcus iniae), rock bream irido virus (RBIV), and immune stimulants (LPS and poly (I:C)). Significant changes in RbSTAT3 transcription were also observed in response to tissue injury. In addition, the transcriptional up-regulation of RbSTAT3 was detected in rock bream heart cells upon recombinant rock bream IL-10 (rRbIL-10) treatment. Subcellular localization and nuclear translocation of rock bream STAT3 following poly (I:C) treatment were also demonstrated. Taken together, the results of the current study provide important evidence for potential roles of rock bream STAT3 in the immune system and wound healing processes.