Hippocampus Abdominalis Research Articles

Molecular characterization of thioredoxin-like protein 1 (TXNL1) from big-belly seahorse Hippocampus abdominalis in response to immune stimulation

Thioredoxin is a highly conserved protein found in both prokaryotes and eukaryotes. Reactive oxygen species (ROS) are produced in response to metabolic processes, radiation, metal oxidation, and pathological infections. High levels of ROS lead to cell death via autophagy. However, thioredoxin acts as an active regulatory enzyme in response to excessive ROS. Here, we performed in-silico analysis, immune challenge experiments, and functional assays of seahorse thioredoxin-like protein 1 (ShTXNL1). Evolutionary identification showed that ShTXNL1 protein belongs to the thioredoxin superfamily comprising 289 amino acids. It possesses an N-terminal active thioredoxin domain and C-terminal proteasome-interacting thioredoxin domain (PITH) of ShTXNL1 which is a component of 26S proteasome and binds to the matrix or cell. Pairwise alignment results showed 99.0% identity and 99.7% similarity with the sequence of Hippocampus species. Conserved thiol-disulfide cysteine residue containing Cys-X-X-Cys motif may be found in the first few amino acids in the second beta sheet starting from the N-terminus. This motif can be discovered in ShTXNL1 as 14CRPC17 and comprised two N-linked glycosylation sites at 72NISA75 and 139NESD142. According to the quantitative real-time polymerase chain reaction analysis from healthy seahorses, highest ShTXNL1 mRNA expression was observed in muscle, followed by ovary, brain, gill, and blood tissues. Moreover, significant temporal expression of ShTXNL1 was observed in gill and blood tissues after bacterial stimuli. Thus, the ShTXNL1 gene may be identified as an immunologically important gene in seahorse.

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.

Effects of sea horse (Hippocampus abdominalis)-derived protein hydrolysate on skeletal muscle development

Effects of sea horse (<i>Hippocampus abdominalis</i>)-derived protein hydrolysate on skeletal muscle development

Insights into the multifunctional role of natural killer enhancing factor-A (NKEF-A/Prx1) in big-belly seahorse (Hippocampus abdominalis): DNA protection, insulin reduction, H2O2 scavenging, and immune modulation activity

Natural killer enhancing factor A (NKEF-A), also known as peroxiredoxin 1 (Prx1), is a well-known antioxidant involved in innate immunity. Although NKEF-A/Prx1 has been studied in different fish species, the present study broadens the knowledge of NKEF-A gene in terms of molecular structure, function, and immune responses in fish species. Hippocampus abdominalis NKEF-A (HaNKEF-A) cDNA encoded a putative protein of 198 amino acids containing a thioredoxin_2 domain, VCP motifs, and three conserved cysteine residues including peroxidatic and resolving cysteines. Amino acid sequence comparison and phylogenetic breakdown showed the higher sequence identity and closer evolutionary position of HaNKEF-A to those of other fish counterparts. A recombinant protein of HaNKEF-A was shown to i) protect supercoiled DNA against mixed catalyzed oxidation, ii) reduce insulin disulfide bonds, and iii) scavenge extracellular H2O2. Results of in vitro assays demonstrated the concentration dependent antioxidant function of recombinant HaNKEF-A. In addition, qPCR assessments revealed that the HaNKEF-A transcripts were constitutively expressed in fourteen tissues with the highest expression in liver. As an innate immune response, HaNKEF-A transcripts were up-regulated in liver post injection of LPS, Edwardsiella tarda, Streptococcus iniae, and polyinosinic-polycytidylic acid. Thus, HaNKEF-A can safeguards big-belly seahorse from oxidative damage and pathogenic infections. This study provides insight into the functions of NKEF-A/Prx1 in fish species.

Morphology of brood pouch formation in the pot-bellied seahorse Hippocampus abdominalis

BackgroundThe reproductive strategies of vertebrates are diverse. Seahorses (Pisces: Syngnathidae) possess the unique characteristic of male pregnancy; i.e., males, not females, incubate embryos in a specialized structure called a ‘brood pouch’. The brood pouch is formed along the ventral midline of the tail. The lumen of the brood pouch is surrounded by loose connective tissue, called pseudoplacenta, and dermis.ResultsWe visualized and evaluated the morphology of brood pouch formation in Hippocampus abdominalis to gain generalizable insights into this process in seahorses. First, we employed several staining methods to characterize the pseudoplacenta and dermis of the brood pouch of mature male seahorses. The pseudoplacenta is composed mainly of reticular fibers, while the dermis is composed mainly of collagenous fibers. Further observations showed that pouch formation is initiated by linear projections of epithelia on both ventrolateral sides of the body. These projections elongated toward the ventral midline, eventually fused together, and then formed a baggy structure composed of a single dermis layer with neither smooth muscle nor pseudoplacenta. Finally, the pseudoplacenta was formed, together with two layers of dermis and smooth muscle. Thus, a fully developed brood pouch was established. The morphology of the luminal epithelium also changed during pouch formation. We analyzed the localization of C-type lectins as markers; haCTL II was localized in both the outer and luminal epithelia of the brood pouch throughout development in the male seahorse, whereas haCTL IV, which was not detected in the early stage of seahorse development, became localized only in the luminal epithelium as development proceeded.ConclusionsWe categorized the processes of brood pouch formation during male seahorse development into three stages: (1) the early stage, characterized by formation of a baggy structure from the primordium; (2) the middle stage, characterized by the differentiation and establishment of brood pouch-specific tissues; and (3) the late stage, characterized by a fully formed pouch with developing blood vessels and a pouch fold ultimately capable of carrying and incubating embryos.

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.

Identification and molecular profiling of DC-SIGN-like from big belly seahorse (Hippocampus abdominalis) inferring its potential relevancy in host immunity

Dendritic-cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) is a C-type lectin that functions as a pattern recognition receptor by recognizing pathogen-associated molecular patterns (PAMPs). It is also involved in various events of the dendritic cell (DC) life cycle, such as DC migration, antigen capture and presentation, and T cell priming. In this study, a DC-SIGN-like gene from the big belly seahorse Hippocampus abdominalis (designated as ShDCS-like) was identified and molecularly characterized. The putative, complete ORF was found to be 1368 bp in length, encoding a protein of 462 amino acids with a molecular mass of 52.6 kDa and a theoretical isoelectric point of 8.26. The deduced amino acid sequence contains a single carbohydrate recognition domain (CRD), in which six conserved cysteine residues and two Ca2+-binding site motifs (QPN, WND) were identified. Based on pairwise sequence analysis, ShDCS-like exhibits the highest amino acid identity (94.6%) and similarity (97.4%) with DC-SIGN-like counterpart from tiger tail seahorse Hippocampus comes. Quantitative real-time PCR revealed that ShDCS-like mRNA is transcribed universally in all tissues examined, but with abundance in kidney and gill tissues. The basal mRNA expression of ShDCS-like was modulated in blood cell, kidney, gill and liver tissues in response to the stimulation of healthy fish with lipopolysaccharides (LPS), Edwardsiella tarda, or Streptococcus iniae. Moreover, recombinant ShDCS-like-CRD domain exhibited detectable agglutination activity against different bacteria. Collectively, these results suggest that ShDCS-like may potentially involve in immune function in big belly seahorses.

Manganese-superoxide dismutase (MnSOD), a role player in seahorse (Hippocampus abdominalis) antioxidant defense system and adaptive immune system

Manganese superoxide dismutase (MnSOD) is a metaloenzyme that catalyzes dismutation of the hazardous superoxide radicals into less hazardous H2O2 and H2O. Here, we identified a homolog of MnSOD from big belly seahorse (Hippocampus abdominalis; HaMnSOD) and characterized its structural and functional features. HaMnSOD transcript possessed an open reading frame (ORF) of 672 bp which codes for a peptide of 223 amino acids. Pairwise alignment showed that HaMnSOD shared highest identity with rock bream MnSOD. Results of the phylogenetic analysis of HaMnSOD revealed a close proximity with rock bream MnSOD which was consistent with the result of homology alignment. The intense expression of HaMnSOD was observed in the ovary, followed by the heart and the brain. Further, immune related responses of HaMnSOD towards pathogenic stimulation were observed through bacterial and viral challenges. Highest HaMnSOD expression in response to stimulants Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide (LPS), and polyinosinic-polycytidylic acid (Poly I:C) was observed in the late stage in the blood tissue. Xanthine/xanthine oxidase assay (XOD assay) indicated the ROS-scavenging ability of purified recombinant HaMnSOD (rHaMnSOD). The optimum conditions for the SOD activity of rHaMnSOD were pH 9 and the 25 °C. Collectively, the results obtained through the expressional analysis profiles and the functional assays provide insights into potential immune related and antioxidant roles of HaMnSOD in the big belly seahorse.

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.

Acoustic characterization of sound production by the Pot-Bellied Seahorse (Hippocampus abdominalis)

Sound production is a critical component of predator-prey interactions. In order to understand why seahorses produce sound in various instances (i.e., courtship, feeding, and stress), we must first quantify the acoustic parameters of the signal. Seahorses produce sound with a stridulation of the supraoccipital bone and the coronet by moving their head upwards in a motion that is referred to as a “snick.” The acoustic signal that accompanies this head movement is called a “click.” We set out to analyze the sound parameters of the largest seahorse species, Hippocampus abdominalis, housed at the Newport Aquarium. Adult and juveniles were tested each individually and allowed one hour to acclimate to an isolated tank. Feeding on brine shrimp (Genus: Artemia) was observed with video and audio recordings that were collected for approximately 12 minutes. SpectraPLUS was used to evaluate the frequency, intensity, and number of clicks present in the audio recordings. The video footage allowed for analysis of the presence of the snick. By characterizing the sound production in this species of seahorse we are able to begin to answer the question of the purpose for the click and snick behavior.

Two distinct CXC chemokine receptors (CXCR3 and CXCR4) from the big-belly seahorse Hippocampus abdominalis: Molecular perspectives and immune defensive role upon pathogenic stress

CXC chemokine receptor 3 (CXCR3) and 4 (CXCR4) are members of the seven transmembrane G protein coupled receptor family, involved in pivotal physiological functions. In this study, seahorse CXCR3 and CXCR4 (designated as HaCXCR3 and HaCXCR4) cDNA sequences were identified from the transcriptome library and subsequently molecularly characterized. HaCXCR3 and HaCXCR4 encoded 363 and 373 amino acid long polypeptides, respectively. The HaCXCR3 and HaCXCR4 deduced proteins have typical structural features of chemokine receptors, including seven transmembrane domains and a G protein coupled receptors family 1 profile with characteristic DRY motifs. Amino acid sequence comparison and phylogenetic analysis of these two CXC chemokine receptors revealed a close relationship to their corresponding teleost counterparts. Quantitative real time PCR analysis revealed that HaCXCR3 and HaCXCR4 were ubiquitously expressed in all the tested tissues, with highest expression levels in blood cells. The seahorse blood cells and kidney HaCXCR3 and HaCXCR4 mRNA expressions were differently modulated when challenged with Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide, and polyinosinic:polycytidylic acid, confirming their involvement in post immune responses.

Anti-fatigue activity of a mixture of seahorse (Hippocampus abdominalis) hydrolysate and red ginseng

Seahorse, a syngnathidae fish, is one of the important organisms used in Chinese traditional medicine. Hippocampus abdominalis, a seahorse species successfully cultured in Korea, was validated for use in food by the Ministry of Food and Drug Safety in February 2016; however. the validation was restricted to 50% of the entire composition. Therefore, to use H. abdominalis as a food ingredient, H. abdominalis has to be prepared as a mixture by adding other materials. In this study, the effect of H. abdominalis on muscles was investigated to scientifically verify its potential bioactivity. In addition, the anti-fatigue activity of a mixture comprising H. abdominalis and red ginseng (RG) was evaluated to commercially utilize H. abdominalis in food industry. H. abdominalis was hydrolyzed using Alcalase, a protease, and the effect of H. abdominalis hydrolysate (HH) on the muscles was assessed in C2C12 myoblasts by measuring cell proliferation and glycogen content. In addition, the mixtures comprising HH and RG were prepared at different percentages of RG to HH (20, 30, 40, 50, 60, 70, and 80% RG), and the anti-fatigue activity of these mixtures against oxidative stress was assessed in C2C12 myoblasts. In C2C12 myoblasts, H2O2-induced oxidative stress caused a decrease in viability and physical fatigue-related biomarkers such as glycogen and ATP contents. However, treatment with RG and HH mixtures increased cell viability and the content of fatigue-related biomarkers. In particular, the 80% RG mixture showed an optimum effect on cell viability and ATP synthesis activity. In this study, all results indicated that HH had anti-fatigue activity at concentrations approved for use in food by the law in Korea. Especially, an 80% RG to HH mixture can be used in food for ameliorating fatigue.

Effects of enzymatic hydrolysate from seahorse Hippocampus abdominalis on testosterone secretion from TM3 Leydig cells and in male mice

Protein hydrolysates, the so-called bioactive peptides, are specific protein fragments that have positive effects on several body functions and may improve human health. Marine organism-derived protein hydrolysates and bioactive peptides have shown to possess many physiological functions. Seahorses, used in traditional medicine, are generally ground to powder form either for direct application or for application following dissolution in warm water. In this present study, we purified, hydrolyzed, and characterized two bioactive peptides (ALC and PEP) obtained from seahorse (Hippocampus abdominalis). The hydrolysates derived from seahorse significantly upregulated the expression of cyclin D and cyclin E and increased cell proliferation and testosterone level in the TM3 mouse Leydig cell line. These findings suggest that the hydrolysates stimulate the proliferation of TM3 cells via the AKT, ERK, and JNK pathways. The decline in the circulating testosterone levels in older men is associated with various adverse health effects. Following daily intake of hydrolysates for 12 weeks, the circulating level of testosterone and the sperm count in mice were measured. We found increased sperm motility (sperm count) as well as an increase in the testosterone level in male mice following a 12-week intake of hydrolysates derived from H. abdominalis. Hence, it can be suggested that seahorse-derived hydrolysates play an important role in improving male health by improving the serum testosterone level.

Molecular and functional characterization of caspase-8 from the big-belly seahorse (Hippocampus abdominalis)

Apoptosis is a physiological process that can also participate in host immune defense mechanisms, including tumor growth suppression along with homeostasis and maturation of immune cells. Caspases are known to be involved in cellular apoptotic signaling; among them, caspase-8 plays an important role in the initiation phase of the apoptotic death cascade. In the current study, we molecularly characterized a caspase-8 homolog (designated as HaCasp-8) from Hippocampus abdominalis. The HaCasp-8 gene harbors a 1476 bp open reading frame (ORF) that codes for a protein of 492 amino acids (aa) with a predicted molecular mass of 55 kDa. HaCasp-8 houses the typical domain architecture of known initiator caspases, including the death effector domain and the carboxyl-terminal catalytic domain. As expected, phylogenetic analysis reflected a closer evolutionary relationship of HaCasp-8 with its teleostean similitudes. The results of our qPCR assays confirmed the ubiquitous expression of HaCasp-8 in physiologically important tissues examined, with pronounced expression levels in ovary tissues, followed by blood cells. HaCasp-8 expression at the mRNA level was found to be significantly modulated by lipopolysaccharide, polyinosinic:polycytidylic acid, Streptococcus iniae, and Edwardsiella tarda injection. Overexpression of HaCasp-8 could trigger a significant level of cell death in HEK293T cells, suggesting its putative role in cell death. Taken together, our findings suggest that HaCasp-8 is an important component in the caspase cascade, and its expression can be significantly modulated under pathogen stress conditions in the big-belly seahorse.

Molecular characterization, transcriptional profiling, and antibacterial potential of G-type lysozyme from seahorse (Hippocampus abdominalis)

Lysozymes are a family of enzymes that catalyze the hydrolysis of bacterial cell wall, acting as antimicrobial effectors of the innate immune system. In the present study, an ortholog of goose-type lysozyme (ShLysG) from the big-belly seahorse (Hippocampus abdominalis) was identified and characterized structurally and functionally. The full-length cDNA sequence (1213 bp) of ShLysG is comprised of an open reading frame made up of 552 bp, encoding a polypeptide of 184 amino acid (aa) with a predicted molecular mass of 20 kDa. In silico analysis of ShLysG revealed the absence of signal peptide and the presence of a characteristic bacterial soluble lytic transglycosylase (SLT) domain bearing three catalytic residues (Glu71, Asp84, and Asp95) and seven N-acetyl-d-glucosamine binding sites (Glu71, Asp95, Tyr98, His99, Ile117, Tyr145, and Asn146). Homology analysis demonstrated that the aa sequence of ShLysG shared 60.7–67.4% identity and 72.6–79.3% similarity with the orthologs of other teleosts. Phylogenetic analysis of ShLysG indicated a closest relationship with the ortholog from Gadus morhua. In healthy seahorse, ShLysG mRNA showed a constitutive expression in all the tissues examined, with the highest expression in kidney and the least expression in liver. The ShLysG mRNA levels were also shown significant elevation upon the bacterial and pathogen-associated molecular pattern (PAMPs) challenges. Furthermore, lytic activities of ShLysG recombinant protein were detected against several Gram-negative and Gram-positive bacterial species. Taken together, these results suggest that ShLysG might possess a potential immune defensive role against invading microbial pathogens in seahorse.

빅벨리 해마(Hippocampus abdominalis) 유래 펩신 가수분해물의 In vitro와 In vivo에서의 항산화 효능

빅벨리 해마(Hippocampus abdominalis) 유래 펩신 가수분해물의 In vitro와 In vivo에서의 항산화 효능

Copper-zinc-superoxide dismutase (CuZnSOD), an antioxidant gene from seahorse (Hippocampus abdominalis); molecular cloning, sequence characterization, antioxidant activity and potential peroxidation function of its recombinant protein

Copper-zinc-superoxide dismutase (CuZnSOD) from Hippocampus abdominalis (HaCuZnSOD) is a metalloenzyme which belongs to the ubiquitous family of SODs. Here, we determined the characteristic structural features of HaCuZnSOD, analyzed its evolutionary relationships, and identified its potential immune responses and biological functions in relation to antioxidant defense mechanisms in the seahorse. The gene had a 5′ untranslated region (UTR) of 67 bp, a coding sequence of 465 bp and a 3′ UTR of 313 bp. The putative peptide consists of 154 amino acids. HaCuZnSOD had a predicted molecular mass of 15.94 kDa and a theoretical pI value of 5.73, which is favorable for copper binding activity. In silico analysis revealed that HaCuZnSOD had a prominent Cu-Zn_superoxide_dismutase domain, two Cu/Zn signature sequences, a putative N-glycosylation site, and several active sites including Cu2+ and Zn2+ binding sites. The three dimensional structure indicated a β-sheet barrel with 8 β-sheets and two short α-helical regions. Multiple alignment analyses revealed many conserved regions and active sites among its orthologs. The highest amino acid identity to HaCuZnSOD was found in Siniperca chuatsi (87.4%), while Maylandia zebra shared a close relationship in the phylogenetic analysis. Functional assays were performed to assess the antioxidant, biophysical and biochemical properties of overexpressed recombinant (r) HaCuZnSOD. A xanthine/XOD assay gave optimum results at pH 9 and 25 °C indicating these may be the best conditions for its antioxidant action in the seahorse. An MTT assay and flow cytometry confirmed that rHaCuZnSOD showed peroxidase activity in the presence of HCO3−. In all the functional assays, the level of antioxidant activity of rHaCuZnSOD was concentration dependent; metal ion supplementation also increased its activity. The highest mRNA expressional level of HaCuZnSOD was found in blood. Temporal assessment under pathological stress showed a delay response by HaCuZnSOD. Our findings demonstrated that HaCuZnSOD is an important antioxidant, which might be involved in the host antioxidant defense mechanism against oxidative stress.

Characterization of a 1-cysteine peroxiredoxin from big-belly seahorse (Hippocampus abdominalis); insights into host antioxidant defense, molecular profiling and its expressional response to septic conditions

1-cysteine peroxiredoxin (Prx6) is an antioxidant enzyme that protects cells by detoxifying multiple peroxide species. This study aimed to describe molecular features, functional assessments and potential immune responses of Prx6 identified from the big-belly seahorse, Hippocampus abdominalis (HaPrx6). The complete ORF (666 bp) of HaPrx6 encodes a polypeptide (24 kDa) of 222 amino acids, and harbors a prominent peroxiredoxin super-family domain, a peroxidatic catalytic center, and a peroxidatic cysteine. The deduced amino acid sequence of HaPrx6 shares a relatively high amino acid sequence similarity and close evolutionary relationship with Oplegnathus fasciatus Prx6. The purified recombinant HaPrx6 protein (rHaPrx6) was shown to protect plasmid DNA in the Metal Catalyzed Oxidation (MCO) assay and, together with 1,4-Dithiothreitol (DTT), protected human leukemia THP-1 cells from extracellular H2O2-mediated cell death. In addition, quantitative real-time PCR revealed that HaPrx6 mRNA was constitutively expressed in 14 different tissues, with the highest expression observed in liver tissue. Inductive transcriptional responses were observed in liver and kidney tissues of fish after treating them with bacterial stimuli, including LPS, Edwardsiella tarda, and Streptococcus iniae. These results suggest that HaPrx6 may play an important role in the immune response of the big-belly seahorse against microbial infection. Collectively, these findings provide structural and functional insights into HaPrx6.

Effect of Direct Transfer to Different Salinities on Early Juvenile Pot‐bellied Seahorse, Hippocampus abdominalis, Survival in Culture Conditions

AbstractIn Tasmania, commercial seahorse culture takes place in tank systems in which approximately 75% of the water is exchanged daily from the Tamar River estuary. The aim of this study was to examine the effect on survival of early juvenile pot‐bellied seahorses, Hippocampus abdominalis, directly transferred and cultured for 9 d in salinities of 5, 10, 15, 20, 25, and 32 g/L. Direct transfer of seahorses to 5 g/L salinity resulted in 100% mortality within 7 d. After 9 d, an improvement in survival was recorded in seahorses cultured in 10 and 15 g/L compared to those cultured in 25 and 32 g/L salinities. This study is the first to report on the tolerance of H. abdominalis in captivity at salinities as low as 10 g/L in early life stages without compromising survival.

First comparative characterization of three distinct ferritin subunits from a teleost: Evidence for immune-responsive mRNA expression and iron depriving activity of seahorse (Hippocampus abdominalis) ferritins.

Ferritins play an indispensable role in iron homeostasis through their iron-withholding function in living beings. In the current study, cDNA sequences of three distinct ferritin subunits, including a ferritin H, a ferritin M, and a ferritin L, were identified from big belly seahorse, Hippocampus abdominalis, and molecularly characterized. Complete coding sequences (CDS) of seahorse ferritin H (HaFerH), ferritin M (HaFerM), and ferritin L (HaFerL) subunits were comprised of 531, 528, and 522 base pairs (bp), respectively, which encode polypeptides of 177, 176, and 174 amino acids, respectively, with molecular masses of ∼20-21kDa. Our in silico analyses demonstrate that these three ferritin subunits exhibit the typical characteristics of ferritin superfamily members including iron regulatory elements, domain signatures, and reactive centers. The coding sequences of HaFerH, M, and L were cloned and the corresponding proteins were overexpressed in a bacterial system. Recombinantly expressed HaFer proteins demonstrated detectable invivo iron sequestrating (ferroxidase) activity, consistent with their putative iron binding capability. Quantification of the basal expression of these three HaFer sequences in selected tissues demonstrated a gene-specific ubiquitous spatial distribution pattern, with abundance of mRNA in HaFerM in the liver and predominant expression of HaFerH and HaFerL in blood. Interestingly, the basal expression of all three ferritin genes was found to be significantly modulated against pathogenic stress mounted by lipopolysaccharides (LPS), poly I:C, Streptococcus iniae, and Edwardsiella tarda. Collectively, our findings suggest that the three HaFer subunits may be involved in iron (II) homeostasis in big belly seahorse and that they are important in its host defense mechanisms.