Earticle

We evaluated the effects of dietary supplementation of phytosterol on the growth, feed utilization, immunity, digestibility, wound healing ability and disease resistance of olive flounder Paralichthys olivaceus. We conducted two consecutive feeding trials at different growth stages of the fish: EXP-1 (68.9 g) and EXP-2 (16.5 g). The experimental diets were supplemented with graded levels of phytosterol (1% ecdysteroid extracted from Achyranthis radix) at 0, 0.025, 0.05, 0.1, 0.2 and 0.4% (designated as Con, PHY0.025, PHY0.05, PHY0.1, PHY0.2 and PHY0.4, respectively). Dietary phytosterol did not significantly affect growth and cholesterol concentration. Feed utilization was higher in fish fed phytosterol-supplemented diets than in fish fed the control diet. Dietary phytosterol increased innate immunity and digestibility of protein and dry matter. Wound healing ability was also increased by the phytosterol supplementation. The survival against Edwardsiella tarda challenge was higher in fish fed low-phytosterol diets than in fish fed the control diet. The optimum dietary level of phytosterol seems to be approximately 0.05%. The results in this study indicate that the phytosterol could be used as a functional supplement in diets to improve feed utilization, immunity, digestibility and wound healing ability of olive flounder.

This study was conducted to estimate the optimum dietary DHA oil level and replacement level of enzyme treated fish meal (EFM) with sardine fish meal for juvenile Atlantic bluefin tuna Thunnus thynnus. Four diets were used: 1) EFM75 in which 75% EFM and 4% DHA oil were applied, 2) EFM60, with 60% EFM and 15% sardine fish meal, 3) DHA2 with 2% of DHA oil, and 4) SL as a raw fish feed. In a feeding trial, juvenile bluefin tuna (body weight 30.1 g) were randomly stocked into four experimental tanks (69 tones) and fed the experimental diets for 13 days. Fish weight gain was higher in the EFM75 and SL groups than in the DHA2 and EFM60 groups. The feed conversion ratio was lower in the EFM75 and DHA2 groups than in the EFM60 and SL groups. Survival was higher in fish fed the formulated diet groups (EFM75, EFM60 and DHA2) than in fish fed SL. This study clearly indicates that up to 10% dietary sardine fish meal can be used in juvenile T. thynnus diets, with an optimum dietary DHA oil level of approximately 3%.

This study was conducted to investigate in vivo and in vitro digestibility in juvenile Atlantic Bluefin tuna Thunnus thynnus. In vivo digestibility was compared between four experimental diets to determine the optimum dietary inclusion level of an enzyme-treated sardine fish meal (EFM) and sardine fish meal (FM). The experimental diets were as follows; EFM75 (75% EFM), EFM60 (60% EFM and 15% FM), FM75 (75% FM) and SL (frozen sand lance) as a raw fish feed. Feces of Bluefin tuna (90.3 g) were collected both by siphoning from a 700 L cage and by dissection in 69 ton concrete rearing tanks. For the siphoning method, protein digestibility was higher in the tuna fed SL diet than that of other groups. The lowest protein digestibility was observed in FM75. For the dissection method, protein digestibility was higher in tuna fed EFM75 diet than that of other groups. The lowest protein digestibility was observed in the EFM60 group. In vitro digestibility was compared in six protein sources to find an alternative source of EFM for the tuna feed. The highest in vitro digestibility was observed in EFM (92%) followed by low temperature FM (72%), meat meal (65%), feather meal (60%), sardine fish meal (57%) and poultry by-product meal (55%).

Algae extracts or clay are supplement into animal feed to promote growth, digestive performance and health status. A feeding trial was conducted to evaluate supplemental effects of an Algae-clay powder (sea lettuce, Ulva lactuca and red algae, Solieria chordalis in exfoliated micronized montmorillonite, ACP) into a low fish meal (LFM) diet on growth and feed utilization, digestibility, innate immunity and disease resistance of juvenile olive flounder (26.5 ± 0.02 g). A high fish meal diet (HFM) was prepared to contain 55 % FM and the LFM was prepared to contain 44 % FM. The ACP was added into the LFM diet by 0.2 % (MF2). Negative effects of LFM diet were observed in growth, feed utilization and diet digestibility of fish after 12 weeks of the feeding trial. The ACP inclusion in the LFM diet had positive effects on all parameters showing similar or higher growth performance, feed utilization, digestibility, goblet cell counts and villi lengths than the HFM diet. Non-specific immune response and antioxidant parameters, such as lysozyme, total immunoglobulin, anti-proteases, superoxide dismutase and catalase were also significantly improved by the addition of ACP into the LFM diet. Activities of digestive enzymes were improved by ACP supplementation compared to LFM group. Following the feeding trial, a 21-day long challenge test against Edwardsiella tarda showed that MF2 group had higher cumulative survival compared to that of HFM or LMF group while a decreased survival was found in the LFM group. The findings in this study indicate that ACP supplementation in a LFM diet can improve growth performance, feed utilization efficiency, digestibility with digestive enzyme activities, intestinal development, innate immunity and disease resistance of olive flounder.

The interferon-induced GTP-binding protein Mx is responsible for a specific antiviral state against a broad spectrum of viral infections that are induced by type-I interferons (IFN α/β) in different vertebrates. In this study, the Mx gene was isolated from the constructed mullet cDNA database. Structural features of mullet Mx (MuMx) were analyzed using different in-silico tools. The pairwise comparison revealed that the MuMx sequence was related to Stegastes partitus Mx with an 83.7% sequence identity, whereas MuMx was clustered into the teleost category in the phylogentic analysis. Sequence alignment showed that the dynamin-type guanine nucleotide- binding domain (G_DYNAMIN_2), central interactive domain (CID), and GTPase effector domain (GED) were conserved among Mx counterparts. The transcriptional expression of MuMx was the highest in blood cells from unchallenged fish. The temporal mRNA profile showed that MuMx expression was significantly elevated in all tissues, including blood, spleen, head kidney, liver, and gills after the injection of polyinosinic-polycytidylic acid (poly I:C) at many time points. Moreover, MuMx expression increased slightly, in the blood, spleen, and head kidney at a few time points after the injection of lipopolysaccharide (LPS) and Lactococcus garvieae (L. garvieae). Results of the subcellular localization analysis confirmed that the MuMx protein was highly expressed in the cytoplasm. The analysis of the gene expression of the viral hemorrhagic septicemia virus (VHSV) under conditions of MuMx overexpression confirmed the significant inhibition of viral transcripts. The cell viability (MTT) assay and VHSV titer quantification with the presence of MuMx indicated a significant reduction in virus replication. Collectively, these findings suggest that Mx is a specific immune-related gene that elicits crucial antiviral functions against viral antigens in the mullet fish.

In this study, the saponin-rich fractions of five individual (two Red and three Black) sea cucumbers (Apostichopus japonicus) in South Korea were investigated for their antiproliferative effect against HL-60, B16F10, MCF-7, and Hep3B tumor cell lines. The red sea cucumber saponin-rich fraction (SSC) from Jeju Island (JRe) decreased the growth of HL-60 with an IC50 value of 23.55 ± 3.40 lg/mL, which represented the strongest anticancer activity among the extracts. Further, SSC downregulated B-cell lymphoma extra-large (Bcl-xL), while upregulating, to different degrees, Bcl-2-associated X protein (Bax), caspase-9, caspase-3, PARP cleavage, and apoptotic bodies in cancer cells. Evidence for SSC inducing apoptosis via the mitochondria-mediated pathway was found. The contents of SSCs were determined using ultra high-performance liquid chromatography coupled with a quadrupole orbitrap mass spectrometry to comparatively evaluate the regional influence. In West Sea, the total SSC content of A. japonicus was 15.5 mg/g, representing the highest content, while A. japonicus in the South Sea yielded the lowest content at 8 mg/g. The major saponin constituent in SSC was identified as Holotoxin A1, which may the anti-tumor compound in A. japonicus.

CXCL8 (interleukin-8, IL-8) is a CXC family chemokine that recruits specific target cells and mediates inflammation and wound healing. This study reports the identification and characterization of two cxcl8 homologs from rock bream, Oplegnathus fasciatus. Investigation of molecular signature, homology, phylogeny, and gene structure suggested that they belonged to lineages 1 (L1) and 3 (L3), and designated Ofcxcl8-L1 and Ofcxcl8-L3. While Ofcxcl8-L1 and Ofcxcl8-L3 revealed quadripartite and tripartite organization, in place of the mammalian ELR (Glu-Leu-Arg) motif, their peptides harbored EMH (Glu-Met-His) and NSH (Asn-Ser-His) motifs, respectively. Transcripts of Ofcxcl8s were constitutively detected by Quantitative Real-Time PCR (qPCR) in 11 tissues examined, however, at dierent levels. Ofcxcl8-L1 transcript robustly responded to treatments with stimulants, such as flagellin, concanavalin A, lipopolysaccharide, and poly(I:C), and pathogens, including Edwardsiella tarda, Streptococcus iniae, and rock bream iridovirus, when compared with Ofcxcl8-L3 mRNA. The dierences in the putative promoter features may partly explain the dierential transcriptional modulation of Ofcxcl8s. Purified recombinant OfCXCL8 (rOfCXCL8) proteins were used in in vitro chemotaxis and proliferation assays. Despite the lack of ELR motif, both rOfCXCL8s exhibited leukocyte chemotactic and proliferative functions, where the potency of rOfCXCL8-L1 was robust and significant compared to that of rOfCXCL8-L3. The results, taken together, are indicative of the crucial importance of Ofcxcl8s in inflammatory responses and immunoregulatory roles in rock bream immunity.

Miamiensis avidus causes scuticociliatosis in cultured olive flounders (Paralichthys olivaceus), leading to economic losses in aquaculture in Korea. Quantitative evaluation of the viability of M. avidus is important to develop an effective vaccine or chemotherapeutic agent against it. We used a colorimetric assay based on the reduction of 2- (4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) to quantify the viability of M. avidus. Using this method, we investigated the effect of protease inhibitors on the viability of M. avidus. The assay showed a clear difference in the optical density (OD) of over 104 ciliates, and the metalloprotease inhibitors 1, 10-phenanthroline and ethylenediaminetetraacetic acid (EDTA) reduced the viability of M. avidus by more than 90% when used at concentration of 5 mM and 100 μM, respectively. However, different morphological changes in the parasite were observed when exposed to these two inhibitors. These results indicate that the WST-1 assay is a simple and reliable method to quantify the viability of M. avidus, and metalloproteases are excellent targets for the development of agents and vaccines to control M. avidus infection.

Interferon regulatory factors (IRFs) are among the most important transcription mediators and have multiple biological functions, such as antiviral and antimicrobial defense, cell differentiation, immune modulation, and apoptosis. Three IRF family members (HaIRF4-like, HaIRF6, and HaIRF8) of the big belly seahorse (Hippocampus abdominalis) were molecularly and functionally characterized at the sequence and transcriptional level. The coding sequences of HaIRF4-like, HaIRF6, and HaIRF8 were 1214, 1485, and 1266 bp in length, encoding proteins of size 46.21, 55.32, and 47.56 kDa, respectively. Potential viral transcription and replication was detected against VHSV infection using qPCR in HaIRFs-transfected FHM cells. IRFs significantly reduced viral gene expression at 24 h and 48 h post infection and the expression of interferon-stimulated genes (ISGs) was modulated at transcriptional level upon HaIRF overexpression in FHM cells. Subcellular HaIRF localization was observed using GFP-tagged expression vectors in FHM cells. HaIRF4-like and HaIRF8 were localized to the nucleus, whereas HaIRF6 was observed in the cytoplasm. All three IRFs were ubiquitously expressed in all analyzed tissues of the big belly seahorse. The mRNA expression of IRF4-like, IRF6, and IRF8 increased significantly post injection in the blood and gills following LPS, poly (I:C), and Streptococcus iniae challenge. These findings demonstrate that seahorse IRFs are involved in host defense mechanisms against immune stimulants and HaIRFs induce interferon and ISGs which trigger antiviral activity against viral infections in the host.

Marine bivalves are often used as a sentinel species in coastal environmental monitoring since changes in the environmental quality are often well preserved in their cells and tissues. Anadara and Tegillarca species of Arcidae, the blood cockles, are considered to be good sentinel species in monitoring coastal pollution and ecosystem health because they are distributed widely in the subsurface of intertidal mudflats. Internal cellular defense of the blood cockles to physical and biological stresses is mediated by the circulating hemocytes, while their hemocyte types and functions are poorly studied. In this study, we first characterized morphology and immune-related activities of hemocytes of three common blood cockles Anadara broughtonii, A. kagoshimensis, and Tegillarca granosa using flow cytometry. Based on cell morphology and immunological functions, we described five types of hemocytes identically in the three blood cockles: erythrocytes type-I (erythrocytes-I), erythrocytes type-II (erythrocytes-II), granulocytes, hyalinocytes, and blast-like cells. Erythrocytes were round cells containing hemoglobin with numerous granules in the cytoplasm and these cells consist of two central populations. Erythrocytes-I were the most abundant cells accounting for 80–89% of the total circulating hemocytes and exhibited a certain level of lysosome and oxidative capacity. Erythrocytes-II were the largest cells and displayed high lysosome content and the most active oxidative capacity. Both erythrocytes-I and erythrocytes- II did not show phagocytosis capacity. Granulocytes were intermediated-sized hemocytes characterized by granules in the cytoplasm and long pseudopodia on the cell surface, and these cells were mainly engaged in the cellular defense exhibiting the largest lysosome content, the most active phagocytosis, and high oxidative capacity. Contrary to granulocytes, hyalinocytes were comparatively small and round cells and exhibited no granules in the cytoplasm. Hyalinocytes displayed a certain level of lysosome and phagocytosis and oxidative capacities. Blast-like cells characterized by the smallest size and small quantity of cytoplasm and exhibited an absence of phagocytosis and extremely low oxidative capacity, suggesting that this population is not directly involved in the cell-mediated immune activities. In conclusion, flow cytometry indicated that three blood cockles had five types of hemocytes, and the erythrocytes and granulocytes were mainly involved in the immunological activities.

In our previous study, the anticancer effect of the active fucoidan (JHCF4) isolated from acid-processed Sargassum fusiformis was evaluated. In this study, the liver-protective effects of JHCF4 against ethanol-induced Chang liver cell damage and apoptosis-related responses were investigated. Furthermore, the low cytotoxicity and high cell viability of JHCF4 against ethanol-induced cell damage, as well as its protective effect against ethanol-induced cell apoptosis, were observed via nuclear staining with Hoechst 33342 in Chang liver cells. Additionally, the treatment of the 72 h post-fertilization (hpf) zebrafish model with JHCF4 increased the ethanol-stimulated survival rates as well as decreased oxidative stress, lipid peroxidation, and cell death levels. JHCF4 was found to significantly decrease steatosis production in the 128 hpf zebrafish model by Oil Red O staining, as well as attenuate the malondialdehyde and increase the glutathione contents, compared with the untreated group. These results demonstrate that JHCF4 has a potential hepato-protective effect against ethanol-induced damage both in vitro and in vivo.

Interleukin 11 (IL-11) is a secretory cytokine with pleotropic properties, including anti-inflammatory and antiapoptotic functions. This study aimed to functionally characterize a teleostean IL-11a ortholog from redlip mullet (LhIL-11a) through bioinformatic analysis, transcriptional expression profiling and protein function assays. The deduced LhIL-11a protein sequence is 200 amino acids long, with a predicted molecular weight of 23.168 kDa. Multiple sequence alignment indicates that LhIL-11a has a typical four-bundle architecture of α- helixes as observed in other IL-11s. The identity–similarity matrix show a higher identity between LhIL-11a and other fish IL-11a sequences. Phylogenetic analysis demonstrated that LhIL-11a falls within a clade including other fish counterparts. In the tissue distribution analysis, the highest constitutive expression of LhIL-11a mRNA was observed in the mullet gastrointestinal tract and brain tissues. Following the challenges with LPS, poly I:C and Lactococcus garvie, the transcription levels of LhIL-11a were significantly upregulated in both PBCs and liver. In the biological functional assay, recombinant LhIL-11a protein showed strong activities of suppressing proinflammatory cytokines and apoptotic gene expression in mullet kidney cells and reducing LPS stimulated NO production in murine macrophage cells. Overall, the findings in this study provide the experimental clues to understanding the functional roles of fish IL-11a in inflammation and apoptosis regulation during host defense against invading microbial pathogens.

Calreticulin (CRT) is a multifunctional ubiquitous protein that is widely presented in all cells in eukaryotes except erythrocytes. CRT is well known for diverse cellular functions such as endoplasmic reticulum (ER)- specialized protein quality control during protein synthesis and folding, in-vivo Ca2+ homeostasis, antigen presentation, phagocytosis, wound-healing, proliferation, adhesion, and migration of cells. In the current study, we identified CRT from Hippocampus abdominalis (HaCRT) and analyzed expression profiles and functional properties. The cDNA sequence of HaCRT was identified with an open reading frame of 1226 bp. The molecular weight of HaCRT was estimated as 49 kDa. The in-silico study revealed conserved sequence arrangements such as two CRT signature motifs (5′-KHEQSIDCGGGYVKVF-3′ and 5′ -LMFGPDICG-3′ ), triplicate repeats (5′ -IKDPEAKKPEDWD- 3′ , 5′-IPDPDDTKPEDWD-3′ , 5′-IPDPDAKKPDDWD-3′ ), signal peptide and an ER-targeting 5′ - KDEL-3′ sequence of HaCRT. Close sequence similarity of HaCRT was observed with Hippocampus comes from phylogenetic analysis and pairwise sequence comparison. From quantitative polymerase chain reaction (qPCR) results, HaCRT was ubiquitously distributed in all tested tissues and expression levels of HaCRT were significantly modulated in blood, liver and gill tissues after stimulation with Streptococcus iniae, Edwardsiella tarda, polyinosinic:polycytidylic acid, and lipopolysaccharides. Bacterial- and pathogen-associated molecular patternsbinding activities were observed with recombinant HaCRT (rHaCRT). The treatment of murine macrophages with rHaCRT induced the expression of immune genes, such as tumor necrosis factor-α (TNF-α), interleukin 6 (IL- 6), inducible nitric oxide synthase (iNOS), and interleukin-1β (IL-1β). Furthermore, rHaCRT exhibited woundhealing ability. Based on the results from the above study, we suggest that HaCRT play an indispensable role in the immunity of big-belly seahorses by recognition and elimination of pathogens as well as the tissue repairing process.

As a preliminary investigation into the effects of environmental factors on gonadal development using adaptive physiological characteristics, we examined the involvement of photoperiod and water temperature in the ovarian development of the redspotted grouper, Epinephelus akaara. During October 2014 (non-breeding season), the photoperiod and water temperature were adjusted to 12L:12D and 18°C respectively. Every 3 weeks, daylight was increased as follows a 13L:11D and 14L:10D in the photo-thermal treatment group, and the control group was maintained under natural conditions. After 9 weeks, water temperature was increased to 23°C in the photo-thermal treatment group. In photo-thermal treatment group, GSI increased significantly after 9 weeks (1.36 ± 0.19) and 12 weeks (3.31 ± 0.20) compared with the control group. At 12 weeks after photo-thermal treatment, all fishes matured into  ovaries containing late vitellogenic oocytes. The expression of FSHβ and LHβ mRNAs also increased significantly. However, in the control group, the ovarian development was maintained at an immature stage. Based on these results, we demonstrate that exposure to long photoperiod and high water temperature can induce the sexual maturation of immature red-spotted grouper during the non-breeding season.

Leptin, a well-known appetite hormone, plays a role in fat metabolism in peripheral tissues including the adipose, liver, and muscle tissues. In this study, we investigated the antiobesity and fat accumulation regulatory effects of Ishophloroglucin A, derived from the brown seaweed Ishige okamurae, which acts via the leptin signaling pathway in the peripheral tissues of a high-fat-diet-induced obese mouse model. Obesity in C57BL/6J mice was induced by feeding them with a high-fat diet for 10 weeks and Ishophloroglucin A (2.5 mg/kg) was orally treated for the last 4 weeks. Body weights were monitored once per week during the experimental period. After the experiment, several serum biochemical parameters were measured using commercial kits and the white adipose, liver, and muscle tissues were observed using immunohistochemistry methods. Ishophloroglucin A significantly reduced glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and leptin level, which increase as a result of high-fat diet. Also, Ishophloroglucin A clearly activated the leptin signaling pathway in all examined peripheral tissues, reduced the adipose tissue size, and alleviated steatosis in the liver and muscle tissues. These results implied that Ishophloroglucin A treatment for 4 weeks positively induced molecular mechanisms and histologic changes related with leptin signaling. These findings suggested that constant Ishophloroglucin A treatment clearly regulates obesity and peripheral fat accumulation via the leptin signaling pathway in high-fat-diet-induced obese mice.

The previous study suggested that the sulfated polysaccharides from Hizikia fusiforme (HFPS) possess strong antioxidant activity. The purpose of this study is to isolate fucoidan from HFPS and to investigate its antioxidant activity. Afucoidan (HFPS-F4) with a molecular weight of 102.67 kDa was isolated from HFPS. HFPS-F4 contains 99.01% of fucoidan (71.79 ± 0.56% of carbohydrate and 27.22 ± 0.05% of sulfate content). The fucoidan increased the viability of H2O2-treated Vero cells by 5.41, 11.17, and 16.32% at the concentration of 12.5, 25, and 50 μg/mL, respectively. Further results demonstrated that this effect act diminishing apoptosis by scavenging intracellular reactive oxygen species (ROS) via increasing the expression of the endogenous antioxidant enzymes, which was induced by elevating total nuclear factor (erythroid-derived 2)-like 2 (Nrf2) levels. In addition, the in vivo test results displayed that the pretreatment of fucoidan improved the survival rates and decreased heart-beating rate, ROS, cell death, and lipid peroxidation in H2O2-stimulated zebrafish. Taken together, these results demonstrated that fucoidan isolated from HFPS has strong in vitro and in vivo antioxidant activities and it could be utilized in pharmaceutical, nutraceutical, and cosmeceutical industries.

Thioredoxin (Trx) is a small ubiquitous multifunctional protein with a characteristic WCGPC thiol-disulfide active site that is conserved through evolution. Trx plays a crucial role in the antioxidant defense system. Further, it is involved in a variety of biological functions including gene expression, apoptosis, and growth regulation. Trx exists in several forms, with the cytosolic (Trx-1) and mitochondrial (Trx-2) forms being the most predominant. In this study, the mitochondrial Trx protein (HaTrx-2), from the big-belly seahorse (Hippocampus abdominalis) was characterized, and its molecular features and functional properties were investigated. The cDNA sequence of HaTrx-2 consists of a 519 bp ORF, and it encodes a polypeptide of 172 amino acids. This protein has a calculated molecular mass of 18.8 kDa and a calculated isoelectric point (pI) of 7.80. The highest values of identity (78.7%) and similarity (86.2%) were observed with Fundulus heteroclitus Trx-2 from the pairwise alignment results. The phylogenetic analysis revealed that HaTrx-2 is closely clustered with teleost fishes. The qPCR results showed that HaTrx-2 was prevalently expressed at various levels in all the tissues examined. The ovary showed the highest expression, followed by the brain and kidney. HaTrx-2 showed varying mRNA expression levels during the immune challenge experiment, depending on the type of tissue and the time interval. Our results confirmed the antioxidant property of HaTrx-2 by performing the MCO assay, DPPH radical scavenging activity, and cell viability assays. Further, an insulin disulfide reduction assay revealed the dithiol remove the enzymatic activity of HaTrx-2. Altogether these results indicate that HaTrx-2 plays indispensable roles in the regulation of oxidative stress and immune response in the seahorse.

Malectin is a carbohydrate-binding lectin protein found in the endoplasmic reticulum (ER). It selectivity binds to Glc2-N-glycan and is involved in a glycoprotein quality control mechanism. Even though malectin may play a role in immunity, its role in innate immunity is not fully known. In the present study, we identified and characterized the malectin gene from Hippocampus abdominalis (HaMLEC). We analyzed sequence features, spatial expression levels, temporal expression profiles upon immune responses, bacterial and carbohydrate binding abilities and anti-viral properties to investigate the potential role of HaMLEC in innate immunity. The molecular weight and isoelectric point (pI) were estimated to be 31.99 kDa and 5.17, respectively. The N-terminal signal peptide, malectin superfamily domain and C-terminal transmembrane region were identified from the amino acid sequence of HaMLEC. The close evolutionary relationship of HaMLEC with other teleosts was identified by phylogenetic analysis. According to quantitative PCR (qPCR) results, HaMLEC expression was observed in all the examined tissues and high expression was observed in the ovary and brain, compared to other tested tissues. Temporal expression of HaMLEC in liver and blood tissues were significant modulated upon exposure to immunogens Edwardasiella tarda, Streptococcus iniae, polyinosinic:polycytidylic and lipopolysaccharide. The presence of carbohydrate binding modules (CBMs) of bacterial glycosyl hydrolases were functionally confirmed by a bacterial binding assay. Anti-viral activity significantly reduced viral hemorrhagic septicemia virus (VHSV) replication in cells overexpressing HaMLEC. The observed results suggested that HaMLEC may have a significant role in innate immunity in Hippocampus abdominalis.

Recently, a putative new hyperparasitic haplosporidian in the genus Urosporidium was identified from metacercariae of the trematode Parvatrema duboisi infecting Manila clam Ruditapes philippinarum on the west coast of Korea. In this study, we applied small subunit ribosomal DNA (SSU rDNA) sequences as a marker to substantiate the phylogenetic relationship of the unidentified Urosporidium within the Order Haplosporida. In our phylogenetic analysis, the 1890 bp of SSU rDNA sequences obtained were closely related to a haplosporidian parasite forming a sister clade to Urosporidium group, although the gene sequences were only 89.22–89.70% similar to Urosporidium spp. Such molecular phylogenetic distance within the genus suggested that the unidentified Urosporidium is a new member of the genus. Accordingly, we report the unidentified haplosporidian hyperparasite as Urosporidium tapetis sp. nov.

Syndecan-2, also known as CD362, is a transmembrane heparan sulfate proteoglycan which regulates cell growth, proliferation, cell adhesion, wound healing, and recruits immune cells. In the present study, we performed bioinformatics, spatial and temporal expression analyses of Hippocampus abdominalis syndecan-2 (HaSDC-2). Additionally, functional assays were conducted. HaSDC-2 has five major domains; an extracellular heparan sulfate attachment domain, a co-receptor binding domain, a transmembrane domain, two conserved domains (C1 domain, C2 domain), and a variable (V) domain. The ectodomain contained a signal peptide and GAG attachment sites. In-silico analysis revealed that HaSDC-2 contained a 798 bp long ORF and protein sequence of 265 amino acid residues. Further analysis of the amino acid sequence predicted a 28.9 kDa molecular weight and a 4.13 theoretical isoelectric point. The spatial expression of HaSDC-2 was ubiquitous in all tested tissues. HaSDC-2 expression in the liver was upregulated 24 h post-injection in response to all stimuli. Further, HaSDC-2 expression in blood cells was upregulated at 12 and 72 h post-injection in response to all the stimuli. HaSDC-2 + pcDNA™3.1(+) transfected cells exhibited significant survival in response to cell stressors such as H2O2 and HED. The ectodomain of recombinant HaSDC-2 treated cells showed significant cell proliferation in a concentration-dependent manner. The scratch wound healing assay showed significant Δ gap closures with increasing concentrations of HaSDC-2. Collectively, these results indicated that syndecan-2 was involved in regulating immune responses and cell stress conditions.

Peroxiredoxins (Prxs) are cysteine-dependent antioxidant proteins that play a leading part in oxidative stress response. Peroxiredoxin 4 (Prx4) is located in the endoplasmic reticulum, where it is primarily involved in regulating the concentration of H2O2, generated during protein folding. Prx4 is also located in the extracellular space, where it activates the JAK/STAT-mediated stress response. Here, we focus on the identification and characterization of the sequence and function of Prx4 from the big-belly seahorse (Hippocampus abdominalis) (HaPrx4). The size of the HaPrx4 coding sequence was 777 bp, which encoded a 258 amino acid protein of 28.8 kDa molecular weight. The amino acid sequence comprises a signal peptide, two active sites with peroxidatic cysteine and resolving cysteine, catalytic triad, and peroxiredoxin superfamily domain. According to the tissue distribution results, ovaries exhibited the highest HaPrx4 expression level within fourteen examined tissues. The HaPrx4 transcriptional responses to four immune stimulants (lipopolysaccharides, polyinosinic: polycytidylic acid, Edwardsiella tarda, and Streptococcus iniae) were evaluated in the blood and liver tissues. Additionally, the functions of recombinant HaPrx4 protein were evaluated by metal ion-catalyzed oxidation assay, peroxidase activity assay, insulin reduction assay, cell viability assay, and Hoechst staining. The assay results confirmed that the functions of HaPrx4 involved DNA protection, hydrogen peroxide (H2O2) elimination, oxidoreductase activity, enhancing cell survival, and cell protection. The results of the current study propose that HaPrx4 is effectively involved in H2O2 scavenging activity during stress conditions and in innate immune responses of the big-belly seahorse.

Particulate matters (PM), the main contributor to air pollution, have become a serious issue that threatens human’s health. Skin is the largest organ in humans, as well as the primary organ exposed to PM. Overexposure of PM induces skin damage. Diphlorethohydroxycarmalol (DPHC), an algal polyphenol with the potential of skin protection, has been isolated from the edible brown seaweed Ishige okamurae. The purpose of the present study is to investigate the protective eect of DPHC against PM (ERM-CZ100)-induced skin damage in human dermal fibroblasts (HDF) cells. The results indicated that DPHC significantly and dose-dependently reduced intracellular reactive oxygen species generation in HDF cells. In addition, DPHC significantly induced collagen synthesis and inhibited collagenase activity in ERM-CZ100-stimulated HDF cells. Further study demonstrated that DPHC remarkably reduced the expression of human matrix metalloproteinases through regulation of nuclear factor kappa B, activator protein 1, and mitogen-activated protein kinases signaling pathways in ERM-CZ100-stimulated HDF cells. This study suggested that DPHC is a potential candidate to protect skins against PM-induced damage, and it could be used as an ingredient in pharmaceutical and cosmeceutical industries.

Peroxiredoxins (Prxs) are cysteine-dependent antioxidant proteins that play a leading part in oxidative stress response. Peroxiredoxin 4 (Prx4) is located in the endoplasmic reticulum, where it is primarily involved in regulating the concentration of H2O2, generated during protein folding. Prx4 is also located in the extracellular space, where it activates the JAK/STAT-mediated stress response. Here, we focus on the identification and characterization of the sequence and function of Prx4 from the big-belly seahorse (Hippocampus abdominalis) (HaPrx4). The size of the HaPrx4 coding sequence was 777 bp, which encoded a 258 amino acid protein of 28.8 kDa molecular weight. The amino acid sequence comprises a signal peptide, two active sites with peroxidatic cysteine and resolving cysteine, catalytic triad, and peroxiredoxin superfamily domain. According to the tissue distribution results, ovaries exhibited the highest HaPrx4 expression level within fourteen examined tissues. The HaPrx4 transcriptional responses to four immune stimulants (lipopolysaccharides, polyinosinic: polycytidylic acid, Edwardsiella tarda, and Streptococcus iniae) were evaluated in the blood and liver tissues. Additionally, the functions of recombinant HaPrx4 protein were evaluated by metal ion-catalyzed oxidation assay, peroxidase activity assay, insulin reduction assay, cell viability assay, and Hoechst staining. The assay results confirmed that the functions of HaPrx4 involved DNA protection, hydrogen peroxide (H2O2) elimination, oxidoreductase activity, enhancing cell survival, and cell protection. The results of the current study propose that HaPrx4 is effectively involved in H2O2 scavenging activity during stress conditions and in innate immune responses of the big-belly seahorse.

Galectins are well-known β-galactoside-binding proteins, which play vital roles in innate immune responses of both vertebrates and invertebrates. However, knowledge regarding invertebrate galectins is still in its infancy. With the intention of filling the knowledge gap, here we identified a quadruple domain-containing galectin from marine invertebrate disk abalone, Haliotis discus discus (AbGalec), and characterized it. AbGalec consisted of four distinct carbohydrate-recognition domains (CRDs) and lacked a signal peptide. Expression analysis revealed AbGalec to be ubiquitously expressed in all the examined early embryonic stages of abalone, with highest expression in the 16-cell stage, suggesting the importance of AbGalec in early developmental processes. Tissue distribution analysis revealed the highest expression of AbGalec in abalone mantle, followed by that in gills and hemocytes. Immune challenge experiments revealed significant upregulation of AbGalec at 24 h and 48 h post injection (p.i.) with bacterial and viral components. These results suggested the possible involvement of AbGalec in host defense mechanisms. Polyinosinic: polycytidylic acid (Poly I:C) and viral hemorrhagic septicemia virus (VHSV) injections were capable of inducing AbGalec transcript expression more prominently than bacterial stimulants, thus providing evidence for its role in viral infections. We determined the virus-neutralizing ability of a quadruple domain-containing galectin for the first time, by analyzing the downregulation of VHSV transcripts during the overexpression of AbGalec. Significant downregulation of VHSV transcripts was observed after 24 h and 48 h of post infection. Collectively, our findings reveal the potent antiviral responses of molluscan quadruple domain-containing galectin, AbGalec, along with its involvement in innate immunity.

Growth of fish is controlled by various environmental factors, including water temperature (WT). WT is also a major factor that affects the eating behavior of fish. In this study, we studied the relationship between WT and expression profiles of factors involved in appetite regulation and digestion in red spotted grouper. Fish were divided into three groups with 30 fish per group and reared for 2 weeks in a recirculating system at three different WT (natural WT, 20 °C, and 25 °C). After 2 weeks, they were sacrificed immediately after food intake (+ 0 h), 3 h after (+ 3 h), 6 h after (+ 6 h), and 3 h before being fed the next day (− 3 h). Fish samples were obtained for analysis of mRNA expression levels of cholecystokinin (CCK), leptin A (LepA), leptin B (LepB), and neuropeptide Y (NPY) in the brain, liver, and intestine. Expression of CCK and NPY mRNA was significantly higher at 25 °C compared to that at natural WT and 20 °C, but no difference was observed for leptin. This suggests that the red spotted grouper shows characteristics typical of subtropical fish and has higher levels of feeding and digestion when reared at 25 °C water temperature condition.

Background: Fish body color is one of the major factors that determine the commercial value of farmed fish, to understand for coloration mechanisms. The expression of melanin-related genes is according to the developmental stage and light intensity in the red spotted grouper, Epinephelus akaara. Methods: To investigate changes in melanin formation and melanin-related genes in the larval development stage, fish larvae were reared from fertilized egg stage to 50 days after hatching (DAH). Experiment of body color changes was performed under each different light intensity conditions. Melanin-related genes expression was analyzed by real-time qPCR, and body coloration difference was represented by RGB (red, green, blue) code value. Results: Expression levels of melanin-concentrating hormone (MCH) mRNA, pro-opiomelanocortin (POMC) mRNA, and melanocortin 1 receptor (MC1R) mRNA were at their highest 5 DAH (days after hatching). Expression levels of agoutisignaling protein (ASIP) mRNA were at their highest 10 DAH. Results of body color changes according to changes in light intensity conditions showed that the expression level of MCH mRNA in the 2000 lx group was the highest at 6 weeks. The expression levels of POMC mRNA and MC1R mRNA in the 1000 lx group were at their lowest at 9 weeks. RGB color code value under different light intensities were the brightest under 1000 lx and the darkest under 0 lx. Conclusion: Our research suggests that POMC mRNA and MC1R mRNA in the red spotted grouper are involved in melanin synthesis, and these genes are thought to be controlled by light intensity. To better understand the molecular mechanism of coloration in the red spotted grouper, further studies are needed to clarify the relationship between melanophore development and melanin-related genes.

Glutaredoxins are a group of heat stable oxidoreductases ubiquitously found in prokaryotes and eukaryotes. They are widely known for GSH (glutathione)-dependent protein disulfide reduction and cellular redox homeostasis. This study was performed to identify and characterize rockfish (Sebastes schlegelii) glutaredoxin 1 (SsGrx1) at molecular, transcriptional, and functional levels. The coding sequence of SsGrx1 was 318 bp in length and encoded a protein containing 106 amino acids. The molecular weight and theoretical isoelectric point of the putative SsGrx1 protein were 11.6 kDa and 6.71 kDa, respectively. The amino acid sequence of SsGrx1 comprised a CPYC redox active motif surrounded by several conserved GSH binding sites. The modeled protein structure was found to consist of five α-helices and four β-sheets, similar to human Grx1. SsGrx1 showed a tissue specific expression in all the tissues tested, with the highest expression in the kidney. Immune stimulation by lipopolysaccharides (LPS), polyinosinic:polycytidylic acid (polyI:C), and Streptococcus iniae (S. iniae) could significantly modulate the SsGrx1 expression pattern in the blood and gills. Analysis of its subcellular localization disclosed that SsGrx1 was prominently localized in the cytosol. Recombinant SsGrx1 (rSsGrx1) exhibited significant activity in insulin disulfide reduction assay and HED (β-Hydroxyethyl Disulfide) assay. Furthermore, transient overexpression of SsGrx1 in FHM (fathead minnow) cells significantly enhanced cell survival upon H2O2-induced apoptosis. Collectively, our findings strongly suggest that SsGrx1 plays a crucial role in providing rockfish immune protection against pathogens and oxidative stress.