Earticle

The thioredoxin domain containing 5 (TXNDC5) is a recently discovered member of the protein disulfide isomerase family (PDI), which is mainly involved in the proper folding of and the correct formation of disulfide bonds in newly synthesized proteins via its disulfide isomerase and chaperone activities. Although the structural and functional features of mammalian TXNDC5 have been explored in previous studies, no studies have reported the functional characteristics of TXNDC5 in teleost fish. In this study, we report the identification and characterization of TXNDC5 from big-belly seahorse (Hippocampus abdominalis) (ShTXNDC5) accompanied by functional studies. The in-silico analysis revealed that the gene encodes a 433 amino acid (aa) long polypeptide chain with a predicted molecular weight of 49.3 kDa. According to homology analysis, ShTXNDC5 shares more than 55% sequence similarity with other teleost TXNDC5 proteins, and the alignment of the gene sequence convincingly reflects the accepted phylogeny of teleost. Analysis of the spatial distribution of ShTXNDC5 expression showed that its highest expression was observed in the ovary, gill, and pouch of seahorses. Moreover, significant upregulation of ShTXNDC5 transcription was noted in seahorse blood and kidney tissues in a timedependent manner upon viral and bacterial immune challenges. Furthermore, considerable NADPH turnover, insulin reduction ability and significant cell survival effects of ShTXNDC5 were determined by the functional assay, revealing its capability to overcome cellular oxidative stress. Altogether, these findings expand our understanding of TXNDC5 at the molecular and functional levels, and its putative role in seahorse immunity.

Overexposure to ultraviolet B (UVB) causes skin damage. The purpose of this studywas to evaluate the protective effect of a fucoidan with a molecular weight of 102.67 kDa, isolated from Hizikia fusiforme, against UVB-induced photodamage in vitro in human dermal fibroblasts (HDFs) and in vivo in zebrafish. Fucoidan remarkably inhibited commercial collagenase. Additionally, it significantly and dose-dependently decreased the intracellular reactive oxygen species (ROS) levels and increased the viability of UVB-irradiated HDFs. Furthermore, fucoidan remarkably improved collagen synthesis, inhibited intracellular collagenase, and reduced the expression of matrix metalloproteinases and pro-inflammatory cytokines in UVB-irradiated HDFs. Further research demonstrated that these effects occurred through the regulation of the activator protein 1, nuclear factor kappa B, and mitogenactivated protein kinase signaling pathways. Furthermore, the in vivo results showed that fucoidan protected zebrafish larvae against UVB-induced photodamage by decreasing cell death via the suppression of lipid peroxidation and inflammatory response through ROS clearance. In conclusion, fucoidan isolated fromHizikia fusiforme exhibits strong in vitro and in vivo photoprotective effects, and can be used as an ingredient in the cosmeceutical and pharmaceutical industries.

Protein hydrolysates and krill meal (KM) are used as protein sources in aquafeeds. The study was conducted to examine the supplemental effects of shrimp protein hydrolysates (SH) or KM in a high‐plant‐protein diet for red seabream (Pagrus major). A fish meal (FM)-based diet (40%) was considered as the high-FM diet (HFM) and a diet containing 25% FM and soy protein concentrate, in the expense of FM protein from HFM diet, was considered as the low fish meal (LFM) diet. Two other experimental diets (SH and KM) were prepared by including SH and KM into LFM diet at 5% inclusion levels in exchange of 5% FM from the LFM diet. A feeding trial was conducted for fifteen weeks using triplicate group of fish (Initial mean body weight, 8.47 ± 0.05 g) for a diet. Growth performance and feed efficiency of fish were significantly enhanced by HFM, KM and SH supplemented diets over those of fish fed LFM diet. Interestingly, these parameters of fish fed SH diet showed better performance than KM and HFM groups. Liver IGF-I expression of fish fed SH diet was comparable to HFM group and higher than KM and LFM diets. Protein digestibility of SH diet was significantly higher than KM, HFM, and LFM diets. Dry matter digestibility of SH diet was comparable to HFM diet and significantly higher than KM and LFM diets. Nitro blue tetrazolium and superoxide dismutase activities of HFM, SH and KM groups were significantly elevated than the LFM group and SH diet increased catalase and glutathione peroxidase activities of fish compared to KM and LFM groups. Hemoglobin level and hematocrit of fish fed SH and KM diets were significantly higher than LFM group. A diet containing 20% FM with KM is comparable to a HFM diet which contains 40% FM for red seabream. SH can be used to replace FM from red seabream diet down to 20% and fish performance can be improved better than a diet containing 40% FM. Overall, it seems that SH is more effective ingredient in red seabream diet compared to KM.

The anti-inflammatory effect of a fucoidan with a molecular weight of 102.67 kDa isolated from an enzymatic digest of Sargassum fusiforme was investigated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and zebrafish. The results indicated that the fucoidan significantly and dose-dependently inhibited the production of inflammatory molecules, including tumor necrosis factor-alpha (TNF-α), nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), as well as improved the viability of LPS-stimulated RAW 264.7 cells. Moreover, the fucoidan suppressed the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) by regulating the nuclear factor kappa B (NF-κB) pathway in LPS-stimulated RAW 264.7 cells. Furthermore, in vivo test results suggested that the fucoidan remarkably reduced reactive oxygen species (ROS), cell death, and NO levels in LPS-stimulated zebrafish in a dose-dependent manner. Taken together, these results demonstrated that the fucoidan isolated from S. fusiforme possesses strong anti-inflammatory activities in vitro and in vivo, and could prove as an important candidate to be used to develop anti-inflammatory agents in pharmaceutical and cosmeceutical industries.

Glutathione-S-transferase (GST) is a key enzyme in the phase-II detoxification process and is a biomarker of oxidative stress. In this study, we analyzed the molecular, biochemical, and antioxidant properties of GST alpha-4 from Hippocampus abdominalis (HaGSTA-4). Also, the spatial and temporal expression of HaGSTA-4 upon immune challenge with abiotic and biotic stimulants were evaluated. The HaGSTA-4 ORF encodes 223 amino acids with a molecular weight of 25.7 kDa, and an estimated isoelectric point (pI) of 8.47. It consists of the GST_C superfamily and thioredoxin-like superfamily domain. The phylogenetic tree revealed that HaGSTA-4 is evolutionarily conserved with its GST alpha class counterparts. From pairwise alignment, the highest values of identity (78.5%) and similarity (85.7%) were with Parambassis ranga GSTA-4. Protein rHaGSTA-4 exhibited the highest conjugation activity towards 1-chloro-2,4-dinitrobenzene (CDNB) at pH 7 and 20 ◦C. A disk diffusion assay showed that rHaGSTA-4 significantly protects cells from the stress of exposure to ROS inducers such as CuSO4, CdCl2, and ZnCl2. Furthermore, overexpressed HaGSTA-4 defended cells against oxidative stress caused by H2O2; evidence of selenium-independent peroxidase activity. From qPCR, the tissue-specific expression profile demonstrates that HaGSTA-4 is most highly expressed in the kidney, followed by the intestine and stomach, among fourteen different tissues extracted from healthy seahorses. The mRNA expression profile of HaGSTA-4 upon immune challenge varied depending on the tissue and the time after challenge. Altogether, this study suggests that HaGSTA-4 may be involved in protection against oxidative stress, in immune defense regulation, and xenobiotic metabolism.

Glutathione S-transferases (GSTs) are important enzymes involved in phase II detoxification and function by conjugating with the thiol group of glutathione. In this study, we isolated an omega class GST from the big-belly seahorse (Hippocampus abdominalis; HaGSTO1) to study the putative xenobiotic responses and defense ability against viral and bacterial infections in this animal. The isolated HaGSTO1 gene, with a cording sequence of 720 bp, encodes a peptide of 239 amino acids. The predicted molecular mass and theoretical isoelectric point of HaGSTO1 was 27.47 kDa and 8.13, respectively. In-silico analysis of HaGSTO1 revealed a characteristic N-terminal thioredoxin-like domain and a C-terminal domain. Unlike other GSTs, the C-terminal of HaGSTO1 reached up to the N-terminal, and the N-terminal functional group was cysteine rather than tyrosine or serine, as observed in other GSTs. Phylogenetic analysis showed the evolutionary proximity of HaGSTO1 with other identified vertebrate and invertebrate GST orthologs. For the first time, we demonstrated the viral defense capability of HaGSTO1 against viral hemorrhagic septicemia virus (VHSV) infection. All six nucleoproteins of VHSV were significantly downregulated in HaGSTO1-overexpressing FHM cells at 24 h after infection compared with those in the control. Moreover, arsenic toxicity was significantly reduced in HaGSTO1-overexpressing FHM cells, and cell viability increased. Real-time polymerase chain reaction analysis showed that HaGSTO1 transcripts were highly expressed in the pouch and gill when compared with those in other tissues. Blood HaGSTO1 transcripts were significantly upregulated after Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide, and polyinosinic: polycytidylic acid challenge experiments. Collectively, these findings suggest the involvement of HaGSTO1 in the host defense mechanism of seahorses.

이 연구는 붉바리(Epinephelus akaara)의 사육 관리 효율성을 제고하기 위하여 암반지역에 서식하는 붉바리 서식행동특성을 고려하여 사육 수조에 쉼터(shelter)을 제공하였을 때 붉바리의 성장에 미치는 영향을 조사하였다. 실험기간은 2018년 9월부터 2019년 1월까지로, 실험 그룹은 쉼터를 설치하지 않은 대조구와 쉼터를 2개 설치한 실험구 I , 쉼터를 4개 설치한 실험구 II로 구분하여 사육하였다. 사육수조 내 쉼터 설치에 따른 체중 변화를 조사한 결과 대조구에 비해 실험구에서 체중의 증가하였고, 특히 실험구 II에서 체중이 가장 높게 나타났으며, 일간성장율과 사료효율 또한 대조구에 비해 실험구에서 높게 나타났다. 쉼터 설치에 따른 스트레스 반응을 조사하기 위해 CRH mRNA 발현량을 분석 한 결과 제한된 공간에 적은 수의 쉼터 설치는 사육 초기에 CRH mRNA 발현량이 높게 나타났으나 사육환경에 적응하면서 감소하는 경향을 보였다. 쉼터 설치 후 붉바리가 쉼터에 숨어 은신하는 행동이 관찰되었으며, 쉼터의 설치가 붉바리에게 안정적인 사육환경을 제공 할 수 있을 것으로 생각된다. 추후 적정 사육조건에서 쉼터에 반응하는 붉바리의 적응 반응 특성을 토대로 한 생산성 평가가 요구된다.

To enhance the efficiency of the breeding control of the red spotted grouper, Epinephelus akaara, this study investigated effect on the behavioral characteristics, growth and stress response of red spotted grouper, when providing a rest area (artificial fish shelter) in a rearing tank, taking into account the ecological habits of the fish inhabiting the rock-bottomed areas. The fish was divided into the control group (no shelter), treatment I (two shelters) and treatment II (four shelters), respectively. The experiment was conducted from September 2018 to January 2019. The body weight was higher in treatment II than in control and treatment I (p<0.05). The daily growth rate and feed efficiency rate were higher in treatment I and II than in control. Most of the red spotted grouper's behavior in the rearing tank showed a tendency to form a group under the shelter. The corticotropin-releasing hormone (CRH) mRNA expression in the brain was increased in the treatment I, but then decreased. These results indicate that the shelter may be provide a stable management condition in rearing tank for the red spotted grouper.

Peroxiredoxins (Prxs) are ubiquitously expressed antioxidant proteins that can protect aerobic organisms from oxidative stress. Here, we characterized the HaPrx3 homolog at the molecular level from big-belly seahorse (Hippocampus abdominalis) and analyzed its functional activities. The coding sequence of HaPrx3 consists of 726 bp, which encodes 241 amino acids. The predicted molecular weight and theoretical isoelectric point (pI) of HaPrx3 was 26.20 kDa and 7.04, respectively. Multiple sequence alignments revealed that the arrangements of domains, catalytic triads, dimers, and decamer interfaces of HaPrx3 were conserved among Prx sequences of other organisms. According to the phylogenetic analysis, HaPrx3 is clustered with the teleost Prx3 subclade. The highest transcript level of HaPrx3 was detected in the ovary tissue among fourteen healthy fish tissues. The mRNA levels of HaPrx3 in blood and liver tissues were significantly (P < 0.05) upregulated in response to lipopolysaccharide (LPS), polyinosinic-polycytidylic (poly I:C), Edwardsiella tarda, and Streptococcus iniae, suggesting its involvement in immune responses. Under functional properties, insulin disulfide reduction assay confirmed the oxidoreductase activity of recombinant HaPrx3. A cell viability assay and Hoechst staining indicated cell survival ability and reduction of apoptotic activity, respectively. Moreover, a peroxidase activity assay verified peroxidase activity, while a metal-catalyzed oxidation (MCO) assay indicated the DNA protection ability of HaPrx3. Collectively, it is concluded that HaPrx3 may play a significant role in oxidative stress and immune responses against pathogenic infections in big-belly seahorses.

The tetraspanin superfamily proteins are transmembrane proteins identified in a diverse range of eukaryotic organisms. Tetraspanins are involved in a variety of essential biological functions, including cell differentiation, adhesion, migration, signal transduction, intracellular trafficking, and immune responses. For an infection to occur, viruses must interact with various cell surface components, including receptors and signaling molecules. Tetraspanin CD63 is involved in the organization of the cell membrane and trafficking of cellular transmembrane proteins that interact with many viruses. In this study, the cd63 gene was characterized by studying its expression and function in a zebrafish model. The functional domains and structural features of Cd63, such as the Cys-Cys- Gly (CCG) motif in the large extracellular loop and cysteine residues, are conserved in zebrafish. We confirmed that cd63 was expressed in immune system organs, such as the axial vein and pronephric duct, during the embryonic development of zebrafish. To better understand the role of cd63 in the zebrafish immune system, we established cd63-deficient zebrafish lines using the clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9 (CRISPR/Cas9) system. A 19 bp insertion mutation was generated in single guide RNA (sgRNA) target sequence of exon 3 of the cd63 gene, to create a pre-mature stop codon. We then analyzed the expression of cd63-related genes cxcr4a and cxcr4b in wild type (WT) and cd63-deficient zebrafish. We believe our study provides an important model that could be used to investigate the roles of cd63 in viral infection in vivo.

Cystatins are reversible inhibitors of cysteine proteases which show an omnipresent distribution in the life on earth. Although, cystatins with mammalian origin were well characterized and their roles in physiology were reported in details, those from teleostean origin are still underrepresented in literature. However, role of cystatins in fish physiology and immune defense is highlighted in few recent reports. In this study, a cystatin C holmologue from rock bream (Oplegnathus fasciatus); termed RbCytC was identified and molecularly characterized. The complete coding sequence of RbCytC was 387 bp in length, which codes for a polypeptide with 129 amino acids, including a signal peptide of 19 amino acids. The consensus cystatin family signatures including a G residue, turning up towards the N-terminus region, QVVAG motif, locating at the middle of the sequence and the PW motif at the c terminal region was found to be well conserved in RbCytC. Phylogenetic analysis using different cystatin counterparts affirmed the close evolutionary relationship of RbCytC with its teleostan homologs which belong to family 2 cystatins. The predicted molecular model of RbCytC resembled most of the structural features of empirically elucidated tertiary structures for chicken egg white cystatin. According to the qPCR assays, RbCytC showed detectable expression in all fish tissues used in the experiment, with markedly pronounced expression level in liver. Moreover, its basal mRNA expression was up-regulated in liver and spleen tissues by experimental rock bream iridovirus infection, whereas down regulated in the same tissues, post live Edwardsiella tarda injection. Collectively, outcomes of our study validate the structural homology of RbCytC with known cystatin C similitudes, especially those of teleosts and suggest its potential roles in proteolytic processes of rock bream physiology as well as in host immune defense mechanisms.

In the present study, the in vitro and in vivo anti-inflammatory effects of the sulfated polysaccharides isolated from Sargassum fulvellum (SFPS) were evaluated in lipopolysaccharide (LPS)- stimulated RAW 264.7 macrophages and zebrafish. The results indicated that SFPS improved the viability of LPS-stimulated RAW 264.7 macrophages from 80.02 to 86.80, 90.09, and 94.62% at the concentration of 25, 50, and 100 μg/mL, respectively. Also, SFPS remarkably and concentrationdependently decreased the production levels of inflammatory molecules including nitric oxide (NO), tumor necrosis factor-alpha, prostaglandin E2, interleukin-1 beta, and interleukin-6 in LPStreated RAW 264.7 macrophages. In addition, SFPS significantly inhibited the expression levels of cyclooxygenase-2 and inducible nitric oxide synthase in LPS-treated RAW 264.7 macrophages. Furthermore, the in vivo test results indicated that SFPS improved the survival rate of LPS-treated zebrafish from 53.33 to 56.67, 60.00, and 70.00% at the concentration of 25, 50, and 100 μg/mL, respectively. In addition, SFPS effectively reduced cell death, reactive oxygen species, and NO levels in LPS-stimulated zebrafish. Taken together, these results suggested that SFPS possesses strong in vitro and in vivo anti-inflammatory activities, and could be used as an ingredient to develop anti-inflammatory agents in the functional food and pharmaceutical industries.

This study aimed to evaluate how fish meal (FM) replacement in diets with a mixture of animal and plant protein sources affect growth performance, feed utilization, hematological parameters and innate immunity of red seabream Pagrus major. A control FM diet was formulated to contain 65% FM (Con). Two other diets were prepared replacing FM in the control diet with a mixture of protein sources (wheat gluten, soy-protein concentrate, tankage meal, and poultry by-product meal) by 30 and 40% (FM30 and FM40, respectively). Total 300 red seabream (body weight, 77.6±0.3g) were distributed to 12 tanks (300 L) in 4 replicates per diet. The fish were fed the diets to apparent satiation for 19 weeks. After the feeding trial, no significant differences could be observed in growth performance, feed utilization, hematological parameters, innate immunity, and survivals among all the dietary treatments. This longterm feeding trial at low water temperature (13.8-17.5°C) indicates that a proper mixture ratio of wheat gluten, soy protein concentrate, tankage meal, and poultry by-product meal can replace FM up to 40% in red seabream diets.

The supplementary effects of an emulsifier mixture (polyoxyethylene (20) sorbitan monolaurate and distilled monoglycerides) were investigated on feed utilization, growth performance, and immunity of olive flounder, Paralichthys olivaceus, replacing fish oil with beef tallow. A fish oil containing diet was considered as positive control (PC) and a diet containing beef tallow instead of fish oil was considered as negative control (TW). Two other diets (EM01 and EM02) were prepared by adding 0.01 and 0.02% of the emulsifier into the TW diet. After 8 weeks of a feeding trial, growth performance and feed utilization were significantly higher in fish fed EM01, EM02, or PC diets compared to those fed the TW diet. Dry matter digestibility was higher in EM01 and EM02 diets than the TW diet and comparable to PC diet. Lipid digestibility was significantly higher in PC diet compared to TW diet. Liver EPA and DHA levels were lower, and oleic acid level was higher in fish fed diets containing tallow compared to PC group. Significantly increased aspartate aminotransferase and triglyceride levels were observed in TW group. Dietary supplemented emulsifier restored the reduced performance of olive flounder fed diets containing tallow instead of fish oil. However, the optimum inclusion level of the emulsifier should be elucidated in future studies.

This study was conducted to evaluate the effects of dietary supplementation with shrimp soluble extract (SSE) on growth performance, feed utilization, innate immunity and digestibility of Pacific white shrimp Litopenaeus vannamei. A basal diet (Con) was formulated and three other diets were prepared with SSE supplementation at different levels of 1, 2 and 4% (designated as SSE1, SSE2 and SSE4, respectively). Triplicate groups of shrimp (1.20±0.01 g) were fed one of the experimental diets for 6 weeks. At the end of the feeding trial, growth performance and feed utilization of the shrimp were significantly improved by dietary supplementation of SSE4 compared to those of shrimp fed the Con diet. Shrimp fed SSE4 diet had significantly higher phagocytic activity than shrimp fed the Con diet. Dietary supplementation of SSE improved the protein and dry matter digestibility of shrimp. These results indicate that SSE can be used as a functional additive in the diets for Pacific white shrimp.

Loss of L-gulonolactone oxidase (GULO), which catalyzes the last step of the ascorbic acid (AA) biosynthesis pathway, results in a complete lack of AA in several Osteichthyes fish species, including zebrafish. In this study, sGULO, the active GULO gene from cloudy catshark (Scyliorhinus torazame) was cloned into zebrafish using the Gateway cloning method. The resulting Tg(b-actin:sGULO:mCherry) fish were analyzed for the effects of a reestablished AA pathway. Fluorescent microscopy and PCR were used to analyze the integration of the construct into the zebrafish genome. Catalytic activity of sGULO, AA production, growth-related characteristics, and gene expression were investigated to evaluate the effects of AA production in Tg fish. The mCherry fluorescent protein indicated the proper integration and expression of the sGULO construct in zebrafish. The sGULO gene was ubiquitously expressed in all the studied tissues and the enzyme activity indicated an increased AA production in Tg fish. The growth of Tg fish was also increased, and antioxidant system analysis suggests that reactive oxygen species production was reduced in Tg fish compared with wild type. Expression of the AA transporter slc23a1 was significantly downregulated in Tg homozygous fish. These results collectively indicate the effects of reestablished AA synthesis in zebrafish.

This study was conducted to evaluate the dietary supplementation of two Bacillus spp. on digestibility, growth performance, innate immunity, and water ammonia level of Litopenaeus vannamei. A control diet was made without the probiotic supplementation, and four other diets were prepared by including B. subtilis (BS) alone (0.1 × 1010 and 0.2 × 1010) or a mixture of BS and B. pumilus (BP) (0.1 × 1010 and 0.2 × 1010). Quadruplicate groups of shrimp (0.14 g) were hand-fed one of the diets for 8 weeks. Shrimp fed diets containing Bacillus spp. showed significantly higher apparent digestibility coefficient of protein and dry matter than shrimp fed the control diet. The growth performance was significantly improved in shrimp fed higher dose (0.2 × 1010) of the Bacillus spp. compared to the control group. Innate immunity of shrimp fed the probiotic diets was significantly enhanced compared to the control group. In a water ammonia test, culture water of the probiotic diets had lower total ammonia concentration than that of the control diet. The results in this study suggest that Bacillus spp. can positively affect digestibility, growth performance, feed efficiency, innate immunity, and water quality for L. vannamei.

Thioredoxin domain-containing protein 17 (TXNDC17) is an important, highly conserved oxidoreductase protein, ubiquitously expressed in all living organisms. It is a small (~14 kDa) protein mostly co-expressed with thioredoxin 1 (TRx1). In the present study, we obtained the TXNDC17 gene sequence from a previously constructed yellowtail clownfish (Amphiprion clarkii) (AcTXNDC17) database and studied its phylogeny as well as the protein’s molecular characteristics, antioxidant, and antiapoptotic effects. The full length of the AcTXNDC17 cDNA sequence was 862 bp with a 372 bp region encoding a 123 amino acid (aa) protein. The predicted molecular mass and isoelectric point of AcTXNDC17 were 14.2 kDa and 5.75, respectively. AcTXNDC17 contained a TRX-related protein 14 domain and a highly conserved N-terminal Cys43-Pro44-Asp45-Cys46 motif. qPCR analysis revealed that AcTXNDC17 transcripts were ubiquitously and differently expressed in all the examined tissues. AcTXNDC17 expression in the spleen tissue was significantly upregulated in a time-dependent manner upon stimulation with lipopolysaccharide (LPS), polyinosinic-polycytidylic (poly I:C), and Vibrio harveyi. Besides, LPSinduced intrinsic apoptotic pathway (TNF-α, caspase-8, Bid, cytochrome C, caspase-9, and caspase-3) gene expression was significantly lower in AcTXNDC17-overexpressing RAW264.7 cells, as were NF-κB activation and nitric oxide (NO) production. Furthermore, the viability of H2O2-stimulated macrophages was significantly improved under AcTXNDC17 overexpression. Collectively, our findings indicate that AcTXNDC17 is involved in the innate immune response of the yellowtail clownfish.

이 연구는 제주도 연안에 서식하는 큰열매모자반(Sargassum macrocarpum)의 종 동정을 위한 기초자료를 제공하기 위해 형태적 특성과 계통 유연관계를 조사하였다. 형태학적 분석 결과, 수집된 큰열매모자반은 원추상 부착기, 직립하는 줄기 그리고 주지의 치상돌기 등 형태적으로 기존에 보고된 연구결과와 유사한 양상을 보였다. 하지만 암생식기탁의 모양은 긴 주걱형과 도피침형에서 드물게 Y자형도 나타나 형태적 다양성이 관찰되었다. 부착기의 모양은 생육기간이나 부착기질에 적응하여 전형적인 원추상 에서 뭉툭한 다각형까지 여러 가지 형태적 변이를 보였다. 조천읍 조천리와 북촌리 연안에서 수집된 큰열매모자반의 mtDNA cox3 서열은 한경면 용당리에서 보고된 서열과 100% 일치하였는데, 이는 제주도 전 해역의 큰열매모자반이 유전적으로 동일한 기원 에서 유래된 것으로 판단되었다. 또한 큰열매모자반의 mtDNA cox3 서열이 Sargassum yamadae, S. siliquastrum의 서열들과 동일하여, 분류체계의 재조명이나, 교잡가능성과 생식적 격리에 대한 후속연구의 필요성을 시사하고 있다. 이번 연구결과는 해양생태계에서 해중림을 구성하는 주요 종 중 하나인 큰열매모자반의 형태적 특성과 계통 유연관계에 대한 정보를 통하여 모자반류의 종 동정 자료에 적용될 것이다.

The purpose of this study was to investigate the morphological characteristics and phylogenetic relationship to provide information for the classification of Sargassum macrocarpum inhabiting Jeju Island, Korea. The morphological characteristics of S. macrocarpum such as conical holdfast, erect stem and denticle protuberances on both edges of the main branches were similar to those of previously reported for Sargassum morphology. However, morphological variation was observed as the female receptacle is long spatulate or oblanceolate and rarely Y-shaped. The shape of holdfast showed various morphological variations from a typical cone to a blunt polygon by adapting to the age or substrate shape. The nucleotide sequences of mtDNA cox3 gene obtained from Jocheon-ri and Bukchon-ri, Jocheon-up in this study were identical to those previously reported in Yongdang-ri, Hangyeong-myeon, suggesting that the S. macrocarpum populations over whole Jeju Island seacoast might be genetically derived from the same origin. In addition, the cox3 sequences of S. macrocarpum were identical to those of S. yamadae and S. siliquastrum, suggesting that it is necessary to review their taxonomic position and to carry out future studies on inter-specific hybridization and reproductive isolation among related species. The results of the present study will be applied to Sargassum species classification through the morphological characteristics and phylogenetic relationship of S. macrocarpum, as one of main species that form forests in marine ecosystems.

Reproductive cycle of the blue-striped angelfish, Chaetodontoplus septentrionalis were histologically investigated. Fish were monthly collected in the coastal waters of Munseom, Seogwipo, Jeju-Island from February to December 2018. The gonadosomatic index (GSI) increased from May and maintained high values in August. The reproductive cycle of female fish can be classified by the characteristics observed during gonadal development as followed: growing stage (November to June), early mature stage (May to June), mature and spawning stage (June to September), and degenerative and recovery stage (September to December). In the male, testicular development period was similar to that of ovarian development period, but mature and spawning period was one month longer from June to October. Fecundity of mature female ranged from 4,601 to 22,840 and was correlated positively with total length and body weight. The histological analysis of gonadal development indicated that the C. septentrionalis was summer-spawning type and is considered a multiple spawner during spawning season.

CD63, a member of the tetraspanin family, is involved in the activation of immune cells, antiviral immunity, and signal transduction. The economically important anemonefishes Amphiprion sp. often face disease outbreaks, and the present study aimed to characterize CD63 in Amphiprion clarkii (denoted AcCD63) to enable better disease management. The in-silico analysis revealed that the AcCD63 transcript is 723 bp long and encodes 240 amino acids. The 26.2 kDa protein has a theoretical isoelectric point of 5.51. Similar to other tetraspanins, AcCD63 consists of four domains: short N-/C-terminal domains and small/large extracellular loops. Pairwise sequence alignment revealed that AcCD63 has the highest identity (100%) and similarity (99.2%) with CD63 from Amphiprion ocellaris. Multiple sequence alignment identified a conserved tetraspanin CCG motif, PXSCC motif, and C-terminal lysosome-targeting GYEVM motif. The quantitative polymerase chain reaction analysis showed that AcCD63 was highly expressed in the spleen and head kidney tissue, with low levels of expression in the liver. Temporal expression patterns of AcCD63 were measured in the head kidney and blood tissue after injection of polyinosinic:polycytidylic acid (poly (I:C)), lipolysacharides (LPS), or Vibrio harveyi (V. harveyi). AcCD63 was upregulated at 12 h post-injection with poly (I:C) or V. harveyi, and at 24 h post-injection with all stimulants in the head kidney. At 24 h post-injection, poly (I:C) and LPS upregulated, whereas V. harveyi downregulated AcCD63 expression in the blood. All viral hemorrhagic septicemia virus transcripts (M, G, N, RdRp, P, and NV) were downregulated in response to AcCD63 overexpression, and removal of viral particles occurred via the involvement of AcCD63. The expression of antiviral genes MX dynamin-like GTPase 1, interferon regulatory factor 3, interferon-stimulated gene 15, interferon-gamma, and viperin in CD63-overexpressing fathead minnow cells was downregulated. Collectively, our findings suggest that AcCD63 is an immunologically important gene involved in the A. clarkii pathogen stress response.

Interferon-induced protein 35 kDa (IFP35) has been demonstrated to play important roles in antiviral defense, inflammatory response and cancer progression. However, its precise function in teleost fish remains to be elucidated. Herein, we functionally characterized the rock bream (Oplegnathus fasciatus) IFP35 (OfIFP35) to understand its expression pattern, subcellular localization, antiviral activity, and regulation of downstream genes. OfIFP35 consists of an 1107 bp open reading frame encoding 368 amino acids, including two N-mycinteractor (Nmi)/IFP35 domains (NIDs). The predicted molecular weight of OfIFP35 was 42 kDa, with a theoretical isoelectric point (pI) of 5.10. Evolutionary conservation of IFP35 was analyzed using multiple, pairwise alignments and phylogenetic tree analysis. OfIFP35 in rock bream was found to be highest expressed in the gills. Immune challenges with iridovirus, polyinosinic:polycytidylic acid, lipopolysaccharide, and live bacteria (Streptococcus iniae and Edwardsiella tarda) significantly upregulated its mRNA expression in gill and liver tissues of the rock bream. GFP-tagged OfIFP35 was localized in the cytoplasm of FHM cells, and its overexpression significantly suppressed VHSV transcription in vitro. Moreover, the analysis of downstream gene expression revealed that OfIFP35 could activate the type I interferon pathway. Collectively, these findings indicate that OfIFP35 is important for the immune system of rock bream as it promotes defense responses during viral infections.

The study was conducted to investigate the dietary supplementation of citrus by-product (CBP) and CBP fermented with Bacillus subtilis (BS) or B. pumilus (BP) on growth performance, feed utilization, innate immune responses and disease resistance of Korean rockfish Sebastes schlegelii. Triplicate groups of juvenile fish (initial weight of 22.9 g) were fed one of five experimental diets which were formulated to replace a synthetic vitamin C (L-ascorbyl- 2-polyphosphate, LAPP) with CBP or fermented CBP. A basal diet without LAPP and CBP was used as the control diet (Con). At the end of the 13 weeks feeding trial, growth performance, feed utilization and survival of fish fed the Con were significantly lower than those of fish fed the LAPP, CBP and fermented CBP diets. Fish fed the Con were significantly lower in lysozyme activity than fish fed LAPP, CBP and fermented CBP diets. In a challenge test against Edwardsiella tarda, cumulative mortality of fish fed LAPP, CBP and fermented CBP diets was significantly lower (20-30%) than that of the Con (60%). The findings in this study indicate that the CBP and/or fermented CBP can be a promising vitamin C source for LAPP replacement in diet for Korean rockfish.

This study was conducted to determine the supplemental effects of two insect meals, mealworm (MW) and black soldier fly (BSF), with high or low lipid levels in diets, on Pacific white shrimp Litopenaeus vannamei. Sardine and tuna by-product meals were used as the fish meal source in a control (Con) diet. The fish meals were replaced with MW, defatted MW (deMW), BSF or defatted BSF (deBSF), respectively. The shrimp (body weight: 0.47 g) were stocked into 20 acryl tanks (215 L) and fed the diets six times a day. After 45 days of the feeding trial, the shrimp that were fed insect meals had significantly higher phenoloxidase and superoxide dismutase activities than the shrimp fed Con diet. The gene expressions of prophenoloxidase, crustin and penaeidine-3c in shrimp hepatopancrease were also higher in shrimp that were fed the insect diets, regardless of defatting than those in shirmp that were fed Con diet. The survival against Vibrio parahaemolyticus was higher in shrimp that were fed the diets containing defatted insect meals than in shrimp that were fed Con diet. These results indicate that MW and BSF, regardless of lipid levels, could be good protein sources for the enhancement of innate immunity and anti-oxidant capacity of the shrimp.

Air pollution is a process that mixes pollutants into the atmosphere, which is potentially harmful to humans and causes negative impacts on the surrounding environment (biotic and abiotic). The negative health effects associated with air pollution have been reported from both indoor and outdoor environments. Specifically, dust storms originating in Chinese and Mongolian desert areas introduce significant amounts of particulate matter (PM) to the Korean atmosphere. Previously, several studies reported that urban PM (UPM) is a potential agent that causes inflammation in the lungs by altering multiple signal transduction pathways; therefore, screening and identification of anti-inflammatory compounds against UPM-induced inflammation is an urgent requirement. In the present study, we attempted to study the anti-inflammatory properties of 3-Hydroxy-5,6-epoxy-β- ionone (HEBI), a pure compound isolated from invasive brown seaweed, Sargassum horneri (brown edible seaweed), against UPM-stimulated lung macrophages (MH-S). Anti-inflammatory parameters of HEBI were evaluated usingWestern blots, ELISA, RT-qPCR, and MTT assays. According to the results, HEBI at concentrations between 31.3 and 125 μg/mL reduced UPM-induced NO, PGE2, and pro-inflammatory cytokine production via blocking the downstream signal transduction of NF-κB and MAPKs. Specifically, HEBI down-regulated the mRNA expression levels of Toll-like receptors 2 and 4, which are well-known NF-κB and MAPKs stimulators. Taken together, HEBI is a potential candidate to develop functional foods and active ingredients in cosmeceuticals because of its profound effects against UPM-induced inflammation in MH-S macrophages.

β-catenin is a structural protein that makes the cell-cell connection in adherence junctions. Besides the structural functions, it also plays a role as a central transducer of the canonical Wnt signaling cascade, regulating nearly four hundred genes related to various cellular processes. Recently the immune functions of β-catenin during pathogenic invasion have gained more attention. In the present study, we elucidated the immune function of fish β-catenin by identifying and characterizing the β-catenin homolog (PhCatβ) from redlip mullet, Planiliza haematocheila. The complete open reading frame of PhCatβ consists of 2352 bp, which encodes a putative β-catenin homolog (molecular weight: 85.7 kDa). Multiple sequence alignment analysis revealed that β-catenin is highly conserved in vertebrates. Phylogenetic reconstruction demonstrated the close evolutionary relationship between PhCatβ and other fish β-catenin counterparts. The tissue distribution analysis showed the highest mRNA expression of PhCatβ in heart tissues of the redlip mullet under normal physiological conditions. While in response to pathogenic stress, the PhCatβ transcription level was dramatically increased in the spleen and gill tissues. The overexpression of PhCatβ stimulated M2 polarization and cell proliferation of murine RAW 264.7 macrophage. In fish cells, the overexpression of PhCatβ resulted in a significant upregulation of antiviral gene transcription and vice versa. Moreover, the overexpression of PhCatβ could inhibit the replication of VHSV in FHM cells. Our results strongly suggest that PhCatβ plays a role in macrophage activation and antiviral immune response in redlip mullet.

This study was conducted to examine digestibility of insect meals for Pacific white shrimp (Litopenaeus vannamei) and their utilization as fish meal substitutes. The tested insect meals were mealworm, silkworm, black soldier fly, rice grasshopper, two-spotted cricket, dynastid beetle and white-spotted flower chafer. Apparent digestibility coefficients of the tested insect meals were 83–89% for protein, 91–98% for lipid, 84–90% for energy, 77–81% for dry matter, 28–36% for chitin, 76–96% for amino acids and 89–93% for fatty acids. The amino acid availability of insect meals was high in taurine (93–96%), arginine (91–95%) and lysine (90–95%). Availability of fatty acids were 89–93% for saturated fatty acids, 90–93% for monounsaturated fatty acids and 88–93% for polyunsaturated fatty acids. For a feeding trial, a control diet was formulated using 27% tuna byproduct meal as a fish meal source and seven other diets were prepared replacing 10% tuna byproduct meal in the control diet with each insect meal. Triplicate groups of shrimp (initial body weight: 0.17 g) were fed the diets for 65 days. The growth performance was significantly improved when the shrimp were fed black soldier fly or dynastid beetle included diet. Dietary supplementation of insect meals significantly improved non-specific immune responses and antioxidant enzyme activity in the shrimp. These results indicate that the tested insect meals have high potentials to be used as a protein source that could replace fish meal in diets for the shrimp.

Skin is the largest organ of humans. Overexposure to ultraviolet (UV) is the primary environmental factor that causes skin damage. The compound, (-)-loliode, isolated from the brown seaweed Sargassum horneri, showed strong antioxidant and anti-inflammatory activities in in vitro and in vivo models. To further explore the potential of (-)-loliode in cosmetics, in the present study, we investigated the photoprotective effect of (-)-loliode in vitro in skin cells and in vivo in zebrafish. The results indicated that (-)-loliode significantly reduced intracellular reactive oxygen species (ROS) level, improved cell viability, and suppressed apoptosis of UVB-irradiated human keratinocytes. In addition, (-)-loliode remarkably attenuated oxidative damage, improved collagen synthesis, and inhibited matrix metalloproteinases expression in UVB-irradiated human dermal fibroblasts. Furthermore, the in vivo test demonstrated that (-)-loliode effectively and dose-dependently suppressed UVB-induced zebrafish damage displayed in decreasing the levels of ROS, nitric oxide, lipid peroxidation, and cell death in UVB-irradiated zebrafish. These results indicate that (-)-loliode possesses strong photoprotective activities and suggest (-)-loliode may an ideal ingredient in the pharmaceutical and cosmeceutical industries.

Remedies toward sustainable aquaculture rely upon research that unveils the molecular mechanisms behind host immunity and their interactions with pathogens. Antiviral defense is a major innate immune response in fish. The antiviral protein GCHV-induced gene-2 (Gig2), a member of the interferon-stimulated gene (ISG), was identified and characterized from rockfish (Sebastes schlegelii). Gig2 exists in two isoforms, namely, SsGig2-I1 and SsGig2-I2, in rockfish with lengths of 163 and 223 bp, respectively. Bioinformatic analysis indicated the availability of poly (ADP-ribose) polymerase domain in both proteins, and 51.3% identity and 71.3% similarity between both isoforms were observed. The basal expression pattern revealed the highest tissue-specific expression in rockfish gills for both isoforms. The immune challenge experiment disclosed a distinctive and strong expression of each transcript in the presence of poly I:C. Both isoforms are localized in the endoplasmic reticulum. Interferon (IFN) pathway gene analysis revealed no significant upregulation of IFN related genes. Viral hemorrhagic septicemia virus (VHSV) gene expression analysis revealed strong downregulation of viral transcripts after 48 h of infection in the presence of Gig2 isoforms. Collectively, these results indicate the protective role of Gig2 in rockfish against VHSV infection and help broaden our understanding of the innate immunity of fish.

In the rocky intertidal environment, the frequency and duration of heatwaves have increased over the last decade, possibly due to global climate change. Heatwaves often result in lethal or sub-lethal disturbances in benthic animals by changing their metabolic activities. In this study, we investigated the impacts of extreme heatwave stress on the hemocyte functions of Mytilisepta virgata and subsequent mortality to gain a better understanding of the potential causes and consequences of mass mortality events in this mussel during summer. We discriminated three types of hemocytes in the hemolymph, granulocytes, hyalinocytes, and blast-like cells, using flow cytometry and revealed that granulocytes were the major hemocyte involved in cellular defensive activities, such as phagocytosis and reactive oxygen species (ROS) production. For the experiment, mussels were exposed to a 40◦C air temperature for 12 h per day over 5 days under laboratory conditions as a simulated semi-diurnal tidal cycle. Mortality began to occur within 3 days after beginning the experiment, and all mussels had died by the end of the experiment. Flow cytometry indicated that the mussels exposed to high air temperatures produced significantly more ROS than did the control mussels within 2 days after the onset of the experiment, which may have caused oxidative stress. Such high levels of ROS in the hemolymph increased DNA damage in hemocytes after 3 days of exposure and decreased the phagocytosis of hemocytes 4 days after the experiment began. The observed mortality and decline in immune capacity suggested that an extreme heat event occurring in the rocky intertidal ecosystem during summer could exert sublethal to lethal impacts on macrobenthic animals.