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In contrast to the well-documented motility of monopisthocotyleans within the monogenean group, the mobility of polyopisthocotyleans, such as Microcotyle sebastis, has largely been undocumented with empirical evidence. This study aimed to investigate the locomotion of the polyopisthocotylean parasite of M. sebastis, which infests Korean rockfish (Sebastes schlegelii), thereby providing tangible evidence of early behavior in polyopisthocotyleans. The oncomiracidium of M. sebastis demonstrated movement facilitated by the hamulus and bilateral cilia, aiding in directional changes and propulsion. In the gill filaments of M. sebastis larvae utilized both the hamulus and their entire body for initial movements, effectively navigating between gill filaments. The hamulus played a crucial role in the locomotion of the early larval stages before the development of three clamp pairs, after which it began to degenerate. This study presents concrete evidence of early mobility in polyopisthocotyleans like M. sebastis, offering insights that could inform aquaculture management strategies, particularly in the mitigation of parasite-induced damages.

Abstract Background Salinomycin, an antibiotic, have potential as a veterinary drug for fish due to its anti-parasitic activity against several fish parasites. Thus the residual levels of salinomycin in muscles of two significant aquaculture species in Korea, olive flounder and black rockfish, were analyzed using HPLC-MS-MS. Results The proper method to analyze the residual salinomycin in fish muscles using LC-MS-MS was settled and the method was validated according to CODEX guidelines. The residues in three distinct groups for two fish species were analyzed using the matrix match calibration curves at points of five different times following oral administration. After oral administration, salinomycin rapidly breaks down in both olive flounder and black rockfish. After 7th days, the average residue in all groups of two fish spp. decreased below limit of quantitation (LOQ). Conclusion Due to low residue levels in fish muscles, salinomycin may therefore be a treatment that is safe for both fish and humans. This result could contribute to establishment of MRL (minimal residual limit) for approval of salinomycin for use in aquaculture.

ABSTRACT Streptococcus parauberis is the dominant etiological agent of streptococcosis, the most devastating bacterial disease in the olive flounder farming industry in South Korea. In this study, the distribution of serotypes, antimicrobial susceptibility, and presence of antimicrobial resistance genes (ARGs) in S. parauberis isolates obtained between 1999 and 2021 was thoroughly investigated to gain insight into the dynamics of their presence and the relationship between serotypes and antimicrobial resistance. Disk diffusion testing of 103 isolates against 10 antimicrobial agents was performed, and epidemiological cut-off values generated through normalized resistance interpretation analysis were used to classify wild-type (WT) and non-wild-type (NWT) populations. Principal component analysis and hierarchical clustering were implemented to achieve an understanding on the relationship between serotypes and antimicrobial resistance patterns. PCR-based serotyping showed that serotype Ia (67.1%) was the most prevalent in South Korea, followed by serotypes Ib/Ic (25.2%) and II (7.7%). The highest proportion of isolates was assigned to NWT against amoxicillin (80.6%), followed by oxytetracycline (77.7%) and erythromycin (48.5%). The time-scale data showed that recently obtained serotypes Ib/Ic and II isolates tended to be categorized as NWT populations resistant to more antibiotics, possibly due to microbial adaptation to antibiotic pressure. ARGs responsible for resistance to oxytetracycline and erythromycin were found only in NWT populations in serotype Ia [tet(S) and erm(B), respectively], and serotype II [tet(M) and mef(J)-msr(I), respectively]. We also found that the mef-msr gene pair in S. parauberis serotype II might be involved in low-level resistance to erythromycin. IMPORTANCE This study presents serotype distribution and antimicrobial susceptibility data along with the antimicrobial resistance genes (ARGs) of Streptococcus parauberis, which is an important bacterial fish pathogen worldwide. In particular, almost all oxytetracycline and erythromycin non-wild-type (NWT) populations harbored tet(S) or tet(M), and erm(B) or mef(J)-msr(I), respectively. Interestingly, these ARGs were distributed in a highly serotype-dependent manner, resulting in a clear correlation between the antibiogram and serotype distribution. Moreover, recent isolates belonging to serotypes Ib/Ic and II tended to be more frequently categorized as NWT against antimicrobials, including amoxicillin and cefalexin compared to old isolates, while a dramatic decrease in erythromycin and clindamycin NWT frequencies was observed in recent serotype Ia isolates, which lacked erm(B). These variations might be attributed to shifts in the antibiotics employed in South Korean aquaculture over time. The overall findings would provide important background knowledge for understanding the epidemiology of S. parauberis infection in aquaculture.

CD96 is a membrane-bound receptor discovered in humans in 1992 that is mainly present in natural killer cells and T cells derived from haematopoietic cells and performs immune functions. Based on the sequence of CD96 obtained from red seabream (Pagrus major), phylogenetic analysis with other species, infections of normal fish, Streptococcus iniae and red sea bream iridovirus (RSIV), and expression analysis was conducted using real-time polymerase chain reaction. Phylogenetic analysis showed the highest homology with Sparus aurata, and multiple sequence analysis confirmed the conservation of major domains between different fish species. Normal fish high expression results were confirmed in the head kidney, and spleen, which are the haematopoietic organs of the fish. High expression levels were confirmed in the gills, liver, spleen, and kidney on day three after RSIV infection. After S. iniae infection, high expression was confirmed in the gills and liver on day one, and high expression was confirmed in the spleen from 12 hours. These results show that PmCD96 functions as an immune gene in P. major and is considered a basic research case for CD96 in fish’s hematopoietic organ immune system.

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease in salmonids and rainbow trout fry syndrome. This pathogen has attained a global presence and can spread both horizontally and vertically. However, it was not documented in Korea before September 2018. In this study, the objectives were to characterize Flavobacterium psychrophilum strain FPRT1, isolated from diseased rainbow trout genotypically and phenotypically. We also conducted various investigations to better understand its impact and assess potential control measures. We acquired fifty rainbow trout (approximately 70 g in weight) and transferred them to a laboratory aquarium. During the initial acclimation period, we observed mortality and examined affected fish for clinical signs. We isolated the bacterium from the spleen of infected rainbow trout using tryptone yeast extract salts agar supplemented with glucose, naming this FPRT1. Antibiotic susceptibility testing was carried out, and from the result, we selected enrofloxacin to administer to the trout orally to reduce mortality. To evaluate pathogenicity, we exposed the trout to FPRT1 at different water temperatures (8, 15, and 22 °C). Genomic analysis was conducted to identify the serotype and relatedness of FPRT1 to European strains. Affected fish displayed clinical signs, such as ulcerative lesions in the mandible, anemia with pale gills, exophthalmia, and increased mucus secretion. Internal symptoms included pale liver and enlarged spleen. FPRT1 was susceptible to erythromycin, enrofloxacin, florfenicol, oxytetracycline, and gentamicin, but resistant to oxolinic acid and sulfamethoxazole/trimethoprim. Oral administration of enrofloxacin resulted in a decrease in mortality from 28% to 6%. Pathogenicity tests revealed varying mortality rates due to FPRT1 at different temperatures. The highest rates were observed at 8 °C (ranging from 43% to 100%) for both intraperitoneal and intramuscular injections, and lower rates occurred at 22 °C (ranging from 0% to 30%), with intramuscular injections displaying higher susceptibility. Genomic analysis identified FPRT1 as serotype 2 and indicated its close genetic relationship with European strains based on the core genome and dispensable genome. The substantial genomic similarity between our strain and European strains suggests the possibility of bacterial spread through the importation of fertilized eggs from Europe. In conclusion, this study highlights the introduction of the previously undocumented pathogen (F. psychrophilum) into Korean rainbow trout populations. The detection of this pathogen and its pathogenicity assessment is not only important for understanding its impact on local aquaculture but also for establishing surveillance and control measures to prevent further transmission and outbreaks in the region.

This study detected two potential pathogens, Vibro parahaemolyticus, which causes acute hepatopancreatic necrosis disease (AHPND), and white spot syndrome virus (WSSV), in fishing bait in South Korea. However, their infectious nature was not confirmed, possibly due to the degradation caused by freezing/thawing or prolonged storage under frozen conditions. While infectivity was not confirmed in this study, there is still a significant risk of exposure to these aquatic products. Furthermore, fishing bait and feed should be handled with caution as they are directly exposed to water, increasing the risk of disease transmission. In Australia, cases of WSSV infection caused by imported shrimp intended for human consumption have occurred, highlighting the need for preventive measures. While freezing/thawing is a method for inactivating pathogens, there are still regulatory and realistic issues to be addressed.

Aquaculture, a crucial sector of the global food industry, faces a myriad of issues due to parasitic invasions. One such parasite, Microcotyle sebastis, which afflicts Korean rockfish in South Korea, has a significant economic impact. The impending danger of resistance to traditional anthelmintics necessitates the exploration of new antiparasitic candidates. Although the efficacy of salinomycin against aquatic parasites such as ciliates and sporozoans is known, its influence on monogeneans has yet to be studied. Therefore, this study investigated the efficacy and safety of salinomycin for the treatment of M. sebastis infections, presenting the first exploration of salinomycin’s therapeutic potential against monogeneans. In vitro examinations revealed a minimum effective concentration of salinomycin of 5 mg/kg, which led to necrosis of the haptor upon dislodging from the gill filaments. The one-time oral administration of the drug at concentrations of 5 mg/kg and 10 mg/kg showed a significant dose-dependent reduction in parasite counts, with no apparent behavioral side effects in Korean rockfish. Biochemical analyses monitored the liver, heart, and kidney enzymes, specifically aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatine kinase–myocardial band (CK-MB). At both 20 °C and 13 °C, no significant differences were observed in the levels of AST and ALT. However, at 20 °C, alterations in BUN levels were evident on Day 14, a deviation not observed at 13 °C. The CK-MB analysis revealed elevated enzyme levels at both temperatures when compared to the control group, reflecting the similar changes observed in terrestrial animals administered salinomycin. The biochemical data suggest that the oral administration of salinomycin is potentially more favorable at 13 °C than at 20 °C. Although our findings warrant further comprehensive studies, including on the long-term and potential effects on nontarget species and water quality, they also suggest that salinomycin could be considered as an alternative or adjunctive treatment if resistance to the currently used praziquantel against M. sebastis is confirmed.

Seahorses, which have been cultivated since the 2000s, are economically very important. Gas bubble disease (GBD) is a significant concern in the cultivation of seahorses; therefore, this study aimed to determine the cause of GBD-induced death in two species of Syngnathidae (Syngnathus schlegeli and Hippocampus haema). Rod-shaped bacteria were observed histopathologically and identified as Vibrio splendidus by conventional and real-time PCR analyses. The lethality of V. splendidus varies depending on the host’s immune status, and the disease can be prevented through water quality management or improvement of the breeding environment. In this study, the GBD lesions (gas bubbles) were observed at 12°C, 8.0 mg/L of dissolved oxygen, 30 ppt of salinity, and pH 7.7. In addition, rod-shaped bacteria, infiltration of inflammatory cells, and extensive serous exudate were confirmed in the lesions where gas bubbles were found. PCR analysis was able to detect V. splendidus, possibly a secondary infection of the immunocompromised syngnathid fish. Understanding the risk of immunity control and the correlation between these lesions and causal agents will be of great help to the aquaculture industry and the ornamental fish market.

The oomycete Aphanomyces invadans causes epizootic ulcerative syndrome (EUS), a World Organization for Animal Health (WOAH)-listed disease that has seriously impacted a wide range of fish worldwide. Currently, only three conventional polymerase chain reaction (PCR) assays are recommended for the detection of A. invadans. The robust quantitative PCR (qPCR) assay has recently become more important due to its highly accurate nature and the applicability of qPCR-based environmental DNA (eDNA) detection in the monitoring of pathogens in aquatic environments. Therefore, in this study, we developed a novel TaqMan probe-based qPCR method to sensitively and quantitatively detect A. invadans. The assay limit of detection was determined using 10-fold serial dilutions of linearized A. invadans plasmid. Assay sensitivity was assessed in the presence of interfering substances and compared to three WOAH-listed primers using the mycelia and zoospores of A. invadans with and without fish muscle tissue. The assay specificity was also theoretically and experimentally assessed against other oomycetes, fish muscle tissue, and water samples. The assay's repeatability and reproducibility were determined. In this study, the limit of detection of the developed assay was 7.24 copies of A. invadans genomic DNA per reaction (95% confidence interval (CI): 2.75 to 19.05 copies/reaction). The assay showed the same sensitivity in the presence of other substances. Compared to the WOAH-recommended PCR assays, this assay had 10-times higher sensitivity for all tested samples. There were no cross-reactions with other closely related oomycetes, fish muscle, or water samples, indicating that the assay was highly specific for A. invadans. The repeatability and reproducibility tests showed little variation, ranging from 0.1-0.9% and 0.04-1.1%, respectively, indicating the high consistency, repeatability, and reliability of the developed assay. This highly rapid, sensitive, specific, and consistent EUS qPCR assay would be of importance in transboundary disease management and the monitoring of pathogens in aquatic environments.

Interferon regulatory factor (IRF) 3 and IRF7 are important innate immune-related genes that induce type I interferon (IFN). In this study, PmIRF3 and PmIRF7 were identified from red sea bream (Pagrus major) infected by red sea bream iridovirus (RSIV), and expression and functional analyses were performed. PmIRF3 and PmIRF7 consisted of 465 and 436 amino acids, respectively, and contained a well-conserved IRF domain, IRF3 domain, and serine-rich domain (SRD). PmIRF3 and PmIRF7 are ubiquitously expressed in the liver and gills in normal fish, and with a high expression being recorded in kidney and spleen during RSIV infection. In P. major fin cells (PMF cells), pAcGFP-PmIRF3 and pAcGFP-PmIRF7 were localized to the cytoplasm, and overexpression of PmIRF3 and PmIRF7 using the pcDNA3.1 vector induced type I IFN. In addition, in the experiment in vitro, PMF cells overexpressed with PmIRF3 or PmIRF7 exhibited antiviral activity against RSIV and significantly reduced viral genome copies and virus titer. Therefore, PmIRF3 and PmIRF7 may have antiviral activity through the induction of type I IFN against RSIV infection of red sea bream.

Parasitic infections pose significant challenges in aquaculture, and the increasing resistance to conventional anthelmintics necessitates the exploration of alternative treatments. Levamisole hydrochloride (HCl) has demonstrated efficacy against monogenean infections in various fish species; however, research focused on Microcotyle sebastis infections in Korean rockfish (Sebastes schlegelii) remains limited. Therefore, this study aimed to evaluate the efficacy of levamisole HCl against M. sebastis infections in Korean rockfish with the goal of optimizing anthelmintic usage in aquaculture. In this study, we first assessed the susceptibility of M. sebastis to levamisole HCl in vitro. Subsequently, in vivo evaluations were conducted to assess the drug’s efficacy, safety, and to identify optimal administration methods. In vitro experiments revealed concentration-dependent sensitivity of M. sebastis to levamisole HCl, with a minimum effective concentration (MEC) of 100 mg/L. In vivo experiments employed oral administration, intraperitoneal injection, and immersion treatments based on the MEC. Oral administration proved to be a safe method, yielding efficacy rates of 27.3% and 41.6% for 100 mg/kg and 200 mg/kg doses, respectively, in contrast to the immersion and injection methods, which induced symptoms of abnormal swimming, vomiting, and death. Biochemical analyses conducted to assess the safety of levamisole HCl revealed a transient, statistically significant elevation in the levels of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) on day three post-administration at 20 °C. Following this, no substantial differences were observed. However, at 13 °C, the enzyme levels remained relatively consistent, emphasizing the role of water temperature conditions in influencing the action of levamisole HCl. Our research findings substantiate the efficacy of levamisole HCl against M. sebastis in Korean rockfish, underscoring its potential for safe oral administration. These results provide valuable insights for developing parasite control strategies involving levamisole HCl in Korean rockfish populations while minimizing adverse impacts on fish health and the environment. However, this study bears limitations due to its controlled setting and narrow focus. Future research should expand on these findings by testing levamisole HCl in diverse environments, exploring different administration protocols, and examining wider temperature ranges.

Red sea bream iridovirus (RSIV) causes significant economic losses in the aquaculture industry. We analyzed the pathogenicity of RSIV in flathead grey mullets (Mugil cephalus), the correlation of histopathological lesions, and interspecies horizontal transmission, through immersion infection and cohabitation challenges. Flathead grey mullets, which were challenged by immersion infection, exhibited mortality at 14 and 24 days after RSIV exposure. Viral shedding in seawater peaked 2–3 days before or after the observed mortality. Specific lesions of RSIV were observed in the spleen and kidney, and the correlation between histopathological grade and viral load was the highest in the spleen. In a cohabitation challenge, flathead grey mullets were the donors, and healthy rock bream, red sea bream, and flathead grey mullets were the recipients. Viral shedding in seawater was the highest in flathead grey mullet and rock bream at 25 °C, with 106.0 RSIV copies L/g at 14 dpi. No mortality was observed in any group challenged at 15 °C, and no RSIV was detected in seawater after 30 dpi. The virus shed from RSIV-infected flathead grey mullets caused horizontal transmission through seawater. These findings suggest that rapid decision-making is warranted when managing disease in fish farms.

Ubiquitin-40S ribosomal protein S27a (RPS27A), ubiquitin-like protein Fubi, and ribosomal protein (S30FAU) are ubiquitin-related proteins that are involved in the regulation of immune-related functions such as cell cycle, protein expression, and apoptosis. This study aimed to confirm the molecular characteristics, gene expression analysis, and antibacterial activity of RPS27A and S30FAU identified from the starry flounder (15 starry flounders of 128.7 ± 18.2 g). An expression analysis using a normal fish showed that RPS27A was highly expressed in the head kidney, heart, and stomach. In contrast, S30FAU exhibited high expression in the stomach, heart, and head kidney. Upon simulating an artificial pathogen infection, RPS27A was highly expressed in the heart at 1 h and 3 days post-viral hemorrhagic septicemia (VHSV) infection, and had a high expression in the kidney, liver, and heart at 7 days post-Streptococcus parauberis (S. parauberis) infection. S30FAU was highly expressed in the spleen and gills at 1 day and 12 h post-VHSV infection, respectively, and exhibited a high expression in the kidney at 7 days post-S. parauberis infection. In an MIC analysis, RPS27A and S30FAU showed antimicrobial activity against all bacteria used in this study. In the biofilm assay, S30FAU was removed from S. parauberis in a concentration-dependent manner, and the cytotoxicity test showed no hemolytic activity in both RPS27A and S30FAU. Therefore, RPS27A and S30FAU of the starry flounder were confirmed to possess antimicrobial peptide abilities without limitations of cytotoxicity. This study provides valuable information on the antibacterial ability and molecular biology of the ubiquitin family isolated from the starry flounder.

Red sea bream iridovirus (RSIV) causes significant economic losses in aquaculture. Here, we analyzed the pathogenicity, viral shedding, and transmission dynamics of RSIV in rock bream (Oplegnathus fasciatus) by employing immersion infection and cohabitation challenge models. Rock bream challenged by immersion exposure exhibited 100% mortality within 35 days post RSIV exposure, indicating that the viral shedding in seawater peaked after mortality. At 25 °C, a positive correlation between the viral loads within infected rock bream and virus shedding into the seawater was observed. Specific RSIV lesions were observed in the spleen and kidney of the infected rock bream, and the viral load in the spleen had the highest correlation with the histopathological grade. A cohabitation challenge mimicking the natural transmission conditions was performed to assess the virus transmission and determine the pathogenicity and viral load. The RSIV-infected rock breams (donors) were cohabited with uninfected rock bream, red sea bream (Pagrus major), and flathead grey mullet (Mugil cephalus) (recipients) at both 25 °C and 15 °C. In the cohabitation challenge group maintained at 15 °C, no mortality was observed across all experimental groups. However, RSIV was detected in both seawater and the recipient fish. Our results provide preliminary data for further epidemiological analyses and aid in the development of preventive measures and management of RSIVD in aquaculture.

Interferon regulatory factors (IRFs) play crucial roles in antiviral processes, such as in the transcriptional induction of interferon (IFN) and IFN-stimulated genes (ISGs). In this study, the genes encoding IRF5 and IRF6 were identified in Pagrus major, and their expression in various organs after pathogen infection was analyzed. In the coding sequences of P. major (Pm)IRF5 and PmIRF6, the DNA binding domain, IRF association domain, and viral-activated domain were found to be highly conserved. Phylogenetic analysis revealed that PmIRF5 and PmIRF6 were most closely related to IRF5 and IRF6 of large yellow croakers. The mRNAs for PmIRF5 and PmIRF6 were constitutively expressed in all organs analyzed but were highly expressed in the liver and gills. As a result of an infection with red sea bream iridovirus, a major pathogen of red sea bream, PmIRF5 and PmIRF6 expression was significantly upregulated in the spleen and kidney. On the basis of these results, it can be concluded that IRF5 and IRF6 expression play an influential role in the immune system of red sea bream infected with viruses.

Background: The role of dominant frequency (DF) in tracking the efficiency of a stepwise catheter ablation (step-CA) in persistent atrial fibrillation (peAF) remains poorly studied. We hypothesized that the DF time-course during step-CA displays divergent patterns between patients in whom a step-CA successfully restores long-term sinus rhythm (SR) and those with recurrence.Methods: This study involved 40 consecutive patients who underwent a step-CA for peAF (sustained duration 19 ± 11 months). Dominant frequency was computed on electrograms recorded from the right and left atrial appendages (RAA; LAA) and the coronary sinus before and during the step-CA synchronously to the 12-lead ECG. Dominant frequency was defined as the highest peak within the power spectrum.Results: Persistent atrial fibrillation was terminated by a step-CA in 28 patients [left-terminated (LT)], whereas 12 patients remaining in AF after ablation [not left-terminated (NLT)] were cardioverted. Over a mean follow-up of 34 ± 14 months, all NLT patients had a recurrence. Among the 28 LT patients, 20 had a recurrence, while 8 remained in SR throughout follow-up. The RAA and V1 DF had the best predictive values of the procedural failure to terminate AF (area under the curve; AUC 0.84, p < 0.05). A decision tree model including a decrease in LAA DF ≥ 6.61% during the first 20 min following pulmonary vein isolation (PVI) and a baseline RAA DF

The analytical and diagnostic performances of methods for detecting red sea bream iridovirus (RSIV), which infects marine fish, have not been evaluated. As disease management and transmission control depend on early and reliable pathogen detection, rapid virus detection techniques are crucial. Herein, we evaluated the diagnostic performance of a TaqMan-based real-time polymerase chain reaction (PCR) assay that detects RSIV rapidly and accurately. The assay amplified the RSIV, infectious spleen and kidney necrosis virus, and turbot reddish body iridovirus genotypes of Megalocytivirus and the detection limit was 10.96 copies/reaction. The assay’s performance remained uncompromised even in the presence of nine potential PCR inhibitors, including compounds commonly used in aquaculture. The variation of the cycle threshold values between assays performed by three technicians was evaluated using a plasmid DNA containing the major capsid protein gene sequence. The variation between replicates was low. The diagnostic sensitivity and specificity of the developed assay were evaluated using fish samples (n = 510) and were found to be 100% and 99.60%, respectively. Two technicians evaluated the reproducibility of the assay using fish samples (n = 90), finding a high correlation of 0.998 (p < 0.0001). Therefore, the newly developed real-time PCR assay detects RSIV both accurately and rapidly.

Ichthyophthirius multifiliis is a major pathogen that causes a high mortality rate in trout farms. However, systemic responses to the pathogen and its interactions with multiple organs during the course of infection have not been well described. In this study, dual-organ transcriptomic responses in the liver and head kidney and hemato-serological indexes were profiled under I. multifiliis infection and recovery to investigate systemic immuno-physiological characteristics. Several strategies for massive transcriptomic interpretation, such as differentially expressed genes (DEGs), Poisson linear discriminant (PLDA), and weighted gene co-expression network analysis (WGCNA) models were used to investigate the featured genes/pathways while minimizing the disadvantages of individual methods. During the course of infection, 6,097 and 2,931 DEGs were identified in the head kidney and liver, respectively. Markers of protein processing in the endoplasmic reticulum, oxidative phosphorylation, and the proteasome were highly expressed. Likewise, simultaneous ferroptosis and cellular reconstruction was observed, which is strongly linked to multiple organ dysfunction. In contrast, pathways relevant to cellular replication were up-regulated in only the head kidney, while endocytosis- and phagosome-related pathways were notably expressed in the liver. Moreover, interestingly, most immune-relevant pathways (e.g., leukocyte trans-endothelial migration, Fc gamma R-mediated phagocytosis) were highly activated in the liver, but the same pathways in the head kidney were down-regulated. These conflicting results from different organs suggest that interpretation of co-expression among organs is crucial for profiling of systemic responses during infection. The dual-organ transcriptomics approaches presented in this study will greatly contribute to our understanding of multi-organ interactions under I. multifiliis infection from a broader perspective.

Red sea bream iridoviral disease (RSIVD) causes serious economic losses in the aquaculture industry. In this paper, we evaluated RSIV kinetics in rock bream under various rearing water temperatures and different RSIV inoculation concentrations. High viral copy numbers (approximately 103.7–106.7 RSIV genome copies/L/g) were observed during the period of active fish mortality after RSIV infection at all concentrations in the tanks (25 °C and 20 °C). In the group injected with 104 RSIV genome copies/fish, RSIV was not detected at 21–30 days post-infection (dpi) in the rearing seawater. In rock bream infected at 15 °C and subjected to increasing water temperature (1 °C/d until 25 °C) 3 days later, the virus replication rate and number of viral copies shed into the rearing seawater increased. With the decrease in temperature (1 °C/d) from 25 to 15 °C after the infection, the virus replicated rapidly and was released at high loads on the initial 3–5 dpi, whereas the number of viral copies in the fish and seawater decreased after 14 dpi. These results indicate that the number of viral copies shed into the rearing seawater varies depending on the RSIV infection level in rock bream.

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus that causes high mortality in cultured flounder. Naturally occurring VHSV strains vary greatly in virulence. Until now, little has been known about genetic alterations that affect the virulence of VHSV in flounder. We recently reported the full-genome sequences of 18 VHSV strains. In this study, we determined the virulence of these 18 VHSV strains in flounder and then the assessed relationships between differences in the amino acid sequences of the 18 VHSV strains and their virulence to flounder. We identified one amino acid substitution in the phosphoprotein (P) (Pro55-to-Leu substitution in the P protein; PP55L) that is specific to highly virulent strains. This PP55L substitution was maintained stably after 30 cell passages. To investigate the effects of the PP55L substitution on VHSV virulence in flounder, we generated a recombinant VHSV carrying PP55L (rVHSV-P) from rVHSV carrying P55 in the P protein (rVHSV-wild). The rVHSV-P produced high level of viral RNA in cells and showed increased growth in cultured cells and virulence in flounder compared to the rVHSV-wild. In addition, rVHSV-P significantly inhibited the induction of the IFN1 gene in both cells and fish at 6 h post-infection. An RNA-seq analysis confirmed that rVHSV-P infection blocked the induction of several IFN-related genes in virus-infected cells at 6 h post-infection compared to rVHSV-wild. Ectopic expression of PP55L protein resulted in a decrease in IFN induction and an increase in viral RNA synthesis in rVHSV-wild-infected cells. Taken together, our results are the first to identify that the P55L substitution in the P protein enhances VHSV virulence in flounder. The data from this study add to the knowledge of VHSV virulence in flounder and could benefit VHSV surveillance efforts and the generation of a VHSV vaccine.

The data presented here are related to the research article entitled “Temperature-dependent immune response of olive flounder (Paralichthys olivaceus) infected with viral hemorrhagic septicemia virus (VHSV)” [1]. In the cited article, we sequenced the whole transcriptome of the olive flounder using Illumina RNA-Seq. Differentially expressed genes (DEG) analysis of VHSV infected head kidney samples showed perturbations in gene expression. Herein we made a comparison of DEGs at early stage of VHSV infection of olive flounder (4 h post infection) in colder (13 °C) and warmer (20 °C) temperatures. The analysis of signaling pathways showed that several major immune pathways were altered. The gene ontology terms associated with the genes differentially expressed are also presented. Keywords: Paralichthys olivaceus, Olive flounder, Transcriptome, RNA-Seq, Viral hemorrhagic septicemia virus (VHSV), Head kidney

Antimicrobial peptides (AMPs) are known to play a role as a first line of defence against microbial invasion. Liver Expressed Antimicrobial Peptides-2 (LEAP-2) is one of the AMPs. LEAP-2 includes four highly conserved cysteine residues and belongs to a cysteine-rich peptides group. We identified and characterized the molecular properties of LEAP-2 in rock bream. The expression levels of rock bream LEAP-2 (RbLEAP-2) in the 12 different tissues of healthy fish and the RbLEAP-2 expression pattern after infections with Edwardsiella piscicida (E. piscicida), Streptococcus iniae (S. iniae) and red seabream iridovirus (RSIV) were examined. This data provide that RbLEAP-2 plays an important role in innate immunity when rock bream is infected with a pathogen. Keywords: Rock bream, Liver expressed antimicrobial Peptide-2, Innate immune system, Antimicrobial peptides, Antimicrobial activity

There is technology available for anti-thrombus with earthworms, but the procedure is complex and extracts protein with inferior purity. In order to develop a simplified process with a stronger purity of protease, we investigated the Lumbricus rubellus earthworm and Perinereis linea lugworm. We purified water extracts cut off at 10 kDa of molecular weight using ultrafiltration because proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues. We purified EW1 (raw earthworm extract), EW2 (molecular weight (m.w) > 10 kDa of earthworm extract), and EW3 (m.w 10 kDa), and LW3 (m.w EW1 > EW3 > LW2 > LW1 > LW3. In particular, EW2 and LW2 showed a potent fibrinolytic effect in two different worm specimens. The protein content of each sample was detected as 2.34 (EW1), 3.03 (EW2), 2.80 (LW1), 3.71 (LW2) mg/ml respectively, and their molecular weights were measured using SDS-PAGE. The samples contained the following amounts of total fatty acids: EW1, 3.61%; EW2, 0.48%; LW1, 4.96%; and LW2, 0.23%.We developed a process to increase the thrombolytic effect with a higher purity protein. The study results demonstrate this procedure and provide basic data for developing an anti-thrombolytic agent. Keywords: Lumbricus rubellus, Perinereis linea, Fibrinolytic effect, Fatty acid

C-reactive protein (CRP) is a member of the pentraxin family and is an acute-phase response to plasma protein; its level in blood increases rapidly in response to trauma, inflammation, and infection. In the present study, we analysed the molecular characteristics of the Oplegnathus fasciatus CRP (RbCRP) gene sequence using multiple alignments and phylogenetic analyses of the deduced amino acids. In addition, we also examined RbCRP gene expression in rock bream infected with the pathogens Edwardsiella piscicida (E. piscicida), Streptococcus iniae (S. iniae) or red sea bream iridovirus (RSIV) and in healthy rock bream individuals. In healthy individuals, RbCRP was ubiquitously expressed in all 14 tested tissues, mainly in the trunk kidney and head kidney. Expression of RbCRP was notably upregulated in the spleen and whole kidney after RSIV infection. This study can provide basic data on the innate immune system of the rock bream to viral and microbial infections. Keywords: C-reactive protein (CRP), Rock bream, Edwardsiella piscicida, Streptococcus iniae, Red sea bream iridovirus (RSIV)

Streptococcus parauberis is a known etiologic agent that causes damage leading to death in flatfish (paralichthys olivaceus). Liposomes were used to deliver streptococcal oral vaccines to the intestinal mucous membranes of paralichthys olivaceus. The liposomes were coated for stabilization, and stability was measured with high performance liquid chromatography (HPLC). The liposomes were stable until day nine and were orally administered to flatfish as a vaccine. The resultant antibody titers were analyzed. The titers resulting from the uncoated liposomes were highest two weeks after the oral administration, and those resulting from the coated liposomes were highest one week after boosting. In addition, the bacteria were subcutaneously injected to artificially infect flatfish and the survival rates and relative survival rates were analyzed. The coated liposomes were found to yield the highest survival rate.

We provide raw data from a transcriptomic analysis of olive flounder in response to changes in water temperature. At the time of this analysis, the olive flounder genome was not yet available in China, and there were no related references. Therefore, assembly was carried out using the de novo method to reveal the entire nucleotide sequence based on the nucleotide sequence information of the sequenced reads. The functions of expressed genes based on Gene Ontology analysis are also categorized and presented. Keywords: RNA-seq data, Olive flounder, Water temperature stimulation

The interferon-related developmental regulator 1 (IFRD1) protein is expected to play a role in the regulation of inflammatory responses in adult mice, since it is known to repress transcription of NF-κB in myoblasts that regenerate skeletal muscle after traumatic injury Micheli et al., 2011. The IFRD2 gene is expressed in many tissues including skeletal muscle, kidney, heart, brain, lung, placenta and liver in adult humans and is highly expressed in adult human skeletal muscle and heart. In mice, interferon-related developmental regulator 2 (IFRD2) may be associated with early haematopoiesis after gastrulation and in the hepatic primordium Buanne et al., 1998. In this study, we analysed the molecular characteristics of the IFRD2 gene identified from Pagrus major (PmIFRD2) and performed multiple alignments and phylogenetic analyses of the protein sequence. In addition, we examined the expression pattern of IFRD2 in healthy red sea bream tissues and the temporal expression pattern after challenging with various pathogens [Edwardsiella piscicida (E. piscicida), Streptococcus iniae (S. iniae) and red sea bream iridovirus (RSIV)]. This study characterises the non-specific immune response of the red sea bream after viral and microbial infections. Keywords: Interferon-related developmental regulator 2 (IFRD2), Red sea bream, Edwardsiella piscicida, Streptococcus iniae, Red sea bream iridovirus (RSIV)

Secretory carrier membrane proteins (SCAMPs) are widely distributed integral membrane proteins implicated in membrane trafficking. Secretory carrier membrane protein 5 (SCAMP5) is expected to be involved in regulation of the immune response because it is expressed in a variety of immune tissues and promotes the secretion of cytokines in monocytes and macrophages. In this study, we performed an analysis of the molecular characteristics and phylogenetic of the SCAMP5 gene identified in Pagrus major (PmSCAMP5). In addition, we analysed PmSCAMP5 gene expression levels in the tissues of red sea bream infected with various pathogens [Edwardsiella piscicida (E. piscicida), Streptococcus iniae (S. iniae) and Red sea bream iridovirus (RSIV)], and we analysed PmSCAMP5 gene expression levels in the tissues of healthy red sea bream. This study was carried out to provide basic data on the non-specific immune system of the red sea bream. Keywords: Secretory carrier membrane protein 5, Red sea bream, Edwardsiella piscicida, Streptococcus iniae, Red sea bream iridovirus

CXCL10 plays an important role in angiogenesis and inhibits the differentiation of endothelial cells into capillaries. It also plays an important role in the generation and transmission of effector T cell responses and the recruitment of T cells to inflammatory sites. In this article, we constructed cDNAs to identify and analyse the CXCL10 domain, and performed multiple alignments and a phylogenetic analysis to determine homology with other animals. Real-time PCR was performed to confirm construction and expression after bacterial and viral infection.

This data article reports the expression level of T-cell surface antigen CD2 gene in organs from normal rock bream through quantitative real-time PCR. We also report the expression level of CD2 gene when anthropogenic infection with bacterial or viral pathogens was induced. The expression pattern of CD2 gene in normal rock bream was proved to be highly expressed in hematopoietic cells involved in the production and development of peripheral blood leukocytes (PBLs) and immune cells. It also proved that it maintains high expression for normal immunity in gills, skin, and intestines exposed directly to the environment. The expression pattern of CD2 gene in pathogenic infection has proven that CD2 is a factor involved in immunity. These data are considered to be a basic study of teleost immune system and will contribute to the study of fish blood cells.

Nuclear factor-kappaB (NF-κB) repressing factor (NKRF) specifically inhibits the transcriptional activity of NF-κB protein. The PmNKRF cDNA is composed of 757 amino acid residues. Alignment analysis revealed that the G-patch and R3H domains are conserved in different organisms. We aimed to analyse red sea bream NKRF (PmNKRF) gene expression after infection with pathogens [Streptococcus iniae or red sea bream iridovirus (RSIV)] and in healthy individuals. In healthy individuals, PmNKRF was ubiquitously expressed in all 12 tested tissues, predominantly in the head kidney and spleen. Expression of PmNKRF was significantly up-regulated in the gills, kidney, liver and spleen after RSIV infection. After S. iniae infection, PmNKRF expression was significantly down-regulated in the gills and significantly up-regulated in the kidney, liver and spleen. Keywords: NF-κB repressing factor, Pagrus major, Streptococcus iniae, Red sea bream iridovirus

This article provides efficacy and safety data of ceftiofur antibiotics against streptococcal infection in starry flounder. Ceftiofur, which is a veterinary antibiotics, is effective against fishery bacteria. Ceftiofur can be prescribed and sold by veterinarians. However, it is illegal in South Korea for fishery disease managers to prescribe and sell ceftiofur. Therefore, in order to utilize available antibiotics and prevent illegal use of veterinary antibiotics, it is necessary to perform research to determine the recommended effective dose and administration methods of antibiotics for fisheries. In this article, the appropriate concentration and injection method of antibiotics to treat starry flounder infected with S. parauberis PH0710 were provided. In addition, histopathological examination results were provided to confirm the effect of antibiotics on the host tissue. Accordingly, these data could be used as basic data for the application of ceftiofur antibiotics in disease management for fisheries. Keywords: Antibiotics, Ceftiofur, Starry flounder, Streptococcus parauberis PH0710, Efficacy, Safety

Galectin (Gal) is a member of a family of β-galactoside-binding lectin. The members of this family play important roles in the recognition of carbohydrate ligands and in various other biological processes. In this study, we identified the gene encoding Gal-9 in Pagrus major (PmGal-9) and analyzed its expression in various tissues after pathogen challenge. Alignment analysis revealed that the two galactose-binding lectin domains of the deduced protein were highly conserved among all the teleosts. Phylogenetic analysis revealed that PmGal-9 is most closely related to the Gal-9 gene of gilthead sea bream. PmGal-9 was ubiquitously expressed in all tissues analyzed but was predominantly expressed in the spleen, head kidney, and intestine. After challenges with major microbial pathogens (Edwardsiella piscicida, Streptococcus iniae, or red sea bream iridovirus) of red sea bream, PmGal-9 mRNA expression was significantly regulated in most immune-related tissues. These results suggested that PmGal-9 not only plays an important role in the immune system of red sea bream but is also a possible inflammatory marker for pathogenic diseases.

The consumption of fish and shellfish worldwide is steadily increasing, and tuna is a particularly valuable fish species. However, infection caused by Kudoa spp. is causing problems in many fish including the Pacific bluefin tuna (Thunnus orientalis), and there is much controversy about the association of these infections with foodborne disease. In this study, using haematological and histological analyses of the blood and internal organs (liver, spleen, kidney, heart, stomach, intestine, gill, and muscle) of Pacific bluefin tuna cultured in South Korea, infection with Myxosporea was first identified, and molecular biological analysis was conducted. In this study, Kudoa hexapunctata was finally identified. The Pacific bluefin tunas analysed in this study did not show any gross pathology lesions, such as visible cysts and/or myoliquefaction, of infection with this species. The histological analytical results can provide guidelines for the identification of K. hexapunctata. In the case of K. hexapunctata-induced infection, unlike other countries, such as Japan, there have been no reports in South Korea, and this study is the first to detect K. hexapunctata infection in Pacific bluefin tuna cultured in South Korea. The correlation between K. hexapunctata and food poisoning is not yet clear, however, it is thought that continuous observation of its infection is necessary.

Although over-nutrition from overfeeding-induced obesity is known to be highly associated with metabolic and immunological disorders in humans, little is known about overfeeding-induced obesity in fish farming. The purpose of this study was to investigate changes in immuno-physiological parameters, to better understand the potential risk of overfeeding–induced obesity in fish. Commercial feed was provided to fish in the overfed group until they refuse to eat, but fish in the control group was fed with the feed at 1% bodyweight per day. The hemato-serological, histological, and immunological changes were observed at weeks 2 and 8. Rainbow trout leukocytes were co-incubated with oxidized low-density lipoprotein (OxLDL), and the phagocytes engulfing the OxLDL and the presence of apoptotic cells were evaluated. The body weight, body mass index (BMI), and hepatosomatic index (HSI) index were significantly higher in the overfed group, and high lipid accumulation and fatty changes were also observed in their livers, indicating that the feeding regime used in this study led to overfeeding-induced obesity. Likewise, much higher numbers of and larger vacuoles were observed in overfed fish macrophages, showing unclear boundaries between the cytoplasm and extracellular space. In the overfed group, the expression of IL-10, HSP70, TLR2, and CD36 was significantly higher, and lymphocyte apoptosis was more evident, indicating that overfeeding-induced obese fish might have immunologic disorders. This was the first study to demonstrate that overfeeding-induced obesity could cause an immune-physiological imbalance in rainbow trout, making them more vulnerable to infectious diseases and various stressful conditions. This study will contribute to improvements in fish nutrition, feeding practices, fish nutrition, and disease prevention in the aquaculture industry.

Vibrio harveyi is one of the pathogens that threaten the shrimp farming industry. However, metabolic changes induced by V. harveyi infection in shrimp remain unknown. In this study, we first conducted high resolution-magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR)-based metabolomics studies on gill, hepatopancreas, and haemolymph of V. harveyi-infected white leg shrimp, Litopenaeus vannamei. Using multivariate statistical analysis, we observed a clear separation between the early (3 and 9 h post-injection (hpi)) and late phases (24, 72 and 144 hpi) of the infection in all tissues. Moreover, metabolic changes in response to V. harveyi infection were faster in the haemolymph in the early phase and significantly changed in the late phase of the infection in the gills. Extensive changes were observed in the hepatopancreas, with 24 hpi being the turning point of progression from early to late phase infection in the hepatopancreas. V. harveyi infection increased the energy demand in L. vannamei and the amino acid and carbohydrate metabolism pathways also exhibited significant changes depending on the tissue. Thus, each tissue displayed different metabolic changes, depending on the progress of the infection.

The use of probiotics is considered an attractive biocontrol method. It is effective in growth promotion in aquaculture. However, the mode of action of probiotics in fish in terms of growth promotion remains unclear. The objective of the present study was to investigate growth promotion effect of dietary administration of host-derived probiotics, Lactococcus lactis WFLU12, on olive flounder compared to control group fed with basal diet by analyzing their intestinal and serum metabolome using capillary electrophoresis mass spectrometry with time-of flight (CE-TOFMS). Results of CE-TOFMS revealed that 53 out of 200 metabolites from intestinal luminal metabolome and 5 out of 171 metabolites from serum metabolome, respectively, were present in significantly higher concentrations in the probiotic-fed group than those in the control group. Concentrations of metabolites such as citrulline, tricarboxylic acid cycle (TCA) intermediates, short chain fatty acids, vitamins, and taurine were significantly higher in the probiotic-fed group than those in the control group. The probiotic strain WFLU12 also possesses genes encoding enzymes to help produce these metabolites. Therefore, it is highly likely that these increased metabolites linked to growth promotion in olive flounder are due to supplementation of the probiotic strain. To the best of our knowledge, this is the first study to show that dietary probiotics can greatly influence metabolome in fish. Findings of the present study may reveal important implications for maximizing the efficiency of using dietary additives to optimize fish health and growth.

Reactor internals are sensitive to dynamic loads such as earthquakes and flow induced vibration. Thus, it is essential to identify the dynamic characteristics to evaluate the seismic integrity of the structures. However, a full-sized system is too large to perform modal experiments, making it difficult to extract data on its modal characteristics. In this research, we constructed a finite element model of the APR1400 reactor internals to identify their modal characteristics. The commercial reactor was selected to reflect the actual boundary conditions. Our FE model was constructed based on scale-similarity analysis and fluid-structure interaction investigations using a fabricated scaled-down model.