Association Between Muscle Growth and Transcription of a Mutant MSTN Gene in Olive Flounder (Paralichthys olivaceus).

Myostatin (MSTN, also known as growth differentiation factor-8 (GDF-8)), a member of the transforming growth factor ß (TGF-ß) superfamily, functions as a negative regulator of skeletal muscle development and growth. However, it is also expressed in a wide range of tissues in fish and thus may have more diverse roles in this group than in mammals. In this study, we assessed the genome-wide transcriptional expression pattern associated with the CRISPR/Cas9-mutated MSTN gene in the olive flounder (Paralichthys olivaceus) in association with changes in cell proliferation and transportation processes. There were no differences in the hepatosomatic index, and the growth of male and female fish increased in the F1 progeny of the MSTN mutants. Furthermore, the histopathological analysis showed that myostatin editing resulted in a 41.24% increase in back muscle growth and 46.92% increase in belly muscle growth in male flounder compared with normal flounder, and a 16.01% increase in back muscle growth and 14.26% increase in belly muscle growth in female flounder compared with normal flounder. This study demonstrates that editing of the myostatin gene enhances muscle growth in olive flounder, with a notably more pronounced effect observed in males. Consequently, myostatin-edited male flounder could represent a valuable asset for the flounder aquaculture industry.

Integrated Augmented and Virtual Reality Technologies for Realistic Fire Drill Training.

In this article, we propose a novel fire drill training system designed specifically to integrate augmented reality (AR) and virtual reality (VR) technologies into a single head-mounted display device to provide realistic as well as safe and diverse experiences. Applying hybrid AR/VR technologies in fire drill training may be beneficial because they can overcome limitations such as space-time constraints, risk factors, training costs, and difficulties in real environments. The proposed system can improve training effectiveness by transforming arbitrary real spaces into real-time, realistic virtual fire situations, and by interacting with tangible training props. Moreover, the system can create intelligent and realistic fire effects in AR by estimating not only the object type but also its physical properties. Our user studies demonstrated the potential of integrated AR/VR for improving training and education.

Characterization and Pathogenicity of Flavobacterium psychrophilum Isolated from Rainbow Trout (Oncorhynchus mykiss) in Korea.

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.

First Report of Enterocytozoon hepatopenaei Infection in Giant Freshwater Prawn (Macrobrachium rosenbergii de Man) Cultured in the Republic of Korea.

In the Republic of Korea, Enterocytozoon hepatopenaei (EHP) was first isolated from Pacific whiteleg shrimp in April 2020; however, there are no existing reports of EHP infection in other shrimp or prawns. Here, we aimed to investigate EHP infection and its prevalence in giant freshwater prawn farms in the Republic of Korea. We tested prawns from 22 farms for EHP infection, and samples from eight farms showed positive EHP infection results in 2021. In EHP-infected prawn farms, the prevalence ranged from 4.9% to 18.2%. The prevalence of EHP infection in the Republic of Korea, derived from the prevalence in prawn farms, was estimated to be 0.8% in 2021. The proliferation of EHP was observed within the hepatopancreatic epithelial cells of prawns using H&E and Giemsa staining. Mature EHP was observed in the sinus between epithelial cells of the digestive tubules. Phylogenetic analysis revealed a clade distinct from the previously reported EHP in Pacific whiteleg shrimps. This is the first report of EHP infection in a giant freshwater prawn in the Republic of Korea, where the prevalence of EHP infection is not high, but it is recognized as an emerging disease that requires periodic monitoring and quarantine management in giant freshwater prawns.

Identification and characterization of warm temperature acclimation proteins (Wap65s) in rainbow trout (Oncorhynchus mykiss).

Hemopexin is a vital glycoprotein for processing excessive iron in blood and functions as an iron scavenger in mammals. Teleosts however, unlike mammals, have two known hemopexin paralogs called warm temperature acclimation-related 65 kDa protein (Wap65-1 and Wap65-2, collectively termed Wap65s). Although Wap65s in rainbow trout have been considered notable biomarkers with significantly higher and/or lower expression under conditions of stress or disease, the individual roles, similarities and differences between the two paralogs are not well known. The aim of this study was to gain an understanding of the characteristics and functions of trout Wap65s from the perspective of iron-metabolism, physiological roles, and relevant immunological responses. The expression of Wap65-1 and -2 in this study was determined in the face of challenges by Aeromonas salmonicida, infectious hematopoietic necrosis virus (IHNV), and iron-dextran. Immuno-histochemistry (IHC) was employed to localize the major cell types for Wap65-2 expression, and trout leukocytes were isolated and incubated with LPS and OxLDL for comprehending the immunological characteristics of Wap65-2. We demonstrate that Wap65-1 is expressed only in the liver but Wap65-2 is systemically expressed in most organs and tissues. Interestingly, Wap65-1 expression was not significantly changed under A. salmonicida and iron-dextran administration, but was significantly decreased under IHNV. In contrast, Wap65-2 was up-regulated in all challenged groups, however with different expression patterns in the blood and liver. These results suggested that the two paralogs may participate in different biological roles. IHC showed that Wap65-2 antibody had high affinity for leukocyte-like cells, and macrophages but not lymphocytes significantly increased expression under LPS and OxLDL stimulation. These results support the conclusion that trout Wap65-2, not Wap65-1 may have conventional hemopexin functions such as reported in mammals including effects on iron metabolism, inflammation, and acute-phase protein.

First Report of Enterocytozoon hepatopenaei Infection in Pacific Whiteleg Shrimp (Litopenaeus vannamei) Cultured in Korea.

The consumption of cultured crustaceans has been steadily increasing, and Pacific whiteleg shrimp (Litopenaeus vannamei) are major cultivated invertebrates worldwide. However, shrimp productivity faces a variety of challenges, mainly related to outbreaks of lethal or growth retardation-related diseases. In particular, hepatopancreatic microsporidiosis caused by the microsporidian parasite Enterocytozoon hepatopenaei (EHP) is an important disease associated with growth retardation in shrimp. Here, we report the detection of EHP through histopathological, molecular and electron microscopy methods in the hepatopancreas of Pacific whiteleg shrimp with growth disorder in a South Korean farm. Phylogenetic analysis showed a clade distinct from the previously reported EHP strains isolated in Thailand, India, China and Vietnam. An EHP infection was not associated with inflammatory responses such as hemocyte infiltration. Although EHP infection has been reported worldwide, this is the first report in the shrimp aquaculture in Korea. Therefore, an EHP infection should be managed and monitored regularly for effective disease control and prevention.

Dual-Organ Transcriptomic Analysis of Rainbow Trout Infected With Ichthyophthirius multifiliis Through Co-Expression and Machine Learning.

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.

The Complete Genome of Nocardia seriolae MH196537 and Intra-Species Level as Analyzed by Comparative Genomics Based on Random Forest Algorithm.

Nocardiosis is a major problem affecting fish that are farmed in seacages as well as freshwater fish; therefore, deciphering the bacteriological features of Nocardia seriolae is crucial. In particular, a number of studies over the past two years have reported the genome sequence of N. seriolae, and a comparative genomics approach is expected to yield valuable information on its epidemiological characteristics. The purpose of this study was to perform whole-genome sequence analysis of N. seriolae MH196537 from the Japanese eel and to investigate the significant differences noted between strains isolated from freshwater fish and marine fish by using Random Forest, a reliable machine learning algorithm. The Pacbio platform was employed to sequence the MH196537 strain, and genomic information from the other 16 strains was used for comparative analyses. All coding sequences of the 17 strains were categorized in RASTtk Sub-systems. The MH196537 strain had one contig, and it shared a high average nucleotide identity (ANI) with the freshwater strains (0.9994 - 0.9999) rather than the seawater strains (0.9985 - 0.9994). Moreover, 22 RASTtk subsystems carried a different number of genes from each N. seriolae. The fatty acids, lipids, and isoprenoids subsystem showed the highest mean decrease in the Gini index of over 1.5. Interestingly, freshwater strains were found to harbor all of the genes for both the mevalonate (MVA) and non-mevalonate pathways (MEP), whereas only the MEP existed in strains from diseased marine fish. Considering the differences in the byproducts of isoprenoids from the different pathways, it is likely that this will affect host-pathogen interactions; therefore, harboring the different pathways for the synthesis of isoprenoids could be an important pathogenic factor of N. seriolae.

Overfeeding-Induced Obesity Could Cause Potential Immuno-Physiological Disorders in Rainbow Trout (Oncorhynchus mykiss).

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.

Genome-wide comparison of Carnobacterium maltaromaticum derived from diseased fish harbouring important virulence-related genes.

Although Carnobacterium maltaromaticum has been used as a probiotic in fish, it was reported to cause disease for the first time in Korea. The objective of this study was to understand the differences between pathogenic and non-pathogenic strains. Pathogenicity was tested by challenging rainbow trout with C. maltaromaticum ATCC35586 and 18ISCm isolated from diseased fish, and DSM20342 isolated from a dairy product. We also compared 24 genomes of C. maltaromaticum strains plus the genome of our isolate 18ISCm sequenced in this study. Only the strains from diseased fish caused high mortality with severe histopathological changes. Although all strains shared more than 90% of Ko_id, wecC and xtmA were found only in strains from diseased fish. Interestingly, only strains from diseased fish harboured two wecC paralogs involved in the production of D-mannosaminuronic acid which is a major component of a well-known virulence factor, teichuronic acid. Two wecC paralogs of 18ISCm were increased when they were co-cultured with trout blood cells, suggesting that wecC genes might play a role in virulence. The results of this study show that strains isolated from diseased fish are different from strains derived from food in terms of pathogenicity to fish and the presence of virulence-related genes.

Effects of Lactococcus lactis subsp. lactis I2 with ß-Glucooligosaccharides on Growth, Innate Immunity and Streptococcosis Resistance in Olive Flounder (Paralichthys olivaceus).

To identify and quantify the effects of a combination of dietary 1 × 108 CFU/g Lactococcus lactis subsp. lactis I2 (LI2) and 0.1% ß-glucooligosaccharides (BGO) on the growth and immunity of olive flounder (Paralichthys olivaceus), a feeding experiment was conducted. Flounder (14 ± 0.5 g) were divided into two groups and fed control and synbiotic feeds for 8 weeks. Investigations were carried out on growth and feed utilization, innate immunity, serum biochemical parameters, intestinal lactic acid bacterial (LAB) viability, microvillus length, and changes in the expression levels of genes encoding pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1ß, and IL-6). Results demonstrated the synbiotic diet had significantly better (p < 0.05) responses in terms of weight gain and specific growth rate, three innate immune parameters (respiratory burst, serum lysozyme, and superoxide dismutase), intestinal LAB viability, and the relative TNF-α expression level (p < 0.05). Moreover, after challenge with Streptococcus iniae (1 × 108 CFU/ml), the synbiotically fed group exhibited significantly higher (p < 0.05) protection against streptococcosis, validating the observed changes in immune parameters and induction of the cytokine-encoding gene. Therefore, according to the results of the present study, synbiotic feed (LI2 + BGO) increased growth, modulated innate immune parameters and protected olive flounder against streptococcosis.

Above 30% external quantum efficiency in green delayed fluorescent organic light-emitting diodes.

Highly efficient green thermally activated delayed fluorescent organic light-emitting diodes with an external quantum efficiency of 31.2% were investigated by using 3-(3-(carbazole-9-yl)phenyl) pyrido[3',2':4,5]furo[2,3-b]pyridine (3CzPFP) derived from carbazole and pyrido[3',2':4,5]furo[2,3-b]pyridine. The host material showed well-matched photoluminescence emission with absorption of the green dopant material, (4s,6s)-2,4,5,6-tetra(9H-carbazol-9-yl)isophthalonitrile (4CzIPN) and harvested all excitons of 4CzIPN. The 3CzPFP:4CzIPN film exhibited high photoluminescence quantum yield of 100%, and the green delayed fluorescence device employing the 3CzPFP host showed high maximum quantum efficiency of 31.2 ± 0.5% at 1% doping after optimization of the device structure.

Above 20% external quantum efficiency in novel hybrid white organic light-emitting diodes having green thermally activated delayed fluorescent emitter.

High efficiency hybrid type white organic light-emitting diodes (WOLEDs) combining a green thermally activated delayed fluorescent (TADF) emitting material with red/blue phosphorescent emitting materials were developed by manipulating the device architecture of WOLEDs. Energy transfer between a blue phosphorescent emitting material and a green TADF emitter was efficient and could be managed by controlling the doping concentration of emitters. A high quantum efficiency above 20% was achieved in the hybrid WOLEDs by optimizing the device structure of the hybrid type WOLEDs for the first time and the device performances of the hybrid WOLEDs were comparable to those of all phosphorescent WOLEDs.

Phosphine oxide type bipolar host material for high quantum efficiency in thermally activated delayed fluorescent device.

Highly efficient thermally activated delayed fluorescence devices were developed using a bipolar host material, 2,7-bis(diphenylphosphoryl)-9-phenyl-9H-carbazole (PPO27), derived from carbazole and diphenylphosphine oxide. The PPO27 host was effective for good charge balance and energy transfer from the PPO27 host to thermally activated delayed fluorescence dopant. The PPO27 device doped with (4s,6s)-2,4,5,6-tetra(9H-carbazol-9-yl)isophthalonitrile dopant could realize a high quantum efficiency of 24.2%.

Synthesis of dimesitylborane-substituted phenylcarbazoles as bipolar host materials and the variation of the green PHOLED performance with the substituent position of the boron atom.

Two structural isomers of the bipolar host material containing the dimesitylborane moiety were synthesized and their device performance was investigated. The quantum efficiency of the devices depends on the substituent position of the dimesitylborane moiety. The maximum external quantum efficiency of the device reached as high as 23.8% with a green color coordinate of (0.30, 0.63).

Expression analysis and enzymatic characterization of phospholipase Cδ4 from olive flounder (Paralichthys olivaceus).

Phospholipase Cδ4 (PLCδ4) plays a significant role in cell proliferation, tumorigenesis, and in an early stage of fertilization. Despite the characterization of the mammalian PLCδ4, extensive study in aquatic organisms has not been carried out so far. Here, we performed the molecular and biochemical characterization of flatfish Paralichthys olivaceus PLCδ4 (PoPLCδ4) to understand its enzymatic properties and physiological functions. The olive flounder PLCδ4 cDNA has an open reading frame (ORF) of 2,268 bp, and encodes a 755 amino acid polypeptide with a predicted molecular weight of 86 kDa. All the characteristic domains found in mammalian PLCδ isoforms (PH domain, EF hands, an X-Y catalytic region, and a C2 domain) were found to be present in PoPLCδ4. The mRNA expression analysis of PoPLCδ4 showed that PoPLCδ4 is predominantly expressed in the brain, eye and heart tissues. Like other mammalian PLCδ proteins, the enzyme activity of recombinant PoPLCδ4 to phosphatidylinositol-4,5-bis-phosphate (PIP2) was noted to be concentration- and Ca(2+)-dependent. The structural features and biochemical characteristics of PoPLCδ4 were found to be similar to those of mammalian PLCδ4. This is the first demonstration of the expression analysis and enzymatic characterization of piscine PLCδ4.

PLC-δ1-Lf, a novel N-terminal extended phospholipase C-δ1.

Phospholipase C-δ (PLC-δ), a key enzyme in phosphoinositide turnover, is involved in a variety of physiological functions. The widely expressed PLC-δ1 isoform is the best characterized and the most well understood phospholipase family member. However, the functional and molecular mechanisms of PLC-δ1 remain obscure. Here, we identified that the N-terminal region of mouse PLC-δ1 gene has two variants, a novel alternative splicing form, named as long form (mPLC-δ1-Lf) and the previously reported short form (mPLC-δ1-Sf), having exon 2 and exon 1, respectively, while both the gene variants share exons 3-16 for RNA transcription. Furthermore, the expression, identification and enzymatic characterization of the two types of PLC-δ1 genes were compared. Expression of mPLC-δ1-Lf was found to be tissue specific, whereas mPLC-δ1-Sf was widely distributed. The recombinant mPLC-δ1-Sf protein exhibited higher activity than recombinant mPLC-δ1-Lf protein. Although, the general catalytic and regulatory properties of mPLC-δ1-Lf are similar to those of PLC-δ1-Sf isozyme, the mPLC-δ1-Lf showed some distinct regulatory properties, such as tissue-specific expression and lipid binding specificity, particularly for phosphatidylserine.

Functional analysis of duplicated genes and N-terminal splice variant of phospholipase C-δ1 in Paralichthys olivaceus.

Phosphoinositide-specific phospholipase C δ (PLC δ) plays an important role in many cellular responses and is involved in the production of second messenger. Here, we describe the presence of novel N-terminal extended alternative splice form of PLC-δ1B in Paralichthys olivaceus, which differs from the reported mammalian PLC-δ1 isoform. The two variants PoPLC-δ1B-Lf and PoPLC-δ1B-Sf share exon 3 (including the PH domain) to exon 16, but differ at the exon 1 (Short form: Sf) and novel exon 2 (Long form: Lf) of the transcript. For the characterization of the novel duplicated gene variant of PLC-δ1B in P. olivaceus, tissue-specific expression with RT-PCR and real-time PCR, and purification and enzymatic characterization of native and recombinant proteins of all the three-types of PLC-δ1 isoforms (PoPLC-δ1A, PoPLC-δ1B-Lf and PoPLC-δ1B-Sf) of P. olivaceus were studied. The PoPLC-δ1A was ubiquitously distributed in gill, kidney and spleen. The PoPLC-δ1B-Lf gene was widely detected in various tissues, especially in the digestive system, while PoPLC-δ1B-Sf was highly expressed in the stomach. The recombinant PoPLC-δ1A, PoPLC-δ1B-Lf and PoPLC-δ1B-Sf proteins were expressed as a histidine-tagged fusion protein in Escherichia coli. The PLC activity of the PoPLC-δ1 isoform proteins showed a concentration-dependent activity to phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP(2)). In addition, U73122, the PLC inhibitor, effectively inhibited PLC activities of PoPLC-δ1A, PoPLC-δ1B-Lf and PoPLC-δ1B-Sf proteins. However, PoPLC-δ1A and PoPLC-δ1B-Lf were sensitive at pH 7.5, while PoPLC-δ1B-Sf was relatively sensitive at pH 7. These results might be useful for the study of phospholipase C-mediated signal transduction in fish.