Efficacy of a recombinant M-like protein, SimA as a subunit vaccine candidate against Streptococcus parauberis infection in olive flounder, Paralichthys olivaceus.

Streptococcus parauberis, a gram-positive cocci, causes bacterial disease in farmed fish. The recent increase in S. parauberis infection in aquatic farms in South Korea has justified the importance of vaccine development for the prevention of this disease. In this study, we evaluated the effect of subunit vaccines prepared from recombinant M-like protein (SimA) and fibrinogen-binding protein (FBP) candidates with an aluminum hydroxide adjuvant against S. parauberis infection in olive flounder Paralichthys olivaceus. For the in vivo experiment, fish (average length, 7.18 cm; average weight, 3.5 g) were injected intraperitoneally with: phosphate buffer saline (PBS, group 1), PBS/aluminum hydroxide (group 2), FBP/aluminum hydroxide (group 3), SimA/aluminum hydroxide (group 4), and SimA/FBP/aluminum hydroxide (group 5). After 3 weeks, the fish in each group were boosted using PBS (group 1 and 2), FBP (group 3), SimA (group 4), and SimA/FBP (group 5) without adjuvant. We found that the relative percent survival of fish after S. parauberis exposure in group 2, 3, 4, and 5 was 6.25%, 18.75%, 50%, and 12.5%, respectively, whereas the mortality in groups 1 was 80%, respectively. We performed Western blot, ELISA, and quantitative real time RT-PCR (qRT-PCR) after vaccination to investigate the further efficacy of the vaccine. Western blot and ELISA of vaccinated fish serum confirmed the production of specific antibodies against SimA and FBP. Furthermore, results of qRT-PCR showed that recombinant protein SimA induced a remarkably specific-antibody response compared with that in FBP or control and increased the expression of various immune response-related genes including interleukin-8 (IL-8), toll-like receptor 2 (TLR2), tumor necrosis factor-α (TNF-α), CD4-1, and MHC II. Thus, these results indicate that SimA is a potent vaccine candidate for protection against S. parauberis infection.

Red sea bream interleukin (IL)-1ß and IL-8 expression, subcellular localization, and antiviral activity against red sea bream iridovirus (RSIV).

Interleukin-1 beta (IL-1ß) is transcribed by monocytes, macrophages, and dendritic cells in response to activation of toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs) or cytokine signalling and causes a rapid inflammatory response to infection. IL-8, also known as chemokine C-X-C motif ligand (CXCL)-8, is regulated by IL-1ß and affects the chemotaxis of macrophages and neutrophils upon pathogen infection. In healthy red sea bream, rsbIL-1ß is most highly distributed in the liver, and rsbIL-8 is most highly distributed in the head kidney. In response to RSIV infection, rsbIL-1ß and rsbIL-8 mRNA are significantly upregulated in the kidney and spleen. This may be because the primary infection targets of RSIV are the kidney and spleen. In the gills, both genes were significantly upregulated at 7 days after RSIV infection and may be accompanied by a cytokine storm. In the liver, both genes were significantly downregulated at most observation points, which may be because the immune cells such as macrophages and dendritic cells expressing rsbIL-1ß or rsbIL-8 migrated to other tissues because the degree of RSIV infection was relatively low. Using a GFP fusion protein, it was confirmed that rsbIL-1ß and rsbIL-8 were localized to the cytoplasm of Pagrus major fin (PMF) cells. RsbIL-1ß overexpression induced the expression of interferon gamma (IFN-γ), myxovirus-resistance protein (Mx) 1, IL-8, IL-10, TNF-α, and MyD88, while rsbIL-8 overexpression induced the expression of IFN-γ, Mx1, rsbIL-1ß and TNF-α. In addition, overexpression of both genes significantly reduced the genome copies of RSIV and significantly reduced the viral titers. Therefore, rsbIL-1ß and rsbIL-8 in red sea bream play an antiviral role against RSIV through their normal signalling.

Functional characterization and gene expression profile of perforin-2 in starry flounder (Platichthys stellatus).

The membrane attack complex/perforin (MACPF) superfamily consists of multifunctional proteins that form pores on the membrane surface of microorganisms to induce their death and have various immune-related functions. PFN2 is a perforin-like protein with an MACPF domain, and humans with deficient PFN2 levels have increased susceptibility to bacterial infection, which can lead to fatal consequences for some patients. Therefore, in this study, we confirmed the antimicrobial function of PFN2 in starry flounder (Platichthys stellatus). The molecular properties were confirmed based on the verified amino acid sequence of PsPFN2. In addition, the expression characteristics of tissue-specific and pathogen-specific PsPFN2 mRNA were also confirmed. The recombinant protein was produced using Escherichia coli, and the antimicrobial activity was then confirmed. The coding sequence of PFN2 (PsPFN2) in P. stellatus consists of 710 residues. The MACPF domain was conserved throughout evolution, as shown by multiple sequence alignment and phylogenetic analysis. PsPFN2 mRNA is abundantly distributed in immune-related organs such as the spleen and gills of healthy starry flounder, and significant expression changes were confirmed after artificial infection by bacteria or viruses. We cloned the MACPF domain region of PFN2 to produce a recombinant protein (rPFN2) and confirmed its antibacterial effect against a wide range of bacterial species and the parasite (Miamiensis avidus).

Development of loop-mediated isothermal amplification method for detection of Kudoa septempunctata (Myxozoa: Multivalvulida) in olive flounder (Paralichthys olivaceus).

A loop-mediated isothermal amplification (LAMP) assay was developed and validated for early, rapid, and sensitive detection of Kudoa septempunctata, a myxosporean parasite found in olive flounder (Paralichthys olivaceus). Recently, several outbreaks associated with ingestion of raw olive flounder muscles harboring mature K. septempunctata spores have been reported, and it is becoming obvious that fresh K. septempunctata spores can cause problems in humans when ingested. Thus, it is necessary to develop reliable detection method of K. septempunctata, to prevent outbreaks and ensure food safety. The LAMP assay has advantages over other molecular detection methods for detecting K. septempunctata in olive flounder muscle, in terms of simplicity, rapidity, and sensitivity. The reaction condition was optimized as 63 °C, 45 min, with three sets of specific primers. The results can be simply confirmed with the naked eye after adding SYBR Green I or by conventional electrophoresis followed by ethidium bromide staining. This LAMP assay did not show any cross-reaction with other kudoid myxosporeans (Kudoa lateolabracis, Kudoa thyrsites) can be found in olive flounder muscles and was validated by testing Kudoa septempunctata spore-spiked samples and field samples. The results showed that the LAMP assay is ten times more sensitive than the conventional polymerase chain reaction in this study and can be applied for early detection for monitoring and epidemiological studies of K. septempunctata in olive flounder aquaculture farms.

Method for 3D atomic structure determination of multi-element nanoparticles with graphene liquid-cell TEM.

Determining the 3D atomic structures of multi-element nanoparticles in their native liquid environment is crucial to understanding their physicochemical properties. Graphene liquid cell (GLC) TEM offers a platform to directly investigate nanoparticles in their solution phase. Moreover, exploiting high-resolution TEM images of single rotating nanoparticles in GLCs, 3D atomic structures of nanoparticles are reconstructed by a method called "Brownian one-particle reconstruction". We here introduce a 3D atomic structure determination method for multi-element nanoparticle systems. The method, which is based on low-pass filtration and initial 3D model generation customized for different types of multi-element systems, enables reconstruction of high-resolution 3D Coulomb density maps for ordered and disordered multi-element systems and classification of the heteroatom type. Using high-resolution image datasets obtained from TEM simulations of PbSe, CdSe, and FePt nanoparticles that are structurally relaxed with first-principles calculations in the graphene liquid cell, we show that the types and positions of the constituent atoms are precisely determined with root mean square displacement values less than 24 pm. Our study suggests that it is possible to investigate the 3D atomic structures of synthesized multi-element nanoparticles in liquid phase.

Non-specific defensive factors of the Pacific oyster Crassostrea gigas against infection with Marteilioides chungmuensis: a flow-cytometric study.

In order to assess changes in the activity of immunecompetency present in Crassostrea gigas infected with Marteilioides chungmuensis (Protozoa), the total hemocyte counts (THC), hemocyte populations, hemocyte viability, and phagocytosis rate were measured in oysters using flow cytometry. THC were increased significantly in oysters infected with M. chungmuensis relative to the healthy appearing oysters (HAO) (P<0.05). Among the total hemocyte composition, granulocyte levels were significantly increased in infected oysters as compared with HAO (P<0.05). In addition, the hyalinocyte was reduced significantly (P<0.05). The hemocyte viability did not differ between infected oysters and HAO. However, the phagocytosis rate was significantly higher in infected oysters relative to HAO (P<0.05). The measurement of alterations in the activity of immunecompetency in oysters, which was conducted via flow cytometry in this study, might be a useful biomarker of the defense system for evaluating the effects of ovarian parasites of C. gigas.

Green-Light-Selective Organic Photodiodes with High Detectivity for CMOS Color Image Sensors.

Stacked structures employing wavelength-selective organic photodiodes (OPDs) have been studied as promising alternatives to the conventional Si-based image sensors because of their color constancy. Herein, novel donor (D)-π-acceptor (A) molecules are designed, synthesized, and characterized as green-light-selective absorbers for application in organic-on-Si hybrid complementary metal-oxide-semiconductor (CMOS) color image sensors. The p-type molecules, combined with two fused-type heterocyclic donors and an electron-accepting unit, exhibit cyanine-like properties that are characterized by intense and sharp absorption. This molecular design leads to improved absorption properties, thermal stability, and higher photoelectric conversion compared to those of a molecular design based on a nonfused ring. A maximum external quantum efficiency of 66% (λmax = 550 nm) and high specific detectivity (D*) of 8 × 1013 cm Hz1/2/W are achieved in an OPD consisting of a bulk heterojunction blend with two transparent electrodes on both sides. Finally, the green-light-detection capability of the narrow-band green-selective OPD is demonstrated by the optical simulation of an organic-on-Si hybrid, stacked-type, full-color photodetector comprising the green-light-selective OPD and a bottom Si photodiode with only blue and red color filters. Based on this molecular design, further optimization of the OPDs can allow the development of various optoelectronic sensors including 3D-stacked image sensors with enhanced sensitivities to replace the conventional Si-based CMOS image sensors.

The molecular characterization, expression analysis and antimicrobial activity of theromacin from Asian polychaeta (Perinereis linea).

Antimicrobial peptides (AMPs) are molecular factors in innate immunity and are believed to play a key role in invertebrate host defence. We identified theromacin (TM) from an Asian polychaeta, Perinereis linea, using de novo RNA-seq analysis. TM, a typical AMP of invertebrates, is a cysteine-rich AMP with five disulfide bonds consisting of ten cysteine residues. In gene expression analysis, TM genes were constantly upregulated after lipopolysaccharide (LPS) stimulation. In contrast, after peptidoglycan (PGN) stimulation, it was upregulated initially and downregulated after 12 h. We synthesized a peptide based on the macin AMP in the TM amino acid sequence. The synthetic peptide showed antibacterial activity against some Gram-positive and Gram-negative bacteria. Therefore, the AMPs of P. linea might have broad roles in host defence and exhibit different degrees of activity.

Genetic variability comparison of cultured Israeli carp (Cyprinus carpio) from Korea using microsatellites.

In aquaculture, cultured fish often undergo continuous cross-fertilization without any inflow of new broodstock. This lowers genetic diversity, leading to increased disease rates and decreased survival rates. To improve the mass production and easy culture of Israeli carp, it is essential to investigate the population structure and genetic diversity of these fish. However, such a survey has not yet been performed on Korean Israeli carp. In this study, we used seven microsatellite markers to analyze the genetic diversity and association of cultured Israeli carp from Korea and China. The average numbers of alleles per locus (N A ) for two Korean (KorA and KorB) and two Chinese (ChA and ChB) populations were as follows: KorA (10.42), KorB (14.43), ChA (20.57) and ChB (20.71). The expected heterozygosity (H e ) ranged from 0.672 to 0.897 and from 0.827 to 0.938 in the Korean sample and Chinese sample respectively. The genetic diversity of the Korean Israeli carp was about half that of the Chinese carp. The diversity of the Korean Israeli carp was very low, suggesting that the immunity of this population could be weak, and that diversity-recovery studies are urgently needed. Therefore, our results may therefore form the foundation for future research efforts towards genetic monitoring and selective breeding, continuous research needs to be conducted in order to recover the genetic diversity of the Korean Israeli carp.

Growth Comparison of Israeli Carp (Cyprinus carpio) to Different Breeding Combination.

The purpose of this study was to investigate the improvement of growth in Israeli carp (Cyprinus carpio), and the cross experiment was carried out with two strains of Israeli carp. Four combinations of Israeli carp from Jeonbuk fisheries farm and Songpu mirror carp from Heilong Jiang, China (KK; Jeonbuk ♀ × Jeonbuk ♂, KC; Jeonbuk ♀ × China ♂, CC; China ♀ × China ♂ and CK; China ♀ × Jeonbuk ♂) were developed and reared. Body length, body weight and condition factor were determined at 20, 40, 60 and 170 days post-hatch (DPH). The results showed that there were differences in growth rate of the four groups. Body length of four groups were CK > CC > KC > KK and body weight were CC > CK > KC > KK at 170 DPH. The growth perfomance of four groups were statistically significant difference (P<0.05). During the rearing, CC group had longer length and higher weight at 170 DPH compared to other three groups and also condition factor was highest in the CC group, but there was no significant difference in a survival rate. These results indicated that the growth performance mainly depended upon brooder combination but survival rate could not significantly affect brooder.

Gonadogenesis and Sex Differentiation in the Korean Rose Bitterling, Rhodeus uyekii).

This report describes the sex differentiation of the Korean rose bitterling, Rhodeus uyekii, from hatching to 170 days post-hatch (DPH) in relation to total length (TL), body weight (BW), and integral water temperature (IWT). The growth curve of TL from just hatching to 83 DPH was 5.144e0.045t (R² = 0.961; t, time), and that of BW was 2.398e0.086t (R² = 0.725). Primordial germ cells (PGCs) were observed at 17 DPH (7.9 mm TL, 3.74 mg BW, 374°C IWT), and thereafter began to protrude into the peritoneal cavity. At 21 DPH (9.2±0.14 mm TL, 4.8±0.07 mg BW, 462°C IWT), some PGCs contained condensed chromatin and oocyte were observed in meiotic prophase. In contrast to the ovaries, which grew gradually after sexual differentiation, testes began multiplying at 25 DPH (10.1 mm TL, 5.42 mg BW, 550°C IWT), when testicular differentiation was first identified, and multiplied continuously thereafter. At 33 DPH (11.2 mm TL, 10.5 mg BW, 726°C IWT), the developing testes contained spermatogonia that exhibited mitotic activity. No spermatocyte or sperm cell was observed until 83 DPH (18.9 TL, 48.2 mg BW, 1,826°C IWT). At 170 DPH (32.5 mm TL, 270.1 mg BW, 3,740°C IWT), which was the end point of this study, the mature ovaries showed germinal vesicle breakdown, while the mature testes contained observable spermatocytes and sperm cells. These results allow us to identify the sex differentiation type of the Korean rose bitterling as differentiated gonochoristic.

Expression of Immune-Related Genes during Loach (Misgurnus anguillicaudatus) Embryonic and Early Larval Development.

Early life stage mortality in fish is one of the problems faced by loach aquaculture. However, our understanding of immune system in early life stage fish is still incomplete, and the information available is restricted to a few fish species. In the present work, we investigated the expression of immune-related transcripts in loach during early development. In fishes, recombination-activating gene 1 (RAG-1) and sacsin (SACS) have been considered as immunological function. In this study, the expression of the both genes was assessed throughout the early developmental stages of loach using real-time PCR method. maRAG-1 mRNA was first detected in 0 dph, observed the increased mostly until 40 dph. Significant expression of maRAG-1 was detected in 0 to 40 dph. These patterns of expression may suggest that the loach start to develop its function after hatching. On the other hand, maSACS was detected in unfertilized oocyte to molura stages and 0 to 40 dph. maSACS mRNA transcripts were detected in unfertilized oocytes, suggesting that they are maternally transferred.

Molecular identification and expression analysis of a natural killer cell enhancing factor (NKEF) from rock bream Oplegnathus fasciatus and the biological activity of its recombinant protein.

Natural killer cell enhancing factor (NKEF) belongs to the defined peroxiredoxin (Prx) family. Rock bream NKEF cDNA was identified by expressed sequence tag (EST) analysis of rock bream liver that was stimulated with the LPS. The full-length RbNKEF cDNA (1062 bp) contained an open reading frame (ORF) of 594 bp encoding 198 amino acids. RbNKEF was significantly expressed in the gill, liver, and intestine. mRNA expression of NKEF in the head kidney was examined under viral and bacterial challenge via real-time RT-PCR. Experimental challenge of rock bream with Edwardsiella tarda, Streptococcus iniae, and RSIV resulted in significant increases in RbNKEF mRNA in the head kidney. To obtain a recombinant NKEF, the RbNKEF ORF was expressed in Escherichia coli BL21 (DE3), and the purified soluble protein exhibited a single band corresponding to the predicted molecular mass. When kidney leucocytes were treated with a high concentration of rRbNKEF (10 µg/mL), they exhibited significantly enhanced cell proliferation and viability under oxidative stress.