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The structures of fish serum immunoglobulin differ among different fish species. In this study, we accidently isolated a rabbit immunoglobulin (Ig) light chain bound to serum immunoglobulin from different marine fish species using phage display. Fish Ig was separated using a protein A column.The phage library was generated from variable regions of rabbit spleen B cells immunized with bluefin tuna Thunnus orientalis Ig. Fish Ig–specific phages were enriched using two rounds of bio-panning with yellowtail Seriola quinqueradiata serum Ig, followed by two rounds of bio-panning with red seabream Pagrus major serum Ig.The enriched phages demonstrated an increase in binding specificity to the tuna, yellowtail, and red seabream Igs compared to the phages listed in the unpanned library.A recombinant protein of a single clonal phage, which encodes the rabbit Ig light chain, was produced, and the binding specificities to fish Igs were analyzed using enzyme-linked immunosorbent assay (ELISA) and western blotting.The recombinant protein exhibited binding properties to fish Igs in the ELISA. However, the recombinant protein that bound to serum protein(s), but not IgM, was detected via western blotting.The recombinant protein may provide a novel information on the common structural feature in the fish immunoglobulins., 公開日: 2023-07-21

Red sea bream iridovirus (RSIV) is the pathogen that causes red sea bream iridoviral disease. It causes a huge loss to the Japanese aquaculture industry. In 2021, outbreaks of red sea bream iridovirus occurred in South Japan. This study analysed nine whole-genome sequences of RSIV isolated in Oita and Ehime Prefectures in 2021 using a short-read next-generation sequencer. Nine isolates had highly uniform sequences, and there was no variant depending on locations or host species. Phylogenetic analyses with other reported megalocytivirus isolates showed that RSIV isolated in 2021 was genetically different from RSIV previously isolated in Oita and Ehime Prefectures in 2017-2019. These results suggest that RSIV isolated in Oita and Ehime Prefectures in 2021 might spread from a common ancestor different from the recent one. Additionally, it was found that RSIV isolated in 2021 had sequence mutations on protein-coding sequences that may be involved in viral pathogenicity and infectivity.

Virus like particles (VLPs) are non-infectious nanoparticles containing repetitive, high density viral epitopes on the surface and can prevent viral infections in aquatic animals. Here, we evaluated the immuno-stimulation effect of infectious hypodermal and hematopoietic necrosis virus like particle (IHHNV-VLP) using a next generation sequencing in Fenneropenaeus merguiensis to identify the important immune-related genes that may prevent viral infection. The in situ target of IHHNV was predominantly found in gill tissue following IHHNV-VLP administration in juvenile shrimp. Comparative transcriptome analysis in the injected gills showed that there were 326 unigenes expressed differently than the mock-injected samples. One of the most differential genes between the two animal groups was the antioxidative gene, peroxiredoxin (FmPrx), that was up-regulated after 6 h post-VLP injection. Phylogenetic tree analysis showed that this gene could be found among many shrimp species and was closely clustered among Prx families. The expression of FmPrx was also detected in all tissues examined, thus suggesting the multi-functional roles of this gene in many tissues. Administration of IHHNV-VLP in vivo led to a significant increase in peroxidase activity in gill tissue-approximately two-fold versus control animals; the WSSV copy number was significantly reduced. These data suggest that IHHNV-VLP exerts an immune-stimulating effect by enhancing the level of immune-related genes including FmPrx and its corresponding peroxidase activity, which are a well-known part of the shrimp innate immune system.

The digestive organs of terrestrial isopods harbor bacteria of the recently proposedmollicute family Hepatoplasmataceae. The only complete genome available for Hepatoplasmataceae is that of “Candidatus Hepatoplasma crinochetorum”. The scarcity of genome sequences has hampered our understanding of the symbiotic relationship between isopods and mollicutes. Here, we present four complete metagenome-assembled genomes (MAGs) of uncultured Hepatoplasmataceae members identified from shotgun sequencing data of isopods. We propose three novel species “Candidatus Tyloplasma littoralis” identified from the semiterrestrial isopod Tylos granuliferus, “Candidatus Hepatoplasma vulgare” identified from the common pill bug Armadillidium vulgare, and “Candidatus Hepatoplasma scaber” identified from the common rough woodlouse Porcellio scaber. Phylogenetic analysis of 16S ribosomal RNA sequences showed that “Candidatus Tyloplasma littoralis” and other semiterrestrial isopod-associated mollicutes form a sister clade to terrestrial Hepatoplasma members, justifying their assignment to a novel genus. Phylogenomic analysis of 151 mollicutes confirmed that Hepatoplasmataceae is a sister clade of Metamycoplasmataceae in the order Mycoplasmoidales. Our analysis also revealed that that Hepatoplasmataceae lack major metabolic pathways but has a likely intact type IIA CRISPR-Cas9 machinery, indicating that these mollicutes have an ectosymbiotic lifestyle with high nutritional dependence on their host. We did not find any evidence that Hepatoplasmataceae encode digestive enzymes that could provide nutritional benefits to the host, which suggests that they may act as defensive symbionts.

Crustacean genomes harbor sequences originating from nimaviruses, a family of large double-stranded DNA viruses infecting crustaceans. In this study, we recovered metagenome-assembled genomes of 25 endogenous nimaviruses from crustacean genome data. Phylogenetic analysis revealed four major lineages withinNimaviridae, and for three of these lineages, we propose novel genera of endogenous nimaviruses: “Majanivirus” and “Pemonivirus” identified from penaeid shrimp genomes, and “Clopovirus” identified from terrestrial isopods. Majanivirus genomes contain multiple eukaryotic-like genes such as baculoviral inhibitor of apoptosis repeat-containing genes, innexins, and heat shock protein 70-like genes, some of which contain introns. An alignment of long reads revealed that that each endogenous nimavirus species specifically inserts into host microsatellites or within 28S rDNA. This insertion preference was associated with the type of virus-encoded DNA recombination enzymes, the integrases. Majaniviruses, pemoniviruses, some whispoviruses, and possibly clopoviruses specifically insert into the arthropod telomere repeat motif (TAACC/GGTTA)n and all possessed a specific tyrosine recombinase family. Pasiphaea japonica whipovirus and Portunus trituberculatus whispovirus, the closest relatives of white spot syndrome virus, integrate into the host 28S rDNA and are equipped with members of another family of tyrosine recombinases that are distantly related to telomere-specific tyrosine recombinases. Endogenous nimavirus genomes identified from sesarmid crabs, which lack tyrosine recombinases and are flanked by a 46-bp inverted terminal repeat, integrate into (AT/TA)n microsatellites through the acquisition of a Ginger2-like cut- and-paste DDE transposase. These results suggest that endogenous nimaviruses are giant transposable elements that occupy different sequence niches through the acquisition of different integrase families.ImportanceCrustacean genomes harbor sequences originating from a family of large DNA viruses called nimaviruses, but it is unclear why they are present. We show that endogenous nimaviruses selectively insert into repetitive sequences within the host genome, and this insertion specificity was correlated with different types of integrases, which are DNA recombination enzymes encoded by the nimaviruses themselves. This suggests that endogenous nimaviruses have colonized various genomic niches through the acquisition of integrases with different insertion specificities. Our results point to a novel survival strategy of endogenous large DNA viruses colonizing the host genomes. These findings may clarify the evolution and spread of nimaviruses in crustaceans and lead to measures to control and prevent the spread of pathogenic nimaviruses in aquaculture settings.

Red sea bream iridovirus (RSIV) belonging to the genus Megalocytivirus of the family Iridoviridae is the cause of serious mass mortality of cultured marine fishes. RSIV-type megalocytiviruses show extremely high nucleotide sequence identities. Thus, epidemiological studies on this virus are limited. This study developed two primer sets amplifying the regions possessing single nucleotide polymorphism (SNP) to determine the relationships and divergence of RSIV-type megalocytiviruses isolated from cultured marine fishes in Japan. The two regions were designed according to the genome sequences of the representative RSIV genotype II of megalocytivirus members in GenBank. The SNP 1 and 2 regions have sequences homologous to hypothetical protein ORF 24 and ORF 31, respectively, of RSIV (accession no. AP017456.1). By sequencing the regions, 53 polymorphic sites were identified. The phylogenetic analysis of 25 RSIV-type megalocytivirus isolates, classified into RSIV cluster, was clustered into eight haplotypes (seven haplotypes from Oita, two haplotypes from Ehime, and one haplotype shared between Oita and Ehime). These findings suggested that SNP in the RSIV genome is a powerful application for the detection and identification of RSIV-type megalocytiviruses.

Here, we investigated the effect of dietary cysteic acid on the growth performance, sulfur amino acid content, and gene expression levels of taurine-synthesizing enzymes, growth hormone (GH), and insulin-like growth factor 1 (IGF-1) in Japanese flounder Paralichthys olivaceus. Juvenile flounder (0.9 g) were fed one of four diets for 30 days: with 0.25, 0.5, or 1.0% cysteic acid (C0.25, C0.5, C1.0) supplementation and without supplementation (control). Fish in the C0.25 and C0.5 groups showed significantly better growth than those in the control group (P 0.05), the expression level of IGF-1 in C1.0 fish was significantly higher than that in controls (P

Gene expression profiles during the transition from fasting to refeeding were investigated in the gut and liver of Masu salmon Oncorhynchus masou masou. Fish were starved for 3 days and then fed at 3% of body weight. The gut and liver were resected before and after feeding, and the tissues were used for transcriptome analyses. Twenty-nine and 45 genes were more than eightfold differently expressed in the gut and liver, respectively. Genes involved in fatty acid and carbohydrate metabolism were differentially expressed in the liver tissues. Genes involved in protein folding, such as GRP78 and endoplasmin, were significantly upregulated in both organs at 6, 12, and 24 h after feeding. Furthermore, glycogen synthase kinase (GSK) 3-binding protein, a negative regulator of GSK with pivotal roles in stress response, was downregulated in both organs 6 h after feeding. These results suggest that the change in nutritional status from fasting to refeeding is accompanied by cellular stress.

Vibrio penaeicida (family Vibrionaceae ) is an important bacterial pathogen that affects Japanese shrimp aquaculture. Only two whole-genome sequences of V. penaeicida are publicly available, which has hampered our understanding of the pathogenesis of shrimp vibriosis caused by this bacterium. To gain insight into the genetic features, evolution and pathogenicity of V. penaeicida, we sequenced five V. penaeicida strains (IFO 15640T, IFO 15641, IFO 15642, TUMSAT-OK1 and TUMSAT-OK2) and performed comparative genomic analyses. Virulence factors and mobile genetic elements were detected. Furthermore, average nucleotide identities (ANIs), clusters of orthologous groups and phylogenetic relationships were evaluated. The V. penaeicida genome consists of two circular chromosomes. Chromosome I sizes ranged from 4.1 to 4.3 Mb, the GC content ranged from 43.9 to 44.1 %, and the number of predicted protein-coding sequences (CDSs) ranged from 3620 to 3782. Chromosome II sizes ranged from 2.2 to 2.4 Mb, the GC content ranged from 43.5 to 43.8 %, and the number of predicted CDSs ranged from 1992 to 2273. All strains except IFO 15641 harboured one plasmid, having sizes that ranged from 150 to 285 kb. All five genomes had typical virulence factors, including adherence, anti-phagocytosis, flagella-related proteins and toxins (repeats-in-toxin and thermolabile haemolysin). The genomes also contained factors responsible for iron uptake and the type II, IV and VI secretion systems. The genome of strain TUMSAT-OK2 tended to encode more prophage regions than the other strains, whereas the genome of strain IFO 15640T had the highest number of regions encoding genomic islands. For comparative genome analysis, we used V. penaeicida (strain CAIM 285T) as a reference strain. ANIs between strain CAIM 285T and the five V. penaeicida strains were >95 %, which indicated that these strains belong to the same species. Orthology cluster analysis showed that strains TUMSAT-OK1 and TUMSAT-OK2 had the greatest number of shared gene clusters, followed by strains CAIM 285T and IFO 15640T. These strains were also the most closely related to each other in a phylogenetic analysis. This study presents the first comparative genome analysis of V. penaeicida and these results will be useful for understanding the pathogenesis of this bacterium.

The white spot syndrome virus (WSSV) has been considered a serious threat to shrimp aquaculture. Besides, the activation of cell metabolism as an immune reaction to the virus is now recognized as a piece of the pivotal puzzle of the antiviral responses. Hence, this study explores the relationship between metabolic gene expression and antiviral responses in shrimp using transcriptome analysis. The RNA-seq libraries of Fenneropenaeus merguensis hemocytes after WSSV challenge at early (6 hpi) and late (24 hpi) stages of infection were analyzed to identify differentially expressed genes (DEGs) that the WSSV subverted the expression. One-hundred-thirty-three DEGs that were expressed in response to WSSV infection at both stages were identified. Based on the GO annotation, they were related to innate immunity and metabolic pathway. The expression correlation between "full term" (NGS) and qRT-PCR of 16 representative DEGs is shown. Noticeably, the expression profiles of all the selected metabolic genes involved in glucose metabolism, lipid metabolism, amino acid metabolism, and nucleotide metabolism showed a specific correlation between NGS and qRT-PCR upon WSSV infection. Of these, we further characterized the function related to the WSSV response of glutamine: fructose-6-phosphate aminotransferase (FmGFAT), the rate-limiting enzyme of the hexosamine biosynthesis pathway, which was found to be up-regulated at the late stage of WSSV infection. Suppression of FmGFAT by RNA interference resulted in postponing the death of WSSV-infected shrimp and reduction of viral copy number. These results suggested that the FmGFAT is linked between metabolic change and WSSV responses in shrimp, where the virus-induced metabolic rewiring hijack biological compounds and/or energy sources to benefit the viral replication process.

Here, we present the genome sequence of lymphocystis disease virus 2 LCDV-JP_Oita_2018 (genus Lymphocystivirus , family Iridoviridae ), which was isolated from a diseased Japanese flounder ( Paralichthys olivaceus ) in Japan. The LCDV-JP_Oita_2018 genome was assembled into a circular contig of 186,627 bp, with 140 predicted protein-coding genes and a GC content of 27%.

Moritella sp. strains F1 and F3 are lipid-degrading bacteria that were isolated from intermediate water from the Sagami Trough, in Japan. We present the draft genome sequences of these two strains, which have 4,983,334 bp and 4,967,310 bp, respectively.

Streptococcus agalactiae is a causative agent of streptococcosis disease in various fish species, including Nile tilapia (Oreochromis niloticus Linn.). Vaccination is an effective disease prevention and control method, but limitations remain for protecting against catastrophic mortality of fish infected with different strains of streptococci. Immunoproteomics analysis of S. agalactiae was used to identify antigenic proteins and construct a chimeric multiepitope vaccine. Epitopes from five antigenic proteins were shuffled in five helices of a flavodoxin backbone, and in silico analysis predicted a suitable RNA and protein structure for protein expression. 45F2 and 42E2 were identified as the best candidates for a chimeric multiepitope vaccine. Recombinant plasmids were constructed to produce a recombinant protein vaccine and DNA vaccine system. Overexpressed proteins were determined to be 30 kDa and 25 kDa in the E. coli and TK1 systems, respectively. The efficacy of the chimeric multiepitope construct as a recombinant protein vaccine and DNA vaccine was evaluated in Nile tilapia, followed by S. agalactiae challenge at 1 × 107 CFU/mL. Relative percentage survival (RPS) and cumulative mortality were recorded at approximately 57–76% and 17–30%, respectively. These chimeric multiepitope vaccines should be applied in streptococcosis disease control and developed into a multivalent vaccine to control multiple diseases.

The genomes of Streptococcus agalactiae (group B streptococcus; GBS) collected from diseased fish in Thailand and Vietnam over a nine-year period (2008–2016) were sequenced and compared (n = 21). Based on capsular serotype and multilocus sequence typing (MLST), GBS isolates are divided into 2 groups comprised of i) serotype Ia; sequence type (ST)7 and ii) serotype III; ST283. Population structure inferred by core genome (cg)MLST and Bayesian clustering analysis also strongly indicated distribution of two GBS populations in both Thailand and Vietnam. Deep phylogenetic analysis implied by CRISPR array's spacer diversity was able to cluster GBS isolates according to their temporal and geographic origins, though ST7 has varying CRISPR1-spacer profiles when compared to ST283 strains. Based on overall genotypic features, Thai ST283 strains were closely related to the Singaporean ST283 strain causing foodborne illness in humans in 2015, thus, signifying zoonotic potential of this GBS population in the country.

Lectins are found in most living organisms, providing immune surveillance by binding to carbohydrate ligands. In fishes, C-type lectins were isolated from mucus of respiratory organs (skin and gills), where they aid the mucosal immune response in regulating microbiota and suppressing pathogens. In shrimp, however, no mucosal immunity or any form of gill-specific immune defense has been reported, and most identified C-type lectins are associated with hemocyte cellular and humoral responses. Interestingly, our microarray analysis revealed the localization of highly expressed novel biodefense genes in gills, among which is Marsupenaeus japonicus gill C-type lectin (MjGCTL), which we previously reported. Gill mucus collected from M. japonicus displayed similar bacterial agglutination ability as observed with recombinant MjGCTL. This agglutinating ability can be attributed to endogenous MjGCTL (nMjGCTL) detected in gill mucus, which was confirmed with an agglutination assay using purified nMjGCTL from gills. In addition, nMjGCTL also promoted in vivo bacterial phagocytosis by hemocytes. In vivo knockdown of MjGCTL resulted in a compromised immune system, which was manifested by impaired agglutination capacity of gill mucus and downregulation of the gill antimicrobial peptides, crustin and penaeidin. Shrimp immunocompromised by MjCGTL knockdown, apparently lost the ability to respond to attaching and penetrating bacteria. This was evident as increased total bacteria and Vibrio counts in both gills and hemolymph, which were correlated with low survival during a bacterial challenge. These results reveal immune defense by shrimp gills resembling a primitive form of mucosal immunity.

Acute hepatopancreatic necrosis disease (AHPND), caused by a toxin-producing Vibrio parahaemolyticus strain, has become a serious threat to shrimp aquaculture. The need to regulate antibiotic use prompted the development of alternative ways to treat infections in aquaculture including the use of chicken egg yolk immunoglobulin (IgY) for passive immunization. This study evaluated the protective effect of IgY against AHPND infection in Litopenaeus vannamei (Boone). IgY was isolated from eggs laid by hens immunized with recombinant PirA-like (rPirA) and PirB-like (rPirB) toxins. Whole-egg powders having IgY specific to rPirA (anti-PirA-IgY) and rPirB (anti-PirB-IgY) and IgY from non-immunized hen (control-IgY) were mixed with basal diets at 20% concentrations and used to prefeed shrimp 3 days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti-PirA-IgY, anti-PirB-IgY and control-IgY diets were 86%, 14% and 0%, respectively. Only the feed containing anti-PirA-IgY protected shrimp against AHPND. Increasing the concentration of rPirA antigen to immunize hens and lowering the amount of egg powder in feeds to 10% consistently showed higher survival rates in shrimp fed with anti-PirA-IgY (87%) compared with the control (12%). These results confirm that addition of anti-PirA-IgY in feeds could be an effective prophylactic method against AHPND infection in shrimp.

Crustacean hemocytes, which have usually been classified morphologically based on methods using Giemsa or May-Giemsa stains, have recently been categorized using monoclonal antibodies or marker genes. However, these latter techniques are not yet widely used, and different classification methods are used for hemocytes among laboratories. Therefore, we aimed to develop a molecular classification method that can be widely used by researchers. The method we have developed uses lectins and magnetic-activated cell sorting (MACS) to isolate sub-populations of hemocytes. Two lectins, wheat germ agglutinin (WGA) and tomato lectin (Lycopersicon esculentum lectin; LEL), characteristically bind to hemocytes, which allows them to be classified into three sub-populations. Furthermore, different sub-populations of hemocyte can be isolated by using LEL and MACS. These sub-populations were characterized as non-granular and granular hemocytes, and the accumulation patterns of the gene transcripts were consistent with the results of a functional analysis reported previously. The lectin-based hemocyte isolation method developed in this study has good reproducibility.

Fc receptors (FcRs) are specific to the Fc portion of immunoglobulin (Ig) molecules. Here, four Fc receptor-like proteins, JF-FcR-like protein 1-4, were identified in Japanese flounder. Their open reading frames encoded 358, 255, 519 and 441 amino acid residues, respectively. JF-FcR-like protein mRNAs were mainly detected in kidney and spleen of healthy fish. Injection of formalin-killed cells (FKCs) of Edwardsiella tarda significantly increased the spleen mRNA levels of JF-FcR-like protein 1 but not the other JF-FcR-like proteins. Injection of FKC of Streptococcus iniae did not significantly affect any of the JF-FcR-like protein mRNAs. These findings suggest that the FcR-like proteins have different involvements in pathogen responses.

We examined lysozyme activities in the serum and the leukocyte extracts of the banded houndshark Triakis scyllium.The serum exhibited lytic activity, but not the leukocyte extracts.The lytic substance in the serum was of approximately 14 kDa and the N-terminal amino acid sequence was YVYSK.cDNA cloning identified a C-type lysozyme (TsLysC) gene and two G-type lysozyme (TsLysG) cDNA clones of different lengths.The TsLysC gene encodes 149 amino acids residues, and the sequence derived from the N-terminal amino acid sequencing was displayed at position 17–21. TsLysG, on the other hand, contains two ORFs that are homologous to the N- and C-terminal regions of G-type lysozyme of other fish species.TsLysC mRNA levels were high in the liver. TsLysG mRNA level was significantly lower than TsLysC mRNA in the liver., 公開日: 2023-06-04

Antibodies of cartilaginous fish are of scientific interest due to their phylogenetic position. In the present study, we developed antiserum against IgM of the banded houndshark, Triakis scyllium, and characterized binding activity of the IgM against fish pathogenic bacteria.Pentameric and monomeric IgM antibodies were separated by gel filtration chromatography using high performance liquid chromatography and SDS–PAGE. Antisera were developed by immunizing rabbits with unfractionated IgM antibodies separated by SDS–PAGE electrophoresis.Shark serum antibodies were found to have binding affinity for Aeromonas hydrophila, Vibrio anguillarum, Edwardsiella tarda, and Pseudomonas plecoglossicida antigens but not Lactococcus garvieae by enzyme-linked immunosorbent assay.We speculate the binding activities of shark antibodies may confer protection against certain bacterial pathogens., 公開日: 2022-07-08

Although microorganisms in pond water and shrimp gut have been thought to be important factors in shrimp health, knowledge of the microbiota surrounding the shrimp is limited. In this study, we analyzed bacterial and eukaryotic microbiota in the pond water of whiteleg shrimp Litopenaeus vannamei, and bacterial compositions in the stomach and midgut of shrimp that are reared in the ponds.Microorganisms were collected at three sites in each of two ponds using filter membranes for 7 consecutive days.Amplicons of 16S rDNA and 18S rDNA were generated. Shrimp were also collected from the ponds on the same days and 16S rDNA was amplified from their stomachs and midguts and analyzed.Results showed that the bacterial communities in the surface water were distributed uniformly and were relatively stable, and the differences between the ponds were small. However, a principal coordinate analysis indicated significant differences in the composition of bacteria in the water of each pond and the microbiota shifted continuously during the sampling period.The eukaryotic plankton in the water changed dynamically and consecutively including the alternations of the dominant families in 1 to 2 days.The bacterial compositions of the digestive tracts, especially the stomach, clearly differed between the two ponds.Also, a bacterium, classified as Candidatus Bacilloplasma in class Mollicutes which was found in the stomachs might be a novel lineage of Mollicutes.Analysis of the combination of bacterial and eukaryotic microbiota, and not only the intestine (midgut) but also the stomach microbiota would be useful to understand microbiome in shrimp aquaculture., 公開日: 2024-03-20

16S rRNA sequences of the lipid-degrading bacteria Moritella sp., strains F1 and F3

The Gram-negative bacterium Vibrio nigripulchritudo is an important shrimp pathogen. Here, we present the genome sequence of Vibrio nigripulchritudo TUMSAT-TG-2018, which was isolated from a diseased pacific white shrimp ( Litopenaeus vannamei ). The assembly totaled 6.8 Mbp, consisting of two chromosomes and four plasmids.