Kuchijirosho virus (KJV) infects the brain of the tiger puffer, Takifugu rubripes, causing lethal disease in cultured populations. Next-generation sequencing identified eight RNA genome segments encoding proteins of 562, 498, 454, 369, 370, 164, 207, and 83 amino acids. BLAST analysis showed that segment 1 encodes a polymerase basic protein 1 (PB1)-like RNA-dependent RNA polymerase homologous to those of members of the family Amnoonviridae. Segment 5 encodes a protein that shows similarity to envelope proteins with two predicted transmembrane domains, suggesting a function as an envelope glycoprotein. The remaining segments could not be annotated due to the limited homology of their encoded proteins to known proteins. These findings indicate that KJV represents a novel lineage within the family Amnoonviridae.

Abstract Teleost display remarkable species diversity despite relatively conserved karyotypes, suggesting an important role for chromosomal rearrangements in speciation. Yet this hypothesis remains poorly tested in pelagic marine fishes, as genomic studies have predominantly focused on freshwater and coastal taxa. In this study, we investigated the genomic basis of hybrid incompatibility between two parapatric Pacific mackerels, Scomber japonicus and S. australasicus , hypothesizing chromosomal rearrangements as the major driving forces. We generated haplotype-resolved de novo genome assemblies for both Pacific species and assessed genomic rearrangements along with the genome of the Atlantic species, S. scombrus, reconstructed from publicly available data. Comparative genomic analyses revealed extensive chromosomal rearrangements across the genomes. Notably, the rate of chromosomal inversions was approximately sevenfold higher between the two Pacific species than between allopatric lineages. These rearrangements included complex structural changes involving megabase-scale inversions and associated translocations. Our analyses also showed that the sex chromosomes of the three species evolved independently. In the two Pacific species, large recombination-suppression regions (>10 Mb) arose convergently but through distinct mechanisms: tandem chromosomal inversions in S. japonicus and, most likely, transposable element–mediated sequence divergence in S. australasicus . By contrast, recombination suppression in S. scombrus is restricted to a ∼14 kb hemizygous region containing amhr2Y , generated by duplication and translocation. Population-level analyses further revealed ongoing evolution of recombination-suppressed regions in the Pacific species and uncovered multiple Y chromosome lineages in S. australasicus that differ markedly in the extent of recombination suppression. Together, these results demonstrate rapid structural genome evolution in Scomber and provide a genomic framework for understanding how chromosomal rearrangements contribute to reproductive isolation, sex chromosome turnover, and the diversification of pelagic marine fishes.

Anisakis simplex complex is a major causative agent of human anisakiasis, but species composition and tissue distribution, including intra-vitam muscle parasitism, remain unclear. We investigated the prevalence and tissue localization of Anisakis larvae in live-caught Pacific chub mackerel (Scomber japonicus) from four Japanese regions. A high-resolution melting (HRM) assay targeting the ITS region enabled high-throughput discrimination of A. simplex sensu stricto (s.s.), A. pegreffii, A. berlandi, and A. simplex s.s. × A. pegreffii hybrids. In Iwate and Shizuoka (Pacific coast), A. simplex s.s. was predominant in both viscera and muscle tissues, with a positive correlation between tissue burdens and a ventral bias of muscle larvae. In Fukui (Sea of Japan), overall prevalence was high, and the viscera showed a mixed sibling-species composition, whereas muscle larvae were predominantly A. simplex s.s. In Nagasaki (East China Sea), A. pegreffii dominated the viscera, but only A. simplex s.s. occurred in muscle. These findings indicate that A. simplex s.s. can parasitize muscle of live mackerel and that heavier visceral infections are associated with greater muscle involvement. This study underscores the importance of sibling-species monitoring, proper cold-chain and evisceration management, and HRM assays for effective surveillance to mitigate anisakiasis risk.

The initial detection of host-derived chemical cues is a key physiological event that strongly influences infection success in parasitic copepods such as Caligus fugu. Although ionotropic receptors were previously identified in C. fugu as potential host-sensing molecules, their ligands and specific roles in host recognition remain unknown. Since the infective copepodid I stage preferentially attaches to the fins of the puffer host, genes expressed at higher levels in fins may contribute to host recognition by C. fugu. In this study, differentially expressed genes between the pectoral fins and skin of Takifugu rubripes were comprehensively identified through RNA-seq to expand the list of candidate host-recognition genes beyond those detected earlier. To refine this list, genes expressed at higher levels in pectoral fins that were annotated with the gene ontology term "extracellular region" (Gene Ontology:0005576) were examined for predicted protein features, with emphasis on secreted, soluble molecules that the parasite could encounter in the surrounding water. From an initial set of 126 genes, intracellular and membrane-associated proteins were removed, and those possessing signal peptides or globular domains were retained. The remaining 92 proteins were considered likely secreted, water-soluble host-derived ligands. Relative qPCR analysis highlighted col10a1a, stc2a, mmp9, fibinb, bmp5, c4b, bmp6, and lipg as potential secreted cues for C. fugu. Antimicrobial peptides such as hamp and db1 were also identified as candidates. Overall, this study expands the pool of molecules that may function as chemoattractants for C. fugu and provides targets for future experimental validation.

Euryhaline fishes acclimate to various osmotic environments by changing the direction of water and ion transport between body fluids and environmental waters. Ionocytes in the gills are one of the most important cells for the active ion transport. This study aimed to identify the molecules responsible for apical Cl− transport in rainbow trout ionocytes. Tissue distribution and time-course changes after seawater transfer were analyzed for mRNA expression of slc26a6, cftr1, and cftr2. slc26a6 was specifically expressed in the freshwater gills and decreased after seawater transfer. Both cftr genes were expressed in the gills and higher in seawater; however, the magnitude of expression increase after seawater transfer was greater in cftr1 than in cftr2. These results suggest that Cftr1 is mainly functioned in hypo-osmoregulation and that Cftr2 may also be involved in ion transport under freshwater conditions, such as acid–base regulation. Slc26a6 was localized at the apical membrane of Nkcc1-negative ionocytes only in freshwater-acclimated trout. Apical Cftr1 localization was also identified in most of ionocytes in seawater-acclimated fish. These results indicate that Slc26a6 in freshwater and Cftr1 in seawater contribute to osmoregulatory Cl− transport across the apical membrane of ionocytes in rainbow trout.

CD4 and LAG-3 are related molecules that are receptors for MHC class II molecules. Their major functional differences are situated in their cytoplasmic tails, in which CD4 has an activation motif and LAG-3 an inhibitory motif. Here, we identify shark LAG-3 and show that a previously identified shark CD4-like gene has a genomic location, expression pattern, and motifs similar to CD4 in other vertebrates. In nurse shark (Ginglymostoma cirratum) and cloudy catshark (Scyliorhinus torazame), the highest CD4 expression was consistently found in the thymus whereas such was not the case for LAG-3. Throughout jawed vertebrates, the CD4 cytoplasmic tail possesses a Cx(C/H) motif for binding kinase LCK, and the LAG-3 cytoplasmic tail possesses (F/Y)xxL(D/E) including the previously determined FxxL inhibitory motif resembling an immunoreceptor tyrosine-based inhibition motif (ITIM). On the other hand, the acidic end of the mammalian LAG-3 cytoplasmic tail, which is believed to have an inhibitory function as well, was acquired later in evolution. The present study also identified CD4-1, CD4-2, and LAG-3 in the primitive ray-finned fishes bichirs, sturgeons, and gars, and experimentally determined these sequences for sterlet sturgeon (Acipenser ruthenus). Therefore, with CD4-1 and CD4-2 already known in teleosts (modern ray-finned fish), these two CD4 lineages have now been found within all major clades of ray-finned fish. Although different from each other, the cytoplasmic tails of ray-finned fish CD4-1 and chondrichthyan CD4 not only contain the Cx(C/H) motif but also an additional highly conserved motif which we expect to confer a function. Thus, although restricted to some species and gene copies, in evolution both CD4 and LAG-3 molecules appear to have acquired functional motifs besides their canonical Cx(C/H) and ITIM-like motifs, respectively. The presence of CD4 and LAG-3 molecules with seemingly opposing functions from the level of sharks, the oldest living vertebrates with a human-like adaptive immune system, underlines their importance for the jawed vertebrate immune system. It also emphasizes the general need of the immune system to always find a balance, leading to trade-offs, between activating and inhibiting processes.

See GitHub - sapporo-wes/test-workflow - #broadinstitute/gatk/MitochondriaPipeline.

See GitHub - sapporo-wes/test-workflow - biosciencedbc/jga-analysis - per-sample workflow.

Rainbow trout fry syndrome (RTFS) and bacterial coldwater disease (BCWD) is a globally distributed freshwater fish disease caused by Flavobacterium psychrophilum. In spite of its importance, an effective vaccine is not still available. Manipulation of the microbiome of skin, which is a primary infection gate for pathogens, could be a novel countermeasure. For example, increasing the abundance of specific antagonistic bacteria against pathogens in fish skin might be effective to prevent fish disease. Here, we combined cultivation with 16S rRNA gene amplicon sequencing to obtain insight into the skin microbiome of the rainbow trout (Oncorhynchus mykiss) and searched for skin bacteria antagonistic to F. psychrophilum. By using multiple culture media, we obtained 174 isolates spanning 18 genera. Among them, Bosea sp. OX14 and Flavobacterium sp. GL7 respectively inhibited the growth of F. psychrophilum KU190628-78 and NCIMB 1947(T), and produced antagonistic compounds of<3 kDa in size. Sequences related to our isolates comprised 4.95% of skin microbial communities, and those related to strains OX14 and GL7 respectively comprised 1.60% and 0.17% of the skin microbiome. Comparisons with previously published microbiome data detected sequences related to strains OX14 and GL7 in skin of other rainbow trout and Atlantic salmon.

Kuchijirosho is a lethal infectious disease of fugu Takifugu rubripes, and the causative pathogen has been predicted to be an RNA virus. Although the homogenate of kuchijirosho-affected brain is pathogenic to fugu, the suspected viral particles have not been found in the brain and the viral genome has not been isolated. We attempted to clone the cDNA of the kuchijirosho virus genome using the Rapid Determination System for Viral RNA Sequence method. Three cDNA segments of ca. 1,000 nt each, which could be parts of the viral genome, were obtained from total RNA extracted from the brains of fugu artificially infected with kuchijirosho. According to RT-qPCR, the brain had more of these three kuchijirosho-associated RNAs (KARs) than any other tissues. KARs in the brain were detected 1-2 days after injecting the homogenate of kuchijirosho-affected brain and KARs expression levels were increased rapidly until death. These results show that the detection of KARs can be sufficiently effective for the molecular diagnosis of kuchijirosho. Even if KARs are parts of the viral genome, it is unclear to which taxonomic family the kuchijirosho virus belongs, because the nucleotide sequences of KARs did not correspond to those of any other organisms including viruses.

Heterobothrium okamotoi, a monogenean gill parasite, exhibits high host specificity for the tiger puffer, Takifugu rubripes, and it has been experimentally verified that the parasite cannot colonize either closely related species such as the grass puffer Takifugu niphobles or distantly related fish such as the red seabream Pagrus major. Previously, we demonstrated in T. rubripes that immunoglobulin M (IgM) with d-mannose affinity induced deciliation of the oncomiracidia, the first step of parasitism, indicating that the parasite utilizes the molecule as a receptor for infection. In the present study, we purified mannose-specific IgM from 2 nonhost species, T. niphobles and P. major, by affinity and gel-filtration chromatography techniques and compared their deciliation-inducing activity against H. okamotoi oncomiracidia. The IgM of the former showed activity, whereas the latter had no effect, suggesting that in addition to d-mannose-binding ability, the crystallizable fragment domain of IgM, which is not part of the antigen-binding domain, plays an important role in host recognition by the oncomiracidia, such as direct binding to the parasites. It also suggests that the host specificity of H. okamotoi is relatively low upon initial recognition, and the specificity is established by exclusion in nonhosts during a later stage.

Rapid radiation associated with phenotypic divergence and convergence provides an opportunity to study the genetic mechanisms of evolution. Here we investigate the genus Takifugu that has undergone explosive radiation relatively recently and contains a subset of closely-related species with a scale-loss phenotype. By using observations during development and genetic mapping approaches, we show that the scale-loss phenotype of two Takifugu species, T. pardalis Temminck & Schlegel and T. snyderi Abe, is largely controlled by an overlapping genomic segment (QTL). A search for candidate genes underlying the scale-loss phenotype revealed that the QTL region contains no known genes responsible for the evolution of scale-loss phenotype in other fishes. These results suggest that the genes used for the scale-loss phenotypes in the two Takifugu are likely the same, but the genes used for the similar phenotype in Takifugu and distantly related fishes are not the same. Meanwhile, Fgfrl1, a gene predicted to function in a pathway known to regulate bone/scale development was identified in the QTL region. Since Fgfr1a1, another memebr of the Fgf signaling pathway, has been implicated in scale loss/scale shape in fish distantly related to Takifugu, our results suggest that the convergence of the scale-loss phenotype may be constrained by signaling modules with conserved roles in scale development.