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A large number of farmed red sea bream (Pagrus major) and their gametes have escaped and dispersed into the wild from net-cage aquaculture since the advent of the technology in the mid-20th century. These escapees have likely been recruited into wild populations, affecting their genetic diversity. In this study, we used assignment methods with microsatellite markers to assess the frequency of escapee farmed red sea bream within the wild populations in a major aquaculture production region (UWJ) and in two regions with relatively lower production (IWG and SIJ) in Ehime Prefecture, Japan. The frequency of escapees in UWJ ranged from 14.1% to 30.2%, as shown by three methods of assignment. Escapees were frequently identified among specimens caught by angling (ranging from 26.7% to 46.7%), suggesting that they stay in the same area even after escape or are less cautious than wild individuals. No escapee was found in IWG and SIJ, areas with less extensive aquaculture. Some wild-caught fish appeared to have hybridized with farmed fish, as indicated by the threshold of membership coefficients obtained by simulation. These results clearly indicate that large numbers of farmed individuals have escaped from UWJ, a major aquaculture area, since the inception of red sea bream farming in Japan. Genetic reshuffle has possibly occurred following hybridization of wild and farmed individuals.

Medaka Oryzias latipes is a small freshwater fish widely distributed in Japan. It is a popular ornamental fish and now has been recognized as an important model organism in many areas of biological research. Here we report microsporidian infections for the first time in medaka, from 2 research facilities and a wild population. Infected medaka exhibited abnormal appearance with whitish trunk muscle, and microsporidian spores were detected from the affected tissue. The size of spores was similar in all the three cases: 7.0 μm in length and 3.7-4.2 μm in width. In the histological observation, numerous sporophorous vesicles containing spores or other developmental stages were observed within the myocytes of the trunk muscle. Nucleotide sequence of the ribosomal RNA gene was determined and it was identical among all three cases. A BLAST search revealed it shared 99.5-99.6% identity with Pleistophora hyphessobryconis, a microsporidian known to infect >20 freshwater fish species. Light microscopic observation of spores and histological features also indicated the microsporidian infection in medaka is caused by P. hyphessobryconis. This is the first record of the microsporidian species from medaka and from Japan., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Female Japanese flounder Paralichthys olivaceus grow more rapidly than the male. The goal of all-female commercial production requires an efficient method of genetic sex identification. We conducted genome-wide association analysis of female and male farmed Japanese flounder (n = 24 per phenotypic sex) and found all regions of chromosome 24 to be significantly associated with phenotypic sex, suggesting it as the sex chromosome. Genetic sex was identified based on single nucleotide polymorphisms (SNP) on chromosome 24 (n = 3568) using multidimensional scaling analysis, and individuals were clearly separated according to sex by the first dimension. The 61 SNPs most highly associated with sex were selected, and an amplicon-based SNP panel was developed. This was used to determine genetic sex of 39 females and 40 males. Eleven phenotypic males were assigned as female with XX genotype, suggesting sex reversal. Genetic sex was also assessed based on the indel of the amh gene promoter, which is the major candidate sex gene of Japanese flounder. We found four SNPs perfectly associated with genotypic sex in the sex-associated SNP panel, one of which was located in exon 2 of the amh gene. Along with the indel of the amh gene promoter, the sex-associated SNP panel will be of value in identifying genetic sex of farmed Japanese flounder. Molecular sexing will facilitate all-female production by breeding sex-reversed males., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The morphology of farm-reared fish often differs from that of their wild counterparts, impacting their market value. Two caudal fin tip shapes, acutely angled and blunted, are recognized in farmed populations of red sea bream Pagrus major. The angled form is preferred by consumers over the blunt since it resembles that of wild fish. Discovering the cause of the blunted tip is crucial to maximizing the commercial value of farmed red sea bream. We hypothesized that the blunt fin tip is the result of opportunistic bacteria and conducted partial 16S rRNA metagenomic barcoding and generated a clone library of the 16S rRNA gene to compare bacterial communities of the 2 fin forms. Metagenomic barcoding revealed an abundance of 5 bacterial genera, Sulfitobacter, Vibrio, Tenacibaculum, Psychrobacter, and an unknown genus of Rhodobacteraceae, on the caudal fin surface. Sulfitobacter was significantly more common on the angled caudal fin than the blunted. Vibrio is the dominant genus on the blunted caudal fin. The clone library identified these genera to species level, and Sulfitobacter sp., Vibrio harveyi, Tenacibaculum maritimum, and Psychrobacter marincola were frequently observed in blunt caudal fins. Our results suggest that opportunistic pathogenic bacteria such as V. harveyi and T. maritimum are not the primary cause of caudal fin malformation, and multiple factors such as combinations of injury, stress, and pathogenic infection may be involved. The reason for the significantly greater occurrence of Sulfitobacter sp. in the angled caudal fin is unknown, and further investigation is needed.

The processes by which animals become genetically isolated in an open environment such as the ocean have not yet been fully elucidated. Morphologically different populations of Pacific white-sided dolphin Lagenorhynchus obliquidens are observed sympatrically in the Sea of Japan. However, genetic studies that exclusively used limited mitochondrial loci or microsatellite DNA have failed to demonstrate the existence of genetically distinct populations. Here, to reveal the population structure, we analysed genome-wide population genetic data using single nucleotide polymorphisms (SNPs) gathered in 2018-2021 from all domestic captive individuals in aquaria, the majority of which originated from the wild, as well as from some stranded individuals, together covering a wide area of coastal water around Japan (n = 123). Multiplexed intersimple sequence repeat genotyping-by-sequencing analysis was performed to obtain the SNP data. Principal coordinate analysis and the clustering method structure indicated that two genetically-distinct populations exist, with little interpopulation gene flow revealed. In addition, the genotypic segregation was reflected in differences in external morphotype. Furthermore, a population demographic analysis based on the whole-genome sequences of an individual from each population indicated that sea-level changes during the Last Glacial Period probably led to allopatric divergence of this species in a limited area of the Sea of Japan, with that group subsequently sharing a distribution area with the other population. These findings yield insights into the formation of genetically isolated sympatric populations in the ocean., (© 2022 John Wiley & Sons Ltd.)

Parentage sibship-inference analyses were conducted using mtDNA sequencing and six microsatellite genotypes of 182 Japanese eel preleptocephali that were collected from one net-tow near the West Mariana Ridge in May 2014. At least 328 parents were involved in producing the 182 preleptocephali, and several parents may have spawned a few times during 3 days of a spawning period. Half-sibs suggested that a few parents mated with 1-3 partners, indicating that the Japanese eel can form spawning aggregations in which several parents mate with each other in the ocean., (© 2022 Fisheries Society of the British Isles.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Deformities in cultured fish species may be genetic, and identifying causative genes is essential to expand production and maintain farmed animal welfare. We previously reported a genetic deformity in juvenile red sea bream, designated a transparent phenotype. To identify its causative gene, we conducted genome-wide linkage analysis and identified two single nucleotide polymorphisms (SNP) located on LG23 directly linked to the transparent phenotype. The scaffold on which the two SNPs were located contained two candidate genes, duox and duoxa, which are related to thyroid hormone synthesis. Four missense mutations were found in duox and one in duoxa, with that in duoxa showing perfect association with the transparent phenotype. The mutation of duoxa was suggested to affect the transmembrane structure and thyroid-related traits, including an enlarged thyroid gland and immature erythrocytes, and lower thyroxine (T 4 ) concentrations were observed in the transparent phenotype. The transparent phenotype was rescued by T 4 immersion. Loss-of-function of duoxa by CRISPR-Cas9 induced the transparent phenotype in zebrafish. Evidence suggests that the transparent phenotype of juvenile red sea bream is caused by the missense mutation of duoxa and that this mutation disrupts thyroid hormone synthesis. The newly identified missense mutation will contribute to effective selective breeding of red sea bream to purge the causative gene of the undesirable phenotype and improve seed production of red sea bream as well as provide basic information of the mechanisms of thyroid hormones and its related diseases in fish and humans., (© 2021. The Author(s), under exclusive licence to The Genetics Society.)

The complement system plays an important role in immune regulation and acts as the first line of defense against any pathogenic attack. To comprehend the red sea bream (Pagrus major) immune response, three complement genes, namely, pmC1r, pmMASP and pmC3, belonging to the classical, lectin and alternative complement cascade, respectively, were identified and characterized. pmC1r, pmMASP, and pmC3 were comprised of 2535, 3352, and 5735 base mRNA which encodes 732, 1029 and 1677 aa putative proteins, respectively. Phylogenetically, all the three studied genes clustered with their corresponding homologous clade. Tissue distribution and cellular localization data demonstrated a very high prevalence of all the three genes in the liver. Both bacterial and viral infection resulted in significant transcriptional alterations in all three genes in the liver with respect to their vehicle control counterparts. Specifically, bacterial challenge affected the pmMASP and pmC3 expression, while the viral infection resulted in pmC1r and pmC3 mRNA activation. Altogether, our data demonstrate the ability of pmC1r, pmMASP and pmC3 in bringing about an immune response against any pathogenic encroachment, and thus activating, not only one, but all the three complement pathways, in red sea bream., (Copyright © 2018. Published by Elsevier Ltd.)

Beginning with the larval stages, marine pufferfish such as Takifugu niphobles contain tetrodotoxin (TTX), an extremely potent neurotoxin. Although highly concentrated TTX has been detected in adults and juveniles of these fish, the source of the toxin has remained unclear. Here we show that TTX in the flatworm Planocera multitentaculata contributes to the toxification of the pufferfish throughout the life cycle of the flatworm. A species-specific PCR method was developed for the flatworm, and the specific DNA fragment was detected in the digesta of wild pufferfish adults. Predation experiments showed that flatworm larvae were eaten by the pufferfish juveniles, and that the two-day postprandial TTX content in these pufferfish was 20-50 μg/g. Predation experiments additionally showed flatworm adults were also eaten by pufferfish young, and after two days of feeding, TTX accumulated in the skin, liver and intestine of the pufferfish.

We previously reported a body color deformity in juvenile red sea bream, which shows transparency in the juvenile stage because of delayed chromatophore development compared with normal individuals, and this finding suggested a genetic cause based on parentage assessments. To conduct marker-assisted selection to eliminate broodstock inheriting the causative gene, developing DNA markers associated with the phenotype was needed. We first conducted SNP mining based on AFLP analysis using bulked-DNA from normal and transparent individuals. One SNP was identified from a transparent-specific AFLP fragment, which significantly associated with transparent individuals. Two alleles (A/G) were observed in this locus, and the genotype G/G was dominantly observed in the transparent groups (97.1%) collected from several production lots produced from different broodstock populations. A few normal individuals inherited the G/G genotype (5.0%), but the A/A and A/G genotypes were dominantly observed in the normal groups. The homologs region of the SNP was searched using a medaka genome database, and intron 12 of the Nell2a gene (located on chromosome 6 of the medaka genome) was highly matched. We also mapped the red sea bream Nell2a gene on the previously developed linkage maps, and this gene was mapped on a male linkage group, LG4-M. The newly found SNP was useful in eliminating broodstock possessing the causative gene of the body color transparency observed in juvenile stage of red sea bream.

It is known that tetrodotoxin (TTX), also known as pufferfish toxin, is an extremely potent neurotoxin and had been detected in various taxa. However, the exact function of the toxin in TTX-bearing organisms has remained unclear. In Takifugu pufferfish species, it has been suggested that TTX is utilized to protect larvae from predators but no experimental proof exists. In the present study, we used pufferfish Takifugu alboplumbeus larvae from wild and cultured parents to determine the effects of the maternal TTX on the survival of toxic and non-toxic pufferfish larvae, respectively. TTX contents in the larval pufferfish differed between the larvae derived from wild and cultured parents (1.23 ± 0.20 ng/individual vs. undetectable levels, respectively). Immunohistochemical staining with anti-TTX monoclonal antibody demonstrated that the TTX-specific signals were primarily observed at the body surface of the larvae of wild parents, but not of cultured parents. Predation experiments demonstrated that the juveniles of Girella punctata and Chaenogobius gulosus, used as predator fish, ingested the pufferfish larvae derived from either type of parents, but instantly spat out those from wild parents only. These results indicate that larvae, which are at the most vulnerable stage in the life of pufferfish, are protected by maternal TTX., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Red sea bream iridoviral disease (RSIVD) is a major viral disease in red sea bream farming in Japan. Previously, we identified one candidate male individual of red sea bream that was significantly associated with convalescent individuals after RSIVD. The purpose of this study is to identify the quantitative trait loci (QTL) linked to the RSIVD-resistant trait for future marker-assisted selection (MAS). Two test families were developed using the candidate male in 2014 (Fam-2014) and 2015 (Fam-2015). These test families were challenged with RSIV, and phenotypes were evaluated. Then, de novo genome sequences of red sea bream were obtained through next-generation sequencing, and microsatellite markers were searched and selected for linkage map construction. One immune-related gene, MHC class IIβ, was also used for linkage map construction. Of the microsatellite markers searched, 148 and 197 were mapped on 23 and 27 linkage groups in the female and male linkage maps, respectively, covering approximately 65% of genomes in both sexes. One QTL linked to an RSIVD-resistant trait was found in linkage group 2 of the candidate male in Fam-2014, and the phenotypic variance of the QTL was 31.1%. The QTL was closely linked to MHC class IIβ. Moreover, the QTL observed in Fam-2014 was also significantly linked to an RSIVD-resistant trait in the candidate male of Fam-2015. Our results suggest that the RSIVD-resistant trait in the candidate male was controlled by one major QTL closely linked to the MHC class IIβ gene and could be useful for MAS of red sea bream.