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Crustacean aquaculture is expected to be a major source of fishery commodities in the near future. Hemocytes are key players of the immune system in shrimps; however, their classification, maturation, and differentiation are still under debate. To date, only discrete and inconsistent information on the classification of shrimp hemocytes has been reported, showing that the morphological characteristics are not sufficient to resolve their actual roles. Our present study using single-cell RNA sequencing revealed six types of hemocytes of Marsupenaeus japonicus based on their transcriptional profiles. We identified markers of each subpopulation and predicted the differentiation pathways involved in their maturation. We also predicted cell growth factors that might play crucial roles in hemocyte differentiation. Different immune roles among these subpopulations were suggested from the analysis of differentially expressed immune-related genes. These results provide a unified classification of shrimp hemocytes, which improves the understanding of its immune system.

Aquaculture production is expected to increase with the help of genomic selection (GS). The possibility of performing GS using only a small number of SNPs has been examined in order to reduce genotyping costs; however, the practicality of this approach is still unclear. Here, we tested whether the effects of reducing the number of SNPs impaired the prediction accuracy of GS for standard length, body weight, and testes weight in the tiger pufferfish ( Takifugu rubripes ). High values for predictive ability (0.563–0.606) were obtained with 4000 SNPs for all traits under a genomic best linear unbiased predictor (GBLUP) model. These values were still within an acceptable range with 1200 SNPs (0.554–0.588). However, predictive abilities and prediction accuracies deteriorated using less than 1200 SNPs largely due to the reduced power in accurately estimating the genetic relationship among individuals; family structure could still be resolved with as few as 400 SNPs. This suggests that the SNPs informative for estimation of genetic relatedness among individuals differ from those for inference of family structure, and that non-random SNP selection based on the effects on family structure (e.g., site- F ST , principal components, or random forest) is unlikely to increase the prediction accuracy for these traits.

Heterogametic sex chromosomes have evolved independently in various lineages of vertebrates. Such sex chromosome pairs often contain nonrecombining regions, with one of the chromosomes harboring a master sex-determining (SD) gene. It is hypothesized that these sex chromosomes evolved from a pair of autosomes that diverged after acquiring the SD gene. By linkage and association mapping of the SD locus in fugu (Takifugu rubripes), we show that a SNP (C/G) in the anti-Müllerian hormone receptor type II (Amhr2) gene is the only polymorphism associated with phenotypic sex. This SNP changes an amino acid (His/Asp384) in the kinase domain. While females are homozygous (His/His384), males are heterozygous. Sex in fugu is most likely determined by a combination of the two alleles of Amhr2. Consistent with this model, the medaka hotei mutant carrying a substitution in the kinase domain of Amhr2 causes a female phenotype. The association of the Amhr2 SNP with phenotypic sex is conserved in two other species of Takifugu but not in Tetraodon. The fugu SD locus shows no sign of recombination suppression between X and Y chromosomes. Thus, fugu sex chromosomes represent an unusual example of proto-sex chromosomes. Such undifferentiated X-Y chromosomes may be more common in vertebrates than previously thought.

Two large abalone species prevalent in the Japanese archipelago, Haliotis discus discus and H. madaka , are considered to have different morphological characteristics, and previous population genomics analyses have suggested that they have distinct genomic features. However, integrated analyses comparing the genetic and morphological data from these species are lacking; thus, it remains unclear whether these two species are distinct biological species. In this study, we performed integrated genetic and morphological analyses on individuals of the two species captured in Tokushima Prefecture, Japan. Genetic analysis based on 118 species-diagnostic single-nucleotide polymorphism (SNP) loci showed that H. discus discus and H. madaka were clearly assigned to distinct genetic clusters corresponding to the two species, with the exception of one H. madaka individual showing evidence of genomic introgression from H. discus discus . Multiple statistical analyses based on quantitative characteristics of shell shape and foot muscle color suggested that this H. madaka individual with genomic introgression from H. discus discus had H. discus discus -like quantitative characteristics consistently. In summary, this study suggests that H. discus discus and H. madaka are distinct biological species reproductively isolated in natural environments, but genomic introgression possibly blurs the species boundary between H. discus discus and H. madaka .

Studies using genome-wide single nucleotide polymorphisms (SNPs) have become commonplace in genetics and genomics, due to advances in high-throughput sequencing technologies. Since the numbers of required SNPs and samples vary depending on each research goal, genotyping technologies with high flexibility in the number of SNPs/samples and high repeatability have been intensively investigated. For example, the ultrahigh-multiplexed amplicon sequencing, Ion AmpliSeq, has been used as a high-throughput genotyping method mainly for diagnostic purposes. Here, we designed a custom panel targeting 3,187 genome-wide SNPs of fugu, Takifugu rubripes , and applied it for genotyping farmed fugu to test its feasibility in aquaculture studies. We sequenced two libraries consisting of different pools of individuals ( n  = 326 each) on the Illumina MiSeq sequencer. Consequently, over 99% target regions (3,178 SNPs) were amplified and 2,655 SNPs were available after filtering steps. Strong correlation was observed in the mean depth of coverage of each SNP between duplicate runs ( r  = 0.993). Genetic analysis using these genotype data successfully detected the known population structure and the sex determining locus of fugu. These results show the method is superior in repeatability and flexibility, and suits genetic studies including molecular breeding, such as marker assisted and genomic selection.

Genetic and phenotypic analyses of independent secondary contact zones between certain pairs of divergent populations offer powerful opportunities to assess whether the consequences vary with different environmental backgrounds. Populations of the ice goby Leucopsarion petersii are distributed throughout the Japanese archipelago and comprise genetically and phenotypically divergent groups in the Japan Sea and the Pacific Ocean. In particular, populations in the Japan Sea have a larger body size and numbers of vertebrae than those in the Pacific Ocean. Herein, we performed integrated analyses of genotypes and phenotypes of two independent secondary contact zones and investigated their consequences. Population genetic analyses revealed asymmetric introgression of the mitochondrial genome of either lineage relative to little admixture of nuclear genomes in both secondary contact zones. On phenotype analyses, vertebral numbers were clearly explained by nuclear genomic ancestry in both secondary contact zones, whereas body size was not, suggesting that a little introgression of nuclear genes regulates body size. Actually, we observed biased introgression of a candidate gene, neuropeptide Y (NPY), which potentially controls body size in the ice goby. Moreover, the body size changes in the introgressed populations possibly affect the introgression patterns of mitochondrial genomes across these zones. Collectively, our results demonstrated that genomic and phenotypic consequences of secondary contact varied in marine variable environments.

Subarachnoid hemorrhage (SAH) is a catastrophic form of stroke responsible for significant morbidity and mortality. Oxidative stress, inflammation, and neuronal apoptosis are important in the pathogenesis of early brain injury (EBI) following SAH. Preconditioning exercise confers neuroprotective effects, mitigating EBI; however, the basis for such protection is unknown. We investigated the effects of preconditioning exercise on brain damage and sensorimotor function after SAH. Male rats were assigned to either a sham-operated (Sham) group, exercise (Ex) group, or no-exercise (No-Ex) group. After a 3-week exercise program, they underwent SAH by endovascular perforation. Consciousness level, neurological score, and sensorimotor function were studied. The expression of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), 4-hydroxynonenal (4HNE), nitrotyrosine (NT), ionized calcium-binding adaptor molecule 1 (Iba1), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), interleukin 1β (IL-1β), 14–3-3γ, p -β-catenin Ser37, Bax, and caspase-3 were evaluated by immunohistochemistry or western blotting. The terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) assay was also performed. After SAH, the Ex group had significantly reduced neurological deficits, sensorimotor dysfunction, and consciousness disorder compared with the No-Ex group. Nrf2, HO-1, and 14–3-3γ were significantly higher in the Ex group, while 4HNE, NT, Iba1, TNF-α, IL-6, IL-1β, Bax, caspase-3, and TUNEL-positive cells were significantly lower. Our findings suggest that preconditioning exercise ameliorates EBI after SAH. The expression of 4HNE and NT was reduced by Nrf2/HO-1 pathway activation; additionally, both oxidative stress and inflammation were reduced. Furthermore, preconditioning exercise reduced apoptosis, likely via the 14–3-3γ/ p -β-catenin Ser37/Bax/caspase-3 pathway.

Serum and tissue miR-21 expression in patients with breast cancer (BC) is a useful biomarker for cancer diagnosis, progression, and treatment. Matrix metalloproteinase-1 (MMP-1) is also important in breast cancer carcinogenesis. However, miR-21 and MMP-1/CD63 in urine exosomes in these patients have not been examined. Urine samples were collected from patients with BC and 26 healthy females. Urinary exosomes were isolated and confirmed by western blotting with anti-CD63 antibody and electron microscopy observation. MiR-21 and MMP-1/CD63 expression was examined by quantitative RT-PCR and western blotting, respectively. Patients with very early stage breast cancer were evaluated. MiR-21 expression in the patients was 0.26 [95% CI: 0.20–0.78], which was significant lower than in the 26 controls (1.00 [95% CI: 1.01–3.37], p = 0.0947). MMP-1/CD63 expression in patients was significantly higher than in controls (1.74 [95% CI: 0.86–5.08] vs 0.535 [95% CI: −0.01–2.81], p = 0.0001). Sensitivity and specificity were 0.708 and 0.783 for miR-21 and 0.792 and 0.840 for MMP-1/CD63, respectively. Sensitivity and specificity of combined expression were 95% and 79%, respectively. The sensitivity of MMP-1/CD63 expression in urinary exosomes was better than that of miR-21 expression. Thus, miR-21 and MMP/CD63 may be useful markers for BC screening.

The tiger puffer Takifugu rubripes is one of the most popular aquacultural fish; however, there are two major obstacles to selective breeding. First, they have a long generation time of 2 or 3 years until maturation. Second, the parental tiger puffer has a body size (2–5 kg) much larger than average market size (0.6–1.0 kg). The grass puffer Takifugu niphobles is closely related to the tiger puffer and matures in half the time. Furthermore, grass puffer can be reared in small areas since their maturation weight is about 1/150 that of mature tiger puffer. Therefore, to overcome the obstacles of maturation size and generation time of tiger puffer, we generated surrogate grass puffer that can produce tiger puffer gametes through germ cell transplantation. Approximately 5000 tiger puffer testicular cells were transplanted into the peritoneal cavity of triploid grass puffer larvae at 1 day post hatching. When the recipient fish matured, both males and females produced donor-derived gametes. Through their insemination, we successfully produced donor-derived tiger puffer offspring presenting the same body surface dot pattern, number of dorsal fin rays, and DNA fingerprint as those of the donor tiger puffer, suggesting that the recipient grass puffer produced functional eggs and sperm derived from the donor tiger puffer. Although fine tunings are still needed to improve efficiencies, surrogate grass puffer are expected to accelerate the breeding process of tiger puffer because of their short generation time and small body size.

Acute ischemic stroke (AIS) is a major cause of mortality and disability and remains a serious and significant global health problem. The development of neurovascular protectants to treat AIS successfully has been beset by disappointments and setbacks. Many promising candidates have lacked significant pleiotropic protective activity for brain tissue and cerebral blood vessels in clinical trials, while those with protective activity have had poor bioavailability or high toxicity. Moreover, the majority of agents did not confer significant neurovascular protection or clinical efficacy, as measured by standard behavioral endpoints in clinical trials of heterogeneous populations of patients with AIS. The recombinant tissue plasminogen activator alteplase is approved in many countries for the treatment of AIS in the first 3 h after symptom onset. Many drug candidates have been subject to clinical trials, including those with anti-excitotoxic, anti-inflammatory, antioxidant, antiapoptotic/regenerative, calcium/adrenergic-modulating/antihypertensive, thrombolytic, nootropic/stimulant, fluid regulatory, or oxygen-delivering mechanisms of action. Some agents, such as tenecteplase, edaravone and minocycline, may be approved for global use in the future. This review evaluates almost all neurovascular protectants subject to clinical trial evaluation for the treatment of AIS, and includes 241 studies conducted between 1978 and 2014. The development of agents that reduce brain injury after AIS will require new and different approaches based on a deeper understanding of the pathophysiology of AIS. Moreover, the future treatment for AIS is likely to lie in combination therapy rather than monotherapy. Additional approaches to the testing and use of neurovascular protectants should be considered.