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The overload of nutrients of anthropogenic origin, including phosphate, onto coastal waters has been reported to have detrimental effects on corals. However, to the best of our knowledge, the phosphate concentration threshold for inhibiting coral calcification is unclear owing to a lack of information on the molecular mechanisms involved in the inhibitory effect of phosphate. Therefore, in this study, we prepared a new phosphate analogue, fluorescein-4-isothiocyanate (FITC)-labelled alendronic acid (FITC-AA), from commercially available reagents and used it as a novel probe to demonstrate its transfer pathway from ambient seawater into Acropora digitifera. When the juveniles at 1 d post-settlement were treated with FITC-AA in a laboratory tank, this phosphate analogue was found in the subcalicoblastic extracellular calcifying medium (SCM) and was absorbed on the basal plate in the juveniles within a few minutes. When the juveniles bear zooxanthellae at 3 months post-settlement, FITC-AA was observed on the corallite walls within a few minutes after adding ambient seawater. We concluded that FITC-AA in ambient seawater was transferred via a paracellular pathway to SCM and then absorbed on the coral CaCO

Many studies have investigated the ability of environmental DNA (eDNA) to identify the species. However, when individual species are to be identified, accurate estimation of their abundance using traditional eDNA analyses is still difficult. We previously developed a novel analytical method called HaCeD-Seq (haplotype count from eDNA by sequencing), which focuses on the mitochondrial D-loop sequence for eels and tuna. In this study, universal D-loop primers were designed to enable the comprehensive detection of multiple fish species by a single sequence. To sequence the full-length D-loop with high accuracy, we performed nanopore sequencing with unique molecular identifiers (UMI). In addition, to determine the D-loop reference sequence, whole genome sequencing was performed with thin coverage, and complete mitochondrial genomes were determined. We developed a UMI-based Nanopore D-loop sequencing analysis pipeline and released it as open-source software. We detected 5 out of 15 species (33%) and 10 haplotypes out of 35 individuals (29%) among the detected species. This study demonstrates the possibility of comprehensively obtaining information related to population size from eDNA. In the future, this method can be used to improve the accuracy of fish resource estimation, which is currently highly dependent on fishing catches.

While microbial biogeochemical activities such as those involving denitrification and sulfate reduction have been considered to play important roles in material cycling in various aquatic ecosystems, our current understanding of the microbial community in groundwater ecosystems is remarkably insufficient. To assess the groundwater from three locations in the Ryukyu limestone aquifer of Okinawa Island, which is located in the southernmost region of Japan, we performed shotgun metagenomic analysis on the microbial communities at the three sites and screened for functional genes associated with nitrogen metabolism. Shotgun analysis showed that bacteria accounted for 90–98% of the microbial communities, which included archaea, eukaryotes and viruses, at all three sampling sites. The bacterial communities associated with nitrogen metabolism shifted by month at each site, indicating that this metabolism was accomplished not via specific bacteria but rather by the bacterial community as a whole. Interestingly, site 3 contained much higher levels of the denitrification genes narG/narH/nxrB, narG/narZ/nxrA, napA, nirK, norB and nosZ than the other two sites. This site was thought to have undergone denitrification that was driven by high quantities of dissolved organic carbon (DOC). In contrast, site 2 was characterized by a high nitrate-nitrogen (NO3-N) content and a low amount of DOC, and this site yielded a moderate amount of denitrification genes. Site 1 showed markedly low amounts of all nitrogen metabolism genes. Overall, nitrogen metabolism in the Ryukyu limestone aquifer was found to change based on environmental factors.

Bacterial communities are important factors governing changes in river ecology. We evaluated the diversity of bacterial communities in sediment and size-fractionated water samples collected from the Tama River, Tokyo, Japan, to verify their taxonomy and functional classes. Samples were collected from three river areas, namely up-, mid- and downstream, and analyzed using MiSeq shotgun metagenomic sequencing. Bacterial diversity in the river water was highest midstream for the free-living bacterial communities. Dominant bacterial classes upstream were Alphaproteobacteria and Betaproteobacteria, and there was a gradual shift to dominance by Actinobacteria mid- and downstream. Potential pathogenic genera, such as Flavobacterium, Mycobacterium and Bacteroides, also dominated mid- and downstream. Interestingly, Mycobacterium was most abundant midstream, whereas Pseudomonas was observed upstream. The gene possibly associated with sulfur metabolism, sigB, and those associated with purine metabolism, purF, purD, purN, purH, guaB and guaA, dominated in the bacterial communities found mid- and downstream and are thought to ensure survival and virulence in their environments. NO2-N, NH4-N and PO4-P were found to influence bacterial populations in water but not those residing in the sediment.

Ofunato Bay is located in the northeastern Pacific Ocean area of Japan, and it has the highest biodiversity of marine organisms in the world, primarily due to tidal influences from the cold Oyashio and warm Kuroshio Currents. Our previous results from performing shotgun metagenomics indicated that Candidatus Pelagibacter ubique and Planktomarina temperata were the dominant bacteria (Reza et al., 2018a, 2018b). These bacteria are reportedly able to catabolize dimethylsulfoniopropionate (DMSP) produced from phytoplankton into dimethyl sulfide (DMS) or methanethiol (MeSH). This study was focused on seasonal changes in the abundances of bacterial genes (dddP, dmdA) related to DMSP catabolism in the seawater of Ofunato Bay by BLAST+ analysis using shotgun metagenomic datasets. We found seasonal changes among the Candidatus Pelagibacter ubique strains, including those of the HTCC1062 type and the Red Sea type. A good correlation was observed between the chlorophyll a concentrations and the abundances of the catabolic genes, suggesting that the bacteria directly interact with phytoplankton in the marine material cycle system and play important roles in producing DMS and MeSH from DMSP as signaling molecules for the possible formation of the scent of the tidewater or as fish attractants.

Small photosynthetic eukaryotes play important roles in oceanic food webs in coastal regions. We investigated seasonal changes in the communities of photosynthetic picoeukaryotes (PPEs) of the class Mamiellophyceae, including the genera Bathycoccus, Micromonas and Ostreococcus, in Ofunato Bay, which is located in northeastern Japan and faces the Pacific Ocean. The abundances of PPEs were assessed over a period of one year in 2015 at three sampling stations, KSt. 1 (innermost bay area), KSt. 2 (middle bay area) and KSt. 3 (bay entrance area) at depths of 1 m (KSt. 1, KSt. 2 and KSt. 3), 8 m (KSt. 1) or 10 m (KSt. 2 and KSt. 3) by employing MiSeq shotgun metagenomic sequencing. The total abundances of Bathycoccus, Ostreococcus and Micromonas were in the ranges of 42–49%, 35–49% and 13–17%, respectively. Considering all assayed sampling stations and depths, seasonal changes revealed high abundances of PPEs during the winter and summer and low abundances during late winter to early spring and late summer to early autumn. Bathycoccus was most abundant in the winter, and Ostreococcus showed a high abundance during the summer. Another genus, Micromonas, was relatively low in abundance throughout the study period. Taken together with previously suggested blooming periods of phytoplankton, as revealed by chlorophyll a concentrations in Ofunato Bay during spring and autumn, these results for PPEs suggest that greater phytoplankton blooming has a negative influence on the seasonal occurrences of PPEs in the bay.

In an effort to determine genus- or species-level taxonomic profiles and diversity of bacterial consortia in the Tama River around urban Tokyo, next-generation sequencing technology targeting a 16S ribosomal RNA (rRNA) gene amplicon was employed. Metagenomic analysis performed by an Ion Personal Genome Machine after sequentially filtering samples through 5-, 0.8- and 0.2-μm filters yielded 1.48 Gb of 16S sequences (average 2.38 M reads/sample). The results indicated that half of the bacterial sequences belonged to Proteobacteria, followed by Bacteroidetes, Actinobacteria and Cyanobacteria. Flavobacterium (Bacteroidetes), possibly including a potential fish pathogen, was the most numerous genera in the Tama River metagenome, and accounted for ~ 16% of assigned 16S reads, followed by Mycobacterium. Other dominant bacterial genera including Zoogloea, Sediminibacterium, Hyphomicrobium, Sphingopyxis, Thiothrix and Lysobacter, were thought to be associated with waste water and sludge. MiSeq metagenomic analysis revealed that environmental factors, particularly water temperature, influenced the bacterial composition throughout the year, with a strong negative correlation observed for Proteobacteria and a positive correlation for Bacteroidetes. In terms of bacterial genera, Flavobacterium was positively correlated with temperature, while Polaromonas, Pseudomonas and Bradyrhizobium were negatively correlated with this, suggesting dynamic change in the free-living bacterial population throughout the year and versatile adaptation strategies in relation to environmental factors.

Medaka (Oryzias latipes) is a temperate eurythermal fish that is able to survive over a wide range of water temperatures ranging from near zero to over 30°C throughout the year; it maintains its normal physiological and biochemical processes through temperature acclimation. To determine the mechanisms involved in temperature acclimation of fish, the fast skeletal muscle tissues of medaka underwent global gene expression analysis using next-generation sequencing. Ten individuals were placed into two aquariums at 24°C. While the water temperature of one aquarium was decreased to 10°C, that of the other aquarium was increased to 30°C; these temperatures were subsequently maintained for 5weeks. RNA sequencing (RNA-Seq) analyses revealed that 11 genes involved in energy metabolism and muscle atrophy were significantly highly expressed in the 10°C-acclimated fish. Meanwhile, significantly higher expression levels were observed for 20 genes encoding myofibrillar proteins and heat shock proteins in the 30°C-acclimated fish. Moreover, 1103 genes had at least fourfold differential expression between the acclimation groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses provided important information: although the expression of genes related to metabolic processes were activated, muscle atrophy occurred in the 10°C-acclimated fish, and muscle cells divided actively in the 30°C-acclimated fish and avoided thermal stress by expressing heat shock proteins. Therefore, RNA-Seq analyses with the available genome database will be useful for better understanding the molecular mechanisms involved in the temperature acclimation of fish.

It is common to count the numbers of specified fish in the field after speciation of captured fish according to their morphology and to subject these counts to appropriate statistical analyses. In recent years, a non-invasive method to estimate the abundance of a particular fish species using environmental DNA (eDNA) has been developed. However, it is still difficult to determine accurate numbers of fish species using such method. We predict that the estimation of individuals of certain fish species in the field is more accurate and easier by using haplotypes of DNA in the fast evolutionary region. Therefore, we focused on the regulatory region (D-loop) in mitochondrial DNA, which is known to have a high genetic variation at the intraspecific level of the targeting eel. We investigated haplotype diversity in eel at first and then determined the number of D-loop haplotypes contained in their exfoliated cells in breeding water. Finally, we developed a novel analytical method, HaCeD-Seq, to estimate the number of individuals based on the abovementioned data.

Development of high-throughput DNA sequencing technologies has enabled scientists to generate vast amounts of genetic information that may provide a comprehensive understanding of key roles played by environmental microorganisms. Generally the microorganisms inhabit a particular niche and correlate well with environmental changes. It is accepted that the read counts obtained through metagenomic analyses correlate semi-quantitatively with the relative abundance of bacterial species. In our marine metagenomic study conducted on the Ofunato Bay, Iwate Prefecture, Japan, we observed such correlation which exists for bacterioplankton Candidatus Pelagibacter ubique, identified as the dominant bacterial species of the bay. Shotgun metagenomic analyses identified three strains of Ca. Pelagibacter in the bay, viz., dmdA-HTCC1062, dmdA-HTCC9022, and O19-dmdA, that showed a dynamic change throughout the year particularly in the 10-m depth zone. Interestingly, the total abundances of those strains that fall in the Ca. Pelagibacter genus were found to correlate well with the read counts per g seawater samples used for analyses. It is assumed that whole-genome sequence (WGS) reads for members of the metagenome would show similar trend provided that proper precautions are taken to ensure collection of representative sample from the environment.

In this article, methods or techniques of metagenomics including targeted 16S/18S rRNA analyses and shotgun sequencing will be discussed. It is sometimes difficult, especially for beginners, to follow the manufacturer’s recommendation as mentioned in the protocol and to go through different steps from the preparation of starting material (e.g., DNA), library preparation, and so on. We will try to explain all the steps in detail and share our experience here. It all starts with collection of samples and collection of ecological/environmental metadata followed by sample fractionation (optional), extraction of DNA, sequencing, and finally data analyses to interpret results. Sample collection has always been the most important part of a study as it requires proper planning, a good workforce to execute, permission(s) of sampling from appropriate authority, and precaution(s) about endangered species during sampling. Here, we first describe methodology for a shallow river and in the later section methodology for a deep marine bay. In either case, slight modifications can be made to succeed in sampling. Determination of physicochemical parameters as metadata simultaneously is also an important task. These samples are then processed to extract DNA which needs to be representative of all cells present in the sample. Finally, sequencing is done by a next-generation sequencer, and data analyses are completed. Through these methods, scientists are now able to overcome the unculturability problem of more than 99% of environmental microorganisms and uncovered functional gene diversity of environmental microorganisms.

The Ofunato Bay in the northeastern Pacific Ocean area of Japan possesses the highest biodiversity of marine organisms in the world and has attracted much attention due to its economic and environmental importance. We report here a shotgun metagenomic analysis of the year-round variation in free-living bacterioplankton collected across the entire length of the bay. Phylogenetic differences among spring, summer, autumn and winter bacterioplankton suggested that members of Proteobacteria tended to decrease at high water temperatures and increase at low temperatures. It was revealed that Candidatus Pelagibacter varied seasonally, reaching as much as 60% of all sequences at the genus level in the surface waters during winter. This increase was more evident in the deeper waters, where they reached up to 75%. The relative abundance of Planktomarina also rose during winter and fell during summer. A significant component of the winter bacterioplankton community was Archaea (mainly represented by Nitrosopumilus), as their relative abundance was very low during spring and summer but high during winter. In contrast, Actinobacteria and Cyanobacteria appeared to be higher in abundance during high-temperature periods. It was also revealed that Bacteroidetes constituted a significant component of the summer bacterioplankton community, being the second largest bacterial phylum detected in the Ofunato Bay. Its members, notably Polaribacter and Flavobacterium, were found to be high in abundance during spring and summer, particularly in the surface waters. Principal component analysis and hierarchal clustering analyses showed that the bacterial communities in the Ofunato Bay changed seasonally, likely caused by the levels of organic matter, which would be deeply mixed with surface runoff in the winter.

The Ofunato Bay in Iwate Prefecture, Japan is a deep coastal bay located at the center of the Sanriku Rias Coast and considered an economically and environmentally important asset. Here, we describe the first whole genome sequencing (WGS) study on the microbial community of the bay, where surface water samples were collected from three stations along its length to cover the entire bay; we preliminarily sequenced a 0.2 μm filter fraction among sequentially size-fractionated samples of 20.0, 5.0, 0.8 and 0.2 μm filters, targeting the free-living fraction only. From the 0.27–0.34 Gb WGS library, 0.9 × 106–1.2 × 106 reads from three sampling stations revealed 29 bacterial phyla (~80% of assigned reads), 3 archaeal phyla (~4%) and 59 eukaryotic phyla (~15%). Microbial diversity obtained from the WGS approach was compared with 16S rRNA gene results by mining WGS metagenomes, and we found similar estimates. The most frequently recovered bacterial sequences were Proteobacteria, predominantly comprised of 18.0–19.6% Planktomarina (Family Rhodobacteraceae) and 13.7–17.5% Candidatus Pelagibacter (Family Pelagibacterales). Other dominant bacterial genera, including Polaribacter (3.5–6.1%), Flavobacterium (1.8–2.6%), Sphingobacterium (1.4–1.6%) and Cellulophaga (1.4–2.0%), were members of Bacteroidetes and likely associated with the degradation and turnover of organic matter. The Marine Group I Archaea Nitrosopumilus was also detected. Remarkably, eukaryotic green alga Bathycoccus, Ostreococcus and Micromonas accounted for 8.8–15.2%, 3.6–4.9% and 2.1–3.1% of total read counts, respectively, highlighting their potential roles in the phytoplankton bloom after winter mixing.

Ofunato Bay, in Japan, is the home of buoy-and-rope-type oyster aquaculture activities. Since the oysters filter suspended materials and excrete organic matters into the seawater, bacterial communities residing in its vicinity may show dynamic changes depending on the oyster culture activities. We employed a shotgun metagenomic technique to study bacterial communities near oyster aquaculture facilities at the center of the bay (KSt. 2) and compared the results with those of two other localities far from the station, one to the northeast (innermost bay, KSt. 1) and the other to the southwest (bay entrance, KSt. 3). Seawater samples were collected every month from January to December 2015 from the surface (1 m) and deeper (8 or 10 m) layers of the three locations, and the sequentially filtered fraction on 0.2-μm membranes was sequenced on an Illumina MiSeq system. The acquired reads were uploaded to MG-RAST for KEGG functional abundance analysis, while taxonomic analyses at the phylum and genus levels were performed using MEGAN after parsing the BLAST output. Discrimination analyses were then performed using the ROC-AUC value of the cross validation, targeting the depth (shallow or deep), locality [(KSt. 1 + KSt. 2) vs. KSt 3; (KSt. 1 + KSt. 3) vs. KSt. 2 or the (KSt. 2 + KSt. 3) vs. KSt. 1] and seasonality (12 months). The matrix discrimination analysis on the adjacent 2 continuous seasons by ROC-AUC, which was based on the datasets that originated from different depths, localities and months, showed the strongest discrimination signal on the taxonomy matrix at the phylum level for the datasets from July to August compared with those from September to June, while the KEGG matrix showed the strongest signal for the datasets from March to June compared with those from July to February. Then, the locality combination was subjected to the same ROC-AUC discrimination analysis, resulting in significant differences between KSt. 2 and KSt. 1 + KSt. 3 on the KEGG matrix. These results suggest that aquaculture activities markedly affect bacterial functions.

Shrimps inhabiting the coastal water can survive in a wide range of salinity. However, the molecular mechanisms involved in their acclimation to different environmental salinities have remained largely unknown. In the present study, we acclimated kuruma shrimp Marsupenaeus japonicus at 1.7 %, 3.4 % and 4.0 % salinities. After acclimating for 6, 12, 24 and 72 h, we determined free amino acid concentrations in their abdominal muscle, and performed RNA-seq analysis on this muscle. The concentrations of free amino acids were clearly altered depending on salinity after acclimating for 24 h. Glutamine and alanine concentrations were markedly increased following the increase of salinity. In association with such changes, many genes related to amino acid metabolism changed their expression levels. In particular, the increase of the expression level of the gene encoding glutamate-ammonia ligase which functions in the glutamine metabolism appeared to be relevant to the increased glutamine concentration at high salinity. Furthermore, the alanine concentration increased at high salinity was likely to be associated with the decrease in the expression levels of the alanine-glyoxylate transaminase gene. Thus, there is a possibility that changes in the concentration of free amino acids for osmoregulation in kuruma shrimp are regulated by changes in the expression levels of genes related to amino acid metabolism.

Shrimps inhabiting around the coastal area can survive in a wide range of salinity. However, the molecular mechanisms involved in their adaptation to different environmental salinity have remained largely unknown. In the present study, we reared kuruma shrimpMarsupenaeus japonicusat 1.7 %, 3.4 % and 4.0 % salinity. After rearing for 6, 12, 24 and 72 h, we determined free amino acid concentrations in their abdominal muscle, and performed RNA-seq analysis on this muscle. The concentrations of free amino acids were clearly altered depending on salinity after rearing for 24 h. Glutamine and alanine concentrations were markedly increased following the increase of salinity. In association with such changes, many genes related to amino acid metabolism changed their expression levels. Notably, the increased glutamine content at high salinity appeared to be relevant to the increase of the expression level of the gene encoding glutamate-ammonia ligase which functions in the glutamine metabolism. Furthermore, the alanine content increased at high salinity was likely to be associated with the decrease in the expression levels of the alanine-glyoxylate transaminase gene. Thus, the changes in the concentration of free amino acids for osmoregulation in kuruma shrimp are considered to be regulated by the changes in the expression levels of genes related to amino acid metabolism.Summary statesmentKuruma shrimpMarsupenaeus japonicuschanges free amino acid contents and associated gene expression levels in their muscle to adjust effectively to different salinity.

Shrimps inhabiting coastal waters can survive in a wide range of salinity. However, the molecular mechanisms involved in their acclimation to different environmental salinities have remained largely unknown. In the present study, we acclimated kuruma shrimp (

The stress-related corticotropin-releasing hormone (CRH) was first identified by isolation of its cDNA from the brain of the Japanese eel Anguilla japonica. CRH cDNA encodes a signal peptide, a cryptic peptide and CRH (41 amino acids). The sequence homology to mammalian CRH is high. Next, the distribution of CRH-immunoreactive (ir) cell bodies and fibers in the brain and pituitary were examined by immunohistochemistry. CRH-ir cell bodies were detected in several brain regions, e.g., nucleus preopticus pars magnocellularis, nucleus preopticus pars gigantocellularis and formatio reticularis superius. In the brain, CRH-ir fibers were distributed not only in the hypothalamus but also in various regions. Some CRH-ir fibers projected to adrenocorticotropic hormone (ACTH) cells in the rostral pars distalis of the pituitary and also the α-melanocyte-stimulating hormone (α-MSH) cells in the pars intermedia of the pituitary. Finally, the neuroanatomical relationship between the CRH neurons and gonadotropin-releasing hormone (GnRH) neurons was examined by dual-label immunohistochemistry. CRH-ir fibers were found to be in close contact with GnRH-ir cell bodies in the hypothalamus and in the midbrain tegmentum and GnRH-ir fibers were in close contact with CRH-ir cell bodies in the nucleus preopticus pars magnocellularis. These results suggest that CRH has some physiological functions other than the stimulation of ACTH and α-MSH secretion and that reciprocal connections may exist between the CRH neurons and GnRH neurons in the brain of the Japanese eel.

Nucleotide sequences in internal transcribed spacer (ITS)-1 region derived from dried nori products produced in Japan, China, and the Republic of Korea were compared. Thalli contained in the Japanese products were genetically homogenous, and their nucleotide sequences in ITS-1 were identical to those of the reference strains of Pyropia yezoensis f. narawaensis. In Chinese products, the thalli were related to P. yezoensis strain Minomiasakusa. In contrast, the thalli in the Korean products were genetically heterogeneous, and several different P. yezoensis strains and other Pyropia spp. were used for dried nori products. In some thalli produced in both China and Korea, the DNA sequences of the ITS-1 region were identical with that of Japan, suggesting that the cultivar strains might have been transplanted from Japan to China in recent years. The 432-bp-long nucleotide sequences in the ITS-1 region of thalli derived from Japanese origin were cleaved to two restriction fragments at 154 and 278 bp by cleavage of PCR-amplified products using MspI. Conversely, almost all of the corresponding sequences derived from China and Korea were lacking MspI or other restriction patterns, except for nori products from some areas that cultivate a closely related strain to the Japanese cultivar.

Autophagy is well established as a starvation-induced process in yeast and mammalian cells and tissues. To elucidate the cellular mechanisms induced by starvation in fish, we characterized the induction of autophagy in cultured zebrafish cells under starvation conditions. As an autophagic marker protein, the microtubule-associated protein 1-light chain 3B protein (MAP1-LC3B) was cloned from the fish cells, and its expression and localization were characterized. In zebrafish embryonic (ZE) cells, posttranslational modifications produced two distinct forms of MAP1-LC3B, i.e., a cytosolic form and a membrane-bound form (types I and II, respectively). Immunofluorescence microscopy revealed fluorescently labeled autophagosomes in cells stably transfected with a green fluorescent protein (GFP)–MAP1-LC3B fusion protein and showed that this protein accumulated in punctate dots in a time-dependent manner in response to amino acid starvation. Starvation also induced the degradation of long-lived proteins. Treatment with 3-methyladenine and wortmannin, two class-III inhibitors of phosphoinositide 3-kinase (PI3K), repressed autophagy under starvation conditions, indicating that the PI3K class-III pathway regulates starvation-induced autophagy in fish.

SUMMARYThe pro-apoptotic caspase-3 gene has been shown to have key functions in the execution of apoptosis (programmed cell death) in vertebrate cells. However, the central role of caspase-3 in morphogenesis during development remains unclear. In this study, transgenic zebrafish that overexpress full-length pro-caspase-3 were generated to determine the effects of caspase genes on vertebrate morphogenesis and stress tolerance. The enhanced expression of the full-length pro-caspase-3 cDNA induced extremely high levels of caspase activity and extensive apoptosis in the transgenic embryos, and 33–46% of F2 embyos in the transgenic lines exhibited some form of morphological abnormality. Pro-caspase-3 transgenic zebrafish exhibited abnormal morphogenesis in the eyes, notochord, heart and yolk sac, suggesting that enhanced processing of pro-caspase-3 triggers significant apoptotic responses in the specific target tissues that are undergoing morphogenesis during development. The transgenic fish had reduced eye size and showed degeneration of the retina, including the photoreceptor cell layers, whereas pigmentation and lens formation were not affected. In addition, heart failure due to a weakened heartbeat and reduced circulation was noted in the pro-caspase-3 transgenic embryos. The transgenic embryos were markedly sensitive to stress conditions, such as UV irradiation at 2 or 5 mJ cm–2. On the other hand, caspase-3 deficiency through injection of antisense morpholino oligo into embryos repressed apoptosis and enhanced stress tolerance after UV irradiation. Therefore, the caspase-3-mediated pro-apoptotic signalling pathway and its activation play critical roles in the induction of apoptosis and stress tolerance during zebrafish embryogenesis.

The selenium (Se)-containing antioxidant selenoneine (2-selenyl-Nα,Nα,Nα-trimethyl-l-histidine) has recently been discovered to be the predominant form of organic Se in tuna blood. Although dietary intake of fish Se has been suggested to reduce methylmercury (MeHg) toxicity, the molecular mechanism of MeHg detoxification by Se has not yet been determined. Here, we report evidence that selenoneine accelerates the excretion and demethylation of MeHg, mediated by a selenoneine-specific transporter, organic cations/carnitine transporter-1 (OCTN1). Selenoneine was incorporated into human embryonic kidney HEK293 cells transiently overexpressing OCTN1 and zebrafish blood cells by OCTN1. The Km for selenoneine uptake was 13.0 μM in OCTN1-overexpressing HEK293 cells and 9.5 μM in zebrafish blood cells, indicating high affinity of OCTN1 for selenoneine in human and zebrafish cells. When such OCTN1-expressing cells and embryos were exposed to MeHg–cysteine (MeHgCys), MeHg accumulation was decreased and the excretion and demethylation of MeHg were enhanced by selenoneine. In addition, exosomal secretion vesicles were detected in the culture water of embryos that had been microinjected with MeHgCys, suggesting that these may be responsible for MeHg excretion and demethylation. In contrast, OCTN1-deficient embryos accumulated MeHg, and MeHg excretion and demethylation were decreased. Furthermore, Hg accumulation was decreased in OCTN1-overexpressing HEK293 cells, but not in mock vector-transfected cells, indicating that selenoneine and OCTN1 can regulate MeHg detoxification in human cells. Thus, the selenoneine-mediated OCTN1 system regulates secretory lysosomal vesicle formation and MeHg demethylation. Electronic supplementary material The online version of this article (doi:10.1007/s10126-013-9508-1) contains supplementary material, which is available to authorized users.