Molecular Localization of Health-Promoting Peptides Derived from Fish Protein Hydrolyzates on Fish Muscle Proteins.

The four previously reported health-promoting dipeptides, valine-tyrosine, lysine-tryptophan, methionine-phenylalanine, and arginine-isoleucine, found in the fish muscle hydrolyzates, were mainly located in the myosin subfragment-1 heavy chain, whereas the health-promoting tripeptide, alanine-lysine-lysine, was found in the fibrous rod consisting of the myosin subfragment-2 and light meromyosin with a regular coiled-coil structure of α-helix, irrespective of the fish species. Furthermore, the localization of these peptides either in the random coil, ß-sheet, or α-helix was also examined in the three-dimensional image, showing no specific tendency. Surprisingly, the same trend was observed even for the mammalian rabbit fast muscle myosin heavy chain. Since a trade-off between myofibrillar ATPase and structural stability has been reported for fish living at low environmental temperatures, it is speculated that fish muscle proteins, when ingested, are easily digested by various proteases in the human digestive tract and provide various health-promoting peptides also in vivo. While fish actin contained only two dipeptides, methionine-phenylalanine and valine-tyrosine, glyceraldehyde 3-phosphate dehydrogenase, one of the major components of fish muscle water-soluble protein, contained all of the four dipeptides and one tripeptide mentioned above.

Single-polyp metabolomics for coral health assessment.

Coral reef ecosystems supported by environmentally sensitive reef-building corals face serious threats from human activities. Our understanding of these reef threats is hampered by the lack of sufficiently sensitive coral environmental impact assessment systems. In this study, we established a platform for metabolomic analysis at the single-coral-polyp level using state-of-the-art mass spectrometry (probe electrospray ionization/tandem mass spectrometry; PESI/MS/MS) capable of fine-scale analysis. We analyzed the impact of the organic UV filter, benzophenone (BP), which has a negative impact on corals. We also analyzed ammonium and nitrate samples, which affect the environmental sensitivity of coral-zooxanthella (Symbiodiniaceae) holobionts, to provide new insights into coral biology with a focus on metabolites. The method established in this study breaks new ground by combining PESI/MS/MS with a technique for coral polyps that can control the presence or absence of zooxanthellae in corals, enabling functions of zooxanthellae to be assessed on a polyp-by-polyp basis for the first time. This system will clarify biological mechanisms of corals and will become an important model system for environmental impact assessment using marine organisms.

Metagenomic analysis of the microbial communities and associated network of nitrogen metabolism genes in the Ryukyu limestone aquifer.

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 in the Ryukyu limestone aquifer of Okinawa Island, which is located in the southernmost region of Japan, we performed metagenomic analysis on the microbial communities at the three sites and screened for functional genes associated with nitrogen metabolism. 16S rRNA amplicon analysis showed that bacteria accounted for 94-98% of the microbial communities, which included archaea at all three sites. The bacterial communities associated with nitrogen metabolism shifted by month at each site, indicating that this metabolism was accomplished by the bacterial community as a whole. Interestingly, site 3 contained much higher levels of the denitrification genes such as narG and napA 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.

Pilot study of a comprehensive resource estimation method from environmental DNA using universal D-loop amplification primers.

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.

Visualisation of Phosphate in Subcalicoblastic Extracellular Calcifying Medium and on a Skeleton of Coral by Using a Novel Probe, Fluorescein-4-Isothiocyanate-Labelled Alendronic Acid.

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 CaCO3 skeletons because FITC-AA with a high polarity group cannot permeate through cell membranes.

Seasonal and annual changes in the microbial communities of Ofunato Bay, Japan, based on metagenomics.

Five years of datasets from 2015 to 2019 of whole genome shotgun sequencing for cells trapped on 0.2-µm filters of seawater collected monthly from Ofunato Bay, an enclosed bay in Japan, were analysed, which included the 2015 data that we had reported previously. Nucleotide sequences were determined for extracted DNA from three locations for both the upper (1 m) and deeper (8 or 10 m) depths. The biotic communities analysed at the domain level comprised bacteria, eukaryotes, archaea and viruses. The relative abundance of bacteria was over 60% in most months for the five years. The relative abundance of the SAR86 cluster was highest in the bacterial group, followed by Candidatus Pelagibacter and Planktomarina. The relative abundance of Ca. Pelagibacter showed no relationship with environmental factors, and those of SAR86 and Planktomarina showed positive correlations with salinity and dissolved oxygen, respectively. The bacterial community diversity showed seasonal changes, with high diversity around September and low diversity around January for all five years. Nonmetric multidimensional scaling analysis also revealed that the bacterial communities in the bay were grouped in a season-dependent manner and linked with environmental variables such as seawater temperature, salinity and dissolved oxygen.

Phosphate bound to calcareous sediments hampers skeletal development of juvenile coral.

To test the hypothesis that terrestrial runoff affects the functions of calcareous sediments in coral reefs and hampers the development of corals, we analysed calcareous sediments with different levels of bound phosphate, collected from reef areas of Okinawajima, Japan. We confirmed that phosphate bound to calcareous sediments was readily released into ambient seawater, resulting in much higher concentrations of phosphorous in seawater from heavily polluted areas (4.3-19.0 µM as compared with less than 0.096 µM in natural ambient seawater). Additionally, we examined the effect of phosphate released from calcareous sediments on the development of Acropora digitifera coral juveniles. We found that high phosphate concentrations in seawater clearly inhibit the skeletal formation of coral juveniles. Our results demonstrate that calcareous sediments in reef areas play a crucial role in mediating the impact of terrestrial runoff on corals by storing and releasing phosphate in seawater.

Interleukin-17A/F1 Deficiency Reduces Antimicrobial Gene Expression and Contributes to Microbiome Alterations in Intestines of Japanese medaka (Oryzias latipes).

In mammals, interleukin (IL)-17A and F are hallmark inflammatory cytokines that play key roles in protection against infection and intestinal mucosal immunity. In the gastrointestinal tract (GI), the induction of antimicrobial peptide (AMP) production via Paneth cells is a fundamental role of IL-17A and F in maintaining homeostasis of the GI microbiome and health. Although mammalian IL-17A and F homologs (referred to as IL-17A/F1-3) have been identified in several fish species, their function in the intestine is poorly understood. Additionally, the fish intestine lacks Paneth cells, and its GI structure is very different from that of mammals. Therefore, the GI microbiome modulatory mechanism via IL-17A/F genes has not been fully elucidated. In this study, Japanese medaka (Oryzias latipes) were used as a teleost model, and IL-17A/F1-knockout (IL-17A/F1-KO) medaka were established using the CRISPR/Cas9 genome editing technique. Furthermore, two IL-17A/F1-deficient medaka strains were generated, including one strain containing a 7-bp deletion (-7) and another with an 11-bp addition (+11). After establishing F2 homozygous KO medaka, transcriptome analysis (RNA-seq) was conducted to elucidate IL-17A/F1-dependent gene induction in the intestine. Results of RNA-seq and real-time PCR (qPCR) demonstrated down-regulation of immune-related genes, including interleukin-1ß (IL-1ß), complement 1q subunit C (C1qc), transferrin a (Tfa), and G-type lysozyme (LyzG), in IL-17A/F1-KO medaka. Interestingly, protein and lipid digestive enzyme genes, including phospholipase A2, group IB (pla2g1b), and elastase-1-like (CELA1), were also downregulated in the intestines of IL-17A/F1-KO medaka. Furthermore, to reveal the influence of these downregulated genes on the gut microbiome in IL-17A/F1-KO, 16S rRNA-based metagenomic sequencing analysis was conducted to analyze the microbiome constitution. Under a non-exposed state, the intestinal microbiome of IL-17A/F1-KO medaka differed at the phylum level from wild-type, with significantly higher levels of Verrucomicrobia and Planctomycetes. Additionally, at the operational taxonomic unit (OTU) level of the human and fish pathogens, the Enterobacteriaceae Plesiomonas shigelloides was the dominant species in IL-17A/F1-KO medaka. These findings suggest that IL-17A/F1 is involved in the maintenance of a healthy gut microbiome.

HaCeD-Seq: a Novel Method for Reliable and Easy Estimation About the Fish Population Using Haplotype Count from eDNA.

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.

Seasonal changes in the communities of photosynthetic picoeukaryotes in Ofunato Bay as revealed by shotgun metagenomic sequencing.

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.

Metagenome-based diversity analyses suggest a strong locality signal for bacterial communities associated with oyster aquaculture farms in Ofunato Bay.

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.

Taxonomic profiles in metagenomic analyses of free-living microbial communities in the Ofunato Bay.

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.

Basin-scale seasonal changes in marine free-living bacterioplankton community in the Ofunato Bay.

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.

Seasonal changes in the abundance of bacterial genes related to dimethylsulfoniopropionate catabolism in seawater from Ofunato Bay revealed by metagenomic analysis.

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.

Changes in free amino acid concentrations and associated gene expression profiles in the abdominal muscle of kuruma shrimp (Marsupenaeus japonicus) acclimated at different salinities.

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 (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 sequencing analysis on this muscle. The concentrations of free amino acids were clearly altered depending on salinity after 24 h of acclimation. 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 glutamine metabolism, appeared to be associated with the increased glutamine concentration at high salinity. Furthermore, the increased alanine concentration at high salinity was likely associated with the decrease in the expression levels of the the gene encoding alanine-glyoxylate transaminase. 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.

Global gene expression analysis of the muscle tissues of medaka acclimated to low and high environmental temperatures.

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.

Cloning of corticotropin-releasing hormone (CRH) precursor cDNA and immunohistochemical detection of CRH peptide in the brain of the Japanese eel, paying special attention to gonadotropin-releasing hormone.

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.

Selenoneine, a novel selenium-containing compound, mediates detoxification mechanisms against methylmercury accumulation and toxicity in zebrafish embryo.

The selenium (Se)-containing antioxidant selenoneine (2-selenyl-N α,N α,N α-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 K m 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.

Induction of autophagy by amino acid starvation in fish cells.

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.

Extensive apoptosis and abnormal morphogenesis in pro-caspase-3 transgenic zebrafish during development.

The 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.