Program

Scombrids, which include commercially important fish species such as bluefin tuna Thunnus orientalis, generally do not spawn in captivity without hormonal treatments. To induce spawning of various marine fish species, goserelin ([des-Gly10, D-Ala6]-GnRHa) is often administered by injections or pellet-implantations. However, a noninvasive method is needed for spawning induction in scombrids, which are sensitive to stress. In our previous study, spawning induction by oral administration of goserelin was successful in chub mackerel Scomber japonicus. However, we also revealed that this method required the use of high doses of GnRHa because of its poor intestinal absorbance. Therefore, we focused on another type of GnRHa, nafarelin ([des-Gly10, 3-(2-naphthyl)-D-Ala6]-GnRHa), which was expected to show higher intestinal absorbance than goserelin because 3-(2-naphthyl)-D-Ala is hydrophobic and facilitates cell membrane penetration by hydrophobic interaction. First, the receptor-binding potency of nafarelin was determined by an in vitro reporter gene assay using a mammalian cell line expressing chub mackerel GnRH-1 receptor. Second, its resistance to digestive enzymes was examined by comparing the degradation of nafarelin, goserelin, and native GnRH-1 after chymotrypsin digestion. Finally, the plasma nafarelin level of the mackerel was measured following oral intubation of fluorescent-labeled nafarelin to examine its intestinal absorbance. Native GnRH-1 of chub mackerel activated reporter gene expression at concentrations of 0.01–0.1 pM, whereas both nafarelin and goserelin activated it at 0.001–0.01 fM. Moreover, nafarelin showed significantly higher tolerance to enzyme digestion than goserelin. Further, orally intubated nafarelin had approximately 2-fold higher intestinal absorbance than that of goserelin. Therefore, nafarelin exhibits higher oral delivery efficiency for spawning induction than goserelin and is expected to become a powerful tool for spawning induction in mackerels as well as tunas.

Abnormal water temperatures caused by climate change may affect the reproduction of species that have temperature-dependent sex determination (TSD), for example, by causing extremely unbalanced sex ratios. We have recently shown that the cobaltcap silverside, a species from the order Atheriniformes, where several cases of TSD have been documented, possesses the Y-chromosome-linked anti-Müllerian hormone (amhy). Species that present both a chromosomal sex marker and TSD may be useful indicators of the effects of climate change on fish reproduction, for example by monitoring mismatches between genotypic and phenotypic sex (sex-reversal). In this context, the primary goal of this study was to clarify the effects of temperature on sex determination of H.tsurugae. In laboratory experiments, newly hatched larvae were reared at constant, environmentally relevant temperatures of 18, 22, or 26°C and their amhy-based sex genotype and histologically-determined sex phenotype were assessed and compared at the end of 13 weeks. We also screened a wild population in Tokyo Bay for the presence of sex-reversals and estimated the hatching date (by otolith increment analysis) and the temperature each individual experienced shortly after hatching (from water temperature charts for the region). Comparison of the phenotypic and genotypic sex of reared individuals revealed that the percentages of XX-males and XY-females at 18, 22 and 26°C were 0, 11, and 42%, and 67, 10, and 8%, respectively. Screening of wild H.tsurugae collected in 2014 (n=337), 2015 (n=243), and 2016 (n=100) revealed the presence of 25XX-males and 13XY-females, 18XX-males and 14XY-females, and 18XX-males in the three years, respectively. The birth date/early life water temperature estimation suggests that exposure to relatively low and high water temperatures short after hatching induced the formation of sex reversed XX-males and XY-females, respectively, in a manner consistent with laboratory results. These results suggest the occurrence of TSD in this species.

Sex determination in pejerrey is temperature-dependent (TSD) at low and high temperatures and genetically-prescribed at intermediate temperatures (24-25°C). Pejerrey is typically gonochoristic and gonadal differentiation is characterized by a cephalocaudal/left-to-right histological gradient in both sexes and by the occurrence of apoptosis in the anterior half of the right gonad in putative males. Apoptosis, the anti-Müllerian hormone, and aromatase are conceivably implicated in these processes but their possible interplay and relation with the differentiation gradient are still unclear. To shed light on these points, we examined the spatiotemporal patterns of apoptosis by TUNEL analysis and of amh and cyp19a1a gene expression by in situ hybridization in gonads during the period of gonadal sex determination/histological differentiation of pejerrey. Progeny from a single XY/XX cross was reared for 14 weeks at 25°C and fish were analyzed weekly for apoptosis, gene expression, degree of histological gonadal differentiation, and sex ratios. Ovarian and testicular differentiation began at 4 and 7 weeks, respectively. XX and XY individuals were 36% and 96% male, respectively. Apoptosis was detected with increasing intensity from 1 and 4 weeks in XY and XX fish, respectively, and was virtually limited to the anterior region of the right gonads. amh expression was detected from 1 and 2 weeks in the left and right gonads, respectively, of all XY fish and about 1/3 of the XX fish; it was typically stronger in the left gonad and areas without apoptosis. cyp19a1a expression was observed from 2 weeks in both genotypes but was maintained only in XX gonads without amh expression and/or apoptosis. The results suggest that apoptosis per se is not the cause of masculinization in pejerrey but rather may be a mechanism to delay differentiation in the right gonads until it is clearly stablished in the left, preventing discrepant (intersex) differentiation within an individual.

Fish cells have cellular mechanisms that allow adaptation to a wide range of thermal environments. Previously, we identified that a homolog of the mammalian valosin-containing protein (VCP), p97 and the yeast cell division cycle gene 48 (CDC48) was the major cold-inducible protein in cold-treated fish cultured cells, and enhanced cell growth promotion under cold temperature. To elucidate the molecular mechanism to compensate reduced cellular processes under cold-temperature in ectotherms, we characterized that CDC48 was essential for protein degradation in both ubiquitin-proteasome system and autophagy in neural development in zebrafish in vivo. When we introduced antisense morpholino oligo in zebrafish embryos, CDC48 morphant were growth retardation and abnormal morphology only at 15˚C under cold conditions as compared with control embryos. We also generated CDC48 knockout lines using Crispr/Cas9 system. Those lines recapitulated the morpholino phenotype previously reported, and homozygous mutants were also growth retardation under cold conditions. The CDC48-deficient embryos showed defects in neuronal formation, cell death, ROS production and the accumulation of poly-ubiquitinated proteins in the central nervous system under cold conditions, suggesting dysfunction of the ubiquitin-proteasome system (UPS) under cold conditions. In addition, under cold conditions, the autophagosomes accumulating autophagic marker protein p62 were formed, and autolysosomes were not produced irrespective of the presence of CDC48, indicating that cold conditions reduced autophagic functions. Therefore, CDC48 is essential for the neural development through the UPS function under cold conditions during early embryogenesis.

Chum salmon catch has been decreasing for nearly two decades in the Sanriku-region, northern Honshu, Japan. Understanding of the genetic characteristics in chum salmon is important for their conservation and fisheries management for sustainable use. We investigated the genetic population structure of chum salmon returned to the Tsugaruishi River with leading salmon run in Iwate Prefecture using 16 polymorphic microsatellite loci, for better understanding of the genetic characteristics of seasonal chum collections in relation to the age composition within the river. More than 1000 fish of 12 collections were obtained from early September in 2015 to middle January in 2016. Early and late runs were tentatively defined for salmon homing to the Tsugaruishi River from early September to late October and from early November to late January next year, respectively. The year classes of chum salmon returned included unusually high rate of older fish, about 34% 5-year olds, 30% 4-year olds, 19% 3-years-olds, 16% 6-year olds, and 1% 2-year olds, with 3-years olds predominantly in the early-run, 4- and 6-years olds mostly in the late-run, and 5-years olds through the homing periods. The observed mean allelic richness in all collections elevated in the early-run (12.21-12.58) compared with the late-run (11.27-11.72). But, the expected and observed heterozygosities were similar in early- (0.845-0.855, 0.835-0.850) and late-run (0.835-0.842, 0.818-0.841), respectively, without significant departure from Hardy-Weinberg equilibrium at all the examined loci in each collection. Pairwise F_ST estimates after Bonferroni corrections suggested genetic differentiation between early- and late-run fish in all collections. Such differentiation between early- and late-run cohorts was also observed within and/or among 3-, 4-, and 5-years olds, but not in 6-years old fish. These findings suggest a genetic heterogeneity owing to temporal breeding isolation even within the same year classes of homing chum salmon in the Tsugaruishi River.

Sex chromosomes are thought to be evolved from a pair of autosomes. Most mammals and birds have heteromorphic sex chromosomes with highly degenerated Y and W chromosome, respectively, due to suppression of recombination. Unlike mammals and birds, however, many fish present homomorphic sex chromosomes. In particular, a previous study in our laboratory showed that sex in fugu (Takifugu rubripes) is determined by a combination of the Amhr2X and Amhr2Y alleles, and implied that recombination between X and Y chromosomes has yet stopped. Thus, fugu provides a rare opportunity to study the evolution of SD gene in the very early phase.
To obtain insights into the early stage of allelic divergence of the sex-determining (SD) gene, we characterized the patterns of polymorphism within and between the Amhr2 gene of the X and Y chromosome in fugu. As the Amhr2X and Amhr2Y alleles differ in a single nucleotide in exon 9, we took advantage of this SNP site to obtain allele-specific DNA sequences from wild male individuals. We then performed population genetics analyses within and between the Amhr2X and Amhr2Y.
Our analysis revealed that the nucleotide diversity around the SNP site in exon9 among Amhr2Y alleles was very low compared to autosomal alleles. In the case of Amhr2X alleles, the diversity around the SD-SNP was relatively low. However, the magnitude was not extreme as that of Y alleles. We also found high peaks of divergence between X and Y alleles in a narrow window around the SNP site in exon 9. Taken together, these results imply that the intensity of selections acting on the Amhr2X and Amhr2Y alleles are different and that the affected region has been narrowed due to ongoing recombination between X and Y chromosomes.

In shrimp and other decapod crustaceans, ovarian maturation is under the control of various hormones, including vitellogenesis-inhibiting hormone (VIH), which inhibits vitellogenin (Vg) synthesis. Therefore, information on fluctuations in VIH and Vg levels in relation to ovarian maturation is important for understanding reproductive mechanisms in Crustacea. In previous reports, we measured VIH and Vg levels in the hemolymph in relation to molting, and examined the effects of eyestalk ablation in the whiteleg shrimp, Litopenaeus vannamei. It was found that hemolymph Vg levels increased after eyestalk ablation; however, VIH levels in the hemolymph did not decrease in a significant manner.
VIH is a member of the crustacean hyperglycemic hormone (CHH) family, and the existence of multiple CHH family peptides has been reported in several crustacean species. Our group previously isolated seven CHH family peptides in L. vannamei, which were named sinus gland peptides (SGP)-A to -G, and six peptides among these were reported to have VIH activity. SGP-G was presumed to be the major VIH in L. vannamei because it predominated in the eyestalks; it was also found to increase under conditions of stress.
In order to further clarify how VIH acts in accordance with maturation, we deemed it necessary to examine gene expression patterns together with hemolymph peptide levels. In this study, we aimed to determine the expression levels of five selected VIH genes in the eyestalks in relation to ovarian maturation in L. vannamei. Expression patterns for these VIH genes did not change significantly in adults; however, three VIH genes were significantly decreased in subadults after unilateral ablation. These results suggest that VIH acts with more efficacy in subadults than in adults, and that the endocrine pathways controlling reproduction in shrimp involve a number of factors in addition to VIH.

Ocean warming has been extending the geographic range of commercially important sea urchins to the high latitudes. In Toga Bay along Oga Penisula, Mesocentrotus nudus was replaced to Heliocidaris crassispina as a predominant sea urchin species. In this study, growth, gonad size, gonad development, gonad color and year-class composition of 106 individuals of H. crassispina collected in August 2014 were examined. Compared to the aboriginal H. crassispina in western regions, the growth was slightly slow, but the gonad indices were almost the same. While the ovaries of the species were in partly spawned or spent stages, the testes were in pre-matured stage. This implies the spawning asynchronous between both sexes. In addition, the acini with two to four different developmental stages were present in the testis of an individual. This was firstly observed in sea urchins. Lightness (L^*) of the gonads increased with the increase in gonad indices. Yellowness (b^*) of the gonads decreased with increasing ages. The population was consisted of the year classes from 2006 to 2012, suggesting the continuous juvenile recruitment. Increasing seawater temperature could be the cause of increased juvenile recruitment. It seems that marked high water temperature over 26°C in the summer of 2010 and 2012 caused mass mortality of M. nudus, and resulted in the predominance of H. crassispina. The results of the gonad examination suggest the possibility of the juvenile recruitment by the transport of larvae from the population in southwestern regions of the Sea of Japan.

The common East Asian octopus, Octopus sinensis d’Orbigny, 1841 (‘madako’), is very attractive for development as a commercially farmed species because of its highly prized taste and rapid growth rate, but its planktonic paralarval phase and subsequent settling stage are currently barriers to closing its life cycle for successful mariculture. Hemigrapsus is a brachyuran crustacean genus including small crabs (‘isogani,’ ‘iwagani’) endemic to Japanese coasts. Attention recently has been drawn particularly to H. sanguineus (de Haan, 1835) and H. takanoi Asakura & Watanabe, 2005, because in the last few decades they have become highly successful invasive species in both Europe and North America. Their effects are devastating on both endemic edible crab species and on mussel beds: they apparently out-compete much larger species such as Cancer pagurus; and they feed actively on coastal molluscs such as Mytilus spp. It is judged that Hemigrapsus species are a potential source of crab larvae and juvenile crabs to supply as feed to paralarvae and recently settled juveniles of O. sinensis. Reliable production of crab larvae on a year-round basis has appeal as a prey source superior to the use of organisms such as Artemia (on which octopus paralarvae will feed, but with which there seems to be major nutritional mis-match). Advantages of Hemigrapsus species as a crustacean feed source for octopuses include a lack of competition as target items for human seafood because of their small size, and their presumed hardiness in being able to out-compete larger species of crab. In addition, research on species in this genus may uncover ways to mitigate their invasive prowess. We here report on experiments (1) to raise them in captivity; (2) to extend their spawning season; and (3) to test their suitability as prey organisms for O. sinensis mariculture.

Sargassum seaweeds often form dense canopy beneath the water surface. We hypothesized that the canopy works as a huge trap of neustonic or planktonic larvae of benthic animals. Our preliminary survey showed the occurrence of high-density bivalve spats in the canopy of Sargassum horneri at Kitsunezaki coast of Oshika Peninsula, Miyagi, Japan in June 2015. Therefore, we conducted further surveys on the occurrence pattern of bivalve spats in the canopy of S. horneri in the area with time in 2016. Epifauna samples were collected by scuba diving once a month from January to June 2016. Samples were taken from four vertically different parts of S. horneri; Surface: constantly remained at sea surface, Intermediate: periodically exposed to the air or submerged underwater, Underwater: constantly underwater and Bottom: around the holdfast at the bottom. All epiphytic animals (> 0.1 mm) were classified into ten taxonomic groups and bivalve spats were sorted out from them. Seaweed substrates were dried at 80 °C for two days. Density of each taxonomic group was calculated as individual number per seaweed dry weight (g). For the bivalve spats, identification of species was conducted by the examination of DNA sequences. The total abundance of epifauna was rapidly increased from May 2016. Bivalve spats were observed in high density at ‘Surface’ along with the abundant copepod. The most dominant bivalve species was Mytilus galloprovincialis and only one individual of Clinocardium californiense, which is one of the fishery target species along the Tohoku coast, was also occurred. Our results suggested that the canopy of S. horneri sweeping around at the sea surface provided bivalve larvae and other epifaunal animals with the substrate for larval settlement. There could be a possibility of Sargassum beds to connect the planktonic or neustonic larval stages and the benthic stages of bivalves.

To recover the declining population of pen shells in nature, production of pen shell juveniles has been started by several research groups in Japan aiming for their release or aquaculture. However, artificial spawning induction of broodstocks by temperature manipulation is often unsuccessful, and development of artificial fertilization methods for pen shells are awaited. For successful fertilization, fully developed oocytes in an ovary should undergo oocyte maturation, which includes meiosis resumption. But artificial induction of pen shell oocyte maturation had been challenging since methods used in other bivalves were ineffective. In our study aimed to identify an oocyte maturation-inducing substance in pen shells, we have discovered retinoic acid (RA) to possess strong activity to induce oocyte maturation. This study was aimed to clarify the activity in detail and apply RA for artificial fertilization of pen shells. For the maturation-inducing activity assay, ovary fragments from pen shells in the spawning season were digested with 0.05% collagenase. Following digestion, fully developed oocytes were recovered by filtration and centrifugation. Treatment with 1.0 microM All-trans-RA (At-RA) induced germinal vesicle break down (GVBD), a typical morphological sign of the oocyte maturation, in approximately 60% of the oocytes. In contrast, 1.0 microM At-retinol, 1.0 microM At-retinal, 2 mM ammonia and 1.0 microM serotonin were ineffective. Treatment with At-RA for 30 min was sufficient to induce GVBD and was more potent than its isomers, 9-cis- and 13-cis-RA. Furthermore, oocytes obtained from ovaries in the ripe stage came to respond to 1.0 microM At-RA with more than 90% GVBD during the final development of the ovary. The treatment of oocytes with At-RA was found to be indispensable for successful artificial fertilization. Fertilized eggs also developed to the veliger stage without any apparent abnormality. These results indicate the possible application of RA for the artificial fertilization of pen shells.

Taurine is a major component in the tissues of marine invertebrates. Although functions of taurine in marine invertebrates are not fully understood yet, its involvement in osmotic adaptation has been suggested. In addition, hypotaurine, a precursor of taurine, is also suggested to play a role in adaptation to sulfide-rich environment, by binding to toxic hydrogen sulfide and producing non-toxic thiotaurine. We have studied the mechanisms of taurine and hypotaurine accumulation mainly using the hydrothermal-vent specific mussel Bathymodiolus septemdierum, and also the shallow-sea mussel Mytilus galloprovincialis for comparison. We have demonstrated that “taurine-transporter (TAUT)”, actively expressed in the gill of the vent mussel, can collect hypotaurine as well as taurine. We also found that cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSAD) genes are expressed at high levels in the gill of the vent mussel, suggesting active synthesis of hypotaurine there. In addition, we established a reverse HPLC system to detect taurine and its precursors with common free amino acids simultaneously, and found that hypotaurine content is higher in the vent species than in shallow-sea species. It was also found that free cysteine, which is recognized to be a starting material for hypotaurine/taurine synthesis in mammals, is undetectable in the gill of the vent mussel whereas it is detectable in shallow-sea species, suggesting higher CDO activity in the vent mussel. It has been shown that vent-specific mussels have been evolved from a shallow-sea mussel lineage. The vent species have utilized the taurine accumulation system that had existed in the shallow-sea ancestor but may have modified them to accumulate hypotaurine efficiently.

Bivalves inhabit a wide variety of environments including metal-deficient freshwater to metal-rich deep-sea hydrothermal vents. We hypothesize that metal regulation system is an important key for their high environmental adaptability, and DMT, divalent metal transporter 1, which is known that conserved many species from bacteria to mammals, and transport various divalent metal ions from outside to inside cells, plays important role. We have searched DMT genes in the hydrothermal vent-specific mussel Bathymodiolus septemdierum and discovered two DMT1-like cDNA fragments: one was a potential homolog of mammalian DMT1 but the other encoded new protein that is similar to but different from DMT and named the latter DMTRP (DMT-related protein). Both DMT and DMTRP have 12 transmembrane region and their N- and C- terminal protrude to cytoplasm. These are common characters among known DMTs. The sequence similarity between DMT and DMTRP are 50%. We also discovered these two genes from the genome database of the oyster Crassostrea gigas, snail lottia gigantea, amphioxus Branchiostoma floridae, sea urchin Strongylocentrotus purpuratus, acorn worm Saccoglossus kowalevskii, octopus Octopus bimaculoides, squid Loligo pealeii, and brachiopod Lingula anatine, but not found from Arthropoda and Vertebrata. Phylogenetic analysis revealed that DMT and DMTRP are diverged before divergence of deuterostomes and protostomes. These results suggest that retention of two types of DMTs is important to live in marine environment. We are trying to clarify the functions of DMT and DMTRP, looking at their differences in functions and usages. We will report functional differences of the two transporters, examined through the functional expression experiments using frog oocytes, tissue specificity of expression and metal accumulation, and comparative studies with shallow-water species.

As a common pathogen of abalone, Vibrio infection caused mass deaths of abalone and great economic losses. Among them, Vibrio parahaemolyticus is the most harmful pathogen in abalone aquaculture. It is well known that the disease resistance of abalone, as an invertebrate, is entirely dependent on the innate immune systems, including cellular and humoral responses. To date, little is known about the functional genes and proteins in anti-bacterial immune response of abalone. Our study showed that the expression level of matrix metalloproteinase 1 (MMP-1) gene was significantly upregulated in abalone hemocytes when challenged with Vibrio parahaemolyticus, indicating that MMP-1 might be engaged in the anti-Vibrio immune response of abalone. Besides, by using RNA interference technology, the effects of MMP-1 on Vibrio parahaemolyticus infection and immune related-physiological activities of abalone were explored. The results showed that MMP-1 deficiency significantly decreased infection and replication of Vibrio parahaemolyticus in vivo, while the apoptosis of abalone hemocytes was increased after MMP-1 knock-down. Furthermore, MMP-1 was also engaged in the humoral immunity of abalone. Therefore, our study was helpful to elucidate the mechanism of anti-pathogen immune response of abalone and served as an important pathogen-control reference for abalone aquaculture.

Hermaphrodite Corbicula fluminea clam have unusual reproduction mode, androgenesis. In the fertilized eggs, the all maternal chromosomes and centrosomes were extruded as two first polar bodies simultaneously. To reveal the process of unusual polar body formation we observed the oocytes labeled with α-,γ-tubulin antibody and rhodamine-phalloidine by confocal microscopy. The meiotic spindle organized near the animal pole was parallel to the oocyte surface at the Metaphase-I. Actin thin layer was uniformly distributed along the membrane of oocyte. At the anaphase the circular actin poor regions were appeared. In these two actin poor regions the meiotic half spindle were docked. The two bulges formed at anaphase on the actin poor region and all chromosomes and centrosomes were located within the bulges. The centrosome was located at the apical region of the bulge. The microtubules were radiated for the bulge base. At late anaphase the around the actin poor region and two actin rings became distinct. The diameter of these rings gradually decreased and the cytokinesis was occurred at the base of bulges. Thus, all centrosomes and chromosomes were extruded together with polar body. The present study suggests that centrosomes at the first meiosis could have same ability to approach and attach the membrane and induce polar body formation.